CN117946270A

CN117946270A - Anti-CD 93 antibodies and uses thereof

Info

Publication number: CN117946270A
Application number: CN202311817000.6A
Authority: CN
Inventors: 张苗; 周祥山; 董洁娴; 李静荣; 孙乐桥; 苏杭; 张婵; 于飞雁; 马领弟
Original assignee: China Resources Biomedical Co ltd
Current assignee: China Resources Biomedical Co ltd
Priority date: 2023-12-26
Filing date: 2023-12-26
Publication date: 2024-04-30

Abstract

The present disclosure provides an antibody or antigen binding fragment thereof that specifically binds CD93. The invention aims to screen and obtain an anti-CD 93 antibody molecule with stronger interaction activity between CD93 and IGFBP7 and/or MMRN2 through an antibody discovery technology, so as to obtain an anti-tumor medicament with better tumor inhibiting effect by promoting the normalization of tumor tissue blood vessels, inhibiting vascular leakage and increasing immune cell infiltration.

Description

Anti-CD 93 antibodies and uses thereof

Reference to sequence Listing

The present application comprises a sequence listing in computer readable form, which is incorporated herein by reference.

Technical Field

The present disclosure relates to the field of antibodies, and more particularly to antibodies to CD93 and uses thereof.

Background

CD93, cluster of Differentiation 93, is encoded by the CD93 gene. The human CD93 protein consists of 629 amino acids, the sequence of which is shown in SEQ ID NO:1 (Uniprot Q9NPY 3). CD93 belongs to the type I transmembrane glycoprotein of a family XIV protein, comprising a C-type lectin-like domain, an EGF-like repeat, a highly glycosylated mucin domain, a transmembrane domain and an intracellular domain. CD93 is expressed predominantly on endothelial cells, platelets, immature B cells and monocytes. CD93 is thought to be a downstream signaling factor for VEGF, playing an important role in regulating vascular normalization. Ligands that bind CD93, mainly IGFBP7 and MMRN-2 proteins. IGFBP7 is a secreted protein of the IGFBP family consisting of an IGF binding domain (IB) at the N-terminus, a Kazal-like serine protease inhibitor domain and an Ig-like C2 class domain (IgC 2) at the C-terminus. IGFBP7 is rarely expressed in human normal tissue blood vessels, but expression of tumor tissue vasculature is upregulated, e.g., head and neck cancerous tumor tissue. The up-regulation of the VEGF signaling pathway results in an anaerobic environment that induces IGFBP7 expression. MMRN-2 protein binds to a variety of group 14C lectin proteins, while IGFBP7 binds only CD 93. IGFBP7 does not affect CD93 expression, and MMRN-2 acts to stabilize the CD93 protein by inhibiting CD93 degradation. IGFBP7 and MMRN-2 did not have a competing relationship when bound to CD 93. The interaction of IGFBP7 with CD93 is mediated by the IB domain of IGFBP7 and the Epidermal Growth Factor (EGF) -like domain of CD93, respectively, whereas MMRN-2 interacts with the C-type lectin-like domain (CTLD) of CD 93. Through research of tumor-bearing mice animal models, the tumor infiltration of immune cells can be increased by blocking the interaction pathway of CD93 and IGFBP7, the normalization of blood vessels in tumor tissues is promoted, and the effect of inhibiting the growth of tumors is achieved. Meanwhile, the CD93 channel blocking and anti-tumor small molecule drugs or immune checkpoint drugs are combined to obtain the effect of enhancing tumor growth inhibition.

The VEGF channel targeted therapy plays a role in inhibiting tumor growth by taking inhibition of tumor angiogenesis as a core, and the CD93 signal channel is taken as a VEGF downstream signal channel molecule, so that an important role in normalizing blood vessels of tumor cells is played. In order to achieve the aim, the anti-tumor value of the combination can be increased by promoting the delivery of small molecular drugs in the blood vessels of solid tumors when the combination is combined with chemotherapeutic drugs. The blocking effect of the CD93 pathway is the expansion of VEGF through the vascular regulation of tumor cells.

Currently DyanmiCure Biotechnology LLC reports have found anti-human CD93 antibodies that block the interaction of CD93 and IGFBP7 or CD93 and MMRN 2. Although these antibodies showed blocking activity at high concentrations, the tumor inhibitory effect on tumor-bearing mice was not significant (WO 2022067262 A1). The technology aims at screening and obtaining anti-CD 93 antibody molecules with stronger interaction activities of blocking CD93 and IGFBP7 and/or MMRN2 through an antibody discovery technology, so as to obtain an anti-tumor drug with better tumor inhibiting effect by promoting the normalization of tumor tissue blood vessels, inhibiting vascular leakage and increasing immune cell infiltration.

Disclosure of Invention

In response to the above-described problems, the present disclosure provides antibodies, methods of making the same, compositions, and the like. The benefits provided by the present disclosure are broadly applicable to the fields of antibody therapy and diagnostics, and can be used in conjunction with antibodies that react with a variety of targets.

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

The heavy chain variable region comprises:

(i) HCDR1 comprising a sequence corresponding to SEQ ID NO: 12. 15, 18, 20, and 23, or a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity to SEQ ID NO: 12. 15, 18, 20 and 23;

(ii) HCDR2 comprising an amino acid sequence corresponding to SEQ ID NO: 13. 16, 19, 21, and 24, or a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity or consisting of SEQ ID NO: 13. 16, 19, 21 and 24; and

(Iii) HCDR3 comprising an amino acid sequence corresponding to SEQ ID NO: 14. 17, 22, and 25 or a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity or consisting of SEQ ID NO: 14. 17, 22, and 25;

the light chain variable region comprises:

(i) LCDR1 comprising a sequence corresponding to SEQ ID NO: 26. 29, 32 and 35 or a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity or consisting of SEQ ID NO: 26. 29, 32 and 35; and

(Ii) LCDR2 comprising a sequence corresponding to SEQ ID NO: 27. 30, 33 and 36 or a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity or consisting of SEQ ID NO: 27. 30, 33 and 36; and

(Iii) LCDR3 comprising a sequence corresponding to SEQ ID NO: 28. 31, 34 and 37 or a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity or consisting of SEQ ID NO: 28. 31, 34 and 37.

In some embodiments of the invention, the antibodies or fragments thereof include the following combinations:

(i) SEQ ID NO:12, HCDR1, SEQ ID NO:13, and HCDR2 as set forth in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:26, LCDR1, SEQ ID NO:27, and LCDR2 as set forth in SEQ ID NO: LCDR3 as shown at 28; or (b)

(Ii) SEQ ID NO:15, HCDR1, SEQ ID NO:16, and HCDR2 as set forth in SEQ ID NO:17, HCDR3, SEQ ID NO:29, LCDR1, SEQ ID NO:30, and LCDR2 as set forth in SEQ ID NO: LCDR3 as indicated at 31; or (b)

(Iii) SEQ ID NO:18, HCDR1, SEQ ID NO:19, and HCDR2 as set forth in SEQ ID NO:17, HCDR3, SEQ ID NO:29, LCDR1, SEQ ID NO:30, and LCDR2 as set forth in SEQ ID NO: LCDR3 as indicated at 31; or (b)

(Iv) SEQ ID NO:20, HCDR1 shown in SEQ ID NO:21, and HCDR2 as set forth in SEQ ID NO:22, HCDR3, SEQ ID NO:32, LCDR1, SEQ ID NO:33, and LCDR2 as set forth in SEQ ID NO: LCDR3 as shown at 34; or (b)

(V) SEQ ID NO:23, HCDR1, SEQ ID NO:24, and HCDR2 as set forth in SEQ ID NO:25, HCDR3 shown in SEQ ID NO:35, LCDR1, SEQ ID NO:36, and LCDR2 as set forth in SEQ ID NO: LCDR3 as shown at 37.

In some embodiments of the invention, wherein the heavy chain variable region comprises a sequence identical to SEQ ID NO: 2. 4, 6, 8 and 10, or 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 another or consisting of SEQ ID NO: 2. 4, 6, 8 and 10;

Wherein the light chain variable region comprises a sequence identical to SEQ ID NO: 3.5, 7, 9 and 11, or 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: 3.5, 7, 9 and 11.

In some embodiments of the invention, the antibody or fragment comprises one of the following combinations:

(i) A heavy chain variable region comprising a sequence identical to SEQ ID NO:2 or 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, composing;

A light chain variable region comprising a sequence identical to SEQ ID NO:3 or 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, composing;

(ii) A heavy chain variable region comprising a sequence identical to SEQ ID NO:4 or 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, the composition is formed;

A light chain variable region comprising a sequence identical to SEQ ID NO:5 or 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, composing;

(iii) A heavy chain variable region comprising a sequence identical to SEQ ID NO:6, or 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, composition;

a light chain variable region comprising a sequence identical to SEQ ID NO:7 has 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 or consists of SEQ ID NO:7, forming;

(iv) A heavy chain variable region comprising a sequence identical to SEQ ID NO:8 has 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 or consists of SEQ ID NO:8, the composition is formed;

A light chain variable region comprising a sequence identical to SEQ ID NO:9 has 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 or consists of SEQ ID NO:9, composition;

(v) A heavy chain variable region comprising a sequence identical to SEQ ID NO:10 or 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, the composition is as follows;

A light chain variable region comprising a sequence identical to SEQ ID NO:11 or 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.

In some embodiments of the 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 of antibodies and the light chain constant region is selected from the kappa or lambda chain.

In some embodiments of the invention, the antibody or fragment, wherein the antibody heavy chain constant region is preferably selected from one of IgG1, igG2c, igG2b, and the light chain constant region is preferably a kappa chain.

In some embodiments of the invention, the antibody or fragment, wherein the antibody is selected from the group consisting of: whole antibodies, bispecific antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies;

The fragment is selected from the group consisting of: fab fragments, fab' fragments, F (ab) ₂ fragments, fv fragments and ScFv.

In another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding said antibody or fragment.

In another aspect, the invention provides a vector comprising said nucleic acid molecule.

In another aspect, the invention provides a host cell comprising said nucleic acid molecule or said vector.

In another aspect, the invention provides a conjugate comprising said antibody or fragment coupled to at least one detectable label.

In another aspect, the invention also provides an antibody drug conjugate comprising an antibody comprising one or more drug moieties covalently linked to the antibody or fragment directly or via a linker.

In another aspect, the invention provides a multispecific molecule comprising an antibody or antigen-binding fragment of the invention; preferably, the multispecific molecule specifically binds CD93, and additionally specifically binds one or more other targets; further preferred, the multispecific molecule further comprises at least one molecule having a second binding specificity for a second target.

In another aspect, the invention provides a pharmaceutical composition or kit comprising said antibody or fragment, or said nucleic acid molecule, or said vector, or said host cell, or said conjugate, or said antibody drug conjugate, or said multispecific molecule, and a pharmaceutically acceptable carrier.

In another aspect, the invention also provides the use of said antibody or fragment, or said nucleic acid molecule, or said vector, or said host cell, or said conjugate, or said antibody drug conjugate, or said multispecific molecule, or said pharmaceutical composition or kit, in the preparation of a kit for diagnosing, detecting, or monitoring a disease associated with CD93 expression.

In another aspect, the invention also provides the use of said antibody or fragment, or said nucleic acid molecule, or said vector, or said host cell, or said conjugate, or said antibody drug conjugate, or said multispecific molecule, or said pharmaceutical composition or kit, in the manufacture of a medicament for treating a disease associated with CD93 expression or determining the prognosis thereof.

In another aspect, the invention provides said use, wherein said disease associated with CD93 expression is a cancer selected from the group consisting of: carcinoma species such as kidney cancer, pancreatic cancer, breast cancer, gastric cancer, glioma, etc.

Drawings

The accompanying drawings are included to provide a further understanding of the disclosure, and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure, without limitation to the disclosure.

FIG. 1 is a graph of the binding activity of an anti-human CD39 antibody to human CD93 overexpressing cells detected by flow cytometry, wherein the abscissa mAb coc. Log is the concentration of the antibody of the invention in nM and the ordinate MFI-PE is the mean fluorescence intensity;

FIG. 2 is a graph of the binding activity of anti-human CD39 antibodies to cynomolgus monkey CD93 overexpressing cells detected by flow cytometry, wherein the abscissa mAb coc.log is the concentration of the antibodies of the invention in nM and the ordinate MFI-PE is the mean fluorescence intensity;

FIG. 3 is a graph showing the binding activity of an anti-human CD39 antibody to a mouse CD93 overexpressing cell by flow cytometry, wherein the abscissa indicates the number of the antibody of the present invention and the ordinate indicates the mean fluorescence intensity of MFI-PE;

FIG. 4 is a flow cytometry detection of the binding specificity of an 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;

FIG. 5 is a graph of flow cytometry detection of anti-human CD39 antibodies blocking human CD 93/human IGFBP7 binding activity, wherein mAb coc.Log on the abscissa is the concentration of the antibodies of the invention in nM and MFI-PE on the ordinate is the mean fluorescence intensity;

FIG. 6 is a graph of flow cytometry detection of anti-human CD39 antibodies blocking human CD 93/human MMRN-2 binding activity, wherein the abscissa mAb coc. Log is the concentration of the antibodies of the present invention, in nM, and the ordinate MFI-PE is the mean fluorescence intensity.

Detailed Description

In order that those skilled in the art will better understand the present invention, a clear and complete description of the technical solutions of the examples of the present invention will be provided below, it being apparent that the examples described are merely illustrative of some, but not all, embodiments of the present invention. Therefore, the invention is not limited to the specific embodiments illustrated. Furthermore, any section headings used herein are not to be construed as limiting the subject matter described.

Unless otherwise defined herein, scientific and technical terms used in connection with the present application will have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, terms in the singular shall include the plural and terms in the plural shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In the present application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "include" and other forms (such as "include" and "contain") is not limiting. Furthermore, the scope provided in the specification and the appended claims includes all values between the endpoints and between the endpoints.

Definition of the definition

For a better understanding of the present invention, definitions and explanations of related terms are provided below.

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 chain of an antibody can be classified as either a kappa or lambda light chain. Heavy chains can be classified as mu, delta, gamma, alpha or epsilon, 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 linked to the constant region by a "J" region of about 12 or more amino acids, and the heavy chain also comprises 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 (CH 1, CH2 and CH 3). 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 complementarity determining regions, CDRs, for short) separated by relatively conserved regions (called framework regions, FR, for short). Each VH and VL consists of 3 CDRs and 4 FR in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from the N-terminal to the C-terminal. CDRs on VH are HCDR1, HCDR2 and HCDR3; the CDRs on VL are LCDR1, LCDR2, LCDR3. The variable regions (VH and VL) of each heavy/light chain pair form antigen binding sites/portions, respectively. The distribution of amino acids in various regions or domains follows the numbering definitions in common systems such as Kabat, IMGT or Chothia, and in particular embodiments of the present disclosure, the determination of CDR sequences uses the numbering definitions in Kabat systems.

Antibodies in the present disclosure also include antigen-binding portions (interchangeably used with the term "antigen-binding fragment"). An antigen binding portion refers to a polypeptide comprising a fragment of an intact antibody that retains the ability to specifically bind to an antigen to which the full length or intact antibody specifically binds, and/or that competes with the full length antibody for binding to the same antigen. In some cases, the antigen binding portion includes Fab, fab ', F (ab') ₂, fd, fv, dAb, and Complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and at least a portion of antibodies comprising sufficient antigen binding capacity to confer specificity 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 the specificity can be screened in the same manner as for an intact antibody.

The term "isotype" refers to the class of antibodies (e.g., igM or IgG 1) encoded by the heavy chain constant region gene.

The term "monoclonal antibody" or "mAb" refers to an antibody molecule/preparation consisting of single molecules. Monoclonal antibodies exhibit a single binding specificity and affinity for a particular epitope. Antibodies of the invention may be derived from different species including, but not limited to, mice, rats, rabbits, guinea pigs, and humans.

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

The term "chimeric antibody" as used herein refers to antibodies whose variable region sequences are from one species and constant region sequences are from another species, e.g., antibodies in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

The term "humanized antibody" is intended to refer to an antibody 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 a human framework sequence. In addition, additional framework region modifications can be made within the human framework sequence.

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

The terms "CD93" and "CD93 antigen" are used interchangeably herein to include any variant, isoform and species homolog of human CD93 that is naturally expressed by a cell or expressed on a cell transfected with the CD93 gene. In some embodiments, binding of an antibody of the present disclosure to CD93 antigen mediates killing of cells expressing CD93 (e.g., tumor cells) by inactivating CD 93. Killing of CD93 expressing cells may occur through one or more of the following mechanisms: cell death/apoptosis induction, ADCC and CDC.

The term "anti-CD 93 antibody" or "CD93 antibody" refers to an antibody as defined herein that is capable of binding to a CD93 antigen or to a cell expressing CD 93.

The term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed.

The term "isolated" refers to a state obtained from a natural state by manual means. If a certain "isolated" substance or component occurs naturally, it may be due to a change in its natural environment, or the substance is separated from the natural environment, or both. For example, a polynucleotide or polypeptide that is not isolated naturally occurs in a living animal, and the same polynucleotide or polypeptide in high purity isolated from that natural state is referred to as an isolated polynucleotide or polypeptide. The term "isolated" does not exclude mixed artificial or synthetic substances nor other impure substances that do not affect the activity of the isolated substances. For example, the isolated antibody may be substantially free of other cellular material and/or chemicals.

The term "vector" refers to a nucleic acid vector into which a polynucleotide may be inserted. When a vector allows expression of a protein encoded by a polynucleotide inserted therein, the vector is referred to as an expression vector. The vector may be transformed, transduced or transfected into a host cell to express the carried genetic material element in the host cell. Vectors are well known to those of skill in the art and include, but are not limited to, plasmids, phages, cosmids, artificial chromosomes such as Yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs); phages such as lambda phage or M13 phage and animal viruses. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV 40). The vector may contain a number of 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 comprise an origin of replication. For the vector expressing the antibody, a vector type in which the heavy chain and the light chain of the antibody are present in different vectors or a vector type in which the heavy chain and the light chain are present in the same vector may be used.

The term "host cell" refers to a cellular system that can be engineered to produce a protein, protein fragment, or peptide of interest. Host cells include, but are not limited to, cultured cells, e.g., mammalian cultured cells derived from rodents (rat, mouse, guinea pig, or hamster), such as CHO, BHK, NSO, SP/0, YB2/0; or human tissue or hybridoma cells, yeast cells, and insect cells, and cells contained within a transgenic animal or cultured tissue. The term encompasses not only the particular subject cell, but also the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term "host cell".

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 as determined by aligning and comparing the sequences. "percent identity" refers to the percentage of identical residues between amino acids or nucleotides in a comparison molecule and is calculated based on the size of the smallest molecule being compared. For these calculations, the gaps in the alignment (if any) are preferably addressed by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that may be used to calculate identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology,(Lesk,A.M.,ed.),1988,New York:Oxford University Press;Biocomputing Informatics and Genome Projects,(Smith,D.W.,ed.),1993,New York:AcademicPress;Computer Analysis of Sequence Data,Part I,(Griffin,A.M.,and Griffin,H.G.,eds.),1994,New Jersey:Humana Press;von Heinje,G.,1987,Sequence Analysisin Molecular Biology,New York:Academic Press;Sequence Analysis Primer,(Gribskov,M.and Devereux,J.,eds.),1991,New York:M.Stockton Press; and Carillo etal,1988,SIAMJ.Applied Math.48:1073.

The term "immunogenicity" refers to the ability to stimulate the formation of specific antibodies or sensitized lymphocytes in an organism. It refers not only to the nature 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 to the fact that specific immune responses of antibodies or sensitized T lymphocytes can develop in the immune system of an organism after stimulation of the organism with an antigen. Immunogenicity is the most important property of an antigen. Whether an antigen is able to 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 immunization.

The term "transfection" refers to the process of introducing nucleic acid into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipofection and chemical and physical methods such as electroporation. Numerous transfection techniques are well known in the art and are 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.

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.

The term "immune effector function" includes any function mediated by a component of the immune system that results in inhibition of tumor growth and/or inhibition of tumorigenesis, as well as inhibition of tumor dissemination and metastasis. Preferably, 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 bearing tumor-associated antigens (e.g., by binding of antibodies to surface antigens), and/or inhibition of proliferation of cells bearing tumor-associated antigens, preferably ADCC and/or CDC. Antibodies can also function simply by binding to tumor-associated antigens on the surface of tumor cells. For example, an antibody may block the function of a tumor-associated antigen or induce apoptosis simply by binding to a tumor-associated antigen on the surface of a tumor cell.

The term "cancer" refers to solid and non-solid tumors such as leukemia mediated by growth, proliferation or metastasis of any tumor or malignant cell that is responsible for a medical condition. For example, cancers associated with or caused by abnormal expression of CD93, including but not limited to: b-cell lymphomas, including NHL, pre-B-cell lymphocytic leukemia/lymphoma and mature B-cell neoplasms, 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-, medium-and high-grade FL, cutaneous follicular central lymphoma, marginal zone B-cell lymphoma (MALT type, intranode and spleen type), hairy cell leukemia, diffuse large B-cell lymphoma, burkitt's lymphoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, fahrenheit macroglobulinemia and Anaplastic Large Cell Lymphoma (ALCL)

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.

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 active agent, which 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 modifiers, surfactants, adjuvants, and ionic strength enhancers, e.g., pH modifiers include, but are not limited to, phosphate buffers, surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, e.g., tween-80, ionic strength enhancers include, but are not limited to, sodium chloride.

The term "adjuvant" refers to a non-specific immunopotentiator that, when delivered to an organism with an antigen or delivered to an organism in advance, can enhance the immune response to an antigen or alter the type of immune response in an organism. There are various adjuvants including, but not limited to, aluminum adjuvants (e.g., aluminum hydroxide), freund's adjuvants (e.g., freund's complete adjuvant and Freund's incomplete adjuvant), corynebacterium parvum, lipopolysaccharide, cytokines, and the like. Freund's adjuvant is the most commonly used adjuvant in current animal experiments. Aluminum hydroxide adjuvants are more commonly used in clinical trials.

Anti-CD 93 antibodies

In some aspects, the invention includes an isolated antibody or antigen-binding fragment thereof.

In the context of the present application, "antibody" may include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized and primate 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 muteins and variants thereof, modified antibodies; and derivatives thereof (including Fc fusion proteins and other modifications), as well as any other immunoreactive molecules, so long as they exhibit preferential association or binding with CD93 protein. Furthermore, unless the context dictates otherwise, the term also includes all classes of antibodies (i.e., igA, igD, igE, igG and IgM) and all subclasses (i.e., igG1, igG2, igG3, igG4, igA1, and IgA 2). 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 a modified chimeric monoclonal antibody.

The variable regions and CDRs in an antibody sequence can be identified according to the general rules already developed in the art (as described above, e.g., kabat) numbering system or by aligning the sequences with a database of known variable regions.

Regardless of how the antibody is produced, methods for testing the ability of the antibody to bind to an antigen (e.g., CD 93) are known in the art and include any antibody-antigen binding assay, such as Radioimmunoassays (RIA), ELISA, western blots, immunoprecipitation, SPR, and competitive inhibition assays (see, e.g., janeway et al, below and U.S. patent application publication No. 2002/0197266, and the above section on competitive assays).

According to the invention, in standard assays (e.g., the assays described in the present invention), if an antibody has significant affinity to a predetermined target (e.g., CD93 protein or CD93 expressing cells), the antibody is able to bind to the predetermined target, flow Cytometry (FCM) may be used in order to test the binding of monoclonal antibodies to living cells expressing CD 93. Preferably, in flow cytometric fluorescence sorting (FACS) analysis, the binding of the antibody to a target expressed on the cell surface is determined, and the antibody is capable of binding to the target with an "affinity" if it binds detectably to the target (CD 93 protein or CD93 expressing cells).

The CD93 specificity according to the present invention means that it is capable of binding to one or more CD93 epitopes, in particular CD93 epitopes of the native conformation, in particular human CD93 specificity.

The engineering of desired properties of an antibody without altering the properties is varied in the art by various means, such as the manner in which the light and heavy chains of an antibody are recombined, amino acid substitutions made, etc., as employed in the present disclosure. For example, sequences of the invention, including chimeric or humanized antibody sequences, may be subjected to conservative amino acid substitutions.

Antibodies interact with target antigens primarily through amino acid residues located in the Complementarity Determining Regions (CDRs) of the six heavy and light chains. 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 particular naturally occurring antibody by constructing expression vectors that contain CDR sequences from the particular naturally occurring antibody that are 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) Nature321:522-525; and Queen, C. Et al (1989) Proc. Natl. Acad. Sci. U.S.A.86:10029-10033). Such framework sequences may be obtained from public DNA databases including germline antibody gene sequences. These lines are sequences that differ from the mature antibody gene sequence in that they do not contain the fully assembled variable genes that are formed by V (D) J ligation during B cell maturation. Germline gene sequences will also have sequences that differ from the high affinity second antibody repertoire (secondary repertoire antibody) at individual points that are uniform across the variable region.

Mouse antibodies are highly immunogenic in humans, resulting in reduced therapeutic efficacy when repeatedly applied, with major immunogenicity mediated by the heavy chain constant region. If the individual antibodies are chimeric or humanized, the immunogenicity of the mouse antibodies in humans can be reduced or completely avoided.

Chimeric antibodies refer to antibodies in which different portions are derived from different animal species, e.g., antibodies having variable regions derived from mouse antibodies and human immunoglobulin constant regions. The variable regions of the heavy and light chains of the mouse antibodies are joined to the constant regions of the human heavy and light chains to obtain chimerism of the antibodies (e.g., as described in Kraus et al in Methods in MolecularBiology series, recombinant antibodies for CANCER THERAPY ISBN-0-89603-918-8). In a preferred embodiment, the chimeric antibody is produced by ligating a human kappa light chain constant region to a mouse light chain variable region. In another preferred embodiment, chimeric antibodies can be produced by linking a human lambda light chain constant region to a mouse light chain variable region.

By humanized antibody is meant an antibody in which CDR sequences/antigen binding portions or sites derived from the germline of another mammalian species, such as a mouse, are grafted onto a human framework sequence.

In order to reduce immunogenicity of antibodies to humans, humanized anti-CD 93 antibodies are produced using the sequences of the CD93 antibodies of the present disclosure, utilizing the CDR regions of murine anti-CD 93 antibodies in combination with human framework regions (e.g., human immunoglobulins) to form the humanized anti-CD 93 antibodies of the present disclosure, the humanized antibodies are expected to retain the function of binding to human CD93 as well as the function of binding to monkey CD 93.

Preparation or production of antibodies

Antibodies of the invention can be produced by a variety of techniques, including conventional monoclonal antibody methods, such as Kohler AND MILSTEIN, nature256:495 The standard somatic hybridization technique of (1975), and other techniques for producing monoclonal antibodies, such as viral or oncogene transformed B lymphocytes or phage display techniques using antibody gene libraries, somatic hybridization, and in turn, for example, by genetic engineering recombinant techniques, may be employed. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the invention are obtained by chemical synthesis or PCR amplification, the resulting DNA molecules are inserted into an expression vector, then the host cells are transfected, then the transfected host cells are cultured under specific conditions, and the antibodies of the invention are expressed.

Other preferred animal systems for the preparation of monoclonal antibody secreting hybridomas are the rat and rabbit systems (e.g., described in Spieker-Poletetal, proc. Natl. Acad. Sci. U.S. A.92:9348 (1995), see also Rossietal. Am. J. Clin. Pathol.124:295 (2005)). Hybridoma production in mice is a very well established method. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion methods are also known.

Monoclonal antibodies may be prepared using a variety of techniques known in the art, including hybridoma techniques, recombinant techniques, phage display techniques, transgenic animals, or some combination thereof. For example, hybridomas and art-recognized biochemical and genetic engineering techniques can be used to produce monoclonal Antibodies, as described in detail in An,Zhiqiang(ed.)Therapeutic Monoclonal Antibodies:From Bench to Clinic,JohnWiley and Sons,1st ed.2009;Shire et.al.(eds.)Current Trends in Monoclonal Antibody Development and Manufacturing,Springer Science+Business Media LLC,1sted.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, n.y., 1981), each of which is incorporated herein by reference in its entirety.

It will be appreciated that the selected binding sequences may be further altered, e.g., to increase affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to produce multispecific antibodies, and the like, and antibodies comprising altered target binding sequences are also antibodies of the invention.

In some embodiments, the method of producing an antibody or fragment described in the present disclosure comprises the steps of:

(i) Expressing the antibody or fragment in a host cell; and optionally

(Ii) Isolating the antibody or antigen binding fragment thereof from the host cell.

In a preferred embodiment, anti-CD 93 monoclonal antibodies are prepared by using hybridomas.

To obtain hybridomas producing antibodies of the invention, e.g., 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, e.g., a mouse myeloma cell line. The hybridomas produced are screened for the production of antigen-specific antibodies. The generation of hybridomas is well known in the art. See, e.g., harlow and Lane (1988) Antibodies, A Laboratory Manual, cold Spring Harbor Publications, new York.

Antibodies of the invention may also be produced in host cell transfectomas using, for example, a combination of recombinant DNA techniques and gene transfection methods well known in the art (e.g., morrison, s. (1985) Science 229:1202). In some embodiments, DNA encoding part or full length light and heavy chains obtained by standard molecular biology techniques is inserted into one or more expression vectors, such 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 genes are linked into a vector such that transcriptional and translational control sequences within the vector perform their intended functions of regulating the transcription and translation of the antibody genes.

The antibody light chain gene and the antibody heavy chain gene may be inserted into the same or different expression vectors. In some embodiments, the variable region is used to generate a full length antibody gene of any antibody isotype by inserting it into an expression vector that already encodes the heavy and light chain constant regions of the desired isotype, such that the VH segment is operably linked to the CH segment and the VL segment within the vector, and the CL segment within the vector. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene may be cloned into a vector such that the signal peptide is linked to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

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 the various techniques commonly used for introducing exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Antibodies of the invention can be expressed in prokaryotic or eukaryotic host cells, such as mammalian host cells, which can assemble and secrete properly folded and immunocompetent antibodies.

Mammalian host cells for expression of 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), NSO myeloma cells, COS cells and SP2 cells for use with a DHFR selection marker (e.g., as described in R.J. Kaufman and P.A. Sharp (1982) J.MoI.biol.159:601-621). 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 expression of the antibody in the host cell or by secreting the antibody into the medium in which the host cell is grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

In another preferred embodiment, transgenic or transchromosomal mice with a partially human immune system (rather than a mouse system) may be used to generate human monoclonal antibodies to CD 93.

Another strategy for generating monoclonal antibodies is to isolate the genes encoding the antibodies directly from the antibody-producing lymphocytes of the defined strategy, see for example Babcocketal.,1996;Anovel strategy for generating monoclonal antibodies from single,isolated lymphocytes producing antibodies of defined strategy. for details of recombinant antibody engineering and also see Welschof and Krau,Recombinant antibodes for cancer therapy ISBN-0-89603-918-8and Benny K.C.Lo Antibody Engineering ISBN 1-58829-092-1.

To prepare chimeric antibodies, the murine immunoglobulin variable region can be linked to a human immunoglobulin constant region using methods known in the art (see, e.g., U.S. Pat. No. 4,816,567 to Capilli et al). An isolated nucleic acid encoding a VH region can be converted to a full length heavy chain gene by operably linking the nucleic acid encoding the VH region to another DNA molecule encoding a heavy chain constant region (CH 1, CH2, and CH 3). The sequence of a human heavy chain constant region gene is known in the art (see, e.g., kabat et al (1991),Sequences Of Proteins of Immunological Interest,Fifth Edition,U.S.Department of Health and Human Services,NIH Publication No.91-3242). heavy chain constant region may 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 may be converted to a full-length light chain gene (and Fab light chain gene) by operably linking the DNA encoding the VL to another DNA molecule encoding the light chain constant region CL, the sequence of a human light chain constant region gene is known in the art (see, e.g., kabat et al supra), and DNA fragments comprising these regions may be obtained by standard PCR amplification.

To prepare humanized antibodies, murine CDR regions can be inserted into human framework sequences using methods known in the art (see Winter, U.S. Pat. nos. 5,225,539; U.S. Pat. No. 5,530,101 to Queen et al, U.S. Pat. No. 5,585,089, U.S. Pat. No. 5,693,762, and Lo,Benny,K.C.,editor,in Antibody Engineering:Methods and Protocols,volume 248,Humana Press,New Jersey,2004)., or alternatively transgenic animals that are capable of producing no endogenous immunoglobulins after immunization and that are capable of producing a complete human antibody repertoire have been reported, for example, to completely inhibit endogenous antibody production in chimeric and germ line mutant mice, and then transferring an array of human germ line immunoglobulin genes to the germ line mutant mice would result in the mice producing human antibodies upon antigen stimulation (see, e.g., jakobovits et al, 1993,Proc.Natl.Acad.Sci.USA 90:2551;Jakobovits et al, 1993, nature362:255-258; brugmann et al, 1993,Year in Immunology 7:33; and Duchosal et al, 1992,Nature 355:258). Non-limiting examples of such transgenic animals include, humab mice (Medarex, inc.), which contain human immunoglobulin gene loci encoding unrearranged heavy (μ and γ) and kappa chain immunoglobulin sequences, and a gene locus (see also WO 35:3535:3535, and F.19935, et al, and a gene locus (see, e.g., moomu.19935:3535, 1997, F.1997) carrying a gene map (see, J.35:3593, F.1997, F.),3535, and F.),murine (see, J.1991, J.),35F.),35, F.),35, and F. (see also F.),1996, F.), nature Biotech 14: 309).

Nucleic acid molecules encoding antibodies of the invention

In some aspects, the invention relates to isolated nucleic acid molecules comprising a nucleic acid sequence encoding an isolated antibody or fragment thereof as described above in the present disclosure.

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

To prepare chimeric antibodies, the murine immunoglobulin variable region can be linked to a human immunoglobulin constant region using methods known in the art (see, e.g., U.S. Pat. No.4,816,567 to Capilli et al). By operably linking a nucleic acid encoding a VH to another DNA molecule encoding heavy chain constant regions (CH 1, CH2 and CH 3), the isolated nucleic acid encoding the VH region can be converted to a full length heavy chain gene, and DNA fragments comprising these regions can be obtained by standard PCR amplification. The isolated nucleic acid encoding the VL region can be converted to 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. 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 genes into full-length antibody chain genes, fab fragment genes or scFv genes. In these operations, a DNA fragment encoding a VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or flexible linker.

Conjugate(s)

In one aspect, the present disclosure provides a conjugate comprising an antibody or fragment thereof as described above coupled to at least one detectable label. Detectable labels include, but are not limited to: (i) providing a detectable signal; (ii) Interaction with a second label to modify a detectable signal provided by the first or second label, such as FRET (fluorescence resonance energy transfer ); (iii) The mobility (e.g., electrophoretic mobility) is affected by charge, hydrophobicity, shape, or other physical parameters, or (iv) a capture moiety is provided, such as affinity, antibody/antigen, or ion complexation.

Suitable structures for the label are e.g. fluorescent labels, luminescent labels, chromophore labels, radioisotope labels, isotopic labels, preferably stable isotopic labels, isobaric labels (isobaric label), enzymatic labels (e.g. horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), particle labels (especially metal particle labels, magnetic particle labels, polymer particle labels), organic small molecules (e.g. biotin, ligands or binding molecules of receptors (e.g. cell adhesion proteins or lecithins), label sequences comprising nucleic acid and/or amino acid residues detectable by use of binding agents, etc. labels include, but are not limited to, barium sulfate, ioxitic acid, iodic acid, calcium amiodarone, sodium diatrizoate, meglumine, methoxamine, caseinate and radiodiagnostic agents (including positron emitters, (e.g. fluoro-18 and carbon-11), gamma emitters (e.g. iodo-123, iodo-125, iodo-99 m, iodo-111) and fluoro-131), fluorescent substances (e.g. g. fluoro-co-1, and co-channel-luminescent substances) and fluorescent substances (e.g. g. gold-shadow-emitting substances).

The detectable labels described above can be detected by methods known in the art. For example, fluorescent markers may be detected using a photodetector to detect the emitted light. Enzyme labels are typically detected by providing a substrate to an enzyme and detecting a 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 immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In certain embodiments, a detectable label as described above may be attached to an antibody or antigen binding fragment thereof of the invention by linkers of different lengths to reduce potential steric hindrance.

Antibody drug conjugates/immunoconjugates

In one aspect, the present disclosure provides an antibody drug conjugate comprising an antibody comprising one or more drug moieties/therapeutic agents linked (e.g., covalently linked) directly or via a linker to an antibody or fragment thereof as described previously. In the antibody-drug conjugate of the present application, the linker structure for conjugating the anti-CD 93 antibody to the drug is not particularly limited, as long as the resulting antibody-drug conjugate can be used.

Because of the ability of the antibody-drug conjugates to selectively deliver one or more drugs to a target tissue (e.g., a tumor-associated antigen, such as a CD 93-expressing tumor), the antibody-drug conjugates can increase the therapeutic efficacy of the antibodies or antigen-binding fragments thereof of the invention in treating a disease (e.g., cancer).

Multispecific molecules

The antibodies or antigen binding fragments thereof of the invention can be used to form multispecific molecules (e.g., bispecific molecules). The antibodies or antigen binding fragments thereof of the invention may be part of a multispecific molecule (e.g., a bispecific molecule) that comprises a second functional moiety (e.g., a second antibody) or a third functional moiety (e.g., a third antibody) having a binding specificity different from the antibodies or antigen binding fragments thereof of the invention, thereby being capable of binding to at least two different binding sites and/or target molecules. For example, an antibody or antigen-binding fragment thereof of the invention may be linked to a second antibody or antigen-binding fragment thereof capable of specifically binding any protein that may be used as a potential target for combination therapy. To produce the bispecific or multispecific molecules, an antibody or antigen-binding fragment thereof of the invention may be linked (e.g., by chemical coupling, gene fusion, non-covalent association, or other means) to one or more other binding molecules (e.g., additional antibodies, antibody fragments, peptides, or binding mimics).

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

In certain preferred embodiments, the multispecific molecule specifically binds CD93 (e.g., human CD93 or monkey CD 93) and specifically binds one or more other targets.

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

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

Pharmaceutical composition

In some aspects, the present disclosure provides a pharmaceutical composition or kit comprising an antibody or fragment as described above, a nucleic acid molecule as described above, a vector as described above, a host cell as described above, a conjugate as described above, an antibody drug conjugate as described above, a multispecific molecule as described above; and a pharmaceutically acceptable carrier.

The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical compositions of the invention may also be administered in combination with, for example, another immunostimulant, anticancer agent, antiviral agent, or vaccine, such that the anti-CD 93 antibody enhances the immune response to the vaccine. Pharmaceutically acceptable carriers can include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous media, nonaqueous 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.

Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavouring agents, thickening agents, colouring agents, emulsifying agents or stabilizing agents such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylmethylanisole, butylated hydroxytoluene and/or propyl arsenate. As disclosed herein, the antibodies or antigen-binding fragments thereof of the disclosed compositions may be oxidized in a solvent containing one or more antioxidants, such as methionine, that reduce the antibodies or antigen-binding fragments thereof. Redox can prevent or reduce the decrease in binding affinity, thereby enhancing antibody stability and extending shelf life. Thus, in some embodiments, the invention provides compositions comprising one or more antibodies or antigen binding fragments thereof and one or more antioxidants, such as methionine. The invention further provides methods wherein the antibody or antigen-binding fragment thereof is admixed with one or more antioxidants, such as methionine, such that the antibody or antigen-binding fragment thereof may be protected from oxidation to extend its shelf life and/or increase activity.

For further illustration, pharmaceutically acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactate ringer's injection, non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil or peanut oil, antimicrobial agents of bacteriostatic or fungistatic concentration, isotonic agents such as sodium chloride or glucose, 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 carboxymethyl cellulose, hydroxypropyl methylcellulose or polyvinylpyrrolidone, emulsifying agents such as polysorbate 80 (TWEEN-80), sequestering or chelating agents such as EDTA (ethylenediamine tetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. The antimicrobial agent used as a carrier may be added to a pharmaceutical composition in a multi-dose container containing phenols or cresols, mercuric preparations, benzyl alcohol, chlorobutanol, methyl and propyl parahydroxybenzoates, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.

Administration, formulation and dosage

The pharmaceutical compositions of the invention may be administered to a subject in need thereof in vivo by a variety of routes including, but not limited to, oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal and intrathecal, or by implantation or inhalation. The compositions of the present invention may be formulated as solid, semi-solid, liquid or gaseous forms of formulation; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants and aerosols. The appropriate formulation and route of administration may be selected depending upon the intended application and treatment regimen.

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

Formulations suitable for parenteral administration (e.g., by injection) include aqueous or nonaqueous, 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, thickening agents and solutes which render the formulation isotonic with the blood (or other relevant body fluids) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of isotonic carriers suitable for use in such formulations include sodium chloride injection, ringer's solution or lactated ringer's injection. Similarly, the particular dosage regimen (including dosage, time and repetition) will depend on the particular individual and medical history of the individual, and empirical considerations such as pharmacokinetics (e.g., half-life, clearance rate, etc.).

The requirements for effective pharmaceutical carriers for injectable formulations/compositions are well known to those of ordinary skill in the art (see, e.g., pharmaceuticals AND PHARMACY PRACTICE, J.B.LIPPINCOTT Company, philadelphia, pa., banker and Chalmers editions, pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, toissel, 4 th edition, pages 622-630 (1986)).

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

Those skilled in the art will appreciate that the appropriate dosage may vary from patient to patient. Determining the optimal dose generally involves balancing the level of therapeutic benefit with any risk or adverse side effects. The dosage level selected will depend on a variety of factors including, but not limited to, the activity of the particular compound, the administration, the time of administration, the rate of clearance of the compound, the duration of treatment, other co-administered drugs, compounds and/or materials, the severity of the condition, as well as the species, sex, age, weight, condition, general health and previous medical history of the patient. The dosage is typically selected to achieve a local concentration at the site of action that achieves the desired effect without causing substantial deleterious or adverse side effects.

In general, the antibodies or antigen-binding fragments thereof of the invention may be administered in a variety of ranges.

In certain preferred embodiments, the course of treatment involving an antibody or antigen-binding fragment thereof of the invention will comprise 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 invention may be administered daily, every two days, every four days, weekly, every ten days, every two weeks, every three weeks, monthly, every six weeks, every two months, every ten weeks, or every three months. In this regard, it is understood that the dosage or adjustment interval may be varied based on patient response and clinical practice.

A compatible formulation for parenteral administration (e.g., intravenous injection) will comprise an antibody or antigen-binding fragment thereof as disclosed herein at a concentration of about 5 μg/mL to about 100 mg/mL.

The antibodies of the invention may be co-administered with one or more other therapeutic agents (e.g., cytotoxic agents, radiopharmaceuticals, antineoplastic agents, anti-angiogenic agents, or and immunosuppressants) to reduce induction of an immune response against the antibodies of the invention. The antibody may be linked to the therapeutic agent (as an immune complex) or may be administered separately from the therapeutic agent.

In the context of administration of 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 of treatments or therapeutic agents administered to a subject. The treatment or therapeutic agent may be administered prior to, concurrently with, or after the administration of the second treatment or therapeutic agent to the patient. Preferably, the therapeutic or therapeutic agents are administered to the subject in a sequence, amount, and/or over a time interval such that the therapeutic or therapeutic agents may act together. In a particular embodiment, the therapeutic or therapeutic agents are administered to the subject in a sequence, amount, and/or over a time interval such that they provide increased benefits over if administered otherwise (particularly independently of each other). Preferably, the added benefit is a synergistic effect.

Medical application

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

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, cattle, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., and farm animals such as zoo animals.

Treatment of disorders associated with CD93 expression

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

As described herein, the antibodies of the present disclosure have one or more activities that can therapeutically apply killer cells and/or inhibit cells. In particular, killing cells, inhibiting proliferation of cells, and/or inhibiting colony formation of cells can be used to treat or prevent cancer (including cancer metastasis). Inhibition of cell proliferation, colony formation and/or metastasis may be employed, inter alia, for the treatment or prevention of cancer metastasis and metastatic spread of cancer cells.

In some aspects, the disclosure provides a method for treating or determining prognosis of a disease associated with CD93 expression in a subject, comprising administering to a subject in need thereof 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.

In some aspects, the 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 of treating a disease associated with CD93 expression or determining the prognosis thereof in a subject.

In some aspects, the disclosure provides the 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 a reagent (or drug) for treating a disease associated with CD93 expression or determining the prognosis thereof.

In one embodiment, the disease associated with CD93 expression comprises a neoplastic disease, such as cancer.

The antibody or antigen binding fragment thereof may be used alone as a monotherapy or may be used in combination with chemotherapy or radiation therapy.

The antibody or antigen binding fragment thereof may be used in combination with an anticancer agent, a cytotoxic agent, or a chemotherapeutic agent.

The term "anti-cancer agent" or "antiproliferative agent" means any agent that can be used to treat cell proliferative disorders such as cancer, and includes, but is not limited to, cytotoxic agents, cytostatic agents, anti-angiogenic agents, radiation therapy and radiation therapeutic agents, targeted anti-cancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormonal therapy, radiation therapy, anti-metastatic agents and immunotherapeutic agents. It will be appreciated that in selected embodiments as described above, such anti-cancer agents may comprise conjugates and may be conjugated to the disclosed site-specific antibodies prior to administration. More specifically, in certain embodiments, a selected anti-cancer agent is linked to a unpaired cysteine of an engineered antibody to provide an engineered conjugate as described herein. Thus, such engineered conjugates are expressly contemplated as being within the scope of the present invention. In other embodiments, the disclosed anti-cancer agents will be administered in combination with site-specific conjugates comprising different therapeutic agents as described above.

Diagnosis of

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

In some aspects, the disclosure provides a method of diagnosing, detecting, or monitoring a disease associated with CD93 expression, comprising administering to a subject in need thereof 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.

In some aspects, the 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 of diagnosing, detecting, or monitoring a disease associated with CD93 expression in a subject.

In yet another aspect, the present disclosure provides the 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 a reagent (or drug) for diagnosing, detecting, or monitoring a disease associated with CD93 expression.

Samples may be analyzed by a variety of assays, such as radioimmunoassays, enzyme immunoassays (e.g., ELISA), competitive binding assays, fluorescent immunoassays, immunoblot assays, western blot analysis, and flow cytometry assays. Compatible in vivo diagnostic or diagnostic assays may include imaging or monitoring techniques known in the art, such as magnetic resonance imaging, computerized tomography (e.g., CAT scan), positron emission tomography (e.g., PET scan), radiography, ultrasound, and the like, as known to those skilled in the art.

The methods described herein for detecting or monitoring CD93 expression or the level of cells expressing CD93 in vitro may also be used for non-diagnostic purposes.

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

The sample from the subject is blood, fecal matter (urine or faeces), oral or nasal secretions, or alveolar lavage, interstitial fluid, sweat or extracts thereof from the subject.

Pharmaceutical package and kit

Pharmaceutical packages and kits comprising one or more containers of one or more doses of the antibodies or antigen binding fragments thereof are also provided. In certain embodiments, unit doses are provided, wherein the unit doses contain a predetermined amount of a composition comprising, for example, an antibody or antigen-binding fragment thereof, with or without one or more other agents. For other embodiments, such unit doses are supplied in single use, pre-filled syringes. In other embodiments, the compositions contained in the unit dose may comprise saline, sucrose, or the like; buffers such as phosphates and the like; and/or formulated in a stable and effective pH range. Or in certain embodiments, the conjugate composition may be provided as a lyophilized powder, which may be reconstituted upon addition of a suitable liquid (e.g., sterile water or saline solution). 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 to be used to treat the selected neoplastic disease condition.

Such kits typically comprise a pharmaceutically acceptable formulation of the engineered conjugate in a suitable container, and optionally one or more anticancer agents or other agents in the same or different containers. The kit may also contain other pharmaceutically acceptable formulations for diagnostic or combination therapy.

More specifically, the kits may have a single container containing the antibodies or antigen binding fragments thereof of the disclosure, with or without additional components, or they may have different containers for each desired agent. Where a combination therapeutic agent is provided for conjugation, a single solution may be pre-mixed in molar equivalent combination or with more of one component than the other. Or the conjugate of the kit and any optional anticancer agent may be stored separately in separate containers prior to administration to the patient. The kit may further comprise a second/third container means for holding a sterile pharmaceutically acceptable buffer or other diluent, such as bacteriostatic water for injection (BWFI), phosphate Buffered Saline (PBS), ringer's solution and dextrose solution.

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 a dry powder. When the reagents or components are provided in dry powder form, the powder may be reconstituted by the addition of a suitable solvent. It is contemplated that the solvent may also be provided in another container.

Examples

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

Example 1 production of mouse anti-human CD93 antibodies

50-25 Μg human CD93 extracellular domain fusion protein carrying human Fc tag (supplied by Ruiti Chemie) was intraperitoneally injected with 5 SJL mice (supplied by Ruiti Chemie) and 5 Balb/c mice (supplied by Ruiti Chemie), together with Mn ²⁺, immunized once a week, serum was collected from the mice after immunization twice, and serum titer was measured. Gene gun immunization was performed using 5 SJL mice and 5 Balb/c mice, with 4. Mu.g of plasmid pCP-hCD93FL (supplied by Dairy Chemie) encoding full-length human CD93, together with granulocyte-macrophage colony-stimulating factor GM-CSF (R & D, cat.215-GM-050/CF) and Mn ²⁺, once every week or two weeks, and after two immunizations, mouse serum was collected for serum titer detection. When the serum titer detection meets the fusion requirement, taking the lymph node, spleen or bone marrow of the mouse for cell fusion.

The collected spleen cells were mixed with mouse myeloma cells SP2/0 (supplied by Ruiy Chemie) and subjected to cell fusion by a high-efficiency electrofusion technique. The fused cells were diluted into DMEM medium containing HT, plated in 96-well plates in proportion, and the 96-well plates were incubated overnight in a 5% co ₂, 37 ℃ incubator, after 24 hours, with DMEM medium containing 2 xHAT.

After 10-14 days, the supernatant of the hybridoma cells of the 96-well plate is taken for an ELISA binding experiment. The procedure is briefly described below, the human CD93 recombinant protein was diluted to 1. Mu.g/ml with CB buffer (Sigma, cat. C3041), mixed well, added to 96-well plates at 100. Mu.l/well, and left to stand at 2-8℃for coating overnight. After washing the plate 3 times with 1xPBS containing 0.05% Tween-20, i.e., 1xPBST, 1xPBS blocking solution containing 1% BSA was added at 300. Mu.l/well, and incubated at 37℃for 2 hours. After washing the plates 3 times with 1xPBST, hybridoma cell supernatants were added, 100. Mu.l/well, and incubated at 37℃for 1 hour. After washing 3 times with 1xPBST, HRP-labeled goat anti-mouse IgG (Invitrogen, cat. G-21040) diluted with 1xPBS was added at 100. Mu.l/well, and incubated at 37℃for 1 hour. After 3 washes with 1xPBST, TMB color development solution (Biyun day, cat. P0209) was added at 100. Mu.l/well, and after incubation at 37℃for 10min, ELISA stop solution (Soy pal, cat. C1058) was added at 100. Mu.l/well. The 96-well plate was placed in an microplate reader for OD450nm reading. Positive clones obtained from the primary screening were expanded to 24-well plates and subjected to human CD93 binding rescreening. Clones showing positive in this experiment will be preferentially used for subcloning in order to obtain stable monoclonal hybridoma cell lines, and the subcloning plates are again subjected to primary and secondary screening.

The selected hybridoma cell line was further expanded into a 100ml flask for production, and the supernatant was collected and purified by a protein A affinity column. Antibody concentration was measured by a280 and purity by polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions and size exclusion chromatography (SEC-HPLC). The analysis of purity of anti-human CD93 monoclonal antibodies using SEC-HPLC is shown in table 1.

TABLE 1 purity analysis of anti-human CD93 monoclonal antibodies using SEC-HPLC

Subtype identification was performed against human CD93 monoclonal antibodies using an enzyme-linked immune reaction, and the experimental procedure is briefly described below. Anti-mouse IgG capture antibody (Invitrogen, cat. A16080) diluted to 1. Mu.g/ml with 1 xBS was added at 50. Mu.l/well to 96-well plates and incubated at 2-8℃overnight. After washing once with 1xPBST, 1xPBS blocking solution containing 1% BSA was added at 250. Mu.l/well, and incubated at room temperature for 2 hours. After 3 dilutions using 1xPBST, hybridoma cell culture supernatants were added at 50. Mu.l/well and incubated at 37℃for 1 hour. After washing 3 times with 1xPBST, SBA clonotyping System-HRP (SouthernBiotech, cat.5300-05) diluted with 1xPBS containing 1% BSA was added at 50. Mu.l/well, and incubated at 37℃for 30 minutes. After 3 washes with 1xPBST, TMB color development solution (Biyun day, cat. P0209) was added at 100. Mu.l/well, and after incubation at 37℃for 10-15 min, ELISA stop solution (Soxhobao, cat. C1058) was added at 100. Mu.l/well. The anti-human CD93 monoclonal antibody subtypes are shown in table 2.

TABLE 2 subtype identification of anti-human CD93 monoclonal antibodies using ELISA reactions

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

EXAMPLE 2 cloning and sequencing analysis of CD93 monoclonal antibodies

The total RNA sample was obtained by extraction as follows: 1x10 ⁷ hybridoma cells were prepared, the cell culture medium was removed, and at least 1ml NucleoZOL cell lysate (MACHEREY-NAGEL, cat.740406.200) was added to the petri dish for cell lysis, and pipetting was repeated to ensure complete cell lysis. To the lysate, 200. Mu.l of RNase-free water was added according to 500. Mu. l NucleoZOL, the sample was vigorously shaken for 15 seconds, incubated at room temperature for 5 minutes, centrifuged at 12,000Xg for 15 minutes at room temperature, and 500. Mu.l of the supernatant was transferred to a new 1.5ml centrifuge tube. Leaving a layer of supernatant on the DNA/protein pellet. To each 500. Mu.l of supernatant was added 500. Mu.l of isopropanol to precipitate RNA. After incubating the samples at room temperature for 10 minutes, the samples were centrifuged at 12,000Xg for 10 minutes. The supernatant was removed and discarded. Mu.l of 75% ethanol was added for precipitation and centrifuged at 8,000Xg for 3 minutes. After the ethanol in the precipitate was removed by pipetting, and washing with 75% ethanol was repeated once, the RNA precipitate was dissolved in RNase-free water to obtain about 1. Mu.g/. Mu.l RNA.

Synthesis and amplification of cDNA heavy and light chain variable regions: according toRACE 5'/3' kit. The first cDNA strand was synthesized using random primers. The heavy chain and light chain variable region PCR amplification reaction is carried out by using RACE to amplify the heavy chain and light chain variable region, namely using cDNA as a template and using a mouse Ig-PRIMER SET primer as a Gene Specific Primer (GSP). The PCR products were stained and analyzed by electrophoresis in a 1.2% agarose gel. The desired DNA fragment was collected for purification.

TA cloning of VH and VL fragments: transferring the target gene into a pMD 18-T construction vector, and converting the connection product into DH5 alpha competent cells. One tube of DH 5. Alpha. TM. Cells was thawed on ice, gently mixed, 50. Mu.l of cells were transferred to a 1.5ml centrifuge tube, 1-5. Mu.l (1-10 ng) of DNA was added and gently mixed. The tubes were incubated on ice for 30 minutes. The shock cells were heated in a water bath at 42℃for 20 seconds. The tube was placed on ice for 2 minutes. Mu.l of pre-warmed LB medium was added to each centrifuge tube. Incubate at 37℃at 225rpm for 1 hour. 20-200. Mu.l of the mixture was applied to a pre-heated LB plate containing 100. Mu.g/ml ampicillin. Incubate overnight at 37 ℃.

Cloning and sequencing: from each plate, 15 colonies of transformants were selected and the sequence of the insert was analyzed by DNA sequencing. And heavy and light chain CDR regions were analyzed for sequences. Table 3 shows the anti-CD 93 monoclonal antibody heavy chain variable region and light chain variable region sequences. 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-CD 93 monoclonal antibodies, respectively.

TABLE 3 heavy and light chain variable region sequences of anti-human CD93 monoclonal antibodies

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

TABLE 4 heavy chain variable region VH CDRs of anti-human CD93 monoclonal antibodies

/>

TABLE 5 light chain variable region VL CDRs of anti-human CD93 antibodies

Example 3 detection of the binding Activity of anti-human CD93 monoclonal antibodies to CD93 by flow cytometry

The binding activity of the anti-human CD93 monoclonal antibodies to CD93 overexpressing cell lines was detected by flow cytometry. Briefly, after human CD93, cynomolgus CD93 and mouse CD93 overexpress CHO-K1 cell lines, i.e., CHO-K1-CD93 (Gibby organism, cat. GM-C22126), CHO-K1-cynoCD93 (Gibby organism, cat. GM-C22128) and CHO-K1-mCD93 (Gibby organism, cat. GM-C22125) were grown to log phase, the cells were digested with Ackutase solution (Invitrogen Cat. 00-4555-56), neutralized with complete medium, washed, centrifuged, resuspended with FASC buffer and added to 96-well plates at 2X10 ⁵/well. The antibody is prepared by FACS buffer solution, diluted by 3 times, mixed evenly, 100 mul/hole is added into the corresponding CHO-K1 over-expression cell strain, mixed evenly, and then kept stand for incubation for 30 minutes at 2-8 ℃. After centrifugation at 300Xg for 5 minutes, the supernatant was removed. Cells were washed with FACS buffer. PE-labeled goat anti-mouse IgG fluorescent secondary antibody (Biolegend, cat.405305) diluted with FACS buffer was added at 100 μl/well, mixed, and incubated at 2-8 ℃ for 30 min. After centrifugation at 300Xg for 5 minutes, the supernatant was removed. Cells were washed with FACS buffer. Cells were resuspended with 1xPBS and examined using BD lyric flow cytometer. The results are shown in fig. 1,2 and 3. Binding affinities are shown in table 6. Experimental results show that 20F1B9, 32D10G7 and 61C6A1 of 5 anti-human CD93 antibodies can be combined with high affinity with human CD93 and cynomolgus monkey CD93 over-expression cell lines, and 11A11B6 and 86B2E10 can be combined with low affinity with human CD93 and cynomolgus monkey CD93 over-expression cell lines. All 5 anti-human CD93 antibodies did not bind to the mouse CD93 overexpressing cell line.

TABLE 6 binding affinity of anti-human CD93 antibodies to human CD93 and cynomolgus monkey CD93 overexpressing cell lines

Example 4 flow cytometry detection of binding specificity of anti-human CD93 antibodies

The binding specificity of the anti-human CD93 antibody to CHO cells is outlined below. After CHOZN-K1 cells (Sigma, cat.) were grown to the logarithmic growth phase, appropriate amounts of the cells were taken in 15ml centrifuge tubes, centrifuged at 200Xg for 7 minutes, and the cells were resuspended in FASC buffer and added to 96-well plates at 2X10 ⁵/well. The antibody is prepared by FACS buffer solution, 100 μl/well of the antibody is added into CHOZN-K1 cell strain after mixing, and the mixture is incubated at 2-8deg.C for 30 min. After centrifugation at 300Xg for 5 minutes, the supernatant was removed. Cells were washed with FACS buffer. PE-labeled goat anti-mouse IgG fluorescent secondary antibody (Biolegend, cat.405305) diluted with FACS buffer was added at 100 μl/well, mixed, and incubated at 2-8 ℃ for 30 min. After centrifugation at 300Xg for 5 minutes, the supernatant was removed. Cells were washed with FACS buffer. Cells were resuspended with 1xPBS and examined using BD lyric flow cytometer. The results are shown in FIG. 4. Experimental results show that none of the 5 anti-human CD93 antibodies bind to CHOZN-K1 cells in a non-specific manner.

Example 5 detection of the binding affinity of anti-human CD93 antibodies to CD93 by Biacore

Anti-human CD93 antibody affinity was detected using Biacore, and the experimental procedure is briefly described below. The coupling of An-mFc was performed using a murine anti-capture kit (Cytiva, cat. BR100838) and An amino coupling kit (Cytiva, cat. BR100633), i.e., 50mM NHS and 200mM EDC obtained by 1:1 mixing activated the 1-8 channel surface of the CM5 chip (Cytiva, cat. BR100530) at a flow rate of 10 μl/min for 420 seconds. Then, the anti-mFc antibody diluted with a sodium acetate solution pH5.0 was injected at a flow rate of 10. Mu.l/min at a concentration of 30. Mu.g/ml for 420 seconds. 1M ethanolamine was injected at 10. Mu.l/min for 420 seconds to block excess active carboxyl groups on the chip. In affinity KD assays, the running buffer is a 1xHBS-EP+ (pH 7.4) solution (Cytiva, cat. BR100669). The anti-human CD93 antibody was diluted to a concentration of 2. Mu.g/ml using running buffer, and 2. Mu.g/ml of anti-human CD93 antibody was injected into the detection channel flow cell at a flow rate of 10. Mu.l/min, followed by sample injection for 60 seconds, and capturing was performed. Human CD93 extracellular domain fusion protein carrying an hFc tag was diluted with running buffer at 400, 200, 100 or 25nM and diluted in a multiple ratio. The human CD93 ECD-hFc samples to be tested were injected into the test channels at a flow rate of 30. Mu.l/min, respectively. The binding and dissociation times of the antigen-antibody were set to 180 seconds and 400 seconds, respectively. Finally, 10mM glycine pH 1.5 was injected into the detection channel at a flow rate of 30. Mu.l/min for 30 seconds. Data analysis was performed using Biacore Insight Evaluation Software (Version 2.0.15.12933). Curve fitting was chosen with the 1:1 binding model and kinetic parameters were calculated. As shown in Table 7, the experimental results show that the binding affinity of 5 anti-human CD93 monoclonal antibodies with human CD93 is 1.0E-09-1.0E-10.

TABLE 7 results of anti-human CD93 monoclonal antibody binding kinetics

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

EXAMPLE 6 flow cytometry detection of anti-human CD93 antibody-mediated human CD 93/human IGFBP7 binding blocking Activity

Anti-human CD93 antibodies mediate human CD 93/human IGFBP7 binding blocking activity, and the experimental procedure is outlined below. After labeling the hFc tagged recombinant human IGFBP7 protein (supplied by Dairy chemical) using EZ Link Sulfo-NHS-Biotin (Thermo scientific, cat. 21217), uncoupled Biotin was removed using a Zeba ^TM desalting column (Thermo Scientific, cat. 89882). After human CD93 overexpressing cell line CHO-K1-hCD93 was grown to log phase, cells were digested with Actutase solution (Invitrogen Cat.00-4555-56), neutralized with complete medium, washed, centrifuged, resuspended in FASC buffer and plated into 96-well plates at 2X10 ⁵/well. The biotin-labeled human IGFBP7 recombinant protein was diluted with FACS buffer at a concentration of 40nM, added to cells at 50 μl/well, and incubated at 2-8deg.C for 30min at rest. Anti-human CD93 monoclonal antibody was diluted with FACS buffer, diluted 3-fold, added to cells at 50. Mu.l/well after mixing, and incubated at 2-8deg.C for 30min at rest. After centrifugation at 300Xg for 5 minutes, the supernatant was removed. Cells were washed with FACS buffer. Streptavidin-PE (Jackson ImmunoResearch, cat. 016-110-084) diluted in FACS buffer was added at 100 μl/well, mixed well, and incubated at 2-8deg.C for 30min. After centrifugation at 300Xg for 5 minutes, the supernatant was removed. Cells were washed with FACS buffer. Cells were resuspended with 1xPBS and examined using BD lyric flow cytometer. The results are shown in FIG. 5. The blocking activity is shown in Table 8. Experimental results indicate that 20F1B9, 32D10G7 and 61C6A1 of 5 anti-human CD93 antibodies block the binding of human CD93 to human IGFBP 7.

Example 7 flow cytometry detection of anti-human CD93 antibody mediated human CD 93/human MMRN-2 binding blocking Activity

Anti-human CD93 antibodies mediated human CD 93/human MMRN-2 binding blocking activity, the experimental procedure is briefly described below. After labeling the hFc tagged recombinant human MMRN-2 protein (supplied by Dairy chemical) using EZ Link Sulfo-NHS-Biotin (Thermo scientific, cat. 21217), uncoupled Biotin was removed using a Zeba ^TM desalting column (Thermo Scientific, cat. 89882). After human CD93 overexpressing cell line CHO-K1-hCD93 was grown to log phase, cells were digested with Actutase solution (Invitrogen Cat.00-4555-56), neutralized with complete medium, washed, centrifuged, resuspended in FASC buffer and plated into 96-well plates at 2X10 ⁵/well. The biotin-labeled human MMRN-2 recombinant protein was diluted with FACS buffer at a concentration of 70nM, added to the cells at 50 μl/well and incubated at 2-8deg.C for 30 min at rest. Anti-human CD93 monoclonal antibody was diluted with FACS buffer, diluted 3-fold, added to cells at 50. Mu.l/well after mixing, and incubated at 2-8deg.C for 30 min at rest. After centrifugation at 300Xg for 5 minutes, the supernatant was removed. Cells were washed with FACS buffer. Streptavidin-PE (Jackson ImmunoResearch, cat. 016-110-084) diluted in FACS buffer was added at 100 μl/well, mixed well, and incubated at 2-8deg.C for 30 min. After centrifugation at 300Xg for 5 minutes, the supernatant was removed. Cells were washed with FACS buffer. Cells were resuspended with 1xPBS and examined using BD lyric flow cytometer. The results are shown in FIG. 6. The blocking activity is shown in Table 8. Experimental results show that 11A11B6 and 86B2E10 can block the binding of human CD93 and human MMRN-2 in 5 anti-human CD93 antibodies.

TABLE 8 anti-human CD93 monoclonal antibody blocking Activity

/>

Example 8 epitope grouping experiments at protein level

Epitope grouping of anti-human CD93 antibodies was analyzed at the protein level using an enzyme-linked immunosorbent assay. The experimental procedure is briefly described below, using 1xPBS to dilute anti-human CD93 monoclonal antibodies to a concentration of 1. Mu.g/ml, split into 96-well plates at 100. Mu.l/well, and incubated at 2-8℃overnight. After washing 3 times with 1xPBS, 1xPBS blocking solution containing 1% BSA was added at 300. Mu.l/well and blocked at 37℃for 2 hours. Human CD93 recombinant protein (supplied by Rui Zhi chemical) diluted with 1xPBS was added at an initial concentration of 10. Mu.g/ml, diluted 5-fold in a gradient, mixed well, and incubated at 100. Mu.l/Kong Jiaru at 37℃for 1 hour. After 3 washes with 1xPBST, HRP-labeled goat anti-human IgG (Sigma, cat. A0170) diluted with 1xPBS was added at 100. Mu.l/well and incubated at 37℃for 1 hour. After 3 washes with 1xPBST, TMB color development solution (Biyun day, cat. P0209) was added at 100. Mu.l/well, and after incubation at 37℃for 10min, ELISA stop solution (Soy pal, cat. C1058) was added at 100. Mu.l/well. The 96-well plate was placed in an microplate reader for OD450nm reading. And four parameter fits were performed with GRAPHPAD PRISM to calculate EC80 values.

Anti-human CD93 monoclonal antibody was diluted to a concentration of 1. Mu.g/ml using 1xPBS, split into 96-well plates at 100. Mu.l/well, and incubated at 2-8℃overnight. After washing 3 times with 1xPBS, 1xPBS blocking solution containing 1% BSA was added at 300. Mu.l/well and blocked at 37℃for 2 hours. Mu.l of a second anti-human CD93 monoclonal antibody (supplied by Rui Zhi Chemie) and 50. Mu.l of hFc-tagged human CD93 recombinant protein (concentration 2 xEC. Mu.l/well) were added, and incubated at 37℃for 1 hour. After 3 washes with 1xPBST, HRP-labeled goat anti-human IgG (Sigma, cat. A0170) diluted with 1xPBS was added at 100. Mu.l/well and incubated at 37℃for 1 hour. After 3 washes with 1xPBST, TMB color development solution (Biyun day, cat. P0209) was added at 100. Mu.l/well, and after incubation at 37℃for 10 min, ELISA stop solution (Soy pal, cat. C1058) was added at 100. Mu.l/well. The 96-well plate is placed in an enzyme labeling instrument for OD450nm reading, the inhibition rate is calculated, and epitope grouping of the anti-human CD93 monoclonal antibody is carried out according to the result. The experimental results are shown in Table 9, and the results show that 11A11B6 and 86B2E10 have the same epitope, 20F1B9 and 32D10G7 have the same recognition epitope, and 61C6A1 and 11A11B6 and 32D10G7 recognition epitopes are different.

TABLE 9 Epitope binding assay for anti-human CD93 monoclonal antibodies

Inhibition rate, percent	11A11B6	20F1B9	32D10G7	61C6A1	86B2E10
						11A11B6	87.49	1.81	0.27	15.11	71.24
20F1B9	-2.08	89.01	82.40	ND^*	ND^*
						32D10G7	-6.60	92.71	84.76	5.46	-3.42
61C6A1	-3.25	ND^*	8.07	93.43	ND^*
						86B2E10	79.36	ND^*	26.78	ND^*	78.38

ND ^*: indicating that no detection was performed.

Incorporated by reference

The entire contents of each of the patent documents and scientific documents mentioned herein are incorporated by reference for all purposes.

Equivalency of

The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The above embodiments should therefore be regarded as illustrative in all respects, rather than limiting on the invention described herein. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

1. An isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds CD93 and comprises a heavy chain variable region (VH) and a light chain variable region (VL),

The heavy chain variable region comprises:

the light chain variable region comprises:

2. The antibody or fragment of claim 1, comprising a combination of:

3. The antibody or fragment of claim 1 or 2, wherein the heavy chain variable region comprises a sequence identical to SEQ ID NO: 2. 4, 6, 8 and 10, or 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 another or consisting of SEQ ID NO: 2. 4, 6, 8 and 10;

4. The antibody or fragment of any one of claims 1-3, comprising one of the following combinations:

5. The antibody or fragment of any one of claims 1-4, further comprising a heavy chain constant region and a light chain constant region, wherein the antibody heavy chain constant region is selected from an IgG series antibody and the light chain constant region is selected from a kappa or lambda chain.

6. The antibody or fragment of claim 5, wherein the antibody heavy chain constant region is preferably selected from one of IgG1, igG2c, igG2b, and the light chain constant region is preferably a kappa chain.

7. The antibody or fragment of any one of the above claims, wherein the antibody is selected from the group consisting of: whole antibodies, bispecific antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies;

8. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding an antibody or fragment according to any one of claims 1-7.

9. A host cell comprising the nucleic acid molecule of claim 8.

10. A multispecific molecule comprising the antibody or antigen-binding fragment of any one of claims 1-7; preferably, the multispecific molecule specifically binds CD93, and additionally specifically binds one or more other targets; further preferred, the multispecific molecule further comprises at least one molecule having a second binding specificity for a second target.