TW201940516A - Novel anti-cd47 antibodies - Google Patents

Abstract

The present disclosure provides anti-CD47 antibodies or antigen-binding fragments thereof, isolated polynucleotides encoding the same, pharmaceutical compositions comprising the same, and the uses thereof.

Description

New anti-CD47 antibody

The present invention relates to a novel anti-human CD47 antibody.

Since the "magic bullet" hypothesis proposed by Paul Ehrlich, rituximab was approved in 1997. In the past decades, monoclonal antibody-based therapies have been used in tumors, autoimmune diseases, and many other diseases. Remarkable progress has been made. The main mechanism of the anti-cancer effect of mAb depends on its interaction with components of the immune system through antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and Alter signal transduction to kill cancer cells. Among these mechanisms, macrophage-mediated phagocytosis has proven to be an important mechanism for eliminating diseased or injured cells.

Cluster of differentiation 47 (CD47), also known as integrin-related protein (IAP), is an immunoglobulin superfamily membrane protein with a size of approximately 50 kDa. CD47 interacts with its ligands on macrophages and signal-regulating protein α (SIRPα) to send anti-phagocytosis signals or "don't eat me" signals, thereby evading immune surveillance. Analysis of patient tumors and matching adjacent normal tissues revealed that CD47 was overexpressed on AML, NHL, breast cancer, NSCC, and ovarian cells, and increased CD47 performance was associated with poor clinical prognosis. These data show that CD47 can be used as a new immune checkpoint for cancer therapies for blocking the CD47-SIRPα interaction. Several anti-CD47 monoclonal antibodies and SIRPα fusion proteins have achieved effective phagocytosis of macrophages against AML, NHL, breast cells and ovarian cells. In addition, the combination of anti-CD47 monoclonal antibodies with approved antibodies (anti-tumor-associated antigens) has effectively enhanced anti-tumor activity. Based on these preclinical studies, two anti-CD47 monoclonal antibodies (Hu5F9-G4 and CC-90002) and a SIRPα engineered fusion protein (TTI-621) are in Phase I or II of an active clinical trial, which covers blood malignancy Tumors and human solid tumors.

There is a great need for new anti-CD47 antibodies.

The articles "a," "an," and "the" throughout this application are used herein to refer to the grammatical objects of one or more (ie, at least one) of the articles. For example, "an antibody" refers to one antibody or more than one antibody.

The application provides an isolated anti-CD47 antibody or antigen-binding fragment thereof, comprising:
a) 1, 2 or 3 heavy chain complementarity determining region (CDR) sequences selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO : 9 and SEQ ID NO: 11; and / or
b) 1, 2, or 3 light chain CDR sequences selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 29.

In certain embodiments, the isolated anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the group consisting of: a) a heavy chain variable region, the heavy chain variable region comprising 1, 2 or 3 CDR sequences selected from SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5; and b) a heavy chain variable region, the heavy chain variable region comprising 1, 2 or 3 selected from SEQ ID NO: 7, CDR sequences of SEQ ID NO: 9 and SEQ ID NO: 11.

In certain embodiments, the isolated anti-CD47 antibody or antigen-binding fragment thereof comprises a light chain variable region selected from the group consisting of: a) a light chain variable region, the light chain variable region comprising 1, 2 or 3 CDR sequences selected from SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6; b) a light chain variable region, the light chain variable region comprising 1, 2 or 3 selected from SEQ ID NO : 29. The CDR sequences of SEQ ID NO: 4 and SEQ ID NO: 6; and c) a light chain variable region comprising 1, 2 or 3 selected from SEQ ID NO: 8, SEQ ID CDR sequences of NO: 10 and SEQ ID NO: 12.

In certain embodiments, the isolated anti-CD47 antibody or antigen-binding fragment thereof comprises: a) a heavy chain variable region comprising 1, 2 or 3 selected from SEQ ID NO: 1, SEQ CDR sequences of ID NO: 3 and SEQ ID NO: 5; and a light chain variable region comprising one, two or three selected from SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID CDR sequence of NO: 6; b) a heavy chain variable region comprising 1, 2 or 3 CDR sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5 And a light chain variable region comprising one, two, or three CDR sequences selected from the group consisting of SEQ ID NO: 29, SEQ ID NO: 4, and SEQ ID NO: 6; and c) the heavy chain may Variable region, the heavy chain variable region comprising 1, 2 or 3 CDR sequences selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11; and a light chain variable region, the light chain may The variable region comprises 1, 2 or 3 CDR sequences selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12.

In certain embodiments, the isolated anti-CD47 antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21 and SEQ ID NO: 25, and homologous sequences that have at least 80% sequence identity but still retain specific binding affinity to CD47

In certain embodiments, the isolated anti-CD47 antibody or antigen-binding fragment thereof comprises a light chain variable region selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23 and SEQ ID NO: 27, and homologous sequences that have at least 80% sequence identity thereto but still retain specific binding affinity for CD47.

In certain embodiments, the isolated anti-CD47 antibody or antigen-binding fragment thereof comprises:
a) a heavy chain variable region comprising SEQ ID NO: 13 and a light chain variable region comprising SEQ ID NO: 15;
b) a heavy chain variable region comprising SEQ ID NO: 17 and a light chain variable region comprising SEQ ID NO: 19;
c) a heavy chain variable region comprising SEQ ID NO: 21 and a light chain variable region comprising SEQ ID NO: 23; and
d) A heavy chain variable region comprising SEQ ID NO: 25, and a light chain variable region comprising SEQ ID NO: 27.

In certain embodiments, the isolated anti-CD47 antibody or antigen-binding fragment thereof further comprises one or more amino acid residue substitutions or modifications, but still retains a specific binding affinity for CD47. In certain embodiments, at least one of the substitutions or modifications is in one or more of the CDR sequences, and / or in one or more of the heavy chain or light chain variable region sequences without any CDR sequence.

In certain embodiments, the isolated anti-CD47 antibody or antigen-binding fragment thereof further comprises an immunoglobulin constant region, optionally a human immunoglobulin constant region, or optionally a human IgG constant region.

In certain embodiments, the isolated anti-CD47 antibody or antigen-binding fragment thereof is humanized.

In some embodiments, the isolated anti-CD47 antibody or antigen-binding fragment thereof is a camelized single chain domain antibody, a diabody, scFv, scFv dimer, BsFv, dsFv, (dsFv) ₂ , Fv fragment, Fab, Fab ', F (ab') 2, ds diabody, nanobody, domain antibody, or bivalent antibody.

In certain embodiments, the isolated anti-CD47 antibody or antigen-binding fragment thereof can be no more than 10 ^-9 M (e.g. no more than 9 × 10 ^-10 M, no more than 8 × 10 ^-10 M, no more than 7 × 10 ^-10 M, no more than 6 × 10 ^-10 M, no more than 5 × 10 ^-10 M, no more than 4 × 10 ^-10 M, no more than 3 × 10 ^-10 M, no more than 2 × 10 ^-10 M, K _D values of no more than 10 ^-10 M, no more than 9 × 10 ^-11 M, no more than 8 × 10 ^-11 M, or no more than 7.5 × 10 ^-11 M) are specific to human CD47 expressed on the cell surface In combination, this K _D value was determined by flow cytometry.

In certain embodiments, the isolated anti-CD47 antibody or antigen-binding fragment thereof is capable of specifically binding to cynomolgus CD47.

In certain embodiments, the isolated anti-CD47 antibody or antigen-binding fragment thereof is linked to one or more conjugated moieties. In certain embodiments, the conjugated moiety comprises a scavenger modulator, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme substrate label, a DNA alkylating agent, and a topoisomerase Inhibitor tubulin adhesive or other anticancer drugs.

The application also provides an antibody or antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment thereof described herein for the same epitope.

The present application also provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described in the present application, and a pharmaceutically acceptable carrier.

The application also provides an isolated polynucleotide encoding the antibody or antigen-binding fragment thereof described in the application. In certain embodiments, the isolated polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO: 28.

The application also provides a vector comprising the isolated polynucleotide described in the application.

The application also provides a host cell comprising the vector described in the application.

The application also provides a method for expressing the antibody or antigen-binding fragment thereof described in the present application, which comprises culturing the host cell described in the application under the condition of expressing the vector described in the application.

The application also provides a method of treating a disease or condition in a subject that may benefit from modulation of CD47 activity, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof or a pharmaceutical composition described herein. In certain embodiments, the disease or condition is a disease or condition associated with CD47. In certain embodiments, the disease or condition is cancer, autoimmune disease, fibrosis, inflammatory disease, or infectious disease. In certain embodiments, the individual is a human. In certain embodiments, the administration is via oral, intranasal, intravenous, subcutaneous, sublingual, or intramuscular administration.

In certain embodiments, the cancer is lung cancer, bronchial cancer, bone cancer, liver and bile duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spinal cancer, Brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary cancer, stomach cancer, vaginal cancer, thyroid cancer , Glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, malignant sarcoma, teratoma, adenocarcinoma, leukemia, myeloma and lymphoma.

In certain embodiments, the disease or condition is a hematologic cancer selected from non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic Lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), diffuse large B-cell lymphoma (DLBCL), Richter syndrome, Burkitt's lymphoma, or follicular lymphoma .

The present application also provides a method for modulating CD47 activity in a cell expressing CD47, which comprises exposing the cell expressing CD47 to an antibody or an antigen-binding fragment thereof described herein.

The application also provides a method for detecting the presence or content of CD47 in a sample, which comprises contacting the sample with the antibody or antigen-binding fragment thereof described in the application, and determining the presence or content of CD47 in the sample.

The present application also provides a method for diagnosing a CD47-related disease or condition in an individual, comprising: a) contacting a sample obtained from the individual with an antibody or antigen-binding fragment thereof described in the application; b) determining where the The presence or content of CD47 in the sample; and c) correlating the presence or content of CD47 with the presence or status of the disease or condition associated with CD47 in the individual.

This application also provides the use of an antibody or antigen-binding fragment thereof described herein for the manufacture of a medicament for treating a disease or condition associated with CD47 in an individual.

The application also provides the use of the antibody or antigen-binding fragment thereof described in the application in the preparation of a diagnostic reagent for the diagnosis of a disease or condition related to CD47.

The present application also provides a set comprising the antibody or antigen-binding fragment thereof described in the present application, which can be used to detect CD47.

Priority claim

This application claims priority from PCT Application No. PCT / CN2018 / 079673 filed on March 20, 2018 and China Application No. 201810530689.7 filed on May 29, 2018.

The following description of this application is only to illustrate various embodiments of this application. Therefore, the specific modifications discussed here should not be construed as limiting the scope of the application. Those skilled in the art can easily derive various equivalents, changes, and modifications without departing from the scope of the present application. It should be understood that such equivalent embodiments are included in the scope of the present invention. All documents cited in this application, including published publications, patents and patent applications, are incorporated by reference in their entirety.

Definitions <br/> The term "antibody" in the present invention includes any antibody that can bind to a specific antigen: immunoglobulin, monoclonal antibody, polyclonal antibody, polyvalent antibody, bivalent antibody, monovalent antibody. A natural intact antibody contains two heavy (H) chains and two light (L) chains. Mammalian heavy chains can be divided into α, δ, ε, γ, and μ. Each heavy chain consists of a variable region (V _H ) and first, second, and third constant regions (C _H1 and C _{H2 respectively).} , C _H3 ); mammalian light chains can be divided into λ or κ, each light chain consists of a variable region (V _L ) and a constant region. The antibody is of "Y" type, and the neck of the "Y" type structure consists of the second and third constant regions of the two heavy chains, which are bound by disulfide bonds. Each arm of the "Y" structure includes the variable region and the first constant region of one of the heavy chains, which are combined with the variable region and the constant region of a light chain. The variable regions of the light and heavy chains determine antigen binding. The variable region of each chain contains three hypervariable regions, called complementarity determining regions (CDRs) (the CDRs of the light chain include LCDR1, LCDR2, LCDR3, and the CDRs of the heavy chain include HCDR1, HCDR2, HCDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed in the present invention can be named or identified by Kabat, IMGT, Chothia or Al-Lazikani nomenclature. (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273 (4), 927 (1997); Chothia, C. et al., J Mol Biol. Dec 5; 186 (3 ): 651-63 (1985); Chothia, C and Lesk, AM, J. Mol. Biol., 196,901 (1987); Chothia, C. et al., Nature. Dec 21-28; 342 (6252): 877-83 (1989); Kabat EA et al., National Institutes of Health, Bethesda, Maryland (1991); Marie-Paule Lefranc et al, Developmental and Comparative Immunology, 27: 55-77 (2003); Marie-Paule Lefranc et al, Immunome Research , 1 (3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (second edition), chapter 26, 481-514, (2015)). Among them, the three CDRs are separated by a side continuous portion called a framework region (FR). The framework region is more highly conserved than the CDR and forms a scaffold to support the hypervariable loop. The constant regions of the heavy and light chains have nothing to do with antigen binding, but have multiple effector functions. Antibodies can be divided into several classes based on the amino acid sequence of the constant region of the heavy chain. Depending on whether they contain α, δ, ε, γ, and μ heavy chains, antibodies can be divided into five main classifications or isomers: IgA, IgD, IgE, IgG, and IgM. Several major antibody classifications can also be divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain) or IgA2 ( α2 heavy chain) and so on.

The term "bivalent" in this application refers to an antibody or antigen-binding fragment having two antigen-binding sites; the term "monovalent" refers to an antibody or antigen-binding fragment having only a single antigen-binding site; and the term "multivalent" "Valence" refers to an antibody or antigen-binding fragment having multiple antigen-binding sites. In some embodiments, the antibody or antigen-binding fragment thereof is bivalent.

The term "antigen-binding fragment" in this application refers to an antibody fragment formed from an antibody portion containing one or more CDRs or any other antibody fragment that binds an antigen but does not have a complete antibody structure. Examples of antigen-binding fragments include, but are not limited to, such as bifunctional antibodies (diabody), Fab, Fab ', F (ab') ₂ , Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv) ₂ , Disulfide stabilized bifunctional antibody (ds diabody), single chain antibody molecule (scFv), scFv dimer (bivalent bifunctional antibody), bivalent single chain antibody (BsFv), camelized single domain antibody (camelized single domain antibody), nanobodies, domain antibodies, and bivalent domain antibodies. The antigen-binding fragment can bind the same antigen as the parent antibody.

An "Fab" fragment of an antibody refers to a portion of an antibody molecule composed of a light chain (including a variable region and a constant region) and a variable region and a constant region of a heavy chain that are bound by disulfide bonds.

"Fab '" fragment refers to a Fab fragment that includes a portion of the hinge region.

"F (ab ') ₂ " refers to the dimer of Fab'. The "Fv" segment of an antibody refers to the smallest antibody fragment that contains a complete antigen-binding site. The Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.

"DsFv" refers to a disulfide-bonded Fv fragment in which the linkage between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond. In some embodiments, "(dsFv) _2" comprises three peptide chains: two portions by V _H polypeptide linker (e.g. long flexible linker) is connected, and two V _L binding moiety through a disulfide bond, respectively.

"Single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region directly connected or connected by a peptide chain sequence (Huston JS, etc., Proc Natl Acad Sci USA, 85 : 5879 (1988)).

The "Fc" of an antibody refers to another part of the antibody consisting of the second and third constant regions of the first heavy chain linked to the second and third constant regions of the second heavy chain via a disulfide bond. The Fc segment of an antibody is responsible for many different effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not play a role in antigen binding.

"Single-chain antibody Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody composed of scFv linked to the Fc segment of an antibody.

"Camelized single domain antibody", "heavy chain antibody" or "HCAb (Heavy-chain-only antibodies, HCAb)" all refer to antibodies containing two V _H domains without light chains ( Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231 (1-2): 25-38 (1999); Muyldermans S., J Biotechnol. Jun; 74 (4): 277-302 (2001); WO94 / 04678; WO94 / 25591; US Patent No. 6,005,079). Heavy chain antibodies were originally derived from the camel family (camel, dromedary, and llama). Although the light chain is missing, camelized antibodies have the full function of confirmed antigen binding (Hamers-Casterman C. et al., Nature. Jun 3; 363 (6428): 446-8 (1993); Nguyen VK. Et al., " Heavy-chain antibodies in Camelidae; a case of evolutionary innovation, "Immunogenetics. Apr; 54 (1): 39-47 (2002); Nguyen VK. Et al., Immunology. May; 109 (1): 93-101 (2003) ). The variable region (VHH domain) of a heavy chain antibody is the smallest antigen-binding unit produced by known adaptive immunity (Koch-Nolte F. et al., FASEB J. Nov; 21 (13): 3490-8. Epub 2007 Jun 15 (2007)).

"Nano antibody" refers to an antibody fragment consisting of a VHH domain from a heavy chain antibody and two constant regions, CH2 and CH3.

A "diabody" or "dAb" contains a small antibody fragment with two antigen-binding sites, where the fragment contains the V _H domain and the V _L domain (V _H -V _L or V _H -V _L ) (see, Holliger P. et al., Proc Natl Acad Sci USA. Jul 15; 90 (14): 6444-8 (1993); EP404097; WO93 / 11161). The adapter between the two domains is very short, so that the two domains on the same chain cannot be paired with each other, thus forcing the two domains to pair with the complementary domains of the other chain to form two antibody binding sites. Antigen binding sites can target the same antigen (or antigenic epitope). In certain embodiments, the "scFv dimer" is a bivalent bifunctional antibody or a bivalent ScFv (BsFv), comprising the dimerization of V _H -V _L with another V _H -V _L moiety (linked by a polypeptide sub-connection), such that a portion of the V _H to form two binding sites other portion of _{the L-V} cooperate, this two binding sites may target the same antigen (or antigenic epitope).

A "domain antibody" refers to an antibody fragment that contains only the heavy or light chain variable regions. In some cases, two or more _VH domains are covalently bound by a peptide linker and form a bivalent or multivalent domain antibody. The two _VH domains of a bivalent domain antibody can target the same or different antigens.

The term "chimeric" as used in this application refers to an antibody or antigen-binding fragment having a portion of the heavy and / or light chain derived from one species and the rest of the heavy and / or light chain derived from a different species. In an illustrative example, a chimeric antibody may include a constant region derived from a human and a variable region derived from a non-human animal, such as a mouse. In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

The term "humanized" as used in this application means an antibody or antigen-binding fragment comprising CDRs derived from non-human animals, FR regions derived from humans, and constant regions derived from humans (if applicable).

"CD47" in this application is derived from any vertebrate source, including mammals, such as primates (eg, humans, monkeys) and rodents (eg, mice and rats). An exemplary sequence of human CD47 includes a human CD47 protein (NCBI Accession No. GI: 4502673). Exemplary sequences of CD47 include Mus musculus (mouse) CD47 protein (NCBI accession number GI: 76364104) and Macaca fascicularis (monkey) CD47 protein (NCBI accession number GI: 544416831).

The term "CD47" in this application is intended to cover any form of CD47, such as 1) a natural unprocessed CD47 molecule, a "full-length" CD47 chain, or a naturally occurring variant of CD47 (including, for example, splice variants or allelic variants) Body); 2) any form of CD47 produced by processing in a cell; or 3) the full length, fragment (e.g., truncated form, extracellular / transmembrane domain) or modified form of CD47 subunit produced by recombinant methods (E.g. mutant form, glycosylated / pegylated form, histidine tag / immunofluorescent fusion form).

The term "anti-CD47 antibody" refers to an antibody capable of specifically binding CD47 (eg, human or monkey or mouse or rat CD47).

The "specific binding" or "specific binding" in this application refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen. In certain embodiments, the antibody or antigen-binding fragment thereof of the present application specifically binds to human and / or monkey CD47, and its binding affinity (K _D ) is ≦ 10 ^-6 M (eg, ≦ 5 × 10 ^-7 M ^{, ≤2 × 10 -7 M, ≤10} -7 M, ≤5 × 10 -8 M, ≤2 × 10 -8 M, ≤10 -8 M, ≤5 × 10 -9 M, ≤4 × 10 - ⁹ M, ≤3 × 10 ^-9 M, ≤2 × 10 ^-9 M, ≤10 ^-9 M, 5 × ≤10 ^-10 M or 5 × 10 ^-11 M). KD in this application refers to the ratio (koff / kon) of dissociation speed to binding speed, which can be determined by using any method commonly used in this technology, including but not limited to surface plasmon resonance method and micro-heat flow method HPLC-MS and flow cytometry (such as FACS). In certain embodiments, K _D values may be appropriately used by flow cytometry.

The ability to "block binding" or "compete for the same epitope" in this application means that the antibody or its antigen-binding fragment inhibits the interaction between two molecules (such as human CD47 and anti-CD47 antibodies) to any detectable Ability to measure degree. In certain embodiments, an antibody or antigen-binding fragment that blocks binding between two molecules can inhibit the interaction between the binding between two molecules by at least 85% or at least 90%. In certain embodiments, such inhibition can be greater than 85% or greater than 90%.

As used herein, an "antigenic determinant" refers to an amino acid or atomic group of an antigen molecule that is bound to an antibody. If two antibodies show competitive binding to an antigen, they may bind the same or closely related epitope on the antigen. For example, if the antibody or antigen-binding fragment thereof blocks the reference antibody from binding to the antigen by at least 85% or at least 90% or at least 95%, the antibody or antigen-binding fragment thereof can be considered to be the same / close to the reference antibody Related epitopes.

Those skilled in the art will recognize that limited experiments can be used to determine whether a given antibody blocks the antibodies described in this application (e.g., rodent monoclonal antibodies W3452-1.164.16 and W3452-2.683.2 and humanization). The antibodies W3452-1.164.16-z11 and W3452-2.683.2-z27) bind to the CD47 antigen polypeptide, thereby determining whether a given antibody binds to the same epitope as the antibody described herein. If the binding of the antibody described in this application to the CD47 antigen polypeptide decreases, indicating that the given antibody competes with the antibody described in this application, then the two antibodies bind to the same or closely related epitope. Alternatively, if the binding of a given antibody to a CD47 antigen polypeptide is inhibited by the antibodies described herein, then the two antibodies bind to the same or closely related epitope.

The name of an antibody in this application may include one or more suffix symbols, which usually indicate the type of antibody or specific modifications made to the antibody. For example, "IgG1" or "IgG4" refers to the human IgG1 isotype or IgG4 isotype human (unless otherwise specified) antibody constant region, "z" refers to a humanized antibody, "K" refers to a kappa light chain, and "P" And "E" refer to S228P and L235E in the constant region of human IgG4, respectively.

In the present application, when "conservative substitution" is used in an amino acid sequence, it means that one amino acid residue is replaced with another amino acid residue having a side chain with similar physicochemical properties. For example, between hydrophobic side chain amino acid residues (e.g. Met, Ala, Val, Leu and Ile), neutral hydrophilic side chain residues (e.g. Cys, Ser, Thr, Asn and Gln), acidic side chains Conservative substitutions are made between residues (such as Asp, Glu), basic side chain amino acids (such as His, Lys, and Arg) or directional side chain residues (such as Trp, Tyr, and Phe). It is known in the art that conservative substitutions usually do not cause significant changes in the conformational structure of the protein and are therefore able to retain the biological activity of the protein.

As used herein, the terms "homologous" and "homologous" are used interchangeably and refer to a nucleic acid sequence (or its complementary strand) or amino acid sequence that has at least 80% (e.g., (At least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%).

When "percent sequence identity" is used for an amino acid sequence (or nucleic acid sequence), it refers to a candidate sequence after sequence alignment, and if necessary, introducing a gap to maximize the number of identical amino acids (or nucleic acids). In this example, the percentage of amino acid (or nucleic acid) residues in the candidate sequence is the same as that of the reference sequence. Conservative substitutions of this amino acid residue may or may not be considered the same residue. Tools disclosed in this technology can be used, such as BLASTN, BLASTp (National Center for Biotechnology Information (NCBI), see also, Altschul SF et al., J. Mol. Biol., 215: 403-410 (1990) Stephen F. et al., Nucleic Acids Res., 25: 3389-3402 (1997)), ClustalW2 (Website of European Bioinformatics Institute, see, Higgins DG et al., Methods in Enzymology, 266: 383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, England), 23 (21): 2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software to align sequences to determine the percentage sequence of amino acid (or nucleic acid) sequences Identity. Those skilled in the art can use the default parameters of the tool or adjust the parameters appropriately according to the comparison needs, for example, by selecting a suitable algorithm.

"Effective function" as used in this application refers to the biological activity of the antibody's Fc region bound to its effectors such as the C1 complex and Fc receptor. Exemplary effector functions include complement-dependent cytotoxicity (CDC) induced by the interaction of antibodies with C1q on the C1 complex, antibody-dependent cell-mediated cytotoxicity induced by binding of the Fc region of the antibody to Fc receptors on effector cells (ADCC) and engulfing.

"Treatment" or "therapy" of a condition includes preventing or reducing a condition, reducing the speed at which a condition arises or developing, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition , Reduce or terminate the symptoms associated with a condition, produce a complete or partial reversal of a condition, cure a condition, or a combination of the above.

"Isolated" matter has been artificially changed from its natural state. If some "isolated" substance or component appears in nature, it has changed or left its original state, or both. For example, a polynucleotide or polypeptide naturally occurring in a living animal is not isolated, but if the polynucleotide or polypeptide is sufficiently separated from substances that coexist in its natural state and exists in a sufficiently pure state, Can be considered "separated."

In the present invention, a "vector" refers to a vehicle that can be used to operably insert a polynucleotide encoding a protein and obtain expression of the protein. Vectors can be used to transform, transduce, or transfect host cells so that the genetic material elements they carry are expressed within the host cells. For example, vectors include: plastids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC), phages such as lambda phage or M13 phage, and animal viruses. Animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (Such as SV40). Vectors can contain a variety of performance-controlling elements, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Alternatively, the vector may contain a replication initiation site. A carrier can also include components that assist it in entering cells, including, but not limited to, viral particles, liposomes, or protein shells. The vector may be a performance vector or a selection vector. The vector (e.g., expression vector) provided in this application contains the nucleic acid sequence encoding the antibody or antigen-binding fragment thereof described in this application, and at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence. , And at least one select mark. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (e.g., SV40) ), Lambda phage and M13 phage, plastid pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM. , PCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

The "host cell" in the present invention refers to a cell into which an exogenous polynucleotide and / or vector is introduced.

A "CD47-related" disease or condition in the present invention refers to any disease or condition caused, exacerbated, or otherwise associated with an increase or decrease in the performance or activity of CD47. In some embodiments, the CD47-related condition is an immune-related disease, such as cancer, an autoimmune disease, an inflammatory disease, or an infectious disease.

"Cancer" as used in this application refers to any medical condition characterized by malignant cell growth or tumor, abnormal proliferation, invasion or metastasis, and includes solid tumors and non-solid cancers (blood malignancies) such as leukemia. "Solid tumor" as used in this application refers to a solid mass of tumor and / or malignant cells. Examples of cancers or tumors include hematological malignancies, oral cancers (e.g., lip, tongue, or throat cancer), digestive organs (e.g., esophagus, stomach, small intestine, colon, large intestine, or rectum), peritoneal cancer, liver, or biliary tract Cancer, pancreatic cancer, respiratory system such as laryngeal or lung (small cell or non-small cell) cancer, bone cancer, connective tissue cancer, skin cancer (e.g. melanoma), breast cancer, reproductive organs (fallopian tube, uterus, cervix, Testicular, ovarian or prostate) cancer, urethral (eg bladder or kidney) cancer, brain and endocrine gland (eg thyroid) cancer. In certain embodiments, the cancer is selected from the group consisting of ovarian cancer, breast cancer, head and neck cancer, kidney cancer, bladder cancer, hepatocellular carcinoma, and colorectal cancer. In certain embodiments, the cancer is a blood cancer. In certain embodiments, the hematologic cancer is selected from non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic Myeloid leukemia (CML), multiple myeloma (MM), diffuse large B-cell lymphoma (DLBCL), Richter syndrome, Burkitt's lymphoma, or follicular lymphoma.

"Pharmaceutically acceptable" means that the carrier, solvent, diluent, excipient, and / or salt referred to are generally chemically and / or physically compatible with the other ingredients in the formulation and are physiologically compatible with the recipient Compatible.

Anti- CD47 antibodies <br/> This application provides anti-CD47 antibodies and antigen-binding fragments thereof, which comprise anti-CD47 antibodies W3452-1.164.16, W3452-1.164.16-z11, W3452-2.683.2 or W3452-2.683.2 -one or more (eg 1, 2, 3, 4, 5, or 6) CDR sequences of z27.

"W3452-1.164.16" in this application refers to a rodent monoclonal antibody having a heavy chain variable region as shown in SEQ ID NO: 13 and a kappa light chain variable region as shown in SEQ ID NO: 15 .

In the present application, "W3452-1.164.16-z11" refers to humanization with a heavy chain variable region as shown in SEQ ID NO: 17 and a kappa light chain variable region as shown in SEQ ID NO: 19 Monoclonal antibodies.

"W3452-2.683.2" in this application refers to a rodent monoclonal antibody having a heavy chain variable region as shown in SEQ ID NO: 21 and a lambda light chain variable region as shown in SEQ ID NO: 23 .

"W3452-2.683.2-z27" in this application refers to humanization with a heavy chain variable region as shown in SEQ ID NO: 25 and a lambda light chain variable region as shown in SEQ ID NO: 27 Monoclonal antibodies.

Table 1 shows the CDR sequences of these anti-CD47 antibodies. The heavy and light chain variable region sequences are also provided below in Tables 2 and 3.
Table 1. CDR amino acid sequences
Table 2. Variable region amino acid sequences
Table 3. Variable region nucleic acid sequences

The CDRs are known to be responsible for antigen binding, however, not all 6 CDRs have been found to be indispensable or invariable. In other words, one or more of the CDRs of the anti-CD47 antibody W3452-1.164.16, W3452-1.164.16-z11, or W3452-2.683.2 can be replaced or changed or modified, while substantially maintaining affinity for CD47 specific binding.

In certain embodiments, the anti-CD47 antibodies and antigen-binding fragments thereof described herein comprise the heavy chain CDR3 sequences of anti-CD47 antibodies W3452-1.164.16, W3452-1.164.16-z11, or W3452-2.683.2. In certain embodiments, the anti-CD47 antibodies and antigen-binding fragments described herein comprise a heavy chain CDR3 sequence as shown in SEQ ID NOs: 5 and 11. The heavy chain CDR3 region is located at the center of the antigen-binding site and is therefore considered to have the most contact with the antigen and provides the most free energy for the affinity of the antibody to the antigen. In addition, heavy chain CDR3 is also considered to be the most diverse CDR of antigen binding sites in terms of length, amino acid composition, and conformation due to various diversification mechanisms (Tonegawa S., Nature. 302: 575-81). The divergence of the heavy chain CDR3 is sufficient to generate most antibody specificities (Xu JL, Davis MM. Immunity. 13: 37-45) and desired antigen-binding affinity (Schier R et al., J Mol Biol. 263: 551-67).

In certain embodiments, the antibodies and antigen-binding fragments thereof described herein include appropriate framework region (FR) sequences, so long as the antibodies and antigen-binding fragments thereof can specifically bind to CD47. The CDR sequences shown in Table 1 are obtained from rat or mouse antibodies, but they can be transplanted to any suitable species (such as mouse, human, rat, rabbit, and Other) of any suitable FR sequence.

In certain embodiments, the antibodies and antigen-binding fragments thereof described herein are humanized. Humanized antibodies or antigen-binding fragments desirably have reduced immunogenicity in the human body. A humanized antibody or antigen-binding fragment thereof is chimeric in its variable region because non-human CDR sequences are grafted into human or substantially human FR sequences. Humanization of antibodies or antigen-binding fragments can basically be accomplished by replacing non-human (e.g., mouse) CDR genes with corresponding human CDR genes on human immunoglobulin genes (see, for example, Jones et al. (1986) Nature 321 : 522-525; Riechmann et al. (1988) Nature 332: 323-327; Verhoeyen et al. (1988) Science 239: 1534-1536).

The methods known in the art can be used to select suitable human heavy and light chain variable domains to achieve this purpose. In an illustrative example, a best-fit method can be used in which non-human (e.g., rodent) antibody variable domain sequences are screened or BLAST aligned with a database of known human variable domain sequences And identify the human sequence closest to the non-human query sequence for use as a human framework for transplanting non-human CDR sequences (see, eg, Sims et al. (1993) J. Immunol. 151: 2296; Chothia et al. (1987) J. Mot. Biol. 196: 901). Alternatively, a framework derived from the consensus sequence of all human antibodies can be used to transplant non-human CDRs (see, eg, Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89: 4285; Presta et al. (1993) J. Immunol ., 151: 2623).

In certain embodiments, the humanized antibodies or antigen-binding fragments described herein are composed essentially of human sequences except for non-human CDR sequences. In some embodiments, the variable region FR and the constant region (if present) are all or substantially derived from human immunoglobulin sequences. The human FR sequence and human constant region sequence may be derived from different human immunoglobulin genes. For example, the FR sequence is derived from one human antibody and the constant region is derived from another human antibody. In some embodiments, the humanized antibody or antigen-binding fragment comprises human FR1-4.

In certain embodiments, the humanized antibodies and antigen-binding fragments thereof described herein comprise one or more FR sequences of W3452-1.164.16-z11 or W3452-2.683.2-z27.

Exemplary humanized anti-CD47 antibodies W3452-1.164.16-z11, and W3452-2.683.2-z27 retain affinity for specific binding to cells expressing CD47, and are at least comparable to the parent rat antibody in this regard, Even better than the parent rat antibody. These two exemplary humanized antibodies retain their functional interactions with CD47-expressing cells (such as CCRF-CEM cells), as reflected in their ability to block CD47-SIRPα interaction activity and induce efficient macrophage-mediated Induced tumor cell phagocytosis.

In some embodiments, a human-derived FR region may comprise the same amino acid sequence as the human immunoglobulin from which it is derived. In some embodiments, one or more amino acid residues of the human FR are substituted with corresponding residues from a parent non-human antibody. This is desirable in certain embodiments to bring the humanized antibody or fragment thereof in close proximity to a non-human parent antibody structure. In certain embodiments, the humanized antibody or antigen-binding fragment described in this application contains no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amine in each human FR sequence Acid residue substitutions, or no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in all FRs in the heavy or light chain variable domains. In some embodiments, this amino acid residue change may exist only in the heavy chain FR region, only in the light chain FR region, or on both chains.

In certain embodiments, the antibodies and antigen-binding fragments thereof described herein comprise a heavy chain variable domain sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, or SEQ ID NO: 25. In certain embodiments, the antibodies and antigen-binding fragments thereof described herein comprise a light chain variable domain sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23, or SEQ ID NO: 27.

In some embodiments, the anti-CD47 antibodies and antigen-binding fragments described herein comprise all or part of a heavy chain variable domain and / or all or part of a light chain variable domain. In one embodiment, the anti-CD47 antibodies and antigen-binding fragments described herein are single-domain antibodies consisting of all or part of the heavy chain variable domains described herein. More information on this single domain antibody is available in the prior art (see, e.g., U.S. Patent No. 6,248,516).

In certain embodiments, the anti-CD47 antibodies and antigen-binding fragments thereof described herein further comprise an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region comprises a heavy chain and / or light chain constant region. The heavy chain constant region comprises the CH1, hinge and / or CH2-CH3 regions. In certain embodiments, the heavy chain constant region comprises an Fc region. In certain embodiments, the light chain constant region comprises Cκ or Cλ.

In some embodiments, the anti-CD47 antibodies and antigen-binding fragments thereof described herein have constant regions of immunoglobulin (Ig), preferably human Ig, and preferably human IgG. In certain embodiments, the anti-CD47 antibodies and antigen-binding fragments thereof described herein include a constant region of the IgG1 isotype, which may cause ADCC or CDC, or a constant region of the IgG4 or IgG2 isotype, which has a reducing or eliminating effect Features. Effector functions (such as ADCC and CDC) can be cytotoxic to cells expressing CD47. Various assays can be used to assess effector functions, such as Fc receptor binding assays, C1q binding assays, and cell lysis assays.

The binding affinities of the antibodies and antigen-binding fragments described in this application can be expressed by the K _D value, which represents the ratio of the dissociation rate to the binding rate (k _off / k _on ) when the binding between the antigen and the antigen-binding molecule reaches equilibrium. Using techniques well known in the suitable methods, including flow cytometry, for example, may be appropriately determined antigen binding affinity (e.g., K _D). In some embodiments, the binding of the antibody to the antigen at different concentrations can be determined by flow cytometry. The determined average fluorescence intensity (MFI) can be first plotted against the antibody concentration, at this time by using Prism 5th Edition ( GraphPad Software, San Diego, CA), fitting the correlation between the specific binding fluorescence intensity (Y) and the antibody concentration (X) to a single site saturation formula: Y = B _max * X / (K _D + X) K _D value can be calculated, where B _max refers to the maximum specific binding of the test antibody to the antigen.

In some embodiments, the anti-CD47 antibodies and antigen-binding fragments thereof described in this application can be no more than 10 ^-9 M, no more than 9 × 10 ^-10 M, no more than 8 × 10 ^-10 , no more than 7 × 10 ^-10 , no more than 6 × 10 ^-10 , no more than 5 × 10 ^-10 M, no more than 4 × 10 ^-10 M, no more than 3 × 10 ^-10 M, no more than 2 × 10 ^-10 M, no more than 10 The binding affinity (K _D ) of ^-10 M, no more than 9 × 10 ¹¹ M, no more than 8 × 10 ^-11 M, or no more than 7.5 × 10 ^-11 M specifically binds to human CD47, and the K _D value is determined by Flow cytometry assay.

In certain embodiments, the anti-CD47 antibodies and antigen-binding fragments thereof described herein cross-react with cynomolgus CD47.

The binding to human CD47 antibodies can also be used as a "half maximal effect concentration" (EC ₅₀₎ value indicates that the maximum effect is observed means (e.g., binding or inhibition, etc.) when the antibody concentration of 50%. EC ₅₀ values may be known in the art by the methods as measured, for example, a sandwich method (e.g., ELISA, Western mark), flow cytometry, and other binding assays. In certain embodiments, the antibodies and fragments thereof described herein are no more than 0.45 nM, no more than 0.5 nM, no more than 0.55 nM, no more than 0.6 nM, no more than 0.65 nM, no more than 0.7 nM, and no more than 0.75. An EC ₅₀ value (ie, 50% binding concentration) of not more than 0.8 nM specifically binds to human CD47, and the EC ₅₀ value is determined by flow cytometry.

In certain embodiments, the antibodies and antigen-binding fragments thereof bind to cynomolgus CD47 with a lower binding affinity than human CD47. For example, the exemplary antibodies W3452-1.164.16-z11 less than the binding of human CD47 EC ₅₀ values cynomolgus CD47 binding.

In certain embodiments, the antibodies and fragments described herein are no more than 0.1 nM, no more than 0.15 nM, no more than 0.2 nM, no more than 0.25 nM, no more than 0.3 nM, no more than 0.35 nM, no more than 0.4 nM, or no more than 0.45 nM EC ₅₀ values of specifically binding CD47 and recombinant cynomolgus monkey, the EC ₅₀ value was measured by flow cytometry.

In certain embodiments, the antibodies and fragments thereof described herein have an affinity for specific binding to human CD47 sufficient for diagnostic and / or therapeutic use.

In certain embodiments, the antibodies and fragments thereof described herein block the binding of human CD47 to its ligand and thereby promote (e.g., induce or increase) phagocytosis, and antitumor activity without promoting (e.g., induce or increase) Erythrocyte agglutination reaction.

The antibody or antigen-binding fragment thereof described in this application may be a monoclonal antibody, a polyclonal antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a labeled antibody, a bivalent antibody, or an anti-idiotypic antibody. Recombinant antibodies are antibodies made using recombinant methods in vitro, not in animals.

Antibody variants <br/> The antibodies and antigen-binding fragments thereof described herein also encompass various variants thereof. In certain embodiments, the antibody and its antigen-binding fragments encompass the exemplary antibodies described in this application (i.e., W3452-1.164.16, W3452-1.164.16-z11, W3452-2.683.2, or W3452-2.683.2 -z27).

In certain embodiments, the antibody variant is in one or more of the CDR sequences shown in Table 1, one or more of the variable region sequences shown in Table 2 (but not in any of the CDR sequences), and / Or the constant region (e.g., the Fc region) contains one or more modifications or substitutions. These variants retain their affinity for the specific binding of CD47 to their parents, but possess one or more of the required characteristics brought about by this modification or substitution. For example, antibody variants can have improved antigen-binding affinity, improved productivity, improved stability, improved glycosylation pattern, reduced risk of glycosylation, reduced deamination, reduced or eliminated effector functions, improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and / or compatibility with conjugation (eg, one or more introduced cysteine residues).

Methods known in the art, such as "alanine scanning mutagenesis", can be used to screen parental antibody sequences to identify suitable or better residues to be modified or substituted (see, for example, Cunningham and Wells, (1989) Science, 244: 1081-1085). In short, target residues (such as positively charged residues such as Arg, Asp, His, Lys, and Glu) can be recognized and replaced by uncharged or negatively charged amino acids (such as alanine or polyalanine), Modified antibodies are generated and screened for target characteristics. If a substitution at a particular amino acid position exhibits a target functional change, that position can be identified as a potential residue for modification or substitution. This potential residue can be further evaluated by substitution with another residue (eg, a cysteine residue, a positively charged residue, etc.).

Affinity variants <br/> Affinity variants can be contained in one or more of the CDR sequences shown in Table 1, one or more FR sequences, or the heavy or light chain variable region sequences shown in Table 2 Modifications or substitutions. It is well known in the art that the CDR region is flanked by two FR regions in the variable region, so those skilled in the art can easily identify the FR sequence based on the CDR sequences in Table 1 and the variable region sequences in Table 2. . This affinity variant maintains the affinity of the parent antibody to specifically bind to CD47, or even has an improved affinity to CD47 that specifically binds to the parent antibody. In certain embodiments, at least one (or all) substitutions in a CDR sequence, a FR sequence, or a variable region sequence include conservative substitutions.

Those skilled in the art will understand that in the CDR sequences and variable region sequences shown in Tables 1 and 2, one or more amino acid residues may be substituted, and the obtained antibody or antigen-binding fragment still remains related to CD47 binding affinity, or even has improved binding affinity. Various methods known in the art can be used for this purpose. For example, a library of antibody variants (such as Fab or scFv variants) can be generated and expressed using phage display technology, and subsequently screened for affinity for binding to human CD47. As another example, computer software can be used to bind the antibody to human CD47 and recognize the amino acid residues on the antibody that form the binding interface. These residues can be avoided in substitutions to prevent a decrease in binding affinity, or they can be used as replacement targets to obtain stronger binding.

In certain embodiments, the humanized antibodies or epitope fragments described herein include one or more amino acid residue substitutions in one or more CDR sequences and / or one or more FR sequences. In certain embodiments, the affinity variant comprises no more than 10, 9, 8, 7, 6, 6, 5, 3, 2, or 1 substitutions in the CDR sequence and / or the FR sequence.

In certain embodiments, the anti-CD47 antibody and its antigen-binding fragment comprises 1, 2, or 3 sequences with at least 80% (e.g., at least 85%, 88%, 90%, 91%, (92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity CDR sequences, while maintaining similar or higher levels of binding to CD47 relative to their parent antibody Affinity.

In certain embodiments, the anti-CD47 antibody and antigen-binding fragment thereof comprises one or more sequences that are at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%) of the sequences listed in Table 2. , 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity of the variable region sequence, while maintaining similar or higher levels relative to its parent antibody binding to CD47 Affinity. In some embodiments, in a variable region sequence selected from Table 2, a total of 1 to 10 amino acids are substituted, inserted, or deleted. In some embodiments, the substitution, insertion, or deletion occurs in a region outside the CDR (eg, in the FR).

Glycosylated variants The anti-CD47 antibodies and antigen-binding fragments described herein also include glycosylated variants. The glycosylated variant can be obtained to increase or decrease the degree of glycosylation of the antibody or antigen-binding fragment.

The antibody or antigen-binding fragment thereof may comprise one or more amino acid residues with a side chain to which a carbohydrate moiety (eg, an oligosaccharide structure) may be attached. Glycosylation of antibodies is usually N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to a side chain of an aspartic acid residue, for example, an aspartic acid residue in a tripeptide sequence, such as aspartic acid-X-serine and asparagine Acid-X-threonine, where X is any amino acid other than proline. O-linked glycosylation refers to the attachment of a sugar of one of N-acetylgalactosamine, galactose, or xylose to a hydroxyl amino acid, most commonly to serine or threonine. Removal of natural glycosylation sites can be conveniently accomplished, for example, by changing the amino acid sequence so that the above-mentioned tripeptide sequence (for N-linked glycosylation site) or serine is present in the sequence Or one of the threonine residues (for O-linked glycosylation sites) is substituted. In a similar manner, new glycosylation sites can be created by introducing such tripeptide sequences or serine or threonine residues.

Cysteine engineered variants <br/> The anti-CD47 antibodies and antigen-binding fragments described herein also encompass cysteine engineered variants that include one or more introduced free cysteine amines Acid residues.

Free cysteine residues are cysteine residues that are not part of the disulfide bond. Cysteine engineered variants can be used, for example, at sites of engineered cysteine and, for example, cytotoxic and / or imaging compounds, tags, or radioisotopes by, for example, cis , And other substances. Methods for engineering antibodies or antigen-binding fragments to introduce free cysteine residues are well known in the art, see for example WO2006 / 034488.

Fc variants <br/> The anti-CD47 antibodies and antigen-binding fragments described herein also include Fc variants, which contain one or more amino acid residue modifications or substitutions in their Fc region and / or hinge region.

In certain embodiments, the anti-CD47 antibody or antigen-binding fragment comprises one or more amino acid substitutions that improve pH-dependent binding to neonatal Fc receptors (FcRn). This variant binds FcRn at an acidic pH, which protects it from degradation in the lysosome, and is subsequently transferred and released outside the cell. Therefore, this variant can have a longer pharmacokinetic half-life. Methods of engineering antibodies and antigen-binding fragments thereof to increase the binding affinity to FcRn are well known in the art, see, for example, Vaughn, D. et al., Structure, 6 (1): 63-73, 1998; Kontermann, R. Et al., Antibody Engineering, Volume 1, Chapter 27: Engineering of the Fc region for improved PK, Springer Publishing, 2010; Yeung, Y. et al., Cancer Research, 70: 3269-3277 (2010); and Hinton, P. et al. J. Immunology, 176: 346-356 (2006).

In certain embodiments, the anti-CD47 antibody or antigen-binding fragment comprises one or more amino acid substitutions that alter antibody-dependent cytotoxicity (ADCC). Certain amino acid residues in the CH2 domain of the Fc region can be substituted to increase ADCC activity. Alternatively or additionally, the carbohydrate structure on the antibody can be altered to increase ADCC activity. Methods for altering ADCC activity by antibody engineering have been described in the prior art, see, for example, Shields RL, et al., J Biol Chem. 2001. 276 (9): 6591-604; Idusogie EE et al., J Immunol. 2000.164 (8): 4178-84; Steurer W. et al., J Immunol. 1995, 155 (3): 1165-74; Idusogie EE. Et al., J Immunol. 2001, 166 (4): 2571-5; Lazar GA. Et al., PNAS, 2006 , 103 (11): 4005-4010; Ryan MC. Et al., Mol. Cancer Ther., 2007, 6: 3009-3018; Richards JO. Et al., Mol Cancer Ther. 2008, 7 (8): 2517-27; Shields RL et al., J. Biol. Chem, 2002, 277: 26733-26740; Shinkawa T. et al., J. Biol. Chem, 2003, 278: 3466-3473.

In certain embodiments, the anti-CD47 antibody or antigen-binding fragment comprises one or more amino acid substitutions that alter complement-dependent cytotoxicity (CDC), such as by enhancing or decreasing C1q binding and / or CDC (for other For examples of Fc region variants, see, for example, WO99 / 51642; Duncan & Winter Nature 322: 738-40 (1988); US Patent No. 5,648,260; US Patent No. 5,624,821; and WO94 / 29351). In certain embodiments, the anti-CD47 antibody polypeptide comprises a human IgG4 constant region, wherein the 228th amino acid residue is changed, such as Ser228Pro (S228P which may prevent or reduce chain exchange), and / or the 235th amine is changed Acid residues, such as Leu235Glu (L235E, which may alter Fc receptor interactions). In certain other embodiments, the anti-CD47 antibody polypeptide comprises a human IgG1 constant region.

In certain embodiments, the anti-CD47 antibody or antigen-binding fragment comprises one or more amino acid substitutions at the interface of the Fc region to facilitate and / or promote heterodimerization. These modifications include the introduction of a protrusion in the first Fc polypeptide and the introduction of a cavity in the second Fc polypeptide, wherein the protrusion can be located in the cavity to promote the interaction between the first and second Fc polypeptide to form a heterodimer. Body or complex. Methods of generating antibodies with such modifications are well known in the art, for example, as described in US Patent No. 5,731,168.

Antigen-binding fragment <br/> This application also provides an anti-CD47 antigen-binding fragment. Various types of antigen-binding fragments are well known in the art and can be developed based on the anti-CD47 antibodies described herein, including, for example, exemplary antibodies whose CDRs and variable sequences are shown in Tables 1 and 2, and Different variants (such as affinity variants, glycosylated variants, Fc variants, cysteine engineered antibodies, etc.).

In certain embodiments, the anti-CD47 antigen-binding fragments described herein are camelized single chain domain antibody, diabody, single-chain Fv fragment (scFv), scFv dimer , BsFv, dsFv, (dsFv) ₂ , Fv fragment, Fab, Fab ', F (ab') ₂ , ds diabody, nanobody, domain antibody, single domain antibody or bivalent domain antibody.

A variety of techniques can be used to generate this antigen-binding fragment. Exemplary methods include enzymatic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); and Brennan et al., Science, 229: 81 (1985)). E. coli) recombinant expression (e.g. for Fab, Fv and ScFv antibody fragments), phage display library screening (e.g. for ScFv) as discussed above, and chemical coupling of two Fab'-SH fragments to form F (ab ') ₂ fragments (Carter et al., Bio / Technology 10: 163-167 (1992)). Other techniques for producing antibody fragments will be apparent to those skilled in the art.

In certain embodiments, the antigen-binding fragment is a scFv. The generation of scFv is described in, for example, WO 93/16185; US Patent No. 5,571,894; and No. 5,587,458. scFv can be fused to an effector protein at the amine-terminus or carboxy-terminus to obtain a fusion protein (see, for example, Antibody Engineering, Borrebaeck, eds.).

Conjugates <br/> In certain embodiments, the anti-CD47 antibody and antigen-binding fragment thereof further comprise a conjugate portion. The conjugate portion can be linked to the antibody and its antigen-binding fragment. The conjugate portion is a non-protein portion that can be attached to the antibody or antigen-binding fragment thereof. It is envisaged that the antibodies or antigen-binding fragments thereof of the present invention may be linked to various conjugate moieties (see, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. (eds.), Carger Press, New York (1989)). These conjugate moieties can be linked to the antibody or antigen-binding fragment by other means such as covalent binding, affinity binding, embedding, coordinate binding, complexing, binding, mixing, or addition.

In certain embodiments, the antibodies and antigen-binding fragments disclosed herein can be engineered to contain a specific site outside the epitope-binding portion, which can be used to bind to one or more conjugate portions. For example, the site may contain one or more reactive amino acid residues (such as cysteine or histidine residues) to facilitate covalent attachment to the conjugate moiety.

In certain embodiments, the antibody may be indirectly attached to the conjugate moiety or through another conjugate moiety. For example, the antibody or antigen-binding fragment thereof can bind biotin and then indirectly bind a second conjugate moiety, which is linked to avidin. The conjugate portion can be a clearance modulator, a toxin (eg, a chemotherapeutic agent), a detectable label (eg, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, or an enzyme substrate) or a purified portion.

A "toxin" may be any agent that is harmful to the cell or may damage or kill the cell. Examples of toxins include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, turpentine, mitomycin, etoposide, teniposide, vincristine, vinblastine, Colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithromycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine , Tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5 -Fluorouracil dacarbaba), alkylating agents (e.g., nitrogen mustard, cetipine phenylbutyrate mustard, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, bleach Amphetamine, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracycline antibiotics (e.g. daunorubicin (formerly Daunor) Doxorubicin) and doxorubicin), antibiotics (such as dactinomycin (formerly known as actinomycin), bleomycin, mithromycin, and anthromycin (AMC)), antimitotic agents (such as vincin Base and vinblastine), topology Allosteric enzyme inhibitors, as well as tubulin binders.

Examples of detectable labels may include fluorescent labels (e.g., luciferin, rhodamine, salamander, phycoerythrin, or Texas red), enzyme-substrate labels (e.g., horseradish peroxidase, alkaline Alkaline phosphatase, luciferase, glucoamylase, lysozyme, sugar oxidase or β-D-galactosidase), radioisotopes (e.g., ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ³⁵ S , ³ H, ¹¹¹ In, ¹¹² In, ¹⁴ C, ⁶⁴ Cu, ⁶⁷ Cu, ⁸⁶ Y, ⁸⁸ Y, ⁹⁰ Y, ¹⁷⁷ Lu, ²¹¹ At, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹² Bi, and ³² P, Other lanthanides), luminescent labels, chromophores, digoxin, biotin / avidin, DNA molecules or gold for detection.

In certain embodiments, the conjugated moiety can be a clearance modulator that helps increase the half-life of the antibody. Illustrative examples include water-soluble polymers such as PEG, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, ethylene glycol / propylene glycol copolymers, and the like. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can be different, and if more than one polymer is attached, it can be the same or different molecules.

In some embodiments, the conjugated portion may be a purified portion, such as a magnetic bead.

In certain embodiments, the antibodies and antigen-binding fragments thereof described herein are used as a substrate for a conjugation moiety.

Polynucleotides and recombinant methods <br/> This application provides isolated polynucleotides encoding anti-CD47 antibodies and antigen-binding fragments thereof.

As used herein, the term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers in single- or double-stranded form. Unless explicitly defined, this term includes polynucleotides containing known natural nucleotide analogs that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, specific polynucleotide sequences also implicitly include conservatively modified variants thereof (eg, degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as clearly marked sequences. In particular, degenerate codon substitutions can be obtained by generating a sequence in which the third position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994)).

In certain embodiments, the isolated polynucleotide comprises one or more nucleic acid sequences as shown in SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, and / or 28, and / or Homologous sequences having at least 80% (e.g., at least 85%, 88%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and / Or only variants with degenerate substitutions and encoding exemplary antibodies as described herein. The DNA encoding the single antibody can be isolated and sequenced by conventional methods (for example, oligonucleotide probes can be used, which can specifically bind to genes encoding the heavy and light chains of the antibody). The encoded DNA can also be obtained by a synthetic method.

Using the well-known recombinant technology in this technology, a polynucleotide (for example, containing the sequence shown in Table 3) encoding the anti-CD47 antibody and its antigen-binding fragment can be introduced into a vector for pure line (amplified DNA) or gene expression. . A variety of vectors are available. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancement sequence, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence .

The vector (e.g., expression vector) provided in this application contains the nucleic acid sequence encoding the antibody or antigen-binding fragment thereof described in this application, and at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence. ), And at least one selectable tag. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (e.g., SV40) ), Lambda phage and M13 phage, plastid pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM. , PCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

A vector comprising a polynucleotide encoding the antibody and its antigen-binding fragment can be introduced into a host cell for pure line or gene expression. The host cell suitable for colonizing or expressing the DNA in the vector in the present invention is a prokaryotic cell, a yeast, or the above-mentioned higher eukaryotic cell. Prokaryotic cells suitable for use in the present invention include eukaryotes such as Gram-negative or Gram-positive bacteria, for example, Enterobacteriaceae, such as E. coli, Enterobacter, Erwinia, Klebsiella Genus, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescens, and Shigella, and Bacillus, such as Bacillus subtilis and lichen Bacillus, Pseudomonas such as Pseudomonas aeruginosa and Streptomyces.

In addition to prokaryotic cells, eukaryotic microorganisms such as filamentous fungi or yeasts can also be used as host cell lines or express vectors encoding anti-CD47 antibodies. Saccharomyces cerevisiae, or baker's yeast, are the most commonly used lower eukaryotic host microorganisms. However, many other genera, species, and strains are commonly used and applicable in the present invention, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as Kluyveromyces lactis, Kluyveromyces fragilis (ATCC 12,424) Kluyveromyces bulgaricus (ATCC 16,045), Kluyveromyces welchii (ATCC 24,178), Kluyveromyces kluyveri (ATCC 56,500), Kluyveromyces drosophila (ATCC 36,906), Kluyveromyces thermotolerance and Marksk Ruwiomyces; Yarrowia lipolytica (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesei (EP 244,234); Streptomyces; Western Schwann yeast, eg: Schwann yeast; and filamentous fungi, such as: Neurospora, Penicillium, Curvularia and Aspergillus, such as Aspergillus nidulans and Aspergillus niger.

The host cells provided in the present invention and suitable for expressing glycosylated antibodies or antigen-binding fragments thereof are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. A variety of baculoviral strains and their variants and corresponding permissive insect host cells have been discovered from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito) , Aedes albopictus (mosquito), Drosophila melanogaster (Drosophila) and silkworm. A variety of virus strains for transfection are publicly available, such as the alfalfa spodoptera nuclear polyhedrosis virus and the Bm-5 variant of the silkworm nuclear polyhedrosis virus. These viruses can be used in the present invention, especially For transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potatoes, soybeans, petunias, tomatoes and tobacco can also be used as hosts.

However, spinal cells are of most interest, and the culture (tissue culture) of spinal cells has become a well-known operation. Examples of mammalian host cells that can be used are: SV40 transformed monkey kidney cell line CV1 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or 293 cell sub-pure line in suspension culture, Graham et al., J. Gen Virol 36:59 (1977)); young hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980) ); Mouse testis support cells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587 ); Human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse breast tumors (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383: 44-68 (1982)); MRC 5 Cells; FS4 cells; and human liver cancer cell lines (Hep G2). In certain preferred embodiments, the host cell is a 293F cell.

The host cell is transformed with the expression or selection vector that can produce anti-CD47 antibodies, and cultured in a conventional nutrient medium, which is modified to be suitable for inducing a promoter, selecting transformed cells, or amplifying a sequence of interest. gene. In another embodiment, the antibody can be produced by homologous recombination methods known in the art.

The host cells used to produce the antibody or antigen-binding fragment thereof in the present invention can be cultured in a variety of media. Commercially available culture media such as Ham's F10 (Sigma), Minimum Basic Medium (MEM, (Sigma)), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma) can be used to culture the host cells. In addition, any of Ham et al., Meth. Enz. 58:44 (1979); Barnes et al., Anal. Biochem. 102: 255 (1980); US Patent No. 4,767,704; No. 4,657,866; No. 4,927,762; No. 4,560,655; Or No. 5,122,469; WO 90/03430; WO 87/00195; or the medium described in US Patent Application Re. 30,985 can be used as a medium for the host cell. These media can be supplemented with necessary hormones and / or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium chloride, magnesium chloride, and phosphate), and buffers (such as HEPES), nucleotides (such as adenylate and thymine), antibiotics (such as gentamicin), trace elements (defined as inorganic compounds with final concentrations usually in the micromolar range), and glucose or equivalent energy sources . The medium may also contain any other necessary additives at appropriate concentrations known in the art. The conditions of the medium, such as temperature, pH, and the like, are the conditions previously used to select host cells for expression and are well known to those skilled in the art.

When using recombinant technology, the antibody can be produced intracellularly, in the parietal space, or secreted directly into the culture medium. If the antibody is produced intracellularly, the debris of the host cell or lysed fragments is first removed, for example, by centrifugation or ultrasound. Carter et al., Bio / Technology 10: 163-167 (1992) describe a method for isolating antibodies secreted into the membrane space of E. coli. In short, the cell paste was opened in the presence of sodium acetate (pH 3.5), EDTA and benzylsulfonium fluoride (PMSF) for about 30 minutes or more. Centrifuge to remove cell debris. If the antibody is secreted into the culture medium, a commercially available protein concentration filter, such as Amicon or Millipore Pellicon ultrafiltration unit, is usually first used to concentrate the supernatant of the performance system. Protease inhibitors such as PMSF can be added in any of the foregoing steps to inhibit protein degradation, as well as antibiotics to prevent accidental contaminant growth.

Anti-CD47 antibodies prepared from the cells can be purified by purification methods such as hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography columns, ammonium sulfate precipitation, salting out, and affinity Chromatography, with affinity chromatography being the preferred purification technique.

In certain embodiments, protein A immobilized on a solid phase is used for immunoaffinity purification of the antibody and antigen-binding fragments. The type of antibody and the presence of any immunoglobulin Fc domain in the antibody determine whether protein A is suitable as an affinity ligand. Protein A can be used to purify human γ1, γ2, or γ4 heavy chain-based antibodies (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is suitable for all murine isomers and human gamma 3 (Guss et al., EMBO J. 5: 1567 1575 (1986)). Agarose is the most commonly used affinity ligand attachment matrix, but other matrices are also available. Mechanically stabilized matrices such as controlled porosity glass or poly (styrene) benzene can achieve faster flow rates and shorter processing times than with agarose. If the antibody contains a CH3 domain, it can be purified using Bakerbond ABX ^™ resin (JT Baker, Philipsburg, New Jersey). Other techniques for protein purification can also be determined based on the antibodies obtained, such as fractionation in ion exchange columns, ethanol precipitation, reversed-phase HPLC, silica gel chromatography, heparin agarose gel chromatography based on anion or cation exchange resins Polyaspartic acid column), chromatography focus, SDS-PAGE, and ammonium sulfate precipitation.

After any preliminary purification steps, low pH hydrophobic interaction chromatography can be used to treat mixtures containing antibodies and impurities of interest, using a wash buffer pH of about 2.5-4.5, preferably at low salt concentrations (e.g., (About 0 to 0.25 M salt concentration).

Pharmaceutical composition <br/> This application further provides the pharmaceutical composition and the one or more pharmaceutically acceptable carriers described in this application, comprising the anti-CD47 antibody or antigen-binding fragment thereof.

The pharmaceutically acceptable carriers used in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous media, non-aqueous media, antimicrobial substances, isotonic substances , Buffers, antioxidants, anesthetics, suspending agents / dispersing agents, chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art or a combination of the above.

Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers or stabilizers such as sugar And cyclodextrin. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, mercaptoacetic acid, mercaptosorbitol, butylmethyl Anisole, butylated hydroxytoluene and / or propyl gallate. As disclosed in the present invention, including one or more antioxidants such as methionine in a composition containing the antibody or antigen-binding fragment thereof disclosed in the present invention can reduce the oxidation of the antibody or antigen-binding fragment thereof. A reduction in oxidation prevents or reduces a decrease in binding affinity, thereby increasing antibody stability and extending shelf life. Therefore, in certain embodiments, the composition provided by the present invention contains one or more of said antibodies or antigen-binding fragments thereof and one or more antioxidants such as methionine. The present invention further provides various methods. By mixing the antibody or antigen-binding fragment thereof provided in the present invention with one or more antioxidants, such as methionine, the antibody or the antigen-binding fragment can be prevented from being oxidized and prolonged. Shelf life and / or increase its activity.

Further, a pharmaceutically acceptable carrier may include, for example, an aqueous medium such as sodium chloride injection, Ringer's solution injection, isotonic glucose injection solution, sterile water injection solution, or glucose and lactate Ringer Injections, non-aqueous media such as: non-volatile oils of plant origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antibacterial substances at bacterial or fungal inhibitory concentrations, 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, emulsifiers such as polysorbate 80 (Tween-80), chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol bis (2-amine) Ethyl ether) tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. The antibacterial agent as a carrier can be added to the pharmaceutical composition in a multi-dose container, which contains phenols or cresols, mercury preparations, benzyl alcohol, chlorobutanol, methyl and propyl parabens, Thimerosa, ammonium chlorobenzyl and chlorophenethyl ammonium. Suitable excipients may include, for example, water, salt, glucose, glycerol or ethanol. Suitable non-toxic auxiliary substances may include, for example, emulsifiers, pH buffers, stabilizers, solubilizers, or substances such as sodium acetate, sorbitan laurate, triethanolamine oleate, or cyclodextrin .

The pharmaceutical composition may be a liquid solution, a suspension, an emulsion, a pill, a capsule, a tablet, a sustained release preparation or a powder. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, and the like.

In certain embodiments, the pharmaceutical composition is formulated as an injectable composition. Injectable pharmaceutical compositions can be prepared in any conventional form, for example, liquid solvents, suspending agents, emulsifiers or solid forms suitable for producing liquid solvents, suspending agents or emulsifiers. Injectable preparations can include sterile and / or pyrogen-free solutions currently in use, sterile dry solubles that are combined with the solvent before use, such as lyophilized powder, including subcutaneous tablets, sterile suspensions for injection, ready-to-use media Combined sterile dry insoluble products, and sterile and / or pyrogen-free emulsions. The solvent can be an aqueous or non-aqueous phase.

In certain embodiments, a unit dose of the injectable preparation is packaged in an ampoule, a tube, or a syringe with a needle. It is known in the art that all preparations for injection should be sterile and pyrogen-free.

In certain embodiments, a sterile lyophilized powder can be prepared by dissolving the antibody or antigen-binding fragment thereof disclosed in this application in a suitable solvent. The solvent may contain a powder or a reconstituted solution prepared from the powder, or other pharmacological components of the reconstituted solution. Suitable excipients include, but are not limited to, water, glucose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other applicable substances. The solvent may contain a buffer, such as a citrate buffer, a sodium or potassium phosphate buffer, or other buffers known to those skilled in the art. In one embodiment, the pH of the buffer is neutral. The dissolution is followed by sterilization by filtration under standard conditions well known in the art, and then lyophilization to obtain the desired formulation. In one embodiment, the resulting solvent is aliquoted into a vial and lyophilized. Each vial can contain a single dose or multiple doses of the anti-CD47 antibody or antigen-binding fragment or combination thereof. The amount of filling in each vial can be slightly higher than that required for each dose or multiple doses (for example, a 10% excess), thereby ensuring accurate sampling and accurate administration. The lyophilized powder can be stored under appropriate conditions, such as in the range of about 4 ° C to room temperature.

The lyophilized powder was reconstituted with water for injection to obtain a preparation for injection administration. In one embodiment, the lyophilized powder can be redissolved in sterile pyrogen-free water or other suitable liquid carriers. The precise amount is determined by the chosen therapy and can be determined based on experience.

Method of Use This application also provides a method of treatment comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof described herein to an individual in need thereof, thereby treating or preventing a condition or disorder associated with CD47. In some embodiments, the condition or disorder associated with CD47 is cancer, autoimmune disease, fibrosis, inflammatory disease, or infectious disease.

Examples of cancer include, but are not limited to, lung cancer, bronchial cancer, bone cancer, liver and bile duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, Cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary cancer, gastric cancer, vaginal cancer, thyroid cancer, glioblastoma Cell tumor, astrocytoma, melanoma, myelodysplastic syndrome, malignant sarcoma, teratoma, adenocarcinoma, leukemia, myeloma and lymphoma.

Examples of autoimmune diseases include, but are not limited to, acquired immunodeficiency syndrome (AIDS, which is a viral disease with autoimmune components), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease , Autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphocytosis syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), shellfish Chet's disease, cardiomyopathy, celiac celiac dermatitis; chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), scarring pemphigoid, cold agglutinin, CREST Syndrome, Crohn's disease, Degos' disease, juvenile dermatomyositis, discoid lupus, primary mixed cryoglobulinemia, fibromyalgia fibromyositis, Graves disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (Stear's disease), juvenile rheumatoid arthritis, Meni Ehrlich disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, refractory anemia, nodular polyarteritis, polychondritis, polyglandular syndrome, rheumatic polymyalgia, polymyositis, and dermatomyositis , Primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Wright's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (progressive Systemic Sclerosis (PSS), also known as Systemic Sclerosis (SS), Sjogren's Syndrome, Systemic Myotonia Syndrome, Systemic Lupus Erythematosus, Gao'an Arteritis, Temporal Arteritis / Giant Cell Arteritis, Ulcerative Colitis, uveitis, vitiligo, and Wegener's granulomatosis.

Inflammatory diseases include, for example, chronic and acute inflammatory diseases. Examples of inflammatory diseases include Alzheimer's disease, asthma, atopic allergies, allergies, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft versus host disease, hemolytic anemia, bones and joints Inflammation, sepsis, stroke, tissue and organ transplantation, vasculitis, diabetic retinopathy, and lung damage caused by ventilator.

Examples of infectious diseases include, but are not limited to, fungal infections, parasitic / protozoal infections, or chronic viral infections, such as malaria, coccidiosis, histoplasmosis, onychomycosis, aspergillosis, blastomycosis, white rosary Mycosis, Paracoccidiosis, Microsporidiosis, Acanthamoeba keratitis, Amoebiasis, Ascariasis, Babesiasis, Pouch disease, Bellissiasis, American Trypanosomiasis, China Clonorchiasis, trypanosomiasis, cryptosporidiosis, schistosomiasis, dragon nematode, echinococcus, elephantiasis, tsutsugamushi, schistosomiasis, ginger schistosomiasis, silk Worm disease, giardiasis, maxillofacial disease, hydatid disease, isosporidiosis, oncomelania fever, leishmaniasis, Lyme disease, trematodes disease, fly maggot disease, onchocerciasis Disease, tick disease, scabies, schistosomiasis, lethargy, roundworm disease, ascariasis, toxoplasmosis, toxoplasmosis, trichinellosis, whipworm disease, trypanosomiasis, helminth infection, hepatitis B (HBV) , Hepatitis C (HCV), herpes virus, EB virus, HIV, cytomegalovirus, herpes simplex virus type I, herpes simplex disease Virus type II, human papilloma virus, adenovirus, human immunodeficiency virus I, human immunodeficiency virus II, Kaposi's sarcoma-associated herpes virus epidemic, thin ring virus (ring virus), human T lymphotrophic virus type I Human T lymphotrophic virus type II, Varicella zoster, JC virus or BK virus infection.

The therapeutically effective dose of the antibody or antigen-binding fragment thereof provided in this application depends on a variety of factors known in the art, such as weight, age, past medical history, current treatment, subject's health status and potential for cross-infection, allergies, hypersensitivity And side effects, as well as the route of administration and the extent of disease development. Those skilled in the art (eg, a doctor or veterinarian) can reduce or increase the dose proportionally according to these or other conditions or requirements.

In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein can be administered at a therapeutically effective dose of between about 0.01 mg / kg and about 100 mg / kg. In certain embodiments, the antibody or antigen-binding fragment thereof is administered at a dose of about 50 mg / kg or less, and in certain embodiments, the dosage is 10 mg / kg or less, 5 mg / kg kg or less, 3 mg / kg or less, 1 mg / kg or less, 0.5 mg / kg or less or 0.1 mg / kg or less. A particular dose can be administered at multiple intervals, such as once a day, twice a day or more, twice a month or more, once a week, once every two weeks, once every three weeks, once a month, or every two Once a month or more. In certain embodiments, the dosage administered may vary with the course of treatment. For example, in certain embodiments, the initial dose may be higher than the subsequent dose. In certain embodiments, the dose administered is adjusted during the course of treatment based on the response of the subject being administered.

The dosing schedule can be adjusted to achieve an optimal response (such as a therapeutic response). For example, a single dose may be administered or divided into multiple divided doses over a period of time.

The antibodies and antigen-binding fragments disclosed in the present invention can be administered by known methods of administration in the art, such as injection (for example, subcutaneous injection, intraperitoneal injection, intravenous injection, including intravenous drip, intramuscular injection or intradermal injection). Injection) or non-injection (eg, oral, nasal, sublingual, rectal or topical).

In some embodiments, the antibodies and antigen-binding fragments disclosed herein can be administered alone or in combination with one or more other therapeutic means or substances. For example, the antibodies and antigen-binding fragments disclosed herein can be administered in combination with another therapeutic agent, such as a chemotherapeutic agent or an anticancer drug.

In certain such embodiments, when the antibodies and antigen-binding fragments disclosed herein are used in combination with one or more of the above-mentioned therapeutic substances, they may be administered simultaneously with the one or more of the therapeutic substances. In some such embodiments, In the above, the antibody and the antigen-binding fragment can be administered simultaneously as part of the same pharmaceutical composition. However, antibodies and antigen-binding fragments that are "combined" with other therapeutic substances need not be administered simultaneously or in the same composition as the therapeutic substance. The meaning of "combined use" in the present invention also includes antibodies and antigen-binding fragments that are administered before or after another therapeutic substance is also considered to be "combined with" the therapeutic substance, even if the antibody or antigen-binding fragment thereof and the first The two substances are administered by different modes of administration. Where possible, other therapeutic substances used in combination with the antibodies or antigen-binding fragments thereof disclosed in the present invention can be used in accordance with the methods of the product description of the other therapeutic substances, or refer to the surgeon's desk reference book 2003 (Physicians' Desk Reference 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th Edition (November 2002)), or refer to other methods known in the art.

The application further provides a method for using the anti-CD47 antibody or an antigen-binding fragment thereof.

In some embodiments, the application provides a method for detecting the presence or content of CD47 in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof, and determining the presence or content of CD47 in the sample.

In some embodiments, the application provides a method for diagnosing a disease or condition associated with CD47 in an individual, comprising: a) contacting a sample obtained from the individual with the antibody or antigen-binding fragment thereof; b) determining the The presence or amount of CD47 in the sample; and c) correlating the presence of CD47 with the CD47-related disease or condition in the individual.

In some embodiments, the present application provides a set comprising the antibody or antigen-binding fragment thereof described in the present application, which is conjugated to a detectable moiety as appropriate. This kit can be used to detect CD47 or diagnose CD47-related diseases.

In some embodiments, the present application also provides the use of the antibody or antigen-binding fragment thereof described in the present application in the manufacture of a medicament for treating a disease or condition related to CD47 in an individual, and in the preparation of a CD47-related diagnosis. Use in diagnostic reagents for diseases or conditions.

Advantages The antibodies and antigen-binding fragments described herein have high affinity for binding to CD47. It can block the CD47-SIRPα interaction and induce efficient macrophage-mediated tumor cell phagocytosis in vitro and in vivo. Due to the widespread expression of CD47 in human normal cells, antibodies in the prior art have been reported to have side effects such as anemia and platelet depletion. It is worth noting that the antibodies and antigen-binding fragments described in this application do not produce anemia and platelet depletion, indicating that they may not produce side effects and have advantages in clinical trials.

The following examples are intended to better illustrate the invention and should not be construed as limiting the scope of the invention. All the specific compositions, materials and methods described below, in whole or in part, are within the scope of the present invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate that specific embodiments are within the scope of the invention. Those skilled in the art can develop equivalent compositions, materials, and methods without adding creativity and without departing from the scope of the invention. It should be understood that various modifications made to the method of the present invention may still be included within the scope of the present invention. The inventors intend to include such variations within the scope of the present invention.

Examples Example 1 : Production of hybridoma antibodies
1.1 Animal Immunity <br/> The extracellular domain (ECD) protein of human CD47 was used as an immunogen for animal immunity. SD rats were purchased from Shanghai SLAC Experimental Animal Co., Ltd. and were housed in animal facilities approved by IACUC. Animal blood was collected before and after immunization, and the serum titer against the target protein was detected by ELISA according to a general ELISA procedure.

1.2 Production of hybridomas <br/> SD rats with the highest serum titers were selected for cell fusion. B cells from the spleen and lymph nodes were fused with SP2 / 0 myeloma cells by electrofusion according to a general electrofusion procedure. After cell fusion, the cells were plated in 96-well plates in DMEM medium supplemented with 20% FBS and 1% HAT selective reagent.

1.3 Humanization of antibodies <br/> The "BestFit" method was used to humanize the light and heavy chains of antibodies. For the light chain, the amino acid sequences of the corresponding V genes of W3452-1.164.16 and W3452-2.683.2 were compared with the internal human germline V-gene database of the company. The sequence of the humanized VL-gene was derived from the use of the Kabat CDR definition, replacing the human CDR sequence with the highest hit with the mouse CDR sequence. For heavy chains, humanized sequences are derived. The first sequence is derived from the light chain. Additional sequences were generated by aligning the mouse framework with the human germline V-gene database. An extended CDR definition was used to define the framework, in which Kabat CDR1 was extended by 5 amino acids at the N-terminus. The highest three hits were used to obtain humanized VH gene sequences. Humanized genes were back-translated, codon optimized for mammalian expression, and synthesized by GeneWiz (Suzhou). The synthetic genes were re-selected into human IgG expression vectors, expressed and purified.

Two humanized pure lines W3452-2.683.2-z27 and W3452-1.164.16-z11 were obtained and expressed in different human constant regions. The "W3452-2.683.2-z27-IgG1L" described in this application refers to the antibody W3452-2.683.2-z27 which further comprises a constant region of human IgG1. The "W3452-2.683.2-z27-IgG4PE, L" described in this application refers to the antibody W3452-2.683.2-z27 which further comprises a human IgG4 constant region containing S228P and L235E mutations. The "W3452-1.164.16-z11-IgG1K" described in this application refers to the antibody W3452-1.164.16-z11 further comprising a constant region of human IgG1.

Reference antibodies were also prepared and used as controls for characterization assays and studies. A total of 4 reference antibodies were prepared, namely: WBP345-BMK1.uIgG4PE.K (sequences are disclosed in WO 2016/109415 A1), WBP345-BMK2.uIgG4P.K (in Liu J. et al., PLoS One, 2015, 10: e0137345 is disclosed as Hu5F9-G4), the sequence of WBP345-BMK3.uIgG1K (C47B222-IgG1, C47B222 is disclosed in WO 2016/081423 A1), WBP345-BMK4.uIgG4.SPK (in US 2017/0081407 A1 is 2.3 D11 IgG4).

Isotype control antibodies W332-1.80.12xAb.hIgG1 and W332-1.80.12xAb.hIgG4 did not bind to CD47 and were used as negative controls.

Example 2 : In vitro characterization
1. Targeted binding (ELISA / FACS)
1.1 Targeted Binding (FACS)
A cell line expressing human CD47 (W345-CHOK1.hPro1.D1), CCRF-CEM (ATCC # CRM-CCL-119) was prepared or purchased from WuXi Biologics.

The binding of CD47 antibodies to cells expressing CD47 (W345-CHOK1.hPro1.D1, CCRF-CEM) was determined by flow cytometry. Briefly, 1 × 10 ⁵ cells / well were added to each well of a 96-well plate, and centrifuged at 1500 rpm at 4 ° C. for 5 minutes, and then the supernatant was removed. Serial dilutions of test antibodies and positive and negative control antibodies were added to the pelleted cells and incubated for 1 hour at 4 ° C. Cells were washed twice with 200 μl PBS containing 1% BSA. PE-conjugated goat anti-human IgG Fc (Jackson, # 109-115-098) diluted 1: 100 with FACS buffer was added to the cells and incubated at 4 ° C for 30 minutes. Two additional washing steps were performed with 200 μl of FACS buffer and then centrifuged at 1500 rpm for 5 minutes at 4 ° C. Finally, the cells were resuspended in 100 μl FACS buffer, the fluorescence values were measured by flow cytometry and analyzed by FlowJo.

Using CHOK1 cells stably transfected with human CD47, the antigen-binding activities of W3452-1.164.16-z11-IgG1K and W3452-2.683.2-z27-IgG1L and W3452-2.683.2-z27-IgG4PE, L were determined by FACS. As shown in Figure 1, W3452-1.164.16-z11-IgG1K and W3452-2.683.2-z27-IgG1L and W3452-2.683.2-z27-IgG4PE, L bind to CD47-transfected cells, but not to the parent CHOK1 blank cells bind. The specific information is shown in Table 4 below.
Table 4 shows the results of human CD47 binding by FACS

1.2 Cross-Combination (FACS)
Cynomolgus monkey PBMC is used for the expression cells of cynomolgus CD47, which is isolated from the fresh blood of cynomolgus monkey.

The binding of CD47 antibody to CD47-expressing cells (cynomolgus monkey PBMC) was determined by flow cytometry. Briefly, 1 × 10 ⁵ cells / well were added to each well of a 96-well plate, and centrifuged at 1500 rpm at 4 ° C. for 5 minutes, and then the supernatant was removed. Serial dilutions of test antibodies and positive and negative control antibodies were added to the pelleted cells and incubated at 4 ° C for 1 hour. Cells were washed twice with 200 μl PBS containing 1% BSA. PE-conjugated goat anti-human IgG Fc (Jackson, # 109-115-098) diluted 1: 100 with FACS buffer was added to the cells and incubated at 4 ° C for 30 minutes. Two additional washing steps were performed with 200 μl of FACS buffer and then centrifuged at 1500 rpm for 5 minutes at 4 ° C. Finally, the cells were resuspended in 100 μl FACS buffer, the fluorescence values were measured by flow cytometry and analyzed by FlowJo.

Using cynomolgus monkey PBMC, the antigen-binding activities of W3452-1.164.16-z11-IgG1K and W3452-2.683.2-z27-IgG1L and W3452-2.683.2-z27-IgG4PE, L were determined by FACS. The results show that W3452-1.164.16-z11-IgG1K binds well to cynomolgus monkey PBMC, has a low EC ₅₀ and a maximum MFI. W3452-2.683.2-z27-IgG1L and W3452-2.683.2-z27-IgG4PE, L show similar EC ₅₀ and MFI (see Table 5 and Figure 2 below). W332-1.80.12.xAb.hIgG1 and W332-1.80.12.xAb.hIgG4 in the table are IgG1 and IgG4 isotype control antibodies, respectively.
Table 5 shows the results of cynomolgus CD47 binding by FACS.

2. Affinity (FACS)
Collect cells and count live cells (W345.CHO-K1.hPro1.FL.G4 (P1)), centrifuge cells and resuspend in an appropriate volume of 1% BSA / 1XPBS, with a density of 1 × 10 ⁶ cells / ml, And 50 μl of cell suspension was added to each well of a 96-well U-shaped plate. It was then centrifuged at 1500 rpm for 4 minutes and the supernatant was discarded.

Dilute W3452-1.164.16-z11-IgG1K and W3452-2.683.2-z27-IgG1L and W3452-2.683.2-z27-IgG4PE, L with 1% BSA / 1XPBS (initial concentration is 10 μg / ml, diluted twice ), And added to the FACS plate at 100 μl / well, and incubated at 4 ° C. for 1 hour. It was then centrifuged at 1500 rpm for 4 minutes and the supernatant was discarded. Goat anti-human IgG Fc FITC (diluted to 14 μg / ml with 1% BSA / 1XPBS) was added at 100 μl / well to resuspend the cells and incubated at 4 ° C for 30 minutes. minute. After that, the cells were washed once with 180 μl / well of 1% BSA / 1XPBS, and the supernatant was discarded. After the final wash, the cells were resuspended in 100 μl / well of 1% BSA / 1XPBS. Store the cells in the dark at 4 ° C until run. Fluorescence intensity was measured by flow cytometry (BD Canto II) and analyzed by FlowJo.

The antigen-binding affinity of W3452-1.164.16-z11-IgG1K and W3452-2.683.2-z27-IgG1L and W3452-2.683.2-z27-IgG4PE, L was determined by FACS using CHOK1 cells stably transfected with human CD47 ( Table 6). All three antibodies showed high affinity to human CD47.
Table 6 shows the results of human CD47 binding affinity measured by FACS

3. Cell-based ligand competition assay <br/> Cell line expressing human CD47 (W345-CHOK1.hPro1.D1), CCRF-CEM (ATCC # CRM-CCL-119) by Wuming Biotechnology Co., Ltd. ( WuXi Biologics). Human SIRPα (W345-hPro1L1.His (sino)) was purchased from Sino Biological (Cat # 11612-H08H-50).

Competition between CD47 antibody and human SIRPα ligand on CD47 expressing cells (W345-CHOK1.hPro1.D1, CCRF-CEM) was determined by flow cytometry. Briefly, cells were added to each well of a 96-well round-bottomed culture plate at 1 × 10 ⁵ cells / well, and centrifuged at 1500 rpm at 4 ° C. for 5 minutes, and then the supernatant was removed. Serial dilutions of the test antibody and positive and negative control antibodies with 1 μg / ml human SIRPα were added to the pelleted cells and incubated at 4 ° C for 2 hours. Cells were washed twice with 200 μl PBS containing 1% BSA. Biotin-conjugated antihistidine-tagged antibody (GenScript, # A00613) diluted 1: 400 with FACS buffer was added to the cells and incubated at 4 ° C for 30 minutes. PE-conjugated streptavidin (Affymetrix, # 12-4317) diluted 1: 200 with FACS buffer was added to the cells and incubated at 4 ° C for 30 minutes. Two additional washing steps were performed with 200 μl of FACS buffer and then centrifuged at 1500 rpm for 5 minutes at 4 ° C. Finally, the cells were resuspended in 100 μl FACS buffer, the fluorescence values were measured by flow cytometry and analyzed by FlowJo.

In order to evaluate the blocking activity of the ligands (SIRPα) of W3452-1.164.16-z11-IgG1K and W3452-2.683.2-z27-IgG1L and W3452-2.683.2-z27-IgG4PE, L, stable transfection of human CD47 was used CHOK1 was tested by FACS using competitive binding to test the above three antibodies for blocking the CD47-SIRPα interaction activity. The results showed that W3452-1.164.16-z11-IgG1K and W3452-2.683.2-z27-IgG1L and W3452-2.683.2-z27-IgG4PE, L blocking rates were more than 90% (see Table 7 and Figure 3 below). W332-1.80.12.xAb.hIgG1 and W332-1.80.12.xAb.hIgG4 in the table are IgG1 and IgG4 isotype control antibodies, respectively.
Table 7 shows the results of the ligand (SIRPα) competition test by FACS

4. Human RBC Hemagglutination Assay (HA)
In order to evaluate the blood agglutination ability of the CD47 antibodies (i.e. Human RBC obtained after three washes and centrifuged at 2000 rpm for 5 minutes, diluted to 4% with PBS, and incubated with a certain titer of CD47 antibody and positive and negative control antibodies in a round-bottomed 96-well plate at 37 ° C. 1 hour. The results are recorded with a camera. Hemagglutination was confirmed by the presence of unsettled red blood cells. Unsettled red blood cells appeared as a mist, while non-agglutinated RBCs were spot-shaped red dots. The results showed that at any concentration tested, W3452-1.164.16-z11-IgG1K, W3452-2.683.2-z27-IgG1L, and W3452-2.683.2-z27-IgG4PE did not show hemocoagulant activity (see Figure 4) . In contrast, the baseline control antibody WBP345-BMK2.uIgG4P.K showed hemocoagulant activity.

5. Cell-based phagocytosis assay <br/> PBMCs were isolated from human blood, and CD14-positive monocytes were isolated from PBMCs by hCD14 microbeads (Miltenyi Biotec, # 130-050-201). Seven angels were differentiated into macrophages by culturing CD14-positive monocytes in 10% FBS RPIM1640 medium containing 100 ng / ml rhM-CSF (R & D Systems, # 216-MC / CF). These monocyte-derived macrophages (MDM) become able to attach, while other cells are washed away. MDM was scraped off and replated in a 96-well plate. Jurkat. 2B8, CCRF-CEM, Raji and hRBC were selected as target cell types. Target cells were labeled with 1 μM CFSE (Life-technology, # C34570) at 37 ° C. for 30 minutes, then washed and added to MDM at a ratio of tumor cells to phagocytes 1: 1, and then CD47 antibodies of different concentrations were added. The phagocytosis of the target cells continued for 2 hours. Subsequently, it was stained with an antibody against CD14, a macrophage marker conjugated to APC (BD Pharmingen, # 561708), and analyzed by flow cytometry. Phagocytosis was measured by screening FL4-positive living cells (CD14 +), and then the percentage of FL1 (CFSE +)-positive cells was evaluated.

Human PBMC-derived macrophages and Jurkat. 2B8 and Raji cells were used to determine phagocytic activity, and the ratio of the number of macrophages taken up by tumor cells to the total number of macrophages was calculated. The results show that W3452-1.164.16-z11-IgG1K, W3452-2.683.2-z27-IgG1L, and W3452-2.683.2-z27-IgG4PE, L induce potent phagocytosis of tumor cells (see Table 8 and Table 9 below, and 5A and 5B). W332-1.80.12.xAb.hIgG1 and W332-1.80.12.xAb.hIgG4 in the table are IgG1 and IgG4 isotype control antibodies, respectively.
Table 8 shows Jurkat.2B8 cell phagocytosis test results
Table 9 shows the results of Raji cell phagocytosis test

6. ADCC and CDC determination
6.1 ADCC Assay Briefly, human PBMCs were cultured overnight in RPMI1640 medium containing 10% fetal bovine serum, 1% penicillin / streptomycin solution and 50 units / mL hIL-2. The next day, PBMCs were used as effector cells, and CCRF-CEM or Raji were used as target cells. In a U-bottomed 96-well plate, each well was added with 2 × 10 ⁴ target cells in 50 μL of RPMI1640 (phenol-free) medium containing 1% FBS. Then, 10 μL of a series of diluted antibodies dissolved in RPMI1640 (phenol-free) medium containing 1% FBS was added to each well. After 15 minutes of incubation at 37 ° C, 4 × 10 ⁵ PBMCs in 40 μL of RPMI 1640 (phenol-free) medium containing 1% FBS were added to each well to obtain an E / T ratio of 20: 1. After 4 hours of incubation at 37 ° C, the mixture was centrifuged at 1500 rpm for 5 minutes and 70 μL of the supernatant was transferred. Use the LDH Cytotoxicity Detection Kit (Roche) to evaluate cell death according to the manufacturer's instructions.

6.2 CDC determination In a U-bottomed 96-well plate, 5 × 10 ⁴ target cells were added to each well in 50 μL of RPMI1640 (phenol-free) medium containing 1% FBS. Then, serial dilution antibodies in 10 μL of RPMI1640 (phenol-free) medium containing 1% FBS were added to each well. After 15 minutes of incubation at 37 ° C, human serum dissolved in 40 μL of RPMI1640 (phenol-free) medium containing 1% FBS was added to each well to obtain a final concentration of 20%. After 2 hours of incubation at 37 ° C, 10 μL of propidium iodide (Invitrogen) was added to each well and incubated for 10 minutes at room temperature. Cell death was evaluated by FACS analysis.

6.3 Results As shown in Figure 6A-6D, W3452-1.164.16-z11-IgG1K and W3452-2.683.2-z27-IgG1L induce effective ADCC activity on both CCRF-CEM and Raji cells, while only W3452-1.164.16- z11- IgG1K induces CDC activity in tumor cells. W3452-2.683.2-z27-IgG4PE, L did not induce or induce weak ADCC and CDC activities.

7. Thermal stability <br/> Differential Scanning Fluorescence (DSF) measurement using 7500 Fast Real-Time PCR System (Applied Biosystems) to detect W3452-1.164.16-z11-IgG1K, W3452-2.683.2- The thermal stability of z27-IgG1L and W3452-2.683.2-z27-IgG4PE, L. Briefly, 19 μL of antibody solution was mixed with 1 μL of 62.5 X SYPRO Orange solution (Invitrogen) and added to 96-well plates (Biosystems). The plate was heated from 26 ° C to 95 ° C at a rate of 2 ° C / min, and the obtained fluorescence data was collected. Calculate the negative derivative of the fluorescence change at different temperatures, and define the maximum value as the melting temperature T _h . If the protein has multiple unfolding transitions, the first two T _h are reported, named T _h 1 and T _h 2. T _h 1 is often interpreted as the formal melting temperature T _m to facilitate comparison between different proteins. Data collection and T _h calculation are performed automatically by its operating software. Once the software reports negative derivative plots at different temperatures, the point on the curve where the curve begins to fall from the pre-transition baseline can be roughly estimated as the starting temperature _Ton .

As shown in Figure 7 and Table 10 below, compared with the normal values of human mAb, W3452-1.164.16-z11-IgG1K, W3452-2.683.2-z27-IgG1L, and W3452-2.683.2-z27-IgG4PE, L show Good thermal stability and as good or better as the benchmark control antibodies (WBP345-BMK1.uIgG4PE.K and WBP345-BMK2.uIgG4P.K).
Table 10 Thermal stability results

8. Serum stability <br/> In order to facilitate in vivo studies, W3452-1.164.16-z11-IgG1K, W3452-2.683.2-z27-IgG1L, and W3452-2.683.2-z27-IgG4PE were performed on human serum, L's serum stability test. Freshly collected human blood was placed in polystyrene tubes without anticoagulants and incubated at room temperature for 30 minutes. The blood was collected after centrifuging the blood at 4000 rpm for 10 minutes. Repeat the centrifugation step until the serum is clear. The antibodies were mixed gently with the serum, and aliquots were taken at 37 ° C at the indicated times (days 0, 1, 4, 7 and 14). Samples were quickly frozen in liquid nitrogen at the indicated times and stored at -80 ° C until use. The samples were evaluated by FACS for their ability to bind to W345-CHOK1.hPro1.D1 cells and CCRF-CEM cells. Briefly, serial dilutions of antibodies were added to W345-CHOK1.hPro1.D1 cells or CCRF-CEM cells and incubated at 4 ° C for 1 hour. Cells were washed twice with 200 μl of PBS containing 1% BSA. PE-conjugated goat anti-human IgG Fc (Jackson, # 109-115-098) diluted 1: 100 with FACS buffer was added to the cells and incubated at 4 ° C for 30 minutes. Two additional washing steps were performed with 200 μl FACS buffer and then centrifuged at 1500 rpm for 4 minutes at 4 ° C. Finally, the cells were resuspended in 100 μl FACS buffer, the fluorescence values were measured by flow cytometry and analyzed by FlowJo.

As shown in Figures 8A-8C, the 14-day serum culture had no effect on the human CD47 binding of the test antibody, which was determined by FACS.

9. The anti-CD47 monoclonal antibody W3452-1.164.16-z11-IgG1K impact evaluation <br/> Raji cells of the B-NSG mouse lymphoma of the B-NSG mouse model of Raji-Luc, W3452- 2.683.2-z27-IgG1L and W3452-2.683.2-z27-IgG4PE, L antitumor activity. 500,000 Raji cells / mouse were implanted into mice by intravenous injection. Mice were imaged in vivo to determine fluorescence levels, and the mice were randomly divided into 9 groups (6 mice each, day 0), at 3 mg / kg or at days 0, 4, 7, and 11 0.5 mg / kg was given twice a week.

As shown in Figure 9, W3452-1.164.16-z11-IgG1K, W3452-2.683.2-z27-IgG1L, and especially W3452-2.683.2-z27-IgG4PE, L show the strongest resistance in this animal model of lymphoma Tumor activity. 3 mg / kg treatment showed the best antitumor ability, much better than WBP345-BMK2.uIgG4P.K (3 mg / kg). When the dosage was 0.5 mg / kg, W3452-2.683.2-z27-IgG4PE, L showed better antitumor ability than W3452-2.683.2-z27-IgG1L. Since CD47 is commonly expressed in normal human cells, especially in human red blood cells, the IgG1 form may cause serious side effects such as anemia. In order to further reduce the Fc-based effector function, we designed the IgG4 version using site mutation technology. W3452-1.164.16-z11-IgG1K induced mild anti-tumor ability, which showed its ability to bind CD47 well and block the interaction between CD47 and its ligand, and even induce better phagocytosis ability, which indicated that The in vivo antitumor ability is inconsistent with the in vitro efficacy.

10. In the non-male juvenile cynomolgus monkeys W3452-2.683.2-z27-IgG1L and W3452-2.683.2-z27-IgG4PE, L pharmacokinetics of
10.1 Clinical Toxicity Study To determine the pharmacokinetics of W3452-2.683.2-z27-IgG1L and W3452-2.683.2-z27-IgG4PE, L in non-juvenile male cynomolgus monkeys after a single intravenous bolus administration, 8 One animal (2 animals / group) was divided into 4 groups: a low dose group and a high dose group (30 and 150 mg / kg), and two antibodies were administered in a single dose (see Table 11). W3452-2.683.2-z27-IgG1L and W3452-2.683.2-z27-IgG4PE, L were administered to animals intravenously. The antibody was formulated in a 20 mM histidine, 5% sucrose solution at pH 5.0. Collected before administration (1 day), 0.25 hours, 0.5 hours, 1 hour, 4 hours, 8 hours, 24 hours, 3 days, 7 days, 14 days, 21 days, and 28 days after administration Blood samples were tested for antibody concentration using ELISA, and pharmacokinetic (PK) data were analyzed using WinNonlin software. Regular cage side observations of general health and appearance, especially skin irritation. Hematology (CBC) and serum chemistry of whole blood samples were analyzed by a hematology analyzer (ADVIA2120) and chemistry (HITACHI 7180), respectively.
Table 11 Grouping and dosage information

After early antibody administration, no abnormal signs regarding general health, appearance, and skin irritation were found. Animals in W3452-2.683.2-z27-IgG4PE, L group (IgG4 form) showed normal signs and normal RBC counts in clinical observation. However, on day 7, the RBC and HGB counts decreased, indicating that both the low and high dose groups had mild anemia. As shown in Figures 10A-10B, red blood cells (RBC) and hemoglobin (HGB) decreased in 7 days after antibody administration. However, RBC and HGB counts began to recover as early as day 7 and reached normal ranges around day 28. In addition, reticulocyte (RET) counts increased significantly as early as day 3 and compensated for RBC loss in the blood (Figure 10D). I therefore conclude that anemia is transient and well tolerated or compensable.

Compared with the IgG4 form, the parameters RBC, HGB, and hematocrit (HCT) were reduced on day 7 for hematological studies of W3452-2.683.2-z27-IgG1L at 30 and 150 mg / kg, and in all Anemia was caused in the animals, and one animal died on the 8th day. Due to severe anemia, 2 animals were euthanized on day 12 (Figures 10A-10C). This severe anemia may be caused by IgG form or Fc function.

10.2 The pharmacokinetic parameters of W3452-2.683.2-z27-IgG4PE, L are shown in Figure 11 and Table 12. For the two groups of 30 mg / kg and 150 mg / kg, W3452-2.683.2-z27-IgG4PE, The average half-lives of L were 147 and 79 hours, respectively. When the dose was increased to 150 mg / kg from 30 mg / kg, W3452-2.683.2-z27 -IgG4PE, L C ₀ of the system exposed to increased 5.99 times. The half-life of W3452-2.683.2-z27-IgG4PE, L is better than Hu5F9-G4 (Hu5F9-G4 is such as Liu, J. et al. (2015) Pre-Clinical Development of a Humanized Anti-CD47 Antibody with Anti-Cancer Therapeutic Potential. The antibody described in PLoS ONE 10, e0137345, which is also used in this application as WBP345-BMK2.uIgG4P.K test) has a long half-life. Fair or better (as shown in Table 13).
Table 12 shows a summary of the PK parameters
Table 13 shows the pharmacokinetic parameters of Hu5F9-G4

Remarks: An initial dose of 1 or 3 mg / kg (PD) was administered on the first day, followed by a weekly maintenance dose (MD) of 30 mg / kg for a period of 6 weeks.

Abbreviations

Figure 1 shows the results of a FACS binding assay for W3452-1.164.16-z11-IgG1K, W3452-2.683.2-z27-IgG1L, and W3452-2.683.2-z27-IgG4PE, L and CHOK1 transfected with human CD47. With the reference antibodies WBP345-BMK1.uIgG4PE.K, WBP345-BMK2.uIgG4P.K and the isotype control antibody W332-1.80.12xAb.hIgG1 (antibody isotype IgG1 negative control, produced by WuXi Biologics) and W332-1.80.12xAb.hIgG4 (Antibody IgG4 negative control, produced by WuXi Biologics).

Figure 2 shows the results of the FACS binding assay of W3452-1.164.16-z11-IgG1K, W3452-2.683.2-z27-IgG1L, and W3452-2.683.2-z27-IgG4PE, L and cynomolgus monkey PBMC, which are compared with the benchmark The antibodies WBP345-BMK1.uIgG4PE.K, WBP345-BMK2.uIgG4P.K and isotype control antibodies W332-1.80.12xAb.hIgG1 and W332-1.80.12xAb.hIgG4 were compared.

Fig. 3 shows W3452-1.164.16-z11-IgG1K, W3452-2.683.2-z27-IgG1L, and W3452-2.683.2-z27-IgG4PE using CHOK1 transfected with human CD47, and L was measured by FACS. Ligand (SIRPα) competition assay results compared with the benchmark antibodies WBP345-BMK1.uIgG4PE.K, WBP345-BMK2.uIgG4P.K and isotype control antibodies W332-1.80.12xAb.hIgG1 and W332-1.80.12xAb.hIgG4 .

Figure 4 shows the hemagglutinating activity of W3452-1.164.16-z11-IgG1K, W3452-2.683.2-z27-IgG1L, and W3452-2.683.2-z27-IgG4PE, L, which are compared with the reference antibody WBP345-BMK1.uIgG4PE Comparison of .K, WBP345-BMK2.uIgG4P.K and isotype control antibodies W332-1.80.12xAb.hIgG1 and W332-1.80.12xAb.hIgG4.

Figures 5A and 5B show W3452-1.164.16-z11-IgG1K, W3452-2.683.2-z27-IgG1L, and W3452-2.683 on Jurkat.2B8 cells (Figure 5A) and Raji cells (Figure 5B), respectively. Macrophage-mediated phagocytic activity of 2-z27-IgG4PE, L, which is comparable to the reference antibodies WBP345-BMK1.uIgG4PE.K, WBP345-BMK2.uIgG4P.K, WBP345-BMK3.uIgG1K, WBP345-BMK4.uIgG4.SPK Compared with isotype control antibodies W332-1.80.12xAb.hIgG1 and W332-1.80.12xAb.hIgG4.

Figures 6A-6D show the ADCC of W3452-1.164.16-z11-IgG1K, W3452-2.683.2-z27-IgG1L, and W3452-2.683.2-z27-IgG4PE, L for Raji cells and CCRF-CEM cells, respectively. (Figures 6A and 6B) and CDC (Figures 6C and 6D) activity, which is comparable to the reference antibodies WBP345-BMK1.uIgG4PE.K, WBP345-BMK2.uIgG4P.K and isotype control antibodies W332-1.80.12xAb.hIgG1 and W332-1.80 .12xAb.hIgG4 compared.

Figure 7 shows the results of the thermal stability of W3452-1.164.16-z11-IgG1K, W3452-2.683.2-z27-IgG1L, and W3452-2.683.2-z27-IgG4PE, L.

Figures 8A, 8B, and 8C show the human serum stability of W3452-1.164.16-z11-IgG1K, W3452-2.683.2-z27-IgG1L, and W3452-2.683.2-z27-IgG4PE, L, respectively.

Figure 9 shows W3452-1.164.16-z11-IgG1K, W3452-2.683.2-z27-IgG1L, and W3452-2.683.2-z27-IgG4PE, with L at 0.5 mg / kg and 3 mg / kg, respectively. Its antitumor activity is compared with the benchmark antibody WBP345-BMK2.uIgG4P.K at a dose of 3 mg / kg.

Figures 10A-10D show red blood cells (RBC, Figure 10A), hemoglobin (HGB, Figure 10B), hematocrit (HCT, Figure 10C), and reticulocyte ( RET, Fig. 10D). Each male cynomolgus monkey was administered in IgG4 form (W3452-2.683.2-z27-IgG4PE, L) or IgG1 form (W3452-2.683.2-z27-IgG1L) at different doses (30 mg / kg or 150 mg / kg). Of antibodies. C1001 and C1002 represent two individual animals administered W3452-2.683.2-z27-IgG1L, C2001 and C2002 represent two individual animals administered W3452-2.683.2-z27-IgG1L, and C3001 and C3002 represent administration of W3452- 2.683.2-z27-IgG4PE, L of 2 individual animals, and C4001 and C4002 represent administration of W3452-2.683.2-z27-IgG4PE, L of 2 individual animals.

Figure 11 shows the serum concentrations of antibodies after administering W3452-2.683.2-z27-IgG4PE, L to individual cynomolgus monkeys at different doses (30 mg / kg or 150 mg / kg).

Claims (36)

An isolated anti-CD47 antibody or antigen-binding fragment thereof, comprising: 1, 2 or 3 heavy chain complementarity determining region (CDR) sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 And SEQ ID NO: 11; and / or 1, 2, or 3 light chain CDR sequences selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 29. The antibody or antigen-binding fragment of claim 1, comprising a heavy chain variable region selected from the group consisting of: a) a heavy chain variable region comprising one, two or three CDR sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5; and b) a heavy chain variable region comprising one, two or three CDR sequences selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, comprising a light chain variable region selected from the group consisting of: a) a light chain variable region comprising one, two, or three CDR sequences selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6; b) a light chain variable region comprising one, two or three CDR sequences selected from the group consisting of SEQ ID NO: 29, SEQ ID NO: 4 and SEQ ID NO: 6; and c) a light chain variable region comprising one, two, or three CDR sequences selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12. An antibody or antigen-binding fragment thereof according to any preceding claim, comprising: a) a heavy chain variable region comprising one, two or three CDR sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5; and a light chain variable region The light chain variable region comprises 1, 2 or 3 CDR sequences selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6; b) a heavy chain variable region comprising one, two or three CDR sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5; and a light chain variable region The light chain variable region comprises 1, 2 or 3 CDR sequences selected from the group consisting of SEQ ID NO: 29, SEQ ID NO: 4 and SEQ ID NO: 6; and c) a heavy chain variable region comprising 1, 2 or 3 CDR sequences selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11; and a light chain variable region The light chain variable region comprises 1, 2 or 3 CDR sequences selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, comprising a heavy chain variable region selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21 And SEQ ID NO: 25, and a homologous sequence having at least 80% sequence identity thereto but still retaining a specific binding affinity for CD47. The antibody or antigen-binding fragment thereof according to any of the preceding claims, comprising a light chain variable region selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23 And SEQ ID NO: 27, and a homologous sequence having at least 80% sequence identity thereto but still retaining specific binding affinity for CD47. An antibody or antigen-binding fragment thereof according to any preceding claim, comprising: a) a heavy chain variable region comprising the SEQ ID NO: 13 and a light chain variable region comprising the SEQ ID NO: 15; b) a heavy chain variable region comprising SEQ ID NO: 17 and a light chain variable region comprising SEQ ID NO: 19; c) a heavy chain variable region comprising SEQ ID NO: 21, and a light chain variable region comprising SEQ ID NO: 23; and d) a heavy chain variable region comprising SEQ ID NO: 25 and a light chain variable region comprising SEQ ID NO: 27. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, further comprising one or more amino acid residue substitutions or modifications, but still retains a specific binding affinity for CD47. The antibody or antigen-binding fragment thereof according to claim 8, wherein at least one of the substitutions or modifications is in one or more of the CDR sequences, and / or one or more of the heavy chain or light chain variable region sequences But not in any of the CDR sequences. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, further comprising an immunoglobulin constant region, optionally a human immunoglobulin constant region, or optionally a human IgG constant region. An antibody or antigen-binding fragment thereof according to any of the preceding claims, which is humanized. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, which is a camelized single domain antibody, bifunctional antibody, scFv, scFv dimer, BsFv, dsFv, (dsFv) ₂ , Fv fragment, Fab, Fab ', F (ab ') 2, ds bifunctional antibody, nanobody, domain antibody or bivalent antibody. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, which can be specifically related to a K _D value of not more than 10 ^-9 M, 5 × 10 ^-10 M, 10 ^-10 M, or 7.5 × 10 ^-11 M human CD47 binding, the K _D value measured by flow cytometry. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, which is capable of specifically binding to cynomolgus monkey CD47. An antibody or antigen-binding fragment thereof according to any of the preceding claims, which is linked to one or more conjugated moieties. The antibody or antigen-binding fragment thereof according to claim 15, wherein the conjugated part contains a scavenger modulator, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme substrate, and a DNA alkylating agent. , Topoisomerase inhibitors, tubulin adhesives or other anticancer drugs. An antibody or antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment of any preceding claim for the same epitope. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of the preceding claims, and a pharmaceutically acceptable carrier. An isolated polynucleotide encoding an antibody or antigen-binding fragment thereof as claimed in claims 1 to 17. The isolated polynucleotide of claim 19, which comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO : 22, SEQ ID NO: 24, SEQ ID NO: 26, and SEQ ID NO: 28, and / or have at least 80% (e.g., at least 85%, 88%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) homologous sequences with sequence identity, and / or variants that have only degenerate substitutions. A vector comprising an isolated polynucleotide as claimed in claim 19 or 20. A host cell comprising a vector as claimed in claim 21. A method of expressing an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 17, comprising culturing a host cell according to claim 22 under conditions such that the vector of claim 21 is expressed. A method of treating a disease or condition in an individual that may benefit from modulation of CD47 activity, comprising administering to the individual a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, or as claimed The pharmaceutical composition of 18. The method of claim 24, wherein the disease or condition is a disease or condition related to CD47. The method of claim 25, wherein the disease or condition is cancer, autoimmune disease, fibrosis, inflammatory disease, or infectious disease. The method according to claim 26, wherein the cancer is lung cancer, bronchial cancer, bone cancer, liver and bile duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spinal cancer , Brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary cancer, gastric cancer, vaginal cancer, thyroid Cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, malignant sarcoma, teratoma, adenocarcinoma, leukemia, myeloma, and lymphoma. The method of any one of claims 23 to 27, wherein the disease or condition is a hematologic cancer selected from the group consisting of non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), and acute myeloid Cell leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), diffuse large B-cell lymphoma (DLBCL), Richter syndrome, Burkitt's lymph Neoplasm or follicular lymphoma. The method of any one of claims 23 to 28, wherein the individual is a human. The method of any one of claims 23 to 29, wherein the administration is via oral, intranasal, intravenous, subcutaneous, sublingual, or intramuscular administration. A method for modulating CD47 activity in a cell expressing CD47, comprising exposing the cell expressing CD47 to an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 17. A method for detecting the presence or content of CD47 in a sample, comprising contacting the sample with an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 17, and determining the presence or content of CD47 in the sample. A method for diagnosing a CD47-related disease or condition in an individual, comprising: a) contacting a sample obtained from the individual with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 17; b) determining The presence or content of CD47 in the sample; and c) correlating the presence or content of CD47 with the presence or status of the disease or condition associated with CD47 in the individual. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 17 in the manufacture of a medicament for treating a disease or condition related to CD47 in an individual. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 17 in the preparation of a diagnostic reagent for diagnosing a disease or condition related to CD47. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, which can be used to detect CD47.