Monovalent CD47 binding proteins

Abstract

The present invention relates to a binding protein or antibody comprising one antigen binding domain that binds to CD47, said antigen binding domain comprising a heavy chain variable region having three complementarity determining regions (CDRs) and a light chain variable region having three CDRs, where said antigen binding domain binds to Q19, N45, T120, R121, E122 and G123 in CD47 as defined in SEQ ID NO: 19, and where said binding protein or antibody univalently binds to CD47. Binding protein and antibody-based compositions, methods of treatment, and kits are also provided.

Description

[0001] The present invention generally relates to the field of monovalent CD47-binding proteins, particularly antibodies, and more particularly to monovalent human CD47-binding proteins and antibodies, such as antibody fragments. Compositions, methods, and kits based on the binding proteins and antibodies are also provided. Such anti-CD47 antibodies have therapeutic uses, such as for the treatment or diagnosis of cancer. Compositions, methods, and kits based on the binding proteins and antibodies are also provided.

[0002] Cancer treatment remains one of the greatest unmet medical needs to date. Despite the progress made in the field of cancer treatment over the past few decades, cancer remains one of the leading causes of death. With the increasing average life expectancy in industrialized countries, the need for improved or novel cancer therapies has become increasingly urgent.

[0003] Targeting the CD47 signaling axis is a relatively new therapeutic strategy. CD47 is a widely expressed cell surface glycoprotein that functions as a signaling receptor for thrombospondin-1 and as a counter-receptor for signal regulatory protein alpha (SIRP-α). Engagement of SIRP-α on the surface of macrophages inhibits phagocytosis, and thus CD47 serves as a physiological marker of self. However, elevated CD47 expression on the surface of certain cancer cells protects the cancer cells from innate immune surveillance and can prevent phagocytosis of the cancer cells by macrophages expressing SIRPα and other cells of the innate immune system (the so-called "don't eat me" signal).

[0004] These findings have led to the development of antibodies and other biologics to inhibit the CD47 / SIRPα interaction in tumor cells. There are currently several candidate molecules in preclinical and clinical development, such as those from Gilead, FortySeven, ALX Oncology, and Arch Oncology. However, there is still a need for alternative and improved treatment options targeting CD47.

[0005] The present invention provides such an alternative improved treatment option in the form of binding proteins and antibodies / antibody fragments (such as antibody-based binding proteins) that target CD47 and bind to CD47 monovalently.

[0006] As will be described in detail elsewhere herein, the antibodies of the present invention (including humanized antibodies) have been shown to bind to CD47 with high affinity and surprisingly exhibit excellent ability to induce direct killing of tumor cells. The advantage of direct cell killing is that the direct cell killing effect can be observed quickly, even at very low concentrations. The antibodies / antibody fragments of the present invention also surprisingly do not induce agglutination at high concentrations.

[0007] To the knowledge of the inventors, no other anti-CD47 antibodies with this combination of advantageous properties have been disclosed, and antibodies (or binding proteins) having one or more (preferably all) of these properties are preferred.

[0008] Such antibodies of the invention (or other binding proteins of the invention, such as those comprising a CD47 antigen-binding domain described herein) can be conveniently and advantageously used for treating diseases associated with CD47 expression, particularly for cancer treatment.

[0009] In one embodiment, the invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds CD47, the antigen-binding domain comprising a heavy-chain variable region having three complementarity-determining regions (CDRs) and a light-chain variable region having three CDRs; wherein the heavy-chain variable region comprises:

[0010] (i) variable heavy-chain (VH) CDR1, which comprises the amino acid sequence NFGMH (SEQ ID NO:5) or a substantially homologous sequence thereof;

[0011] (ii) VH CDR2, which comprises the amino acid sequence WINTYTGEPTYTDDFKG (SEQ ID NO:6) or a substantially homologous sequence thereof; and

[0012] (iii) VH CDR3, which comprises the amino acid sequence GDYRYGDS (SEQ ID NO:7) or a substantially homologous sequence thereof; and / or

[0013] wherein the light-chain variable region comprises:

[0014] (iv) variable light-chain (VL) CDR1, which comprises the amino acid sequence RSSQSLVHSNGKTYLH (SEQ ID NO:8) or a substantially homologous sequence thereof;

[0015] (v) VL CDR2, which comprises the amino acid sequence RVSNRFS (SEQ ID NO:9) or a substantially homologous sequence thereof; and

[0016] (vi) VL CDR3, which comprises the amino acid sequence SQSTHVPFT (SEQ ID NO:10) or a substantially homologous sequence thereof;

[0017] wherein the substantially homologous sequence refers to a sequence that contains 1, 2, or 3 amino acid substitutions compared to a given CDR sequence; and

[0018] wherein the binding protein binds CD47 monovalently.

[0019] In another embodiment, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds CD47, said antigen-binding domain comprising a heavy-chain variable region having three complementarity-determining regions (CDRs) and a light-chain variable region having three CDRs; wherein the heavy-chain variable region comprises:

[0020] (i) Variable heavy-chain (VH) CDR1, which comprises the amino acid sequence NFGMH (SEQ ID NO:5) or a substantially homologous sequence thereof, wherein the substantially homologous sequence refers to a sequence that contains 1 or 2 amino acid substitutions compared to the given CDR sequence.

[0021] (ii) VH CDR2, which comprises the amino acid sequence WINTYTGEPTYTDDFKG (SEQ ID NO:6) or a substantially homologous sequence thereof, wherein the substantially homologous sequence refers to a sequence that contains 1, 2, 3, 4, 5, or 6 (e.g., 1, 2, 3, or 4) amino acid substitutions compared to the given CDR sequence.

[0022] (iii) VH CDR3, which comprises the amino acid sequence GDYRYGDS (SEQ ID NO:7) or a substantially homologous sequence thereof, wherein the substantially homologous sequence refers to a sequence that contains 1, 2, or 3 amino acid substitutions compared to the given CDR sequence.

[0023] Wherein the light-chain variable region comprises:

[0024] (iv) VL CDR1, which comprises the amino acid sequence RSSQSLVHSNGKTYLH (SEQ ID NO:8) or a substantially homologous sequence thereof, wherein the substantially homologous sequence refers to a sequence that contains 1, 2, 3, 4, 5, or 6 (e.g., 1, 2, 3, or 4) amino acid substitutions compared to the given CDR sequence.

[0025] (v) VL CDR2, which comprises the amino acid sequence RVSNRFS (SEQ ID NO:9) or a substantially homologous sequence thereof, wherein the substantially homologous sequence refers to a sequence that contains 1, 2, or 3 amino acid substitutions compared to the given CDR sequence, and

[0026] (vi) VL CDR3, which comprises the amino acid sequence SQSTHVPFT (SEQ ID NO:10) or a substantially homologous sequence thereof, wherein the substantially homologous sequence refers to a sequence that contains 1, 2, or 3 amino acid substitutions compared to the given CDR sequence; and

[0027] Wherein the binding protein binds CD47 monovalently.

[0028] In another embodiment, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds CD47, said antigen-binding domain comprising a heavy-chain variable region having three complementarity-determining regions (CDRs) and a light-chain variable region having three CDRs; wherein said heavy-chain variable region comprises:

[0029] (i) Variable heavy-chain (VH) CDR1, which comprises the amino acid sequence NFGMH (SEQ ID NO:5);

[0030] (ii) VH CDR2, which comprises the amino acid sequence WINTYTGEPTYTDDFKG (SEQ ID NO:6); and

[0031] (iii) VH CDR3, which comprises the amino acid sequence GDYRYGDS (SEQ ID NO:7); and / or

[0032] wherein said light-chain variable region comprises:

[0033] (iv) Variable light-chain (VL) CDR1, which comprises the amino acid sequence RSSQSLVHSNGKTYLH (SEQ ID NO:8);

[0034] (v) VL CDR2, which comprises the amino acid sequence RVSNRFS (SEQ ID NO:9); and

[0035] (vi) VL CDR3, which comprises the amino acid sequence SQSTHVPFT (SEQ ID NO:10); and

[0036] wherein said binding protein binds CD47 monovalently.

[0037] In a preferred embodiment, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds CD47, said antigen-binding domain comprising a heavy-chain variable region having three complementarity-determining regions (CDRs) and a light-chain variable region having three CDRs; wherein said heavy-chain variable region comprises:

[0038] (i) Variable heavy-chain (VH) CDR1, which comprises the amino acid sequence NFGMH (SEQ ID NO:5);

[0039] (ii) VH CDR2, which comprises the amino acid sequence WINTYTGEPTYTDDFKG (SEQ ID NO:6); and

[0040] (iii) VH CDR3, which comprises the amino acid sequence GDYRYGDS (SEQ ID NO:7); and

[0041] wherein said light-chain variable region comprises:

[0042] (iv) A variable light chain (VL) CDR1 comprising the amino acid sequence RSSQSLVHSNGKTYLH (SEQ ID NO:8);

[0043] (v) A VL CDR2 comprising the amino acid sequence RVSNRFS (SEQ ID NO:9); and

[0044] (vi) A VL CDR3 comprising the amino acid sequence SQSTHVPFT (SEQ ID NO:10); and

[0045] wherein the binding protein binds CD47 monovalently.

[0046] In other words, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds CD47, said antigen-binding domain comprising a heavy chain variable region having three complementarity-determining regions (CDRs) and a light chain variable region having three CDRs; wherein the heavy chain variable region comprises:

[0047] (i) A variable heavy chain (VH) CDR1 comprising the amino acid sequence NFGMH (SEQ ID NO:5) or a substantially homologous sequence thereof;

[0048] (ii) A VH CDR2 comprising the amino acid sequence WINTYTGEPTYTDDFKG (SEQ ID NO:6) or a substantially homologous sequence thereof; and

[0049] (iii) A VH CDR3 comprising the amino acid sequence GDYRYGDS (SEQ ID NO:7) or a substantially homologous sequence thereof; and / or

[0050] wherein the light chain variable region comprises:

[0051] (iv) A variable light chain (VL) CDR1 comprising the amino acid sequence RSSQSLVHSNGKTYLH (SEQ ID NO:8) or a substantially homologous sequence thereof;

[0052] (v) A VL CDR2 comprising the amino acid sequence RVSNRFS (SEQ ID NO:9) or a substantially homologous sequence thereof; and

[0053] (vi) A VL CDR3 comprising the amino acid sequence SQSTHVPFT (SEQ ID NO:10) or a substantially homologous sequence thereof;

[0054] wherein the substantially homologous sequence refers to a sequence that contains 1, 2 or 3 amino acid substitutions compared to a given CDR sequence.

[0055] The other features of the antibodies and binding proteins described herein are equally applicable to this embodiment, mutatis mutandis.

[0056] Such binding proteins (preferably antibodies) of the present invention are monovalent for CD47 (or can bind CD47 monovalently), for example, may comprise one antigen-binding domain that binds CD47 (for example, only one antigen-binding domain that binds CD47). Other exemplary binding proteins (preferably antibodies) of the present invention may also have additional antigen-binding domains that bind other target antigens in addition to CD47. Thus, such binding proteins (or antibodies) can be bispecific, trispecific or multispecific, that is, capable of binding more than one target antigen, where CD47 is one of the target antigens. Therefore, such binding proteins (or antibodies) still require one antigen-binding domain that binds CD47 monovalently. Exemplary such binding proteins (or antibodies) have one (or only one) antigen-binding domain, or six (or only six) CDRs that are specific for CD47 (for example, a set of six CDRs).

[0057] In a further embodiment, the present invention provides a monovalent binding protein, such as an antibody, comprising an antigen-binding domain that binds CD47, said antigen-binding domain comprising a heavy-chain variable region having three complementarity-determining regions (CDRs) and a light-chain variable region having three CDRs; wherein the heavy-chain variable region comprises:

[0058] (i) Variable heavy-chain (VH) CDR1, which comprises the amino acid sequence NFGMH (SEQ ID NO:5) or a substantially homologous sequence thereof;

[0059] (ii) VH CDR2, which comprises the amino acid sequence WINTYTGEPTYTDDFKG (SEQ ID NO:6) or a substantially homologous sequence thereof; and

[0060] (iii) VH CDR3, which comprises the amino acid sequence GDYRYGDS (SEQ ID NO:7) or a substantially homologous sequence thereof; and / or

[0061] wherein the light-chain variable region comprises:

[0062] (iv) Variable light-chain (VL) CDR1, which comprises the amino acid sequence RSSQSLVHSNGKTYLH (SEQ ID NO:8) or a substantially homologous sequence thereof;

[0063] (v) VL CDR2, which comprises the amino acid sequence RVSNRFS (SEQ ID NO:9) or a substantially homologous sequence thereof; and

[0064] (vi) VL CDR3, which comprises the amino acid sequence SQSTHVPFT (SEQ ID NO:10) or a substantially homologous sequence thereof;

[0065] Wherein the basic homologous sequence refers to a sequence containing 1, 2 or 3 amino acid substitutions compared to a given CDR sequence.

[0066] As used herein, the term "monovalent" refers to a binding protein (or antibody) having one antigen-binding domain. The term "monovalent for CD47" or its equivalent refers to a binding protein (or antibody) having one antigen-binding domain that is capable of binding one molecule of the same target antigen (here, CD47). Such a binding protein or antibody may be "monovalent for CD47", but may also include additional antigen-binding domains that bind other non-CD47 target antigens. Thus, such a binding protein (or antibody) may be bispecific, trispecific or multispecific, i.e., capable of binding more than one target antigen, where CD47 is one of the target antigens.

[0067] Thus, the binding proteins (or antibodies) described above and elsewhere herein in the present invention are monovalent for CD47. In other words, they are capable of binding CD47 monovalently.

[0068] Certain embodiments of the present invention provide an antibody (or binding protein) that binds CD47 and comprises a VH domain having the amino acid sequence shown in SEQ ID NO: 3 or a basic homologous sequence thereof, and / or a VL domain having the amino acid sequence shown in SEQ ID NO: 4 or a basic homologous sequence thereof.

[0069] Certain embodiments of the present invention provide an antibody (or binding protein) that binds CD47 and comprises a VH domain having the amino acid sequence shown in SEQ ID NO: 3 or a basic homologous sequence thereof, and a VL domain having the amino acid sequence shown in SEQ ID NO: 4 or a basic homologous sequence thereof.

[0070] Certain embodiments of the present invention provide an antibody (or binding protein) that binds CD47 and comprises a VH domain having the amino acid sequence shown in SEQ ID NO: 3, and / or a VL domain having the amino acid sequence shown in SEQ ID NO: 4.

[0071] Certain embodiments of the present invention provide an antibody (or binding protein) that binds CD47 and comprises a VH domain having the amino acid sequence shown in SEQ ID NO: 3, and a VL domain having the amino acid sequence shown in SEQ ID NO: 4.

[0072] In another embodiment, the present invention provides an antibody (or binding protein) that binds to CD47, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:3, or a sequence having at least 80% sequence identity (such as at least 85%, 90%, 95% or 98%) thereto, and / or the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4, or a sequence having at least 80% sequence identity (such as at least 85%, 90%, 95% or 98%) thereto.

[0073] In another embodiment, the present invention provides an antibody (or binding protein) that binds to CD47, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:3, or a sequence having at least 80% sequence identity (such as at least 85%, 90%, 95% or 98%) thereto, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4, or a sequence having at least 80% sequence identity (such as at least 85%, 90%, 95% or 98%) thereto.

[0074] In another embodiment, the present invention provides an antibody (or binding protein) that binds to CD47, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:3, or a sequence having at least 80% sequence identity (such as at least 85%, 90%, 95% or 98%) thereto, and the heavy chain variable region further comprises three CDRs that comprise the amino acid sequences shown in SEQ ID NO:5, 6 and 7, or substantially homologous sequences as defined elsewhere herein; and / or, wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4, or a sequence having at least 80% sequence identity (such as at least 85%, 90%, 95% or 98%) thereto, and the light chain variable region further comprises three CDRs that comprise the amino acid sequences shown in SEQ ID NO:8, 9 and 10, or substantially homologous sequences as defined elsewhere herein.

[0075] In another embodiment, the present invention provides an antibody (or binding protein) that binds to CD47, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 3, or a sequence having at least 80% sequence identity (such as at least 85%, 90%, 95% or 98%) thereto, and the heavy chain variable region further comprises three CDRs, which comprise the amino acid sequences shown in SEQ ID NO: 5, 6 and 7, or substantially homologous sequences as defined elsewhere herein; and, wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 4, or a sequence having at least 80% sequence identity (such as at least 85%, 90%, 95% or 98%) thereto, and the light chain variable region further comprises three CDRs, which comprise the amino acid sequences shown in SEQ ID NO: 8, 9 and 10, or substantially homologous sequences as defined elsewhere herein.

[0076] In another embodiment, the present invention provides an antibody (or binding protein) that binds to CD47, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 3, or a sequence having at least 80% sequence identity (such as at least 85%, 90%, 95% or 98%) thereto, and the heavy chain variable region further comprises three CDRs, which comprise the amino acid sequences shown in SEQ ID NO: 5, 6 and 7; and / or, wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 4, or a sequence having at least 80% sequence identity (such as at least 85%, 90%, 95% or 98%) thereto, and the light chain variable region further comprises three CDRs, which comprise the amino acid sequences shown in SEQ ID NO: 8, 9 and 10.

[0077] The above (and other) embodiments involving SEQ ID NO: 3 and / or SEQ ID NO: 4 equally apply to alternative heavy chain variable regions of the present invention, such as SEQ ID NO: 22, 23, 24, 25 or 26 and / or alternative light chain variable regions, such as SEQ ID NO: 27, 28 or 29. Preferred paired combinations of such heavy and light chain variable regions are described in Table B herein and elsewhere.

[0078] In another embodiment, the present invention provides an antibody (or binding protein) that binds to CD47, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:3, or a sequence having at least 80% sequence identity (such as at least 85%, 90%, 95% or 98%) thereto, and the heavy chain variable region further comprises three CDRs, which comprise the amino acid sequences shown in SEQ ID NO:5, 6 and 7; and wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4, or a sequence having at least 80% sequence identity (such as at least 85%, 90%, 95% or 98%) thereto, and the light chain variable region further comprises three CDRs, which comprise the amino acid sequences shown in SEQ ID NO:8, 9 and 10.

[0079] In an alternative embodiment of the present invention, the sequence identity can be at least 60%, 65%, 70% or 75%.

[0080] Monovalent anti-CD47 antibodies (and binding proteins) that bind to CD47 based on the CO-1scFv antibody sequence listed in Table A are preferred. The CDR domains, FR domains, VH and VL domains, and an exemplary scFv sequence (variable heavy chain, light chain and linker) are shown in Table A herein. Antibodies (or binding proteins) that monovalently bind to CD47 and comprise these CDR domains or groups of VH and VL domains, or monovalent forms comprising such domains (or substantially homologous sequences thereof), such as scFv, including the full-length heavy and light chain IgG sequences provided in Table A, are preferred embodiments of the present invention. Humanized forms of the antibody are also preferred, such as antibodies that comprise a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region is a humanized version of SEQ ID NO:3 and the light chain variable region is a humanized version of SEQ ID NO:4. Such humanized versions include antibodies (or binding proteins) that comprise heavy and / or light chain variable regions as shown in Table B.

[0081] The CDR sequences of certain antibodies of the present invention are listed in Table A herein. In some other embodiments, the CDR sequences of the antibodies of the present invention may be the CDR sequences in the VH and VL domains of the antibodies of the present invention identified by any suitable method (or tool), such as the CDR sequences identified according to the well-known method of Kabat (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., U.S. Public Health Service, National Institutes of Health, Bethesda, MD, pp. 647-669, 1991) (as shown in Table A), or the CDR sequences identified according to the Chothia method (see Chothia C et al. (1989) Nature 342:877-883, or Al-Lazikani et al. (1997) J. Mol. Biol. 273:927-948), or the CDR sequences identified using the IMGT numbering scheme (see Lefranc, M.-P., Immunologist, 7:132-136 (1999); www.imgt.org), or the CDR sequences identified by AbM numbering (see Abhinandan and Martin, 2008, Mol. Immunol. 45:3832-3839).

[0082] Examples of certain substantially homologous sequences are sequences having at least 55%, 60% or 65% identity to the disclosed amino acid sequences. In certain embodiments, the antibody (or binding protein) of the present invention comprises one (or only one) heavy chain variable region comprising an amino acid sequence region having at least 55%, 60%, 65%, 70% or 75% identity to the amino acid sequence of SEQ ID NO:3, more preferably at least 80% identity, more preferably at least 85% identity; more preferably at least 90% or 95% identity, most preferably at least 96%, 97%, 98% or 99% identity; and / or one (or only one) light chain variable region comprising an amino acid sequence region having at least 55%, 60%, 65%, 70% or 75% identity to the amino acid sequence of SEQ ID NO:4, more preferably at least 80% identity, more preferably at least 85% identity; more preferably at least 90% or 95% identity, most preferably at least 96%, 97%, 98% or 99% identity.

[0083] Other preferred examples of substantially homologous sequences are sequences containing conservative amino acid substitutions of the disclosed amino acid sequences.

[0084] Other preferred examples of substantially homologous sequences are: sequences containing 1, 2, 3, 4, 5 or 6; 1, 2, 3, 4 or 5; 1, 2, 3 or 4; preferably 1, 2 or 3; preferably 1 or 2; (more preferably 1) altered amino acids in one or more of the disclosed CDR regions or one or more of the FR regions. Such alterations can be conservative or non-conservative amino acid substitutions, or combinations thereof.

[0085] Other preferred examples of "substantially homologous" sequences are: sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity to the amino acid sequence of one or more of the CDR regions or one or more of the FR regions shown in Table A or B. Thus, in some embodiments, a "substantially homologous" CDR sequence can be a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity to a particular CDR sequence described herein.

[0086] In some embodiments, in antibodies having a "substantially homologous" sequence or a certain degree of sequence identity compared to a given sequence, the altered amino acid residues are not located within the CDR regions. For example, in some embodiments, in antibodies in which the variable heavy chain (VH) has a certain degree of sequence identity to the given VH domain sequence of a particular antibody of the invention (such as CO-1scFv, the humanized CO-1scFv antibody of the invention, or a monovalent scFv-Fc fusion protein), the altered (or mutated) residues are not located within the CDR regions. Thus, in some embodiments, in antibodies having a "substantially homologous" sequence or a certain degree of sequence identity compared to a given sequence, the altered amino acid residues are located in one or more of the framework regions.

[0087] As described elsewhere herein, in other embodiments, in antibodies having a "substantially homologous" sequence or a certain degree of sequence identity compared to a given sequence, the altered amino acid residues can be located within the CDR regions.

[0088] In some embodiments, in antibodies having a "substantially homologous" sequence or a certain degree of sequence identity compared to a given sequence, a total of six CDRs consisting of three VH CDR amino acid sequences (i.e., all three VH CDR sequences considered together) and three VL CDR amino acid sequences (i.e., all three VL CDR sequences considered together) are regarded as the complete CDR repertoire of the antibody, and the amino acid sequence of the complete CDR repertoire of the antibody has at least 70% identity, preferably at least 80%, at least 85%, at least 90% or at least 95% identity compared to the corresponding complete CDR repertoire of a given starting (or reference) antibody. The starting (or reference) antibody may have the CDR sequences of the CO-1 scFv antibody or humanized CO-1 scFv antibody of the present invention as shown in Table A or B, or may have the CDR sequences of a monovalent scFv-Fc fusion protein (CO201-scFv-Fc-mono).

[0089] The residues that are altered can be conservative or non-conservative amino acid substitutions, or combinations thereof.

[0090] In such embodiments, preferred amino acid changes are conservative amino acid substitutions.

[0091] In all embodiments, a binding protein (such as an antibody) containing a substantially homologous sequence retains the ability to bind CD47 monovalently. Preferably, a binding protein (such as an antibody) containing a substantially homologous sequence retains one or more (preferably all) other properties associated with the antibodies of the present invention, such as the CO-1 scFv antibody, humanized CO-1 scFv antibody described herein, or a monovalent scFv-Fc fusion protein (such as CO201-scFv-Fc-mono).

[0092] Other examples of substantially homologous amino acid sequences of the present invention are detailed in other parts of this document.

[0093] The CDR regions of the antibodies (or binding proteins) of the present invention are preferably separated by appropriate framework regions, such as the framework regions found in natural antibodies and / or effectively engineered antibodies. Thus, the VH, VL and individual CDR sequences of the present invention are preferably arranged or integrated into an appropriate framework or scaffold structure to achieve binding to an antigen (here CD47). Such framework sequences or regions may appropriately correspond to natural framework regions (FR1, FR2, FR3 and / or FR4) to form an appropriate scaffold structure, or may correspond to a consensus framework sequence determined by comparing multiple natural framework regions. In some embodiments, humanized antibodies are provided, in which case human framework regions (or their substantially homologous sequences) can be used. Alternatively, non-antibody scaffolds or frameworks, such as T cell receptor frameworks, can also be used.

[0094] A variety of suitable framework region sequences are known in the art and are documented in the literature, and any of them can be used. Exemplary sequences of the framework region are: those constituting the V H and / or V L domain of the antibody of the present invention, for example, one or more framework regions of the CO-1scFv antibody disclosed in Table A, or one or more framework regions of the humanized CO-1 antibody disclosed in Table B or their substantially homologous framework regions, especially the framework regions that allow maintaining antigen specificity, such as the framework regions that can make the antibody maintain a substantially identical or completely identical three-dimensional structure.

[0095] In certain embodiments, all four heavy chain variable region framework regions (SEQ ID NO: 11-14) and / or light chain variable region framework regions (SEQ ID NO: 15-18) (as appropriate) or their substantially homologous FR regions can be found in the antibody (or binding protein) of the present invention.

[0096] The exemplary CO-1scFv antibody of the present invention comprises murine / mouse VH and VL domains (the source antibody mCO-1 of the scFv is a full-length murine IgG1κ antibody), and CO-1scFv is the single-chain form of this antibody, in which a linker sequence is placed between the VH and VL sequences. Such antibodies generally comprise murine / mouse VH and VL domains, for example, domains having the sequences shown in SEQ ID NO: 3 and 4, or VH and VL domains comprising heavy and light chain CDRs, for example, domains having the sequences shown in SEQ ID NO: 5 to 10, (and related substantially homologous sequences as described herein, for example), and these domains are separated by an appropriate linker sequence. The scFv antibody binds CD47 monovalently, and the present invention also provides any other antibody form that allows such monovalent binding. Some examples are described in other parts of this document.

[0097] In other embodiments of the present invention, a humanized version of the exemplary CO-1 antibody of the present invention is preferred. Therefore, when the antibody (or binding protein) of the present invention is mentioned herein, its preferred embodiments include humanized antibodies (or binding proteins).

[0098] Thus, in some embodiments, the antibodies (or binding proteins) of the present invention can be or comprise humanized antibodies, such as may be referred to as humanized antibodies or humanized binding proteins. A "humanized" antibody is an antibody based on a substantially non-human variable domain structure, in which specific amino acids have been altered to better conform to the amino acids typically present in human antibodies. Methods for preparing humanized antibodies are known in the art. For example, a humanized antibody can be prepared by inserting appropriate CDR regions (e.g., murine CDR regions, such as the CDR regions contained in the antibodies of the present invention) into a human antibody "scaffold" (such as a scaffold comprising a human antibody framework region or a substantially homologous sequence thereof). Thus, in some embodiments, the CDRs of the present invention (e.g., a set of 6 CDRs of the antibodies of the present invention described herein, the CDRs in the exemplary antibodies of the present invention shown in Table A, i.e., the CDRs having the sequences shown in SEQ ID NO: 5-10 or substantially homologous sequences thereof) are placed (or combined, inserted, or transplanted) into a human or humanized antibody framework, such as using an appropriate framework region (FR) in a human antibody or a substantially homologous sequence thereof.

[0099] Exemplary humanized heavy chain variable region (VH) domains of the humanized antibodies (or binding proteins) of the present invention are provided as SEQ ID NO: 22-26, or substantially homologous sequences thereof.

[0100] Exemplary humanized light chain variable region (VL) domains of the humanized antibodies (or binding proteins) of the present invention are provided as SEQ ID NO: 27-29, or substantially homologous sequences thereof.

[0101] Thus, preferred antibodies (or binding proteins) of the present invention, such as humanized antibodies (or binding proteins), comprise any one of the VH domains of SEQ ID NO: 22, 23, 24, 25, or 26, or substantially homologous sequences thereof; and / or (preferably and) any one of the VL domains of SEQ ID NO: 27, 28, or 29, or substantially homologous sequences thereof.

[0102] Thirteen humanized antibodies (or antibodies having substantially homologous sequences thereof) prepared according to the present invention, and binding proteins comprising such antibodies, are all preferred antibodies (or binding proteins) of the present invention. These antibodies are as follows:

[0103] CO201 (comprising the VH domain of SEQ ID NO: 22 and the VL domain of SEQ ID NO: 27);

[0104] CO202 (comprising the VH domain of SEQ ID NO: 23 and the VL domain of SEQ ID NO: 27);

[0105] CO203 (comprising the VH domain of SEQ ID NO: 24 and the VL domain of SEQ ID NO: 27);

[0106] CO204 (comprising the VH domain of SEQ ID NO:25 and the VL domain of SEQ ID NO:27);

[0107] CO205 (comprising the VH domain of SEQ ID NO:22 and the VL domain of SEQ ID NO:28);

[0108] CO206 (comprising the VH domain of SEQ ID NO:23 and the VL domain of SEQ ID NO:28);

[0109] CO207 (comprising the VH domain of SEQ ID NO:24 and the VL domain of SEQ ID NO:28);

[0110] CO208 (comprising the VH domain of SEQ ID NO:25 and the VL domain of SEQ ID NO:28);

[0111] CO209 (comprising the VH domain of SEQ ID NO:22 and the VL domain of SEQ ID NO:29);

[0112] CO210 (comprising the VH domain of SEQ ID NO:23 and the VL domain of SEQ ID NO:29);

[0113] CO211 (comprising the VH domain of SEQ ID NO:24 and the VL domain of SEQ ID NO:29);

[0114] CO212 (comprising the VH domain of SEQ ID NO:25 and the VL domain of SEQ ID NO:29); or

[0115] CO213 (comprising the VH domain of SEQ ID NO:26 and the VL domain of SEQ ID NO:27).

[0116] Other preferred humanized antibodies (or binding proteins) of the present invention comprise the VH domain of SEQ ID NO:26 or a substantially homologous sequence thereof, and the VL domain of SEQ ID NO:28 or a substantially homologous sequence thereof; or the VH domain of SEQ ID NO:26 or a substantially homologous sequence thereof, and the VL domain of SEQ ID NO:29 or a substantially homologous sequence thereof.

[0117] In some embodiments, the humanized VH domain of SEQ ID NO:22 or a substantially homologous sequence thereof is preferably employed.

[0118] In some embodiments, it is preferred to use the humanized VL domain of SEQ ID NO:27 or a substantially homologous sequence thereof.

[0119] In some embodiments, it is preferred to use the humanized VH domain of SEQ ID NO:22 or a substantially homologous sequence thereof, and the humanized VL domain of SEQ ID NO:27 or a substantially homologous sequence thereof.

[0120] A sequence that is substantially homologous to any given sequence in the humanized antibody (or binding protein) is defined elsewhere herein and includes sequences having different numbers of amino acid substitutions or sequences having different percent identities to a given starting sequence, such as a sequence having at least 80% sequence identity thereto.

[0121] In some embodiments, the variant residues may be present in the CDR regions and FR regions of the antibody (or binding protein). In other embodiments, the variant residues may be present in the CDR regions of the antibody (or binding protein). In other embodiments, the variant residues may be present in the FR regions of the antibody (or binding protein).

[0122] The preferred heavy chain FR regions in the humanized antibody (or binding protein) of the present invention are one or more, or all four FR regions (FR1, FR2, FR3, and FR4) shown in SEQ ID NO:22, 23, 24, 25, or 26, or a substantially homologous sequence thereof.

[0123] The preferred light chain FR regions in the humanized antibody (or binding protein) of the present invention are one or more, or all four FR regions (FR1, FR2, FR3, and FR4) shown in SEQ ID NO:27, 28, or 29, or a substantially homologous sequence thereof.

[0124] In certain embodiments, as appropriate, all four heavy chain and / or light chain framework regions (FRs) from SEQ ID NO:22, 23, 24, 25, 26, 27, 28, or 29, or FR regions that are substantially homologous thereto, may be found in the antibody (or binding protein) of the present invention.

[0125] In embodiments using an FR region that is substantially homologous to one or more of the FR regions shown in SEQ ID NO: 22, 23, 24, 25, 26, 27, 28, or 29, each FR region may contain no more than 10 amino acid changes, such as 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 change, compared to the given sequence. Although any amino acid changes may be employed, in some embodiments, the changes may revert the amino acid residue to the corresponding residue in the original murine antibody (here, the CO-1 antibody). Such changes may also be referred to as back mutations. In some embodiments, 1, 2, or 3 back mutations may be present within one or more of the FR regions shown in SEQ ID NO: 22, 23, 24, 25, 26, 27, 28, or 29.

[0126] These humanized VH and VL domains can be provided in any suitable, anti-CD47 monovalent format consistent with the present invention. Exemplary formats are discussed elsewhere herein but include scFv antibodies or Fab or Fab' fragments, and the VH and VL domains of these fragments can be the humanized VH and VL domains, such as as described herein.

[0127] Other antibody (or binding protein) monovalent formats that include an antibody constant region, particularly a human antibody constant region, are also provided, such as an antibody that includes an Fc region, such as an Fc fusion protein. Thus, particularly preferred antibodies (or binding proteins) of the present invention have humanized (or other, such as non-humanized or murine) VH and VL domains as described herein and are conjugated, fused, or linked to the Fc region of an antibody. Such formats remain monovalent for CD47. Thus, a particularly preferred format is a monovalent scFv-Fc format / fusion construct, where an scFv antibody (such as a humanized scFv antibody) is fused or linked to the Fc region. Such formats are known and described in the art and thus preferably include an scFv fragment, such as an anti-CD47 binding scFv fragment of the present invention, that is fused, conjugated, or linked to the Fc region. In these embodiments, the Fc region is preferably a human Fc region, such as an IgG1 or IgG4 Fc region. Exemplary Fc regions include (or consist of, or consist essentially of) CH2 and CH3 domains, optionally further including a hinge region.

[0128] Thus, in some embodiments, the humanized (or other) VH and VL domains described herein are provided in the form of a monovalent scFv to CD47. In other words, such antibodies (or binding proteins) of the invention contain a single scFv fragment capable of binding CD47. The scFv form is well known in the art and comprises (or consists of) a single polypeptide chain in which the VH and VL domains of the antibody are joined by a suitable peptide linker. Preferred and exemplary combinations of VH and VL domains suitable for such monovalent scFv forms are described elsewhere herein (e.g., see Tables A and B), and the VH and / or VL domains of any CD47 antibody of the invention or their CDRs can be used. In some such embodiments, the humanized VH and VL domains described herein are preferred. In some such embodiments, the humanized VH domain of SEQ ID NO:22 or a substantially homologous sequence thereof is preferred. In some such embodiments, the humanized VL domain of SEQ ID NO:27 or a substantially homologous sequence thereof is preferred. In some embodiments, the humanized VH domain of SEQ ID NO:22 or a substantially homologous sequence thereof, and the humanized VL domain of SEQ ID NO:27 or a substantially homologous sequence thereof are preferred.

[0129] Suitable linker sequences for such scFv fragments are well known in the art and have been described. Suitable linker sequences are typically artificially designed flexible linkers, such as GS linkers. An exemplary GS linker sequence is shown in SEQ ID NO:20.

[0130] The monovalent scFv-Fc format (or monovalent scFv-Fc fusion protein) is designed such that the scFv is linked to one chain of the Fc region, while the other chain of the Fc region is not linked to an antibody (or binding protein). Since the Fc region is a dimer structure, the monovalent form is formed by the association of the two chains of the Fc region (i.e., the chain linked to the scFv and the "empty" chain). Exemplary Fc regions include CH2 and CH3 domains and optionally include a hinge region (or other suitable linker, such as an artificial linker or a flexible linker), for example, for linking the scFv to one chain of the Fc region. Preferably, the Fc region is a human Fc region, such as an IgG1 or IgG4 Fc region, but it is obvious that suitable hinge regions and CH2 and CH3 domains can be obtained from other antibody subtypes (such as IgG2, etc.), and such sequences are readily available in the art. An exemplary human IgG4 Fc region (CH2 and CH3 domains) is shown in SEQ ID NO: 30. An exemplary hinge region is shown in SEQ ID NO: 33. However, to facilitate the correct association of the two chains constituting the Fc, the "knobs into hole" mutation can be conveniently used to engineer this fragment. The sequence of one chain ("knob" chain) of the construct is shown in SEQ ID NO: 31, which includes a hinge region-CH2-CH3 and an appropriate "knob" mutation. The sequence of the other chain (i.e., the "hole" chain) of the construct is shown in SEQ ID NO: 32, which includes a hinge region-CH2-CH3 fragment and the corresponding "hole" mutation. The scFv fragment is located at the N-terminus of one of the chains. When these two heavy chain constructs are expressed, the chains will dimerize, and the complex formed by the "knob-hole" dimer can be screened out. This complex includes a monovalent scFv-Fc construct, that is, an Fc region with a single (monovalent) scFv (i.e., a monovalent scFv-Fc fusion protein). Exemplary and preferred fragments containing the CO201 scFv fragment (CO201-scFv-Fc-mono) are SEQ ID NO: 34 (with a knob structure of CO201 scFv) and 35 (with a hole structure of hinge-CH2-CH3). It can be noted that the scFv fragment can also be present on the "hole" chain rather than the "knob" chain.

[0131] In embodiments using an IgG4 hinge region (such as SEQ ID NO: 33), it may be desirable to introduce mutations, such as stabilizing mutations, to prevent Fab arm exchange (Handlogten et al., 2020, MABS 12(1), e1779974). Three exemplary mutations have been discovered: Y219C, G220C, and S228P (see SEQ ID NO: 36), and one or more of these mutations can be used in the IgG4 hinge region.

[0132] Similar to the monovalent scFv-Fc form, other exemplary forms do not contain other antibody constant regions other than the CH2 and CH3 heavy chain regions / domains. Thus, in some embodiments, the CH1 region and / or the CL (or CL1) region is absent or has been removed. In other words, in some embodiments, the antibodies (or binding proteins) of the present invention contain only the CH2 and CH3 regions (or Fc region), optionally also containing a hinge region (or other suitable linker, such as an artificial linker or a flexible linker).

[0133] Thus, preferred such constructs contain an antibody (or binding protein) of the present invention as defined herein, for example having a scFv fragment that contains a VH and / or VL domain of the present invention, or the corresponding three or six (such as six) CDRs as described elsewhere herein.

[0134] A preferred antibody (or binding protein) suitable for such forms is the humanized CO201 antibody described herein. However, any CD47 antibody (or binding protein), such as any CD47 antibody (or binding protein) of the present invention, such as any other humanized antibody of the present invention, or a VH and / or VL domain (or its corresponding CDR regions), or the six CDR regions (and suitable FR regions) shown in Table A, can be incorporated into such form. Antibodies (or binding proteins) employing such form or containing such form (the monovalent scFv-Fc form) are preferred.

[0135] Another aspect of the present invention provides an antibody (or binding protein) that contains an antigen-binding domain that binds CD47 in scFv form, wherein the antigen-binding domain is fused to, linked to, or otherwise attached (such as via a hinge region or a linker) to an Fc region. Thus, in these embodiments, the CH1 region and / or the CL (or CL1) region is absent or removed, and these regions do not participate in the linking of the scFv to the Fc region. Thus, in these embodiments, the one scFv antigen-binding domain can be directly attached to the Fc region, optionally via a hinge region or a linker. In other words, such an antibody (or binding protein) is or consists of, or contains: a monovalent scFv-Fc fragment that contains a scFv antigen-binding domain that binds CD47. As described above, the preferred scFv antigen-binding domain that binds CD47 contains one or more antigen-binding domains of the present invention as defined elsewhere herein. Like other aspects of the present invention, such an antibody (or binding protein) that contains an Fc fusion protein binds CD47 monovalently.

[0136] Such antibodies (or binding proteins) can also be readily constructed as bispecific, trispecific or multispecific constructs as described elsewhere herein. For example, antigen-binding domains (conveniently in the form of scFv fragments) specific for target antigens other than CD47 can be present in such constructs, for example attached to the CH3 portion of the Fc region. Such antibodies (or binding proteins) of the invention, when in the monovalent scFv-Fc form, unexpectedly and advantageously do not induce red blood cell agglutination even at high concentrations. Thus, this particular construct is considered a particularly advantageous form of the CD47 antibody. Preferred antibodies (or binding proteins) of the invention include humanized antibodies which, when in the monovalent scFv-Fc form, do not induce red blood cell agglutination (or significant agglutination) when used at a concentration of up to or not exceeding 1, 2, 5 or 10 μg / ml. Preferred such antibodies (or binding proteins) also do not induce red blood cell agglutination (or significant agglutination) when at a concentration of up to or not exceeding 15, 20, 25, 50, 75 or 100 μg / ml.

[0137] Other features and properties (such as preferred features and properties) of other aspects of the invention (such as binding affinity) can be applied to this aspect of the invention with necessary modifications in detail.

[0138] Advantageously, such forms also have the ability to induce phagocytosis. This approach provides an improvement over other monovalent forms.

[0139] Thus, in a preferred embodiment, the antibodies (or binding proteins) of the invention (including the humanized antibodies or binding proteins of the invention), when provided in the monovalent scFv-Fc form, are capable of causing phagocytosis of tumor cells expressing CD47 by macrophages.

[0140] Thus, in some embodiments, the antibodies (or binding proteins) of the invention (including the humanized antibodies or binding proteins of the invention), when provided in the monovalent scFv-Fc form, are capable of inhibiting (or blocking) the "don't eat me" signal emitted by CD47 expressed on tumor cells, thereby leading to phagocytosis of tumor cells by macrophages or other innate immune cells expressing SIRPα.

[0141] The ability of the antibodies (or binding proteins) of the invention to induce phagocytosis of such tumor cells (such as tumor cells expressing CD47) is advantageous. It has been demonstrated that the antibodies in the monovalent scFv-Fc form of the invention are particularly effective in inducing such phagocytosis, for example, it has been demonstrated that they can induce phagocytosis of a large number / high proportion of tumor cells in the presence of macrophages. In addition, it has been demonstrated that this effect can be exhibited at relatively low doses or concentrations.

[0142] Phagocytosis can be conveniently determined by suitable in vitro assays well known to those skilled in the art. Such assays generally involve contacting target cells (here, tumor cells expressing CD47) with macrophages in the presence of the antibody (or binding protein) to be tested according to the present invention, and assessing the number of tumor cells phagocytosed (e.g., as a percentage). A preferred assay method is described in the Examples, in which macrophages (e.g., murine macrophages such as RAW 264.7 cells) and tumor cells are stained with different dyes and then contacted with each other in the presence of the test concentration of the antibody to be tested. At the end of the assay, the cells are analyzed by flow cytometry. The double-stained cells represent the phagocytosed target cells, and the number thereof can be conveniently expressed as a percentage.

[0143] In some embodiments, the antibodies in the form of monovalent scFv-Fc of the present invention can induce phagocytosis at a concentration of 10 μg / ml. However, the preferred antibodies of the present invention can induce phagocytosis at concentrations lower than 10 μg / ml, 5 μg / ml, 2 μg / ml, or 1 μg / ml. More preferably, the antibodies in the form of monovalent scFv-Fc of the present invention can induce phagocytosis at 0.1 or 1 μg / ml, or lower than 0.1 or 1 μg / ml (e.g., between 0.1 and 1 μg / ml).

[0144] The antibodies (or binding proteins) of the present invention preferably can induce phagocytosis of a variety of cancer cells, such as hematological malignancy cells and solid tumor cells. By way of example only and not by way of limitation, it has been demonstrated that the antibodies (or binding proteins) of the present invention can induce phagocytosis of hematological malignancy cells (such as Jurkat cells).

[0145] The phagocytosis percentage values naturally vary with the relevant cell types. However, the exemplary antibodies in the form of monovalent scFv-Fc of the present invention show good phagocytosis percentage levels for cancer cells, such as at least 10% phagocytosis level can be seen for different types of cancer cells, and at least 20%, 25%, 30%, or 35% levels are observed for some types of cancer cells. By way of example only, the antibodies (or binding proteins) in the form of monovalent scFv-Fc of the present invention can induce at least or not more than 20%, 25%, 30%, or 35% phagocytosis of Jurkat cells (a cancerous T cell line), e.g., when the cells are exposed to the monovalent scFv-Fc antibody (such as CO201-scFv-Fc mono) at a concentration of 10, 1, or 0.1 μg / ml in the presence of macrophages for 2 hours.

[0146] As described above, the present invention provides binding proteins, such as antibodies or binding proteins (including humanized antibodies or binding proteins of the present invention), which comprise the antigen-binding domain of an antibody and bind (or specifically recognize or specifically bind) CD47, such as human CD47, in a monovalent manner (i.e., having one or a single antigen-binding domain capable of binding CD47). Preferred binding proteins of the present invention are antibodies. However, the embodiments described herein involving antibodies are equally applicable to other types of binding proteins with appropriate modifications, and vice versa. Thus, other binding proteins may comprise an antibody of the present invention, or may comprise the antigen-binding domain of an antibody of the present invention, such as the three VL CDR regions and / or the three VH CDR regions of an antibody of the present invention, or the VL and / or VH domains (i.e., the three CDR regions (CDR1, CDR2, and CDR3) and the four FR regions (FR1, FR2, FR3, and FR4) of the VL and / or VH domains of an antibody of the present invention).

[0147] Preferred binding proteins are any polypeptide chains that can bind (e.g., specifically bind) CD47 (such as human CD47) in a monovalent manner (i.e., having one or a single antigen-binding domain capable of binding CD47). Suitable types of binding proteins that can be used in the present invention are known in the art. For example, in some embodiments, immunoglobulin-based polypeptides are used, which typically comprise CDR regions (and optionally FR regions or immunoglobulin-based scaffolds) such that the CDR regions (and optionally FR regions) of an antibody of the present invention can be grafted onto a suitable scaffold or framework, such as an immunoglobulin scaffold. Alternatively, the antigen-binding fragment or antibody of the present invention can be incorporated into any suitable form containing the antigen-binding fragment or antibody, for example, it can be incorporated into a chimeric antigen receptor (CAR) form or a CAR-T cell form.

[0148] As described above, the present invention provides antibodies (or binding proteins), such as isolated antibodies (or binding proteins), which bind (or specifically recognize or specifically bind) CD47. CD47 is sometimes also referred to as integrin-associated protein (IAP), MER6, or OA3. CD47 is expressed on all cell types, but is strongly expressed or overexpressed on the surface of a variety of cancer cells, such as non-Hodgkin lymphoma, Burkitt lymphoma, acute myeloid leukemia (AML), hepatocellular carcinoma, and bladder cancer. High expression is associated with poor prognosis in several cancer types, such as AML (acute myeloid leukemia).

[0149] According to the present invention, the CD47 can be from any species. In a preferred embodiment, the CD47 is human CD47. Thus, in certain embodiments, the antibodies (or binding proteins) of the present invention are capable of binding human CD47. CD47 is a recognized cancer therapeutic target.

[0150] Thus, the binding protein or antibody of the present invention binds or is capable of binding to CD47, such as human CD47.

[0151] The binding proteins and antibodies of the present invention are capable of binding to any suitable form of CD47. Preferred and convenient forms of CD47 to which the binding proteins and antibodies of the present invention can bind include: recombinant CD47 (such as recombinant human CD47), or the native form of CD47 (such as CD47 present on the cell surface (cell surface CD47)), such as CD47 expressed on tumors or cancer cells.

[0152] The sequence of CD47 (such as the sequence of human CD47) is well-known and documented in the art and can be obtained from a variety of sequence databases. For example, the UniProt entry Q08722 provides the sequence of human CD47. Recombinant human CD47 is commercially available.

[0153] A suitable and exemplary human CD47 sequence is shown below as SEQ ID NO:19. Accordingly, the preferred binding proteins or antibodies of the present invention bind or are capable of binding (or specifically binding) to SEQ ID NO:19, or a sequence that is substantially homologous thereto (such as a sequence having at least 80% identity thereto), or a fragment thereof, such as a biologically active fragment.

[0154] MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKF

[0155] KGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELT

[0156] REGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALL

[0157] VAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIA

[0158] ILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQ

[0159] PPRKAVEEPLNAFKESKGMMNDE(SEQ ID NO:19)

[0160] Methods for assessing binding (or binding ability) to a suitable form of CD47 are well known to those skilled in the art and any suitable method can be employed.

[0161] A convenient and suitable method for assessing binding includes in vitro binding assays, such as ELISA assays, for assessing the binding of an antibody (or binding protein) to an immobilized antigen (such as the immobilized form of CD47 as described above, such as recombinant CD47, such as recombinant human CD47, such as comprising SEQ ID NO:19).

[0162] Thus, in certain embodiments, the antibodies (or binding proteins) of the present invention are capable of binding CD47 in an ELISA assay. Those skilled in the art are familiar with ELISA assays and can readily establish suitable conditions to assess the ability of an antibody to bind CD47 in such an assay. For example, CD47 (such as recombinant human CD47) can be captured on an ELISA plate, washed, and then incubated with the anti-CD47 antibody (or binding protein) of the present invention, followed by detection of the bound anti-CD47 antibody (or binding protein). Generally, the antibodies (or binding proteins) of the present invention are capable of binding human CD47 in an ELISA assay.

[0163] In certain embodiments, the binding proteins or antibodies of the present invention bind (as determined by measurement) CD47 (such as recombinant CD47, such as recombinant human CD47, such as comprising SEQ ID NO:19) in a surface plasmon resonance (SPR) assay (such as a BIACore assay, such as using a BIAcore S200 instrument). Suitable SPR (surface plasmon resonance) assays are known in the art and can include, for example, immobilizing an antibody on a solid support and passing different concentrations of CD47 over the antibody. In certain preferred SPR assays, a suitable form of CD47 (such as recombinant CD47, such as recombinant human CD47, such as having SEQ ID NO:19) is captured (or immobilized) on a solid support (such as a sensor chip), and then different concentrations of the binding protein or antibody to be tested (such as a dilution series, such as a two-fold dilution series) are injected. The antibody concentration and RU units are typically selected within certain ranges and levels, respectively, such that the chip is not saturated and allows for a robust fit by the SPR / Biacore software, such as a robust 1:1 fit. Preferred injection concentrations and flow rates and suitable RU units are described in the Examples section.

[0164] Those skilled in the art are aware of the applicable association and dissociation periods in SPR assays. For example, in an SPR assay, the preferred association period is 2 minutes and the preferred dissociation period is 30 minutes (in single-cycle analysis). As described in other parts of this document, the antibodies of the present invention may have a low dissociation rate, and thus a relatively long dissociation period can be employed. Therefore, in a preferred embodiment, the association period can be measured within 2 minutes and / or the dissociation period can be measured within 30 minutes. In certain embodiments, all measurements can be carried out at 25 °C in 20 mM PBS (pH 7.4), 2.7 mM KCl, 137 mM NaCl, 0.05% P20. The kinetic parameters can be determined or calculated by any suitable model or software, for example, by fitting the experimental data of the sensorgram by assuming a 1:1 interaction (in other words, using a 1:1 binding model), such as using single-cycle kinetics software. The particularly preferred SPR assay methods are described in the Examples section herein. Single-cycle analysis is preferably employed.

[0165] Therefore, in certain embodiments, the antibodies (or binding proteins) of the present invention are capable of binding to CD47 (such as recombinant CD47, such as recombinant human CD47) in an SPR assay or an ELISA assay.

[0166] Such SPR assay methods can also be conveniently used to measure the binding kinetics of antibody-antigen interactions, such as determining the association rate (ka), dissociation rate (kd), and affinity (KD).

[0167] In certain preferred embodiments, for example, when in scFv form or as the monovalent scFv-Fc fusion protein of the present invention, or other monovalent forms towards CD47 (such as Fab form), the binding proteins or antibodies of the present invention, including the humanized binding proteins or antibodies of the present invention, have a high binding affinity for CD47 (such as human CD47, such as having SEQ ID NO:19), for example, when determined by an SPR assay, K D (equilibrium dissociation constant) is in the range of 750 pM or lower, such as 750 pM or lower. Therefore, preferably, the binding proteins or antibodies of the present invention, for example, when in scFv form, or other monovalent forms towards CD47 (such as Fab form or monovalent scFv-Fc form), the binding affinity for CD47 (such as human CD47, such as having SEQ ID NO:19) corresponds to a K D less than 750 pM, preferably less than 700 pM, less than 650 pM, less than 600 pM, or less than 550 pM, more preferably less than 500, 450, 400, 350, 300, 250, 200, 150, 125, 100, 75, 50, or 40 pM.

[0168] For example, the binding affinity of the exemplary CO-1 scFv antibody of the present invention is 180 pM, and the binding affinity of the exemplary humanized antibody of the present invention in the scFv form is 61 pM or lower. In addition, the monovalent scFv-Fc fusion protein (CO201-scFv-Fc-mono) shows a binding affinity of 97 pM.

[0169] In some embodiments, the antibodies of the present invention have a higher affinity for human CD47 than certain control antibodies described in WO 2020 / 198370. Preferred affinities of the antibodies of the present invention are discussed elsewhere herein. In other embodiments, the antibodies of the present invention have other advantageous properties, such as improved PCD (e.g., higher levels of PCD, the ability to use lower concentrations of antibody, or faster induction) as described elsewhere herein compared to certain control antibodies described in WO 2020 / 198370.

[0170] In some embodiments, the present invention provides a binding protein, such as an antibody, comprising a humanized binding protein or antibody that comprises an antigen-binding domain that binds CD47, the antigen-binding domain comprising a heavy-chain variable region that comprises three complementarity-determining regions (CDRs) and a light-chain variable region that comprises three CDRs, wherein the binding protein or antibody binds CD47 monovalently and wherein the binding protein or antibody has a binding affinity as defined elsewhere herein. Thus, preferably, such an antibody or binding protein of the present invention, such as when in the scFv form or in other forms that are monovalent for CD47 (e.g., Fab form or monovalent scFv-Fc form), has a binding affinity for CD47 (e.g., human CD47, such as having SEQ ID NO:19) corresponding to a K D less than 750 pM, preferably less than 700 pM, less than 650 pM, less than 600 pM, or less than 550 pM, more preferably less than 500, 450, 400, 350, 300, 250, 200, 150, 125, 100, 75, 50, or 40 pM.

[0171] Any suitable method for determining K D can be employed. However, preferably, K D is determined by a surface plasmon resonance (SPR) assay (e.g., a BIAcore assay), and more preferably, its kinetic parameters are determined in such an assay. The types of SPR assays that are applicable and preferred are described above. Thus, the above K D value can be the K D value determined by the SPR assays described above or elsewhere herein, or the K D value observed when the antibody of the present invention is evaluated in an SPR assay. Particularly preferred methods are described in the Examples section herein.

[0172] The antibodies (or binding proteins) of the present invention (including the humanized antibodies or binding proteins of the present invention) are generally capable of binding CD47 expressed on the cell surface, such as human CD47 expressed on the cell surface (CD47 expressed on the cell surface, or CD47 present on the surface of CD47-expressing cells (such as human cells)). Thus, such cell surface forms represent the native or natural form of CD47 (or the native or natural conformation of CD47) in many cases, for example, the form found on cells that naturally express or overexpress CD47. CD47 is generally expressed on the surface of a variety of tumor cells. In some embodiments, the antibodies (or binding proteins) of the present invention bind CD47 expressed on the surface of human tumor cells. The binding to cell surface CD47 can be evaluated by any suitable method, and preferred methods include flow cytometry assays, as discussed in other parts of this document. In an exemplary flow cytometry method, CD47-expressing cells are incubated or contacted with the anti-CD47 antibody to be tested, and the antibody that binds to CD47 on the cell surface is detected by fluorescence, for example, the antibody is fluorescently labeled by an appropriate means (such as direct or indirect labeling). Thus, if the anti-CD47 antibody to be tested binds to CD47 on the cell surface, the cell will be fluorescently labeled, and such cells, as well as the antibody (or binding protein) that thereby demonstrates the ability to bind to cell surface CD47, can be easily identified by a flow cytometer. A particularly preferred flow cytometry method is described in the Examples section herein. Another method for testing the ability of an antibody to bind to cell surface CD47 is immunohistochemistry.

[0173] EC 50 values can be used to quantify the binding of the antibodies (or binding proteins) of the present invention to CD47 expressed on tumor cells. Methods for calculating EC 50 values are well known to those skilled in the art. However, the EC 50 values herein can be conveniently determined by flow cytometry assays, for example, by incubating or contacting a suitable cell line with the antibodies of the present invention that are directly or indirectly conjugated to a fluorescent label (usually an FITC conjugate) at increasing concentrations, followed by flow cytometry analysis. The exemplary concentration range of the antibodies (or binding proteins) used herein is from 0.1 ng / ml to 100 μg / ml. Curve fitting can then be performed using appropriate software (such as GraphPad Prism).

[0174] The antibodies (or binding proteins) of the present invention (including the humanized antibodies or binding proteins of the present invention) preferably are capable of binding to a variety of cancer cells, such as hematological malignancy cells and solid tumor cells. By way of example only, the antibodies (or binding proteins) of the present invention have been shown to be capable of binding to Jurkat T cells, which are an example of hematological cancer cells (see Figure 2 ). EC 50The value naturally varies with the relevant cell type. However, the exemplary antibodies of the present invention exhibit excellent binding ability to Jurkat cells, which can be reflected by the low (ng / ml) EC reported in the examples. 50 This is worth demonstrating. By way of example only, the antibodies (or binding proteins) of the present invention can bind to Jurkat cells (a cancerous T cell line), and its EC 50 value is 5 ng / ml or lower, preferably 1 ng / ml or lower, or 0.1 ng / ml or lower, or 0.06 ng / ml or lower.

[0175] Therefore, in certain embodiments, the EC 50 value of the antibodies (or binding proteins) of the present invention (e.g., binding to cancer cells, such as CD47-expressing cancer cells, e.g., Jurkat T cells) is 5, 4, 3, 2, or 1 ng / ml or lower, preferably 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 ng / ml or lower, more preferably 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01 ng / ml or lower. In some embodiments, the EC 50 is from 0.01, 0.03, 0.04, 0.05, or 0.1 to 5, 1, or 0.5 ng / ml. Specific exemplary EC 50 values are also shown in the examples. For example, the EC 50 value of CO-1scFv to Jurkat T cells can be as low as 0.05 ng / ml (see Figure 2 ).

[0176] The preferred antibodies (or binding proteins) of the present invention show reduced binding ability to normal cells expressing CD47 compared to tumor cells (such as Jurkat cells). In particular, the preferred antibodies (or binding proteins) of the present invention, including the humanized antibodies (or binding proteins) of the present invention, show reduced binding ability to red blood cells (such as human red blood cells) compared to tumor cells (e.g., Jurkat cells).

[0177] Therefore, in the preferred embodiments of the present invention, the antibodies (or binding proteins) show preferential binding or stronger binding ability to cancer cells (such as Jurkat cells) compared to normal cells (such as red blood cells or human red blood cells) or normal human B cells (such as B cells from the buffy coat of healthy human donors), preferably showing measurable or significantly stronger binding ability. In other words, limited binding to normal cells (especially red blood cells or human red blood cells) or normal human B cells (such as B cells from the buffy coat of healthy human donors) is observed.

[0178] In some embodiments, compared to human red blood cells or normal human B cells, the binding ability of the antibody (or binding protein) of the present invention to Jurkat cells is increased by at least 50%, 75%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750% or 800%. In other words, compared to human red blood cells or normal human B cells, the binding ability of the antibody of the present invention to Jurkat cells is increased by at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold.

[0179] The preferred ECs described above and elsewhere in this document 50 values are preferably determined by appropriate binding assays conducted under appropriate conditions such that the EC 50 values can be measured or determined, for example, by appropriate assays based on flow cytometry, as described above or in the Examples section. These assays can be conducted in any appropriate form of the antibody (or binding protein). Thus, the exemplary values described above and elsewhere in this document can be, for example, values determined when the antibody is in the scFv form or other monovalent forms for CD47 (e.g., the Fab form).

[0180] The antibody (or binding protein) of the present invention, including the humanized antibody or binding protein of the present invention, is capable of killing tumor cells (such as tumor cells expressing CD47), for example, capable of directly killing tumor cells (such as tumor cells expressing CD47). It is believed that this killing effect occurs through the programmed cell death (PCD) pathway. It is further believed that this PCD pathway is caspase-independent.

[0181] Such killing is referred to as direct killing, which means that cell killing can be caused by the antibody (or binding protein) itself, for example, killing can occur without the participation of other entities (such as cells, for example, immune effector cells such as macrophages). This is in contrast to entities that rely on the blockade or inhibition of the CD47-SIRPα interaction, which achieves tumor cell destruction through phagocytosis and involves the recruitment of other cells, such as macrophages or other innate immune cells.

[0182] The ability of the antibodies (or binding proteins) of the present invention to induce such direct cell killing or PCD has significant advantages, particularly for tumor cells. In fact, many previously described anti-CD47 antibodies do not possess this property. The antibodies of the present invention have been demonstrated to be particularly effective in inducing such killing, for example, capable of killing or inducing PCD in a large number / high proportion of tumor cells. In addition, it has been demonstrated that they can rapidly exhibit this effect and / or exhibit this effect at extremely low concentrations. The antibodies of the present invention can also induce such killing (PCD) in soluble form (e.g., as opposed to immobilized form). This is also an advantageous property as it means that such antibodies can function in solution or in soluble form, which is the conventional and convenient form for the administration of therapeutic antibodies.

[0183] PCD can be conveniently determined by Annexin V / 7-AAD assays well-known to those skilled in the art. In fact, kits are commercially available for such assays (e.g., eBioscience TM Annexin V Apoptosis Detection Kit eFluor TM 450, catalog number 88-8006-74) from ThermoFisher. Cells that are Annexin V positive and 7-AAD negative are considered early apoptotic cells, while cells that are Annexin V positive and 7-AAD positive are considered late apoptotic cells. Although the number / percentage of early and late apoptotic cells can be analyzed separately, it is usually convenient to add the data for these two types of apoptotic cells to obtain an overall determination of the total cell death. Such total PCD can be conveniently expressed as a percentage.

[0184] The antibodies (or binding proteins) of the present invention can induce PCD at concentrations below 10 μg / ml, 5 μg / ml, 2 μg / ml, 1 μg / ml, or 0.5 μg / ml. More preferably, the antibodies of the present invention can induce PCD at extremely low concentrations, such as 0.1 μg / ml or lower, for example, between 0.05 or 0.1 and 1, 2, 5, or 10 μg / ml.

[0185] The preferred antibodies of the present invention can induce PCD relatively rapidly, for example, after a contact time as low as 30 minutes. Longer contact times (e.g., 1 hour, 2 hours, or 3 hours) are also effective in inducing PCD. Even longer contact times (e.g., 6 hours or 12 hours) can be equally effective. However, it is clearly more advantageous if the antibody can act quickly.

[0186] The antibodies (or binding proteins) of the present invention preferably are capable of inducing direct killing of a variety of cancer cells, such as hematological malignancy cells and solid tumor cells. By way of example only and not by way of limitation, it has been demonstrated that the antibodies (or binding proteins) of the present invention are capable of killing blood cancer cells, such as CCRF-CEM, Jurkat, and MOLT-4 (seeFigure 3 )。The PCD percentage value naturally varies with the relevant cell type. However, exemplary antibodies of the present invention (including the humanized antibodies of the present inventors) show good levels of killing percentage against a variety of cancer cells. For example, at least 10% killing levels are generally observed for different types of cancer cells, at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60% levels are observed for certain types of cancer cells, such as not exceeding 30%, 35%, 40%, 45%, 50%, 55% or 60%. By way of example only, the antibodies (or binding proteins) of the present invention can induce at least or not more than 20%, 25% or 30% killing of Jurkat cells (a cancerous T cell line), for example when the cells are exposed to the scFv antibodies of the present invention (such as IgG antibodies, for example IgG1 or IgG4) at a concentration of 1 or 0.1 μg / ml for 30 minutes, 1 hour, 2 hours or 3 hours. By way of example only, the antibodies (or binding proteins) of the present invention, such as the humanized antibodies (or binding proteins) of the present inventors, can induce at least or not more than 35%, 40%, 45%, 50%, 55%, 60% or 65% killing of Jurkat cells (a cancerous T cell line), for example when the cells are exposed to the scFv antibodies of the present invention at a concentration of 1 or 0.1 μg / ml for 30 minutes, 1 hour, 2 hours or 3 hours.

[0187] By way of example only, the antibodies (or binding proteins) of the present invention can induce at least or not more than 40%, 45%, 50%, 55% or 60% killing of CCRF-CEM cells, for example when the cells are exposed to the scFv antibodies of the present invention at a concentration of 1 or 0.1 μg / ml for 3 hours.

[0188] This killing effect can be observed at antibody concentrations as high as 1 μg / ml and even as low as 0.1 μg / ml. This direct killing effect can be observed 3 hours after the antibody is incubated with the cells. However, it has been demonstrated that the antibodies of the present invention are still effective at shorter incubation times. In other words, these antibodies are characterized by rapid onset and potency. For example, significant cell death can be observed after incubation times of 30 minutes to 1 hour, 2 hours or 3 hours.

[0189] It is unexpected and advantageous that the antibodies or binding proteins of the present invention have the ability to induce PCD, let alone reach the above levels, because these antibodies or binding proteins monovalently bind CD47, while it has been shown in the prior art that bivalent binding of CD47 may be important for inducing PCD.

[0190] Those skilled in the art are familiar with methods for calculating the PCD percentage value. However, the PCD percentage value in this article can be conveniently determined by in vitro assays (such as AnnexinV / 7-AAD detection). The method is to incubate an appropriate cell line with a specific concentration of the antibody to be tested of the present invention (for example, 0.1 to 1 μg / ml) for a specific duration (for example, 30 minutes to 12 hours, such as about 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours or 12 hours), and then stain with AnnexinV eFluor405 and 7-AAD. The PCD percentage is the sum of Annexin V + 7-AAD - cells and Annexin cells.

[0191] In some embodiments, the antibodies (or binding proteins) of the present invention (including humanized antibodies or binding proteins) do not induce significant killing (PCD) of normal human B cells (such as B cells from the buffy coat of healthy human donors).

[0192] The preferred antibodies (or binding proteins) of the present invention do not induce (or significantly induce) red blood cell (RBC) agglutination. This is a surprising and advantageous property, especially when combined with the ability to induce PCD in tumor cells described in other parts of this article. It can be noted that the humanized antibodies of the present invention can retain the above properties.

[0193] The ability to induce an agglutination reaction can be conveniently determined by in vitro agglutination assays well known to those skilled in the art. Such assays can conveniently involve: contacting increasing concentrations of the antibody to be tested (or control) with a red blood cell preparation, such as human red blood cells, such as freshly isolated human red blood cells, for example 2% (v / v) freshly isolated red blood cells; and incubating the mixture for a specific time (for example, 30 to 60 minutes) or until the cells settle in the well plate or container used (such assays usually use 96-well plates). A diffuse and blurred pattern indicates the presence of agglutination, while a dot pattern indicates no agglutination.

[0194] Preferred antibodies (or binding proteins) of the present invention, including the humanized antibodies of the present invention, do not induce (or significantly induce) red blood cell agglutination when used at a concentration of or at least or not exceeding 1, 2, 5 or 10 μg / ml. Preferred antibodies of the present invention do not induce (or significantly induce) red blood cell agglutination when used at a concentration of or at least or not exceeding 15, 20, 25, 50, 75 or 100 μg / ml. Although any convenient method for evaluating agglutination reactions can be employed, a suitable and preferred detection protocol is described in the above and the Examples section. Therefore, the values described above can be the values determined in the agglutination assay described above (when or if the assay is performed). A particularly preferred method is described in the Examples section of this article.

[0195] Preferably, the above-mentioned abilities and characteristics are observed at a measurable or significant level, more preferably at a statistically significant level, compared to an appropriate control level. Appropriate significance levels are discussed elsewhere in this document. More preferably, compared to the abilities observed with prior art antibodies, the above-mentioned one or more abilities and characteristics are observed at a measurably better or significantly better (preferably statistically significantly better) level.

[0196] For any statistical analysis mentioned herein, the probability value of a statistically significant difference is preferably ≤0.1 or <0.1, more preferably ≤0.05 or <0.05, relative to a relevant control or other comparative entity / measurement. Suitable methods for determining statistical significance are well-known and documented in the art, and any one of them can be used.

[0197] In some embodiments, the binding proteins or antibodies of the present invention, including CO-1 scFv antibodies, humanized CO-1 scFv antibodies, and monovalent scFv-Fc fusion proteins, have one or more, preferably two or more, three or more, four or more, and most preferably all of the functional characteristics described herein, particularly the preferred functional characteristics. Examples and details of the preferred functional characteristics are described elsewhere in this document and include: i) high affinity for CD47, e.g., when measured by SPR (Biacore); ii) the ability to induce direct killing of tumor cells (such as Jurkat cells or CCRF-CEM cells), which ability can be induced at iii) low concentrations (such as an antibody concentration of 0.1 or 1 μg / ml) and / or iv) after a short incubation time (such as 30 minutes or 1 hour); v) the ability not to induce (or severely agglutinate) red blood cell (RBC) agglutination.

[0198] Thus, in some embodiments, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds CD47, wherein the antigen-binding domain comprises a heavy-chain variable region comprising three complementarity-determining regions (CDRs) and a light-chain variable region comprising three CDRs, wherein the binding protein or antibody binds CD47 monovalently, and wherein the binding protein or antibody has one or more, preferably two or more, or three or more, or four or more, and most preferably all of the functional characteristics described herein, particularly the preferred functional characteristics, such as one or more of the above characteristics (i) to (v). In some embodiments, the binding protein or antibody also has the ability to induce phagocytosis of tumor cells.

[0199] In some embodiments, functional feature (ii) is a preferred feature, optionally in combination with one or more of the other features described above. In some embodiments, functional feature (ii) is a preferred feature, optionally in combination with one or more, two or more, three or more, or most preferably all of features (i), (iii), (iv), and (v) described above; or in combination with one or more, two or more, or most preferably all of features (iii), (iv), and (v) described above. In some such embodiments, the binding protein or antibody also has the ability not to induce phagocytosis of tumor cells..

[0200] Throughout the application, the term "a / an" is used to denote "at least one", "at least the first", "one or more", or "multiple" of the recited component or step, unless a specific upper limit is subsequently stated. Thus, "antibody" as used herein means "at least the first antibody".

[0201] Furthermore, when using the terms "comprising", "having", or other equivalent terms, in some more specific embodiments, such as in the definitions of CDR or FR sequences herein, these terms include "consisting of" or "consisting essentially of" or other equivalent terms.

[0202] Nucleic acid molecules (e.g., one or more nucleic acid molecules) comprising a nucleotide sequence encoding a binding protein, antibody, or immunoconjugate of the invention as defined herein, or nucleic acid molecules that are substantially homologous thereto, constitute other aspects of the invention.

[0203] Preferred nucleic acid molecules are nucleic acid molecules encoding an antibody of the invention capable of monovalent binding to CD47 as described elsewhere herein, e.g., an antibody of the invention having CDRs defined in Table A or B and optionally FRs and other regions, or an antibody having a sequence substantially homologous thereto.

[0204] Preferred nucleic acid molecules are nucleic acid molecules encoding an antibody of the invention capable of monovalent binding to CD47 (e.g., comprising a nucleic acid sequence encoding SEQ ID NO:3 and / or SEQ ID NO:4, as SEQ ID NO:1 and / or SEQ ID NO:2, respectively).

[0205] Other preferred nucleic acid molecules comprise sequences encoding the scFv form of the antibodies of the invention or alternative forms that are monovalent for CD47, such as the Fab form, e.g., the sequences (heavy chain variable region and light chain) described in Table A herein. Thus, preferred nucleic acid molecules are nucleic acid molecules encoding the scFv of SEQ ID NO:21, or nucleic acid molecules encoding alternative forms that are monovalent for CD47, such as the VH domain of SEQ ID NO:3 and / or the VL domain of SEQ ID NO:4. Other preferred nucleic acid molecules comprise sequences encoding the humanized antibodies of the invention, e.g., the antibodies (heavy chain and light chain) described in Table B.

[0206] Thus, preferred nucleic acid molecules are molecules encoding the heavy chain of the antibodies of the invention (e.g., molecules encoding SEQ ID NO:22, 23, 24, 25 or 26) and / or molecules encoding the light chain of the antibodies of the invention (e.g., molecules encoding SEQ ID NO:27, 28 or 29). Other preferred nucleic acid molecules comprise sequences encoding the monovalent scFv-Fc fusion proteins of the invention (e.g., CO201-scFv-Fc-mono).

[0207] As used herein, the term "substantially homologous" with respect to an amino acid or nucleic acid sequence includes sequences having at least 60%, 65%, 70% or 75%, preferably at least 80%, more preferably at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the disclosed amino acid or nucleic acid sequence. Thus, substantially homologous sequences of the invention include single or multiple base or amino acid changes (additions, substitutions, insertions or deletions) to the sequences of the invention. At the amino acid level, preferred substantially homologous sequences contain no more than 6 altered amino acids in one or more framework regions and / or one or more complementarity determining regions (CDRs) that make up the sequences of the invention, e.g., only 1, 2, 3, 4, 5 or 6, such as 1, 2, 3, 4 or 5, preferably 1, 2, 3 or 4, preferably 1, 2 or 3, more preferably 1 or 2. Further, at the amino acid level, preferred substantially homologous sequences contain no more than 6 altered amino acids in the combined framework regions (e.g., four framework regions) and / or combined CDRs (e.g., three CDR regions) that make up the VL or VH domain of the antibodies of the invention, e.g., only 1, 2, 3, 4, 5 or 6, such as 1, 2, 3, 4 or 5, preferably 1, 2, 3 or 4, preferably 1, 2 or 3, more preferably 1 or 2. Further, at the amino acid level, preferred substantially homologous sequences contain no more than 6 altered amino acids in the VH domain and / or VL domain of the antibodies of the invention, e.g., only 1, 2, 3, 4, 5 or 6, such as 1, 2, 3, 4 or 5, preferably 1, 2, 3 or 4, preferably 1, 2 or 3, more preferably 1 or 2. The changes can be conservative or non-conservative amino acid changes, or combinations thereof. Preferably the changes are substitutions, preferably conservative amino acid substitutions.

[0208] In certain embodiments, if a given starting sequence is short (e.g., five amino acids in length), the number of amino acid substitutions present in its substantially homologous sequences may be less than the number of optional amino acid substitutions in sequences that are substantially homologous to a longer starting sequence. For example, in certain embodiments, a sequence that is substantially homologous to a starting VH CDR1 sequence of the invention (e.g., a starting VH CDR1 sequence that may be five amino acid residues in length in certain embodiments) preferably has 1 or 2 (more preferably 1) altered amino acids compared to the starting sequence. Thus, in certain embodiments, the number of altered amino acids in a substantially homologous sequence (e.g., a substantially homologous CDR sequence) can be adjusted based on the length of a given starting CDR sequence. For example, different numbers of amino acid alterations may be present depending on the length of a given starting CDR sequence to achieve a particular percent sequence identity in the CDR, such as at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0209] Conventional methods in the art such as alanine scanning mutagenesis and / or deep mutational scanning (designed to make all possible single-site substitutions at all selected residues in a given protein sequence) and / or crystal structure analysis of antigen-antibody complexes can be used to determine which amino acid residues in a CDR do not contribute or contribute insignificantly to antigen binding and thus are ideal candidates for alteration or substitution in embodiments of the invention involving substantially homologous sequences.

[0210] After identification, one or more amino acids can be added, deleted, substituted, or inserted into the amino acid sequence of a parental antibody (such as one of the antibodies of the invention as defined herein) using conventional techniques in the art to form a new antibody, and the resulting new antibody can be tested to identify a CD47-binding antibody that meets the invention. Such methods can be used to construct multiple new antibodies that can be tested for CD47-binding ability. Preferably, the addition, deletion, substitution, or insertion of one or more amino acids occurs in one or more CDR domains.

[0211] For example, the manipulation can be conveniently carried out by genetic engineering at the nucleic acid level, i.e., modifying nucleic acid molecules encoding the appropriate binding protein and its domains such that the amino acid sequence of the ultimately expressed protein is correspondingly modified in an appropriate manner. Any suitable method known in the art and described can be used to test the CD47-binding ability of one or more modified antibodies / binding proteins. Suitable methods are also described in other parts of this document and in the Examples section.

[0212] Antibodies prepared, obtained, or obtainable by these methods constitute another aspect of the invention.

[0213] The term "substantially homologous" also includes modifications or chemical equivalents of the amino acid and nucleotide sequences of the antibodies of the present invention that perform substantially the same functions as the antibody proteins or nucleic acid molecules of the present invention in substantially the same manner. For example, any substantially homologous antibody should retain the ability to bind CD47 monovalently as described above. Preferably, any substantially homologous antibody should retain one or more (or all) of the functional properties of the starting antibody.

[0214] Substantially homologous sequences of the proteins of the present invention include, but are not limited to: conservative amino acid substitutions, or modifications that do not affect the VH, VL, or CDR domains of the antibody, such as the addition of tag sequences, toxins, or other components that do not promote monovalent binding to the CD47 antigen.

[0215] As used herein, "conservative amino acid substitution" refers to the replacement of an original amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include: basic side chains (such as lysine, arginine, histidine), acidic side chains (such as aspartic acid, glutamic acid), uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (such as glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (such as threonine, valine, isoleucine), and aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine). In other embodiments, families of amino acid residues may be grouped based on hydrophobic or hydrophilic side groups.

[0216] Homology or sequence identity can be assessed by any suitable method. However, to determine the degree of homology or identity between sequences, a multiple sequence alignment computer program (e.g., Clustal W (Thompson, Higgins, Gibson, Nucleic Acids Res., 22:4673-4680, 1994)) can be used. If desired, the Clustal W algorithm can be used in conjunction with the BLOSUM 62 scoring matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915–10919, 1992), with a gap opening penalty of 10 and a gap extension penalty of 0.1, to obtain the highest level of alignment between two sequences, where at least 50% of the total length of at least one sequence is involved in the alignment. Other available sequence alignment methods include the Needleman-Wunsch algorithm (Needleman and Wunsch, J. Mol. Biol., 48:443, 1970) and its Smith-Waterman modification (Smith and Waterman, Adv. Appl. Math., 2:482, 1981), to obtain the highest level of match between two sequences and to determine the number of identical amino acids between the two sequences. Other methods for calculating the percent identity of two amino acid sequences are known in the art, including, for example, the method described by Carillo and Lipton (Carillo and Lipton, SIAM J. Applied Math., 48:1073, 1988), and the methods described in Computational Molecular Biology, Lesk, e.d. Oxford University Press, New York, 1988, Biocomputing: Informatics and Genomics Projects.

[0217] Such calculations are usually performed using a computer program. Programs useful for this purpose also include programs that can align sequence pairs, such as ALIGN (Myers and Miller, CABIOS, 4:11-17, 1988), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444-2448, 1988; Pearson, Methods in Enzymology, 183:63-98, 1990), gapped BLAST (Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997), BLASTP, BLASTN or GCG (Devereux, Haeberli, Smithies, Nucleic Acids Res., 12:387, 1984). In addition, the Dali server at the European Bioinformatics Institute provides a structure-based protein sequence alignment service (Holm, Trends in Biochemical Sciences, 20:478-480, 1995; Holm, J. Mol. Biol., 233:123-38, 1993; Holm, Nucleic Acid Res., 26:316-9, 1998).

[0218] For reference, sequences having at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology or sequence identity in the present invention can be determined using the ALIGN program (e.g., the version provided by the GENESTREAM web server of the IGH Institute in Montpellier, France) with default parameters.

[0219] Preferably, any substantially homologous antibody of the present invention should retain the ability to specifically bind to the same epitope recognized by the starting antibody, such as the same epitope recognized by the CDR domains of one or more antibodies of the present invention as described herein or the VH / VL domains of one or more antibodies of the present invention, e.g., binding to the same epitope as one or more of the various antibodies of the present invention (such as the CD47 antibodies shown in Table A or B).

[0220] The binding of the same epitope can be verified by epitope mapping methods well-known in the art, such as: through the crystal structure analysis of the antigen-antibody complex; or through single residue mutation studies (such as alanine scanning and / or deep mutational scanning (DMS), e.g., yeast display combined with DMS technology (see Sierocki et al., 2021, PLoS Negl Trop Dis., 15(3):e0009231; see also Van Blarcom et al., 2015, JMB, 427:6(B):1513-1534 and Medina-Cucurella and Whitehead, 2018, Methods Mol.Biol., 1764:101-121). In certain embodiments, DMS (especially yeast display combined with DMS) is preferably used as the epitope determination method. Any of the above epitope analysis methods can be used in combination with binding assays, such as competition assays, for example as a primary screening step. Therefore, antibodies that recognize the same epitope as one or more of the antibodies of the present invention, such as those determined by crystal structure analysis of the antigen-antibody complex or single residue mutation studies (such as alanine scanning and / or DMS, such as yeast display combined with DMS technology), constitute other aspects of the present invention. In a preferred embodiment, DMS (such as yeast display combined with DMS) is used to evaluate or determine the epitope. The retention of other functional properties (especially binding affinity) can also be conveniently verified by methods well-known in the art or described herein.

[0221] Therefore, those skilled in the art should understand that such methods can be used to test whether any antibody (such as a "substantially homologous" antibody) has the same binding specificity as the antibodies and antibody fragments of the present invention, such as binding to the same epitope, or having the same / equivalent affinity. For example, through crystal structure analysis of the antigen-antibody complex or single residue mutation studies (such as alanine scanning and / or deep mutational scanning (DMS), such as yeast display combined with DMS technology), it is possible to conveniently evaluate whether an antibody (such as a "substantially homologous" antibody) can bind to the same CD47 epitope, and binding assays such as competition assays described elsewhere in this document can be used as an adjunct to determine this. SPR assays (such as the BIAcore assays described elsewhere in this document) can also be conveniently used to determine whether an antibody (such as a "substantially homologous" antibody) can bind to CD47, and optionally to determine the affinity of this binding (such as the K D value). Those skilled in the art should be aware of other applicable methods and variants.

[0222] As described below, a competitive binding assay can be used as a primary or supplementary assay for epitope mapping, to detect whether an antibody (e.g., a "substantially homologous" antibody) retains the ability to specifically bind to the same (or substantially the same) CD47 epitope recognized by one or more antibodies of the present invention (e.g., as shown in the Sequence Listing), or whether it has the ability to compete for binding with multiple antibodies of the present invention as shown in the Sequence Listing. The methods described below are only one example of a suitable competitive assay. Those skilled in the art should be aware of other applicable methods and variations.

[0223] An exemplary competitive assay involves: evaluating the binding of each effective concentration of an antibody of the present invention to CD47 in the presence of different concentrations of the antibody to be tested (e.g., a substantially homologous antibody). Subsequently, the degree of binding inhibition induced by the antibody to be tested can be evaluated. If the antibody to be tested shows enhanced competition with the antibody of the present invention as its concentration increases (i.e., an increase in the concentration of the antibody to be tested results in a corresponding decrease in the amount of the antibody of the present invention bound to CD47), it demonstrates that it binds to the same or substantially the same epitope. Preferably, the antibody to be tested significantly reduces the amount of the antibody of the present invention bound to CD47. More preferably, the antibody to be tested reduces the amount of the antibody of the present invention bound to CD47 by at least about 95%. In such competitive assays, ELISA or flow cytometry can be used to evaluate binding inhibition, but those skilled in the art should be aware of other applicable techniques.

[0224] Such antibodies (or binding proteins) that are capable of binding (or specifically binding) to the same (or substantially the same) CD47 epitope recognized by an antibody of the present invention (such as the antibody shown in Table A), and wherein the binding protein or antibody binds CD47 monovalently, are other embodiments of the present invention. Such antibodies (or binding proteins) optionally also have the ability to compete for binding to CD47 with one or more of the multiple antibodies of the present invention (such as the antibody shown in Table A).

[0225] Accordingly, another aspect of the present invention provides an antibody (or binding protein) that binds (or specifically binds monovalently) to CD47 and has the ability to bind to the same (or substantially the same) epitope as the CO-1scFv (Table A) antibody (i.e., an antibody comprising the VL of SEQ ID NO:4 and the VH of SEQ ID NO:3 as described herein); or has the ability to bind to the same (or substantially the same) epitope as an antibody having the same CDRs as the CO-1scFv (Table A) antibody (i.e., an antibody comprising the VL CDR sequences of SEQ ID NO:8, 9, 10 and the VH CDR sequences of SEQ ID NO:5, 6, 7) to bind CD47. Such antibodies (or binding proteins) optionally also have the ability to compete for binding to CD47 with one or more of the multiple antibodies of the present invention (such as the antibody shown in Table A). The features and properties of other aspects of the present invention are equally applicable to this aspect with necessary modifications in detail.

[0226] As used herein, the term "competitive antibody" refers to an antibody that binds to substantially the same, essentially the same, or exactly the same epitope as the "reference antibody". "Competitive antibodies" include antibodies with overlapping epitope specificities. Thus, a competitive antibody is capable of effectively competing with the reference antibody for binding to CD47. Preferably, the competitive antibody is capable of binding to the same epitope as the reference antibody. In other words, the competitive antibody preferably has the same epitope specificity as the reference antibody.

[0227] As used herein, the "reference antibody" refers to an antibody that can bind to CD47 according to the present invention, which preferably has VH and VL domains as defined in Table A, more preferably comprises the VH domain of SEQ ID NO:3 and the VL domain of SEQ ID NO:4 (or the corresponding three CDR sequences of said sequences) as shown in Table A, or has humanized VH and / or VL domains as shown in Table B.

[0228] Since the sequence listing herein provides the reference antibody, it has become a straightforward technical matter to identify one or more antibodies that bind to the same epitope and optionally competitive antibodies. Epitope mapping can be carried out using standard techniques, some of which are described elsewhere in this document. Such epitope mapping can be supplemented with competition assays (e.g., as a primary or supplementary screening step), which can also be carried out by standard techniques, some of which are described herein. For example, in this regard, CD47 antibodies can be generated by an immunization protocol using the CD47 antigen or preferably cells that express / overexpress the CD47 antigen as an immunogen, and such CD47 antibodies can then be readily screened (e.g., using the methods described herein) to identify antibodies that bind to the same epitope as the reference antibody of the present invention. Alternatively, substantially homologous sequences derived from the antibodies shown in Table A or Table B can be screened in this manner.

[0229] The present invention has indeed carried out epitope mapping on the CO-1 antibody by DMS (especially yeast display in combination with DMS).

[0230] Accordingly, in another embodiment, the present invention provides a binding protein, such as an antibody, or a binding protein or antibody as described elsewhere herein, which comprises an antigen-binding domain that binds to CD47, said antigen-binding domain comprising: a heavy-chain variable region comprising three complementarity-determining regions (CDRs), and a light-chain variable region having three CDRs, wherein said antigen-binding domain binds (or is capable of binding, or specifically binds) to Q19, N45, T120, R121, E122, and G123 in CD47 as defined by SEQ ID NO:19, and wherein said antigen protein or antibody binds to CD47 monovalently. In other embodiments, said antigen-binding domain binds to Q19, L20, N45, T120, R121, E122, and G123 in CD47 as defined by SEQ ID NO:19.

[0231] Alternatively stated, the antigen-binding domain binds to an epitope comprising or consisting of Q19, N45, T120, R121, E122, and G123 in CD47 as defined by SEQ ID NO:19. In other embodiments, the antigen-binding domain binds to an epitope comprising or consisting of Q19, L20, N45, T120, R121, E122, and G123 in CD47 as defined by SEQ ID NO:19.

[0232] In some embodiments, the antigen-binding domain does not bind, or the epitope does not comprise one or more, two or more, three or more, preferably all, of T117, E118, E115, and L21. In some embodiments, the antigen-binding domain does not bind, or the epitope does not comprise T117 in CD47 as defined by SEQ ID NO:19; or T117 and E118; or T117, E118, and E115; or T117, E118, E115, and L21.

[0233] In some embodiments, the antigen-binding domain does not bind, or the epitope does not comprise one or more, two or more, three or more, preferably all, of V54 to A84, M46, or L119 (especially L119) in CD47 as defined by SEQ ID NO:19.

[0234] In some embodiments, the antigen-binding domain does not bind, or the epitope does not comprise one or more, two or more, three or more, four or more, preferably all, of E115 to L119 (especially T117 to L119, more especially T117 and / or L119) in CD47 as defined by SEQ ID NO:19.

[0235] In some embodiments, the antigen-binding domain does not bind, or the epitope does not comprise one or more, preferably two, of T117 and E124 in CD47 as defined by SEQ ID NO:19.

[0236] In some embodiments, the antigen-binding domain does not bind, or the epitope does not comprise one or more, two or more, three or more, preferably all, of L21, E47, E115, and E118 in CD47 as defined by SEQ ID NO:19.

[0237] In some embodiments, the antigen-binding domain does not bind, or the epitope does not comprise one or more, two or more, three or more, four or more, preferably all, of M46, Y55, K57, D69, and L119 in CD47 as defined by SEQ ID NO:19.

[0238] In some embodiments, the antigen-binding domain binds CD47 as defined by SEQ ID NO:19 independently of G70. For example, in such embodiments, mutating G70 to other amino acid residues does not affect (or does not significantly affect) the binding of the antigen-binding domain or antibody of the invention to CD47, or the binding is maintained. In some embodiments, the antigen-binding domain does not bind, or the epitope does not contain residue G70 in CD47 as defined by SEQ ID NO:19.

[0239] In preferred embodiments, the binding protein or antibody that binds to the above-described epitope or residues is evaluated or determined by crystal structure analysis of the antigen-antibody complex or by mutagenesis studies of individual residues (e.g., using alanine scanning and / or deep mutational scanning DMS, such as yeast display-binding DMS). In preferred embodiments, the binding to these epitopes or residues is evaluated or determined by DMS (such as yeast display-binding DMS).

[0240] In preferred embodiments, the binding protein or antibody that binds to the CD47 epitope defined herein has a binding affinity as described elsewhere herein. Accordingly, the invention provides a binding protein (e.g., an antibody) comprising an antigen-binding domain that binds to a CD47 epitope defined herein, the antigen-binding domain comprising: a heavy chain variable region comprising three complementarity-determining regions (CDRs), and a light chain variable region comprising three CDRs, wherein the binding protein or antibody binds CD47 monovalently, and wherein the binding protein or antibody has a binding affinity as defined elsewhere herein. Accordingly, preferably, such an antibody or binding protein, e.g., when in scFv form or monovalent scFv-Fc form, or in other forms that are monovalent for CD47 (such as Fab form), has a binding affinity for CD47 (e.g., human CD47, such as having SEQ ID NO:19) corresponding to a K D less than 750 pM, preferably less than 700 pM, less than 650 pM, less than 600 pM, or less than 550 pM, more preferably less than 500, 450, 400, 350, 300, 250, 200, 150, 125, 100, 75, 50, or 40 pM.

[0241] As used herein, the terms "antibody" and "immunoglobulin" broadly refer to any immunological binding agent that comprises an antigen-binding domain, including polyclonal and monoclonal antibodies. Monoclonal antibodies are preferred. However, the binding proteins and antibodies of the invention have a structure or form that enables them to bind CD47 monovalently, such as antibody fragments that can bind CD47 monovalently.

[0242] As used herein, the term "heavy chain complementarity-determining region" ("heavy chain CDR") refers to the variable region (V HHypervariable regions within the (domain). The variable region of the heavy chain sequentially has three CDRs from the amino terminus to the carboxyl terminus, which are respectively called heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3. The variable region of the heavy chain also has four framework regions (FR1, FR2, FR3, and FR4 in sequence from the amino terminus to the carboxyl terminus). These framework regions separate each CDR.

[0243] As used herein, the term "variable region of the heavy chain" (V H (domain) refers to the variable region of the heavy chain of an antibody molecule.

[0244] As used herein, the term "light chain complementarity determining region" ("light chain CDR") refers to the hypervariable region within the variable region of the light chain (V L (domain) of an antibody molecule. The variable region of the light chain sequentially has three CDRs from the amino terminus to the carboxyl terminus, which are respectively called light chain CDR1, light chain CDR2, and light chain CDR3. The variable region of the light chain also has four framework regions (FR1, FR2, FR3, and FR4 in sequence from the amino terminus to the carboxyl terminus). These framework regions separate each CDR.

[0245] As used herein, the term "variable region of the light chain" (V L (domain) refers to the variable region of the light chain of an antibody molecule.

[0246] As described in other parts of this application, the binding proteins and antibodies of the present invention have structures or forms that enable them to bind CD47 monovalently. Any suitable monovalent form can be adopted, such as any antibody or antibody fragment form that contains only one antigen-binding domain capable of binding CD47. Exemplary and preferred forms or fragments are scFv antibodies, Fab or Fab' fragments. Other exemplary monovalent forms or fragments include single-domain antibodies (nanobodies), monobodies, monovalent scFv-Fc fusion proteins, and monovalent IgG (half-antibodies). As described in other parts of this application, a particularly preferred form is a monovalent scFv-Fc fusion protein.

[0247] The antibody or binding protein can be naturally occurring or wholly or partially synthetic.

[0248] The antigen-binding domain of the antibody or binding protein of the present invention generally comprises: the variable region of the light chain of the antibody (V L ) that contains three CDR domains, and the variable region of the heavy chain of the antibody (V H ) that contains three CDR domains.

[0249] However, it is well documented in the art that it is not always necessary for the three CDRs of the variable region of the light chain of the antibody and the three CDRs of the variable region of the heavy chain to achieve antigen binding. Therefore, constructs known to be smaller than the above classical antigen-binding domain can also effectively bind antigens.

[0250] For example, camelid VHH antibodies and other single-domain antibodies containing only the VH domain have demonstrated that these domains alone can also bind antigen with acceptably high affinity. Thus, three CDRs (or even a single CDR) can effectively bind antigen and form an antigen-binding domain.

[0251] Although the preferred antigen-binding domain of the antibodies of the present invention may contain six CDR regions (three in the light chain + three in the heavy chain), the present invention also encompasses antibodies having an antigen-binding domain with fewer than six CDR regions (such as 3 CDR regions). The present invention also contemplates antibodies having an antigen-binding domain that contains only heavy-chain CDRs or light-chain CDRs.

[0252] Preferred light-chain CDR regions that combine with the designated heavy-chain CDR regions to form an antigen-binding domain are described elsewhere herein. However, the present invention also contemplates other light-chain variable regions containing three CDRs for use in combination with the heavy-chain variable regions of the present invention. Those skilled in the art can readily identify light-chain variable regions that can be used in combination with the heavy-chain variable regions of the present invention and that can produce a monovalent CD47-binding antibody that meets the present invention.

[0253] For example, the heavy-chain variable region of the present invention can be combined with a single light-chain variable region, or a set of light-chain variable regions, and the resulting antibodies can be tested for CD47 binding.

[0254] If desired, a similar method can be used to identify alternative heavy-chain variable regions for use in combination with the preferred light-chain variable regions of the present invention.

[0255] The antibodies, binding proteins, and nucleic acid molecules of the present invention are generally "isolated" or "purified" molecules, i.e., distinct from any such components that may be present in the human or animal body or in tissue samples derived from the human / animal body. However, the sequences can be identical or substantially homologous to sequences present in the human / animal body. Thus, the terms "isolated" or "purified" as used herein, when referring to nucleic acid molecules or sequences and proteins or polypeptides (such as antibodies), refer to molecules that are separated or purified or substantially removed from their natural environment, e.g., isolated or purified from the human or animal body (if they are indeed naturally produced), or molecules produced by technical processes, i.e., including recombinantly and synthetically produced molecules.

[0256] Thus, when used to describe protein or polypeptide molecules (such as CDR 1, 2, and 3 of the light chain, CDR 1, 2, and 3 of the heavy chain, light-chain variable region, heavy-chain variable region, and binding protein or antibody of the present invention), the terms "isolated" or "purified" generally refer to a protein that is substantially free of cellular material from its source or other proteins. In some embodiments, particularly when the protein will be produced by recombinant techniques and administered to a human or animal, such isolated or purified protein is substantially free of culture medium; or when produced by chemical synthesis, is substantially free of chemical precursors or other chemicals.

[0257] It should be noted that in certain embodiments, the antibodies and the like of the present invention are not naturally occurring and are artificial constructs in the sense that they do not correspond to naturally occurring molecules. For example, preferred antibodies may be engineered or recombinantly produced, or induced experimentally in animal species, such as by immunization. In other words, in the embodiments, the antibodies and the like of the present invention are non-native or non-naturally produced.

[0258] Those skilled in the art will understand that the proteins and polypeptides of the present invention (such as heavy and light chain CDRs, heavy and light chain variable regions, antibodies and antibody fragments) can be prepared by a variety of ways known and described in the art, but are most preferably prepared by recombinant methods.

[0259] The nucleic acid fragments encoding the heavy chain and / or light chain regions of the antibodies of the present invention, as appropriate, can be derived or produced by any suitable method, such as by cloning or synthesis.

[0260] Once the nucleic acid fragments encoding the heavy chain and / or light chain regions of the antibodies of the present invention are obtained, these fragments can be further manipulated by standard recombinant DNA techniques. Generally, or as part of this further manipulation procedure, the nucleic acid fragments encoding the antibody molecules of the present invention are usually integrated into one or more suitable expression vectors to facilitate the production or manipulation of the antibodies of the present invention.

[0261] Possible expression vectors include, but are not limited to, cosmids, plasmids or modified viruses (such as replication-defective retroviruses, adenoviruses and adeno-associated viruses), provided that the vector is compatible with the host cell used. Transposons can also be used. The expression vector "is suitable for transformation of the host cell", meaning that the expression vector contains the nucleic acid molecule of the present invention and regulatory sequences selected based on the host cell chosen for expression, and the regulatory sequences are operatively linked to the nucleic acid molecule. "Operatively linked" is intended to mean that the nucleic acid is linked to the regulatory sequences in a manner that permits its expression.

[0262] Accordingly, the present invention contemplates an expression vector (such as a recombinant expression vector) that contains or comprises the nucleic acid molecule of the present invention or a fragment thereof, and the regulatory sequences necessary for transcription and translation of the protein sequence encoded by the nucleic acid molecule.

[0263] An expression vector can be introduced into a host cell to produce a transformed host cell. The terms "transformed with", "transfected with", "transformation" and "transfection" are intended to cover the introduction of a nucleic acid (e.g., a vector) into a cell by a variety of possible techniques known in the art. Methods suitable for the transformation and transfection of host cells can be found in Sambrook et al. (1989) (Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989) and other laboratory textbooks.

[0264] As is well known to those skilled in the art, suitable host cells include a variety of eukaryotic host cells and prokaryotic cells. For example, the proteins of the present invention can be expressed in yeast cells or mammalian cells (such as HEK or CHO cells). In addition, if applicable, the proteins of the present invention can also be expressed in prokaryotic cells (such as Escherichia coli (E. coli)). Cell-free expression systems can also be used.

[0265] The proteins of the present invention can also be prepared by chemical synthesis using techniques well known in protein chemistry, such as solid-phase synthesis.

[0266] On the other hand, there is provided an expression construct, expression vector or expression system (such as a virus, bacterium or other expression construct, vector or system), for example, one or more expression constructs or vectors containing one or more nucleic acid fragments, segments or molecules of the present invention. Preferably, the expression construct, vector or system is recombinant. Preferably, the construct, vector or system further comprises regulatory sequences necessary for the transcription and translation of the protein sequence encoded by the nucleic acid molecule of the present invention.

[0267] On the other hand, there is provided a host cell (such as a mammalian, bacterial or yeast host cell) or virus, for example, one or more host cells or viruses containing one or more expression constructs or vectors of the present invention. There are also provided host cells (such as mammalian, bacterial or yeast host cells) or viruses, for example, one or more host cells or viruses containing one or more nucleic acid molecules of the present invention. Host cells (such as mammalian host cells, bacterial host cells or yeast host cells) or viruses expressing the antibodies (or binding proteins) of the present invention constitute another aspect.

[0268] Another aspect of the present invention provides a method for producing (or manufacturing) the antibody (or binding protein) of the present invention, the method comprising the step of culturing the host cell of the present invention. The preferred method comprises the following steps: (i) culturing a host cell comprising one or more expression vectors or one or more nucleic acid sequences of the present invention under conditions suitable for expressing an antibody or binding protein; and optionally (ii) isolating or obtaining the antibody or binding protein from the host cell or the growth medium / supernatant. Such production (or manufacturing) methods may also include the steps of purifying the antibody or protein product and / or formulating the antibody or product into a composition (such as a pharmaceutical composition), the composition comprising at least one additional component, such as a pharmaceutically acceptable carrier, excipient or diluent.

[0269] In some embodiments of the present invention, when the antibody or binding protein is composed of multiple polypeptide chains (such as in Fab form, monovalent scFv-Fc fusion protein), it is preferred that all polypeptides are expressed in the host cell, and these polypeptides may be from the same or different expression vectors, so that the complete protein (such as the antibody protein of the present invention) can be assembled in the host cell and isolated or purified therefrom.

[0270] On the other hand, the present invention provides a method for binding CD47, comprising contacting a composition comprising CD47 with the antibody or binding protein of the present invention.

[0271] On the other hand, the present invention provides a method for detecting CD47, comprising contacting a composition suspected of containing CD47 with the antibody or binding protein of the present invention under effective conditions allowing the formation of a CD47 / antibody complex, and detecting the formed complex.

[0272] A therapeutic (or diagnostic) method is to use an antibody capable of targeting a specific antigen expressed on cancer cells, which antigen is not expressed or is expressed at a low level on normal cells. Such target antigens exemplified by CD47 can be utilized, and the antibody can be used to specifically kill tumor cells carrying the antigen through various mechanisms, including by delivering immunolabeled or radiolabeled conjugates, which can specifically kill target cells when delivered to cells carrying the antigen. Such targeting can also be used for diagnosis.

[0273] Accordingly, the present invention also provides a series of conjugated antibodies and binding proteins (immunoconjugates), wherein the anti-CD47 antibody (or binding protein) of the present invention is effectively linked to at least one other therapeutic or diagnostic agent. The term "immunoconjugate" is widely used to define the effective binding of an antibody (or binding protein) to another active agent (such as a therapeutic or diagnostic agent), and does not refer only to a specific type of effective binding mode, and is particularly not limited to chemical "conjugation". Fusion proteins, such as recombinant fusion proteins, are particularly contemplated. As long as the delivery or targeting agent (the anti-CD47 component) can bind to the target and the therapeutic or diagnostic agent remains sufficiently functional after delivery, the mode of linkage is applicable.

[0274] For example, in some preferred embodiments, the antibody (or binding protein) of the present invention forms part of an immunotoxin, or (e.g., therapeutically) is used as part of an immunotoxin, wherein the antibody itself effectively binds or combines with a toxic agent (such as a chemotherapeutic agent, a toxin, or a radioactive substance such as a radioactive tracer, e.g., for radioimmunotherapy). Effective linkage includes all direct and indirect linkage forms described herein and known in the art.

[0275] Suitable chemotherapeutic agents or toxins are well-known and described in the art, and for example, cytotoxic proteins derived from bacteria or plants can be used. The toxin should be capable of killing the target cells after being taken up by the cells. Accordingly, a preferred immunoconjugate of the present invention is an immunotoxin (sometimes also referred to as an antibody-drug conjugate, ADC) comprising the antibody of the present invention linked or otherwise conjugated to a toxin. In a preferred immunoconjugate, an active ingredient such as a radionuclide, a toxin (such as diphtheria toxin), or other cell inhibitors can be bound (conjugated) or otherwise linked to the corresponding antibody.

[0276] Immunoconjugates involving conjugation to RNA molecules (such as siRNA) or DNA molecules can also be used.

[0277] In some embodiments, the antibody of the present invention is used in an "unconjugated" "naked" form (e.g., therapeutically).

[0278] In another aspect, there is provided a method for diagnosing or imaging a subject, comprising administering to the subject a suitable amount of the antibody or binding protein of the present invention as defined herein, and detecting the presence and / or amount and / or location of the antibody or binding protein of the present invention in the subject.

[0279] Suitable diseases that can be imaged or diagnosed according to the present invention are cancers, such as cancers described elsewhere herein with respect to disease treatment.

[0280] In one embodiment, the present invention provides a method for diagnosing mammalian cancer, comprising the following steps:

[0281] (a) Contacting a test sample taken from the mammal with one or more antibodies (or binding proteins) of the present invention.

[0282] In another embodiment, the present invention provides a method for diagnosing cancer in a mammal, comprising the steps of:

[0283] (a) Contacting a test sample taken from the mammal with one or more antibodies (or binding proteins) of the present invention;

[0284] (b) Detecting the presence and / or amount and / or location of antibody-antigen complexes in the test sample; and, optionally

[0285] (c) Comparing the presence and / or amount of antibody-antigen complexes in the test sample with a control.

[0286] In the above method, the contacting step is carried out under conditions permitting the formation of antibody-antigen complexes. Those skilled in the art can readily determine suitable conditions.

[0287] In the above method, any suitable test sample can be employed, such as biopsy cells, tissues or organs suspected of being affected by a disease, or tissue sections.

[0288] In certain of the above methods, the presence of any amount of antibody-antigen complexes in the test sample indicates the presence of the disease. Preferably, for a positive diagnosis, the amount of antibody-antigen complexes in the test sample should be more (preferably significantly more) than the amount detected in a suitable control sample. More preferably, the above "significantly more" level is statistically significant, preferably with a p-value < 0.05. Suitable methods for determining statistical significance are well-known and documented in the art, and any one of them can be employed.

[0289] Those skilled in the art can readily select a suitable control sample. For example, in diagnosing a specific disease, a suitable control can be a sample from a subject not suffering from the disease. Suitable control "values" can also be readily determined without running a control "sample" each time a test is performed, for example by referring to the range of normal subjects known in the art.

[0290] For diagnostic or imaging applications, the antibodies (or binding proteins) of the present invention can be labeled with detectable markers, including: radioisotopes, such as 3 H, 14 C, 32 P, 35 S, 123 I, 125 I, 131I; radioactive emitters (such as α, β or γ emitters); fluorescent (fluorophore) or chemiluminescent (chromophore) compounds such as fluorescein isothiocyanate, rhodamine, fluorescein or europium; enzymes such as alkaline phosphatase, β-galactosidase or horseradish peroxidase; imaging agents; metal ions; or chemical moieties such as biotin, which can be detected by binding to a specific detectable counterpart (such as labeled avidin / streptavidin). Methods for attaching labels to binding proteins (such as antibodies) are known in the art. Such detectable labels can be used to detect the presence, amount or location of a binding protein-antigen complex in a test sample.

[0291] Preferred detectable labels for in vivo detection include: X-ray detectable compounds such as bismuth(III), gold(III), lanthanum(III) or lead(II); radioactive ions such as copper 67 , gallium 67 , gallium 68 , indium 111 , indium 113 , iodine 123 , iodine 125 , iodine 131 , mercury 197 , mercury 203 , rhenium 186 , rhenium 188 , rubidium 97 , rubidium 103 , technetium 99 m or yttrium 9 0; nuclear magnetic resonance spin isotopes such as cobalt(II), copper(II), chromium(III), dysprosium(III), erbium(III), gadolinium(III), holmium(III), iron(II), iron(III), manganese(II), neodymium(III), nickel(II), samarium(III), terbium(III), vanadium(II) or ytterbium(III); or rhodamine or fluorescein.

[0292] The present invention also includes a diagnostic agent or imaging agent comprising an antibody of the present invention linked to a label or detectable label capable of directly or indirectly generating a detectable signal. Suitable labels or detectable labels are as described elsewhere herein.

[0293] In one embodiment, the cancer diagnostic method is an in vitro method.

[0294] In one embodiment, the cancer diagnostic method is an in vivo method.

[0295] In other words, the present invention provides a method for screening a subject for cancer.

[0296] Compositions comprising at least one antibody (or binding protein) or immunoconjugate of the invention, or at least one nucleic acid molecule or expression vector of the invention, or at least one host cell of the invention, constitute other aspects of the invention. Preparations (compositions) comprising one or more antibodies etc. of the invention, optionally mixed with suitable diluents, carriers or excipients, constitute preferred embodiments of the invention. Such preparations can be used for medical purposes, and thus the compositions of the invention are preferably pharmaceutically acceptable or acceptable for administration to humans or non-human animals (especially humans). Suitable diluents, excipients and carriers are known to those skilled in the art.

[0297] Any suitable mode of administration can be employed. The compositions according to the invention can be suitable, for example, for oral, nasal, parenteral (such as intravenous, intraperitoneal, subcutaneous, intradermal, intramuscular), topical or rectal administration, or can be provided in a form suitable for mucosal delivery, and can be administered by any of the above-mentioned modes or any other suitable mode of administration. In a preferred embodiment, the compositions of the invention are provided in a form suitable for intravenous administration. In some embodiments, the compositions of the invention are provided in a form suitable for intraperitoneal (i.p.) administration. In some embodiments, the compositions of the invention are provided in a form suitable for direct injection into the tumor (intratumoral).

[0298] The active compounds (such as the antibodies of the invention) as defined herein can be provided in conventional pharmaceutical dosage forms, including tablets, coated tablets, nasal sprays, solutions, emulsions, liposomes, exosomes, powders, capsules or sustained-release formulations. The preparation of these dosage forms can be carried out using conventional pharmaceutical excipients and standard production methods. In addition, nucleic acids or nucleic acid-based carriers (such as mRNA-based carriers) or virus-based carriers can be used for the administration of the active compounds of the invention, for example, by encoding the antibodies or binding proteins of the invention.

[0299] Injection solutions can be prepared in a conventional manner, for example, by adding preservatives (such as parabens) or stabilizers (such as EDTA). Subsequently, the solution can be filled into injection bottles or ampoules.

[0300] The pharmaceutical compositions (preparations) of the invention are preferably administered by the parenteral route. Intravenous administration is the preferred mode. In some embodiments, the mode of administration is intraperitoneal (i.p.) administration. In some embodiments, administration is by intratumoral injection. Parenteral administration can be carried out by subcutaneous, intramuscular, intraperitoneal or intravenous injection using a syringe. Alternatively, parenteral administration can be carried out by an infusion pump. Another option is a powder or liquid formulation for administering the antibody in the form of a nasal or pulmonary spray. Yet another option is that the antibodies of the invention can also be administered transdermally (such as by a patch, optionally an iontophoretic patch) or transmucosally (such as buccal mucosal administration).

[0301] Those skilled in the art can determine suitable dosage units.

[0302] Another aspect of the invention provides an anti-CD47 antibody (or binding protein) or immunoconjugate as defined herein for therapeutic use, particularly for the treatment or prevention of cancer. In other embodiments, the nucleic acid molecule, expression vector, host cell or virus of the invention can also be used in the treatment methods described herein.

[0303] According to the invention, the antibody can target CD47-positive cells, such as tumour cells.

[0304] In one embodiment, solid tumours are treated.

[0305] In one embodiment, haematological or blood cancers are treated.

[0306] In some embodiments, tumours or cancers characterized by the expression or overexpression of CD47 (e.g., on their surface) are treated.

[0307] Accordingly, another aspect of the invention provides an anti-CD47 antibody (or binding protein) as defined herein for the treatment or prevention of cancers or tumours characterized by (or associated with) CD47 expression or overexpression, such as cancers or tumours characterized by undesired, inappropriate, abnormal, increased or overexpressed CD47 expression.

[0308] In some embodiments, the cancer is characterized by (or associated with) CD47 signalling (e.g., abnormal, inappropriate or undesired CD47 signalling).

[0309] In some embodiments, the antibody (or binding protein) of the invention can induce direct killing of cancer or tumour cells expressing CD47.

[0310] Preferred cancers to be treated according to the present invention include: lung cancer, such as non-small cell lung cancer (NSCLC, such as squamous non-small cell lung cancer); small cell lung cancer (e.g., extensive stage small cell lung cancer); melanoma (e.g., metastatic melanoma, such as BRAF-negative metastatic melanoma or multiple melanomas); lymphoma (e.g., acute T cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma (such as B cell or T cell non-Hodgkin lymphoma or Burkitt lymphoma) or chronic lymphocytic lymphoma); leukemia (e.g., acute myeloid leukemia, acute lymphocytic leukemia or myelodysplastic syndrome); renal cell carcinoma (RCC, such as clear cell renal cell carcinoma); colorectal cancer; bladder urothelial carcinoma; urethral cancer; head and neck cancer (e.g., recurrent or metastatic head and neck squamous cell carcinoma); breast cancer (e.g., metastatic HER-2 negative breast cancer); advanced liver cancer; brain cancer (e.g., glioblastoma or astrocytoma); gastric cancer; esophageal cancer; pancreatic cancer; adenocarcinoma; mesothelioma; peritoneal cancer; fallopian tube cancer; cervical cancer; ovarian cancer; sarcoma (e.g., metastatic sarcoma); hematological malignancies; thyroid cancer; salivary gland cancer; laryngeal cancer; neuroblastoma; retinoblastoma; and testicular cancer.

[0311] Without wishing to be bound by theory, it is believed that the antibodies of the present invention may be superior to prior art antibodies in terms of therapeutic efficacy, particularly in terms of speed of action and concentration required (compared to prior art antibodies, the antibodies of the present invention have been shown to rapidly induce significant direct tumor cell killing at low concentrations / doses). In some embodiments, the antibodies of the present invention have the ability not to induce red blood cell agglutination.

[0312] Without wishing to be bound by theory, it is believed that the antibodies of the present invention may inhibit tumor growth by blocking the cell cycle progression. Thus, in some embodiments, the antibodies (or binding proteins) of the present invention are capable of causing cell growth arrest.

[0313] The efficacy of the antibody has been demonstrated in a relevant cancer model of the present invention (a xenograft model of acute lymphocytic leukemia). In this model, a single administration of the CO201-scFv at a concentration / dose of 1.33 nM significantly delayed tumor progression. Thus, the antibodies (and binding proteins) of the present invention represent a significant advance in the field of anti-CD47 therapeutic agents. Without wishing to be bound by theory, it is believed that the antibodies of the present invention may inhibit tumor growth by blocking the cell cycle progression. Thus, in some embodiments, the antibodies (or binding proteins) of the present invention are capable of causing cell growth arrest. In some embodiments, the antibodies (or binding proteins) of the present invention are capable of delaying cancer progression.

[0314] In the treatment methods and uses of the present invention, the binding protein or antibody is administered to a subject in need of treatment (such as an animal, such as a human or non-human mammal) in a pharmaceutically, therapeutically or physiologically effective amount. Accordingly, the methods and uses may include an additional step of identifying a subject in need of treatment. A person skilled in the art can readily determine the appropriate and effective concentration / dose to be administered. Based on the animal models currently in use, exemplary concentrations / doses can be from 0.05, 0.1, 1 or 5 nM to 10, 20, 30, 40 or 50 nM, for example, being or about 0.05, 0.1, 1, 2, 4, 6, 10, 20, 30 or 40 nM. Treatment of a disease or disorder according to the present invention (such as treatment of an existing disease) includes curing the disease or disorder, or any alleviation or mitigation of the disease, such as reducing the severity of the disease or disease symptoms.

[0315] The treatment methods and uses of the present invention are applicable both to the prevention of diseases and to the active treatment of diseases (such as treatment of existing diseases). Accordingly, the present invention also encompasses prophylactic treatment. Based on this, in the methods and uses of the present invention, "treatment" also includes prevention or prophylaxis where appropriate.

[0316] Such prophylactic (or protective) aspects can be conveniently carried out in healthy, normal or at-risk subjects and can include complete prevention and significant prevention. Similarly, significant prevention can include cases where the severity of the disease or symptoms is reduced (such as measurably or significantly reduced) compared to the severity of the disease or symptoms expected without treatment.

[0317] Accordingly, subjects suitable for treatment according to the present invention include any animal that may develop cancer, and more specifically cancers that contain tumor cells expressing CD47.

[0318] Accordingly, the in vivo methods and uses described herein are generally carried out in mammals. Any mammal can be treated, such as humans and any livestock, domestic animals or laboratory animals. Specific examples include mice, rats, pigs, cats, dogs, sheep, rabbits, horses, cattle and monkeys. However, preferably, the mammal is a human.

[0319] Accordingly, the terms "animal", "patient" or "subject" as used herein include any mammal, such as humans and any livestock, pets or laboratory animals. Specific examples include mice, rats, pigs, cats, dogs, sheep, rabbits, horses, cattle and monkeys. However, preferably, the animal, patient or subject is a human. Accordingly, the subject or patient being treated according to the present invention is preferably a human.

[0320] In another embodiment, the subject is a subject having, suspected of having (or developing), or likely to have (or develop) a relevant disease or disorder as described above.

[0321] In other words, the present invention provides a method for treating or preventing cancer, which method comprises administering to a patient in need thereof a therapeutically effective amount of an antibody (or binding protein) of the present invention as defined herein. Each of the embodiments of the therapeutic use of the present invention described herein is, mutatis mutandis, applicable to this aspect.

[0322] The therapeutically effective amount will be determined based on clinical evaluation and can be readily monitored. Preferred cancer treatment regimens are as described in other parts of this document.

[0323] In yet other words, the present invention provides the use of an antibody (or binding protein) of the present invention as defined herein in the preparation of a medicament for treatment. Preferred treatment regimens are cancer treatment or prevention as described in other parts of this document.

[0324] Each of the embodiments of the therapeutic use of the present invention described herein is, mutatis mutandis, applicable to this aspect.

[0325] In some embodiments, the antibodies (or binding proteins) of the present invention can be used in monotherapy. In other embodiments, they can be used in combination with other standard cancer therapeutics.

[0326] The present invention also includes kits that contain one or more antibodies or compositions of the present invention, or one or more nucleic acid molecules encoding the antibodies of the present invention; or one or more expression vectors containing the nucleic acid sequences of the present invention; or one or more host cells or viruses containing the expression vectors or nucleic acid sequences of the present invention. Preferably, the kits are for the methods and uses described herein, such as the therapeutic, diagnostic or imaging methods described herein. Preferably, the kits contain instructions for use of the kit components. Preferably, the kits are for diagnosing, imaging, or treating / preventing diseases or disorders as described in other parts of this document, and optionally contain instructions for diagnosing, imaging, treating or preventing such diseases or disorders using the kit components. Equivalent embodiments of the binding proteins of the present invention are also provided.

[0327] The antibodies (or binding proteins) of the present invention as defined herein can also be used as molecular tools for in vitro or in vivo applications and detection, such as binding assays or diagnostic assays. Since the antibody (and binding protein) has an antigen-binding site that binds CD47, it can be a member of a specific binding pair, and these molecules can be used in any detection that requires a specific CD47 binding pair member.

[0328] Accordingly, other aspects of the present invention also provide reagents containing the antibodies (or binding proteins) of the present invention as defined herein, and the use of such antibodies (or binding proteins) as molecular tools, for example for in vitro or in vivo detection, particularly for detecting CD47 (such as in a target sample).

[0329] As used herein, the term "reduce" or "decrease" (or equivalent terms) includes any measurable decrease or reduction compared to an appropriate control. A person skilled in the art can readily determine an appropriate control, which may include untreated or placebo-treated subjects, healthy subjects, samples or assays without the antibody (or binding protein) of the present invention, or samples or assays containing a control antibody (or binding protein), such as samples or assays containing an isotype control antibody (or binding protein). Preferably, the reduction or decrease will be significant, e.g., clinically or statistically significant.

[0330] As used herein, the term "increase" (or equivalent terms) includes any measurable elevation or enhancement compared to an appropriate control. A person skilled in the art can readily determine an appropriate control, which may include untreated or placebo-treated subjects, healthy subjects, samples or assays without the antibody (or binding protein) of the present invention, or samples or assays containing a control antibody (or binding protein), such as samples or assays containing an isotype control antibody (or binding protein). Preferably, the increase will be significant, e.g., clinically or statistically significant.

[0331] Preferably, such an increase (and other increases, improvements, or positive effects described herein) or such a reduction (and other reductions, decreases, or negative effects described herein) is a measurable increase, reduction, etc. (as appropriate); more preferably a significant increase, reduction, etc.; most preferably an increase with clinical or statistical significance, e.g., a probability value ≤ 0.05 or < 0.05 compared to an appropriate control level or value (such as compared to untreated or placebo-treated subjects, or to healthy / normal subjects, or to the same subject before treatment).

[0332] Methods for determining the statistical significance of differences between groups of subjects tested or differences in the levels of specific parameters are well known in the art and are documented in the literature. For example, when a statistical comparison is made using an appropriate significance test (such as the Student t-test, Mann-Whitney U rank sum test, chi-square test, Fisher's exact test, one-way analysis of variance, or two-way analysis of variance), a reduction or increase in the level of a specific parameter or a difference between groups of subjects tested is generally considered to be statistically significant if the probability value ≤ 0.05 or < 0.05.

[0333] Amino acid sequences disclosed herein and their sequence identifiers (SEQ ID NO)

[0334] All amino acid sequences described herein are listed from the N-terminus to the C-terminus in accordance with the conventions of the art.

[0335]

[0336]

[0337] Table B: Humanized Antibodies

[0338] huCO-mAb Heavy chain variable sequence (SEQ ID NO:) Light chain variable sequence (SEQ ID NO:) CO201 22 27 CO202 23 27 CO203 24 27 CO204 25 27 CO205 22 28 CO206 23 28 CO207 24 28 CO208 25 28 CO209 22 29 CO210 23 29 CO211 24 29 CO212 25 29 CO213 26 27

[0339] Humanized variable heavy chain domain (VH)

[0340] huCO-1VH-v1

[0341] QVQLVQSGSELKKPGASVKVSCKASGYTFT NFGMH WVRQAPGQGLEWMG WINTYT

[0342] GEPTYTDDFKG RFVFSLDTSVSTAYLQISSLKAEDTAVYYCAR GDYRYGDS WGQGTTVTVSS(SEQ IDNO:22)

[0343] huCO-1VH-v2

[0344] QVQLVQSGSELKKPGASVKVSCKASGYTFT NFGMH WVRQAPGQGLEWMG WINTYT

[0345] GEPTYTDDFKG RFVFSLDTSVSTAYLQISSLKAEDTAVYYCTR GDYRYGDS WGQGTTVTVSS(SEQ IDNO:23)

[0346] huCO-1VH-v3

[0347] QVQLVQSGSELKKPGASVKVSCKASGYTFT NFGMH WVRQAPGQGLKWMG WINTYT

[0348] GEPTYTDDFKG RFVFSLDTSVSTAYLQISSLKAEDTAVYYCTR GDYRYGDS WGQGTTVTVSS(SEQ IDNO:24)

[0349] huCO-1VH-v4

[0350] QVQLVQSGSELKKPGASVKVSCKASGYTFT NFGMH WVRQAPGKGLKWMG WINTYT

[0351] GEPTYTDDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCTR GDYRYGDS WGQGTTVTVSS(SEQ IDNO:25)

[0352] huCO-1VH-v5

[0353] QVQLVQSGSELKKPGASVKVSCKASGYTFT NFGMH WVRQAPGQGLKWMG WINTYT

[0354] GEPTYTDDFKG RFVFSLDTSVSTAYLQISSLKAEDTAVYYCAR GDYRYGDS WGQGTTVTVSS(SEQ IDNO:26)

[0355] (CDR underlined)

[0356] Humanized variable light chain domain (VL)

[0357] huCO-1VL-v1

[0358] DIVMTQTPLSLSVTPGQPASISC RSSQSLVHSNGKTYLH WYLQKPGQPPQLLIY RVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC SQSTHVPFT FGQGTKLEIK(SEQ ID NO:27)

[0359] huCO-1VL-v2

[0360] DIVMTQTPLSLSVTPGQPASISC RSSQSLVHSNGKTYLH WYLQKPGQSPQLLIY RVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC SQSTHVPFT FGQGTKLEIK(SEQ ID NO:28)

[0361] huCO-1VL-v3

[0362] DIVMTQTPLSLSVTPGQPASISC RSSQSLVHSNGKTYLH WYLQKPGQSPKLLIY RVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC SQSTHVPFT FGQGTKLEIK(SEQ ID NO:29)

[0363] (CDR underlined)

[0364] IgG4 CH2-CH3

[0365] PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA

[0366] KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE

[0367] PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS

[0368] FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:30)

[0369] IgG4-knob

[0370] SKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY

[0371] VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT

[0372] ISKAKGQPREPQVYTLPPSQEEMTKNQVSL W CLVKGFYPSDIAVEWESNGQPENNYK

[0373] TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:31)

[0374] (knob mutation underlined)

[0375] IgG4–hole

[0376] SKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY

[0377] VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT

[0378] ISKAKGQPREPQVYTLPPSQEEMTKNQVSL S C A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL V SRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:32)

[0379] (hole mutation is underlined)

[0380] hinge region

[0381] SKYGPPCPSC(SEQ ID NO:33)

[0382] scFv-Fc(mono)CO201 Fc knob

[0383] QVQLVQSGSELKKPGASVKVSCKASGYTFTNFGMHWVRQAPGQGLEWMGWINTYT

[0384] GEPTYTDDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARGDYRYGDSWGQGTT

[0385] VTVSSGGGGSGGGGSGGGGSDIVMTQTPLSLSVTPGQPASISCRSSQSLVHSNGKTYLH

[0386] WYLQKPGQPPQLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQST

[0387] HVPFTFGQGTKLEIKSKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV

[0388] DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

[0389] KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL WCLVKGFYPSDIA

[0390] VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH

[0391] YTQKSLSLSLGK(SEQ ID NO:34)

[0392] (The knob mutations are underlined)

[0393] scFv-Fc(mono)CO201 Fc hole

[0394] SKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY

[0395] VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT

[0396] ISKAKGQPREPQVYTLPPSQEEMTKNQVSL S C A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL V SRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:35)

[0397] (The hole mutations are underlined)

[0398] Alternative hinge region

[0399] SKYGPPCPPC(SEQ ID NO:36)

[0400] The present invention will be further illustrated by the following non-limiting examples with reference to the accompanying drawings:

[0401] Figure 1: Even at high concentrations, CO-1scFv does not cause agglutination. (Upper panel) Red blood cells (2% v / v in PBS) were co-incubated with CO-1scFv (scFv), anti-CD47 (CO-1.1, CO-1.4, CC2C6, B6H12, MABL-1 or 2D3), or isotype controls (human IgG or MOPC-21) at the indicated concentration ranges of 0.0005 - 1 μg / ml. (Lower panel) Red blood cells (2% v / v in PBS) were co-incubated with CO-1scFv (scFv) at concentration ranges of 0.005 - 10 μg / ml. (Upper and lower panels) Small dot-like circles indicate no agglutination, while a diffuse foggy pattern indicates agglutination. Figure 2 : Binding of CO-1scFv to Jurkat cells. Jurkat cells (2×10 5 cells) were co-incubated with increasing concentrations (range: 0.1 ng / ml - 100 μg / ml) of CO-1scFv or human IgG isotype control, and then co-incubated with 1 μg / ml of His6-FITC. Binding was determined by measuring the percentage of FITC-positive cells. Each data point represents the mean fluorescence intensity (MFI) ± standard deviation (S.D.) from two replicate experiments. The EC 50 value of CO-1scFv = 0.05 ng / ml.

[0402] Figure 3 : CO-1scFv induces potent programmed cell death in cell lines derived from hematological malignancies. Jurkat (A), MOLT-4 (B), or CCRF-CEM (C) cells were treated with CO-1, CO-1scFv, or human IgG isotype control at the indicated time points and concentrations, and then stained with Annexin V and 7-AAD. Data represent the total number of cells undergoing early (Annexin V+7-AAD-) and late (Annexin V+7-AAD+) PCD. (A and C) n = 1; (B) Data represent the mean ± standard deviation from two independent experiments.

[0403] Figure 4 : Surface mapping of the CD47 residue epitope bound by the CO-1 antibody In the crystal structure of the CD47 protein, residues Q19, N45, T120, R121, and E122 were labeled as specific residues forming the epitope bound by the CO-1 antibody (red-labeled (R)). Figure 5 : Binding of huCO-scFv to Jurkat cells. Jurkat cells were co-incubated with increasing concentrations of FITC-conjugated huCO-scFv. FITC fluorescence intensity was analyzed by flow cytometry.

[0404] Figure 6:Agglutination and binding of huCO-scFv to red blood cells (RBCs). (A) 2% RBCs (v / v in PBS) were incubated with increasing concentrations of the indicated antibodies or fragments for 30 minutes. Small dotted circles indicate no agglutination, while a diffuse foggy pattern indicates agglutination. (B) 2% red blood cells (v / v in PBS) were incubated with increasing concentrations of FITC-conjugated CO201-scFv and analyzed by flow cytometry. (C and D) B cells were isolated from the buffy coat of healthy human donors using CD19 magnetic beads and stimulated with 1 μg / ml CpG and RP105 for 24 hours. (C) Binding was detected by flow cytometry after incubation of B cells with increasing concentrations of the indicated antibodies or fragments. (D) B cells were incubated with the indicated antibodies or fragments at the indicated concentrations (μg / ml) for 3 hours, followed by Annexin V and 7-AAD staining. % PCD = proportion of Annexin V-positive cells.

[0405] Figure 7 :Induction of PCD by CO scFv fragments. Jurkat cells were treated with 0.1 or 1 μg / ml of the indicated fragments or CO-1 for 3 hours, followed by Annexin V and 7-AAD staining. % PCD = proportion of Annexin V-positive cells. Data are presented as mean ± standard error of the mean (SEM), n = 2.

[0406] Figure 8 :Agglutination and binding of huCO201-scFv-Fc-mono to red blood cells (RBCs). (A) 2% RBCs (v / v in PBS) were incubated with increasing concentrations of the indicated antibodies or fragments for 30 minutes. Small dotted circles indicate no agglutination, while a diffuse foggy pattern indicates agglutination. (B) 2% red blood cells (v / v in PBS) from three donors were incubated with increasing concentrations of FITC-conjugated CO201-scFv-mono and analyzed by flow cytometry.

[0407] Figure 9 :Induction of phagocytosis by huCO-scFv-Fc-mono. Jurkat cells (5×10 5 cells / ml) were co-cultured with RAW264 cells and treated with the indicated antibody for 2 hours. % phagocytosed cancer cells = % Jurkat-CFSE + RAW-DiO + cell proportion. Data are from one experiment.

[0408] Figure 10: CO201-scFv delays the progression of BCP-ALL in vivo. NSG mice at 6 - 8 weeks of age were injected intrathecally (IT) with lentivirus-transduced Reh cells. As described in Materials and Methods, the progression of cancer was non-invasively tracked in vivo by luminescence signals emitted by Reh cells. The xenograft model was confirmed to be established on the 8th day after IT injection, and then 1.33 nM of CO201-scFv or human IgG4 isotype control was injected. The therapeutic effect was evaluated on the 11th day after IT injection, and cancer progression was measured on the 14th day. The luciferase activity [photons per second, (p / s)] of xenografts in each treatment group was measured at the indicated time points. Each point represents the average luminescence signal (p / s) of mice in the indicated treatment group, and the vertical bars represent the standard error of the mean of 5 xenograft mice in each treatment group. Example

[0409] Example 1: Functional Characterization of Monovalent Anti-CD47 Antibody

[0410] Materials and Methods

[0411] Reagents and antibodies

[0412] The reagents and antibodies used in this study are listed in Tables 1 and 2, respectively.

[0413] The nucleotide and amino acid sequences of the heavy and light chain variable regions of a preferred CD47 antibody of the present invention are shown in Table A. This antibody is a scFv monomer fragment named CO-1scFv. This antibody is derived from a murine full-length IgG1 kappa antibody named CO-1 (mCO-1) and obtained by hybridoma. The CDRs and framework regions of the light and heavy chains of the scFv fragment of mCO-1 are shown in Table A.

[0414] Cell lines and culture conditions

[0415] The human cancer cell lines used in this project were purchased from the American Type Culture Collection (ATCC). Jurkat (clone E6-1), MOLT-4, and CCRF-CEM cell lines were cultured in RPMI1640 medium (Lonza cat. no. BE-12-702F / 12). All media were supplemented with: 10% (v / v) fetal bovine serum (FBS, BioNordika cat. no. FB-1001 / 500) and 1% (v / v) penicillin / streptomycin (P / S, ThermoFisher cat. no. 15140122). All cell lines were cultured in a humidified atmosphere of 37 °C, 95% air, and 5% CO2.

[0416] Red blood cell (RBC) isolation

[0417] Collect human whole blood into heparin-coated tubes (about 5 ml) and dilute it to a total volume of 50 ml with red blood cell washing buffer (PBS containing 0.05% BSA and 1 mM EDTA). Isolate red blood cells from human whole blood by centrifugation at 1800×g for 10 minutes, resuspend in the washing buffer to a total volume of 50 ml. Repeat the washing 3 times. After the last wash, resuspend the red blood cell pellet in PBS to prepare a 2% (v / v) red blood cell suspension.

[0418] Agglutination assay

[0419] Add CD47 antibody (or control) with increasing concentrations (up to 1 μg / ml) to round-bottom 96-well plates. Subsequently, add 2% (v / v) freshly isolated red blood cell suspension to each well and incubate in a standard cell culture incubator for 30 - 60 minutes, or until the cells settle to the bottom of the well. A diffuse, blurred pattern indicates the presence of agglutination, while a small, dot-like circle indicates no agglutination.

[0420] Flow cytometry

[0421] All flow cytometry analyses were performed on a NovoCyte (Agilent Technologies, Inc.) equipped with 3 lasers (405, 488, 605 nm) and 13 detection channels. Data were analyzed using NovoExpress software (Agilent Technologies, Inc.).

[0422] Antibody binding assay Centrifuge the cancer cell line or red blood cells after washing with PBS and wash with washing buffer (PBS containing 3% (w / v) BSA and 1% (w / v) sodium azide). Subsequently, incubate with Human BD Fc Block for 10 minutes and then transfer to round-bottom 96-well plates (2.5×10 5 cells per well). The cells were then incubated with different concentrations of CO-1 scFv or the appropriate isotype control shown on ice for 1 hour with gentle shaking. All samples were prepared in duplicate. After washing the cells twice, resuspend in cold His staining solution (washing buffer containing 1 μg / ml His6-FITC) and incubate on ice for 30 minutes with gentle shaking. After washing the cells twice, perform flow cytometry analysis. Excitation wavelength for FITC: 488 nm, detection: 530 / 30 nm.

[0423] Annexin V and 7-AAD staining assay

[0424] For programmed cell death (PCD) analysis, dilute the cells to 5×10 5cells / ml were seeded in 24-well plates at 1 ml per well. Subsequently, the cells were incubated with different concentrations of the CO-1scFv fragment or appropriate isotype controls for different durations (30 minutes, 1 hour, 2 hours, 3 hours) in a standard cell culture environment. After incubation, the cells were harvested and stained with Annexin V eFlour TM 450 and 7-AAD. Flow cytometry analysis of the cells was performed immediately. Annexin V + 7-AAD- cells were considered early apoptotic cells (in the early stage of PCD), while late apoptotic cells (in the late stage of PCD) were Annexin V + and 7-AAD + . The excitation wavelength of Annexin V is 405 nm, and the detection wavelength is 445 / 45 nm, while the excitation wavelength of 7-AAD is 488 nm, and the detection wavelength is 675 / 30 nm.

[0425] Statistical analysis

[0426] Data in the figures are presented as the mean of independent experiments, and the details can be found in the figure legends. Error bars represent the standard deviation (S.D.).

[0427] Statistical analysis was performed using GraphPad Prism 9 software (GraphPad Software Inc.) with paired two-tailed Student's t-test. A P-value less than 0.05 was considered statistically significant for differences between groups.

[0428] Curve fitting was performed using four-parameter non-linear regression in GraphPad Prism.

[0429]

[0430] Results

[0431] Evaluation of the effect of CO fragment on red blood cell agglutination

[0432] Since anti-CD47 antibodies such as CC2C6 can induce agglutination of red blood cells, we evaluated the ability of the CO-1scFv fragment and several other CD47 antibodies to induce agglutination in red blood cells from healthy human donors. A diffuse, blurred pattern indicates the presence of agglutination, while small punctate circles indicate no agglutination. As Figure 1 shown, even at a concentration as high as 10 μg / ml, the CO-1scFv fragment did not induce agglutination; while the full-length bivalent CO antibodies used as controls (CO-1.1, which is the full-length IgG1 form of the CO-1 antibody, and CO-1.4, which is the full-length IgG4 form of the CO-1 antibody) and other CD47 antibodies induced agglutination.

[0433] Binding of CO-1scFv to Jurkat cells

[0434] After confirming that CO-1scFv did not induce red blood cell agglutination, we further elucidated the binding characteristics of CO-1scFv in the human T cell leukemia cell line Jurkat. Cells were co-incubated with increasing concentrations of CO-1scFv or isotype control, and then co-incubated with anti-His6 FITC secondary antibody. As Figure 2 shown, CO-1scFv bound to Jurkat cells with high affinity. By performing four-parameter logistic curve fitting analysis using GraphPad software, the EC 50 value of CO-1scFv was measured to be 0.05 ng / ml.

[0435] Induction of PCD by CO-1scFv

[0436] It is known that multiple anti-CD47 antibodies can induce programmed cell death (PCD) in a variety of tumor cells. Therefore, we examined the ability of this fragment to induce PCD by incubating cancer cells with the CO-1scFv fragment at different time points and concentrations, followed by Annexin V and 7-AAD staining. Cells that were positive for Annexin V staining and negative for 7-AAD (Annexin V + 7-AAD-) were considered early apoptotic cells; cells that were positive for both dyes (Annexin V + 7-AAD + ) were considered late apoptotic cells. The total percentage of the cell population undergoing PCD was obtained by summing the early and late apoptotic cells. Jurkat cells treated with CO-1scFv showed a strong PCD-inducing effect 30 minutes after treatment with the fragment ( Figure 3 A), and it persisted 3 hours later ( Figure 3 A). PCD was also observed in MOLT-4 cells ( Figure 3 B) and CCRF-CEM cells ( Figure 3 C). For comparison, the effect of the full-length bivalent CO-1 antibody was shown. The ability of the scFv fragment of CO-1 to induce PCD was both unexpected and advantageous, as the prior art suggests that PCD may require bivalent interactions to occur. This property is particularly beneficial in combination with the HA deletion observed above.

[0437] Example 2: Analysis of the binding affinity of antibodies to recombinant CD47 by surface plasmon resonance (SPR)

[0438] Using the Biacore S200 system, the affinity of CO-1 scFv for CD47 was determined by surface plasmon resonance (SPR) technology. Recombinant biotinylated CD47 was immobilized on an SA streptavidin chip to a level of 100 RU (in 10 mM NaAc 5.0 solution). CO-1 scFv was reacted with recombinant CD47 at gradient concentrations.

[0439] Analysis 1

[0440] Instrument: Biacore S200

[0441] Materials:

[0442] SA chip (streptavidin)

[0443] Recombinant CD47 (recCD47): SinoBiological (Cat. No. 12283-H27H-B); 17 kDa, C-terminal His-tag and AVI-tag, biotinylated

[0444] Antibody fragment: scFv (0.11 mg / ml)

[0445] All proteins were stored at -80 °C.

[0446] Buffer: 20 mM phosphate buffer, pH 7.4 / 2.7 mM KCl / 137 mM NaCl / 0.05% P20 Single cycle step:

[0447] Immobilize recCD47 to 100 RU (dissolved in 10 mM NaAc, pH 5.0)

[0448] Regeneration condition: Treat with 10 mM glycine (pH 1.5) for 60 seconds

[0449] Analyte (antibody fragment) was run in single cycle mode

[0450] Injection order (from low concentration to high concentration): 10 nM / 5 nM / 2.5 nM / 1.25 nM / 0.625 nM / 0.3125 nM / 0.1562 nM / 0.07813 nM / 0.039 nM

[0451] Flow rate: 30 μl / min; Injection time: 120 seconds; Dissociation time: 180 seconds (1800 seconds after the last injection).

[0452] Temperature: 25 °C

[0453] Results

[0454] A 1:1 binding model was used to fit the data. All the binding curves could well fit the 1:1 model. Table 3 lists the measured values of the association rate ka (the second column) and the dissociation rate kd (the third column), respectively. The affinity constant KD was calculated as kd / ka (the fourth and fifth columns) and was 180 pM. Therefore, the scFv fragment showed a high binding affinity for CD47.

[0455] Table 3

[0456] Analyte <![CDATA[k a (1 / Ms)]]> <![CDATA[k d (1 / s)]]> <![CDATA[K D (M)]]> scFv <![CDATA[3.1×10 7 > <![CDATA[5.6×10 -3 > <![CDATA[1.79×10 -10 > 180 pM

[0457] Example 3: Epitope Mapping of CO-1

[0458] The epitope mapping of the IgG form of the CO-1 antibody to human CD47 was completed by DeepTope SAS, France, through deep mutational scanning (DMS), see Sierocki et al., 2021, PLoS Negl Trop Dis., 15(3):e0009231. See also Van Blarcom et al., 2015, JMB, 427:6(B):1513-1534; Medina-Cucurella and Whitehead, 2018, Methods Mol. Biol., 1764:101-121.

[0459] Principle of Epitope Mapping by Deep Mutational Scanning (DMS)

[0460] DMS is a mutagenesis method aimed at making all possible single amino acid substitutions for all selected residues in a given protein sequence. The DMS library is obtained in the form of DNA encoding the target protein. In this library, each DNA strand contains a codon mutated relative to the parental sequence.

[0461] This DMS DNA library is integrated into an expression plasmid specifically designed for expressing recombinant proteins on the yeast surface. Subsequently, the yeast is transformed and induced to express the single mutant proteins on its surface. This new library (referred to as the display library) is screened by flow cytometry using a fluorescent reporter molecule to reveal the expression of the protein (anti-tag fluorescent antibody) and the binding of the protein to its partner (fluorescent partner).

[0462] For epitope mapping, ideally, there are two antibodies with compatible epitopes that can bind to the same antigen together. In this way, each of the above two antibodies serves as a conformational control for the mutant antigen with respect to the other antibody. In fact, a single substitution made on this antigen can have 4 types of effects:

[0463] 1. Loss of affinity for the first antibody while retaining binding to the second antibody: This is a mutation in the epitope of the first antibody.

[0464] 2. Loss of affinity for the second antibody while retaining binding to the first antibody: This is a mutation in the epitope of the second antibody.

[0465] 3. Loss of affinity for both antibodies: This is a so-called "destructive" mutation that affects the conformation of the antigen and thus prevents binding of both antibodies.

[0466] 4. No effect: This mutation is not present in the epitope of one of the two antibodies and does not cause a significant change in the conformation of the antigen.

[0467] After flow cytometry analysis, yeast populations that have lost affinity for the target antibody but retained binding to the second antibody are sorted. The plasmids contained in this yeast population are extracted and sequenced by high-throughput sequencing. Analysis of the sequencing data allows identification of mutations that have affected the binding of the antibody to its target. Thus, this analysis allows identification of the positions on the antigen that are important for binding of the target antibody: i.e., its epitope.

[0468] Materials and Methods

[0469] The antigen used for DMS analysis was human CD47 (123 amino acids expressed from glutamine 19 to glutamate 141), see SEQ ID NO:19. The mutant version of this antigen was expressed in yeast in the form of a DMS DNA library. The first antibody used was CO-1. The second antibody was 2D3 (ThermoFisher, catalog number 14-0478-82), which is another anti-CD47 antibody that has been shown not to compete with CO-1 for binding to CD47. Thus, these are antibody pairs suitable for DMS analysis.

[0470] DMS analysis was performed in 2 regions of CD47: library 1 [amino acids 19 to 80] and library 2 [amino acids 81 to 141]. These 2 libraries were transformed into yeast. Unsorted yeast from these 2 generated libraries were sequenced to verify mutagenesis efficiency. 100% of the expected single mutants in all libraries were sequenced. These 2 DMS libraries were successfully generated and cloned into yeast. Each library contains approximately 1200 single amino acid mutants and 2000 DNA codon mutants. Library members encode each of the 20 appropriate amino acids at each mutation site.

[0471] Results

[0472] A DMS map was generated, see Table 4. Each position that affects the binding of the CO-1 antibody by mutation was classified into three categories:

[0473] High impact: 19 to 14 mutations are prohibited (red / R). Positions classified into this category are considered likely to directly interact with IgG (here CO-1).

[0474] Medium impact: 13 to 7 mutations are prohibited (orange / O).

[0475] Low impact: 2 to 6 mutations are prohibited (yellow / Y).

[0476] The CD47 structure (AF-Q08722-F1, Alphafold) was analyzed to distinguish "structural residues" buried inside the structure and epitope residues exposed to the solvent. Residues marked in gray in Table 4 were classified as "structural residues" and were not considered part of the epitope. Mutations in them caused minor structural changes that were sufficient to result in loss of binding to the target antibody but not sufficient to result in loss of binding to the second (so-called "conformational control" antibody, here 2D3).

[0477] Residues marked with * (corresponding to high-impact -R residues but not structural residues) were considered to form the epitope of CO-1. The epitope is also described in Figure 4 in.

[0478]

[0479] Table 4 - Classification of Mutation Sites with Significant Effects on CO-1 Antibody Binding

[0480] Top row: Numbering of amino acid residues in the CD47 molecule of SEQ ID NO:19.

[0481] Second row: Amino acid residues; gray shading = structural residues.

[0482] Third row: Number of mutations prohibited at this site, ranging from 2 to 19.

[0483] Fourth row: R = red, high impact, 19 to 14 mutations are prohibited; O = orange, medium impact, 13 to 7 mutations are prohibited; Y = yellow, low impact, 2 to 6 mutations are prohibited; W = white, no impact.

[0484] Bottom row: * Indicates residues considered to be part of the CO-1 epitope.

[0485] Example 4: Functional Characterization of Monovalent Humanized Anti-CD47 scFv Fragments

[0486] Materials and Methods

[0487] Reagents and antibodies

[0488] The reagents and antibodies used in this study are listed in Table 5 and Table 2, respectively.

[0489] The nucleotide and amino acid sequences of the heavy and light chain variable regions of a preferred CD47 antibody of the present invention are shown in Table A. The antibodies characterized in this example are exemplary humanized scFv fragments targeting CD47, named CO201-scFv, CO209-scFv, CO213-scFv (collectively referred to as huCO-scFv below). All fragments were constructed using the CDR regions from the murine full-length IgG1 kappa antibody CO-1 (mCO-1), which was derived from a hybridoma (the CDR regions and framework regions of this murine antibody are shown in Table A). The CDR and framework regions of the light and heavy chains of the humanized fragments are as shown in SEQ ID NO: 22 to 26 (heavy chain) and 27 to 29 (light chain) and Table B. CO201-scFv was also engineered with an Fc fragment (IgG4 Fc) and named CO201-scFv-Fc-mono. This fragment has one Fc region but only one scFv fragment (i.e., monovalent), and this scFv fragment is linked to one chain of the Fc. This fragment was conveniently designed using the "knobs into hole" mutation to ensure proper association of the two chains. The sequence of one chain (the "knob" chain) of this construct is as shown in SEQ ID NO: 34, which contains the CO201 scFv fragment - hinge region - CH2 - CH3 and the appropriate "knob" mutation. The sequence of the other chain (i.e., the "hole" chain) of this construct is as shown in SEQ ID NO: 35, which contains the hinge region - CH2 - CH3 fragment and the corresponding "hole" mutation. When these two heavy chain constructs are expressed, the chains will dimerize, and the complex formed by the "knob-hole" dimer will be screened out. This complex contains a monovalent scFv-Fc construct, i.e., an Fc region with a single (monovalent) scFv (i.e., a monovalent scFv-Fc fusion protein). Human IgG4 isotype control was purchased from SinoBiological (Catalog No.: HG4K).

[0490] Cell lines and culture conditions

[0491] Jurkat (clone E6-1) cells were purchased from the American Type Culture Collection (ATCC) and cultured in RPMI 1640 medium supplemented with 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin / streptomycin (PS). The cell density was maintained at 0.4 to 1.6 × 10 6 cells / ml, and placed in a humidified atmosphere of 37 °C, 95% air and 5% CO2.

[0492] Red blood cell (RBC) isolation

[0493] Same as Example 1.

[0494] Isolation of normal B cells

[0495] According to the manufacturer's instructions, B cells were isolated from the buffy coat of healthy human donors using CD19 magnetic beads and DETACHaBEADs. After isolation, the B cells were seeded into 24-well plates and stimulated with 1 μg / ml CpG and RP105 for 24 hours, followed by downstream experiments.

[0496] Agglutination assay

[0497] Increasing concentrations (up to 100 μg / ml) of huCO-scFv (or control) were added to round-bottom 96-well plates. Subsequently, 2% (v / v) freshly isolated red blood cell suspension was added to each well and incubated in a standard cell culture incubator for 30 - 60 minutes, or until the cells settled to the bottom of the well. A diffuse, blurred pattern indicates the presence of agglutination, while a small, dot-like circle indicates no agglutination.

[0498] Flow cytometry

[0499] Same as Example 1.

[0500] Antibody binding assay

[0501] The cancer cell line or red blood cells after washing with DPBS were centrifuged and washed with wash buffer (DPBS containing 3% (w / v) BSA, 1% (w / v) sodium azide), then incubated with Human BD Fc Block for 10 minutes and transferred to round-bottom 96-well plates (2.5×10 5 cells per well). The cells were then incubated with different concentrations of FITC-conjugated huCO-scFv, or the indicated human IgG4 isotype control on ice for 1 hour with gentle shaking. All samples were prepared in duplicate. After washing the cells three times with wash buffer, flow cytometry analysis was performed. Excitation wavelength for FITC: 488 nm, detection: 530 / 30 nm.

[0502] Annexin V and 7-AAD staining assay

[0503] Same as Example 1, but incubated with huCO-scFv or control for 3 hours.

[0504] Statistical analysis

[0505] Data in the figures are presented as the mean of independent experiments, and the specific details can be found in the figure legends. Error bars represent the standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism 9 software (GraphPad Software Inc.) with paired two-tailed Student's t-test. A P-value less than 0.05 was considered statistically significant. Curve fitting was completed using four-parameter nonlinear regression in GraphPad Prism.

[0506]

[0507] Results

[0508] Binding of huCO-scFv to cancer cells and human red blood cells

[0509] The binding ability of huCO-scFv to Jurkat cells was detected by flow cytometry and compared with human IgG4 isotype control. huCO-scFv could specifically and with high affinity bind to Jurkat cells ( Figure 5 ).

[0510] Since anti-CD47 antibodies (such as CC2C6) can induce agglutination of red blood cells, we evaluated the ability of huCO-scFv and other CO antibodies to induce agglutination of red blood cells from healthy donor sources. A diffuse and blurred pattern indicates the presence of agglutination, while a small dot-like circle indicates no agglutination. As Figure 6 shown in A, when the concentration did not exceed 1 μg / ml, these humanized fragments did not induce agglutination, while the fully humanized (CO201, CO209, CO213) and chimeric (CO-1.4) IgG4 molecules did the opposite. Flow-based binding experiments also confirmed this low binding affinity of CO201-scFv ( Figure 6 in B). We also evaluated the binding ability of huCO-scFv to B cells from healthy donor sources ( Figure 6 in C), and at the same time showed that these fragments did not induce PCD in the same cell population ( Figure 6 in D).

[0511] Induction of PCD by huCO-scFv

[0512] After 3 hours with the huCO-scFv fragment, PCD induction was detected by Annexin V and 7-AAD staining. Cells with positive Annexin V staining were considered to have undergone PCD. As Figure 7 shown, after Jurkat cells were treated with 0.1 or 1 μg / ml for 3 hours, approximately 60% of the cells were induced to die.

[0513] Since the huCO-scFv fragment did not induce phagocytosis (possibly due to the lack of the Fc portion, data not shown), we designed a monovalent scFv based on CO201 and conjugated it with Fc. This fragment is hereinafter referred to as CO201-scFv-Fc-mono. We evaluated the ability of this fragment to induce agglutination and bind to red blood cells. CO201-scFv-Fc-mono did not induce agglutination at concentrations up to 100 μg / ml ( Figure 8 A), and had low binding to red blood cells from three different donors ( Figure 8 B).

[0514] Effect of huCO-scFv-Fc-mono on phagocytosis

[0515] Materials and methods

[0516] Phagocytosis assay

[0517] All cell lines were purchased from the American Type Culture Collection (ATCC). The Jurkat cell line (clone E6-1) was cultured in RPMI 1640 medium, and RAW264.7 cells were cultured in DMEM medium. Both media were supplemented with 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin-streptomycin (PS).

[0518] Staining of RAW264.7 macrophages

[0519] RAW264.7 cells were rinsed once with supplemented DMEM and then stained with 40 nM Vybrant TM DiO cell labeling solution diluted in supplemented DMEM for 20 minutes in a standard cell culture incubator (37 °C, humid atmosphere, 95% air, 5% CO2). After staining, the cells were rinsed three times with supplemented DMEM.

[0520] Staining and antibody treatment of Jurkat cells

[0521] Jurkat cells were collected by centrifugation, resuspended in DPBS, and stained with 1 μl / ml CellTrace TM Violet cell proliferation dye for 20 minutes in a standard cell culture incubator. Unbound CFSE was quenched by adding 5 volumes of supplemented RPMI, and then the cells were centrifuged and resuspended in supplemented DMEM. The target cells were added to RAW264.7 tissue culture plates and treated with the indicated antibodies for 2 hours in a standard cell culture incubator, followed by washing twice with PBS containing 1 mM EDTA. Cells were analyzed by flow cytometry, and DiO + CellTrace + cells represent the phagocytosed target cells.

[0522] Result

[0523] To determine the induction of phagocytosis by huCO-scFv-Fc-mono, we co-incubated Jurkat cells with the murine macrophage cell line RAW264.7. After treatment with this fragment, the level of induced phagocytosis was comparable to that of the intact IgG4 molecule CO-1.4( Figure 9 ).

[0524] Analysis of the binding affinity of antibodies to recombinant CD47 by surface plasmon resonance (SPR)

[0525] The binding affinity of huCO-mAbs to CD47 was determined by surface plasmon resonance (SPR) using a Biacore S200 system. Recombinant biotinylated CD47 was immobilized on an SA streptavidin chip to a level of 100 RU (in 10 mM NaAc pH 5.0 solution). huCO-mAbs were reacted with recombinant CD47 at gradient concentrations.

[0526] Analysis

[0527] Instrument

[0528] Biacore S200

[0529] Materials and reagents

[0530] SA chip (streptavidin)

[0531] Recombinant CD47 (recCD47): SinoBiological (Catalog No. 12283-H27H-B); 17 kDa, C-terminal His-tag and AVI-tag, biotinylated

[0532] Antibodies: CO201-scFv (1.27 mg / ml), CO209-scFv (1.03 mg / ml), CO213-scFv (1.18 mg / ml). All antibodies were stored at -80 °C.

[0533] Buffer: 20 mM phosphate buffer, pH 7.4 / 2.7 mM KCl / 137 mM NaCl / 0.05% P20 Single cycle Procedure

[0534] Fix recCD47 to 110 - 120 RU (dissolved in 10 mM NaAc, pH 5.0)

[0535] Regeneration conditions: Treatment with 10 mM glycine (pH 2) for 60 seconds

[0536] The analyte (fragment) runs in a single-cycle mode

[0537] Injection order (from low concentration to high concentration): 10 nM / 5 nM / 2.5 nM / 1.25 nM / 0.625 nM / 0.3125 nM / 0.1562 nM / 0.07813 nM / 0.039 nM

[0538] Injection order (from low concentration to high concentration)

[0539] Flow rate: 30 μl / min; injection time: 120 s; dissociation time: 180 s (1800 s after the last injection).

[0540] Temperature: 25 °C

[0541] Results

[0542] The data was fitted using a 1:1 binding model. All binding curves were well fitted to the 1:1 model. Table 6 shows the obtained values of the association rate ka and the dissociation rate kd. The affinity constant K D According to k d / k a Calculated.

[0543]

[0544] Example 5: In vivo activity of CO-201scFv

[0545] Materials and methods

[0546] CO201-scFv antibody fragment

[0547] As described in Example 4, the humanized single-chain variable fragment (scFv) version of CO201, namely CO-201scFv, was prepared by ATUM.

[0548] Cell culture

[0549] All cell lines were purchased from the American Type Culture Collection (ATCC). The B-cell precursor acute lymphoblastic leukemia (BCP-ALL, hereinafter referred to as ALL) cell line Reh was cultured in RPMI 1640 medium (Lonza) at 2×10 5 to 1×10 6HEK293T cells were cultured at a density of 10 cells / ml. HEK293T cells were used for lentiviral production and subcultured every 2 to 3 days in Dulbecco's modified Eagle's medium (DMEM, ThermoFisher) when they reached 70% to 90% confluence. Both cell culture media were supplemented with 10% (v / v) fetal bovine serum (FBS, ThermoFisher) and 1% (v / v) penicillin / streptomycin (PS, ThermoFisher). Cells were cultured in a humidified atmosphere containing 95% air and 5% CO2. Lentivirus in HEK293T cells Production

[0550] HEK293T cells were transfected with 8.3 mg of each plasmid to produce lentiviral vectors containing genes encoding firefly luciferase and enhanced green fluorescent protein (EGFP), the plasmids being: pMD2.G envelope plasmid, pCMVΔ8.91 packaging plasmid, and pSLIEW transfer plasmid. On the day of transfection, cells were cultured to 70% to 90% confluence. Cell culture medium was changed approximately 1 hour before transfection. Transfection mixtures were prepared using a calcium phosphate transfection kit (Invitrogen) according to the manufacturer's instructions. Cell culture medium was changed 4 hours after transfection. Viral supernatants were collected 2 days later and concentrated overnight at 4°C using LentiX concentrator (Takara Bio Inc.), followed by centrifugation at 1500g for 45 minutes (4°C) to collect the virus. The precipitate was resuspended in a small amount (<1 mL) of cell culture medium and stored at -80°C. Frozen lentiviral stocks were titered on Reh cells using a standard transduction protocol (see below).

[0551] Lentiviral transduction of Reh cells

[0552] Reh cells (5×10 per well) 5 Cells) were inoculated into 48-well plates, and the culture medium contained 4 mg / mL polybrene (Merck Millipore). Lentiviral concentrate was added to the cells and spin-infected by centrifugation at 900g for 50 minutes at 34°C. After spin infection, the plate was transferred to a standard cell culture incubator (37°C, 5% CO2, humidified atmosphere) and cultured for 2 days, followed by repeated washing twice by centrifugation at 300g for 10 minutes at 4°C to remove viral particles. A small amount of cells was taken and the proportion of EGFP+ cells was analyzed by flow cytometry. The remaining cells were injected into NSG mice by intratibial (IT).

[0553] Establishment of ALL xenograft model

[0554] Anesthetize 6 - to 8 - week - old female NOD scidIL2Rγ null (NSG) mice (The Jackson Laboratory) with isoflurane (induction: 4% - 5%, maintenance: 2% - 3%, oxygen flow rate: 300 mL / min), and inject transduced Reh cells (5×10 5 cells). Keep the knee joint in a flexed position and expose the proximal tibia. Drill a hole in the tibia using a 23G needle, and then inject Reh cells (40 μL per animal) with a 31G insulin syringe. Mice received systemic and local analgesia before intratibial (IT) injection: 0.05 mg / kg Temgesic (Schlering - Plough) and 1 - 2 mg / kg Marcain (AstraZeneca). Systemic analgesia was repeated 6 - 8 hours after IT injection. Mice received a single intraperitoneal (IP) dose of 1.33 nM of CO201 - scFv or human IgG4 isotype control when their total flux reached more than 10 8 photons per second (p / s). Mice were housed under specific - pathogen - free conditions and had free access to food and water. Health status was monitored daily, and all animal experiments were conducted according to the approval of the Norwegian Food Safety Authority (number: 29016).

[0555] In vivo imaging

[0556] Monitor leukemia progression by non - invasive in vivo imaging using an IVIS Spectrum CT instrument (PerkinElmer). Administer the D - luciferin (150 mg / kg, PerkinElmer) substrate by intraperitoneal (IP) injection. Nine minutes later, record 3 images at 1 - minute intervals using automatic exposure settings. Euthanize the mice when they showed severe engraftment or symptoms exceeding the predetermined humane endpoint.

[0557] Results

[0558] Reh cells were stably transduced with a lentiviral firefly luciferase - EGFP vector and injected intratibially (IT) into NSG mice. Monitor the leukemia progression of Reh cells expressing firefly luciferase by non - invasive in vivo imaging until all mice showed a satisfactory tumor engraftment rate (>10 8 p / s). Mice were divided into two groups and immediately received an intraperitoneal (IP) injection of 1.33 nM of human IgG4 isotype control or CO201 - scFv after the imaging procedure. Further IVIS detection was performed 3 days and 6 days after the treatment injection, and the results showed that the tumor burden in mice treated with CO201 - scFv was significantly reduced compared to mice treated with the isotype control ( Figure 10 ).

[0559] References

[0560] 1. Bomken S, Buechler L, Rehe K, Ponthan F, Elder A, Blair H, et al. Lentiviral marking of patient-derived acute lymphoblastic leukaemic cells allows in vivo tracking of disease progression. Leukemia. 2013;27(3):718 - 21.

[0561] Example 6: Analysis of the binding affinity of CO201 - scFv - Fc - mono for recombinant CD47 by surface plasmon resonance (SPR)

[0562] In the Biacore S200 system, the binding affinity of CO201 - scFv - Fc - mono for CD47 as described in Example 4 was determined by surface plasmon resonance (SPR) technology. Recombinant biotinylated CD47 was immobilized on a SA streptavidin chip to a level of 100 RU (in 10 mM NaAc 5.0 solution). CO201 - scFv - Fc - mono was reacted with recombinant CD47 at gradient concentrations.

[0563] Analysis

[0564] Instrument: Biacore S200

[0565] Materials:

[0566] SA chip (streptavidin)

[0567] Recombinant CD47 (recCD47): SinoBiological (Cat. No. 12283 - H27H - B); 17 kDa, C - terminal His - tag and AVI - tag, biotinylated

[0568] Antibody fragment: CO201 - scFv - Fc - mono (77.3 kDa)

[0569] All proteins were stored at - 80 °C.

[0570] Buffer: 20 mM phosphate buffer, pH 7.4 / 2.7 mM KCl / 137 mM NaCl / 0.05% P20 Single - cycle step:

[0571] Immobilize recCD47 to 100 RU (dissolved in 10 mM NaAc, pH 5.0)

[0572] Regeneration condition: Treated with 10 mM glycine (pH 2) for 60 seconds

[0573] The analyte (CO201-scFv-Fc-mono) was run in a single-cycle mode

[0574] Injection order (from low concentration to high concentration): 20 nM / 10 nM / 5 nM / 2.5 nM / 1.25 nM / 0.625 nM / 0.3125 nM / 0.1562 nM / 0.07813 nM / 0.039 nM

[0575] Flow rate: 30 μl / min; Injection time: 120 seconds; Dissociation time after the last injection was 1800 seconds

[0576] Temperature: 25 °C

[0577] Single-cycle mode

[0578] Results

[0579] The data was fitted using a 1:1 binding model. All binding curves could be well fitted to the 1:1 model. Table 7 shows the obtained association rate (k a ) and dissociation rate (k d ) values. The affinity constant K D was calculated according to the formula k d / k a and was 97 pM. Therefore, the CO201-scFv-Fc-mono fusion protein showed a high binding affinity for CD47

[0580] Table 7

[0581] Analyte <![CDATA[k a (1 / Ms)]]> <![CDATA[k d (1 / s)]]> <![CDATA[K D (M)]]> CO201-scFv-Fc-mono <![CDATA[2.8×10 7 > <![CDATA[2.7×10 -3 > <![CDATA[9.7×10 -11 > 97 pM .

Claims

1. A binding protein or antibody comprising an antigen-binding domain that binds CD47, said antigen-binding domain comprising a heavy-chain variable region having three complementarity-determining regions (CDRs) and a light-chain variable region having three CDRs, wherein said antigen-binding domain binds to Q19, N45, T120, R121, E122, and G123 in CD47 as defined by SEQ ID NO:19, and wherein said binding protein or antibody binds CD47 monovalently.

2. The binding protein or antibody according to claim 1, wherein the heavy-chain variable region comprises: (i) variable heavy-chain (VH) CDR1, comprising the amino acid sequence NFGMH (SEQ ID NO:5) or a substantially homologous sequence thereof; (ii) VH CDR2, comprising the amino acid sequence WINTYTGEPTYTDDFKG (SEQ ID NO:6) or a substantially homologous sequence thereof; and (iii) VH CDR3, comprising the amino acid sequence GDYRYGDS (SEQ ID NO:7) or a substantially homologous sequence thereof; and / or wherein the light-chain variable region comprises: (iv) variable light-chain (VL) CDR1, comprising the amino acid sequence RSSQSLVHSNGKTYLH (SEQ ID NO:8) or a substantially homologous sequence thereof; (v) VL CDR2, comprising the amino acid sequence RVSNRFS (SEQ ID NO:9) or a substantially homologous sequence thereof; and (vi) VL CDR3, comprising the amino acid sequence SQSTHVPFT (SEQ ID NO:10) or a substantially homologous sequence thereof; wherein the substantially homologous sequence refers to a sequence having 1, 2, or 3 amino acid substitutions as compared to a given CDR sequence.

3. The binding protein or antibody according to claim 1 or claim 2, wherein the heavy-chain variable region comprises: (i) variable heavy-chain (VH) CDR1, comprising the amino acid sequence NFGMH (SEQ ID NO:5); (ii) VH CDR2, comprising the amino acid sequence WINTYTGEPTYTDDFKG (SEQ ID NO:6); and (iii) VH CDR3, comprising the amino acid sequence GDYRYGDS (SEQ ID NO:7); and wherein the light-chain variable region comprises: (iv) variable light-chain (VL) CDR1, comprising the amino acid sequence RSSQSLVHSNGKTYLH (SEQ ID NO:8); (v) VL CDR2, comprising the amino acid sequence RVSNRFS (SEQ ID NO:9); and (vi) VL CDR3, comprising the amino acid sequence SQSTHVPFT (SEQ ID NO:10).

4. The binding protein or antibody according to any one of claims 1 to 3, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 3 or a sequence having at least 80% sequence identity thereto, and / or wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 4 or a sequence having at least 80% sequence identity thereto; or wherein the binding protein or antibody comprises a humanized version of SEQ ID NO: 3 and / or SEQ ID NO: 4; or wherein the heavy chain variable region comprises one or more of the amino acid sequences shown in SEQ ID NO: 22, 23, 24, 25 or 26 or a sequence having at least 80% sequence identity thereto, and / or wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27, 28 or 29 or a sequence having at least 80% sequence identity thereto.

5. The antibody according to any one of claims 1 to 4, wherein the antibody is an scFv antibody or a monovalent scFv-Fc form.

6. The binding protein or antibody according to any one of claims 1 to 5, wherein the CD47 binding affinity (K D ) of the binding protein or antibody is less than 250 pM, preferably less than 200 pM.

7. The binding protein or antibody according to any one of claims 1 to 6, wherein the binding protein or antibody is capable of inducing programmed cell death of tumor cells and / or does not induce agglutination of red blood cells.

8. An immunoconjugate comprising the binding protein or antibody according to any one of claims 1 to 7, operably attached to at least one other therapeutic or diagnostic agent.

9. One or more nucleic acid molecules comprising a nucleotide sequence encoding the binding protein or antibody or immunoconjugate according to any one of claims 1 to 8.

10. One or more expression vectors comprising one or more nucleic acid molecules according to claim 9.

11. One or more host cells or viruses comprising the expression vector according to claim 10, or the nucleic acid molecule according to claim 9, or expressing the antibody or binding protein or immunoconjugate according to any one of claims 1 to 8.

12. A method for producing the binding protein or antibody or immunoconjugate according to any one of claims 1 to 8, the method comprising the steps of: (i) culturing a host cell comprising the expression vector according to claim 10 or the nucleic acid molecule according to claim 9 under conditions suitable for expressing the encoded binding protein or antibody or immunoconjugate; and optionally (ii) isolating or obtaining the binding protein or antibody or immunoconjugate from the host cell or the growth medium / supernatant.

13. A composition comprising the binding protein or antibody according to any one of claims 1 to 7, the immunoconjugate according to claim 8, one or more nucleic acid molecules according to claim 9, one or more expression vectors according to claim 10, or one or more host cells or viruses according to claim 11.

14. The binding protein or antibody according to any one of claims 1 to 7, the immunoconjugate according to claim 8, the one or more nucleic acid molecules according to claim 9, the one or more expression vectors according to claim 10, the one or more host cells or viruses according to claim 11, or the composition according to claim 13, for use in therapy, preferably for the treatment or prevention of cancer.

15. A binding protein or antibody comprising an antigen-binding domain that binds CD47, wherein the antigen-binding domain is in the form of a scFv, and wherein the antigen-binding domain is fused, linked or attached to an Fc region, and wherein the binding protein or antibody binds CD47 monovalently.

16. The binding protein or antibody according to claim 15, wherein the binding protein or antibody is defined as in any one of claims 1 to 7.

17. The binding protein or antibody according to claim 15 or claim 16, for use in therapy, preferably for the treatment or prevention of cancer.

18. A method of treating or preventing cancer, the method comprising administering to a patient in need thereof a therapeutically effective amount of the binding protein or antibody according to any one of claims 1 to 7 or claim 15, the immunoconjugate according to claim 8, the one or more nucleic acid molecules according to claim 9, the one or more expression vectors according to claim 10, the one or more host cells or viruses according to claim 11, or the composition according to claim 13.

19. Use of the binding protein or antibody according to any one of claims 1 to 7 or claim 15, the immunoconjugate according to claim 8, the one or more nucleic acid molecules according to claim 9, the one or more expression vectors according to claim 10, the one or more host cells or viruses according to claim 11, or the composition according to claim 13, in the manufacture of a medicament for use in therapy, preferably the medicament for the treatment or prevention of cancer.

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