Antibodies targeting human CD47

Abstract

The present invention provides isolated antibodies that bind and block CD47, vectors containing nucleic acid molecules encoding the amino acid sequences of the binding molecules, and host cells containing the vectors. The present invention also provides methods for preparing the antibodies, pharmaceutical compositions containing the antibodies, and methods of using such antibodies, antibody fragments and derivatives and polypeptides, such as for treating diseases including cancer.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 835,737, filed on April 18, 2019, the disclosure of which is hereby incorporated by reference in its entirety.

[0003] Incorporation of Sequence Listing This application includes a sequence listing named "QLSF002PCT_ST25.txt" submitted in ASCII format via EFS-Web, with a size of 98.7KB and a creation date of April 17, 2020. The contents of the sequence listing are hereby incorporated herein by reference in their entirety. Background of the Invention

[0005] CD47 is a ubiquitously expressed cell surface protein (also known as integrin-associated protein) that interacts with the myeloid inhibitory immune receptor SIRPα (also known as CD172a or SHPS-1). The binding of CD47 to SIRPα negatively constrains the effector functions of innate immune cells, such as host cell phagocytosis (Steven E. Kauder et al., 2018). CD47 expression and / or activity is associated with a variety of diseases and conditions. Therefore, there is a need for therapies that target CD47 and better methods for conducting such therapies.

[0006] CD47 and SIRP-α belong to the immunoglobulin superfamily and both signal inward. SIRP-α is expressed on hematopoietic cells (including macrophages and dendritic cells), while CD47 is ubiquitously expressed (Murata et al., 2014), and when it binds to CD47 on potential phagocytic target cells, phagocytosis is slowed or prevented. The CD47-SIRP-α interaction sends a "don't eat me" signal to phagocytes. Therefore, blocking the interaction of CD47-SIRP-α with monoclonal antibodies can provide effective anti-cancer therapy, namely increasing the phagocytosis of CD47-expressing cells by macrophages (for review, see Chao et al., 2012 Curr Opin Immunol, 24(2): 225-32), which is used to improve the host's immune system's absorption and clearance of cancer cells. This mechanism is effective for leukemias, lymphomas, and various types of solid tumors. In addition, these CD47 blocking antibodies have been shown to be effective in tumor models with other therapeutic antibodies (including and ) synergistic effect.

[0007] Various studies have shown that CD47 antibodies can cause platelet aggregation and red blood cell hemagglutination. When different CD47-expressing cells bind to a bivalent CD47 binding unit (such as an anti-CD47 antibody), they aggregate. This is an example of a homotypic interaction. According to Uno S, Kinoshita Y, Azuma Y, et al. published in 2007, anti-CD47 antibodies can cause red blood cell hemagglutination. Similarly, Dorahy et al. published in 1997 that the CD47 antibody B6H12 caused direct platelet aggregation in some target subjects. Therefore, the main disadvantage of existing CD47-targeted antibodies is the induction of platelet aggregation and RBC hemagglutination.

[0008] Currently clinically approved immunotherapy targeting CD47 has shown good clinical effects. However, the remission rate of these approved agents for patients is still unsatisfactory. Therefore, there is a need in the art to determine a highly effective anti-CD47 antibody that induces low or negative levels of blood coagulation and platelet aggregation. In a drug treatment regimen, such an antibody can be used alone or in combination with other therapeutic agents. Summary of the invention

[0009] The present disclosure provides isolated anti-CD47 monoclonal antagonist antibodies and antigen-binding portions thereof that specifically bind to human CD47. In one aspect of the present invention, an isolated anti-CD47 monoclonal antagonist antibody or an antigen-binding portion thereof comprises a heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 comprises SEQ ID NO: 22. In some embodiments, the anti-CD47 monoclonal antagonist antibody or an antigen-binding portion thereof further comprises a heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 comprises SEQ ID NO: 16, and a heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 comprises SEQ ID NO: 19. In a preferred embodiment, the anti-CD47 monoclonal antagonist antibody or an antigen-binding portion thereof further comprises: (a) a light chain variable region CDR1 comprising SEQ ID NO: 7; (b) a light chain variable region CDR2 comprising SEQ ID NO: 10; and (c) a light chain variable region CDR3 comprising SEQ ID NO: 13.

[0010] In one embodiment, the antibody or portion comprises a light chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 1 and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 4. In another embodiment, the antibody or portion comprises a heavy chain variable region comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO: 4.

[0011] In another aspect of the invention, an isolated anti-CD47 monoclonal antagonist antibody or antigen binding portion thereof comprises a heavy chain variable region CDR3 comprising SEQ ID NO: 23. In some embodiments, the anti-CD47 monoclonal antagonist antibody or antigen binding portion thereof further comprises a heavy chain variable region CDR1 comprising SEQ ID NO: 17, and a heavy chain variable region CDR2 comprising SEQ ID NO: 20. In a preferred embodiment, the anti-CD47 monoclonal antagonist antibody or antigen binding portion thereof further comprises: (a) a light chain variable region CDR1 comprising SEQ ID NO: 8; (b) a light chain variable region CDR2 comprising SEQ ID NO: 11; and (c) a light chain variable region CDR3 comprising SEQ ID NO: 14.

[0012] In one embodiment, the antibody or portion comprises a light chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 2 and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 5. In another embodiment, the antibody or portion comprises a heavy chain variable region comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO: 5.

[0013] In another aspect of the invention, an isolated anti-CD47 monoclonal antagonist antibody or antigen binding portion thereof comprises a heavy chain variable region CDR3 comprising SEQ ID NO: 24. In some embodiments, the anti-CD47 monoclonal antagonist antibody or antigen binding portion thereof further comprises a heavy chain variable region CDR1 comprising SEQ ID NO: 18, and a heavy chain variable region CDR2 comprising SEQ ID NO: 21. In a preferred embodiment, the anti-CD47 monoclonal antagonist antibody or antigen binding portion thereof further comprises: (a) a light chain variable region CDR1 comprising SEQ ID NO: 9; (b) a light chain variable region CDR2 comprising SEQ ID NO: 12; and (c) a light chain variable region CDR3 comprising SEQ ID NO: 15.

[0014] In one embodiment, the antibody or portion comprises a light chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 3 and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 6. In another embodiment, the antibody or portion comprises a heavy chain variable region comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO: 6.

[0015] In another aspect of the invention, an isolated anti-CD47 monoclonal antagonist antibody, or an antigen-binding portion thereof, comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:35 to SEQ ID NO:38; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:25 to SEQ ID NO:27.

[0016] In another aspect of the invention, an isolated anti-CD47 monoclonal antagonist antibody, or an antigen binding portion thereof, comprises: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 39 to SEQ ID NO: 42; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 28 to SEQ ID NO: 31. In another aspect of the invention, an isolated anti-CD47 monoclonal antagonist antibody, or an antigen binding portion thereof, comprises: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 43 to SEQ ID NO: 46; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 32 to SEQ ID NO: 34.

[0017] The antibodies of the disclosed invention can be further engineered into a form suitable for human treatment by modifications that minimize immunogenicity. Suitable antibodies include, but are not limited to, chimeric antibodies and humanized antibodies. The affinity, stability and specificity of the disclosed antibodies can also be further optimized by techniques known to those skilled in the art. Other forms may involve oligomerization, drug conjugation and fusion of the disclosed antibodies with other functional proteins.

[0018] The antibodies of the disclosed invention may be, for example, full-length antibodies, such as IgG1, IgG2, IgG3 or IgG4 isotypes. Alternatively, the disclosed antibodies may be antibody fragments, such as Fab fragments, Fab' fragments and F(ab')2 fragments, diabodies, triabodies, tetrabodies, single-chain variable region fragments (scFv), disulfide-stabilized variable region fragments (dsFv) and half antibodies. Alternatively, the disclosed antibodies may be bispecific antibodies.

[0019] In another aspect of the invention, an isolated monoclonal antibody or antigen binding portion thereof comprises a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:47 to SEQ ID No:56, and a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:57 to SEQ ID No:68.

[0020] In some embodiments, the anti-CD47 antagonist antibody or antigen binding portion thereof binds to and blocks human CD47. Thus, the antibody or antigen binding portion thereof can stimulate an anti-tumor immune response. In some embodiments, the anti-CD47 antagonist antibody or antigen binding portion thereof binds to and blocks CD47.

[0021] In another aspect of the present invention, a composition comprising the isolated anti-CD47 monoclonal antagonist antibody or an antigen-binding portion thereof is also provided.

[0022] In another aspect of the present invention, a pharmaceutical composition comprising the isolated anti-CD47 monoclonal antagonist antibody or antigen binding portion thereof and a pharmaceutically acceptable transporter is also provided. A composition comprising the immunoconjugate of the present invention and a pharmaceutically acceptable transporter is also provided.

[0023] In another aspect of the present invention, a vector comprising an isolated nucleic acid molecule encoding the antibody or antigen-binding portion thereof, and a host cell comprising an expression vector comprising the nucleic acid molecule are also provided.

[0024] The present invention further provides a method of stimulating an immune response using the anti-CD47 antagonist antibodies of the present disclosure. For example, in one embodiment, the present disclosure provides a method of treating a subject in need thereof, comprising the step of administering to the subject an effective amount of the antibody or antigen binding portion of the present disclosure.

[0025] In another aspect, the disclosed invention provides a method of treating cancer in a human, comprising the step of administering to the human an amount of an anti-CD47 antagonist antibody or antigen-binding portion of the disclosed invention effective to treat the cancer.

[0026] In another aspect, the present disclosure provides a method of treating an infectious disease in a human, comprising the step of administering to the human an amount of an anti-CD47 antagonist antibody or antigen-binding portion of the present disclosure effective to treat the infectious disease.

[0027] Other features and advantages of the present disclosure will be apparent from the following description and examples, which should not be construed as limiting.The contents of all references, GenBank entries, patents, and published patent applications cited in this application are expressly incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Exemplary embodiments are shown in the referenced drawings.The embodiments and drawings disclosed herein are to be considered for illustrative rather than restrictive purposes.

[0029] Figure 1 Humanized anti-CD47 antibodies are shown to bind to human MM.1S cancer cells.

[0030] Figure 2 It was shown that the anti-CD47 antibody E24.6 effectively blocked the binding of SIRPα to CD47.

[0031] FIG. 3A to FIG. 3C It was shown that the anti-CD47 antibody purified from the hybridoma induced human monocyte-derived macrophages to phagocytose Raji tumor cells ( Figure 3A )、MM1S cells( Figure 3B ) and SK-OV3 cells ( Figure 3C ).

[0032] FIG. 4A , FIG. 4B , and FIG. 4C show that humanized CD47 antibody and chimeric anti-CD47 antibody induced phagocytosis of MM1S cells.

[0033] Figure 5 It was shown that the lead anti-CD47 antibody did not induce hemagglutination of red blood cells. DETAILED DESCRIPTION

[0034] The embodiments and aspects thereof are described and illustrated below in conjunction with systems, compositions, and methods which are for exemplary and illustrative purposes only and not limiting in scope.

[0035] definition

[0036] As used herein, the terms "include" or "comprising" are used to indicate compositions, methods and their respective components that are useful for the embodiments but may include unspecified elements (whether useful or not). Those skilled in the art will understand that, in general, the terms used herein are generally intended to be "open" terms (e.g., the term "include" should be interpreted as "including but not limited to", the term "have" should be interpreted as "have at least", the term "include" should be interpreted as "include but not limited to", etc.).

[0037] Unless otherwise stated, the terms "a", "an" and "said" and similar quotations used in the context of describing a specific embodiment of the present application (especially in the context of the claims) may be interpreted as covering the singular and plural. References to numerical ranges herein are intended only to be used as a shorthand method of referring to each individual value falling within the range individually. Unless otherwise stated herein, each individual value is incorporated into the specification as if it were individually quoted herein. Unless otherwise stated herein or clearly contradictory to the context, all methods described herein may be performed in any suitable order. The use of all examples or exemplary language (e.g., "such as") associated with a specific embodiment herein is intended only to better illustrate the present application and is not intended to limit the scope of the present application to which protection is otherwise claimed. The abbreviation "eg" is derived from Latin exempli gratia and is used herein to indicate non-limiting examples. Therefore, the abbreviation "eg" is synonymous with the word "for example". Any language in this specification should not be interpreted as representing any unclaimed element that is essential to the practice of the present application.

[0038] As used herein, the term "about" refers to a measurable value, such as an amount, duration, etc., and encompasses variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% of the specified value.

[0039] As used herein, the term "epitope" may include any protein determinant that can specifically bind to an immunoglobulin or T cell receptor. Epitope determinants are usually composed of chemically active molecular surface groups, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics and specific charge characteristics. When the equilibrium dissociation constant is ≤1 μM (preferably ≤100nM, most preferably ≤10nM), the antibody is said to specifically bind to the antigen.

[0040] The term "K D ” may refer to the equilibrium dissociation constant for a specific antibody-antigen interaction.

[0041] As used herein, the term "immune response" may refer to, for example, the actions of lymphocytes, antigen presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by the above cells or the liver, which can cause selective damage to the organism, destroy or eliminate invading pathogens, cells or tissues infected with pathogens, cancer cells, or normal body cells or tissues (in the case of autoimmunity or pathological inflammation) in the organism.

[0042] As used herein, "antigen-specific T cell response" may refer to a T cell response caused by stimulating T cells with a T cell-specific antigen. Non-limiting examples of T cell responses to antigen-specific stimulation include proliferation and production of cytokines (eg, production of IL-2).

[0043] The term "antibody" as used herein refers to a complete immunoglobulin or a monoclonal or polyclonal antigen-binding fragment having an Fc (fragment crystallizable) region or an FcRn binding fragment of an Fc region, referred to herein as an "Fc fragment" or "Fc region". Antigen-binding fragments can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, among others, Fab, Fab', F(ab')2, Fv, dAb and complementary determining region (CDR) fragments, single-chain antibodies (scFv), single-domain antibodies, chimeric antibodies, diabodies and polypeptides comprising at least a portion of an immunoglobulin sufficient to confer specific antigens bound to the polypeptide. The Fc region includes portions of the two heavy chains that constitute two or three classes of antibodies. The Fc region can be produced by recombinant DNA technology or by enzymatic (e.g., papain cleavage) or by chemical cleavage of intact antibodies.

[0044] The term "antibody fragment" as used herein refers to a protein fragment that comprises only a portion of an intact antibody, generally including the antigen binding site of the intact antibody and thus retains the ability to bind antigen. Examples of antibody fragments encompassed by this definition include: (i) a Fab fragment having a VL region, a CL region, a VH region, and a CH1 region; (ii) a Fab' fragment, which is a Fab fragment having one or more cysteine ​​residues at the C-terminus of the CH1 region; (iii) a Fd fragment having a VH region and a CH1 region; (iv) a Fd' fragment having a VH region and a CH1 region and one or more cysteine ​​residues at the C-terminus of the CH1 region; (v) a Fv fragment having a single-arm VL region and a VH region of an antibody; (vi) a dAb fragment consisting of a VH region (Ward et al., Nature 341, 544-546 (1989)); (vii) an isolated CDR region; (viii) a F(ab')2 fragment (a bivalent fragment) comprising two Fab' fragments linked by a disulfide bond at the hinge region; (ix) a single-chain antibody molecule (e.g., single-chain Fv; scFv) (Bird et al., Science 341, 544-546 (1989)); 242:423-426 (1988); and Huston et al., PNAS (USA) 85:5879-5883 (1988)); (x) "diabodies" with two antigen-binding sites, comprising a heavy chain variable region (VH) connected to a light chain variable region (VL) in the same polypeptide chain (see, e.g., EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); (xi) "linear antibodies" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1), which together with complementary light chain polypeptides form a pair of antigen-binding regions (Zapata et al., Protein Eng. 8(10):1057-1062 (1995); and U.S. Pat. No. 5,641,870).

[0045] As used herein, "single chain variable fragment", "single chain antibody variable fragment" or "scFv" antibody refers to an antibody format containing only the variable regions of the heavy chain (VH) and the light chain (VL) connected by a linker peptide. scFv can be expressed as a single chain polypeptide. scFv retains the specificity of the intact antibody from which it is derived. The light chain and heavy chain can be in any order, for example, VH-linker-VL or VL-linker-VH, as long as the specificity of the scFv for the target antigen is maintained.

[0046] As used herein, "isolated antibody" may refer to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to a CD47 protein may be substantially free of antibodies that specifically bind to antigens other than the CD47 protein). However, an isolated antibody that specifically binds to a human CD47 protein may have cross-reactivity with other antigens (such as CD47 proteins from other species). In addition, the isolated antibody may be substantially free of other cellular materials and / or chemicals.

[0047] Anti-CD47 antagonist antibody-producing cells, such as hybridomas, can be selected, cloned, and further screened to obtain desired characteristics, including robust growth, high antibody production, and desired antibody characteristics. Hybridomas can be expanded in isogenic animals, in animals lacking an immune system (e.g., nude mice), or in vitro cell culture. Methods for selecting, cloning, and expanding hybridomas are well known to those of ordinary skill in the art.

[0048] The term "monoclonal antibody" or "monoclonal antibody composition" as used herein may refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0049] As used herein, the term "recombinant human antibody" may refer to all human antibodies prepared, expressed, produced or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom (as described below), (b) antibodies isolated from host cells transformed to express human antibodies, such as antibodies isolated from transfectomas (c) antibodies isolated from recombinant, combinatorial human antibody libraries, and (d) antibodies prepared, expressed, produced or isolated by any other means involving splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, somatic mutagenesis in vivo), so that the amino acid sequences of the VH and VL regions of the recombinant antibodies, although derived from and related to human germline VH and VL sequences, may not exist in the human antibody germline library in vivo under natural conditions.

[0050] The term "isotype" may refer to the antibody class (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. The antibody may be an immunoglobulin G (IgG), IgM, IgE, IgA or IgD molecule, or derived from them.

[0051] Herein, the phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably with the term "an antibody that specifically binds to an antigen."

[0052] As used herein, an antibody that "specifically binds to human CD47" may refer to an antibody that binds to human CD47 protein (and possibly CD47 protein from one or more non-human species) but does not substantially bind to non-CD47 proteins. Preferably, the antibody binds to human CD47 protein with "high affinity", i.e., with a affinity of 1×10 -7 M or less, more preferably 5×10 -8 M or less, more preferably 3×10 -8 M or less, more preferably 1×10 -8 M or less, more preferably 5×10 -9 M or less or even more preferably 1×10 -9 M or less K D .

[0053] As used herein, the term "substantially does not bind" to a protein or cell may mean that it cannot bind or does not bind to a protein or cell with high affinity, i.e., with an affinity of 2×10 -6 M or more, more preferably 1×10 -5 M or more, more preferably 1×10 -4 M or more, more preferably 1×10 -3 M or more, even more preferably 1×10 -2 M or above K D Bind to proteins or cells.

[0054] The term "high affinity" for IgG antibodies may refer to antibodies with a 1×10 -6 M or less, preferably 1×10 -7 M or less, more preferably 1×10 -8 M or less, even more preferably 1×10 -9 M or less, even more preferably 1×10 -10 M or less K D However, "high affinity" binding may vary for other antibody isotypes. The term "pharmaceutical formulation" refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective and does not contain additional ingredients that are unacceptably toxic to a subject to which the preparation would be administered.

[0055] A "therapeutically effective amount" of an agent (e.g., a pharmaceutical preparation or cell) refers to an effective amount that achieves a desired therapeutic outcome, such as a pharmacokinetic or pharmacodynamic effect for treating a disease, condition, or disorder and / or a therapeutic effect, within the necessary dosage and time period. A therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the subject, as well as the cell population being administered. In some embodiments, the methods provided herein involve administering cells and / or compositions in an effective amount (e.g., a therapeutically effective amount).

[0056] As used herein, an "antagonist antibody" is an antibody that blocks or inhibits a biological response by binding to and blocking a receptor (e.g., CD47) to which the antibody binds. For example, an antagonist may inhibit phosphorylation of a receptor or may inhibit signaling / cell activation due to binding of a receptor to a ligand. In one embodiment, an antibody of the invention is an anti-CD47 antagonist antibody.

[0057] A "CDR-grafted antibody" is an antibody that comprises one or more CDRs derived from a particular species or isotype and the framework of another antibody of the same or different species or isotype.

[0058] "Humanized antibodies" have sequences that are different from antibodies derived from non-human species by one or more amino acid substitutions, deletions and / or additions, so that when administered to human subjects, compared to non-human species antibodies, humanized antibodies are less likely to induce an immune response and / or induce a less severe immune response. In one embodiment, specific amino acids in the framework and constant region of the heavy chain and / or light chain of non-human species antibodies are mutated to produce humanized antibodies. In another embodiment, the constant region from a human antibody is fused with the variable region of a non-human species. In another embodiment, the humanized antibody is a CDR-grafted antibody that comprises one or more CDRs derived from antibodies of a specific species or isotype and a human antibody framework. In another embodiment, one or more amino acid residues in one or more CDR sequences of a non-human antibody are changed to reduce the possible immunogenicity of the non-human antibody when it is administered to a human subject, wherein the amino acid residues that are changed are not critical for the immunospecific binding of the antibody to its antigen, or the changes made to the amino acid sequence are conservative changes, so that the binding of the humanized antibody to the antigen is not significantly inferior to the binding of the non-human antibody to the antigen. For examples of how to make humanized antibodies, see U.S. Patent Nos. 6,054,297, 5,886,152, and 5,877,293.

[0059] The term "chimeric antibody" (cAb) refers to an antibody comprising one or more regions from one antibody and one or more regions from one or more other antibodies. In one embodiment, one or more of the CDRs are derived from human anti-CD47 antibodies. In another embodiment, all CDRs are derived from human anti-CD47 antibodies. In another embodiment, CDRs from more than one human anti-CD47 antibody are mixed and matched in a chimeric antibody. For example, a chimeric antibody may comprise CDR1 from the light chain of a first human anti-CD47 antibody, CDR2 and CDR3 from the light chain of a second human anti-CD47 antibody, and CDRs from the heavy chain of a third anti-CD47 antibody. In the context of the present disclosure, cAb represents the variable region of a mouse monoclonal antibody fused to the Fc region of a human antibody. In another embodiment, other combinations are also possible.

[0060] The term "subject" may refer to any human or non-human animal. The subject may be male or female and may be of any suitable age, including infants, juveniles, adolescents, adults, and elderly subjects. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, rabbits, mice, rats, amphibians, and reptiles, although mammals, such as non-human primates, sheep, dogs, cats, cows, and horses, are preferred.

[0061] The binding of the antibodies of the disclosed invention to CD47 can be assessed using one or more well-established techniques in the art. For example, in a preferred embodiment, the antibodies can be tested by ELISA assays, for example using recombinant CD47 protein. Other suitable binding assays include, but are not limited to, flow cytometry assays in which the antibodies react with a cell line expressing human CD47, such as Expi293 cells or ExpiCHO cells that have been transfected to express CD47 (e.g., human CD47) on their cell surfaces. Additionally or alternatively, the binding of the antibodies can be tested in BIAcore binding assays, Octet Red96 (Pall), etc., including binding kinetics (e.g., K D value).

[0062] Preferably, the antibodies of the present invention bind to 5×10 -8 M or less K D Human CD47 protein, binding with 2×10 -8 M or less K D Human CD47 protein, combined with 5×10 -9 M or less K D Human CD47 protein, binding with 4×10 -9 M or less K DHuman CD47 protein, binding with 3×10 -9 M or less K D Human CD47 protein, binding with 2×10 -9 M or less K D Human CD47 protein, binding with 1×10 -9 M or less K D Human CD47 protein.

[0063] The present disclosure relates to isolated monoclonal antibodies or antigen-binding portions thereof that bind to and block CD47 and their uses. In specific embodiments, the antibodies of the present disclosure are derived from identified heavy and light chain germline sequences and / or comprise identified structural features, such as CDR regions comprising identified amino acid sequences. The present disclosure provides isolated antibodies of the present disclosure, methods for preparing such antibodies, and antigen-binding portions thereof. The present disclosure also relates to methods of using antibodies, such as using the anti-CD47 antagonist antibodies of the present disclosure alone or in combination with other immunostimulatory or therapeutic antibodies to stimulate an immune response. Therefore, methods of using the anti-CD47 antagonist antibodies of the present disclosure are also provided, for example, including but not limited to treating human cancers. Various aspects of the present invention relate to antibodies and antibody fragments, pharmaceutical compositions, nucleic acids, recombinant expression vectors, and host cells for preparing such antibodies and fragments. The present invention also includes methods of using the antibodies of the present disclosure to detect human CD47, stimulate CD47 activity in vitro or in vivo, and prevent or treat diseases such as cancer.

[0064] Complementarity determining regions (CDRs) are referred to as hypervariable regions in the light chain variable region and the heavy chain variable region. The more highly conserved portions of the variable region are referred to as frameworks (FRs). The complementary determining regions (CDRs) and framework regions (FRs) of a given antibody can be identified using the systems described by Kabat et al., Lefranc et al., and / or Honegger and Pluckthun above. Those skilled in the art are also familiar with the numbering system described by Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). In this regard, Kabat et al. define a numbering system applicable to the variable region sequences of any antibody. One of ordinary skill in the art can clearly assign this "Kabat numbering" system to any variable region amino acid sequence without relying on any experimental data other than the sequence itself.

[0065] In certain embodiments, the invention provides anti-CD47 antagonist antibodies, or antigen-binding portions thereof. In one embodiment, the mouse antibody or portion comprises (a) a light chain variable region CDR1 comprising SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9; (b) a light chain variable region CDR2 comprising SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12; (c) a light chain variable region CDR3 comprising SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15; (d) a heavy chain variable region CDR1 comprising SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18; (e) a heavy chain variable region CDR2 comprising SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21; (f) a heavy chain variable region CDR3 comprising SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25; NO:24.

[0066] In one embodiment, the present disclosure provides a monoclonal antibody or antigen binding portion thereof that binds to the CD47 epitope, comprising a light chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27; and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 4, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38.

[0067] In another embodiment, the present disclosure provides a monoclonal antibody, or antigen binding portion thereof, that binds to a CD47 epitope, comprising a light chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO:2, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31; and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO:5, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:42.

[0068] In yet another embodiment, the present disclosure provides a monoclonal antibody, or antigen binding portion thereof, that binds to a CD47 epitope, comprising a light chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO:3, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34; and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO:6, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:46.

[0069] Given that each of these antibody Fabs can bind to human CD47, VH sequences and VL sequences can be "mixed and matched" to generate other anti-CD47 binding molecules of the present invention. Preferably, when VH chains and VL chains are mixed and matched, the VH sequence from a specific VH / VL pairing is replaced with a structurally similar VH sequence. Similarly, preferably, the VL sequence from a specific VH / VL pairing is replaced with a structurally similar VL sequence.

[0070] In some embodiments, the humanized antibody or antigen-binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 35 to SEQ ID NO: 38; and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 25 to SEQ ID NO: 27. Preferred heavy chain and light chain combinations include, but are not limited to:

[0071] (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 35 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 25;

[0072] (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 36 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 26;

[0073] (c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 37 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 27;

[0074] (d) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 38 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 27;

[0075] In some embodiments, the humanized antibody or antigen-binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 39 to SEQ ID NO: 42, and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 28 to SEQ ID NO: 31. Preferred heavy chain and light chain combinations include, but are not limited to:

[0076] (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 39 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 28;

[0077] (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 40 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 29;

[0078] (c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:41 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:30;

[0079] (d) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 42 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 31;

[0080] In some embodiments, the humanized anti-CD47 antagonist antibody or antigen binding portion thereof comprises a light chain comprising an amino acid sequence selected from SEQ ID NO: 32 to SEQ ID NO: 34; and a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 43 to SEQ ID NO: 46. Preferred heavy chain and light chain combinations include, but are not limited to:

[0081] (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 43 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 32;

[0082] (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 44 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 33;

[0083] (c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 45 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 34;

[0084] (d) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 46 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 34;

[0085] In some embodiments, the humanized anti-CD47 antagonist antibody, or antigen binding portion thereof, comprises a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:47 to SEQ ID NO:56, and a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:57 to SEQ ID NO:68.

[0086] In one embodiment, the present invention provides an anti-CD47 antibody or an antigen-binding fragment thereof, comprising a heavy chain comprising a CDR3 region as shown in SEQ ID NO:22, and comprising a heavy chain variable region comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in any one of SEQ ID NO:4, SEQ ID NO:35 to SEQ ID NO:38, and SEQ ID NO:57 to SEQ ID NO:60.

[0087] In one embodiment, the present invention provides an anti-CD47 antibody or an antigen-binding fragment thereof, comprising a heavy chain comprising a CDR3 region as shown in SEQ ID NO:23, and comprising a heavy chain variable region comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in any one of SEQ ID NO:5, SEQ ID NO:39 to SEQ ID NO:42, and SEQ ID NO:61 to SEQ ID NO:64.

[0088] In one embodiment, the present invention provides an anti-CD47 antibody or an antigen-binding fragment thereof, comprising a heavy chain comprising a CDR3 region as shown in SEQ ID NO:24, and comprising a heavy chain variable region comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in any one of SEQ ID NO:6, SEQ ID NO:43 to SEQ ID NO:46, and SEQ ID NO:65 to SEQ ID NO:68.

[0089] In one embodiment, the present invention provides an anti-CD47 antibody or an antigen-binding fragment thereof, comprising a light chain comprising a CDR3 region as shown in SEQ ID NO: 13, and having a light chain variable region comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 25 to SEQ ID NO: 27, and SEQ ID NO: 47 to SEQ ID NO: 49.

[0090] In one embodiment, the present invention provides an anti-CD47 antibody or an antigen-binding fragment thereof, comprising a light chain comprising a CDR3 region as shown in SEQ ID NO: 14, and having a light chain variable region comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 28 to SEQ ID NO: 31, and SEQ ID NO: 50 to SEQ ID NO: 53.

[0091] In one embodiment, the present invention provides an anti-CD47 antibody or an antigen-binding fragment thereof, comprising a light chain comprising a CDR3 region as shown in SEQ ID NO: 15, and having a light chain variable region comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 32 to SEQ ID NO: 34, and SEQ ID NO: 54 to SEQ ID NO: 56.

[0092] Thus, in certain embodiments, the CDR3 region is kept constant, while changes can be made to the remaining CDR and / or framework regions of the heavy and / or light chain, and the antibody or antigen-binding fragment thereof retains the ability to bind to CD47 and retains the functional properties of the parent, such as binding affinity.

[0093] In one embodiment, substitutions made within a heavy chain or light chain of at least 95% identity (or at least 96% identity, or at least 97% identity, or at least 98% identity, or at least 99% identity) are conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) of similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage of sequence identity or similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making such adjustments are well known to those skilled in the art. For example, see Pearson (1994) Methods Mol. Biol. 24: 307-331, incorporated herein by reference. Examples of groups of amino acids having side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, tryptophan; (5) basic side chains: lysine, arginine, histidine; (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains: cysteine ​​and methionine. In addition to chemically conserved amino acids, substitutions may include any amino acid that occurs at a similar position in related evolutionarily conserved human variable heavy chain sequences, human variable light chain sequences, and orthologous sequences from non-human species.

[0094] Unless otherwise specified or implied from the context, the following terms and phrases include the meanings provided below. Unless otherwise explicitly stated, or obvious from the context, the following terms and phrases do not exclude the meanings that the term or phrase has acquired in the art to which it belongs. These definitions are provided to help describe specific embodiments and are not intended to limit the claimed invention, as the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the field to which the invention belongs.

[0095] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be helpful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication referenced, whether expressly or implicitly, is prior art.

[0096] Example

[0097] The following examples are not intended to limit the scope of the claims of the present invention, but are intended as examples of specific embodiments. Any variations in the example methods that occur to a skilled person are intended to fall within the scope of the present invention.

[0098] Vector construction:

[0099] The vector pcDNA3.4TOPO (Invitrogen) was connected to a short polylinker comprising EcoRI, XhoI and NotI. The resulting plasmid was digested with EcoRI and NotI restriction enzymes and purified by gel electrophoresis. For heavy chain cloning, we assembled the prepared vector, gblock encoding and VH region (IDT) and human IgG2 gblock encoding XhoI site at the junction of J chain and CH1 domain using the Gibson assembly method. Prepare and digest plasmids with EcoRI and XhoI to adapt to all humanized heavy variable (VH) domains with IgG2 isotypes. All assemblies were completed by the Gibson method (NEB). Light variable regions were constructed by a similar method and using gblocks to assemble the Vkappa region together by gblock fragments encoding constant kappa (Ck).

[0100] Protein expression, purification and binding characterization:

[0101] Plasmids were prepared and transfected into Expi293 or ExpiCHO cells using a transient expression system (Thermo Fisher). Briefly, plasmids were transfected into 3e6 cells / mL of cells at 1 μg total plasmid DNA / mL culture. Heavy and light chain plasmids were mixed in a 1:1 ratio. The cultures were incubated with shaking at 37°C. After 16 hours, we added transfection enhancer 1 and transfection enhancer 2 to the cultures and continued to grow for six days. The supernatant was filtered and the protein titer was determined by the IgG quantification protocol using Octet Red96 (Pall). IgG was purified by Mab Select SureProtein-A column purification on the ACTA PURE system and dialyzed overnight in PBS. The affinity of the purified antibodies to the antigen was characterized by Octet Red96 by loading the purified antibodies onto an anti-human heavy chain (AHC) capture sensor and measuring the binding and dissociation rates of the CD47 histidine-tagged target at three concentrations. (Table 1).

[0102] Table 1. Monovalent binding kinetics of humanized anti-CD47 antibodies compared to a benchmark control and mouse hybridoma as determined by Octet.

[0103]

[0104]

[0105] Flow cytometry

[0106] Human / cynomolgus CD47 transfected CHO cells were harvested by Accutase treatment, washed with culture medium and incubated with serially diluted anti-CD47 antibodies in FACS buffer (DPBS + 2% FBS + 0.05% sodium azide), and then labeled with AF647 F(ab')2 goat anti-human IgG and 7-AAD. The stained samples were analyzed using FlowJo by setting a gate on FSC / SSC and then setting a gate on live / dead cells, and CD47+ cells (mean fluorescence intensity geometric mean) (Table 2).

[0107] MM.1S cells (ATCC CRL-2974) were detached by pipetting, washed with culture medium and incubated with serial dilutions of anti-CD47 antibody in FACS buffer (DPBS + 2% FBS + 0.05% sodium azide), and then labeled with AF647 F(ab')2 goat anti-human IgG and 7-AAD. The stained samples were analyzed using FlowJo by setting gates on FSC / SSC and then on live / dead cells. CD47+ cells (mean fluorescence intensity geometric mean) ( Figure 1 , Table 2).

[0108] Table 2: Anti-CD47 hybridoma binding to human cancer cell lines.

[0109]

[0110]

[0111] SIRP blocking assay using anti-CD47 antibody

[0112] CHO cells expressing human CD47 or Raji cells (ATCC CCL-86) were counted, washed with FACS buffer, and blocked for 10 minutes at room temperature with 20ul human FcR blocker (Miltenyi Biotec cat#130-059-901) per 1E7 cells. Cells were mixed with 1μg / ml rh-SIRPa / Fc chimeric protein and then incubated 1:1 with serially diluted anti-CD47 antibodies (20ug / ml antibody purified from hybridoma, chimeric antibody (cAb) or humanized antibody (hu) 1:4 dilution), positive human IgG2 antibody (C1 or C2), negative control IgG2 antibody (isotype) or no antibody to visualize SIRPa binding to CD47 on cells. Cells were incubated on ice for 20 minutes, washed twice with FACS buffer, and then incubated in anti-hu IgG1 Fc PE secondary antibody diluted 1:500 in FACS buffer for 25 minutes. The samples were washed again and stained with 7-AAD (5ul / 1E6 cells), and finally, flow cytometry analysis was performed using a BD FACS Canto II or LSR Fortessa cell analyzer ( Figure 2 , Table 3).

[0113] Table 3: Anti-CD47 antibodies effectively block SIRPα binding to CD47.

[0114]

[0115] Anti-CD47-mediated phagocytosis of CD47-expressing cells

[0116] Mononuclear cells from human peripheral blood were prepared by density gradient centrifugation and Ficoll cell separation and quantified using a Vi-CellXR cell counter. To isolate monocytes, we cultured 1E8 cells in a T75 flask with RPMI-1640 medium 10% heat-inactivated FBS for 2 hours to allow monocytes to settle and attach. Non-adherent cells were removed and the flasks were cultured with medium and 20ng / ml recombinant human M-CSF for 7 days, and then cultured with medium with 20ng / ml rh-M-CSF and 10ng / ml rh-IL-10 for another 2 days. Monocyte-derived macrophages were washed, harvested by Trypsin-EDTA, resuspended in medium and quantified. These macrophages were mixed with CFSE-labeled target cells (Raji, MM.1S, SK-OV3 cells) and test antibodies in a round-bottom ultra-low attachment 96-well plate at a ratio of 1:4 (effector cells to target cells) and cultured at 37°C for 2 hours. Anti-CD47 antibodies (purified from hybridomas (Figure 3), chimeric antibodies (Figure 4 "cAb"), or humanized antibodies (Figure 4 "hu")), positive (C1 or C2) controls, or negative (isotype) controls were titrated in RPMI-1640 + 10% heat-inactivated FBS medium. Cells were resuspended using a pipette and transferred to V-bottom polypropylene plates for FACS staining using anti-CD36 APC (Thermo Fisher cat#MA1-10210) and 7-AAD in DPBS + 20% FBS. Samples were analyzed using FlowJo by setting a gate on FSC / SSC and then setting a gate on live / dead cells, and CFSE / APC double positive, indicating that macrophages phagocytized the target cells ( Figure 3A , Figure 3B , Figure 3C , Figure 4A, Figure 4B, Figure 4C).

[0117] Hemagglutination of red blood cells on CD47

[0118] After purifying mononuclear cells from whole blood by density gradient centrifugation, red blood cells (RBC) were isolated from the bottom of the tube. RBC were washed 3 times with DPBS and prepared to 2E7 cells / ml using DPBS. The titrated anti-CD47 antibody, positive (C1 or C2) control group and negative (isotype) control group in DPBS were mixed with equal volumes of RBC in a round-bottom 96-well plate and cultured for 2 hours at room temperature. Hemagglutination analysis was performed by image analysis of the wells ( Figure 5 ).

[0119] Sequence Listing

[0120] Mouse Antibodies

[0121] SEQ ID NO:1

[0122]

[0123] Underlined and bold: CDR1, CDR2 and CDR3 (hereinafter the same) are defined according to the Kabat numbering scheme, respectively.

[0124] SEQ ID NO:2

[0125]

[0126] SEQ ID NO:3

[0127]

[0128] SEQ ID NO:4

[0129]

[0130] SEQ ID NO:5

[0131]

[0132] SEQ ID NO:6

[0133]

[0134] Light chain CDR

[0135] CDR1

[0136]

[0137] CDR2

[0138]

[0139] CDR3

[0140]

[0141] Heavy chain CDR

[0142] CDR1

[0143]

[0144]

[0145] CDR2

[0146]

[0147] CDR3

[0148]

[0149] Humanized light chain variable domain

[0150] SEQ ID NO:25

[0151]

[0152] SEQ ID NO:26

[0153]

[0154] SEQ ID NO:27

[0155]

[0156] SEQ ID NO:28

[0157]

[0158] SEQ ID NO:29

[0159]

[0160] SEQ ID NO:30

[0161]

[0162] SEQ ID NO:31

[0163]

[0164] SEQ ID NO:32

[0165]

[0166] SEQ ID NO:33

[0167]

[0168] SEQ ID NO:34

[0169]

[0170] Humanized heavy chain variable domain

[0171] SEQ ID NO:35

[0172]

[0173] SEQ ID NO:36

[0174]

[0175] SEQ ID NO:37

[0176]

[0177] SEQ ID NO:38

[0178]

[0179] SEQ ID NO:39

[0180]

[0181] SEQ ID NO:40

[0182]

[0183] SEQ ID NO:41

[0184]

[0185] SEQ ID NO:42

[0186]

[0187] SEQ ID NO:43

[0188]

[0189] SEQ ID NO:44

[0190]

[0191] SEQ ID NO:45

[0192]

[0193] SEQ ID NO:46

[0194]

[0195] Humanized light chain

[0196] SEQ ID NO:47

[0197]

[0198] SEQ ID NO:48

[0199]

[0200] SEQ ID NO:49

[0201]

[0202] SEQ ID NO:50

[0203]

[0204] SEQ ID NO:51

[0205]

[0206] SEQ ID NO:52

[0207]

[0208] SEQ ID NO:53

[0209]

[0210] SEQ ID NO:54

[0211]

[0212] SEQ ID NO:55

[0213]

[0214] SEQ ID NO:56

[0215]

[0216] Humanized heavy chain

[0217] SEQ ID NO:57

[0218]

[0219] SEQ ID NO:58

[0220]

[0221]

[0222] SEQ ID NO:59

[0223]

[0224] SEQ ID NO:60

[0225]

[0226] SEQ ID NO:61

[0227]

[0228]

[0229] SEQ ID NO:62

[0230]

[0231] SEQ ID NO:63

[0232]

[0233] SEQ ID NO:64

[0234]

[0235] SEQ ID NO:65

[0236]

[0237] SEQ ID NO:66

[0238]

[0239] SEQ ID NO:67

[0240]

[0241] SEQ ID NO:68

[0242]

Claims

1. An isolated anti-CD47 monoclonal antibody or an antigen-binding portion thereof, comprising a heavy chain variable region CDR1, wherein the heavy chain variable region CDR1 is SEQ ID NO: 17; a heavy chain variable region CDR2, wherein the heavy chain variable region CDR2 is SEQ ID NO: 20; and a heavy chain variable region CDR3, wherein the heavy chain variable region CDR3 is SEQ ID NO: 23; and Light chain variable region CDR1, the light chain variable region CDR1 is SEQ ID NO:8, and light chain variable region CDR2, the light chain variable region CDR2 is SEQ ID NO:11; light chain variable region CDR3, the light chain variable region CDR3 is SEQ ID NO:

14.

2. The monoclonal antibody or antigen-binding portion thereof according to claim 1, in, The antibody or portion specifically binds to human CD47.

3. The monoclonal antibody or antigen-binding portion thereof of claim 1, comprising a light chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO: 2 and a heavy chain variable region amino acid sequence that is at least 95% identical to SEQ ID NO:

5.

4. The monoclonal antibody or antigen-binding portion thereof according to claim 1, comprising a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to the sequence shown in SEQ ID NO:

5.

5. The monoclonal antibody or antigen-binding portion thereof according to claim 1, comprising a heavy chain variable region comprising an amino acid sequence that is at least 96% identical to the sequence shown in SEQ ID NO:

5.

6. The monoclonal antibody or antigen-binding portion thereof of claim 1, comprising a heavy chain variable region comprising an amino acid sequence that is at least 97% identical to the sequence shown in SEQ ID NO:

5.

7. The monoclonal antibody or antigen-binding portion thereof of claim 1, comprising a heavy chain variable region comprising an amino acid sequence that is at least 98% identical to the sequence shown in SEQ ID NO:

5.

8. The monoclonal antibody or antigen-binding portion thereof of claim 1, comprising a heavy chain variable region comprising an amino acid sequence that is at least 99% identical to the sequence shown in SEQ ID NO:

5.

9. The monoclonal antibody or antigen-binding portion thereof according to claim 1, comprising: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 39 to SEQ ID NO: 42, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 28 to SEQ ID NO:

31.

10. An isolated monoclonal antibody or antigen binding portion thereof, comprising a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 50 to SEQ ID No: 53, and a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 61 to SEQ ID No:

64.

11. The monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 10, wherein the monoclonal antibody or antigen-binding portion thereof is a Fab fragment, a F(ab')2 fragment, a Fv fragment, or a single-chain antibody.

12. The monoclonal antibody according to any one of claims 1 to 10, which is a chimeric antibody or a humanized antibody.

13. The monoclonal antibody according to any one of claims 1 to 10, which is an immunoglobulin G (IgG), IgM, IgE, IgA or IgD molecule. The monoclonal antibody according to claim 13 , which is IgG1, IgG2, IgG3 or IgG4.

15. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof according to any one of claims 1 to 14 and a pharmaceutically acceptable transporter.

16. Use of the pharmaceutical composition according to claim 15 in the preparation of a medicament for treating cancer in a subject, wherein the cancer is selected from Burkitt's lymphoma, multiple myeloma or ovarian cancer.

Images