Bivalent CD47-binding protein

Abstract

The present invention provides binding proteins or antibodies that bind to CD47 and comprise two antigen-binding domains, the antigen-binding domains comprising a heavy chain variable region comprising three complementarity-determining regions (CDRs) and a light chain variable region comprising three CDRs, and the antigen-binding domains bind to Q19, N45, T120, R121, E122, and G123 in CD47 as defined by SEQ ID NO. 19. Compositions, methods of treatment, and kits based on the binding proteins and antibodies are also provided.

Description

[Technical Field]

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

[0002] Cancer treatment remains one of the greatest unmet medical needs to this day. Despite advances in cancer treatment over the past few decades, cancer remains one of the leading causes of death. Although the population of industrialized countries is benefiting from a longer average expected lifespan, there is an increasing urgency in the search for improved or new cancer treatments.

[0003] A relatively new approach is to target the CD47 axis. CD47 is a ubiquitously expressed cell surface glycoprotein that functions as a signaling receptor for thrombospondin-1 and a counterreceptor for signal regulatory protein-α (SIRP-α). Ligation with SIRP-α on macrophages inhibits phagocytosis, and CD47 thereby serves as a physiological marker of self. However, elevated CD47 expression on some cancer cells can also act to protect cancer cells from innate immune surveillance and prevent their phagocytosis by SIRPα-expressing macrophages and other cells of the innate immune system (the so-called "don't eat me" signal).

[0004] These discoveries have led to the development of antibodies and other types of biologics that block the CD47 / SIRPα interaction in tumor cells. Several candidate molecules, for example from Gilead, FortySeven, ALX Oncology, and Arch Oncology, are in preclinical and clinical development. However, alternative, and preferably improved, therapeutic agents that target CD47 are needed. Summary of the Invention [Means for solving the problem]

[0005] The present invention provides such an alternative and improved therapeutic option in the form of binding proteins and antibodies (eg, antibody-based binding proteins) that are directed to and bind bivalently to CD47.

[0006] As described in more detail elsewhere herein, the antibodies of the present invention, including the humanized antibodies and bivalent scFv-Fc fusion proteins of the present invention, have been shown to be capable of binding to CD47 with high affinity and also exhibit excellent ability to induce direct tumor cell killing. Advantageously, the direct cell killing effect is observed rapidly and at very low concentrations. The antibodies of the present invention have also been shown to be capable of blocking or inhibiting CD47-SIRPα interaction, thereby inhibiting the "don't eat me" signal from tumor cells to macrophages, thereby enabling macrophages to phagocytose CD47-expressing tumor cells. The antibodies of the present invention also exhibit limited binding to normal cells, such as red blood cells.

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

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

[0009] In one embodiment, the invention provides a binding protein, e.g., an antibody, comprising two antigen-binding domains that bind to CD47, wherein the antigen-binding domains comprise a heavy chain variable region comprising three complementarity-determining regions (CDRs) and a light chain variable region comprising three CDRs, wherein the heavy chain variable region comprises: (i) a variable heavy (VH) CDR1 comprising the amino acid sequence of NFGMH (SEQ ID NO. 5) or a sequence substantially homologous thereto; (ii) a VH CDR2 comprising the amino acid sequence WINTYTGEPTYTDDFKG (SEQ ID NO. 6) or a sequence substantially homologous thereto; (iii) a VH CDR3 comprising the amino acid sequence of GDYRYGDS (SEQ ID NO. 7) or a sequence substantially homologous thereto; and / or The light chain variable region (iv) a variable light (VL) CDR1 comprising the amino acid sequence RSSQSLVHSNGKTYLH (SEQ ID NO. 8) or a sequence substantially homologous thereto; (v) a VL CDR2 comprising the amino acid sequence of RVSNRFS (SEQ ID NO. 9) or a sequence substantially homologous thereto; (vi) a VL CDR3 comprising the amino acid sequence of SQSTHVPFT (SEQ ID NO. 10) or a sequence substantially homologous thereto; Said substantially homologous sequences are sequences which contain one, two or three amino acid substitutions compared to the given CDR sequence.

[0010] In another embodiment, the invention provides a binding protein, e.g., an antibody, comprising two antigen-binding domains that bind to CD47, wherein the antigen-binding domains comprise a heavy chain variable region comprising three complementarity-determining regions (CDRs) and a light chain variable region comprising three CDRs, wherein the heavy chain variable region comprises: (i) a variable heavy (VH) CDR1 comprising the amino acid sequence of NFGMH (SEQ ID NO. 5) or a sequence substantially homologous thereto, wherein the substantially homologous sequence contains one or two amino acid substitutions compared to a given CDR sequence; (ii) a VH CDR2 comprising the amino acid sequence of WINTYTGEPTYTDDFKG (SEQ ID NO. 6) or a sequence substantially homologous thereto, wherein the substantially homologous sequence contains one, two, three, four, five or six, e.g., one, two, three or four, amino acid substitutions compared to the given CDR sequence; (iii) a VH CDR3 comprising the amino acid sequence of GDYRYGDS (SEQ ID NO. 7) or a sequence substantially homologous thereto, wherein the substantially homologous sequence comprises one, two, or three amino acid substitutions compared to the given CDR sequence; and / or The light chain variable region (iv) a variable light (VL) CDR1 comprising the amino acid sequence RSSQSLVHSNGKTYLH (SEQ ID NO. 8) or a sequence substantially homologous thereto, wherein the substantially homologous sequence contains one, two, three, four, five or six, e.g., one, two, three or four, amino acid substitutions compared to a given CDR sequence; (v) a VL CDR2 comprising the amino acid sequence of RVSNRFS (SEQ ID NO. 9) or a sequence substantially homologous thereto, wherein the substantially homologous sequence contains one, two, or three amino acid substitutions compared to a given CDR sequence; (vi) a VL CDR3 comprising the amino acid sequence of SQSTHVPFT (SEQ ID NO. 10) or a sequence substantially homologous thereto, wherein the substantially homologous sequence contains one, two, or three amino acid substitutions compared to a given CDR sequence.

[0011] In another embodiment, the invention provides a binding protein, e.g., an antibody, comprising two antigen-binding domains that bind to CD47, wherein the antigen-binding domains comprise a heavy chain variable region comprising three complementarity-determining regions (CDRs) and a light chain variable region comprising three CDRs, wherein the heavy chain variable region comprises: (i) a variable heavy (VH) CDR1 comprising the amino acid sequence of NFGMH (SEQ ID NO. 5); (ii) a VH CDR2 comprising the amino acid sequence WINTYTGEPTYTDDFKG (SEQ ID NO. 6); (iii) a VH CDR3 comprising the amino acid sequence of GDYRYGDS (SEQ ID NO. 7); and / or The light chain variable region (iv) a variable light (VL) CDR1 comprising the amino acid sequence RSSQSLVHSNGKTYLH (SEQ ID NO. 8); and (v) a VL CDR2 comprising the amino acid sequence of RVSNRFS (SEQ ID NO. 9); (vi) a VL CDR3 comprising the amino acid sequence SQSTHVPFT (SEQ ID NO. 10).

[0012] In a preferred embodiment, the invention provides a binding protein, e.g., an antibody, comprising two antigen-binding domains that bind to CD47, wherein the antigen-binding domains comprise a heavy chain variable region comprising three complementarity-determining regions (CDRs) and a light chain variable region comprising three CDRs, wherein the heavy chain variable region: (i) a variable heavy (VH) CDR1 comprising the amino acid sequence of NFGMH (SEQ ID NO. 5); (ii) a VH CDR2 comprising the amino acid sequence WINTYTGEPTYTDDFKG (SEQ ID NO. 6); (iii) a VH CDR3 comprising the amino acid sequence of GDYRYGDS (SEQ ID NO. 7); and The light chain variable region (iv) a variable light (VL) CDR1 comprising the amino acid sequence RSSQSLVHSNGKTYLH (SEQ ID NO. 8); and (v) a VL CDR2 comprising the amino acid sequence of RVSNRFS (SEQ ID NO. 9); (vi) a VL CDR3 comprising the amino acid sequence SQSTHVPFT (SEQ ID NO. 10).

[0013] Exemplary such binding proteins, preferably antibodies, of the present invention can be bivalent or trivalent or more with respect to CD47 (i.e., can bind to CD47 bivalently or trivalently or more), and can include, for example, two antigen-binding domains that bind to CD47 (e.g., only two antigen-binding domains that bind to CD47) or three or more antigen-binding domains that bind to CD47. However, such exemplary binding proteins, preferably antibodies, of the present invention may also have additional antigen-binding domains that bind to target antigens other than CD47. Thus, such binding proteins (or antibodies) can be bispecific, trispecific, or multispecific, i.e., bind to two or more types of target antigens, in which case one of the target antigens is CD47. Thus, such binding proteins (or antibodies) still require two or more antigen-binding domains that bind to CD47. Exemplary such binding proteins (or antibodies) have two or only two antigen-binding domains specific for CD47, or twelve or only twelve CDRs (e.g., two sets of six CDRs).

[0014] In a further embodiment, the invention provides a bivalent binding protein, e.g., an antibody, comprising two antigen-binding domains that bind to CD47, wherein the antigen-binding domains comprise a heavy chain variable region comprising three complementarity-determining regions (CDRs) and a light chain variable region comprising three CDRs, wherein the heavy chain variable region: (i) a variable heavy (VH) CDR1 comprising the amino acid sequence of NFGMH (SEQ ID NO. 5) or a sequence substantially homologous thereto; (ii) a VH CDR2 comprising the amino acid sequence WINTYTGEPTYTDDFKG (SEQ ID NO. 6) or a sequence substantially homologous thereto; (iii) a VH CDR3 comprising the amino acid sequence of GDYRYGDS (SEQ ID NO. 7) or a sequence substantially homologous thereto; and / or The light chain variable region (iv) a variable light (VL) CDR1 comprising the amino acid sequence RSSQSLVHSNGKTYLH (SEQ ID NO. 8) or a sequence substantially homologous thereto; (v) a VL CDR2 comprising the amino acid sequence of RVSNRFS (SEQ ID NO. 9) or a sequence substantially homologous thereto; (vi) a VL CDR3 comprising the amino acid sequence of SQSTHVPFT (SEQ ID NO. 10) or a sequence substantially homologous thereto; The substantially homologous sequences are sequences which contain one, two or three amino acid substitutions compared to the given CDR sequence.

[0015] In a further embodiment, the invention provides a multivalent binding protein, e.g., an antibody, comprising at least two antigen-binding domains that bind to CD47, wherein the antigen-binding domains comprise a heavy chain variable region comprising three complementarity-determining regions (CDRs) and a light chain variable region comprising three CDRs, wherein the heavy chain variable region: (i) a variable heavy (VH) CDR1 comprising the amino acid sequence of NFGMH (SEQ ID NO. 5) or a sequence substantially homologous thereto; (ii) a VH CDR2 comprising the amino acid sequence WINTYTGEPTYTDDFKG (SEQ ID NO. 6) or a sequence substantially homologous thereto; (iii) a VH CDR3 comprising the amino acid sequence of GDYRYGDS (SEQ ID NO. 7) or a sequence substantially homologous thereto; and / or The light chain variable region (iv) a variable light (VL) CDR1 comprising the amino acid sequence RSSQSLVHSNGKTYLH (SEQ ID NO. 8) or a sequence substantially homologous thereto; (v) a VL CDR2 comprising the amino acid sequence of RVSNRFS (SEQ ID NO. 9) or a sequence substantially homologous thereto; (vi) a VL CDR3 comprising the amino acid sequence of SQSTHVPFT (SEQ ID NO. 10) or a sequence substantially homologous thereto; Such substantially homologous sequences are sequences which contain one, two or three amino acid substitutions compared to the given CDR sequence.

[0016] Preferred CDR sequences for such bivalent and multivalent binding proteins (or antibodies) are as defined elsewhere herein.

[0017] As used herein, the term "divalent" refers to a binding protein (or antibody) having two antigen-binding domains. The term "bivalent for CD47" or equivalent terms refers to a binding protein (or antibody) having two antigen-binding domains capable of binding two molecules of the same target antigen, herein CD47. The term "bivalent" can be used as an alternative to divalent. As used herein, the term "multivalent" refers to a binding protein (or antibody) having three or more antigen-binding domains. The term "multivalent for CD47" or equivalent terms refers to a binding protein (or antibody) having three or more antigen-binding domains capable of binding three or more molecules of the same target antigen, herein CD47. Thus, trivalent (having three antigen-binding domains) and tetravalent (having four antigen-binding domains) binding proteins or antibodies are provided. Such binding proteins or antibodies may be "multivalent for CD47" but may contain additional antigen-binding domains that bind to target antigens other than CD47. Thus, such binding proteins (or antibodies) can be bispecific, trispecific or multispecific, i.e., bind to more than one type of target antigen, in which case one of these target antigens is CD47.

[0018] Thus, the binding proteins (or antibodies) of the invention described above and elsewhere herein are at least bivalent, e.g., bivalent or multivalent, with respect to CD47. In other words, they are capable of bivalent or multivalent binding to CD47.

[0019] Certain embodiments of the present invention provide antibodies (or binding proteins) that bind to CD47, comprising a VH domain having the amino acid sequence of SEQ ID NO. 3, or a sequence substantially homologous thereto, and / or a VL domain having the amino acid sequence of SEQ ID NO. 4, or a sequence substantially homologous thereto.

[0020] Certain embodiments of the present invention provide antibodies (or binding proteins) that bind to CD47, comprising a VH domain having the amino acid sequence of SEQ ID NO. 3, or a sequence substantially homologous thereto, and a VL domain having the amino acid sequence of SEQ ID NO. 4, or a sequence substantially homologous thereto.

[0021] Certain embodiments of the present invention provide antibodies (or binding proteins) that bind to CD47, comprising a VH domain having the amino acid sequence of SEQ ID NO.3 and / or a VL domain having the amino acid sequence of SEQ ID NO.4.

[0022] Certain embodiments of the present invention provide an antibody (or binding protein) that binds to CD47, comprising a VH domain having the amino acid sequence of SEQ ID NO.3 and a VL domain having the amino acid sequence of SEQ ID NO.4.

[0023] In another embodiment, the invention provides an antibody (or binding protein) that binds to CD47, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 3 or a sequence having at least 80% (e.g., at least 85%, 90%, 95% or 98%) sequence identity thereto, and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO. 4 or a sequence having at least 80% (e.g., at least 85%, 90%, 95% or 98%) sequence identity thereto.

[0024] In another embodiment, the invention provides an antibody (or binding protein) that binds to CD47, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 3 or a sequence having at least 80% (e.g., at least 85%, 90%, 95%, or 98%) sequence identity thereto, and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 4 or a sequence having at least 80% (e.g., at least 85%, 90%, 95%, or 98%) sequence identity thereto.

[0025] In another embodiment, the invention provides an antibody (or binding protein) that binds CD47, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 3, or a sequence having at least 80% (e.g., at least 85%, 90%, 95% or 98%) sequence identity thereto, and further comprises three CDRs comprising the amino acid sequence of SEQ ID NOs. 5, 6 and 7, as defined elsewhere herein, or sequences substantially homologous thereto; and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO. 4, or a sequence having at least 80% (e.g., at least 85%, 90%, 95% or 98%) sequence identity thereto, and further comprises three CDRs comprising the amino acid sequence of SEQ ID NOs. 8, 9 and 10, as defined elsewhere herein, or sequences substantially homologous thereto.

[0026] In another embodiment, the invention provides an antibody (or binding protein) that binds CD47, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 3, or a sequence having at least 80% (e.g., at least 85%, 90%, 95% or 98%) sequence identity thereto, and further comprises three CDRs comprising the amino acid sequences of SEQ ID NOs. 5, 6 and 7, as defined elsewhere herein, or sequences substantially homologous thereto; and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 4, or a sequence having at least 80% (e.g., at least 85%, 90%, 95% or 98%) sequence identity thereto, and further comprises three CDRs comprising the amino acid sequences of SEQ ID NOs. 8, 9 and 10, as defined elsewhere herein, or sequences substantially homologous thereto.

[0027] In another embodiment, the invention provides an antibody (or binding protein) that binds to CD47, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 3 or a sequence having at least 80% (e.g., at least 85%, 90%, 95% or 98%) sequence identity thereto, and wherein the heavy chain variable region comprises three CDRs comprising the amino acid sequences of SEQ ID NOs. 5, 6 and 7; and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO. 4 or a sequence having at least 80% (e.g., at least 85%, 90%, 95% or 98%) sequence identity thereto, and wherein the light chain variable region comprises three CDRs comprising the amino acid sequences of SEQ ID NOs. 8, 9 and 10.

[0028] The above (and other) embodiments described for SEQ ID NO. 3 and / or SEQ ID NO. 4 apply equally to surrogate heavy chain variable regions of the invention, e.g., SEQ ID NO. 39, 40, 41, 42, or 43, and / or surrogate light chain variable regions of the invention, e.g., SEQ ID NO. 44, 45, or 46. Preferred combinations of such heavy and light chain variable regions are provided in Table E and elsewhere herein.

[0029] In another embodiment, the invention provides an antibody (or binding protein) that binds to CD47, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 3 or a sequence having at least 80% (e.g., at least 85%, 90%, 95% or 98%) sequence identity thereto, and wherein the heavy chain variable region comprises three CDRs comprising the amino acid sequences of SEQ ID NOs. 5, 6 and 7; and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 4 or a sequence having at least 80% (e.g., at least 85%, 90%, 95% or 98%) sequence identity thereto, and wherein the light chain variable region comprises three CDRs comprising the amino acid sequences of SEQ ID NOs. 8, 9 and 10.

[0030] In alternative embodiments of the invention, the sequence identity may be at least 60%, 65%, 70% or 75% sequence identity.

[0031] Other embodiments are immunoglobulin (Ig) forms, such as IgG, IgA, IgD, IgE, or IgM forms, or forms containing all or a portion of an immunoglobulin constant region, such as all or a portion of the IgG, IgA, IgD, IgE, or IgM constant region of the various antibodies (or binding proteins) defined herein, e.g., full-length Ig or IgG, IgM, or IgA forms. IgG forms of the antibodies of the invention described herein, preferably full-length IgG forms (e.g., IgG1, IgG2, IgG3, or IgG4 forms), are preferred, such as the mCO-1 antibody shown in Table A. In some embodiments, IgG1 or IgG4 forms of any of these antibodies are also preferred. In some embodiments, humanized forms of any of these antibodies are also preferred (see, e.g., Table E). It will, of course, be understood that a complete IgG antibody typically comprises two identical or substantially identical heavy chains (with the appropriate variable and constant regions) and two identical or substantially identical light chains (with the appropriate variable and constant regions). In some embodiments, antibody formats containing a portion of an immunoglobulin constant region, e.g., a portion of an IgG, IgA, IgD, IgE, or IgM constant region, are preferred, e.g., in the bivalent scFv-Fc fusion protein format described herein.

[0032] Conveniently, the IgG (or other) form comprises a heavy chain variable region (VH) and a light chain variable region (VL) as described herein, and further comprises appropriate IgG (or other) heavy chain and light chain constant regions. Sequences of such constant regions are well known and described in the art, any of which may be used. Thus, these regions may be derived from any suitable source or species, e.g., mouse or human. Preferably, such IgG (or other) sequences are human IgG (or other) sequences, e.g., human IgG1 or IgG4 sequences.

[0033] A preferred embodiment of the present invention is a full-length IgG1 antibody comprising a heavy chain of SEQ ID NO. 21 or a sequence substantially homologous thereto and / or a light chain of SEQ ID NO. 22 or a sequence substantially homologous thereto. Also preferred is an IgG4 antibody comprising a heavy chain of SEQ ID NO. 23 or a sequence substantially homologous thereto and / or a light chain of SEQ ID NO. 24 or a sequence substantially homologous thereto.

[0034] In a preferred embodiment, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 21 or a sequence having at least 80% (e.g., at least 85%, 90%, 95% or 98%) sequence identity thereto, and / or a light chain comprising the amino acid sequence of SEQ ID NO. 22 or a sequence having at least 80% (e.g., at least 85%, 90%, 95% or 98%) sequence identity thereto. Also preferred are antibodies comprising a heavy chain comprising the amino acid sequence of SEQ ID NO. 23 or a sequence having at least 80% (e.g., at least 85%, 90%, 95% or 98%) sequence identity thereto, and / or a light chain comprising the amino acid sequence of SEQ ID NO. 24 or a sequence having at least 80% (e.g., at least 85%, 90%, 95% or 98%) sequence identity thereto.

[0035] In a preferred embodiment, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 21 and / or a light chain comprising the amino acid sequence of SEQ ID NO. 22. In another preferred embodiment, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 23 and / or a light chain comprising the amino acid sequence of SEQ ID NO. 24.

[0036] In a preferred embodiment, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 21 and a light chain comprising the amino acid sequence of SEQ ID NO. 22. In another preferred embodiment, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 23 and a light chain comprising the amino acid sequence of SEQ ID NO. 24.

[0037] Anti-CD47 antibodies (and binding proteins) based on the mCO-1, CO-1.1, and CO-1.4 antibody sequences set forth in Tables A, B, C, and D are preferred. Tables A, B, C, and D herein set forth the CDR domains, FR domains, VH and VL domains, and IgG (heavy and light chains). Antibodies (or binding proteins) comprising these sets of CDR domains or VH and VL domains, or IgG-containing formats comprising such domains (or sequences substantially homologous thereto), including the full-length heavy and light chain IgG sequences provided in Tables A, B, and C, are preferred embodiments of the invention. Humanized forms of the mCO-1 antibody are also preferred, e.g., antibodies comprising a heavy chain variable domain that is a humanized version of SEQ ID NO. 3 and / or a light chain variable domain that is a humanized version of SEQ ID NO. 4. Such humanized versions include antibodies (or binding proteins) comprising the heavy and / or light chain variable domains set forth in Table E.

[0038] Tables A, B, C and D herein provide the CDR sequences of particular antibodies of the invention. In some other embodiments, the CDR sequences of the antibodies of the invention are identified using any suitable method (or tool), for example, as shown in Tables A, B, C, and D, for example, according to the well-known method of Kabat (e.g., Kabat et al., "Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, pp. 647-669, 1991), or Chothia (e.g., Chothia, C., et al., Nature, 342, 877-883 (1989), or Al-Lazikani et al., Journal of Molecular Biology, 273, 927-948 (1997)), or according to the IMGT numbering system (e.g., Lefranc, M.-P., The Immunologist, 1997). The CDR sequences may be the CDR sequences in the VH domain and VL domain of the antibody of the present invention, as identified using the AbM numbering system (e.g., according to Abhinandan and Martin, Molecular Immunology 45:3832-3839 (2008)), or the AbM numbering system (e.g., according to Abhinandan and Martin, Molecular Immunology 45:3832-3839 (2008)).

[0039] Specific examples of substantially homologous sequences are sequences having at least 55%, 60%, or 65% identity to the disclosed amino acid sequences. In certain embodiments, an antibody (or binding protein) of the invention comprises at least one heavy chain variable region comprising an amino acid sequence region that is at least 55%, 60%, 65%, 70%, or 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% or 95%, and most preferably at least 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO.3, and / or at least one light chain variable region comprising an amino acid sequence region that is at least 55%, 60%, 65%, 70% or 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% or 95%, and most preferably at least 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO.4.

[0040] Other preferred examples of substantially homologous sequences are sequences that contain conservative amino acid substitutions of the disclosed amino acid sequences.

[0041] Other preferred examples of substantially homologous sequences are those that contain one, two, three, four, five, or six, or one, two, three, four, or five, or one, two, three, or four, preferably one, two, or three, preferably one or two (more preferably one) modified amino acids in one or more of the disclosed CDR regions or one or more of the FR regions. Such modifications can be conservative or non-conservative amino acid substitutions or mixtures thereof.

[0042] Other preferred examples of "substantially homologous" sequences are sequences that have 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% amino acid sequence identity with the amino acid sequence of one or more of the CDR regions or one or more of the FR regions disclosed in Tables A or B, C, D, or E. Thus, in some embodiments, a "substantially homologous" CDR sequence may be a sequence that has 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 with a given CDR sequence described herein.

[0043] In some embodiments, in antibodies having a "substantially homologous" sequence to a given sequence or having a particular degree of sequence identity to a given sequence, the modified amino acid residues are not in the CDR regions. For example, in some embodiments, in antibodies having a VH domain that has a particular degree of sequence identity to a given VH domain sequence of a particular antibody of the invention (e.g., mCO-1, CO-1.1, or CO-1.4, a humanized CO-1 antibody of the invention, or a bivalent scFv-Fc fusion protein), the modified (or altered) residues are not in the CDR regions. Thus, in some embodiments, in antibodies having a "substantially homologous" sequence to a given sequence or having a particular degree of sequence identity to a given sequence, the modified amino acid residues are in one or more framework regions.

[0044] As will become clear elsewhere herein, in other embodiments, in antibodies having sequences that are "substantially homologous" to a given sequence, or that have a certain degree of sequence identity to a given sequence, the modified amino acid residues may be in the CDR regions.

[0045] In some embodiments, in antibodies that have sequences that are "substantially homologous" to a given sequence, or that have a particular degree of sequence identity to a given sequence, the three VH CDR amino acid sequences (i.e., all three VH CDR sequences taken together) and the three VL CDR amino acid sequences (i.e., all three VL CDR sequences taken together) that form a set of six total CDRs are considered together to be the complement (or entire CDR set) of the antibody, and the amino acid sequence of said complement (or entire CDR set) of said antibody is at least 70%, preferably at least 80%, or at least 85%, or at least 90%, or at least 95% identical to the corresponding complement (or entire CDR set) of a given original (or reference) antibody. The original (or reference) antibody may have the CDR sequences of the mCO-1, CO-1.1, CO-1.4, CO-1 F(ab')2, or humanized CO-1 antibody of the present invention shown in Tables A, B, CD, and E, or the CDR sequences of the bivalent scFv-Fc fusion protein (CO201-scFv-Fc-bi) (SEQ ID NO. 53).

[0046] The altered residues can be conservative or non-conservative amino acid substitutions or a mixture thereof.

[0047] In such embodiments, preferred modifications are conservative amino acid substitutions.

[0048] In all embodiments, binding proteins, e.g., antibodies, containing substantially homologous sequences retain the ability to bind to CD47. Preferably, binding proteins, e.g., antibodies, containing substantially homologous sequences retain one or more (preferably all) of the other properties described herein for the antibodies of the invention described herein, e.g., mCO-1, CO-1.1, CO-1.4, CO-1 F(ab')2, or humanized CO-1 antibody, or bivalent scFv-Fc fusion proteins (e.g., CO201-scFv-Fc-bi).

[0049] Further examples of substantially homologous amino acid sequences according to the present invention are described elsewhere herein.

[0050] The CDRs of the antibodies (or binding proteins) of the invention are preferably separated by suitable framework regions, such as those found in natural antibodies and / or effective artificial antibodies. H , V L The individual CDR sequences are preferably provided or incorporated within a suitable framework or scaffold to enable antigen (herein, CD47) binding. Such framework sequences or framework regions may correspond to the appropriate naturally occurring framework regions FR1, FR2, FR3, and / or FR4, where appropriate to form a suitable scaffold, or may correspond to consensus framework regions identified, for example, by comparing various naturally occurring framework regions. In some embodiments, humanized antibodies are provided, in which case human framework regions (or sequences substantially homologous thereto) can be used. Alternatively, non-antibody scaffolds or frameworks, such as T-cell receptor frameworks, can be used.

[0051] Suitable sequences that can be used for the framework region are well known and described in the art, and any of these may be used. Exemplary sequences for framework regions are the V H Domain and / or V LOne or more of the framework regions making up the domain, for example, one or more of the framework regions of mCO-1, CO-1.1, CO-1.4, CO-1 F(ab')2 or humanized CO-1 antibody disclosed in Table A or B or C or D or E, or the framework region of a bivalent scFv-Fc fusion protein, for example, CO201-scFv-Fc-bi (SEQ ID NO. 53), or a framework region substantially homologous thereto, and in particular, one or more framework regions that allow maintenance of antigen specificity, for example, one or more framework regions that result in a substantially similar or identical antibody 3D structure.

[0052] In certain embodiments, all four of the framework regions (FRs) of the variable heavy chain (SEQ ID NOs. 11, 12, 13, and 14) and / or variable light chain (SEQ ID NOs. 15, 16, 17, and 18), or FR regions substantially homologous thereto, are found in an antibody (or binding protein) of the invention, as appropriate.

[0053] Exemplary mCO-1, CO-1.1, or CO-1.4 antibodies of the invention comprise murine / mouse VH and VL domains (mCO-1 is a full-length murine (m) antibody (IgG1κ antibody), and CO-1.1 and CO-1.4 are chimeric formats of m(murine)CO-1 in which human IgG1 and IgG4 sequences replace the murine / mouse constant regions of mCO-1, respectively). Thus, in certain embodiments, chimeric antibodies are preferred. Such antibodies typically comprise murine / mouse VH and VL domains, such as those having SEQ ID NOs. 3 and 4 (and related, substantially homologous sequences, e.g., as described herein), together with constant regions from another (non-murine / non-mouse) species, preferably human constant regions.

[0054] In other embodiments of the invention, humanized versions of the exemplary mCO-1, CO-1.1, or CO-1.4 antibodies of the invention are preferred. Thus, when an antibody (or binding protein) of the invention is referred to herein, preferred embodiments include a humanized antibody (or binding protein).

[0055] Thus, in some embodiments, an antibody (or binding protein) of the invention can be or include a humanized antibody, e.g., referred to as a humanized antibody or humanized binding protein. A "humanized" antibody is an antibody based substantially on non-human variable region domains in which certain amino acids have been altered to better correspond to those typically present in human antibodies. Methods for generating humanized antibodies are known in the art. For example, a humanized antibody can be generated by inserting appropriate CDRs (e.g., murine / mouse CDRs such as those present in an antibody of the invention) into a human antibody "scaffold," such as a scaffold comprising human antibody framework regions or sequences substantially homologous thereto. Thus, in some embodiments, the CDRs of the invention, e.g., the set of six CDRs of the antibodies of the invention described herein, e.g., from the exemplary antibodies of the invention shown in Tables A-D, i.e., CDRs having SEQ ID NOs. 5-10 or sequences substantially homologous thereto, are present within (or combined with, inserted into, or grafted onto) a human or humanized antibody framework using appropriate framework (FR) regions, e.g., found in human antibodies, or sequences substantially homologous thereto.

[0056] Exemplary humanized variable heavy (VH) domains for use in the humanized antibodies (or binding proteins) of the invention are provided in SEQ ID NOs. 39-43 or sequences substantially homologous thereto.

[0057] Exemplary humanized variable light (VL) domains for use in the humanized antibodies (or binding proteins) of the present invention are provided in SEQ ID NOs. 44-46 or sequences substantially homologous thereto.

[0058] Thus, a preferred antibody (or binding protein), e.g., a humanized antibody (or binding protein), of the invention comprises any one of the VH domains of SEQ ID NOs. 39, 40, 41, 42 or 43 or sequences substantially homologous thereto, and / or (preferably and) any one of the VL domains of SEQ ID NOs. 44, 45 or 46 or sequences substantially homologous thereto.

[0059] Thirteen humanized antibodies have been produced by the present invention, and these antibodies (or antibodies with sequences substantially homologous thereto), or binding proteins comprising such antibodies, are preferred antibodies (or binding proteins) of the present invention. These antibodies are referred to herein as: CO201 (comprising the VH domain of SEQ ID NO. 39 and the VL domain of SEQ ID NO. 44); CO202 (comprising the VH domain of SEQ ID NO. 40 and the VL domain of SEQ ID NO. 44); CO203 (comprising the VH domain of SEQ ID NO. 41 and the VL domain of SEQ ID NO. 44); CO204 (comprising the VH domain of SEQ ID NO. 42 and the VL domain of SEQ ID NO. 44); CO205 (comprising the VH domain of SEQ ID NO. 39 and the VL domain of SEQ ID NO. 45); CO206 (comprising the VH domain of SEQ ID NO. 40 and the VL domain of SEQ ID NO. 45); CO207 (comprising the VH domain of SEQ ID NO. 41 and the VL domain of SEQ ID NO. 45); CO208 (comprising the VH domain of SEQ ID NO. 42 and the VL domain of SEQ ID NO. 45); CO209 (comprising the VH domain of SEQ ID NO. 39 and the VL domain of SEQ ID NO. 46); CO210 (comprising the VH domain of SEQ ID NO. 40 and the VL domain of SEQ ID NO. 46); CO211 (comprising the VH domain of SEQ ID NO. 41 and the VL domain of SEQ ID NO. 46); CO212 (comprising the VH domain of SEQ ID NO. 42 and the VL domain of SEQ ID NO. 46), or CO213 (comprising the VH domain of SEQ ID NO. 43 and the VL domain of SEQ ID NO. 44); It is called.

[0060] Other preferred humanized antibodies (or binding proteins) of the present invention comprise the VH domain of SEQ ID NO. 43 or a sequence substantially homologous thereto and the VL domain of SEQ ID NO. 45 or a sequence substantially homologous thereto, or the VH domain of SEQ ID NO. 43 or a sequence substantially homologous thereto and the VL domain of SEQ ID NO. 46 or a sequence substantially homologous thereto.

[0061] In some embodiments, a humanized VH domain of SEQ ID NO. 39 or a sequence substantially homologous thereto is preferred, in some embodiments, a humanized VL domain of SEQ ID NO. 44 or a sequence substantially homologous thereto is preferred.

[0062] In some embodiments, a humanized VH domain of SEQ ID NO. 39 or a sequence substantially homologous thereto and a humanized VL domain of SEQ ID NO. 44 or a sequence substantially homologous thereto are preferred.

[0063] Sequences that are substantially homologous to any given sequence in such a humanized antibody (or binding protein) are as defined elsewhere herein and include sequences that have varying numbers of amino acid substitutions or have varying levels of percent identity with the given original sequence, e.g., sequences that have at least 80% sequence identity.

[0064] In some embodiments, the variant residues may be present in both the CDR 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).

[0065] Preferred heavy chain FR regions found in the humanized antibodies (or binding proteins) of the present invention are one or more or all four of the FR regions FR1, FR2, FR3 and FR4 present in SEQ ID NOs. 39, 40, 41, 42 or 43, or sequences substantially homologous thereto.

[0066] Preferred light chain FR regions found in the humanized antibodies (or binding proteins) of the present invention are one or more or all four of the FR regions FR1, FR2, FR3 and FR4 present in SEQ ID NO. 44, 45 or 46, or sequences substantially homologous thereto.

[0067] In certain embodiments, all four of the heavy and / or light chain framework regions (FRs) from SEQ ID NOs. 39, 40, 41, 42, 43, 44, 45, or 46, or FR regions substantially homologous thereto, as appropriate, are found in an antibody (or binding protein) of the invention.

[0068] In embodiments in which FR regions substantially homologous to one or more of the FR regions provided in SEQ ID NOs. 39, 40, 41, 42, 43, 44, 45, or 46 are used, each FR region may contain up to 10 amino acid changes from the given sequence, e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 change. While any amino acid change may be used, in some embodiments, the change may be a change that returns the amino acid residue to a residue found in the original murine antibody, herein the CO-1 antibody. Such changes may also be referred to as backmutations. In some embodiments, there may be 1, 2, or 3 backmutations in one or more of the FR regions provided in SEQ ID NOs. 39, 40, 41, 42, 43, 44, 45, or 46.

[0069] Such humanized VH and VL domains can be provided in any suitable bivalent (with respect to CD47) or at least bivalent or multivalent format (with respect to CD47) according to the invention. Exemplary formats are discussed elsewhere herein, including antibodies (or binding proteins) comprising antibody constant regions, particularly human antibody constant regions, e.g., full-length antibody formats such as IgG1 or IgG4 formats (e.g., with IgG1 and IgG4 heavy chain constant regions and appropriate light chain constant regions as described elsewhere herein). In some embodiments, the humanized (or other, e.g., non-humanized or murine) VH and VL domains described herein are provided in a full-length IgG4 format, e.g., with appropriate human heavy and light chain constant regions. In such antibodies of the invention, an exemplary IgG4 heavy chain constant region is provided in SEQ ID NO. 47 and / or (preferably) an exemplary light chain constant region is provided in SEQ ID NO. 48.

[0070] Other antibody (or binding protein) formats comprising antibody constant regions, particularly human antibody constant regions, are also provided, such as antibodies comprising an Fc region, e.g., Fc fusions. Accordingly, particularly preferred antibodies (or binding proteins) of the invention have the humanized (or other, e.g., non-humanized or murine) VH and VL domains of the invention described herein linked, fused, or attached to the Fc region of the antibody. A particularly preferred format is a bivalent (bivalent) scFv-Fc format / fusion in which two scFv antibodies (e.g., humanized scFv antibodies) are fused or attached to an Fc region. Such formats are well known and described in the art and, therefore, will preferably have two scFv fragments that bind CD47, e.g., two scFv fragments of the invention, fused, linked, or attached to the Fc region. In these embodiments, the Fc region is preferably a human Fc region, such as an IgG1 or IgG4 Fc region. An exemplary Fc region comprises (or consists of, or consists essentially of) a CH2 domain and a CH3 domain, and optionally also comprises (or consists of, or consists essentially of) a hinge domain.

[0071] Thus, in some embodiments, the humanized (or other) VH and VL domains described herein are provided in a format that is bivalent (dual) or at least bivalent (double) with respect to CD47, an scFv format. In other words, such antibodies (or binding proteins) of the invention contain at least two, preferably two, scFv fragments capable of binding to CD47. The scFv format 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 connected by a suitable peptide linker. Preferred and exemplary combinations of VH and VL domains for use in such bivalent (double) scFv formats are described elsewhere herein (see, e.g., Tables A-E); for example, the VH and / or VL domains, or CDRs, from any CD47 antibody of the invention may 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. 39 or a sequence substantially homologous thereto is preferred. In some such embodiments, the humanized VL domain of SEQ ID NO. 44, or a sequence substantially homologous thereto, is preferred. In some embodiments, the humanized VH domain of SEQ ID NO. 39, or a sequence substantially homologous thereto, and the humanized VL domain of SEQ ID NO. 44, or a sequence substantially homologous thereto, are preferred.

[0072] Suitable linker sequences for use in such scFv fragments are well known and described in the art. Suitable linker sequences are typically artificial and flexible linkers such as GS linkers. An exemplary GS linker sequence is provided as SEQ ID NO. 49.

[0073] A bivalent scFv-Fc format (or bivalent scFv-Fc fusion protein) is created in which one scFv is attached to one chain of the Fc region and another scFv is attached to the other chain of the Fc region. Because the Fc region is a dimer, the association of the two chains of the Fc region provides the bivalent format. An exemplary Fc region comprises a CH2 domain and a CH3 domain, and optionally also comprises a hinge domain (or other suitable linker, e.g., an artificial or flexible linker) to connect each scFv to the chain of the Fc region. Preferably, the Fc region is a human Fc region, such as an IgG1 or IgG4 Fc region. An exemplary human IgG4 Fc region (CH2 domain and CH3 domain) is provided in SEQ ID NO. 50. An exemplary hinge region is provided in SEQ ID NO. 51. Although suitable hinges and CH2 and CH3 domains can clearly be derived from antibodies of other subtypes, e.g., IgG1, IgG2, etc., and such sequences are readily available in the art, an exemplary hinge-CH2-CH3 region is provided in SEQ ID NO. 52.

[0074] In embodiments where an IgG4 hinge, such as that of SEQ ID NO. 51, is used, it may be desirable to make mutations, such as stabilizing mutations, to prevent, for example, Fab-arm exchange (Handlogten et al., mAbs, vol. 12, no. 1, e1779974 (2020)). Three exemplary mutations have been identified (see SEQ ID NO. 54), in the form of Y219C, G220C, and S228P, one or more of which may be used in the IgG4 hinge region.

[0075] Similar to the bivalent scFv-Fc format, other exemplary formats contain no other antibody constant regions beyond the CH2 and CH3 heavy chain regions / domains. Thus, in some embodiments, the CH1 and / or CL (or CL1) regions are absent or removed. In other words, the only antibody constant regions present in an antibody (or binding protein) of the invention are the CH2 and CH3 regions (or Fc region), optionally with a hinge domain (or other suitable linker, e.g., an artificial linker or a flexible linker).

[0076] Typically, the scFv in each chain of such a bivalent scFv-Fc molecule will be the same, but they can also be different, e.g., such a construct can contain two different CD47 antibodies (or binding proteins), e.g., two different CD47 antibodies (or binding proteins) of the invention, or one scFv CD47 antibody of the invention, optionally combined with another CD47 scFv antibody. Preferred such constructs therefore contain an antibody (or binding protein) of the invention as defined herein, e.g., an scFv fragment comprising at least two, or preferably two, VH and / or VL domains of the invention, or the corresponding three or six, e.g., six, CDRs of the invention as described elsewhere herein.

[0077] A preferred antibody (or binding protein) for use in such a format is the humanized CO201 antibody described herein. However, any CD47 antibody (or binding protein), such as any CD47 antibody (or binding protein) of the invention, such as any other humanized antibody of the invention, or the VH domain and / or VL domain (or their corresponding CDRs), or the six CDRs shown in Table A (with appropriate FR regions), can be incorporated into this format, and antibodies (or binding proteins) in or comprising this format (bivalent scFv-Fc format) are preferred.

[0078] The sequence of an exemplary bivalent scFv-Fc construct of the invention (CO201scFv-Fc-bi) comprising the CO201 antibody in scFv format is provided in SEQ ID NO. 53. Although a single polypeptide chain is provided, upon expression, a bivalent construct is generated by dimerization of the Fc regions.

[0079] Yet another aspect of the present invention provides antibodies (or binding proteins) comprising two antigen-binding domains that bind to CD47, wherein the antigen-binding domains are in scFv format and are fused, tethered, or otherwise attached (e.g., via a hinge region or linker) to an Fc region. Thus, in these embodiments, the CH1 and / or CL (or CL1) regions are absent or removed, and such regions are not involved in linking or connecting the scFvs to the Fc region. Thus, in these embodiments, the scFv antigen-binding domains can be attached directly to the Fc region, optionally via a hinge region or linker. In other words, such an antibody (or binding protein) is, consists of, or comprises a bivalent scFv-Fc fragment comprising two scFv antigen-binding domains that bind CD47. As noted above, preferred scFv antigen-binding domains that bind CD47 include one or more of the antigen-binding domains of the invention as defined elsewhere herein.

[0080] Such antibodies (or binding proteins) can also readily be made into bispecific, trispecific, or multispecific constructs as described elsewhere herein. For example, in such constructs, an antigen-binding domain (conveniently in the form of an scFv fragment) with specificity for a target antigen other than CD47 can be present attached to the CH3 portion of the Fc region. Such antibodies (or binding proteins) of the invention, when provided in a bivalent scFv-Fc format, have surprisingly and advantageously been shown not to induce hemagglutination of red blood cells, even at high concentrations. This provides an improvement over other bivalent (divalent) formats, including full-length antibody formats. Such antibodies have also been shown to be highly effective as therapeutics. Thus, this particular format is believed to be particularly advantageous for CD47 antibodies, and preferred antibodies (or binding proteins) of the invention, including humanized antibodies of the invention, when provided in a bivalent scFv-Fc format, do not induce hemagglutination (or significant hemagglutination) of RBCs when used at a concentration of 1, 2, 5, or 10 μg / ml, or at least 1, 2, 5, or 10 μg / ml, or up to 1, 2, 5, or 10 μg / ml. Preferred such antibodies (or binding proteins) of the invention do not induce hemagglutination (or significant hemagglutination) of RBCs when used at a concentration of 15, 20, 25, 50, 75, or 100 μg / ml, or at a concentration of at least 15, 20, 25, 50, 75, or 100 μg / ml, or up to 15, 20, 25, 50, 75, or 100 μg / ml. While any convenient method for assessing hemagglutination can be used, suitable and preferred assays are described elsewhere herein and in the Examples section. Thus, the values ​​described above are those as or when quantified in the hemagglutination assays described elsewhere herein. Particularly preferred methods are described in the Examples section herein. Advantageously, such formats preferably retain the ability to induce PCD and / or (optionally) phagocytosis, e.g., at levels described elsewhere herein.

[0081] Other features and characteristics of other aspects of the invention, eg preferred features and characteristics, eg therapeutic use and / or binding affinity, may be applied to this aspect of the invention mutatis mutandis.

[0082] As described above, the present invention provides binding proteins, e.g., antibodies (including humanized antibodies or humanized binding proteins of the invention), or binding proteins comprising an antigen-binding domain of an antibody that binds (or specifically recognizes or specifically binds to) CD47, e.g., human CD47, in an at least bivalent manner, i.e., using two or more antigen-binding domains capable of binding to CD47. A preferred binding protein of the invention is an antibody. However, embodiments described herein relating to antibodies apply equally mutatis mutandis to other types of binding proteins, and vice versa. Thus, other binding proteins can comprise an antibody of the invention, or can comprise the antigen-binding domain of an antibody of the invention, e.g., the three VL CDR regions and / or the three VH CDR regions of an antibody of the invention, or the VL and / or VH domain of the VL and / or VH domain of an antibody of the invention (i.e., the three CDR regions (CDR1, CDR2, and CDR3) and the four FR regions (FR1, FR2, FR3, and FR4)).

[0083] A preferred binding protein is any polypeptide chain that can bind (e.g., specifically bind) to CD47, e.g., human CD47, in at least a bivalent manner, i.e., using two or more antigen-binding domains that can bind to 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, generally comprising CDR regions (and optionally FR regions or an immunoglobulin-based scaffold), are used, whereby the CDR regions (and optionally FR regions) of the antibodies of the present invention can be grafted onto a suitable scaffold or framework, e.g., an immunoglobulin scaffold. Alternatively, the antigen-binding fragments or antibodies of the present invention can be incorporated into any suitable antigen-binding fragment- or antibody-containing format, such as a chimeric antigen receptor (CAR) format or a CAR-T cell format.

[0084] As described above, the present invention provides antibodies (or binding proteins), e.g., isolated antibodies (or binding proteins), that bind to (or specifically recognize or specifically bind to) CD47. CD47 is sometimes also called integrin-associated protein (IAP), MER6, or OA3. CD47 is expressed in all cell types, but is highly expressed or overexpressed on the surface of various cancer cells, e.g., non-Hodgkin's lymphoma, Burkitt's lymphoma, acute myeloid leukemia (AML), primary hepatocellular carcinoma, and bladder cancer. High expression is associated with poor prognosis in some cancer types, e.g., AML.

[0085] According to the present invention, CD47 can be derived from any species. In a preferred embodiment, CD47 is human CD47. Thus, in certain embodiments, the antibody (or binding protein) of the present invention can bind to human CD47. CD47 is a recognized target for cancer therapy.

[0086] Thus, a binding protein or antibody of the invention binds to or is capable of binding to CD47, eg, human CD47.

[0087] The binding proteins and antibodies of the invention can bind to any suitable form of CD47. Preferred and convenient forms of CD47 to which the binding proteins and antibodies of the invention can bind include recombinant CD47, e.g., recombinant human CD47, or native or natural forms of CD47, e.g., CD47 when present on the cell surface (cell surface CD47), e.g., CD47 expressed on tumor or cancer cells.

[0088] The sequence of CD47, e.g., human CD47, is well known and described in the art and can be obtained, e.g., from various sequence databases, e.g., Uniprot entry Q08722 provides the sequence of human CD47. Recombinant human CD47 is commercially available.

[0089] A suitable exemplary human CD47 sequence is provided below as SEQ ID NO. 19. Accordingly, preferred binding proteins or antibodies of the invention bind to or are capable of binding (or specifically binding to) SEQ ID NO. 19 or a sequence substantially homologous thereto (e.g., having at least 80% identity thereto), or a fragment thereof, e.g., a biologically active fragment.

[0090] MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIK TLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE(SEQ ID NO.19)

[0091] Methods for assessing binding to (or ability to bind to) CD47 in a suitable form are well known to those of skill in the art, and any suitable method may be used.

[0092] Convenient and suitable methods for assessing binding will include in vitro binding assays such as ELISA assays to assess binding of the antibody (or binding protein) to an immobilized antigen, e.g., an immobilized form of CD47 as described above, e.g., recombinant CD47, e.g., recombinant human CD47, including SEQ ID NO. 19.

[0093] Thus, in certain embodiments, the antibodies (or binding proteins) of the present invention are capable of binding to CD47 in an ELISA assay. Those skilled in the art are familiar with ELISA assays and can readily establish appropriate conditions for assessing the ability of an antibody to bind to CD47 in such an assay. For example, CD47 (e.g., recombinant human CD47) may be captured on an ELISA plate, followed by washing and incubation with an anti-CD47 antibody (or binding protein) of the present invention, followed by detection of the bound anti-CD47 antibody (or binding protein). Typically, the antibodies (or binding proteins) of the present invention are capable of binding to human CD47 in an ELISA assay.

[0094] In certain embodiments, a binding protein or antibody of the invention binds to CD47 (e.g., recombinant CD47, e.g., recombinant human CD47, e.g., comprising SEQ ID NO. 19) in (as measured in) a surface plasmon resonance (SPR) assay (e.g., a BIACore assay, e.g., using a BIACore S200 instrument). Suitable SPR assays are known in the art and may involve, for example, immobilizing an antibody on a solid support and passing various concentrations of CD47 over the antibody. In certain preferred SPR assays, an appropriate form of CD47, e.g., recombinant CD47, e.g., recombinant human CD47, e.g., having SEQ ID NO. 19, is captured (or immobilized) on a solid support (e.g., a sensor chip), followed by injection of various concentrations (e.g., a dilution series, e.g., a 2-fold dilution series) of the binding protein or antibody to be tested. Generally, antibody concentrations and RU units are chosen in a range and at levels that do not saturate the chip and allow for reliable fitting by the SPR / BIACore software, e.g., a reliable 1:1 fit. The Examples section provides preferred concentrations and flow rates for injection along with appropriate RU units.

[0095] Suitable association and dissociation periods to be used in SPR assays are known to those skilled in the art; for example, a preferred association period in an SPR assay is 2 minutes, and a preferred dissociation period in an SPR assay is 30 minutes (in a single-cycle analysis). As described elsewhere herein, the antibodies of the present invention can have low dissociation rates, allowing for relatively long dissociation periods. Thus, in preferred embodiments, association may be measured for more than 2 minutes, and / or dissociation may be measured for more than 30 minutes. In certain embodiments, all measurements may be performed at 25°C in 20 mM PBS, pH 7.4, 2.7 mM KCl, 137 mM NaCl, 0.05% P20. Kinetic parameters may be determined or calculated by fitting sensogram experimental data using any appropriate model or software, for example, assuming a 1:1 interaction, i.e., a 1:1 binding model, using, for example, Single Cycle Kinetics software. Particularly preferred SPR assays are described in the Examples section of this specification. Preferably, a single cycle analysis is used.

[0096] Thus, in some embodiments, an antibody (or binding protein) of the invention is capable of binding to CD47 (eg, recombinant CD47, eg, recombinant human CD47) in an SPR assay or in an ELISA assay.

[0097] For example, such SPR assay methods can be conveniently used to measure the binding rate of an antibody-antigen interaction to determine the association rate (ka), dissociation rate (kd) and affinity (KD).

[0098] In certain preferred embodiments, the binding proteins or antibodies of the invention, including the humanized antibodies (or binding proteins) of the invention, when in, for example, an IgG format (e.g., IgG1 or IgG4), or an alternative format that is bivalent for CD47 (e.g., a F(ab')2 format or a bivalent scFv-Fc fusion protein format), have high binding affinity for CD47 (e.g., human CD47, e.g., having SEQ ID NO. 19), e.g., a K in the range of 100 pM or less, e.g., 100 pM or less, as measured, e.g., in an SPR assay. D Thus, preferably, a binding protein or antibody of the invention, for example when in an IgG format (e.g., IgG1 or IgG4), or an alternative format that is bivalent for CD47 (e.g., F(ab')2 format or bivalent scFv-Fc fusion protein format), has a K of less than 100 pM, preferably less than 80 pM, 70 pM, 60 pM, or 50 pM, and more preferably less than 45, 40, 35, 30, 25, 20, 15, 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, or 1 pM, for CD47 (e.g., human CD47, e.g., having SEQ ID NO. 19). D or has a binding affinity corresponding thereto.

[0099] In certain embodiments, the binding affinity of a binding protein or antibody of the invention (e.g., an antibody based on mCO-1, CO-1.1, or CO-1.4) for human CD47, e.g., when in an IgG format (e.g., IgG1 or IgG4), or an alternative format that is bivalent for CD47 (e.g., a F(ab')2 format or a bivalent scFv-Fc fusion protein format), is 10 pM or less, e.g., about 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, 4 pM or less, e.g., about 3.0 or 3.5 pM (K D). For example, the exemplary CO-1.1 antibody of the invention exhibits a binding affinity of 3.5 pM, and the CO-1.4 antibody of the invention exhibits a binding affinity of 3.0 pM. The exemplary F(ab')2 fragment of the mCO-1 antibody exhibits a binding affinity of 6.6 pM, and the bivalent scFv-Fc fusion protein (CO201-scFv-Fc-bi) exhibits a binding affinity of 40 pM. Furthermore, exemplary humanized antibodies of the invention exhibit binding affinities of 56 pM or less when in IgG4 format.

[0100] In some embodiments, the antibodies of the invention have higher affinity for human CD47 than the affinity for human CD47 of certain comparison antibodies described in WO 2020 / 198370. Preferred affinities of the antibodies of the invention are discussed elsewhere herein. In other embodiments, the antibodies of the invention have other advantageous properties, such as improved PCD (e.g., higher levels of PCD, use of lower concentrations of antibody, or faster induction) as described elsewhere herein relative to certain comparison antibodies described in WO 2020 / 198370.

[0101] In some embodiments, the invention provides binding proteins, e.g., antibodies, comprising a humanized binding protein or antibody comprising two antigen-binding domains that bind to CD47, wherein said antigen-binding domains comprise a heavy chain variable region comprising three complementarity-determining regions (CDRs) and a light chain variable region comprising three CDRs, and wherein said binding protein or antibody has a binding affinity as defined elsewhere herein. Thus, preferably, such antibodies or binding proteins of the invention have a K of less than 100 pM, preferably less than 80 pM, 70 pM, 60 pM or less than 50 pM, more preferably less than 45, 40, 35, 30, 25, 20, 15, 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5 or 1 pM for CD47 (e.g., human CD47, e.g., having SEQ ID NO. 19), for example, when in an IgG format (e.g., IgG1 or IgG4), or an alternative format that is bivalent for CD47 (e.g., F(ab')2 format or bivalent scFv-Fc fusion protein format). D or has a binding affinity corresponding thereto.

[0102] K D Any suitable method for determining K may be used. However, preferably, D is preferably determined in a surface plasmon resonance assay (e.g., a BIACore assay) in which kinetic parameters are determined. Suitable and preferred types of SPR assays are described above. Thus, the K D The K value may be determined in an SPR assay as described above or elsewhere herein, or may be observed when or if an antibody of the invention is evaluated in an SPR assay. D A particularly preferred method is described in the Examples section of this specification.

[0103] The dissociation rates of the binding proteins or antibodies of the invention are significantly longer / slower, which in turn contributes to the advantageously high binding affinities (KD) observed and may also contribute to good receptor blockade. Thus, in some embodiments, the binding proteins or antibodies of the invention, e.g., when in IgG format (e.g., IgG1 or IgG4), have a binding affinity of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 x 10 -6 k for human CD47 is less than d (or "dissociation rate" or dissociation constant) (seconds -1 x10 -6 For example, the exemplified antibody CO-1.1 of the present invention has a mAb concentration of 4.7×10 -6 CO-1.4 has a dissociation rate measured as 4.4 x 10 -6 Other known CD47 antibodies tested have dissociation rates that are significantly shorter, often an order of magnitude or more shorter, e.g., 1-10×10 -5 , 1 to 10 × 10 -4 or 1 to 10 x 10 -3 The dissociation rate was on the order of .

[0104] Antibodies (or binding proteins) of the invention (including humanized antibodies or binding proteins of the invention) are typically capable of binding to cell surface-expressed CD47, e.g., cell surface-expressed human CD47 (CD47 expressed on the surface of a cell or present at or on the cell surface of a CD47-expressing cell, e.g., a human cell). Thus, such cell surface forms often represent native or natural forms of CD47 (or native or natural configurations of CD47), e.g., the form found on cells that naturally express or overexpress CD47. CD47 is typically expressed on the surface of many tumor cells. In some embodiments, antibodies (or binding proteins) of the invention bind to cell surface-expressed CD47 on human tumor cells. Binding to cell surface CD47 can be assessed by any suitable means, with preferred methods including, for example, flow cytometry assays, as discussed elsewhere herein. In an exemplary flow cytometry method, CD47-expressing cells are incubated with or contacted with the anti-CD47 antibody to be tested, and the antibody bound to CD47 on the cells is detected by fluorescence; for example, the antibody is fluorescently labeled by appropriate means, for example, by direct or indirect labeling. Thus, if the anti-CD47 antibody to be tested binds to CD47 on the cell surface, the cells are fluorescently labeled, and such cells, and therefore antibodies (or binding proteins) capable of binding to cell surface CD47, can be easily identified using a flow cytometer. Particularly preferred flow cytometry methods are described in the Examples section of this specification. Another method for testing the ability of an antibody to bind to CD47 on the cell surface is immunohistochemistry.

[0105] EC2000 was used to quantify the binding of the antibodies (or binding proteins) of the present invention to CD47 expressed on tumor cells. 50 The value can be used. 50 Methods for calculating values ​​will be well known to those skilled in the art. However, for convenience, the EC 50Values ​​can be quantified by flow cytometry assay, for example, by incubating or contacting a suitable cell line with increasing concentrations of an antibody of the invention directly or indirectly conjugated to a fluorescent label (conveniently FITC conjugate), followed by analysis by flow cytometry. An exemplary antibody (or binding protein) concentration range used herein is 0.1 ng / ml to 100 μg / ml. Appropriate curve fitting can then be performed using appropriate software, for example, GraphPad Prism.

[0106] The antibodies (or binding proteins) of the invention (including humanized antibodies or binding proteins of the invention) are preferably capable of binding to a wide range of cancer cells, such as cells from blood cancers and also cells from solid tumors. Purely by way of example, and without any intention of providing an exhaustive list, the antibodies (or binding proteins) of the invention have been shown to be capable of binding to blood cancer cells such as CCRF-CEM, HL-60, Jurkat, K-562, MOLT-4, Raji (Burkitt's lymphoma cell line), Reh (ALL cell line), SUP-T1, and U-937; glioma cells such as A-172, H4, SW1088, U-87-MG, and U-118-MG; bladder cancer cells such as HT-1197, HT-1376, SW780, T24, TCCSUP, and UM-UC-3; and breast cancer cells such as MCF-7 (see Tables 3 and 4). EC 50 Values ​​will, of course, vary depending on the cell type involved, but the exemplified antibodies of the invention have relatively low (ng / ml) EC 50 As can be seen from the values, the antibodies (or binding proteins) of the present invention exhibit extremely good binding with many cancer cells. Purely by way of example, the antibodies (or binding proteins) of the present invention exhibit an EC value of 20 ng / ml or less, preferably 15 ng / ml or less, or 10 ng / ml or less, or 7 ng / ml or less with Jurkat cells (a cancerous T cell line). 50 Can be combined by value.

[0107] Thus, in certain embodiments, the antibodies (or binding proteins) of the invention have an EC of 3.0, 2.5, 2.0, 1.5 or 1.0 μg / ml or less, preferably 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100 ng / ml or less, and more preferably 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, or 10, 9, 8, 7, 6 or 5 ng / ml or less. 50 (e.g., binding to cancer cells, e.g., CD47-expressing cancer cells, e.g., Jurkat T cells). In some embodiments, EC 50 is between 1, 3, 5, or 10 and 950, 750, or 500 ng / ml, or between 1, 3, 5, or 10 and 400, 250, 100, or 50 ng / ml, or between 1, 3, 5, or 10 and 25, 20, or 15 ng / ml. 50 For example, mCO-1 showed low EC values ​​of 5.3 ng / ml for Jurkat T cells, 6.6 ng / ml for U-937 monocytes, and 29.7 ng / ml for MCF-7 breast cancer cells. 50 mCO-1 had an EC value of approximately 1 ng / ml for CCRF-CEM cells. 50 Furthermore, for example, CO-1.1 showed low EC values ​​of 6.2 ng / ml for Jurkat T cells, 12.2 ng / ml for Reh cells (a type of hematological malignant cell), and 0.03 ng / ml for CCRF-CEM cells. 50 The EC values ​​are shown in Table 4. From Table 4, the EC values ​​of the CO-1.1 antibody of the present invention are 50 The values ​​are seen to be significantly (at least an order of magnitude) better than those of the comparative anti-CD47 antibody.

[0108] Preferred antibodies (or binding proteins) of the invention exhibit reduced binding to normal cells expressing CD47 compared to tumor cells, e.g., Jurkat cells. In particular, preferred antibodies (or binding proteins) of the invention exhibit reduced binding to red blood cells (RBCs), e.g., human red blood cells (RBCs), or normal human B cells (e.g., B cells from the buffy coat of a healthy human donor), compared to tumor cells, e.g., Jurkat cells.

[0109] Thus, in a preferred embodiment of the invention, the antibody (or binding protein) exhibits preferential binding, or greater binding, preferably measurably or significantly greater binding, to cancer cells, e.g., Jurkat cells, compared to normal cells, e.g., RBCs or human RBCs, or normal human B cells (e.g., B cells from the buffy coat of a healthy human donor). In other words, limited binding to normal cells, particularly RBCs or human RBCs, or normal human B cells (e.g., B cells from the buffy coat of a healthy human donor), is observed.

[0110] In some embodiments, the antibodies (or binding proteins) of the invention exhibit at least a 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800% increase in binding to Jurkat cells compared to human RBCs or normal human B cells. In other words, the antibodies of the invention exhibit at least a 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold increase in binding to Jurkat cells compared to human RBCs or normal human B cells.

[0111] In certain embodiments, the antibodies (or binding proteins) of the invention have an EC of at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 ng / ml, or at least 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / ml. 50(eg, in the case of binding to normal / healthy cells, such as CD47-expressing normal / healthy cells, such as human erythrocytes or normal human B cells).

[0112] The examples include specific exemplary EC 50 For example, mCO-1 has an EC of 44 ng / ml for human RBCs. 50 values ​​(compared to 5.3 ng / ml for Jurkat cells), and CO1.1 had an EC value of 64 ng / ml for human RBCs. 50 Values ​​are shown (compared to 6.2 ng / ml for Jurkat cells).

[0113] Preferred ECs as described above and elsewhere herein 50 The value is preferably EC 50 The values ​​are those determined in a suitable binding assay, e.g., a suitable flow cytometry-based assay, such as those described above or in the Examples section, performed under appropriate conditions to allow the values ​​to be measured or quantified. These assays can be performed using any suitable antibody (or binding protein) format. Thus, the exemplary values ​​provided above and elsewhere herein can be those that are quantified when, for example, a full-length antibody, e.g., an IgG antibody format of the antibody (e.g., an IgG1 format or an IgG4 format), is evaluated.

[0114] In a preferred embodiment, the antibodies (or binding proteins) of the invention (including humanized antibodies or binding proteins of the invention) inhibit (or block) the interaction between CD47 and SIRPα. Thus, such antibodies can inhibit (or block) the "don't eat me" signal from CD47 expressed on tumor cells, resulting in the phagocytosis of tumor cells by SIRPα-expressing macrophages.

[0115] The ability of an antibody to inhibit (or block) the interaction between CD47 and SIRPα can be quantified (or assessed) using any suitable assay (typically an in vitro assay), such as a binding assay. An exemplary assay is one in which CD47-expressing cells (such as Jurkat cells) are incubated with SIRPα, conveniently recombinant, e.g., recombinant human SIRPα (e.g., increasing concentrations of SIRPα), and then incubated with the anti-CD47 antibody being tested. The ability of SIRPα to inhibit (or block) the binding of the antibody being tested to the CD47-expressing cells can then be measured to provide an indication of the antibody's ability to inhibit (or block) the interaction between CD47 and SIRPα.

[0116] The sequence of SIRPα, eg, human SIRPα, is well known and described in the art and can be obtained, eg, from various sequence databases, for example Uniprot entry P78324 provides the sequence of human SIRPα.

[0117] A suitable exemplary human SIRPα sequence is provided below as SEQ ID NO. 20.

[0118] MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKSPAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFEYASVQVPRK(SEQ ID NO.20)

[0119] Preferably, the inhibition or reduction is measurable or significant, e.g., statistically significant. In certain embodiments, the antibodies of the present invention inhibit or reduce (or block) the interaction between CD47 and SIRPα by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% (e.g., about 99.5%). Typically, such percent inhibition is compared to a control assay or control level, such as a control assay or control level (e.g., a negative control or background level or background assay) in the absence of the antibody (anti-CD47 antibody). Thus, a 0% inhibition (control) level (or conversely, a 100% or maximum interaction level) is typically the level in the absence of the antibody (anti-CD47 antibody). An alternative control can be the use of an isotype control antibody.

[0120] In certain embodiments, the antibodies (or binding proteins) of the invention have an EC50 of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 μg / ml or less for inhibiting the binding or interaction between CD47 (e.g., human CD47, e.g., human CD47 expressed on a cell, preferably a tumor cell, e.g., a Jurkat cell) and SIRPα (e.g., recombinant SIRPα, e.g., recombinant human SIRPα). 50 It has.

[0121] The examples include specific exemplary EC 50 For example, mCO-1 had an EC50 value of 1.9 μg / ml for inhibiting the interaction between CD47 on Jurkat cells and recombinant human SIRPα. 50 The values ​​were shown.

[0122] As described above, in some embodiments, the antibodies (or binding proteins) of the invention can inhibit (or block) the "don't eat me" signal from CD47 expressed on tumor cells, resulting in phagocytosis of the tumor cells by macrophages or other innate immune cells that express SIRPα.

[0123] The ability of the antibodies (or binding proteins) of the invention to induce such phagocytosis of tumor cells (e.g., CD47-expressing tumor cells) is particularly advantageous when combined with the induction of PCD. The antibodies of the invention have been shown to be particularly effective in inducing this phagocytosis, e.g., capable of inducing phagocytosis of a significant number / percentage of tumor cells in the presence of macrophages. Furthermore, they have been shown to exhibit this effect at relatively low doses or concentrations.

[0124] Phagocytosis can be conveniently measured using a suitable in vitro assay that will be well known to those skilled in the art. Such assays generally involve contacting target cells (herein, CD47-expressing tumor cells) with macrophages in the presence of a test antibody (or binding protein) of the present invention and assessing the number of tumor cells that undergo phagocytosis (e.g., as a percentage). The Examples describe a preferred assay in which macrophages (e.g., murine macrophages such as RAW 264.7 cells) and tumor cells are each stained with a different dye and then contacted with each other in the presence of a test concentration of the antibody being tested. At the end of the assay, the cells are analyzed by flow cytometry. Double-stained cells indicate phagocytosed target cells, and these numbers can conveniently be expressed as a percentage.

[0125] In some embodiments, antibodies of the present invention are capable of inducing phagocytosis at a concentration of 10 μg / ml. However, preferred antibodies of the present invention are capable of inducing phagocytosis at concentrations of less than 10 μg / ml, 5 μg / ml, 2 μg / ml, or 1 μg / ml. More preferred antibodies of the present invention are capable of inducing phagocytosis at concentrations of 0.1 or 1 μg / ml or less, for example, between 0.1 and 1 μg / ml.

[0126] The antibodies (or binding proteins) of the invention are preferably capable of inducing phagocytosis of a range of cancer cells, for example, cells from blood cancers and from solid tumors. Purely by way of example, and without the intention of providing an exhaustive list, the antibodies (or binding proteins) of the invention have been shown to be capable of inducing phagocytosis of blood cancer cells such as Jurkat, Reh (ALL cell lines), HL-60 and KG-1a (AML cell lines).

[0127] % phagocytosis values ​​will, of course, vary depending on the cell type involved. However, exemplified antibodies of the invention show good % phagocytosis levels against cancer cells, e.g., levels of at least 10% phagocytosis have been observed against various types of cancer cells, and levels of at least 20%, 25%, 30%, or 35% have been observed against some types of cancer cells. Purely by way of example, antibodies (or binding proteins) of the invention can induce phagocytosis of at least or up to 20%, 25%, 30%, 35%, or 40% of Jurkat cells (a cancerous T-cell line) when, for example, the cells are exposed to a concentration of 10, 1, or 0.1 μg / ml of full-length antibody (e.g., an IgG antibody such as IgG1 or IgG4) in the presence of macrophages for 2 hours.

[0128] The antibodies (or binding proteins) of the invention, including humanized antibodies (or binding proteins) of the invention, are capable of killing tumor cells (e.g., CD47-expressing tumor cells), e.g., directly killing tumor cells (e.g., CD47-expressing tumor cells). This killing is believed to occur via the programmed cell death (PCD) pathway. Furthermore, this PCD pathway is believed to be caspase-independent.

[0129] Such killing is described as direct, meaning that it can result in cell killing by the antibody (or binding protein) itself, and that no other entity, e.g., cell, such as an immune effector cell, such as a macrophage, is required for killing to occur. This is in contrast to, e.g., blocking or inhibiting CD47-SIRPα interaction, where tumor cytolysis occurs by phagocytosis, which involves the recruitment of other cells, e.g., macrophages or other innate immune cells.

[0130] The ability of the antibodies (or binding proteins) of the present invention to induce such direct cell killing or PCD, particularly of tumor cells, is particularly advantageous. Indeed, many previously described anti-CD47 antibodies do not exhibit this property. The antibodies of the present invention have been shown to be particularly effective in inducing this killing, e.g., capable of killing or inducing PCD of a significant number / percentage of tumor cells. Furthermore, they have been shown to demonstrate this effect rapidly and / or at very low concentrations. The antibodies of the present invention are capable of inducing this killing (PCD) even when in a soluble format, as opposed to, for example, when immobilized. Again, this is an advantageous property because it means that such antibodies can function in solution or soluble formats, which would be a common and convenient format for therapeutic antibody administration.

[0131] PCD can be conveniently measured using an Annexin V / 7-AAD assay, which will be well known to those skilled in the art. Indeed, kits for performing such assays are commercially available and can be used (e.g., the eBioscience™ Annexin V Apoptosis Detection Kit (eFluor™ 450 from Thermo Fisher Scientific, catalog number 88-8006-74). Cells that are Annexin V positive and 7-AAD negative are considered early apoptotic cells, and cells that are Annexin V positive and 7-AAD positive are considered late apoptotic cells. The number / % of early and late apoptotic cells can be analyzed separately, but conveniently, these two categories of apoptotic cells are added together to arrive at an overall quantification of the amount of cell death. Such overall PCD can conveniently be expressed as a %.

[0132] Although antibodies of the present invention can induce PCD at relatively high concentrations, such as 10 μg / ml, preferred antibodies of the present invention can induce PCD at concentrations of less than 10 μg / ml, 5 μg / ml, 2 μg / ml, or 1 μg / ml. More preferred antibodies of the present invention can induce PCD at very low concentrations, such as 0.01, 0.03, 0.06, 0.1, or 1 μg / ml or less, such as 0.01, 0.03, 0.06, or between 0.1 and 1 μg / ml.

[0133] Preferred antibodies of the present invention are capable of inducing PCD relatively quickly, for example, after only 30 minutes of contact. Longer contact times, for example, 1, 2, or 3 hours, may also be effective for inducing PCD. Even longer contact times, for example, 6 or 12 hours, may also be effective. Clearly, however, the antibody's ability to act rapidly is advantageous.

[0134] The antibodies (or binding proteins) of the present invention are preferably capable of inducing direct cell death of a wide range of cancer cells, for example, from blood cancers and even from solid tumors. Purely by way of example, and without any intention of providing an exhaustive list, the antibodies (or binding proteins) of the present invention have been shown to be capable of killing blood cancer cells such as CCRF-CEM, Jurkat, MOLT-4, Raji (Burkitt's lymphoma cell line), Reh (ALL cell line), SUP-T1, and U-937; glioma cells such as H4, SW1088, U-87-MG, and U-118-MG; bladder cancer cells such as HT-1197, SW780, T24, and UM-UC-3; and breast cancer cells such as MCF-7 (see Table 3). %PCD values ​​will, of course, vary depending on the cell type involved. However, the exemplified antibodies of the invention, including the humanized antibodies of the invention, show good % killing levels against many cancer cells, e.g., levels of at least 10% killing are consistently seen in various types of cancer cells, and levels of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60%, e.g., up to 50%, 55%, 60% or 65%, have been observed in some types of cancer cells. Purely by way of example, antibodies (or binding proteins) of the invention, including humanized antibodies (or binding proteins) of the invention, may induce killing of at least or up to 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70% of Jurkat cells (a cancer T cell line) when the cells are exposed to, for example, a concentration of 1 or 0.1 μg / ml of full length antibody (e.g., an IgG antibody such as IgG1) or F(ab′)2 fragment or format for 2 or 3 hours.

[0135] Purely by way of further example, an antibody (or binding protein) of the invention may induce killing of at least or up to 40%, 45%, 50%, 55%, 60%, 65% or 70% of Jurkat cells (a cancerous T cell line), for example, when these cells are exposed to a concentration of 1 or 0.1 μg / ml of full length antibody (e.g. an IgG antibody such as IgG1) or F(ab′)2 fragment or format for 30 minutes.

[0136] This killing can be observed when these antibodies are used at 10 μg / ml and even at only 1 μg / ml. However, the antibodies of the present invention have been shown to be advantageously effective at even lower concentrations. For example, maximum cell killing can be observed in the concentration range of 0.06 to 1 μg / ml. Measurable killing can be observed at 0.01 μg / ml or 0.03 μg / ml. Significant killing can be observed at 0.06 μg / ml or 0.1 μg / ml. Advantageously, the antibodies of the present invention exhibit superior cell killing to comparative prior art antibodies at all concentrations tested. Furthermore, as noted above, significant cell killing can be observed using the antibodies of the present invention at a concentration of 0.1 μg / ml, while the comparative antibodies tested do not exhibit substantial killing under the same conditions. While the comparative antibodies are effective when used at higher concentrations, e.g., 10 μg / ml, the antibodies of the present invention are more effective at cell killing under the same conditions.

[0137] This direct killing effect can be observed when these antibodies are incubated with cells for 3 hours. However, these antibodies have been shown to be effective at shorter incubation times, i.e., they are fast-acting and potent. For example, significant cell killing can be observed after incubation times ranging from 30 minutes to, for example, 1, 2, or 3 hours. Advantageously, not only do the antibodies of the present invention exhibit superior cell killing to comparative prior art antibodies at all concentrations tested, but this superior cell killing was observed at all time points tested, particularly after shorter incubation times, e.g., only 30 minutes of exposure.

[0138] Methods for calculating %PCD values ​​will be well known to those skilled in the art. However, conveniently, %PCD values ​​herein can be quantified in an in vitro assay, e.g., an annexin V / 7-AAD assay, by incubating a suitable cell line with a test antibody of the invention at a particular concentration (e.g., 0.01-10 μg / ml) for a particular length of time (e.g., 30 minutes to 12 hours, e.g., about 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, or 12 hours), followed by staining with annexin V eFluor 405 and -7AAD. %PCD can be determined by measuring the %PCD of an appropriate cell line using annexin V eFluor 405 and -7AAD as analyzed by flow cytometry. + 7-AAD - Cells and Annexin V + 7-AAD + The sum of the cells.

[0139] In some embodiments, the antibodies (or binding proteins) of the invention do not induce significant killing (PCD) of normal human B cells (eg, B cells derived from the buffy coat of a healthy human donor).

[0140] The reduced ability of the antibodies (or binding proteins) of the invention to bind to normal cells, such as human red blood cells, is also reflected in the manageable level of hemagglutination observed. For example, the level (or concentration) of a preferred antibody of the invention at which significant hemagglutination is observed is higher than the level (or concentration) required for the induction of programmed cell death. In other words, a preferred antibody of the invention can induce PCD when used at a level (or concentration) lower than the level (or concentration) at which hemagglutination (or significant hemagglutination) is observed. Thus, the level (concentration) of a preferred antibody of the invention that can induce PCD is sufficiently low so as not to induce hemagglutination (or significant hemagglutination). As described elsewhere herein, some formats, such as the bivalent scFv-Fc format of the invention, have been shown to advantageously not induce hemagglutination (or significant hemagglutination).

[0141] The ability to induce hemagglutination can conveniently be measured using an in vitro hemagglutination assay, which will be well known to those skilled in the art. Such an assay can conveniently involve contacting increasing concentrations of a test antibody (or control) with RBCs, e.g., human RBCs, e.g., freshly isolated human RBCs, e.g., a 2% (vol / vol) preparation of freshly isolated RBCs, and incubating the mixture for a specified amount of time (e.g., 30-60 minutes) or until cells settle in the well or receptacle used (conveniently, 96-well plates are used for such assays). A diffuse, fuzzy pattern indicates hemagglutination, while small, punctate circles indicate the absence of hemagglutination. A particularly preferred method is described in the Examples section herein.

[0142] Preferably, the above-described capabilities and properties are observed at a measurable or significant level, more preferably at a statistically significant level when compared to an appropriate control level. Suitable significance levels are discussed elsewhere herein. More preferably, one or more of the above-described capabilities and properties are observed at a level that is measurably better, or more preferably significantly better (preferably statistically significantly better), when compared to the capabilities observed for prior art antibodies.

[0143] In any statistical analysis referred to herein, preferably, a statistically significant difference relative to an appropriate control or other comparison entity or measurement has a probability value of ≦0.1 or <0.1, preferably ≦0.05 or <0.05. Suitable methods for determining statistical significance are well known and described in the art, and any of these may be used.

[0144] In some embodiments, the binding proteins or antibodies of the invention, including the humanized antibodies and bivalent scFv-Fc fusion proteins of the invention, have one or more, preferably two or more, or three or more, or four or more, or five or more, and most preferably all, of the functional properties described herein, particularly preferred functional properties. Examples of preferred functional properties, and further details regarding said properties, are described elsewhere herein and include: i) high affinity for CD47, e.g., as measured by SPR (BIACore); ii) the ability to induce direct killing of tumor cells, e.g., Jurkat cells, which can be induced at iii) low concentrations (e.g., antibody concentrations of 0.01, 0.1, or 1 μg / ml) and / or iv) after short incubation times (e.g., 30 minutes or 1 hour); v) the ability to inhibit or block CD47-SIRPα interaction and thus the "don't eat me" signal; vi) the ability to induce phagocytosis of tumor cells; and vii) limited binding to normal cells, e.g., human RBCs or normal B cells.

[0145] Thus, in some embodiments, the invention provides binding proteins, e.g., antibodies, comprising two antigen-binding domains that bind to CD47, wherein the antigen-binding domains comprise a heavy chain variable region comprising three complementarity-determining regions (CDRs) and a light chain variable region comprising three CDRs, and wherein the binding proteins or antibodies have one or more, preferably two or more, or three or more, or four or more, or five or more, and most preferably all, of the functional properties described herein, particularly the preferred functional properties, such as one or more of properties (i) to (vii) above. In some embodiments, the binding proteins or antibodies also have the ability to not induce hemagglutination (or significant hemagglutination) of red blood cells (RBCs).

[0146] In some embodiments, functional property (ii) is preferred, optionally in combination with one or more of the other properties listed above. In some embodiments, functional property (ii) is preferred, optionally in combination with one or more, two or more, three or more, or most preferably all of the above-listed properties (i), (iii), (iv), and (vi), or one or more, two or more, or most preferably all of the above-listed properties (iii), (iv), and (vi), or one or more, two or more, three or more, four or more, or most preferably all of the above-listed properties (iii), (iv), (v), (vi), and (vii). In some such embodiments, the binding protein or antibody has the ability not to induce hemagglutination (or significant hemagglutination) of red blood cells (RBCs).

[0147] As used throughout this application, the terms "a" and "an" are used with the intention of meaning "at least one," "at least a first," "one or more," or "a plurality" of the referenced components or steps, unless an upper limit is specifically indicated after the term. Thus, "an antibody," as used herein, means "at least a first antibody."

[0148] Furthermore, when the terms "comprise", "comprises", "has" or "having", or other equivalent terms, are used herein, in some more specific embodiments, for example in the definitions of CDR or FR sequences herein, these terms include the terms "consists of" or "consists essentially of", or other equivalent terms.

[0149] Nucleic acid molecules (e.g., one or more nucleic acid molecules) comprising a nucleotide sequence encoding a binding protein or antibody or immunoconjugate of the invention as defined herein, or nucleic acid molecules substantially homologous thereto, form a further aspect of the invention.

[0150] Preferred nucleic acid molecules are those that encode an antibody of the invention described elsewhere herein that is capable of bivalent or multivalent binding to CD47, e.g., an antibody of the invention having CDRs and optionally FRs and other regions as defined in any one of Tables A or B or C or D or E, or an antibody having a sequence substantially homologous thereto.

[0151] Preferred nucleic acid molecules are those that encode antibodies of the invention capable of bivalently or multivalently binding to CD47 (e.g., comprising a nucleic acid sequence encoding SEQ ID NO.3 and / or SEQ ID NO.4, e.g., SEQ ID NO.1 and / or SEQ ID NO.2, respectively).

[0152] Other preferred nucleic acid molecules include sequences encoding IgG forms (e.g., IgG1 or IgG4 forms) of antibodies of the invention, such as those (heavy and light chains) set forth in Tables A, B, and C herein. Accordingly, preferred nucleic acid molecules are those encoding the heavy chains of antibodies of the invention (e.g., those encoding SEQ ID NO. 21 or 23) and / or those encoding the light chains of antibodies of the invention (e.g., those encoding SEQ ID NO. 22 or 24). Other preferred nucleic acid molecules include sequences encoding F(ab')2 formats or fragments of antibodies of the invention, such as those (heavy and light chains) set forth in Table D. Accordingly, preferred nucleic acid molecules are those encoding the heavy chains of antibodies of the invention (e.g., those encoding SEQ ID NO. 25) and / or those encoding the light chains of antibodies of the invention (e.g., those encoding SEQ ID NO. 26). Other preferred nucleic acid molecules include sequences encoding humanized antibodies of the invention, such as those (heavy and light chains) set forth in Table E. Thus, preferred nucleic acid molecules are those encoding the heavy chain of an antibody of the invention (e.g., those encoding SEQ ID NOs. 39, 40, 41, 42, or 43) and / or those encoding the light chain of an antibody of the invention (e.g., those encoding SEQ ID NOs. 44, 45, or 46). Other preferred nucleic acid molecules comprise sequences encoding a bivalent scFv-Fc fusion protein of the invention (e.g., CO201-scFv-Fc-bi).

[0153] The term "substantially homologous" as used herein in connection with an amino acid or nucleic acid sequence includes sequences having at least 60%, 65%, 70%, or 75%, preferably at least 80%, and 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 present invention contain single or multiple base or amino acid modifications (additions, substitutions, insertions, or deletions) to the sequences of the present invention. At the amino acid level, preferred substantially homologous sequences contain up to six, e.g., one, two, three, four, five, or six, e.g., one, two, three, four, or five, preferably one, two, three, or four, preferably one, two, or three, more preferably only one or two modified amino acids in one or more of the framework regions and / or one or more of the CDRs forming the sequences of the present invention. Furthermore, at the amino acid level, preferred substantially homologous sequences contain up to six, for example, 1, 2, 3, 4, 5 or 6, for example, 1, 2, 3, 4 or 5, preferably 1, 2, 3 or 4, preferably 1, 2 or 3, more preferably only 1 or 2, modified amino acids in the combined framework regions (e.g., four framework regions) and / or combined CDRs (e.g., three CDR regions) forming the VL or VH domain of an antibody of the invention. Furthermore, at the amino acid level, preferred substantially homologous sequences contain up to six, for example, 1, 2, 3, 4, 5 or 6, for example, 1, 2, 3, 4 or 5, preferably 1, 2, 3 or 4, preferably 1, 2 or 3, more preferably only 1 or 2, modified amino acids in the VH and / or VL domain of an antibody of the invention. The modifications may be conservative or non-conservative amino acids or a mixture thereof. Preferably, the modifications are substitutions, preferably conservative amino acid substitutions.

[0154] In certain embodiments, when a given original sequence is relatively short (e.g., 5 amino acids in length), a sequence substantially homologous thereto may have fewer amino acid substitutions than may be optionally made in a sequence substantially homologous to a longer original sequence. For example, in certain embodiments, a sequence substantially homologous to an original VH CDR1 sequence according to the present invention, e.g., an original VH CDR1 sequence that may be 5 amino acid residues in length in some embodiments, preferably has one or two (more preferably one) modified amino acids compared to the original sequence. Thus, in some embodiments, the number of modified amino acids in a substantially homologous sequence (e.g., in a substantially homologous CDR sequence) can be adjusted to suit the length of a given original CDR sequence. For example, to achieve a particular % sequence identity in the CDR, e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, a varying number of modified amino acids may be present, e.g., depending on the length of a given original CDR sequence.

[0155] In embodiments of the invention involving substantially homologous sequences, methods routine in the art, such as alanine scanning mutagenesis and / or deep mutational scanning (which aims to make all possible single substitutions at all selected residues in a given protein sequence) and / or analysis of the crystal structure of an antigen-antibody complex, can be used to determine which amino acid residues in the CDRs do not contribute, or do not contribute significantly, to antigen binding and are therefore good candidates for modification or substitution.

[0156] Once identified, the addition, deletion, substitution, or insertion of one or more amino acids into the amino acid sequence of a parent antibody, which is one of the antibodies of the invention as defined elsewhere herein, to form a new antibody, and testing the resulting new antibody to identify antibodies that bind to CD47 in accordance with the invention, can be performed using techniques that are routine in the art. Such methods can be used to generate multiple new antibodies that can all be tested for their ability to bind to CD47. Preferably, the addition, deletion, substitution, or insertion of one or more amino acids is made in one or more of the CDR domains.

[0157] For example, such manipulations may conveniently be carried out by genetic engineering at the nucleic acid level, whereby nucleic acid molecules encoding the appropriate binding proteins and domains thereof are modified, and the amino acid sequence of the resulting expressed protein is in turn modified in an appropriate manner. Testing the ability of one or more of the modified antibodies / binding proteins to bind to CD47 can be carried out by any suitable method well known and described in the art. Suitable methods are described elsewhere herein and in the Examples section.

[0158] The novel antibodies produced, obtained or obtainable by these methods form a further aspect of the present invention.

[0159] The term "substantially homologous" also includes modifications or chemical equivalents of the amino acid and nucleotide sequences of the antibodies of the invention that perform substantially the same function as the protein or nucleic acid molecules of the antibodies of the invention in substantially the same way. For example, any substantially homologous antibody should retain the ability to bind CD47 in a bivalent or multivalent manner as described above. Preferably, any substantially homologous antibody should retain one or more (or all) of the functional capabilities of the original antibody.

[0160] Substantially homologous sequences of the proteins of the invention include, but are not limited to, conservative amino acid substitutions, or modifications that do not affect, for example, the VH, VL, or CDR domains of the antibody, such as antibodies to which tag sequences, toxins, or other moieties have been added that do not contribute to bivalent or multivalent binding of the CD47 antigen.

[0161] As used herein, a "conservative amino acid substitution" refers to a substitution of an amino acid residue with another amino acid residue having a similar side chain. Systems of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In another example, systems of amino acid residues can be classified based on hydrophobic or hydrophilic side chains.

[0162] Homology or sequence identity may be assessed by any convenient method. However, to determine the degree of homology or identity between sequences, computer programs that perform multiple alignments of sequences, such as Clustal W (Thompson, Higgins, Gibson, Nucleic Acids Res. 22:4673-4680 (1994)), are useful. If desired, the BLOSUM62 scoring matrix (Henikoff and Henikoff, Proceedings of the National Academy of Sciences of the United States of America) can be used. The Clustal W algorithm can be used with a gap opening penalty of 10 and a gap extension penalty of 0.1, which obtains the highest order match between two sequences where at least 50% of the total length of one of the sequences is included in the alignment. Another method that may be used to align sequences is the Needleman and Wunsch alignment method (Needleman and Wunsch, Journal of Molecular Biology, 48:443 (1970)), as modified by Smith and Waterman (Smith and Waterman, Advances in Applied Mathematics, 2:482 (1981)), which obtains the highest order match between two sequences and determines the number of identical amino acids between the two sequences.Other methods for calculating the percent identity between two amino acid sequences are generally recognized in the art and include, for example, those described by Carillo and Lipton (Carillo and Lipton, SIAM Journal on Applied Mathematics, 48:1073 (1988)) and in Biocomputing: Informatics and Genomics Projects, Computational Molecular Biology, Lesk (ed.), Oxford University Press, New York, 1988.

[0163] Generally, computer programs are used for such calculations. Examples include ALIGN (Myers and Miller, CABIOS, Computer Applications in the Biosciences, 4, 11-17 (1988)), FASTA (Pearson and Lipman, Proceedings of the National Academy of Sciences of the United States of America, 85, 2444-2448 (1988)), and Pearson, Methods in Enzymology (Pearson and Lipman, Proceedings of the National Academy of Sciences of the United States of America, 85, 2444-2448 (1988)). Programs for comparing and aligning pairs of sequences, such as Gap-BLAST (Altschul et al., Nucleic Acids Research, 25, 3389-3402 (1997)), BLASTP, BLASTN, or GCG (Devereux, Haeberli, Smithies, Nucleic Acids Research, 12, 387 (1984)), are also useful for this purpose. In addition, the Dali server at the European Bioinformatics Institute provides structure-based alignments of protein sequences (Holm, Trends in Biochemical Sciences). Sciences, Vol. 20, pp. 478-480 (1995); Holm, Journal of Molecular Biology, Vol. 233, pp. 123-38 (1993); Holm, Nucleic Acids Research, Vol. 26, pp. 316-319 (1998)).

[0164] As a point of reference, sequences according to the invention having at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology, sequence identity, etc. may be determined using the ALIGN program with default parameters (available, for example, on the internet at the GENESTREAM network server, IGH, Montpellier, France).

[0165] Preferably, any substantially homologous antibody of the present invention will retain the ability to specifically bind the same epitope of CD47 as recognized by the original antibody in question, e.g., the same epitope recognized by one or more CDR domains of an antibody of the present invention or one or more VH and VL domains of an antibody of the present invention described herein, e.g., bind the same epitope as one or more of the various antibodies of the present invention (e.g., one or more of the CD47 antibodies set forth in Tables A, B, C, D or E).

[0166] Binding to the same epitope can be determined by methods well known and described in the art, for example using epitope mapping assays, for example by analyzing the crystal structure of the antigen-antibody complex, or by mutational studies of individual residues (for example alanine scanning and / or deep mutational scanning, DMS, e.g. yeast display combined with DMS, see e.g. Sierocki et al., PLOS Neglected Tropical Diseases, 15(3), e0009231 (2021)), van Braekom et al. See also Blarcom et al., 2015, Journal of Molecular Biology, Vol. 427, No. 6 (B), pp. 1513-1534 and Medina-Cucurella and Whitehead, 2018, Methods in Molecular Biology, Vol. 1764, pp. 101-121. In some embodiments, DMS, particularly yeast display in combination with DMS, is a preferred method for determining epitopes. Any of the above-mentioned assays for determining epitopes can be used in conjunction with binding assays, such as competitive assays, for example, as part of initial screening. Thus, antibodies that bind to the same epitope as one or more of the various antibodies of the present invention, as assessed or determined, for example, by analysis of the crystal structure of the antigen-antibody complex or by examining mutations of individual residues (e.g., using alanine scanning and / or deep mutational scanning (DMS), e.g., yeast display combined with DMS), form a further aspect of the present invention. In a preferred embodiment, DMS, e.g., yeast display combined with DMS, is used to assess or determine the epitope. Possession of other functional properties, particularly binding affinity, can also be readily tested by methods well known in the art or described herein.

[0167] Thus, one skilled in the art will understand that such methods can be used to test whether any antibody, e.g., a "substantially homologous" antibody, has the same binding specificity, e.g., binds to the same epitope, or has the same or equivalent affinity, as the antibodies and antibody fragments of the present invention. For example, analysis of the crystal structure of an antigen-antibody complex, or examination of mutations of individual residues (e.g., using alanine scanning and / or deep mutational scanning (DMS), e.g., yeast display combined with DMS), optionally supplemented with binding assays such as the competition assays described elsewhere herein, can readily be used to assess whether an antibody, e.g., a "substantially homologous" antibody, can bind to CD47, and optionally the affinity of such binding, e.g., K D SPR assays, such as the BIACore assays described elsewhere herein, could also be readily used to determine the . Those skilled in the art will recognize other suitable methods and variations.

[0168] As outlined below, competitive binding assays can be used as initial or auxiliary assays to epitope mapping assays to test whether an antibody, e.g., a "substantially homologous" antibody, retains the ability to specifically bind to the same (or substantially the same) epitope of CD47 recognized by one or more of the antibodies of the invention set forth in the various sequence listings herein, or has the ability to compete with one or more of the various antibodies of the invention set forth in the various sequence listings herein. The method described below is only one example of a suitable competitive assay. Those of skill in the art will recognize other suitable methods and variations.

[0169] An exemplary competition assay involves assessing binding of various effective concentrations of an antibody of the invention to CD47 in the presence of various concentrations of a test antibody (e.g., a substantially homologous antibody). The amount of inhibition of binding induced by the test antibody can then be assessed. A test antibody that exhibits increasing competition with an antibody of the invention with increasing concentration (i.e., increasing concentrations of the test antibody result in a corresponding decrease in the amount of antibody of the invention binding to CD47) is evidence of binding to the same or substantially the same epitope. Preferably, the test antibody significantly reduces the amount of antibody of the invention binding to CD47. Preferably, the test antibody reduces the amount of antibody of the invention binding to CD47 by at least about 95%. ELISA or flow cytometry assays may be used to assess inhibition of binding in such competition assays, although other suitable techniques will be known to those of skill in the art.

[0170] An antibody (or binding protein) that has the ability to bind (or specifically bind) to the same (or substantially the same) epitope of CD47 as recognized by an antibody of the invention (e.g., one or more of the antibodies set forth in Tables A, B, C, D, or E), and which binds to CD47 at least bivalently, is a further embodiment of the invention. Such an antibody (or binding protein) optionally also has the ability to compete for binding to CD47 with one or more of the various antibodies of the invention (e.g., one or more of the antibodies set forth in Tables A, B, C, D, or E).

[0171] Thus, yet another aspect of the present invention provides antibodies (or binding proteins) that bind at least bivalently (or specifically bind at least bivalently) to CD47 and have the ability to bind the same (or substantially the same) epitope as the mCO-1 (Table A) and / or CO-1.1 (Table B) and / or CO-1.4 (Table C) and / or CO-1 F(ab')2 (Table D) antibody, i.e., an antibody comprising a VL of SEQ ID NO. 4 and a VH of SEQ ID NO. 3 as described herein, or an antibody that comprises the same CDRs as the mCO-1 (Table A) and / or CO-1.1 (Table B) and / or CO-1.4 (Table C) and / or CO-1 F(ab')2 (Table D) antibody, i.e., an antibody comprising the VL CDR sequences of SEQ ID NOs. 8, 9, and 10 and the VH CDR sequences of SEQ ID NOs. 5, 6, and 7. Such antibodies (or binding proteins) optionally also have the ability to compete with one or more of the various antibodies of the invention (e.g., one or more of the antibodies set out in Tables A, B, C or D) for binding to CD47. Other features and characteristics of the other aspects of the invention apply to this aspect of the invention mutatis mutandis.

[0172] The term "competing antibody" as used herein refers to an antibody that binds to a similar, substantially or essentially the same epitope as a "reference antibody." A "competing antibody" includes antibodies with overlapping epitope specificity. Thus, a competing antibody is capable of effectively competing with a reference antibody for binding to CD47. Preferably, a competing antibody can bind to the same epitope as the reference antibody. In other words, a competing antibody preferably has the same epitope specificity as the reference antibody.

[0173] As used herein, a "reference antibody" is an antibody capable of binding to CD47 according to the present invention, preferably having a VH domain and a VL domain as defined in Table A, B, C, D or E herein, more preferably having a VH domain comprising SEQ ID NO. 3 and a VL domain comprising SEQ ID NO. 4 (or three related CDR sequences of said sequences) as outlined in Table A, B, C or D, or having a humanized VH domain and / or VL domain as shown in Table E.

[0174] Identifying one or more antibodies that bind the same epitope, and optionally, competing antibodies, is a straightforward technical matter once a reference antibody, such as those outlined in the sequence listing herein, is provided. Epitope mapping can be performed using standard techniques, some of which are outlined elsewhere herein. Such epitope mapping can also be supplemented, for example, as an initial or secondary screening step, with competition assays, which can be performed using standard techniques, some of which are outlined elsewhere herein. In this regard, for example, CD47 antibodies can be generated by immunization protocols using the CD47 antigen, or preferably cells expressing or overexpressing the CD47 antigen, as an immunogen, and then such CD47 antibodies can be readily screened, for example, using the methods described herein, to identify those that bind the same epitope as the reference antibody of the invention. Alternatively, substantially homologous sequences derived from antibodies having sequences set forth in Tables A, B, C, D, or E can be screened in this manner.

[0175] Epitope mapping using DMS, and particularly yeast display in combination with DMS, was actually carried out on the CO-1 antibody of the present invention.

[0176] Accordingly, in another aspect, the invention provides a binding protein, e.g., an antibody, e.g., a binding protein or antibody, as described elsewhere herein, comprising two antigen-binding domains that bind to CD47, wherein the antigen-binding domains comprise a heavy chain variable region comprising three complementarity-determining regions (CDRs) and a light chain variable region comprising three CDRs, and the antigen-binding domains bind (or are capable of binding or specifically bind) to Q19, N45, T120, R121, E122, and G123 in CD47 as defined by SEQ ID NO. 19. In other embodiments, the antigen-binding domains bind to Q19, L20, N45, T120, R121, E122, and G123 in CD47 as defined by SEQ ID NO. 19.

[0177] In other words, 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.

[0178] In some embodiments, the antigen-binding domain does not bind to one or more, two or more, three or more, preferably all, of T117, E118, E115, and L21, or the epitope does not include 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 to T117, or T117 and E118, or T117, E118 and E115, or T117, E118, E115, and L21 in CD47 defined by SEQ ID NO. 19, or the epitope does not include T117, or T117 and E118, or T117, E118 and E115, or T117, E118, E115, and L21 in CD47 defined by SEQ ID NO. 19.

[0179] In some embodiments, the antigen-binding domain does not bind to one or more, two or more, three or more, preferably all of V54 to A84, M46, or L119, particularly L119, in CD47 as defined by SEQ ID NO. 19, or the epitope does not include one or more, two or more, three or more, preferably all of V54 to A84, M46, or L119, particularly L119, in CD47 as defined by SEQ ID NO. 19.

[0180] In some embodiments, the antigen-binding domain does not bind to one or more, two or more, three or more, four or more, preferably all, of E115 to L119, particularly T117 to L119, especially T117 and / or L119, in CD47 as defined by SEQ ID NO. 19, or the epitope does not include one or more, two or more, three or more, four or more, preferably all, of E115 to L119, particularly T117 to L119, especially T117 and / or L119, in CD47 as defined by SEQ ID NO. 19.

[0181] In some embodiments, the antigen-binding domain does not bind to one or more, preferably both, of T117 and E124 in CD47 as defined by SEQ ID NO. 19, or the epitope does not include one or more, preferably both, of T117 and E124 in CD47 as defined by SEQ ID NO. 19.

[0182] In some embodiments, the antigen-binding domain does not bind to 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, or the epitope does not include 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.

[0183] In some embodiments, the antigen-binding domain does not bind to 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, or the epitope does not include 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.

[0184] In some embodiments, binding of the antigen-binding domain to CD47 as defined by SEQ ID NO:19 is independent of G70. For example, in such embodiments, mutation of G70 to another amino acid residue does not affect, does not significantly affect, or maintains binding of the antigen-binding domain or antibody of the invention to CD47. In some embodiments, the antigen-binding domain does not bind to residue G70 in CD47 as defined by SEQ ID NO:19, or the epitope does not include residue G70 in CD47 as defined by SEQ ID NO:19.

[0185] In preferred embodiments, binding proteins or antibodies that bind to the epitopes or residues outlined above are as assessed or determined by analysis of the crystal structure of the antigen-antibody complex or by examining mutations of individual residues (e.g., using alanine scanning and / or deep mutational scanning DMS, e.g., yeast display combined with DMS). In preferred embodiments, binding to such epitopes or residues is as assessed or determined by DMS, e.g., yeast display combined with DMS.

[0186] In a preferred embodiment, a binding protein or antibody that binds to a CD47 epitope as defined herein has a binding affinity as described elsewhere herein. Accordingly, the present invention provides a binding protein, e.g., an antibody, comprising two antigen-binding domains that bind to a CD47 epitope as defined herein, wherein the antigen-binding domains comprise a heavy chain variable region comprising three complementarity-determining regions (CDRs) and a light chain variable region comprising three CDRs, and wherein the binding protein or antibody has a binding affinity as defined elsewhere herein. Thus, preferably, such antibodies or binding proteins, for example when in an IgG format (e.g., IgG1 or IgG4) or an alternative format that is bivalent for CD47 (e.g., a F(ab')2 format or a bivalent scFv-Fc fusion protein format), have a K for CD47 (e.g., human CD47, e.g., having SEQ ID NO. 19) of less than 100 pM, preferably less than 80 pM, 70 pM, 60 pM, or 50 pM, more preferably less than 45, 40, 35, 30, 25, 20, 15, 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, or 1 pM. D or has a binding affinity corresponding thereto.

[0187] As used herein, the terms "antibody" and "immunoglobulin" refer broadly to any immunobinding agent containing an antigen-binding domain, including polyclonal and monoclonal antibodies. Monoclonal antibodies are preferred. However, the binding proteins and antibodies of the present invention have a structure or format such that they can comprise antibodies or antibody fragments that bind bivalently or multivalently to CD47, e.g., antibodies or antibody fragments that bind bivalently or multivalently to CD47. Depending on the type of constant domain in the heavy chain, all antibodies can be assigned to one of five major classes: IgA, IgD, IgE, IgG, and IgM. An antibody of the present invention can fall within any one of these classes. Some of these classes are further divided into subclasses or isotypes, e.g., IgG1, IgG2, IgG3, IgG4, etc. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0188] Generally, when whole antibodies are used in the present invention, IgG is preferred because it is the most common antibody in the physiological situation and is the most easily produced in a laboratory setting.

[0189] The "light chains" of mammalian antibodies are assigned to one of two clearly distinct types, kappa (κ) and lambda (λ), based on the amino acid sequences of the constant domain and some amino acids in the framework regions of the variable domain.

[0190] As used herein, the term "heavy chain complementarity determining region" ("heavy chain CDR") refers to a hypermutable region within the heavy chain variable region (VH domain) of an antibody molecule. The heavy chain variable region has three CDRs, designated heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, from the amino terminus to the carboxy terminus. The heavy chain variable region has four framework regions (FR1, FR2, FR3, and FR4, from the amino terminus to the carboxy terminus). These framework regions separate the CDRs.

[0191] As used herein, the term "heavy chain variable region" (VH domain) refers to the variable region of the heavy chain of an antibody molecule.

[0192] As used herein, the term "light chain complementarity determining region" ("light chain CDR") refers to a region of hypervariability within the light chain variable region (VL domain) of an antibody molecule. The light chain variable region has three CDRs, designated light chain CDR1, light chain CDR2, and light chain CDR3, from the amino terminus to the carboxy terminus. The light chain variable region also has four framework regions (FR1, FR2, FR3, and FR4, from the amino terminus to the carboxy terminus). These framework regions separate the CDRs.

[0193] As used herein, the term "light chain variable region" (VL domain) refers to the variable region of the light chain of an antibody molecule.

[0194] As described elsewhere herein, the binding proteins and antibodies of the invention have a structure or format that allows them to bind to CD47 bivalently or multivalently. Any suitable bivalent or multivalent format may be used, for example, any antibody or antibody fragment format containing at least two antigen-binding domains capable of binding to CD47. Exemplary and preferred formats or fragments are full-length (whole) antibodies, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, IgM, or IgD antibodies, or F(ab')2 fragments, including chimeric antibodies and fragments. Other exemplary bivalent or multivalent formats or fragments include Fab3, diabodies, triabodies, minibodies, 2x scFv conjugates, scFv-Fc, and bivalent nanobodies. As described elsewhere herein, a particularly preferred format is bivalent (two-valent) scFv-Fc.

[0195] In specific embodiments, antibodies or binding proteins of the invention comprise all or a portion of a heavy chain constant region, e.g., an IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, IgM, or IgD constant region. Preferably, the heavy chain constant region is an IgG heavy chain constant region or a portion thereof. IgG1 and IgG4 are examples of suitable formats for antibodies of the invention. Preferably, antibodies (or binding proteins) of the invention comprise or contain a human heavy chain constant region. Preferably, antibodies (or binding proteins) of the invention comprise or contain a human Fc region, e.g., human CH2 and CH3 domains, e.g., a human IgG1 or IgG4 Fc region. Other preferred antibodies (or binding proteins) of the invention do not comprise or contain the CH1 human (or other) heavy chain constant region.

[0196] Additionally, antibodies or binding proteins of the invention can comprise all or a portion of a kappa or lambda light chain constant region, or portions thereof. Some preferred antibodies (or binding proteins) of the invention comprise or contain a human light chain constant region. Other preferred antibodies (or binding proteins) of the invention do not comprise or contain a human (or other) light chain constant region.

[0197] All or part of such constant regions may be naturally occurring, or all or part may be synthetic. Suitable sequences for such constant regions are well known and described in the art. When the antibodies of the present invention contain a complete set of constant regions from the heavy and light chains, such antibodies are typically referred to herein as "full-length" or "whole" antibodies. In some embodiments, such full-length or whole antibodies are preferred. In other embodiments, F(ab')2 formats or fragments, or bivalent scFv-Fc formats or fragments are preferred.

[0198] The antibody or binding protein may be naturally occurring or wholly or partially synthetically produced.

[0199] The antigen-binding domain of an antibody or binding protein of the invention generally comprises an antibody light chain variable region (VL) comprising three CDR domains and an antibody heavy chain variable region (VH) comprising three CDR domains.

[0200] However, it is well documented in the art that the presence of three CDRs from the light chain variable domain and three CDRs from the heavy chain variable domain of an antibody is not always necessary for antigen binding, and therefore constructs smaller than the classical antigen-binding domains described above are known to be effective.

[0201] For example, camelid VHH antibodies and other single domain antibodies containing only a VH domain have shown that these domains can bind antigens with sufficiently high affinity that the three CDRs (or even a single CDR) can effectively bind antigen to form an antigen-binding domain.

[0202] Thus, although a preferred antigen-binding domain in an antibody of the present invention may comprise six CDR regions (three from the light chain and three from the heavy chain), antibodies having an antigen-binding domain with fewer than six CDR regions (e.g., three CDR regions) are encompassed by the present invention. Antibodies having an antigen-binding domain with CDRs from only the heavy chain or only the light chain are also contemplated.

[0203] Preferred light chain CDR regions for use with the designated heavy chain CDR regions to form this antigen-binding domain are described elsewhere herein. However, other light chain variable regions comprising three CDRs for use with the heavy chain variable regions of the present invention are also contemplated. Suitable light chain variable regions that can be used in combination with the heavy chain variable regions of the present invention to generate antibodies that bind bivalently or multivalently to CD47 according to the present invention can be readily identified by one of ordinary skill in the art.

[0204] For example, a heavy chain variable region of the invention can be combined with a single light chain variable region or a repertoire of light chain variable regions, and the resulting antibodies tested for binding to CD47.

[0205] If desired, similar methods could be used to identify alternative heavy chain variable regions for use in combination with the preferred light chain variable regions of the present invention.

[0206] The antibodies, binding proteins, and nucleic acid molecules of the invention are generally "isolated" or "purified" molecules because they are distinguished from all components that may be present in the human or animal body or in tissue samples derived from the human or animal body. However, the sequences may correspond to or be substantially homologous to sequences found in the human or animal body. Thus, the terms "isolated" or "purified," as used herein with respect to nucleic acid molecules or sequences and proteins or polypeptides, e.g., antibodies, refer to such molecules when they are separated, purified, or substantially free from their natural environment, e.g., when separated or purified from the human or animal body (as indeed they exist in nature), or when produced by a technical process, i.e., including recombinantly and synthetically produced molecules.

[0207] Thus, the terms "isolated" or "purified," when used in reference to proteins or polypeptide molecules, such as binding proteins or antibodies of the invention comprising light chain CDRs 1, 2, and 3, heavy chain CDRs 1, 2, and 3, a light chain variable region, a heavy chain variable region, and a full-length antibody, typically refer to proteins that are substantially free of cellular material or other proteins from the original source from which they are derived. In some embodiments, particularly when the protein is to be administered to a human or animal, such isolated or purified proteins are substantially free of culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.

[0208] In embodiments, antibodies and the like of the present invention are not naturally occurring and, in that they are artificial constructs in that they do not correspond to naturally occurring molecules. For example, preferred antibodies can be engineered or recombinantly produced, or can be experimentally induced to be produced in an animal species, for example, by immunization. In other words, in embodiments, antibodies and the like of the present invention are non-native or non-naturally occurring.

[0209] 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, may be prepared in any of several ways well known and described in the art, but are most preferably prepared using recombinant methods.

[0210] Nucleic acid fragments encoding the heavy and / or light chain regions of the antibodies of the invention can be derived or produced by any suitable method, such as cloning or synthesis, as appropriate.

[0211] Once nucleic acid fragments encoding the heavy and / or light chain regions of the antibodies of the invention have been obtained, these fragments can be manipulated by standard recombinant DNA techniques. Typically, or as part of this further manipulation procedure, the nucleic acid fragments encoding the antibody molecules of the invention are generally incorporated into one or more appropriate expression vectors to facilitate the production or manipulation of the antibodies of the invention.

[0212] Possible expression vectors include, but are not limited to, cosmids, plasmids, or modified viruses (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), so long as the vector is compatible with the host cell used. Transposons can also be used. An expression vector is "suitable for transforming a host cell," meaning that the expression vector contains a nucleic acid molecule of the invention and regulatory sequences selected based on the host cell to be used for expression, and the regulatory sequences are operably linked to the nucleic acid molecule. By operably linked, it is meant that the nucleic acid is linked to the regulatory sequences in a manner that allows expression of the nucleic acid.

[0213] Thus, the present invention contemplates expression vectors, e.g., recombinant expression vectors that contain or include a nucleic acid molecule of the invention, or a fragment thereof, and the necessary regulatory sequences for the transcription and translation of the protein sequence encoded by the nucleic acid molecule of the invention.

[0214] 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 encompass the introduction of a nucleic acid (e.g., a vector) into a cell by one of many possible techniques known in the art. Suitable methods for transforming and transfecting host cells can be found in Sambrook et al., 1989 (Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989) and other textbooks.

[0215] Suitable host cells include a wide variety of eukaryotic host cells and prokaryotic cells, as is well known to those skilled in the art. For example, proteins of the invention may be expressed in yeast cells or mammalian cells, such as HEK cells or CHO cells. Furthermore, where appropriate, for example, if the F(ab')2 format is selected, proteins of the invention may be expressed in prokaryotic cells, such as E. coli. Cell-free expression systems may also be used.

[0216] The proteins of the invention may be prepared by chemical synthesis using techniques well known in protein chemistry, such as solid phase synthesis.

[0217] Yet another aspect provides an expression construct or expression vector or expression system (e.g., viral or bacterial or other expression construct, vector or system), e.g., one or more expression constructs or expression vectors comprising one or more of the nucleic acid fragments or segments or molecules of the invention. Preferably, the expression construct or vector or system is recombinant. Preferably, the construct or vector or system further comprises the necessary regulatory sequences for transcription and translation of the protein sequence encoded by the nucleic acid molecule of the invention.

[0218] Yet another aspect provides a host cell (e.g., a mammalian or bacterial or yeast host cell) or virus, e.g., one or more host cells or viruses, comprising one or more expression constructs or expression vectors of the invention. Also provided is a host cell (e.g., a mammalian or bacterial or yeast host cell) or virus, e.g., one or more host cells or viruses, comprising one or more nucleic acid molecules of the invention. A host cell (e.g., a mammalian host cell or a bacterial host cell or a yeast host cell) or virus that expresses an antibody (or binding protein) of the invention forms yet another aspect.

[0219] Yet another aspect of the invention provides methods for producing (or manufacturing) an antibody (or binding protein) of the invention, comprising culturing a host cell of the invention. Preferred methods comprise (i) culturing a host cell comprising one or more expression vectors of the invention or one or more nucleic acid sequences of the invention under conditions suitable for expression of the encoded antibody or binding protein, and optionally (ii) isolating or obtaining the antibody or binding protein from the host cell or from the growth medium / supernatant. Such production (or manufacturing) methods may also include purifying the antibody or protein product, and / or formulating the antibody or product into a composition, e.g., a pharmaceutical composition, comprising at least one additional component, e.g., a pharmaceutically acceptable carrier or excipient or diluent.

[0220] In embodiments, when an antibody or binding protein of the invention is formed from two or more polypeptide chains (e.g., in a F(ab')2 format, a bivalent scFv-Fc format, or a whole antibody), all polypeptides are preferably expressed in a host cell from either the same or different expression vectors, such that the complete protein, e.g., an antibody protein of the invention, is assembled in the host cell and can be isolated or purified therefrom.

[0221] In another aspect, the invention provides a method of binding CD47 comprising contacting a composition comprising CD47 with an antibody or binding protein of the invention.

[0222] In yet another aspect, the invention provides a method of detecting CD47 comprising contacting a composition suspected of containing CD47 with an antibody or binding protein of the invention under conditions effective to allow the formation of a CD47 / antibody complex, and detecting the complex so formed.

[0223] One therapeutic (or diagnostic) approach is the use of antibodies that can target specific antigens that are expressed on cancer cells and not expressed or expressed at low levels on normal cells. These target antigens, exemplified by CD47, can be exploited with antibodies to specifically kill antigen-bearing tumor cells by a variety of mechanisms, including the delivery of immunoconjugates or radiolabeled conjugates that, when delivered to the antigen-bearing cells, specifically kill the target cells. Such targeting can also be used for diagnostic purposes.

[0224] Accordingly, the present invention also provides a range of conjugated antibodies and conjugated binding proteins (immunoconjugates) in which an anti-CD47 antibody (or binding protein) of the present invention is operably attached to at least one other therapeutic or diagnostic agent. The term "immunoconjugate" is used broadly to define an operative association between an antibody (or binding protein) and another active agent (e.g., a therapeutic or diagnostic agent) and does not refer exclusively to any type of operative association, particularly not limited to chemical "conjugation." Fusion proteins, e.g., recombinant fusion proteins, are specifically contemplated. Any mode of attachment is suitable, so long as the delivery or targeting agent (the anti-CD47 component) is capable of binding to the target and is fully functional when the therapeutic or diagnostic agent is delivered.

[0225] For example, in some preferred embodiments, an antibody (or binding protein) of the invention is or is used as part of an immunotoxin (e.g., used as a therapeutic), where the antibody is itself operably associated with or combined with a toxic agent (e.g., a chemotherapeutic agent or toxin or a radioactive material such as a radioactive tracer used in, e.g., radioimmunotherapy). Operable attachment includes all forms of direct and indirect attachment described herein and known in the art.

[0226] Suitable chemotherapeutic agents or toxins are well known and described in the art; for example, cytotoxic proteins derived from bacteria or plants can be used. The toxin should have the ability to kill target cells once it is taken up into said cells. Thus, preferred immunoconjugates of the invention are immunotoxins comprising an antibody of the invention linked or otherwise conjugated to a toxin (sometimes also referred to as antibody-drug conjugates (ADCs)). In preferred immunoconjugates, an active ingredient, such as a radionuclide, toxin (e.g., diphtheria toxin), or other cytostatic agent, can be conjugated (conjugated) or otherwise linked to the corresponding antibody.

[0227] Immunoconjugates involving conjugation with RNA molecules, such as siRNA, or DNA molecules can also be used.

[0228] In some embodiments, the antibodies of the invention are used (eg, as therapeutics) in their "naked," unconjugated form.

[0229] Yet another aspect is a method of diagnosing or imaging a subject, comprising administering to the subject an appropriate amount of an antibody or binding protein of the invention as defined herein, and detecting the presence, and / or amount, and / or location of the antibody or binding protein of the invention in the subject.

[0230] Suitable diseases to be imaged or diagnosed by the present invention are, for example, cancers as described elsewhere herein in connection with disease treatment.

[0231] In one embodiment, the present invention provides a method for diagnosing cancer in a mammal, comprising: (a) contacting a test sample from said mammal with one or more of the antibodies or binding proteins of the invention; The present invention provides a method comprising:

[0232] In a further embodiment, the present invention provides a method of diagnosing cancer in a mammal, comprising: (a) contacting a test sample obtained from said mammal with one or more antibodies or binding proteins of the invention; (b) determining the presence and / or amount and / or location of antibody-antigen complexes in said test sample; and optionally (c) comparing the presence and / or amount of antibody-antigen complexes in the test sample with a control; The present invention provides a method comprising:

[0233] In the above method, the contacting step is carried out under conditions that allow the formation of the antibody-antigen complex. Suitable conditions can be easily determined by one skilled in the art.

[0234] The above methods may use any suitable test sample, such as biopsy cells, tissues or organs, or histological sections suspected to be affected by disease.

[0235] In certain of the above methods, the presence of any amount of antibody-antigen complex in the test sample will indicate the presence of disease. Preferably, for a definitive diagnosis to be made, the amount of antibody-antigen complex in the test sample is greater than, or preferably significantly greater than, the amount found in an appropriate control sample. More preferably, a significantly greater level is statistically significant, preferably statistically significant at a probability value of <0.05. Suitable methods for determining statistical significance are well known and described in the art, and any of these may be used.

[0236] An appropriate control sample can be readily selected by one of skill in the art; for example, in the case of diagnosis of a particular disease, an appropriate control would be a sample from a subject who did not have the disease. An appropriate control "value" could be readily determined without using a control "sample" in every test, for example, by reference to ranges for healthy subjects known in the art.

[0237] For use in diagnostic or imaging applications, the antibodies (or binding proteins) of the invention may be coupled to radioisotopes, e.g. 3 H, 14 C. 32 P, 35 S, 123 I, 125 I, 131 The binding protein may be labeled with a detectable marker such as I, a radiation emitter (e.g., an α-, β-, or γ-emitter), a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine, luciferin, or europium, an enzyme, such as alkaline phosphatase, β-galactosidase, or horseradish peroxidase, an imaging agent, or a metal ion, or a chemical moiety such as biotin that can be detected by binding to a specific cognate detectable moiety, e.g., labeled avidin / streptavidin. Methods for attaching labels to binding proteins, e.g., antibodies, are known in the art. Such detectable markers allow the presence, amount, or location of binding protein-antigen complexes in a test sample to be determined.

[0238] Preferred detectable markers for in vivo use are X-ray detectable compounds such as bismuth(III), gold(III), lanthanum(III) or lead(II), or 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 99m or yttrium 90or a nuclear magnetic spin resonance isotope, 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.

[0239] The present invention also includes diagnostic or imaging agents comprising antibodies of the present invention attached to a label or detectable marker that directly or indirectly produces a detectable signal. Suitable labels or detectable markers are described elsewhere herein.

[0240] In one embodiment, the method for diagnosing cancer is an in vitro method.

[0241] In one embodiment, the method of diagnosing cancer is an in vivo method.

[0242] In another aspect, the present invention provides a method for screening for cancer in a subject.

[0243] Compositions comprising at least a first antibody (or binding protein) or immunoconjugate of the invention, or at least a first nucleic acid molecule or expression vector of the invention, or at least a first host cell of the invention constitute further aspects of the present invention. Formulations (compositions) comprising one or more antibodies of the present invention, optionally in admixture with a suitable diluent, carrier, or excipient, constitute preferred embodiments of the present invention. Such formulations may be for pharmaceutical use, and thus the compositions of the present invention are preferably pharmaceutically acceptable for administration to humans or non-human animals, but particularly humans, or are otherwise acceptable. Suitable diluents, excipients, and carriers are known to those skilled in the art.

[0244] Any mode of administration can be used. Compositions according to the invention can be present in a form suitable for, for example, oral, nasal, parenteral (e.g., intravenous, intraperitoneal, subcutaneous, intradermal, intramuscular), topical or rectal administration, or transmucosal delivery; any of these modes of administration, or indeed any other suitable mode of administration, can be used. In preferred embodiments, compositions according to the invention are present in a form suitable for intravenous administration. In some embodiments, compositions according to the invention are present in a form suitable for intraperitoneal (ip) administration. In some embodiments, compositions according to the invention are present in a form suitable for direct injection into a tumor (intratumoral).

[0245] The active compounds defined herein (e.g., antibodies of the invention) may be present in conventional pharmacological administration forms, such as tablets, coated tablets, nasal sprays, solutions, emulsions, liposomes, exosomes, powders, capsules, or sustained-release forms. Conventional pharmaceutical excipients and conventional production methods may be used for the preparation of these forms. Furthermore, nucleic acids or nucleic acid-based vectors, such as mRNA-based vectors or viral-based vectors, may be used to administer the active compounds of the invention, e.g., by encoding the antibodies or binding proteins of the invention.

[0246] For example, injection solutions may be prepared in a conventional manner, such as by adding preservatives such as p-hydroxybenzoic acid, or stabilizers such as EDTA, etc. The solutions may then be filled into injection vials or ampoules.

[0247] The pharmaceutical compositions (formulations) of the invention are preferably administered parenterally. Intravenous administration is preferred. In some embodiments, administration is intraperitoneal (ip) administration. In some embodiments, administration is by injection into the tumor. Parenteral administration may be by subcutaneous, intramuscular, intraperitoneal, or intravenous injection using a syringe. Alternatively, parenteral administration may be performed using an infusion pump. A further option is a composition that may be a powder or liquid for administration of the antibody in the form of a nasal or pulmonary spray. As a further option, the antibodies of the invention may be administered transdermally, e.g., from a patch, optionally an iontophoretic patch, or transmucosally, e.g., orally.

[0248] The appropriate dosage unit can be determined by one skilled in the art.

[0249] A further aspect of the invention provides an anti-CD47 antibody (or binding protein) or immunoconjugate, as defined herein, for use in therapy, particularly for use in the treatment or prevention of cancer. In other embodiments, the nucleic acid molecules, expression vectors, host cells, or viruses of the invention may also be used in the therapeutic methods described herein.

[0250] According to the present invention, the antibody may target CD47 positive cells, such as tumor cells.

[0251] In one embodiment, a solid tumor is treated.

[0252] In one embodiment, a hematological or blood cancer is treated.

[0253] In some embodiments, tumors or cancers characterized as expressing or overexpressing (eg, on their surface) CD47 are treated.

[0254] Thus, a further aspect of the invention provides an anti-CD47 antibody (or binding protein) as defined herein for use in the treatment or prevention of a cancer or tumor characterized by (or associated with) CD47 expression or overexpression, such as a cancer or tumor characterized by unwanted, inappropriate, aberrant, increased or excessive CD47 expression.

[0255] In some embodiments, cancer is characterized by (or is associated with) CD47 signaling (e.g., aberrant, inappropriate, or unwanted CD47 signaling). For example, in some embodiments, expression of CD47 on the surface of tumor cells and its subsequent interaction with its cognate receptor, SIRPα, on other cells, e.g., phagocytes such as macrophages, triggers an inhibitory signaling pathway in SIRPα-expressing cells such that phagocytosis is inhibited (this signaling is also known as a "don't eat me" signal transmitted from tumor cells to phagocytes such as macrophages). The antibodies (or binding proteins) of the invention act to block, reduce, or inhibit SIRPα-CD47 interaction, thus blocking SIRPα signaling, or "don't eat me" signaling. Blocking this interaction allows phagocytosis of tumor cells by SIRPα-expressing macrophages or other phagocytes.

[0256] In some embodiments, the antibodies (or binding proteins) of the invention are capable of directly inducing the killing of CD47-expressing cancer or tumor cells.

[0257] Preferred cancers to be treated by the present invention include lung cancer, such as non-small cell lung cancer (NSCLC, e.g., squamous NSCLC), small cell lung cancer (e.g., extensive small cell lung cancer), melanoma (e.g., metastatic melanoma such as BRAF-negative metastatic melanoma or multiple melanoma), lymphoma (e.g., acute T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, e.g., B-cell or T-cell non-Hodgkin's lymphoma or Burkitt's lymphoma, or chronic lymphocytic lymphoma), leukemia (e.g., acute myeloid leukemia, acute lymphoblastic leukemia or myelodysplastic syndromes), renal cell carcinoma (RCC, e.g., clear cell renal carcinoma), colorectal cancer, urothelial bladder cancer, 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, hematologic neoplasms, thyroid cancer, salivary gland cancer, laryngeal (larynx) cancer, neuroblastoma, retinoblastoma, and testicular (testicle) cancer.

[0258] Without wishing to be bound by theory, it is believed that the antibodies of the invention may be superior to prior art antibodies in terms of therapeutic efficacy, particularly in terms of speed of action and required concentrations (lower or lower concentrations / doses of the antibodies of the invention have been shown to induce significant direct tumor cell killing at higher rates compared to prior art antibodies). In some embodiments, the antibodies of the invention are also capable of inducing phagocytosis of tumor cells by effector cells such as macrophages.

[0259] The efficacy of the antibodies of the present invention was demonstrated in relevant cancer models (xenograft models of acute lymphoblastic leukemia and Burkitt's lymphoma). In this lymphoblastic leukemia model, concentrations / doses of 10 mg / kg and 25 mg / kg of CO-1.4 resulted in complete inhibition of tumor growth, i.e., cure of the cancer. This effect was observed even at concentrations / doses as low as 1 mg / kg (e.g., after only two injections). This effect was also observed after a single 1.33 nM dose of CO-1.4 or CO201-scFv-Fc-bi. In a Burkitt's lymphoma model, concentrations / doses of 6.67 nM of CO-1.4 and CO201-scFv-Fc-bi resulted in complete inhibition of tumor growth, i.e., cure of the cancer, while a dose of 1.33 nM of CO201-scFv-Fc-bi caused a significant delay in tumor progression. Thus, the antibodies (and binding proteins) of the present invention represent an exciting development in the field of anti-CD47 therapeutics. Without wishing to be bound by theory, it is believed that the antibodies of the present invention may function to arrest the cell cycle and, therefore, the progression of tumor growth. Thus, in some embodiments, the antibodies (or binding proteins) of the present invention are capable of causing the arrest of cell proliferation. In some embodiments, the antibodies (or binding proteins) of the present invention are capable of curing cancer.

[0260] The binding proteins or antibodies in the therapeutic methods and uses of the present invention are administered in a pharmaceutically, therapeutically, or physiologically effective amount to a subject (e.g., an animal, e.g., a human or non-human mammal) in need of treatment. Thus, the methods and uses may include the additional step of identifying the subject in need of treatment. The appropriate and effective concentration / dosage to be administered can be readily determined by one skilled in the art. Based on animal models used to date, exemplary concentrations / dosages can be 0.05, 0.1, or 1 to 30 mg / kg, e.g., at or about 0.05, 0.1, 1, 10, or 25 mg / kg, or 0.05, 0.1, 1, or 5 nM to 10, 20, 30, 40, or 50 nM, e.g., at or about 0.05, 0.1, 1, 2, 4, 6, 10, 20, 30, or 40 nM.

[0261] Treatment of a disease or condition (eg treatment of an underlying disease) according to the present invention includes curing said disease or condition, or any reduction or alleviation of the disease, for example a reduction in disease severity or symptoms of the disease.

[0262] The therapeutic methods and uses of the present invention are suitable for disease prevention as well as for active disease treatment (e.g., treatment of the underlying disease). Therefore, preventive treatment is also encompassed by the present invention. For this reason, in the methods and uses of the present invention, treatment also includes, where appropriate, prevention or prophylaxis.

[0263] Such prophylactic (or preventative) aspects can conveniently be performed on healthy, normal, or at-risk subjects and can include both complete prevention and significant prevention, whereby significant prevention can include scenarios where the severity of a disease or disease symptoms is reduced (e.g., measurably or significantly reduced compared to the severity of the symptoms that would be expected if no treatment were administered).

[0264] Thus, subjects suitable for treatment according to the present invention include any type of animal that can develop cancer, more particularly cancer that contains tumor cells that express CD47.

[0265] Therefore, the in vivo methods and uses described herein are generally carried out in mammals. Any mammal, such as humans and any livestock, pet or laboratory animal, may be treated. Specific examples include mice, rats, pigs, cats, dogs, sheep, rabbits, horses, cows and monkeys. Preferably, however, the mammal is a human.

[0266] Thus, the term "animal" or "patient" or "subject" as used herein includes any mammal, such as humans, and any livestock, pet, or laboratory animal. Specific examples include mice, rats, pigs, cats, dogs, sheep, rabbits, horses, cows, and monkeys. Preferably, however, the animal or patient or subject is a human. Thus, the subject or patient to be treated according to the present invention will preferably be a human.

[0267] In another embodiment, the subject is a subject who has, or is suspected to have (or is developing), or potentially have (or is developing) the disease or condition in question described above.

[0268] Alternatively viewed, the present invention provides a method of 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 invention as defined herein. The therapeutic use embodiments of the invention described herein apply mutatis mutandis to this aspect of the invention.

[0269] A therapeutically effective amount can be determined based on clinical evaluation and can be easily monitored. Preferred cancer treatments are as described elsewhere herein.

[0270] In a further aspect, the present invention provides the use of an antibody (or binding protein) of the invention as defined herein in the manufacture of a medicament for use in a method of therapy, preferably the treatment or prevention of cancer as described elsewhere herein.

[0271] The therapeutic use embodiments of the invention described herein apply mutatis mutandis to this aspect of the invention.

[0272] In some embodiments, the antibodies (or binding proteins) of the invention can be used in monotherapy, hi other embodiments, they can be used in combination with other standard cancer therapeutics.

[0273] The present invention further includes kits comprising one or more antibodies or compositions of the invention, or one or more nucleic acid molecules encoding antibodies of the invention, or one or more expression vectors comprising a nucleic acid sequence of the invention, or one or more host cells or viruses comprising an expression vector or nucleic acid sequence of the invention. Preferably, the kits are for use in the methods and uses described herein, such as the therapeutic, diagnostic, or imaging methods described herein. Preferably, the kits include instructions for use of the kit components. Preferably, the kits are for the diagnosis or imaging, or treatment or prevention of a disease or condition described elsewhere herein, and optionally include instructions for use of the kit components to diagnose, image, treat, or prevent such a disease or condition. Equivalent embodiments involving the binding proteins of the invention are also provided.

[0274] The antibodies (or binding proteins) of the invention as defined herein may also be used as molecular tools for in vitro or in vivo applications and assays, such as binding assays or diagnostic assays. Because antibodies (and binding proteins) have two or more antigen-binding sites that bind to CD47, they can function as members of specific binding pairs, and these molecules can be used in any assay in which a specific CD47-binding pair member is desired.

[0275] Thus, a further aspect of the present invention provides reagents comprising an antibody (or binding protein) of the invention as defined herein, and the use of such an antibody (or binding protein) as a molecular tool for the detection of, e.g., CD47, e.g., in a sample of interest, e.g., in an in vitro or in vivo assay.

[0276] The terms "reduce" or "lowering" (or equivalent terms) as used herein include any measurable decrease or reduction when compared to an appropriate control. Suitable controls would be readily identified by one of skill in the art and could include subjects or healthy individuals who receive no treatment or a placebo, or samples or assays in which an antibody (or binding protein) of the invention is absent or a control antibody (or binding protein), e.g., an isotype control antibody (or binding protein), is present. Preferably, the decrease or reduction is significant, e.g., clinically or statistically significant.

[0277] The term "enhancement" (or equivalent term), as used herein, includes any measurable increase or elevation when compared to an appropriate control. Suitable controls would be readily identified by one of skill in the art and could include untreated or placebo-treated subjects or healthy individuals, or samples or assays in which an antibody (or binding protein) of the invention is absent or a control antibody (or binding protein), such as an isotype control antibody (or binding protein), is present.

[0278] Preferably, the increase is significant, e.g., clinically or statistically significant. Preferably, such increase (and indeed other increases, improvements, or positive effects referred to elsewhere herein) or such decrease (and indeed other decreases, reductions, or negative effects referred to elsewhere herein) is a measurable increase, decrease, etc. (where appropriate), and more preferably, it is a significant increase, decrease, etc., preferably a clinically significant or statistically significant increase, when compared to an appropriate control level or value (e.g., compared to subjects not receiving treatment or receiving a placebo treatment, or compared to healthy or normal subjects, or the same subjects prior to treatment), e.g., having a probability value of ≦0.05 or <0.05 when compared to an appropriate control level or value (e.g., compared to subjects not receiving treatment or receiving a placebo treatment, or compared to healthy or normal subjects, or the same subjects prior to treatment).

[0279] The method of determining the statistical significance of the difference between test groups of subjects or the difference in the level of specific parameters is well known in the art and has been described in the literature.For example, in this specification, the decrease or increase in the level of specific parameters or the difference between test groups of subjects is generally considered to be statistically significant when statistical comparison, using appropriate significance tests such as Student's t-test, Mann-Whitney U-rank-sum test, Chi-square test or Fisher's exact test, one-way analysis of variance (ANOVA) or two-way analysis of variance (ANOVA) test, shows a probability value of ≦0.05 or <0.05.

[0280] Table of Amino Acid Sequences and Sequence Identifiers (SEQ ID NOs) Disclosed herein

[0281] All amino acid sequences herein are listed from N-terminus to C-terminus, as is conventional in the art.

[0282] [Table 1]

[0283] The constant region of mCO-1 shown in Table A is a murine IgG1κ antibody sequence that is well known and described in the art.

[0284] [Table 2]

[0285] [Table 3]

[0286] [Table 4]

[0287] [Table 5]

[0288] AO-A IgG1-full heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGYTDPRDTDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGRVGLGYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO. 27)

[0289] AO-A IgG1-full light chain DIVMTQSPDSLAVSLGERATINCRSSQNIVQSNGNTYLEWYQQKPGQPPKLLIYKVFHRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGSHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO. 28)

[0290] AO-A IgG4-full heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGYTDPRTDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGRVGLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO. 29)

[0291] AO-A IgG4-full light chain DIVMTQSPDSLAVSLGERATINCRSSQNIVQSNGNTYLEWYQQKPGQPPKLLIYKVFHRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGSHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO. 30)

[0292] AO-B IgG1-full heavy chain EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYWIHWVRQMPGKGLEWMGYTDPRTDYTEYNQKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARGGRVGLGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO. 31)

[0293] AO-B IgG1-full light chain DIVMTQSPDSLAVSLGERATINCRSSQNIVQSNGNTYLEWYQQKPGQPPKLLIYKVFHRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGSHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO. 32)

[0294] AO-B IgG4-full heavy chain EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYWIHWVRQMPGKGLEWMGYTDPRTDYTEYNQKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARGGRVGLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO. 33)

[0295] AO-B IgG4-full light chain DIVMTQSPDSLAVSLGERATINCRSSQNIVQSNGNTYLEWYQQKPGQPPKLLIYKVFHRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGSHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO. 34)

[0296] AO-C IgG1-full heavy chain QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGYTDPRTDYTEYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGRVGLGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO. 35)

[0297] AO-C IgG1-full light chain DIVMTQSPDSLAVSLGERATINCRSSQNIVQSNGNTYLEWYQQKPGQPPKLLIYKVFHRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGSHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO. 36)

[0298] AO-C IgG4-full heavy chain QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGYTDPRTDYTEYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGRVGLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO. 37)

[0299] AO-C IgG4-full light chain DIVMTQSPDSLAVSLGERATINCRSSQNIVQSNGNTYLEWYQQKPGQPPKLLIYKVFHRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGSHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO. 38)

[0300] Humanized variable heavy domain (VH)

[0301] huCO-1 VH-v1 QVQLVQSGSELKKPGASVKVSCKASGYTFT NFGMH WVRQAPGQGLEWMG WINTYTGEPTYTDDFKG RFVFSLDTSVSTAYLQISSLKAEDTAVYYCAR GDYRYGDS WGQGTTVTVSS (SEQ ID NO. 39)

[0302] huCO-1 VH-v2 QVQLVQSGSELKKPGASVKVSCKASGYTFT NFGMH WVRQAPGQGLEWMG WINTYTGEPTYTDDFKG RFVFSLDTSVSTAYLQISSLKAEDTAVYYCTR GDYRYGDS WGQGTTVTVSS (SEQ ID NO. 40)

[0303] huCO-1 VH-v3 QVQLVQSGSELKKPGASVKVSCKASGYTFT NFGMH WVRQAPGQGLKWMG WINTYTGEPTYTDDFKG RFVFSLDTSVSTAYLQISSLKAEDTAVYYCTR GDYRYGDS WGQGTTVTVSS (SEQ ID NO. 41)

[0304] huCO-1 VH-v4 QVQLVQSGSELKKPGASVKVSCKASGYTFT NFGMH WVRQAPGKGLKWMG WINTYTGEPTYTDDFKG RFVFSLDTSVSTAYLQISSLKAEDTAVYYCTR GDYRYGDS WGQGTTVTVSS (SEQ ID NO. 42)

[0305] huCO-1 VH-v5 QVQLVQSGSELKKPGASVKVSCKASGYTFT NFGMH WVRQAPGQGLKWMG WINTYTGEPTYTDDFKG RFVFSLDTSVSTAYLQISSLKAEDTAVYYCAR GDYRYGDS WGQGTTVTVSS (SEQ ID NO. 43)

[0306] (CDRs are underlined)

[0307] Humanized variable light domain (VL)

[0308] huCO-1 VL-v1 DIVMTQTPLSLSVTPGQPASISC RSSQSLVHSNGKTYLH WYLQKPGQPPQLLIY RVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC SQSTHVPFT FGQGTKLEIK (SEQ ID NO. 44)

[0309] huCO-1 VL-v2 DIVMTQTPLSLSVTPGQPASISC RSSQSLVHSNGKTYLH WYLQKPGQSPQLLIY RVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC SQSTHVPFT FGQGTKLEIK (SEQ ID NO. 45)

[0310] huCO-1 VL-v3 DIVMTQTPLSLSVTPGQPASISC RSSQSLVHSNGKTYLH WYLQKPGQSPKLLIY RVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC SQSTHVPFTFGQGTKLEIK (SEQ ID NO. 46)

[0311] (CDRs are underlined)

[0312] [Table 6]

[0313] (The CH1 domain is shown in bold and underlined, followed by the hinge (also underlined)-CH2-CH3.)

[0314] IgG4 light steady RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO. 48)

[0315] Linker GGGGSGGGGSGGGGS (SEQ ID NO. 49)

[0316] IgG4 CH2-CH3 PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO. 50)

[0317] hinge SKYGPPCPSC (SEQ ID NO. 51)

[0318] Hinge-CH2-CH3 SKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO. 52)

[0319] [Table 7]

[0320] (CDRs are underlined, linkers are in bold, hinge CH2-CH3 is underlined and italicized)

[0321] Alternative hinge SKYGPPCPPC (SEQ ID NO. 54)

[0322] The invention will now be further described with reference to the following figures and in the non-limiting examples that follow. [Brief explanation of the drawings]

[0323] [Figure 1]Characterization of mCO-1. ​​(A) Jurkat cells (2 × 10 cells) were incubated with increasing concentrations of SIRPα (range 0.1 ng / ml to 100 μg / ml) followed by staining with FITC-conjugated B6H12, 2D3, or mCO-1. ​​Each data point represents the MFI of one sample. (B) Jurkat (upper panel), U-937 (middle panel), or MCF-7 (lower panel) cells (2 × 10 cells) were treated with increasing concentrations of FITC-conjugated mCO-1 (filled circles) or the isotype control MOPC-21 (open circles). Each data point represents the percentage of FITC-positive cells ± SD for duplicate replicates. (C) (Upper panel) A standard curve for quantitative measurement of mCO-1 binding capacity was generated using the QIFIKIT® from Dako according to the manufacturer's instructions. (Lower panel) ABC in Jurkat cells was determined by extrapolation of the standard curve. (D) RBCs (2% v / v in PBS) were incubated with CD47 mAbs B6H12, 2D3, CC2C6, MABL-1, mCO-1, or the isotype control MOPC-21 at concentrations ranging from 0.001 to 1 μg / ml. Small, punctate circles indicate the absence of hemagglutination, while diffuse, fuzzy patterns indicate hemagglutination. (E) RBCs (2% v / v in PBS) were incubated with increasing concentrations (ranging from 0.1 ng / ml to 100 μg / ml) of FITC-conjugated mCO-1 (filled circles) or the isotype control MOPC-21 (open circles). Each data point represents the percentage of FITC-positive cells ± SD for duplicate replicates. (F) Jurkat cells (5 × 105 cells / ml) were incubated for 3 h with mCO-1 or the MOPC-21 isotype control at the concentrations indicated. Cells were then stained with Annexin V eFlour405 and 7-AAD and analyzed by flow cytometry. Data represent mean ± SD, n = 3 (Student's t-test).

[0324] [Figure 2]Characterization of chimeric IgG1 CO-1 (CO-1.1) and AO-176. (A) Jurkat cells (2 × 10 cells) were incubated with increasing concentrations of mCO-1 (range 0.1 ng / ml to 100 μg / ml) followed by staining with FITC-conjugated CO-1.1, AO-176 candidates A, B, and C, or a human IgG isotype control. Each data point represents MFI ± SD of duplicates. (B) Jurkat cells (2 × 10 cells) were incubated with increasing concentrations of SIRPα (range 0.1 ng / ml to 100 μg / ml) followed by staining with FITC-conjugated B6H12, 2D3, or CO-1.1. Each data point represents MFI ± SD of duplicates. (C) Jurkat (top panel), Reh (middle panel), or CCRF-CEM (bottom panel) cells (2 × 10 cells) were treated with increasing concentrations of FITC-conjugated CO-1.1, AO-176 candidates A, B, and C, or a human IgG isotype control. Each data point represents the percentage of FITC-positive cells in duplicate. (D) RBCs (2% v / v in PBS) were incubated with CD47 mAbs B6H12, 2D3, CC2C6, MABL-1, mCO-1, CO-1.1, AO-176 A, B, and C, or human and murine isotype controls at concentrations ranging from 0.0005 to 1 μg / ml. (E) RBCs (2% v / v in PBS) were incubated with AO-176 A, B, and C, or a human IgG isotype control at concentrations ranging from 0.005 to 10 μg / ml. (D and E) Small, dotted circles indicate no hemagglutination, while a diffuse, fuzzy pattern indicates hemagglutination. (F) RBCs (2% v / v in PBS) were incubated with increasing concentrations (range 0.1 ng / ml to 100 μg / ml) of FITC-conjugated CO-1.1, AO-176 A, B, C, or a human IgG isotype control. Each data point represents the percentage of FITC-positive cells ± SD from duplicate replicates.

[0325] [Figure 3]CO-1.1 induces rapid and potent PCD in cancer cells. (A) Jurkat cells (5 × 10 cells / ml) were incubated with CO-1.1 at the indicated concentrations for various durations. (B) Jurkat cells (5 × 10 cells / ml) were treated with low doses of CO-1.1 (0.008–0.12 μg / ml) for 3 hours. (A and B) Data represent mean % PCD, which was the sum of early (Annexin V + 7-AAD-) and late (Annexin V + 7-AAD+) PCD, ±SD, n ≥ 3.

[0326] [Figure 4] CO-1.4 induces PCD in Jurkat cells with negligible hemagglutination in RBCs. (A) RBCs (2% volume / volume in PBS) were incubated with CO-1.1, CO-1.4, or a human IgG isotype control at concentrations ranging from 0.0005 to 1 μg / ml. Small, punctate circles indicate no hemagglutination, while a diffuse, fuzzy pattern indicates hemagglutination. (B) Jurkat cells (5 × 10 cells / ml) were treated for 3 hours with CO-1.4 or a human IgG isotype control (0.12 μg / ml) at the concentrations indicated. Data represent the mean %PCD, which was the sum of early (Annexin V + 7-AAD-) and late (Annexin V + 7-AAD+) PCD, ±SD, n = 3.

[0327] [Figure 5]Induction of programmed cell death by CO-1.1 and AO-176 candidates. (A) Jurkat cells (5 x 10 cells / ml) were treated for 3 hours with 0.1 or 10 μg / ml of CO-1.1, AO-176 A, B, C, or human IgG isotype control. (B) Jurkat cells were treated for the indicated duration with 1 μg / ml of CO-1.1, AO-176 A, B, C, or human IgG isotype control. (C) Jurkat cells (5 x 10 cells / ml) were treated for 3 hours with 1 μg / ml of anti-CD47 antibodies CO-1.1, B6H12, 2D3, or human and mouse isotype controls. (A-C) Data represent the mean % PCD, which was the sum of early (Annexin V + 7-AAD-) and late (Annexin V + 7-AAD+) PCD, ±SD, n ≥ 2.

[0328] [Figure 6] mCO-1 induces phagocytosis in PCD-responsive and non-responsive cell lines. (A) CFSE-labeled Jurkat (upper panel), Reh (middle upper panel), KG-1a (middle lower panel), or HL-60 (lower panel) macrophages (5 × 10 cells / ml) were cultured in the presence or absence of mCO-1 (0.1, 1, or 10 μg / ml) or an isotype control (10 μg / ml) in the presence of DiO-labeled RAW264.7 macrophages. B6H12 (10 μg / ml) was used as a positive control. (B) KG-1a cells were treated with the appropriate isotype control or mCO-1, CO-1.1, and CO-1.4 (1 μg / ml) as indicated. (A and B) After 2 hours of incubation, the percent phagocytosed cells were quantified by flow cytometry analysis and represented the percent of the cell population staining positive for both CFSE and DiO.

[0329] [Figure 7]CO-1 F(ab')2 induces hemagglutination in RBCs at levels comparable to those observed with other CO candidates. RBCs (2% volume / volume in PBS) were incubated with CO-1.1, CO-1.4, F(ab')2, or CD47 control mAbs (CC2C6, B6H12, MABL-1, or 2D3) or a human / mouse (MOPC-21) IgG isotype control at concentrations ranging from 0.0005 to 1 μg / ml. Small, punctate circles indicate the absence of hemagglutination, whereas a diffuse, blurred pattern indicates hemagglutination.

[0330] [Figure 8] CO-1 F(ab')2 induces PCD to a similar extent as CO-1. ​​(A-C) Jurkat cells (A), MOLT-4 (B), or CCRF-CEM (C) (5 x 105 cells / ml) were treated for 3 hours with CO-1, CO-1 F(ab')2, or a human IgG isotype control (1 µg / ml) at the concentrations indicated. 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 ± SD of two independent experiments.

[0331] [Figure 9]CO-1 cures disease in mice in a xenograft model of precursor B-cell acute lymphoblastic leukemia (BCP-ALL). (A) Lentivirally transduced Reh cells were injected intratumorally into 6-8 week-old NSG mice. Tumor progression was tracked by noninvasive in vivo imaging of the luminescence signal emitted by Reh cells as described in Materials and Methods. Tumor engraftment was quantified on day 6 after intratumor injection, followed by injection of CO-1.4 (1-25 mg / kg, as indicated) or a human IgG4 isotype control (25 mg / kg). The effect of treatment was quantified on day 13 after intratumor injection, followed by another injection of the same dose. Treatment was then stopped, but tumor progression was continued to be monitored on days 20 and 27. (B) Xenograft luciferase activity [photons per second (p / s)] over time. Each data point represents the mean ± SEM signal intensity of five xenografted mice in each treatment group.

[0332] [Figure 10] Epitope surface map of CD47 residues bound by the CO-1 antibody. Residues Q19, N45, T120, R121, and E122 in the CD47 protein crystal structure are labeled as some of the specific residues (labeled in red (R)) that form the binding epitope of the CO-1 antibody.

[0333] [Figure 11] Binding of huCO-mAbs to Jurkat cells. Jurkat cells were incubated with increasing concentrations of huCO-mAbs, followed by incubation with goat anti-human FITC. FITC intensity was analyzed by flow cytometry.

[0334] [Figure 12] Hemagglutination of red blood cells (RBCs) by huCO-mAbs. 2% RBCs (vol / vol in PBS) were incubated for 30 minutes with increasing concentrations of the antibodies shown in the figure. Small, punctate circles indicate no hemagglutination, while a diffuse, fuzzy pattern indicates hemagglutination.

[0335] [Figure 13] Induction of PCD in Jurkat cells by huCO-mAbs. Jurkat cells (5x105 cells / ml) were incubated with the indicated antibodies at 10 μg / ml for 3 hours, followed by staining with Annexin V and 7-AAD. %PCD = Annexin V positive cells. Data are shown as mean ± SEM, n=3. *p<0.05, **p<0.01 (paired t-test).

[0336] [Figure 14] Hemagglutination of red blood cells (RBCs) by huCO201-scFv-Fc-bi. 2% RBCs (vol / vol in PBS) were incubated for 30 minutes with increasing concentrations of the antibody or fragment indicated in the figure. Small, punctate circles indicate no hemagglutination, while a diffuse, blurred pattern indicates hemagglutination.

[0337] [Figure 15] Induction of PCD and phagocytosis by CO201-scFv-Fc-bi. (A) Jurkat cells (5x105 cells / ml) were incubated with the indicated fragments or antibodies at the indicated μg / ml concentrations for 3 hours, followed by staining with Annexin V and 7-AAD. %PCD = Annexin V-positive cells. Data are shown as mean ± SEM, n = 2. (B) Jurkat cells (5x105 cells / ml) were cocultured with RAW264.7 and treated with the indicated antibodies at the indicated μg / ml concentrations for 2 hours. %Phagocytosed cancer cells = %JurkatCFSE+RAW-DiO+ cells. Data are shown as mean ± SEM, n = 1.

[0338] [Figure 16] Induction of phagocytosis in Jurkat cells by huCO-mAbs. Jurkat cells (5 x 10 cells / ml) were co-cultured with RAW264.7 and treated with the indicated antibodies for 2 hours. % phagocytosed cancer cells = % JurkatCFSE+RAW-DiO+ cells. Data represent mean ± SEM, n = 2.

[0339] [Figure 17] CO-1 and CO-1bi have potent anti-cancer effects in a xenograft model of BCP-ALL. (A) Six- to eight-week-old NSG mice were intratumorally injected with lentivirally transduced Reh cells. Cancer progression was tracked by noninvasive in vivo imaging of the luminescence signal emitted by Reh cells, as described in Materials and Methods. Establishment of xenografts was confirmed on day 10 after intratumor injection, followed by injection of 1.33 nM chimeric CO-1.4 (referred to in this figure as CO-1), CO201-scFv-Fc-bi (referred to in this figure as CO-1bi), or a human IgG4 isotype control. Treatment efficacy was assessed on day 14 after intratumor injection, followed by monitoring of cancer progression on days 17, 24, and 29. (B) Xenograft luciferase activity [photons per second (p / s)] for each treatment at the indicated time points. Horizontal bars indicate the average luminescence signal (p / s) from mice in the treatment groups indicated in the figure. Each dot represents the luminescence signal (p / s) from one animal, while the vertical bars represent the standard error of the mean for five xenografted mice in each treatment group. (C) Repeat of the experiment described in (A), only in this case mice were treated on day 8 after IT injection. Each dot represents the average luminescence signal (p / s) from mice in the treatment group indicated in the figure, while the vertical bars represent the standard error of the mean.

[0340] [Figure 18]CO-1 and CO-1bi have potent anticancer effects in a xenograft model of Burkitt's lymphoma. Six- to eight-week-old NSG mice were subcutaneously (SC) injected with 1.5 x 10 Raji cells in a 1:2 suspension of RPMI:Vitrogel (100 μl per animal). Cancer progression was monitored by caliper measurement, and tumor volume was calculated as described in Materials and Methods. When tumor volumes reached an average of 190 mm, mice were randomly assigned to different treatment groups and received IP injections of either chimeric CO-1.4 (referred to in this figure as CO-1) (6.67 nM), CO201-scFv-Fc-bi (referred to in this figure as CO-1bi) (1.33 nM or 6.67 nM), or HuIgG4 isotype control (6.67 nM) at the time points indicated by the arrows in this figure. Each dot represents the average tumor volume for the treatment group indicated in the figure. Vertical bars indicate the standard error of the mean. [Example]

[0341] Example 1: Functional properties of bivalent anti-CD47 antibodies (material and method) Reagents and antibodies The reagents and antibodies used in this study are listed in Tables 1 and 2, respectively.

[0342] The nucleotide and amino acid sequences of the heavy and light variable domains of one preferred CD47 antibody of the invention are shown in Table A. This antibody, designated murine CO-1 (mCO-1), was obtained from a hybridoma. It is a murine / mouse IgG1κ antibody that was produced as a full-length chimeric antibody with human IgG1 (CO-1.1, Table B) and IgG4 (CO-1.4, Table C) sequences, as well as an F(ab′)2 fragment based on mCO-1 (CO-1 F(ab′)2, Table D). The CDRs and framework regions of the light and heavy chains of mCO-1 are shown in Table A.

[0343] Cell lines and culture conditions The human cancer cell lines used in this project were purchased from the American Type Culture Collection (ATCC). Jurkat (clone E6-1), MOLT-4, Reh, SUP-T1, U-937, and CCRF-CEM were cultured in RPMI 1640 medium (BioNordika catalog number BE-12-702F / 12). SW-780 and SW1088 cells were cultured in Leibovitz L-15 (ATCC catalog number 30-2008), T24 cells were cultured in McCoy's 5A (ATCC catalog number 30-2007), HT-1197 and MCF-7 cells were cultured in Eagle's Minimum Essential Medium (EMEM, ATCC catalog number 30-2003), and H-4 and U-118-MG cells were cultured in DMEM medium (ThermoFisher catalog number 41965062).

[0344] All cell culture media were supplemented with 10% (vol / vol) fetal bovine serum (FBS, BioNordica, catalog number FB-1001 / 500) and 1% (vol / vol) penicillin / streptomycin (P / S, ThermoFisher, catalog number 15140122), and all cell lines were maintained in a humidified atmosphere at 37 °C. Cell lines cultured in Leibovitz L-15 medium were cultured in 100% air, while other cell lines were incubated in 95% air and 5% CO2.

[0345] Red blood cell (RBC) separation Human whole blood was collected in heparin-coated tubes (approximately 5 ml) and diluted in RBC wash buffer (0.05% BSA, 1 mM EDTA in PBS) to a total volume of 50 ml. RBCs were separated from the human whole blood by centrifugation at 1800 × g for 10 minutes and then resuspended in wash buffer to a total volume of 50 ml. The wash was repeated three times, and after the final wash, the RBC pellet was resuspended in PBS to obtain a 2% (vol / vol) RBC solution.

[0346] Hemagglutination assay Increasing concentrations of CD47 antibody (or control) up to 1 μg / ml were added to a round-bottom 96-well plate. A 2% (vol / vol) solution of freshly isolated RBCs was then added to each well and incubated in a standard cell incubator for 30-60 minutes or until the cells had settled to the bottom of the well. A diffuse, fuzzy pattern indicates hemagglutination, while small, punctate circles indicate the absence of hemagglutination.

[0347] Flow cytometry All flow cytometry analyses were performed on a NovoCyte (Agilent Technologies Inc.) equipped with three lasers (405, 488, and 605 nm) and 13 detection channels, and data were analyzed using NovoExpress software (Agilent Technologies Inc.).

[0348] Antibody binding assay PBS-washed cancer cell lines or RBCs were pelleted, washed with wash buffer (3% (wt / vol) BSA, 1% (wt / vol) sodium azide in PBS), and incubated with Human BD Fc Blosk for 10 min before being added to a round-bottom 96-well plate (2.5 × 10 per well). 5 Cells). Antibodies were conjugated with FITC using a FITC conjugation kit from Abcam. Cells were then incubated with various concentrations of FITC-conjugated CD47 antibodies or the appropriate isotype controls indicated for 1 hour on ice with gentle agitation. All samples were prepared in duplicate. Cells were washed three times and resuspended in cold wash buffer. Cells were kept on ice until analysis by flow cytometry. Excitation wavelength: FITC 488 nm, detection: 530 / 30 nm.

[0349] Antibody binding capacity determination Antibody binding capacity (ABC) was measured on PBS-washed cancer cell lines using a Dako QIFKIT® according to the manufacturer's instructions. Briefly, cells were incubated with saturating concentrations of antibody on ice for 60 minutes, followed by two washes in PBS (pH 7.4) containing 0.1% (wt / vol) BSA and 15 mmol / L NaN3. Next, cells were incubated with FITC-conjugated F(ab')2 fragments of goat anti-mouse immunoglobulin on ice for 45 minutes, after which the cells and beads were washed as described in the protocol. Samples were analyzed by flow cytometry, and the mean fluorescence intensity (MFI) emitted by the beads was used to generate a calibration curve. From this, the ABC of each antibody could be determined by interpolation from the standard curve.

[0350] Blockade of CD47 antibodies by mCO-1 PBS-washed Jurkat cells were seeded into round-bottom 96-well plates (2.5 × 10 cells per well). 5 Cells). Cells were incubated with increasing concentrations of CD47 antibodies (CO-1.1, AO-A / B / C, and mCO-1) or a human IgG isotope control for 1 hour on ice with gentle agitation. Cells were then washed twice with PBS, resuspended in FITC-conjugated CD47 mAb mCO-1 (1 μg / ml), and incubated for an additional 60 minutes on ice (two technical replicates per sample). Cells were then washed, resuspended in PBS, and analyzed by flow cytometry. Excitation 488 nm, emission 530 / 30 nm.

[0351] Blockade of CD47 antibodies by SIRPα PBS-washed Jurkat cells were seeded into round-bottom 96-well plates (2.5 × 10 cells per well). 5Cells). Cells were incubated with increasing concentrations of human recombinant SIRPα on ice for 1 hour with gentle agitation. Cells were then washed twice with PBS, resuspended in 1 μg / ml of FITC-conjugated CD47 mAbs (mCO-1, B6H12, and 2D3) or human IgG isotype control, and incubated on ice for an additional 30 minutes. Each sample was prepared in duplicate. Cells were then washed twice in cold PBS and kept on ice until analysis by flow cytometry. Excitation wavelength: FITC 488 nm, detection: 530 / 30 nm.

[0352] Annexin V and 7-AAD staining For programmed cell death (PCD) analysis, cells were cultured at 5 × 10 per ml in supplemented medium. 5 The cells were diluted and seeded into 24-well plates (1 ml per well). They were then incubated with various concentrations of CO-1 (CO-1.1 or CO-1.4) or a human IgG isotype control for various durations (30 min, 1 h, 2 h, 3 h) under standard cell culture conditions. In some experiments, anti-CD47 mAbs AO-Candidates A, B, and C, CC2C6, B6H12, MABL-1, and 2D3 were added at the indicated concentrations for comparison. Following incubation, cells were harvested and stained with Annexin V eFlour™ 450 and 7-AAD according to the manufacturer's protocol. Cells were immediately analyzed by flow cytometry. Annexin V + 7-AAD - were considered early apoptotic (in the early stage of PCD), while late apoptotic cells (cells in the later stage of PCD) were identified as Annexin V + and 7-AAD + Excitation of Annexin V was at 405 nm and detection at 445 / 45 nm, while 7-AAD was excited at 488 nm and detected at 675 / 30 nm.

[0353] statistical analysis Graphs are presented as mean values ​​from independent experiments as specified in the figure legends. Error bars indicate standard deviation (SD).

[0354] Statistical analysis was performed by performing a paired two-tailed Student's t-test using GraphPad Prism9 software (Graph-Pad Software Inc.). Groups were found to be significantly different from each other when P values ​​were less than 0.05.

[0355] Curve fitting was performed using four-parameter nonlinear regression in GraphPad Prism.

[0356] [Table 8]

[0357] [Table 9]

[0358] result Effect of SIRPα on binding by anti-CD47 antibodies B6H12 is an antibody known to induce blockade of the SIRPα / CD47 interaction, also known as the "don't eat me" signal, between macrophages and tumor cells (3). In contrast, 2D3 has been shown not to inhibit this interaction (3). With this in mind, we incubated Jurkat cells with increasing concentrations of recombinant SIRPα and found that SIRPα binding blocked the interaction of both B6H12 and mCO-1, whereas 2D3 did not appear to be inhibited by this interaction (Figure 1A). Indeed, SIRPα binding appeared to increase the binding of 2D3 to Jurkat cells.

[0359] Binding of mCO-1 to cancer cells and human RBCs The binding of murine CO-1 (mCO-1) to cancer cells was quantified by flow cytometry in comparison with a murine IgG1 isotype control (MOPC-21). The mCO-1 antibody was capable of specifically binding to various cancer cell lines (Table 3), with high affinity for several blood cell lines, such as Jurkat T cells (Figure 1B, upper panel) and U-937 (Figure 1B, middle panel) monocytes, as well as several cell lines derived from solid tumors, such as MCF-7 breast cancer cells (Figure 1B, lower panel). Furthermore, the binding of mCO-1 to the same panel of cancer cell lines was quantified by generating a standard curve of the antibody using calibration beads (Figure 1C, upper panel), followed by interpolation of the FITC signal intensity emitted by the antibody-bound cancer cells (Figure 1C, lower panel and Table 3).

[0360] Because anti-CD47 antibodies such as CC2C6 can induce hemagglutination in RBCs (4), we evaluated the ability of mCO-1 and several other CD47 antibodies to induce hemagglutination in RBCs from healthy human donors. A diffuse, blurred pattern indicates hemagglutination, whereas small, punctate circles indicate the absence of hemagglutination (1). As can be seen in Figure 1D, mCO-1 shows early signs of hemagglutination in RBCs at concentrations between 0.03 and 0.06 μg / ml. The binding of mCO-1 to RBCs was also quantified by flow cytometry, and the EC 50 was 0.04 μg / ml (Fig. 1E), which was determined to correlate well with the hemagglutination assay.

[0361] Induction of PCD by mCO-1 Several known anti-CD47 antibodies have been shown to induce PCD in a wide range of tumor cells (2-4). Therefore, we tested mCO-1 for its ability to induce PCD after incubation with mCO-1 for various durations and concentrations by staining cancer cells with Annexin V and 7-AAD. Cells staining positively with Annexin V and negatively with 7-AAD (Annexin V) were selected. + 7-AAD -) were considered early apoptotic, while cells stained positive for both dyes (Annexin V + 7-AAD + ) were considered late apoptotic. Early and late apoptotic cells were summed to obtain the total percentage of the cell population undergoing PCD. Treatment of Jurkat cells with mCO-1 resulted in a strong induction of PCD as early as 3 hours after treatment with this antibody (Figure 1F). The same trend could be observed in several other tumor-derived cancer cell lines, including CCRF-CEM T cells, Reh acute lymphoblastic leukemia cells, and H4 glioma epithelial cells (Table 3).

[0362] [Table 10]

[0363] Having confirmed mCO-1's ability to bind to several cancer cell lines of human origin and its ability to induce PCD, we set out to create chimeric antibodies to determine their potential as therapeutic agents for treating human cancers. Therefore, we created chimeric IgG1 and IgG4 versions of mCO-1 (sequence information in Tables B and C). These are hereafter referred to as CO-1.1 (chimeric CO-1 IgG1) and CO-1.4 (chimeric CO-1 IgG4). Furthermore, for comparison, we identified three of the best candidate sequences in Arch Oncology's International Publication No. 2020 / 198370 (designated herein as candidates AO-A, AO-B, and AO-C; see SEQ ID Nos. 27–38). Of these, candidate A appears to be the most likely candidate, corresponding to the AO-176 antibody currently under development by Arch Oncology (4; see, e.g., Supplementary Figure S1). AO-176 is an anti-CD47 antibody that claims the ability to induce PCD in several human cancers (5) and is currently undergoing phase 1 and 2 clinical trials (2). We have created IgG1 and IgG4 versions of AO-176 based on all three sequences.

[0364] Characterization of chimeric antibodies (CO-1.1 and AO candidates) After receiving the new antibody, we measured the ability of mCO-1 to block the binding of CO-1.1 and the three AO-IgG1 candidates. Figure 2A shows that mCO-1 potently inhibited CO-1.1 binding at increasing concentrations. However, AO candidates A-C showed little inhibition. This indicates that CO-1 and mCO-1 bind to the same epitope (as expected), but that the AO candidates bind to CD47 in a different manner and to different epitopes than mCO-1 / CO-1.

[0365] As shown in Figure 1A, SIRPα blocked mCO-1 binding. For this reason, the ability of SIRPα to block the binding of chimeric CO-1.1 was evaluated. As shown in Figure 2B, CO-1.1 binding was inhibited by SIRPα at increasing concentrations of this recombinant protein.

[0366] Having determined the similarity between mCO-1 and chimeric CO-1.1 through blocking experiments, we undertook to quantify binding in Jurkat, Reh, and CCRF-CEM cell lines. Figure 2C shows that CO-1.1 binds with high affinity to all three cell lines (see also Table 4). AO candidates A and B also showed significant binding to these cell lines, but at a slower rate than CO-1.1. Candidate C showed poor binding across all three cell lines and poor binding in Reh cells (EC 50 had 10.52 μg / ml (Table 4).

[0367] [Table 11]

[0368] To examine the hemagglutination effect of CO-1.1 on RBCs, CO-1.1, AO-A, B, and C, and various other anti-CD47 antibodies were incubated with freshly isolated RBCs from healthy donors. As can be seen in Figure 2D, CO-1.1 induced hemagglutination at concentrations higher than 0.125 μg / ml and slightly at 0.0625 μg / ml. Candidate AO-A also induced some hemagglutination at the highest doses (1 and 0.5 μg / ml). Candidate AO-B appeared to have slight hemagglutination at the highest dose (1 μg / ml, Figure 2D), while candidate AO-C showed no signs of hemagglutination. Other anti-CD47 mAbs, such as B6H12, 2D3, CC2C6, and MABL-1, were also included as references, while a human IgG isotype control and PBS were used as controls. Since Arch Oncology has published a literature review using 10 μg / ml to determine cell death, we also tested this concentration for hemagglutination effects. As can be seen in Figure 2E, at high concentrations (10 μg / ml), all three AO candidates induce hemagglutination.

[0369] Flow cytometric binding assays were performed to examine the binding affinity of CO-1.1 and the AO candidates (A, B, and C) to RBCs. Binding affinity by FACS appears to correlate well with hemagglutination assays, which show higher affinity for CO-1.1 compared to AO candidates A, B, and C (Figure 2F). Table 4 shows the EC values ​​for all four antibodies. 50 Summarizes values.

[0370] CO-1.1 potently and rapidly induces PCD To evaluate the ability of CO-1.1 to induce PCD in cancer cells, Jurkat cells were treated with CO-1.1 for various durations and doses, followed by Annexin V and 7-AAD staining. Human IgG isotype was used as a control. CO-1.1 induced direct and rapid PCD as early as 30 min of treatment, demonstrating its high potential in eliminating cancer cells (Figure 3A).

[0371] CO-1.1 induces PCD at low concentrations To evaluate the concentration of CO-1.1 that maximally induces PCD in vitro, Jurkat cells were incubated with various concentrations of CO-1.1 as indicated for 3 hours. The maximal cell death signal was achieved at lower concentrations (0.06–0.1 μg / ml) (Figure 3B).

[0372] CO-1.4 induces strong PCD and negligible hemagglutination To evaluate the hemagglutination effect of CO-1.4, we performed an independent experiment in which CO-1.4 was incubated with freshly isolated RBCs. Interestingly, CO-1.4 induced negligible hemagglutination, with no hemagglutination observed at 0.06 μg / ml and 0.1 μg / ml (Figure 4A). These concentrations induce a strong PCD response in Jurkat cells (Figure 4B). Treatment of Jurkat cells with CO-1.4 at a low concentration (0.03 μg / ml) induces over 40% PCD after 3 hours. While the efficacy of CO-1.4 against cancer cells at low concentrations is significant (Figure 3B), CO-1.4 appears even more potent (Figure 4B).

[0373] Comparison of PCD induction by CO-1.1 with other anti-CD47 antibodies Several anti-CD47 antibodies are known for their ability to induce PCD in cancer cells (1, 2, 4, 5). To compare the cell death-inducing ability of CO-1.1 with various anti-CD47 antibodies, Jurkat cells were treated with 0.1 μg / ml and 10 μg / ml of the anti-CD47 mAbs CO-1.1, AO-A, B, and C, or a human isotype control and stained with annexin V and 7-AAD after 3 hours. CO-1.1 significantly induced cell death in Jurkat cells at low concentrations (0.1 μg / ml), whereas AO candidates appeared to induce no or less cell death at this concentration (Figure 5A). However, cell death induced by AO candidates was observed when cells were incubated with a higher concentration (10 μg / ml) for 3 hours (Figure 5A).

[0374] Puro et al. showed that a long (24 h) incubation period was required to induce PCD with AO-176 (4, 5). Therefore, a second experiment was performed in which Jurkat cells were incubated with 1 μg / ml of CO-1.1 or AO candidates A, B, and C for various time points as indicated. Again, the three AO candidates induced less PCD compared with CO-1.1 (Figure 5B).

[0375] We also compared the PCD potential of CO-1.1 with other anti-CD47 antibodies, including B6H12 and 2D3. B6H12 has been shown to induce cell death when immobilized but not when soluble (2). 2D3, to our knowledge, has never been reported to induce cell death. In line with these results, we determined that B6H12 and 2D3 do not induce PCD (Figure 5C).

[0376] References 1. Killian, M.L., "Hemagglutination assay for influenza virus," Methods in Molecular Biology, 1161, 3-9 (2014). 2. Kaur, S., Cicalese, K.V., Banerjee, R., and Roberts, D.D., “Preclinical and Clinical Development of Therapeutic Antibodies Targeting Functions of CD47 in the Tumor Microenvironment,” Antibody Therapeutics, Vol. 3, No. 3, pp. 179–92 (2020). 3. Leclair, P., Liu, CC, Monajemi, M., Reid, GS, Sly, LM, Lim, CJ, "CD47-ligation induced cell death in T-acute lymphoblastic leukemia," Cell Death & Diseases, 2018; 9, 5, 544 (2018). 4. Puro, RJ, Bouchlaka, MN, Hiebsch, RR, Capoccia, BJ, Donio, MJ, Manning, PT, et al., "Development of AO-176, a Next-Generation Humanized Anti-CD47 Antibody with Novel Anticancer Properties and Negligible Red Blood Cell Binding," Molecular Cancer Therapeutics, Vol. 19, No. 3, pp. 835-46 (2020). 5. Uno, S., Kinoshita, Y., Azuma, Y., Tsunenari, T., Yoshimura, Y., Iida, S. et al., “Antitumor activity of a monoclonal antibody against CD47 in xenograft models of human leukemia,” Oncology Reports, Vol. 17, No. 5, pp. 1189-94 (2007).

[0377] Example 2: Effect of CO-1 on phagocytosis (material and method) Reagents and antibodies Murine CO-1 (mCO-1), described above and in Table A, has been sequenced by GenScript. Chimeric IgG1 and IgG4 versions of mCO-1 were generated by GenScript via expression in mammalian expression hosts. The sequences of CO-1.1 (chimeric IgG1 mCO-1) and CO-1.4 (chimeric IgG4 mCO-1) are shown in Tables B and C, respectively. A mouse IgG1κ isotype control (clone MOPC-21) was purchased from StemCell Technologies and purified for preservatives using Zeba™ spin desalting columns from Thermo Fisher Scientific according to the manufacturer's instructions.

[0378] Cell lines and culture conditions Jurkat and Reh cell lines were grown in RPMI 1640 supplemented with 10% (vol / vol) fetal bovine serum (FBS, Thermo Fisher Scientific) and 1% (vol / vol) penicillin / streptomycin (PS, Thermo Fisher Scientific). HL-60 cells were grown in Iscove's Modified Dulbecco's Medium (IMDM, Thermo Fisher Scientific) supplemented with 20% FBS and 1% PS. KG-1a cells were grown in IMDM supplemented with 10% FBS and 1% PS. The murine macrophage cell line RAW264.7 was cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% PS. Jurkat, Reh, HL-60, and KG-1a cells were cultured at 2 × 10 per ml. 5 ~1×10 6 The RAW264.7 cells were maintained at a cell density of 1000 kJ / ml, while the RAW264.7 cells were subcultured every 2–3 days when they reached 70–90% confluence. All cell lines were purchased from the American Type Culture Collection (ATCC) and grown at 37°C in a humidified atmosphere with 95% air and 5% CO2.

[0379] Phagocytosis assay Staining of RAW264.7 macrophages RAW264.7 cells were rinsed once with supplemented DMEM and then stained with 40 nM Vybrant™ DiO cell labeling solution (ThermoFisher) diluted in supplemented DMEM for 20 minutes in a standard cell incubator (humidified atmosphere with 95% air and 5% CO at 37°C). After staining, cells were rinsed three times with supplemented DMEM.

[0380] Staining and antibody treatment of target cells Target cells (Jurkat, Reh, KG-1a, or HL-60) were collected by centrifugation and stained with 1 μl / ml CellTrace™ Violet Cell Proliferation Dye (Thermo Fisher) for 20 minutes in a standard cell incubator. Unincorporated CFSE was quenched by adding supplemented RPMI five times, and cells were pelleted and resuspended in supplemented DMEM. Target cells were added to RAW264.7 tissue culture plates and treated with the indicated antibodies for 2 hours in a standard cell incubator, followed by two washes in PBS containing 1 mM EDTA (Thermo Fisher). Cells were analyzed by flow cytometry and stained with DiO + CellTrace + The cells represented phagocytosed target cells.

[0381] (result) To measure the induction of phagocytosis by mCO-1, CO-1.1, and CO-1.4, four different cell lines were incubated in the presence of the murine macrophage cell line RAW264.7. Previous studies have shown that Jurkat and Reh cells respond to CO-1 antibody with programmed cell death (PCD), whereas KG-1a and HL-60 cells do not respond to CO-1 treatment with PCD. In this study, we found that treatment with mCO-1 at a low concentration of only 0.1 μg / ml induced phagocytosis in all four cell lines (Figure 6A). We also confirmed the ability of chimeric CO-1.1 and CO-1.4 to induce phagocytosis in KG-1a cells (Figure 6B).

[0382] Example 3: Characterization of CO-1 fragment (material and method) Creating a Fragment The mCO-1 fragment was generated by GenScript. Table D shows the sequence of CO-F(ab')2.

[0383] Red blood cell (RBC) separation Same as in Example 1 above.

[0384] Hemagglutination assay Same as in Example 1 above.

[0385] Flow cytometry Same as in Example 1 above.

[0386] Annexin V and 7-AAD staining Same as in Example 1 above.

[0387] (result) Binding of CO fragments to human RBCs Because anti-CD47 antibodies such as CC2C6 can induce hemagglutination in RBCs, the CO-1 F(ab')2 fragment, as well as several other CD47 antibodies, were evaluated for their ability to induce hemagglutination in RBCs from healthy human donors. As can be seen in Figure 7, where a diffuse, fuzzy pattern indicates hemagglutination, while small, punctate circles indicate the absence of hemagglutination, CO-1 F(ab')2 induced hemagglutination at a level comparable to that of the whole CO antibody.

[0388] Induction of PCD by CO fragments Several anti-CD47 antibodies have been shown to induce PCD in a wide range of tumor cells, and therefore the CO-1 F(ab')2 fragment was tested for its ability to induce PCD by staining cancer cells with annexin V and 7-AAD after incubation with this fragment for various durations and at various concentrations. Cells staining positively with annexin V and negatively with 7-AAD (annexin V) were stained negatively with annexin V. + 7AAD - ) were considered early apoptotic, and cells staining positive with both dyes (Annexin V + 7-AAD + ) were designated as late apoptotic. Both early and late apoptotic cells were summed to obtain the total percentage of the cell population undergoing PCD. Treatment of Jurkat cells with the F(ab')2 fragment resulted in a strong induction of PCD as early as 30 min after treatment with this fragment (Fig. 8A). PCD was also observed in MOLT-4 cells (Fig. 8B) and CCRF-CEM cells (Fig. 8C).

[0389] Example 4: SPR analysis of recombinant antibodies to evaluate binding affinity to CD47 (Analysis 1) (material and method) Equipment BIACore S200 material SA chip (streptavidin) recCD47 SinoBiological (12283-H27H-B), 17 kDa, C-terminal His- and AVI-tag, biotinylated Antibodies AO-A, AO-B, AO-C, CO-1.1, all 3mg / ml, 150kDa All proteins were stored at -80°C. Buffer 20mM phosphate buffer, pH7.4 / 2.7mM KCl / 137mM NaCl / 0.05%P20 procedure recCD47 was fixed at a level of 1330 RU (75 nM in 10 mM NaAc, pH 5.0). Regeneration conditions: 60 seconds in 1M NaCl in 50mM NaOH + 60 seconds in 1M NaCl Analytes (antibodies) run in both single-cycle and multiple-cycle modes Injection order: low to high concentration Single cycle: 80nM / 40nM / 20nM / 10nM / 5nM / 2.5nM / 1.25nM / 0.625nM / 0.3125nM. Flow rate: 30μl / min. 120s on - 120s constant - 1800s off. Heavy cycle: 50nM / 25nM x 2 / 12.5nM / 6.25nM / 3.125nM / 1.56nM / 0.78nM / 0.39nM / 0.195nM / 0.1nM. Flow rate: 30µl / min. 120 seconds on, 180 seconds off.

[0390] (result) A 1:1 binding model was used to fit the data. Table 1 shows the values ​​obtained for the binding on and off rates, ka and kd, respectively. The affinity constant, KD, is calculated as kd / ka.

[0391] [Table 12]

[0392] The 1:1 binding model shows a suboptimal fit with the experimental data. This may be due to secondary effects resulting from the high density (high immobilization level) of recCD47 on the chip, which may have some effect on the calculated values. However, this analysis is still useful for comparing binding affinities between antibodies, where CO1.1 is found to have much better binding affinity than the three AO candidates.

[0393] Analysis 2 Equipment BIACore S200 material SA chip (streptavidin) recCD47 Sino Biological (12283-H27H-B), 17 kDa, C-terminal His- and AVI-tag, biotinylated Antibodies CO-1.1 (3mg / ml), CO-1.4 (1.2mg / ml), 2D3 (0.5mg / ml), CC2C6 (0.2mg / ml) All proteins were stored at -80°C. Buffer 20mM phosphate buffer, pH7.4 / 2.7mM KCl / 137mM NaCl / 0.05% P20 procedure recCD47 immobilized to a level of 700RU Regeneration conditions: 60 seconds, 10 mM glycine, pH 1.5 Analyte (antibody) run in single cycle mode Injection order: low to high concentration Temperature 25℃ Single cycle: 20nM / 10nM / 5nM / 2.5nM / 1.25nM / 0.625nM / 0.3125nM / 0.1562nM / 0.07813nM, flow rate: 30μl / min, 120 seconds on, 1800 seconds off

[0394] (result) A 1:1 binding model was used to fit the data. All curves overlapped well with the 1:1 model. Table 6 shows the values ​​obtained for the binding on-rate and off-rate, ka and kd, respectively. The affinity constant, KD, is calculated as kd / ka. The single-cycle (SC) data fit well with the 1:1 model. It can be seen here that CO1.4 has a similar affinity to CO1.1, and both have better binding affinity than the 2D3 and CC2C6 CD47 antibodies.

[0395] [Table 13]

[0396] analysis 3 Equipment and materials: Same as in Analysis 2 except as follows: Antibody fragment F(ab′)2 (2.33 mg / mL) Single Cycle Procedure recCD47 was immobilized to a level of 100 RU (in 10 mM NaAc, pH 5.0). Regeneration conditions: 60 seconds, 10 mM glycine, pH 1.5 Analyte (antibody fragment) run in single cycle mode Injection order: from low to high: 10nM / 5nM / 2.5nM / 1.25nM / 0.625nM / 0.3125nM / 0.1562nM / 0.07813nM / 0.039nM Flow rate: 30 μl / min, 120 seconds on, 180 seconds off - 1800 seconds off after the final injection Temperature 25℃

[0397] (result) A 1:1 binding model was used to fit the data. All curves fit well with the 1:1 model. Table 7 shows the values ​​obtained for the binding on (column 1) and off (column 2) rates, ka and kd, respectively. The affinity constant, KD, was calculated as kd / ka and was found to be 6.6 pM (columns 3 and 4).

[0398] [Table 14]

[0399] Example 5: In vivo activity of CO-1 (material and method) CO-1 antibody Chimeric CO-1.4 was generated by expression in a mammalian expression host by GenScript. The sequence of CO-1.4 is shown in Table C.

[0400] cell culture All cell lines were purchased from the American Type Culture Collection (ATCC). The B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cell line Reh was grown at 2 × 10 cells per ml in RPMI 1640 medium (Lonza). 5 ~1×10 6 Cells were maintained at a density between 100 and 1500 μg / ml. HEK293T cells, used for lentivirus production, were subcultured every 2–3 days in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific) when they reached 70–90% confluence. Both cell culture media were supplemented with 10% (vol / vol) fetal bovine serum (FBS, Thermo Fisher Scientific) and 1% (vol / vol) penicillin / streptomycin (PS, Thermo Fisher Scientific). Cells were cultured in a humidified atmosphere with 95% air and 5% CO2.

[0401] Lentivirus production in HEK293T cells Lentiviral vectors containing genes encoding firefly luciferase and enhanced green fluorescent protein (EGFP) were generated by transfecting HEK293T cells with 8.3 mg of each plasmid from the pMD2.G envelope plasmid, pCMVΔ8.91 packaging plasmid, and pSLIEW transfer plasmid (1). On the day of transfection, cells were cultured until 70%–90% confluent. The cell medium was changed approximately 1 hour before transfection. The transfection mixture was prepared using a calcium phosphate transfection kit (Invitrogen) according to the manufacturer's instructions. The cell medium was changed 4 hours after transfection. Two days later, the viral supernatant was collected and concentrated overnight at 4°C using a LentiX Concentrator (Takara Bio). The virus was then recovered by centrifugation at 1500 g for 45 minutes at 4°C. The pellet was suspended in a small volume (<1 mL) of cell medium and stored at -80°C. Frozen lentiviral stock was titrated in Reh cells using standard transduction protocols (see next section).

[0402] Lentiviral transduction of Reh cells Reh cells (5 x 10 per well) were plated in a 48-well plate with 4 mg / mL polybrene (Merck Millipore) present in the cell culture medium. 5 Cells were inoculated with lentivirus concentrate. Spinfection was performed by adding lentivirus concentrate to the cells and centrifuging the plates at 900 g for 50 min at 34°C. After spinfection, the plates were transferred to a standard cell incubator (37°C, 5% CO2 in a humidified atmosphere) for 2 days, after which viral particles were removed by washing twice at 300 g for 10 min at 4°C. EGFP was detected by flow cytometry. + A small aliquot of these cells was taken to analyze the cell quantity, and the remaining cells were injected intratibially (IT) into NSG mice.

[0403] Establishment of xenograft model Transformed Reh cells (5 × 10 5Reh cells were injected into 6-8 week-old female NOD scid IL2Rγnull (NSG) mice (The Jackson Laboratory) anesthetized with isoflurane (induction 4%-5%, maintenance 2%-3%, oxygen flow 300 mL / min). The knee was kept in a flexed position to expose the proximal end of the tibia. After drilling the tibia with a 23 G needle, Reh cells (40 μL per animal) were injected using a 31 G insulin syringe. Before IT injection, mice were treated with systemic and local analgesia, with 0.05 mg / kg Temgesic (Schlering-Plough) and 1-2 mg / kg Mice were treated with Marcain (AstraZeneca). Systemic analgesia was repeated 6–8 hours after IT injection. On days 6 and 9 after IT injection, mice were injected IP with 10 mg / kg, 25 mg / kg CO-1, or vehicle / saline. Mice were housed under specific pathogen-free conditions and provided with food and water ad libitum. Health status was monitored daily, and all animal procedures were performed in accordance with approval by the Norwegian Food Safety Authority under identification number 29016.

[0404] In vivo imaging Leukemia progression was monitored by noninvasive in vivo imaging using an IVIS Spectrum CT instrument (PerkinElmer). D-luciferin (150 mg / kg, PerkinElmer) substrate was administered by intraperitoneal (IP) injection. After 9 minutes, three images were recorded at 1-minute intervals using an automatic exposure setting. Mice were euthanized if they showed severe engraftment or signs beyond the predetermined humane endpoint.

[0405] (result) Reh cells were stably transfected with a lentiviral firefly luciferase-EGFP vector and injected intravenously into NSG mice. Leukemia progression was tracked by noninvasive in vivo imaging of firefly luciferase-expressing Reh cells, which demonstrated satisfactory tumor engraftment in all mice on day 6 after intravenous injection (Figure 9A). Immediately after the imaging procedure, mice were divided into five groups and IP-injected with either an isotype control (25 mg / kg human IgG4) or CO-1.4 (1, 5, 10, or 25 mg / kg). Subsequent intravenous imaging performed on day 13 after intravenous injection revealed that all mice treated with CO-1.4 exhibited no luminescence signal, while the signal from vehicle-treated mice was almost six-fold greater (Figure 9B). Mice were treated again with the same dose of vehicle / CO-1 as on day 13 and imaged again on days 20 and 27. The signal in both CO-1.4 groups remained absent (Fig. 9A and B).

[0406] References 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, Vol. 27, No. 3, pp. 718-21 (2013).

[0407] Example 6: Epitope mapping of CO-1 Epitope mapping of the IgG form of the CO-1 antibody on human CD47 was performed by Deeptope SAS (France) using DMS (Deep Mutational Scanning) as described, for example, in Sierocki et al., 2021, PLoS Neglected Tropical Diseases, 15(3), e0009231; van Blarcom et al., Journal of Molecular Biology, 427(6), B1513-1534 (2015) and Medina-Cucurella and Whitehead, Methods in Biology. See also Biology, 1764, 101-121 (2018).

[0408] Principles of epitope mapping using DMS (deep mutational scanning) DMS is a mutagenesis method that aims to generate all possible single substitutions at all selected residues in a given protein sequence. A DMS library is obtained in the form of DNA that encodes the protein under study. In this library, each DNA strand contains a mutated codon relative to the parent sequence.

[0409] This DMS DNA library is integrated into expression plasmids specifically designed to express recombinant proteins on the yeast surface. The yeast are then transformed and induced to express the mutated proteins on their surface. This new library (called the display library) is screened by flow cytometry using a fluorescent reporter to reveal the expression of the protein (anti-tag fluorescent antibody) and the binding of the protein to its partner (fluorescent partner).

[0410] The ideal case for epitope mapping is to have two antibodies with compatible epitopes that can bind to the same antigen together. In this way, each of these two antibodies acts as a conformational control for the other antibody's mutated antigen. In fact, a single substitution made in an antigen can have four types of effects:

[0411] 1. Loss of affinity for the first antibody while retaining binding to the second antibody. This is a mutation made within the epitope of the first antibody.

[0412] 2. Loss of affinity for the second antibody while retaining binding to the first. This is a mutation in the epitope of the second antibody.

[0413] 3. Loss of affinity for both antibodies This is a so-called "destructuring" mutation that affects the conformation of the antigen and therefore prevents binding of both antibodies.

[0414] 4. No effect The mutation is not in the epitope of one of the two antibodies and does not result in a significant change in the conformation of the antigen.

[0415] Following flow cytometry analysis, yeast populations that have lost affinity for the antibody of interest but retain binding to the second antibody are sorted. The plasmids contained in these yeast populations are extracted and sequenced by high-throughput sequencing. Analysis of the sequencing data allows for the identification of mutations that affect the binding of the antibody to its target. This analysis therefore allows for the identification of the critical position on the antigen for the binding of the antibody of interest, i.e., its epitope.

[0416] (material and method) The antigen used for DMS analysis was human CD47 (123 expressed amino acids, from glutamine 19 to glutamic acid 141). See SEQ ID NO. 19. Mutated versions of this antigen were expressed on yeast in the form of a DMS DNA library. The first antibody used was CO-1. ​​The second antibody was 2D3 (Thermo Fisher, Cat. No. 14-0478-82), another anti-CD47 antibody that was shown not to compete with CO-1 for binding to CD47. Therefore, these are suitable antibody pairs for use in DMS analysis.

[0417] DMS was performed on two regions of CD47: Library 1 [amino acids 19 to 80] and Library 2 [amino acids 81 to 141]. Yeast was transformed with these two libraries. To verify the efficiency of mutagenesis, unsorted yeast from the two generated libraries was sequenced. 100% of the predicted single mutants for all libraries were sequenced. Two DMS libraries were successfully generated and cloned into yeast. Each library contains approximately 1,200 single amino acid mutants and 2,000 DNA codon mutants. Library components encode the appropriate 20 amino acids at each mutation position.

[0418] (result) A DMS map is generated, see Table 8. All positions that, when mutated, affect the binding of the CO-1 antibody are classified into three categories. High Impact: Mutations between 19 and 14 are forbidden (red / R). Positions that fall into this category are considered likely to directly interact with IgG—herein CO-1. Medium effect: Mutations between 13 and 7 are prohibited (orange / O). Low Impact: Mutations between 2 and 6 are prohibited (yellow / Y).

[0419] Analysis of the CD47 structure (AF-Q08722-F1, Alphafold) was performed to resolve the distinction between "structural residues" buried within the structure and epitope residues exposed to solvent. Residues colored gray in Table 8 are classified as "structural residues" and are not considered part of the epitope. Mutation of these residues induces a moderate structural change sufficient to abolish binding to the antibody in question but not to abolish binding to a second, so-called "conformational control" antibody (in this case, 2D3).

[0420] Residues marked with an * (corresponding to high impact-R residues, not structural residues) are believed to form the CO-1 epitope. The epitope is also shown in Figure 10.

[0421] [Table 15]

[0422] Top line: Number of amino acid residues in the CD47 molecule of SEQ ID NO. 19. Second row: Amino acid residues, grey shading = structural residues. Line 3: The number of forbidden mutations at that position, between 2 and 19. Row 4 R = red, high impact, mutations between 19 and 14 are forbidden, O = orange, medium impact, mutations between 13 and 7 are forbidden, Y = yellow, low impact, mutations between 2 and 6 are forbidden, W = white, no impact. The * at the bottom indicates residues that are believed to be part of the CO-1 epitope.

[0423] Example 7: Functional Characterization of Bivalent Humanized Anti-CD47 Antibodies (material and method) Reagents and antibodies The reagents and antibodies used in this study are listed in Table 9 and Table 2, respectively.

[0424] Table A shows the nucleotide and amino acid sequences of the heavy and light variable domains of one preferred CD47 antibody of the invention. The antibodies characterized in this example are humanized IgG4 anti-CD47 antibodies designated CO201, CO202, CO203, CO204, CO205, CO206, CO207, CO208, CO209, CO210, CO211, CO212, and CO213 (collectively referred to hereafter as huCO-mAbs). They were constructed using CDRs from a murine full-length IgG1κ antibody designated CO-1 (mCO-1) obtained from a hybridoma. (Table A shows the CDR and FR regions of this murine antibody.) The CDR and framework regions of the light and heavy chains of huCO-mAbs are shown in SEQ ID NOS: 39-43 (heavy chain) and SEQ ID NOS: 44-46 (light chain) and Table E, with the CDR sequences underlined. A bivalent single-chain fragment variable (scFv) of CO201 linked to IgG4 Fc was also designed and designated CO201-scFv-Fc-bi. The sequence of one of the chains of this construct is provided as SEQ ID NO. 53 and consists of CO201scFv fragment-hinge-CH2-CH3. Upon expression of this heavy chain construct, the chains dimerize to form an Fc region with one scFv on each chain (i.e., a bivalent scFv-Fc fusion). A human IgG4 isotype control was obtained from Syno Biological (catalog number HG4K).

[0425] Cell lines and culture conditions The human cancer cell line Jurkat (clone E6-1) was purchased from the American Type Culture Collection (ATCC). The cells were cultured in RPMI 1640 medium supplemented with 10% (vol / vol) fetal bovine serum (FBS) and 1% (vol / vol) penicillin / streptomycin (PS). The cells were grown at 0.4–1.6 × 10 per ml in a humidified atmosphere with 95% air and 5% CO at 37 °C. 6 The cells were kept at a density between 100 and 200 cells / well.

[0426] Red blood cell (RBC) separation Same as in Example 1 above.

[0427] Hemagglutination assay Same as in Example 1 above except testing huCO-mAbs (i.e., control).

[0428] Flow cytometry Same as in Example 1 above.

[0429] Antibody binding assay PBS-washed cancer cell lines or RBCs were pelleted, washed with wash buffer (3% (wt / vol) BSA, 1% (wt / vol) sodium azide in DPBS), and incubated with Human BD Fc block for 10 min before being placed in a round-bottom 96-well plate (2.5 × 10 cells per well). 5 Cells were then incubated with various concentrations of huCO-mAbs or human IgG4 isotype control at the indicated concentrations on ice with gentle shaking for 1 hour. All samples were prepared in duplicate. Cells were washed twice in wash buffer and then incubated with goat anti-human FITC staining buffer (goat anti-human FITC diluted 1:200 in wash buffer) protected from light and incubated on ice with gentle shaking for 30 minutes. After washing three times in wash buffer, cells were analyzed by flow cytometry. Excitation wavelength: FITC 488 nm, detection: 530 / 30 nm.

[0430] Annexin V and 7-AAD staining Same as Example 1 above, except incubated with huCO-mAbs or IgG4 human isotype control for 3 hours.

[0431] statistical analysis Graphs are presented as mean values ​​from independent experiments, as specified in the figure legends. Error bars indicate the standard error of the mean (SEM). Statistical analysis was performed by performing a paired, two-tailed Student's t-test using GraphPad Prism 9 software (GraphPad Software, Inc.). Groups were found to be significantly different from each other when P values ​​were less than 0.05. Curve fitting was performed using four-parameter nonlinear regression in GraphPad Prism.

[0432] [Table 16]

[0433] (result) Binding of huCO-mAbs to cancer cells and human RBCs Binding of huCO-mAbs to cancer cells compared to a human IgG4 isotype control was measured by flow cytometry. huCO-mAbs bound specifically and with high affinity to Jurkat cells (Figure 11).

[0434] Because anti-CD47 antibodies such as CC2C6 can induce hemagglutination in RBCs, the huCO-mAbs and several other CD47 antibodies were evaluated for their ability to induce hemagglutination in RBCs from healthy human donors. A diffuse, fuzzy pattern indicates hemagglutination, while small, punctate circles indicate the absence of hemagglutination. As seen in Figure 12, CO201, CO203, CO205, and CO207 showed early signs of hemagglutination at concentrations starting from 0.31 μg / ml. The remaining antibodies all induced hemagglutination from 1.25 μg / ml or higher.

[0435] Induction of PCD by huCO-mAbs Induction of PCD was quantified after a 3-hour incubation with antibody, followed by staining with Annexin V and 7-AAD. Cells staining positive for Annexin V were considered to undergo PCD, and as seen in Figure 13, treatment of Jurkat cells with 10 μg / ml of huCO-mAbs for 3 hours induced 18-47% PCD.

[0436] Induction of hemagglutination by huCO201-scFv-Fc-bi The ability of huCO201-scFv-Fc-bi to induce hemagglutination was evaluated as described above. As can be seen in Figure 14, this fragment did not induce hemagglutination, even at concentrations as high as 100 μg / ml. This finding was surprising but highly advantageous.

[0437] Induction of PCD by CO201-scFv-Fc-bi Induction of PCD by CO201-scFv-Fc-bi was assessed as described above. As seen in Figure 15A, PCD induction was comparable to that of chimeric CO-1.4.

[0438] Effect of bivalent huCO-mAbs on phagocytosis Phagocytosis assay Cell lines were purchased from the American Type Culture Collection (ATCC). Jurkat cell line (clone E6-1) was grown in RPMI 1640, while RAW 264.7 cells were grown in DMEM. Both cell culture media were supplemented with 10% (v / v) FBS and 1% (v / v) PS.

[0439] Staining of RAW264.7 macrophages Same as in Example 2.

[0440] Jurkat cell staining and antibody treatment Same as in Example 2.

[0441] (result) Induction of phagocytosis by huCO-mAbs To quantify the induction of phagocytosis by huCO-mAbs, Jurkat cells were incubated in the presence of the murine macrophage cell line RAW264.7. Treatment with CO201 or CO213 induces phagocytosis of Jurkat cells (Figure 16).

[0442] Induction of phagocytosis by CO201-scFv-Fc-bi Induction of phagocytosis by CO201-scFv-Fc-bi was evaluated as described above. As shown in Figure 15B, the induction of phagocytosis was comparable to that of chimeric CO-1.4.

[0443] SPR analysis to evaluate the binding affinity of antibodies to recombinant CD47 The binding affinity of huCO-mAbs to CD47 was quantified by surface plasmon resonance (SPR) using a BIACore S200 system. Recombinant biotinylated CD47 was immobilized on an SA streptavidin chip at a concentration of 100 RU (in 10 mM NaAc, pH 5.0). huCO-mAbs were reacted with recombinant CD47 at a gradient concentration.

[0444] (analysis) device BIACore S200 Materials and reagents SA chip (streptavidin) recCD47 Sino Biological (12283-H27H-B), 17 kDa, C-terminal His- and AVI-tag, biotinylated Antibodies CO201-CO212 (all 0.5 mg / ml) · Buffer 20mM phosphate buffer, pH7.4 / 2.7mM KCl / 137mM NaCl / 0.05% P20 Single Cycle Procedure Immobilize recCD47 to a level of 100 RU (10 mM NaAc, pH 5.0) Regeneration conditions: 60 seconds, 10 mM glycine, pH 1.5 Analyte (antibody or fragment) used in single cycle mode Injection order: from low to high: 10nM / 5nM / 2.5nM / 1.25nM / 0.625nM / 0.3125nM / 0.1562nM / 0.07813nM / 0.039nM Flow rate 30 μl / min, 120 seconds on, 180 seconds off - 1800 seconds off after last injection, Temperature 25℃

[0445] (result) A 1:1 binding model was used to fit the data. All curves fit well with the 1:1 model. Table 10 shows the values ​​obtained for the binding on-rate and off-rate, ka and kd, respectively. The affinity constant K D is k d / k a Calculate as follows.

[0446] [Table 17]

[0447] Example 8: In vivo activity of CO-1bi (material and method) CO-1 antibody and CO-1bi fusion protein Chimeric CO-1.4 (referred to in this example as CO-1) was generated by GenScript via expression in a mammalian expression host. The sequence of CO-1.4 is shown in Table C. A bivalent scFv of CO201 coupled to Fc (CO201-scFv-Fc-bi) as described in Example 7 was designed and generated by ATUM and referred to in this example as CO-1bi.

[0448] cell culture All cell lines were purchased from the American Type Culture Collection (ATCC). The B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cell line, Reh, was grown at 2 × 10 cells per ml in RPMI 1640 medium (Lonza). 5~1×10 6 The Burkitt lymphoma cell line, Raji, was maintained at a density of between 4 × 10 cells per ml in RPMI 1640 medium. 5 ~2×10 6 HEK293T cells were used for lentivirus generation and were subcultured every 2–3 days when they reached 70–90% confluence in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific). The cell culture medium was supplemented with 10% (vol / vol) fetal bovine serum (FBS, Thermo Fisher Scientific) and 1% (vol / vol) penicillin / streptomycin (PS, Thermo Fisher Scientific). Cells were cultured in a humidified atmosphere with 95% air and 5% CO2.

[0449] Lentivirus production in HEK293T cells Same as in Example 5 above.

[0450] Lentiviral transduction of Reh cells Same as in Example 5 above.

[0451] Establishment of ALL xenograft model Transduced Reh cells (5 × 10 5Reh cells were injected into 6-8 week-old female NOD scid IL2Rγnull (NSG) mice (Jackson Laboratory) anesthetized with isoflurane (induction 4%-5%, maintenance 2%-3%, oxygen flow 300 mL / min). The knee was kept in a flexed position to expose the proximal end of the tibia. After drilling a hole in the tibia with a 23 G needle, Reh cells (40 μL per animal) were injected using a 31 G insulin syringe. Mice were treated with systemic and local analgesics, 0.05 mg / kg Temgesic (Schlering-Plough) and 1-2 mg / kg Marcain (AstraZeneca) before IT injection. The mice were injected intraperitoneally (IP) with a single 1.33 nM dose of either CO-1, CO-1bi, or a human IgG4 isotype control. Their health status was monitored daily, and all animal procedures were performed in accordance with approval by the Norwegian Food Safety Authority under identification number 29016.

[0452] In vivo imaging Same as in Example 5 above.

[0453] Establishment of a Burkitt's lymphoma xenograft model Mice were implanted with 1.5 × 10 guinea pigs in a 1:2 suspension of RPMI:Vitrogel (TheWell Bioscience) in 0.1 mL of RPMI:Vitrogel (TheWell Bioscience) on the right flank. 6 Raji cells were inoculated subcutaneously (SC). The tumor size was an average of 190 mm. 3 Treatment began when tumor volume reached 1000 mg / kg / day, and mice were IP injected with the indicated doses of CO-1 or CO-1bi twice weekly for 3 weeks. Tumor volumes were measured in two dimensions using standard calipers. Tumor volume was calculated using the following formula:

[0454]

number

[0455] Tumor volume ≥ 2.5cm 3 Animals that reached 0.5% or progressed to ulceration in the tumor were sacrificed.

[0456] (result) Reh cells were stably transduced with a lentiviral firefly luciferase-EGFP vector and injected intratumorally into NSG mice. Leukemia progression was tracked by noninvasive in vivo imaging of firefly luciferase-expressing Reh cells, demonstrating sufficient tumor engraftment in all mice at day 10 after intratumor injection (Figure 17A). Mice were divided into three groups and IP-injected with 1.33 nM of either isotype control (human IgG4), CO-1, or CO-1bi immediately after the imaging procedure. IVIS performed at day 14 after intratumor injection revealed no luminescence signal in all mice treated with CO-1 or CO-1bi, while the signal from the isotype control-treated mice was more than fourfold increased compared to day 10 after intratumor injection (Figures 17A and 17B). Mice were imaged again at days 17, 24, and 29 after intratumor injection, and the luminescence signal from CO-1 or CO-1bi-treated mice remained low (Figure 17B). The experiment was repeated, only this time the mice were treated on day 8 post-IT injection, with essentially the same results as on day 14 post-IT injection (FIG. 17C).

[0457] Raji cells were injected SC in suspension with an extracellular matrix gel called Vitrogel. Tumor development was monitored by caliper measurements, and on day 9 after cell injection, mice were randomly divided into four different treatment groups and injected with either a human IgG4 isotype control (6.67 nM), CO-1 (6.67 nM), or CO-1bi (1.33 nM or 6.67 nM). Treatment was repeated twice per week for a total of three weeks. As can be seen in Figure 18, CO-1 and CO-1bi rapidly cured tumors in mice when administered at 6.67 nM, while treatment with CO-1bi at 1.33 nM caused a significant delay in tumor development.

[0458] Example 9 SPR analysis to evaluate the binding affinity of CO-1bi to recombinant CD47 The binding affinity of CO-1bi to CD47 was quantified by surface plasmon resonance (SPR) on a BIACore S200 system. Recombinant biotinylated CD47 was immobilized on an SA streptavidin chip to a concentration of 100 RU (in 10 mM NaAc, pH 5.0). CO-1bi was reacted with recombinant CD47 at a gradient concentration.

[0459] (analysis) Equipment BIACore S200 (material) SA chip (streptavidin) recCD47 Sino Biological (12283-H27H-B), 17 kDa, C-terminal His- and AVI-tag, biotinylated Antibody fragment CO-1bi (103.5 kDa) All proteins were stored at -80°C. Buffer 20mM phosphate buffer, pH7.4 / 2.7mM, KCl / 137mM, NaCl / 0.05%, P20 Single Cycle Procedure recCD47 immobilized to a level of 100 RU (in 10 mM NaAc pH 5.0) Regeneration conditions: 10 mM glycine, pH 2, 60 seconds Analyte (CO-1bi) used in single cycle mode Injection order: from low to high: 20nM / 10nM / 5nM / 2.5nM / 1.25nM / 0.625nM / 0.3125nM / 0.1562nM / 0.07813nM / 0.039nM Flow rate: 30 μl / min, 120 seconds on - 1800 seconds off after last injection Temperature 25℃ Single Cycle Mode

[0460] (result) A 1:1 binding model was used to fit the data. All curves fit well with the 1:1 model. Table 11 shows the values ​​obtained for the binding on and off rates ka and kd, respectively. The affinity constant K D k d / k a , calculated as 40 pM. Therefore, the CO-1bi fusion protein exhibits high binding affinity to CD47.

[0461] [Table 18]

Claims

1. 1. A binding protein or antibody comprising two antigen-binding domains that bind to CD47, 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; A binding protein or antibody, wherein the antigen-binding domain binds to Q19, N45, T120, R121, E122, and G123 in CD47 as defined by SEQ ID NO.

19.

2. The heavy chain variable region (i) a variable heavy (VH) CDR1 comprising the amino acid sequence of NFGMH (SEQ ID NO. 5) or a sequence substantially homologous thereto; (ii) a VH CDR2 comprising the amino acid sequence of WINTYTGEPTYTDDFKG (SEQ ID NO. 6), or a sequence substantially homologous thereto; (iii) a VH CDR3 comprising the amino acid sequence of GDYRYGDS (SEQ ID NO. 7) or a sequence substantially homologous thereto; and / or The light chain variable region (iv) a variable light (VL) CDR1 comprising the amino acid sequence RSSQSLVHSNGKTYLH (SEQ ID NO. 8), or a sequence substantially homologous thereto; (v) a VL CDR2 comprising the amino acid sequence of RVSNRFS (SEQ ID NO. 9) or a sequence substantially homologous thereto; (vi) a VL CDR3 comprising the amino acid sequence of SQSTHVPFT (SEQ ID NO. 10) or a sequence substantially homologous thereto; 2. The binding protein or antibody of claim 1, wherein the substantially homologous sequence is a sequence containing one, two, or three amino acid substitutions compared to the given CDR sequence.

3. The heavy chain variable region (i) a variable heavy (VH) CDR1 comprising the amino acid sequence of NFGMH (SEQ ID NO. 5); (ii) a VH CDR2 comprising the amino acid sequence of WINTYTGEPTYTDDFKG (SEQ ID NO. 6); and (iii) a VH CDR3 comprising the amino acid sequence of GDYRYGDS (SEQ ID NO. 7); and The light chain variable region (iv) a variable light (VL) CDR1 comprising the amino acid sequence RSSQSLVHSNGKTYLH (SEQ ID NO. 8); and (v) a VL CDR2 comprising the amino acid sequence of RVSNRFS (SEQ ID NO. 9); and (vi) a VL CDR3 comprising the amino acid sequence of SQSTHVPFT (SEQ ID NO. 10).

4. the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 3 or a sequence having at least 80% sequence identity thereto, and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO. 4 or a sequence having at least 80% sequence identity thereto; or the binding protein or antibody comprises a humanized version of SED ID NO. 3 and / or SED ID NO. 4, or 4. The binding protein or antibody of any one of claims 1 to 3, wherein the heavy chain variable region comprises one or more amino acid sequences of SEQ ID NO. 39, 40, 41, 42, or 43, or a sequence with at least 80% sequence identity thereto, and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO. 44, 45, or 46, or a sequence with at least 80% sequence identity thereto.

5. 5. The antibody of any one of claims 1 to 4, wherein the antibody is a full length Ig antibody, preferably a full length IgG antibody, more preferably an IgG1 or IgG4 antibody, or wherein the antibody is a bivalent antibody fragment against CD47, preferably an F(ab')2 fragment or a bivalent scFv-Fc format.

6. Binding affinity (K) for CD47 of less than 100 pM, preferably less than 60 pM, more preferably less than 10 pM Ｄ 6. The binding protein or antibody of claim 1, wherein

7. 7. The binding protein or antibody of any one of claims 1 to 6, wherein the binding protein or antibody is capable of inducing programmed cell death of tumor cells and optionally capable of inducing phagocytosis of tumor cells.

8. An immunoconjugate comprising the binding protein or antibody of 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 of any one of claims 1 to 8.

10. 10. One or more expression vectors comprising one or more of the nucleic acid molecules of claim 9.

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

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

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

15. A binding protein or antibody comprising two antigen-binding domains that bind to CD47, wherein the antigen-binding domains are in scFv format, and wherein the antigen-binding domains are fused, linked, or attached to an Fc region.

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

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

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

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

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