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WO2024213119A1 - Domaines de liaison à cd47 sensibles au ph et leurs utilisations - Google Patents

Domaines de liaison à cd47 sensibles au ph et leurs utilisations Download PDF

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Publication number
WO2024213119A1
WO2024213119A1 PCT/CN2024/087509 CN2024087509W WO2024213119A1 WO 2024213119 A1 WO2024213119 A1 WO 2024213119A1 CN 2024087509 W CN2024087509 W CN 2024087509W WO 2024213119 A1 WO2024213119 A1 WO 2024213119A1
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seq
binding domain
antibody
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cdr
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Wangzhi LI
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Adaptocue LLC
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Adaptocue LLC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • Phagocytosis is a major mechanism to clear pathogens, disease cells, dying cells, and cell debris by professional phagocytes such as macrophages, monocytes, dendritic cells, and granulocytes.
  • the trigger of phagocytosis of a target cell or agent by a phagocyte is driven by an integration and imbalance of pro-phagocytic ( “eat me” ) signal and anti-phagocytic ( “don’t eat me” ) signals.
  • CD47 cluster of differentiation 47
  • IAP integrated protein
  • IAP interleukin-associated protein
  • CD47 functions as a major anti-phagocytic signal that inhibits phagocytosis of the cells expressing CD47 through interacting with the Signal Regulatory Protein (SIRP) receptor, most notably SIRP ⁇ , on phagocytes such as macrophages and triggering a "don't eat me” signal.
  • SIRP Signal Regulatory Protein
  • CD47 is ubiquitously expressed in normal tissues and cells, and plays an important role in sparing the normal cells from phagocytosis.
  • Disease cells such as cancer cells, often with upregulation of CD47 expression, however hijack this mechanism to escape normal immune control and clearance by phagocytes.
  • CD47 has been shown to be highly expressed and associated with adverse prognosis in a wide variety of cancers (Zhang et al., Front Immunol, 2020) .
  • Blocking the CD47 “don’t eat me” signal by CD47 binders, together with co-presence of sufficient “eat me” signal, is shown to promote phagocytosis of a variety of CD47-expressing cancer cells, and presents an attractive therapeutic strategy to treat a broad range of cancers.
  • upregulated CD47 is also found in atherosclerotic plaques to prevent effective removal of the diseased tissue, and administration of CD47-blocking antibodies can normalize this defective phagocytic clearance and ameliorates atherosclerosis (Kojima et al., Nature, 2016) .
  • Blocking of CD47 is also shown to reduce lung fibrosis in vivo (Wernig et al., Proc Natl Acad Sci U S A, 2017) .
  • CD47 is reported to prevent elimination of diseased fibroblasts in fibrotic scleroderma and blocking CD47 reversed skin fibrosis in combination with IL-6 blockade (Lerbs et al., JCI Insight, 2020) .
  • CD47 is reported in other diseases such as gaucher diseases, multiple sclerosis and stroke among others (Gheibihayat et al., Molecules, 2021) . Also, upregulation of CD47 expression is found during infection and blockade of CD47 enhances innate and adaptive immune response to infection (Zahavi et al., Antibodies (Basel) , 2020) . Thus, anti-CD47 therapeutics also holds promise for treating non-cancer diseases such as atherosclerosis, fibrosis and infection.
  • the present disclosure provides a CD47-binding domain wherein the domain: a) exhibits higher binding affinity to CD47 at an acidic pH than at physiological pH, wherein the acidic pH is less than 7.0 and the physiological pH is 7.2 to 7.5, and b) is not an IgV extracellular domain of the Signal-regulatory protein (SIRP) family proteins.
  • the acidic pH is 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, or 6.9.
  • the CD47-binding domain binds to CD47 with a binding affinity that is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold higher at an acidic pH than at physiological pH.
  • the pH-sensitive CD47-binding domain binds to human wild type CD47 at physiological pH with a monovalent affinity preferably lower than the monovalent affinity of wild type human SIPR ⁇ V2 IgV (SEQ ID NO: 306) to human wild type CD47, wherein the physiological pH is 7.2, 7.3 or 7.4.
  • the pH-sensitive CD47-binding domain binds to human wild type CD47 at physiological pH with a monovalent affinity preferably lower than the monovalent affinity of a CD47-binding antibody comprising a heavy chain (HC) sequence of SEQ ID NO: 441 and a light chain (LC) sequence of SEQ ID NO: 442 to CD47, wherein the physiological pH is 7.2, 7.3 or 7.4.
  • HC heavy chain
  • LC light chain
  • the pH-sensitive CD47-binding domain exhibits negligible binding to human primary platelets at physiological pH, or exhibiting binding to human primary platelets at physiological pH with an EC50 lower than that of monovalent binding of wild type human SIPR ⁇ V2 IgV (SEQ ID NO: 306) as measured by a FACS binding assay, wherein the physiological pH is 7.2, 7.3 or 7.4.
  • the pH-sensitive CD47-binding domain binds to at least one of the following residues: E29, E35, D46, D51, E97, E100, E104 or E106 of SEQ ID NO: 474, wherein the pH-sensitive CD47-binding domain exhibits higher binding affinity to CD47 at an acidic pH than at physiological pH.
  • the pH-sensitive CD47-binding domain comprises a Fragment antigen-binding domain (Fab) of antibody, a single chain Fab domain (scFab) , a Fragment variable (Fv) domain of antibody, a single chain Fv (scFv) , a disulfide-stabilized Fv (dsFv) , a VHH antibody or nanobody, a heavy chain variable domain (VH) single chain antibody (VH dAb) or a light chain variable domain (VL) single chain antibody (VL dAb) or other single-chain domain antibody, or an anticalin, DARPIN, affibody, affimer, centryin domain, fibronectin domain, D-Domain or other alternative scaffold known in the arts to function as antigen binding domainthat binds to CD47, or other polypeptide or peptide that binds to CD47.
  • Fab fragment antigen-binding domain
  • scFab single chain Fab domain
  • Fv Fragment variable domain
  • the pH-sensitive CD47-binding domain is a variant of a parent CD47-binding domain or fragment thereof a CD47-binding antibody selected from a group of CD47-binding antibodies as set forth in Table 1, and comprises one or more mutations of residues of the parent CD47-binding domain.
  • the pH-sensitive CD47-binding domain comprises a set of three VH CDR sequences and/or three VL CDR sequences at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, or at least 90%identical to the set of VH and/or VL CDR amino acid sequences of a parent anti-CD47 antibody as set forth in Table 2.
  • the set of VH CDR sequences and/or VL CDR sequences of the pH-sensitive CD47-binding domain comprise one or more substitutions selected from the group of substitutions of the CDR sequences of its parent anti-CD47 antibody as set forth in Table 2.
  • the pH-sensitive CD47-binding domain is a variant derived from the parent anti-CD47 antibody B6H12.
  • the CD47-binding domain exhibits higher binding affinity to CD47 at an acidic pH than at physiological pH, wherein the acidic pH is less than 7.0 and the physiological pH is 7.2 to 7.5, and comprises one or more substitutions of the parent CDR residues selected from the group consisting of:G1H, G1E, G1D, Y2H, Y2E, Y2D, G3H, G3E, G3D, M4H, M4E and M4D of HCDR1 SEQ ID NO: 82; T1H, T1E, T1D, I2H, I2E, I2D, T3H, T3E, T3D, S4H, S4E, S4D, G5H, G5E, G5D, G6H, G6E, G6D, T7H, T7E, T7D,
  • the CD47-binding domain comprises one or more substitutions of the parent CDR residues selected from the group consisting of: Y2H of SEQ ID NO: 82; T3H, Y8H and Y10H of SEQ ID NO: 84; A3H of SEQ ID NO: 89; S7H, D8H and Y9H of SEQ ID NO: 91; K1H and Q5H of SEQ ID NO: 95; and G5H, F6H and R8H of SEQ ID NO: 99.
  • the CD47-binding domain exhibits higher binding affinity to CD47 at an acidic pH than at physiological pH, and comprises:
  • VH CDR1 HCDR1 sequence of SEQ ID NO: 76: GX 1 X 2 MS, wherein X 1 is Y, H, E or D, X 2 is G, H, E or D,
  • VH CDR2 (HCDR2) sequence of SEQ ID NO: 77: TIX 1 X 2 GGTX 3 TX 4 YPDSVKG, wherein X 1 is T, H, E or D, X 2 is S, H, E or D, X 3 is Y, H, E or D, X 4 is Y, H, E or D,
  • VH CDR3 HCDR3 sequence of SEQ ID NO: 78: SLX 1 X 2 NAMDY, wherein X 1 is A, H, E or D, X 2 is G, H, E or D,
  • VL CDR1 (LCDR1) sequence of SEQ ID NO: 79: RASQTIX 1 X 2 X 3 , wherein X 1 is S, H, E or D, X 2 is D, H or E, X 3 is Y, H, E or D,
  • LCDR2 VL CDR2 (LCDR2) sequence of SEQ ID NO: 80: X 1 FASX 2 X 3 X 4 X 5 , wherein X 1 is K, H, E or D, X 2 is Q, H, E or D, X 3 is S, H, E, D or R, X 4 is I, H, E, D or A, X 5 is S, H, E, D or T, and
  • VL CDR3 LCDR3 sequence of SEQ ID NO: 81: QNGHX 1 X 2 PX 3 T, wherein X 1 is G, H, E or D, X 2 is F, H, E or D, X 3 is R, H, E or D.
  • the CD47-binding domain exhibits higher binding affinity to CD47 at an acidic pH than at physiological pH wherein the acidic pH is less than 7.0 and the physiological pH is 7.2 to 7.5, and comprises one or more histidine substitutions of the CDR residues of a parent antibody comprising a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 82, 84, 89, 91, 95 and 99 respectively, wherein the histidine substitutions of the parent CDR residues are selected from the group consisting of: T3H, Y8H and Y10H of SEQ ID NO: 84; A3H of SEQ ID NO: 89; S7H and Y9H of SEQ ID NO: 91; K1H and Q5H of SEQ ID NO: 95; and G5H, F6H and R8H of SEQ ID NO: 99.
  • the CD47-binding domain exhibits higher binding affinity to CD47 at an acidic pH than at physiological pH, and comprises:
  • VH CDRs selected from at least one of the group consisting of:
  • a HCDR1 at least 50%, at least 60%, at least 70%, at least 80%or at least 90%identical to a HCDR1 as set forth in one of the sequences selected from the group consisting of SEQ ID NO: 82 to 83 ;
  • a HCDR2 at least 50%, at least 60%, at least 70%, at least 80%or at least 90%identical to a HCDR2 as set forth in one of the sequences selected from the group consisting of SEQ ID NO: 84 to 88 and SEQ ID NO: 111 to 113;
  • a HCDR3 at least 50%, at least 60%, at least 70%, at least 80%or at least 90%identical to a HCDR3 as set forth in one of the sequences selected from the group consisting of SEQ ID NO: 89 to 90;
  • VL CDRs one or more VL CDRs (LCDRs) selected from at least one of the group consisting of:
  • a LCDR1 at least 50%, at least 60%, at least 70%, at least 80%or at least 90%identical to a LCDR1 as set forth in one of the sequences selected from the group consisting of SEQ ID NO: 91 to 94 and SEQ ID NO: 107 to 110;
  • a LCDR2 at least 50%, at least 60%, at least 70%, at least 80%or at least 90%identical to a LCDR2 as set forth in one of the sequences selected from the group consisting of SEQ ID NO: 95 to 98;
  • a LCDR3 at least 50%, at least 60%, at least 70%, at least 80%or at least 90%identical to a LCDR3 as set forth in one of the sequences selected from the group consisting of SEQ ID NO: 99 to 106;
  • the CD47-binding domain comprises a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 82, 85/86/87/88, 89, 91, 95 and 99 respectively; or SEQ ID NO: 82, 84, 90, 91, 95 and 99 respectively; or SEQ ID NO: 82, 84, 89, 92/93/94/107/108/109/110, 95 and 99 respectively; or SEQ ID NO: 82, 84, 89, 91, 96/97/98 and 99 respectively; or SEQ ID NO: 82, 84, 89, 91, 95 and 100/101/102/103/104/105 /106 respectively; or SEQ ID NO: 82, 85/86/87/88, 90, 91, 95 and 99 respectively; or SEQ ID NO: 82, 84, 89, 92/93/94/107/108/109/110, 95 and 102 respectively; or SEQ ID NO: 82
  • the CD47-binding domain comprises a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences as set forth in Table 4.
  • the CD47-binding domain comprises a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 82, 85/86/87/88, 89, 91, 95 and 99 respectively; or SEQ ID NO: 82, 84, 90, 91, 95 and 99 respectively; or SEQ ID NO: 82, 84, 89, 92/93/94, 95 and 99 respectively; or SEQ ID NO: 82, 84, 89, 91, 96/97/98 and 99 respectively; or SEQ ID NO: 82, 84, 89, 91, 95 and 100/101/102/103/104/105/106 respectively; or SEQ ID NO: 82, 86/87/88, 90, 91, 95 and 99 respectively; or SEQ ID NO: 82, 86, 89, 93, 96 and 105 respectively; or SEQ ID NO: 82, 87, 89, 93, 97 and 104 respectively;
  • the CD47-binding domain comprises:
  • the CD47-binding domain comprises a pair of VH and VL sequences selected from the group of pairs of VH and VL sequences consisting of SEQ ID NO: 124 and 133, SEQ ID NO: 125 and 133, SEQ ID NO: 126 and 133, SEQ ID NO: 127 and 133, SEQ ID NO: 128 and 133, SEQ ID NO: 123 and 134, SEQ ID NO: 123 and 135, SEQ ID NO: 123 and 136, SEQ ID NO: 123 and 137, SEQ ID NO: 123 and 138, SEQ ID NO: 123 and 139, SEQ ID NO: 123 and 140, SEQ ID NO: 123 and 141, SEQ ID NO: 126 and 148, SEQ ID NO: 127 and 144, SEQ ID NO: 126 and 146, SEQ ID NO: 127 and 143, SEQ ID NO: 126 and 153, SEQ ID NO: 126 and 150, and SEQ ID NO: 129
  • the CD47-binding domain comprises a pair of VH and VL sequences selected from the group of pairs of VH and VL sequences consisting of SEQ ID NO: 127 and 133, SEQ ID NO: 128 and 133, SEQ ID NO: 123 and 137, SEQ ID NO: 123 and 141, SEQ ID NO: 126 and 148, SEQ ID NO: 127 and 144, SEQ ID NO: 126 and 146, SEQ ID NO: 127 and 143.
  • the CD47-binding domain is a Fab, a Fab’ , a F (ab’ ) 2, a single chain Fab domain (scFab) , a Fragment variable (Fv) domain, a single chain Fv (scFv) , a disulfide-stabilized Fv (dsFv) , a nanobody, or a single chain antibody.
  • the CD47-binding domain comprises a single chain fragment variable (scFv) comprising a VL-linker-VH sequence or preferably a VH-linker-VL sequence, wherein:
  • (ii) comprises an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%identical to a VH sequence selected from the group consisting of SEQ ID NO: 123 to 132; or
  • (iii) comprises an amino acid sequence with addition, deletion and/or substitution of one or more amino acids compared with SEQ ID NO: 123;
  • (i) comprises the amino acid sequence of SEQ ID NO: 121 or 122;
  • (ii) comprises an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%identical to a VL sequence selected from the group comprising SEQ ID NO: 133 to 174 and 266 to 273; or
  • (iii) comprises an amino acid sequence with addition, deletion and/or substitution of one or more amino acids compared with SEQ ID NO: 133;
  • the linker comprises a sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 276) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 277) .
  • the CD47-binding domain comprises a single chain fragment variable (scFv) comprising a sequence of SEQ ID NO: 175 or 176.
  • the CD47-binding domain comprises a scFv comprising a VH-linker-VL sequence, wherein the linker comprises GGGGSGGGGS GGGGS (SEQ ID NO: 276) and the pair of VH and VL sequences comprise SEQ ID NO: 124 and 133, SEQ ID NO: 125 and 133, SEQ ID NO: 126 and 133, SEQ ID NO: 127 and 133, SEQ ID NO: 128 and 133, SEQ ID NO: 123 and 134, SEQ ID NO: 123 and 135, SEQ ID NO: 123 and 136, SEQ ID NO: 123 and 137, SEQ ID NO: 123 and 138, SEQ ID NO: 123 and 139, SEQ ID NO: 123 and 140, SEQ ID NO: 123 and 141, SEQ ID NO: 124 and 133
  • the CD47-binding domain comprises a scFv comprising a VH-linker-VL sequence, wherein the linker comprises GGGGSGGGGSGGGGS (SEQ ID NO: 276) , and the pair of VH and VL sequences comprise SEQ ID NO: 127 and 133, SEQ ID NO: 128 and 133, SEQ ID NO: 123 and 137, SEQ ID NO: 123 and 141, SEQ ID NO: 126 and 148, SEQ ID NO: 127 and 144, SEQ ID NO: 126 and 146, or SEQ ID NO: 127 and 143.
  • the CD47-binding domain comprises a scFv comprising a sequence of SEQ ID NO: 300 or 301.
  • the pH-sensitive CD47-binding domain is a variant derived from the parent anti-CD47 antibody BC31M4 (see (Li et al., J Hematol Oncol, 2023) ) , wherein the variant exhibits improved pH-sensitive CD47-binding with reduced binding at physiological pH and/or increased binding at acidic pH compared to that of the parent antibody BC31M4.
  • the CD47-binding domain comprises three variable VH CDR sequences at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, or at least 80%identical to the three parent VH CDR amino acid sequences of SEQ ID NO: 19, 20 and 21, and/or three VL CDR sequences at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, or at least 80%identical to the three parent VL CDR amino acid sequences of SEQ ID NO: 22, 23 and 24.
  • the CD47-binding domain comprises one or more substitutions of parent CDR residues selected from the group consisting of: Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, Y7H/E/D and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, R2H/E/D, A4H/E/D, V5H/E/D, Y6H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 21, R1H/E/D, S3H/E/D, Q4H/E/D, S7H/E/D, R8H/E/D, and N9H/E/D of SEQ
  • the CD47-binding domain exhibits improved pH-sensitive CD47-binding with reduced binding at physiological pH and/or increased binding at acidic pH compared to that of the parent antibody BC31M4, and comprise a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences selected from the group of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences as set forth in Table 5.
  • the CD47-binding domain comprises a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 177, 20, 21, 22, 23 and 24 respectively; or SEQ ID NO: 178, 20, 21, 22, 23 and 24 respectively; or SEQ ID NO: 179, 20, 21, 22, 23 and 24 respectively; or SEQ ID NO: 19, 20, 180, 22, 23 and 24 respectively; or SEQ ID NO: 19, 20, 181, 22, 23 and 24 respectively; or SEQ ID NO: 19, 20, 182, 22, 23 and 24 respectively; or SEQ ID NO: 19, 20, 21, 183, 23 and 24 respectively; or SEQ ID NO: 19, 20, 21, 184, 23 and 24 respectively; or SEQ ID NO: 19, 20, 21, 185, 23 and 24 respectively; or SEQ ID NO: 19, 20, 21, 186, 23 and 24 respectively; or SEQ ID NO: 19, 20, 21, 187, 23 and 24 respectively; or SEQ ID NO: 19, 20, 21, 188, 23 and 24 respectively; or SEQ ID NO: 19, 20, 21, 189, 23
  • the pH-sensitive CD47-binding domain is a variant derived from the parent anti-CD47 antibody BC27 (see (Li et al., J Hematol Oncol, 2023) ) , wherein the variant exhibits improved pH-sensitive CD47-binding with reduced binding at physiological pH and/or increased binding at acidic pH compared to that of the parent antibody BC27.
  • the CD47-binding domain comprises three variable VH CDR sequences at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, or at least 80%identical to the three parent VH CDR amino acid sequences of SEQ ID NO: 19, 20 and 428, and/or three VL CDR sequences at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, or at least 80%identical to the three parent VL CDR amino acid sequences of SEQ ID NO: 22, 23 and 429.
  • the CD47-binding domain comprises one or more substitutions of parent CDR residues selected from the group consisting of: Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, Y7H/E/D and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, R2H/E/D, K4H/E/D/A, V5H/E/D, Y6H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 428, R1H/E/D, S3H/E/D, Q4H/E/D, S7H/E/D, R8H/E/D, and N9
  • the CD47-binding domain exhibits improved pH-sensitive CD47-binding with reduced binding at physiological pH and/or increased binding at acidic pH compared to that of the parent antibody BC27, and comprise a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences selected from the group of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences as set forth in Table 6.
  • the CD47-binding domain comprises a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 177, 20, 428, 22, 23 and 429 respectively; or SEQ ID NO: 178, 20, 428, 22, 23 and 429 respectively; or SEQ ID NO: 179, 20, 428, 22, 23 and 429 respectively; or SEQ ID NO: 19, 20, 180, 22, 23 and 429 respectively; or SEQ ID NO: 19, 20, 181, 22, 23 and 429 respectively; or SEQ ID NO: 19, 20, 182, 22, 23 and 429 respectively; or SEQ ID NO: 19, 20, 428, 183, 23 and 429 respectively; or SEQ ID NO: 19, 20, 428, 184, 23 and 429 respectively; or SEQ ID NO: 19, 20, 428, 185, 23 and 429 respectively; or SEQ ID NO: 19, 20, 428, 186, 23 and 429 respectively; or SEQ ID NO: 19, 20, 428, 187, 23 and 429 respectively; or SEQ ID NO: 19,
  • the pH-sensitive CD47-binding domain is a variant derived from the parent anti-CD47 antibody lemzoparlimab (TJ-C4) (see US20200140565A1) .
  • the CD47-binding domain exhibits higher binding affinity to CD47 at an acidic pH than at physiological pH, and comprises three variable VH CDR sequences at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, or at least 80%identical to the three parent VH CDR amino acid sequences of SEQ ID NO: 37, 38 and 39, and/or three VL CDR sequences at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, or at least 80%identical to the three parent VL CDR amino acid sequences of SEQ ID NO: 40, 41 and 42.
  • the CD47-binding domain comprises one or more substitutions of parent CDR residues selected from the group consisting of: R1H/E/D, A2H/E/D, W3H/E/D, and N5H/E/D of SEQ ID NO: 37, R2H/E/D, I3H/E/D, K4H/E/D, R5H/E/D, K6H/E/D, T7H/E/D, D8H/E, Y14H/E/D, and K19H/E/D of SEQ ID NO: 38, N2H/E/D, R3H/E/D, A4H/E/D, and F5H/E/D of SEQ ID NO: 39, K1H/E/D, S5H/E/D, Y8H/E/D, N11H/E/D, N12H/E/D, R13H/E/D, N14H/E/D, and Y15H/E
  • the CD47-binding domain exhibits higher binding affinity to CD47 at an acidic pH than at physiological pH, and comprise a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences selected from the group of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences as set forth in Table 7.
  • the CD47-binding domain comprises a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 222, 38, 39, 40, 41 and 42 respectively; or SEQ ID NO: 37, 223, 39, 40, 41 and 42 respectively; or SEQ ID NO: 37, 38, 224, 40, 41 and 42 respectively; or SEQ ID NO: 37, 38, 39, 225, 41 and 42 respectively; or SEQ ID NO: 37, 38, 39, 40, 41 and 226 respectively; or SEQ ID NO: 37, 38, 39, 40, 41 and 227 respectively; or SEQ ID NO: 37, 38, 39, 40, 41 and 228 respectively; or SEQ ID NO: 37, 223, 224, 40, 41 and 42 respectively; or SEQ ID NO: 222, 38, 224, 40, 41 and 42 respectively; or SEQ ID NO: 222, 223, 39, 40, 41 and 42 respectively; or SEQ ID NO: 37, 38, 224, 40, 41 and 42
  • the pH-sensitive CD47-binding domain is a variant derived from the parent anti-CD47 antibody AO-176 (see Blood (2016) 132 (Supplement 1) : 4180) , wherein the variant exhibits improved pH-sensitive CD47-binding with reduced binding at physiological pH and/or increased binding at acidic pH compared to that of the parent antibody AO-176.
  • the CD47-binding domain comprises three variable VH CDR sequences at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, or at least 80%identical to the three parent VH CDR amino acid sequences of SEQ ID NO: 61, 62 and 63, and/or three VL CDR sequences at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, or at least 80%identical to the three parent VL CDR amino acid sequences of SEQ ID NO: 64, 65 and 66.
  • the CD47-binding domain comprises one or more substitutions of parent CDR residues selected from the group consisting of: Y2H/E/D, T3H/E/D, T5H/E/D, N6H/E/D, and Y7H/E/D of SEQ ID NO: 61, Y1H/E/D, T2H/E/D, R5H/E/D, T6H/E/D, Y8H/E/D, T9H/E/D, and Y11H/E/D of SEQ ID NO: 62, G1H/E/D, R3H/E/D, G5H/E/D, G7H/E/D, and Y8H/E/D of SEQ ID NO: 63, R1H/E/D, N5H/E/D, Q8H/E/D, S9H/E/D, N10H/E/D, N12H/E/D, and Y14H/
  • Antibody comprising a pH-sensitive CD47-binding domain
  • the present disclosure provides a protein comprising at least one of the pH-sensitive CD47-binding domains of the present disclosure.
  • the protein comprises a Fc or its functional fragment.
  • the Fc is selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and its modification.
  • the Fc is a human IgG1 with wild-type effector function (SEQ ID NO: 291) .
  • the Fc is a human IgG1 with reduced effector function.
  • the Fc comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 288 to 290.
  • the Fc is a human IgG1 Fc with enhanced antibody-dependent cellular phagocytosis (ADCP) function.
  • the Fc is a human IgG4 Fc with a mutation of S228P (SEQ ID NO: 286) .
  • the Fc is a human IgG2 Fc (SEQ ID NO: 287) .
  • the Fc comprises a homodimeric Fc, wherein the two Fc chains of the Fc comprise mutation that promote formation of heterodimeric Fc.
  • the knobs-into-holes heterodimeric Fc comprises a pair of Fc amino acid sequence selected from the group consisting of SEQ ID NO: 292: 293, 294: 295, 296: 297 and 298: 299, wherein the “: ” indicates pairing of the two sequences from the left to right of the “: ” symbol.
  • the protein is an isolated antibody comprising a full heavy chain and full light chain, wherein the heavy chain and light chain form a pH-sensitive CD47-binding Fab domain of the present disclosure.
  • the present disclosure provides an isolated antibody or the antigen-binding portion thereof, wherein it competes for binding to CD47 with the pH-sensitive CD47-binding domain or the antigen-binding portion thereof the present disclosure.
  • Antibody fusion protein comprising a pH-sensitive CD47-binding domain
  • the present disclosure provides an antibody fusion protein comprising: 1) a full antibody comprising two pH-sensitive CD47-binding Fab domains of the present disclosure, and 2) a CD47-binding SIRP IgV domain, wherein the SIRP IgV domain comprises one, two, three or more SIRP IgV monomers, and is linked preferably through a linker to the N-terminal or C-terminal of the heavy chain or the light chain of the antibody.
  • the antibody fusion protein is configured in a format FV-1, FV-2, FV-3 or FV-4 as set forth in FIG. 1.
  • the SIRP IgV monomer is a SIRP ⁇ , SIRP ⁇ or SIRP ⁇ IgV.
  • the SIRP IgV is a SIRP ⁇ , SIRP ⁇ or SIRP ⁇ IgV comprising one or more mutations selected from the group consisting of K53H, R69H, Q52H, K68H, Q52H+K68H, K53H+K68H, R69H+K68H, K53H+Q52H, R69H+Q52H, K53H+K68H+Q52H, and R69H+K68H+Q52H.
  • the SIRP IgV monomer comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 306 to 311.
  • the CD47-binding Fab domain of the antibody fusion protein comprises a pair of VH and VL sequence selected from the group consisting of SEQ ID NO: 126 and 148, SEQ ID NO: 127 and 144, SEQ ID NO: 126 and 146, and SEQ ID NO: 127 and 143.
  • the antibody fusion protein comprises a heavy chain of SEQ ID NO: 258 and a light chain of SEQ ID NO: 312 or 313.
  • the antibody fusion protein comprises a heavy chain of SEQ ID NO: 259 and a light chain of SEQ ID NO: 314 or 315.
  • the present disclosure provides a fusion protein comprising a CD47-binding domain of the present disclosure, comprises at least one additional domain that binds to a non-CD47 antigen.
  • the additional domain binds to a soluble antigen, an aggregate of antigen, a surface antigen on a cell, a surface antigen on a microbe, or a hapten antigen, wherein the antigen is not CD47.
  • the fusion protein comprising a CD47-binding domain of the present disclosure and at least one additional non-CD47 binding domain is an antibody fusion protein.
  • the antibody fusion protein comprising a CD47-binding domain of the present disclosure and at least one additional non-CD47 binding domain, is configured in a format variant selected from the group consisting of FV-6 to 21 as set forth in FIG. 2 to 5.
  • the additional non-CD47 binding domain of the fusion protein binds to one or more antigens selected from the group consisting of Amyloid Beta, Amyloid fibril, SAP, CD38, SLAMF7, BCMA, GPRC5D, FcRH5, CD138, CD56, CD74, CD26, CD46, CD19, CD20, CD22, CD30, CD33, CD37, CD70, CD117, CD79b, CD123, CD52, CD98, CD205, PD-L1, HER2, EGFR, CD93, TGF ⁇ , IL-6, IL-6R, IL-8, GDF-15, GFRAL, FGFR2, FGFR3, FGFR4, FGFR1, GD2, GD3, B7-H3, B7-H4, phosphatidylserine, DR5, DR4, DR3, TL1A, CD95 (Fas) , TNFR1, TNFR2, CD24, CD31, CD61, CD200, SLAMF3, SLAMF
  • the additional non-CD47 binding domain of the fusion protein binds to one or more antigens selected from the group consisting of PD-L1, CD38, SLAMF7, CD20, CD19, CD30, CD70, CD117, CA-IX, HER2, EGFR, CD93, FGFR2, GD2, Claudin18.2, Claudin 6, Claudin 1, Claudin 2, Claudin 3, Claudin 4, Claudin 7, B7-H3, DLL3, DR5, DR4, CD95, Phosphatidylserine, Nectin-4, CDH3, CDH6, CDH17, CDH2, integrins, CD44, ICAM-1, EpCAM, CEACAM5, CEACAM1, CEACAM6, CD24, HLA-G, FAP, CTGF and TL1A.
  • one or more antigens selected from the group consisting of PD-L1, CD38, SLAMF7, CD20, CD19, CD30, CD70, CD117, CA-IX, HER
  • the additional non-CD47 binding domain of the antibody fusion protein binds to one or more antigens selected from the group consisting of Dectin-1, Dectin-2, CLEC5A, MerTK, TREM1, MARCO, CLEVER-1, PSGL-1, VSIG4, CD40, CD205, CD206, CD36, CD91, DC-SIGN, CLEC9A, TLR5, LILRB1 (ILT2) , LILRB2 (ILT4) , LILRB4 (ILT3) , NKG2D, NKp30, NKp46, NKp80, DNAM-1, PD-1, CTLA-4, TIGIT, LAG3, CD3, 4-1BB, OX40, ICOS, CD27 and CD70.
  • one or more antigens selected from the group consisting of Dectin-1, Dectin-2, CLEC5A, MerTK, TREM1, MARCO, CLEVER-1, PSGL-1, VSIG4, CD40, CD205, CD206, CD36, CD91, DC
  • the additional non-CD47 binding domain of the fusion protein comprises an antibody domain or antigen-binding fragment thereof comprising a set of heavy chain variable (VH) and/or light chain variable (VL) complementarity determining region (CDR) sequences at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%or at least 95%identical to the set of VH and/or VL CDR sequences according to the IMGT numbering scheme (The Immunologist, 7, 132-136 (1999) ; Dev Comp Immunol. 2003 Jan; 27 (1) : 55-77) of an antibody selected from a group of exemplary antibodies as set forth in Table 8 to 10.
  • VH heavy chain variable
  • VL light chain variable complementarity determining region
  • the additional non-CD47 binding domain of the fusion protein comprises a peptide, a receptor or fragment thereof, a ligand or fragment thereof, a cytokine or fragment thereof, or a chemokine or fragment thereof, or a growth factor or fragment thereof.
  • the additional non-CD47 binding domain of the antibody fusion protein comprises an amino acid sequence at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%or at least 95%identical to an amino acid sequence selected from a group of exemplary peptides and polypeptides as set forth in Table 11.
  • an antibody fusion protein comprising: 1) a full anti-CD47 antibody of the present disclosure comprising two heavy chains and two light chains, wherein the two Fc region of the two heavy chains form a homodimeric Fc, and 2) a single-chain domain binding to a non-CD47 antigen, wherein the single-chain domain is linked preferably through a linker to the N-terminal or C-terminal of the heavy chain or the light chain of the anti-CD47 antibody.
  • the antibody fusion protein is configured in a format of FV-6 or 7 as set forth in FIG. 2, wherein the single-chain domain is linked to the N-terminal of the light chain or heavy chain.
  • the single-chain domain of the antibody fusion protein configured in a format of FV-6 or 7 comprises a sequence of SEQ ID NO: 316, 317, 318 or 319.
  • the antibody fusion protein is configured in a format of FV-8 or 9 as set forth in FIG. 2, wherein the single-chain domain is linked to the C-terminal of the heavy chain or light chain.
  • the single-chain domain of the antibody fusion protein configured in a format of FV-8 or 9 comprises a sequence of SEQ ID NO: 320, 321 or 322.
  • an antibody fusion protein comprising: 1) a first heavy chain comprising VH1-CH1-hinge-CH2-CH3 and a second chimeric heavy chain comprising VH2-CL-hinge-CH2-CH3, wherein the Fc region of the two heavy chains form a heterodimeric Fc, 2) a first light chain comprising VL1-CL, wherein it pairs with the first heavy chain to form the first Fab domain that binds to a non-CD47 antigen, and 3) a second chimeric light chain comprising VL2-CH1, wherein it pairs with the second chimeric heavy chain to form a second anti-CD47 Fab domain of the present disclosure.
  • the antibody fusion protein is configured in a format of FV-10 as set forth in FIG. 3.
  • an antibody fusion protein comprising: 1) a first heavy chain comprising VH1-CH1-hinge-CH2-CH3 and a second chimeric heavy chain comprising VH2-CL-hinge-CH2-CH3, wherein the Fc region of the two heavy chains form a heterodimeric Fc, 2) a first light chain comprising VL1-CL, wherein it pairs with the first heavy chain to form a first anti-CD47 Fab domain of the present disclosure, and 3) a second chimeric light chain comprising VL2-CH1, wherein it pairs with the second chimeric heavy chain to form the second Fab domain that binds to a non-CD47 antigen.
  • the antibody fusion protein is configured in a format of FV-11 as set forth in FIG. 3.
  • an antibody fusion protein comprising: 1) a first heavy chain comprising VH1-CH1-hinge-CH2-CH3 and a second chimeric heavy chain comprising VH2-CL-linker-VH1-CH1-hinge-CH2-CH3, wherein the Fc region of the two heavy chains form a heterodimeric Fc, 2) a first light chain comprising VL1-CL, wherein it pairs with the VH1-CH1 part of the first and second heavy chains to form two Fab domains that bind to a non-CD47 antigen, and 3) a second chimeric light chain comprising VL2-CH1, wherein it pairs with the VH2-CL part of the second chimeric heavy chain to form an anti-CD47 Fab domain of the present disclosure.
  • the antibody fusion protein is configured in a format of FV-13 as set forth in FIG. 3.
  • an antibody fusion protein comprising: 1) a first heavy chain comprising VH1-CH1-hinge-CH2-CH3 and a second chimeric heavy chain comprising VH1-CH1-linker-VH2-CL-hinge-CH2-CH3, wherein the Fc region of the two heavy chains form a heterodimeric Fc, 2) a first light chain comprising VL1-CL, wherein it pairs with the VH1-CH1 part of the first and second heavy chains to form two anti-CD47 Fab domains of the present disclosure, and 3) a second chimeric light chain comprising VL2-CH1, wherein it pairs with the VH2-CL part of the second chimeric heavy chain to form a Fab domain that binds to a non-CD47 antigen.
  • the antibody fusion protein is configured in a format of FV-12 as set forth in FIG. 3.
  • an antibody fusion protein comprising: 1) a full antibody comprising two Fab domains binding to a non-CD47 antigen, and 2) a single-chain CD47-binding domain of the present disclosure, wherein the single-chain CD47-binding domain is linked preferably through a linker to the N-terminal or C-terminal of the heavy chain or the light chain of the antibody.
  • the antibody fusion protein is configured in a format FV-14, FV-15, FV-16 or FV-17 as set forth in FIG. 4.
  • an antibody fusion protein comprising: 1) a first polypeptide comprising a Fc region, and a single-chain CD47-binding domain of the present disclosure, wherein the single-chain CD47-binding domain is linked preferably through a linker to the N-terminal of the Fc region, 2) a full heavy chain of an antibody and a full light chain of an antibody, wherein the antibody heavy chain and the light chain pair to form an half-antibody that binds to a non-CD47 antigen, and 3) the Fc region of the first polypeptide and the antibody heavy chain form a heterodimeric Fc.
  • the antibody fusion protein is configured in a format of FV-18 as set forth in FIG. 5.
  • the linker of an antibody fusion protein configured in a format of FV-18 comprises an amino acid sequence of SEQ ID NO: 274 or 275.
  • the antibody fusion protein further comprises a second single-chain CD47-binding domain of the present disclosure, wherein the second single-chain CD47-binding domain is linked preferably through a linker to the N-terminal of first single-chain CD47-binding domain, the light chain, or the heavy chain of the antibody fusion protein.
  • the antibody fusion protein comprising a second single-chain CD47-binding domain is configured in a format of FV-19, 20 and 21 as set forth in FIG. 5.
  • the CD47-binding domain of the antibody fusion protein configured in a format of FV-1 to 21 comprises a VH sequence selected from the group consisting of SEQ ID NO: 123 to 132, and a VL sequence selected from the group consisting of SEQ ID NO: 133 to 174 and 266 to 273.
  • the CD47-binding Fab domain of the antibody fusion protein configured in a format of FV-1 to 13 comprises a VH sequence selected from the group consisting of SEQ ID NO: 196 to 202, and a VL sequence selected from the group consisting of SEQ ID NO: 203 to 221.
  • the CD47-binding Fab domain of the antibody fusion protein configured in a format of FV-1 to 13 comprises a VH sequence selected from the group consisting of SEQ ID NO: 196 to 202 and 433, and a VL sequence selected from the group consisting of SEQ ID NO: 204 to 209, 213 to 218 and 434 to 440.
  • the CD47-binding Fab domain of the antibody fusion protein configured in a format of FV-1 to 13 comprises a VH sequence selected from the group consisting of SEQ ID NO: 229 to 235, and a VL sequence selected from the group consisting of SEQ ID NO: 236 to 240.
  • the CD47-binding Fab domain of the antibody fusion protein configured in a format of FV-1 to 13 comprises a pair of VH and VL sequence of SEQ ID NO: 127 and 133, SEQ ID NO: 128 and 133, SEQ ID NO: 123 and 137, SEQ ID NO: 123 and 141, SEQ ID NO: 126 and 148, SEQ ID NO: 127 and 144, SEQ ID NO: 126 and 146, or SEQ ID NO: 127 and 143.
  • the single-chain CD47-binding domain of the antibody fusion protein configured in a format of FV-14 to 21, comprises a scFv comprising VH-linker-VL or VL-linker-VH, wherein the linker comprises a sequence of SEQ ID NO: 276 or 277.
  • the scFv comprises a sequence of SEQ ID NO: 300 or 301.
  • the single-chain non-CD47 binding domain of the antibody fusion protein configured in a format of FV-6 to 9, comprises a scFv comprising VH-linker-VL or VL-linker-VH, wherein the linker comprises a sequence of SEQ ID NO: 276 or 277.
  • the VH and VL sequences of the scFv of the antibody fusion protein configured in a format of FV-6 to 9, comprise a pair of VH and VL sequences selected from the group of VH and VL sequence pairs consisting of SEQ ID NO: 323 and 324, 325 and 326, 327 and 328, 329 and 330, 331 and 332, 333 and 334, 335 and 336, 337 and 338, 339 and 340, 341 and 342, 343 and 344, 345 and 346, 347 and 348, 349 and 350, 351 and 352, 353 and 354, 355 and 356, 357 and 358, 359 and 360, 361 and 362, 363 and 364, 365 and 366, 367 and 368, 369 and 370, 371 and 372, 373 and 374, 375 and 376, 377 and 378, 379 and 380, 381 and 382, 383 and 384, 385 and 386, 387 and 388
  • the non-CD47 binding Fab domain of the antibody fusion protein configured in a format of FV-10 to 21 comprises a pair of VH and VL sequences selected from the group of VH and VL sequence pairs consisting of SEQ ID NO: 323 and 324, 325 and 326, 327 and 328, 329 and 330, 331 and 332, 333 and 334, 335 and 336, 337 and 338, 339 and 340, 341 and 342, 343 and 344, 345 and 346, 347 and 348, 349 and 350, 351 and 352, 353 and 354, 355 and 356, 357 and 358, 359 and 360, 361 and 362, 363 and 364, 365 and 366, 367 and 368, 369 and 370, 371 and 372, 373 and 374, 375 and 376, 377 and 378, 379 and 380, 381 and 382, 383 and 384, 385 and 386, 387 and 388, 389 and 390
  • an antibody fusion protein configured in a format of FV-10 to 13 and FV-18 to 21, wherein the CD47-binding domain comprising a VH and VL sequence pairs consisting of SEQ ID NO: 126 and 148, SEQ ID NO: 127 and 144, SEQ ID NO: 196 and 203, or SEQ ID NO: 433 and 434, and the non-CD47 binding domain comprising a VH and VL sequence pair of SEQ ID NO: 393 and 394.
  • the homodimeric Fc of the antibody fusion proteins configured in a format of FV-1 to 9 and FV-14 to 17 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 286 to 291. In some embodiments, the homodimeric Fc of the antibody fusion proteins configured in a format of FV-1 to 9 and FV-14 to 17 comprises an amino acid sequence of SEQ ID NO: 291.
  • the heterodimeric Fc of the antibody fusion proteins configured in a format of FV-10 to 13 and FV-18 to 21 comprises a pair of Fc amino acid sequence selected from the group consisting of SEQ ID NO: 292: 293, 294: 295, 296: 297 and 298: 299, wherein the “: ” indicates pairing of the two Fc sequences from the left to right of the “: ” symbol.
  • the heterodimeric Fc of the antibody fusion proteins configured in a format of FV-10 to 13 and FV-18 to 21 comprises a pair of Fc amino acid sequence of SEQ ID NO: 292: 293.
  • the homodimeric or heterodimeric Fc comprises a human IgG1 Fc with wild type or enhanced effector fuction.
  • the linker of an antibody fusion protein configured in a format of FV-1 to 9 and FV-12 to 21, comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 274 to 279.
  • the linker of an antibody fusion protein configured in a format of FV-1 to 4, FV-6 to 8, FV-14 to 16, and FV-18 to 21, comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 277.
  • the linker of an antibody fusion protein configured in a format of FV-5, 12, 13, 18, 19, 20 and 21, comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 275.
  • the linker of an antibody fusion protein configured in a format of FV-9 and 17, comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 279.
  • Protein drug conjugate comprising a pH-sensitive CD47-binding domain
  • the present disclosure provides a protein drug conjugate comprising a pH-sensitive CD47-binding domain of the present disclosure, an antibody comprising a pH-sensitive CD47-binding domain of the present disclosure, an antibody fusion protein comprising a pH-sensitive CD47-binding domain of the present disclosure, or a protein comprising a pH-sensitive CD47-binding domain of the present disclosure.
  • the protein of the protein drug conjugate is configured in a format selected from the group consisting of FV-1 to 21 as set forth in FIG. 1 to 5.
  • the protein drug conjugate comprises at least one conjugated moiety selected from the group consisting of a cytotoxic agent, a cytostatic agent, a radioactive isotope or compound, a chelator, a calreticulin-inducing agent, an immune stimulatory adjuvant moiety, a steroid, an immunosuppressor, DNA, RNA, a photosensitizer, a toxin, and an enzyme/pro-drug converting enzyme.
  • a conjugated moiety selected from the group consisting of a cytotoxic agent, a cytostatic agent, a radioactive isotope or compound, a chelator, a calreticulin-inducing agent, an immune stimulatory adjuvant moiety, a steroid, an immunosuppressor, DNA, RNA, a photosensitizer, a toxin, and an enzyme/pro-drug converting enzyme.
  • the conjugated moiety comprises a cytotoxic agent selected from the group consisting of an auristatins, a topoisomerase inhibitor, a maytansinoids, a tubulysins, a taxane, a trichothecene, a vinca alkaloids, methotrexate, a camptothecin, an etoposide, a calicheamicin, an anthracycline, a duocarmycin, a benzodiazepine, an amatoxin, thailanstatin A and a spliceostatin.
  • a cytotoxic agent selected from the group consisting of an auristatins, a topoisomerase inhibitor, a maytansinoids, a tubulysins, a taxane, a trichothecene, a vinca alkaloids, methotrexate, a camptothecin, an etoposide, a calicheamicin
  • the conjugated moiety comprises a cytotoxic agent selected from the group consisting of SN-38, Dxd, exatecan, MMAE, MMAF, DM1, DM4, eribulin, seco-DUBA, PBD, adriamycin, doxorubicin, daunorubicin, epirubicin, idarubicin, PNU-159682, tautomycin, calyculin A and salubrinal, or fullerenols.
  • a cytotoxic agent selected from the group consisting of SN-38, Dxd, exatecan, MMAE, MMAF, DM1, DM4, eribulin, seco-DUBA, PBD, adriamycin, doxorubicin, daunorubicin, epirubicin, idarubicin, PNU-159682, tautomycin, calyculin A and salubrinal, or fullerenols.
  • the conjugated moiety comprises a cytotoxic agent selected from the group consisting of tubulin inhibitors, DNA topoisomerase inhibitors, DNA minor groove binders, DNA alkylating agents, DNA intercalating agents, RNA polymerase inhibitors, spliceosome inhibitors or nicotinamide phosphoribosyltransferase inhibitors (NAMPTi) .
  • a cytotoxic agent selected from the group consisting of tubulin inhibitors, DNA topoisomerase inhibitors, DNA minor groove binders, DNA alkylating agents, DNA intercalating agents, RNA polymerase inhibitors, spliceosome inhibitors or nicotinamide phosphoribosyltransferase inhibitors (NAMPTi) .
  • the conjugated moiety comprises a MMAE.
  • the MMAE payload is conjugated to the protein with a valine-citrulline (VC) dipeptide linker, as described in patents US7745394 and US6884869.
  • the conjugated moiety comprises Dxd.
  • the Dxd payload is conjugated to the protein with a glycyl-glycyl-phenylalanyl-glycine (GGFG) tetrapeptide linker, as described in patents US10155821B2 and US9808537B2.
  • the conjugated moiety comprises PNU-159682.
  • the PNU-159682 payload is conjugated to the protein with a succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker, as described in patent US8389697B2.
  • SMCC succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate
  • DS-8201 comprising a Dxd payload
  • DB-1303 DualityBio
  • SKB264 comprising a toposiomeriase I inhibitor payload
  • the conjugated moiety comprises a radioactive isotope or compound selected from the group consisting of 225 Ac, 211 At, 212 Bi, 14 C, 62 Cu, 64 Cu, 67 Cu, 18 F, 66 Ga, 67 Ga, 68 Ga, 123 I, 125 I, 131 I, 111 In, 177 Lu, 15 O, 212 Pb, 186 Re, 188 Re, 44 Sc, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 90 Y or 89 Zr.
  • a radioactive isotope or compound selected from the group consisting of 225 Ac, 211 At, 212 Bi, 14 C, 62 Cu, 64 Cu, 67 Cu, 18 F, 66 Ga, 67 Ga, 68 Ga, 123 I, 125 I, 131 I, 111 In, 177 Lu, 15 O, 212 Pb, 186 Re, 188 Re, 44 Sc, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 90 Y
  • the conjugated moiety comprises a chelator.
  • the chelator preferentially comprises DOTA, DOTATATE, or DOTA-Bn.
  • the foregoing chelator optionally chelates with 177 Lu.
  • the conjugated moiety comprises a calreticulin-inducing agent selected from the group consisting of anthracyclin such as doxorubicin, doxorubicin, daunorubicin, epirubicin, idarubicin and mitoxantrone, and a PP1/GADD34 inhibitor such as tautomycin, calyculin A and salubrinal, or fullerenols.
  • anthracyclin such as doxorubicin, doxorubicin, daunorubicin, epirubicin, idarubicin and mitoxantrone
  • a PP1/GADD34 inhibitor such as tautomycin, calyculin A and salubrinal, or fullerenols.
  • the conjugated moiety comprises an agonist to a pattern recognition receptor (PRR) for pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) .
  • PRR pattern recognition receptor
  • the foregoing pattern recognition receptors include but are not limited to, Toll-like receptors (TLRs) , STimulator of INterferon Genes (STING) , C-type lectin receptors (CLRs) , Rig-I-like receptors (RLRs) and NOD-like receptors (NLRs) , as described in reference such as (Li et al., Signal Transduct Target Ther, 2021) .
  • the conjugated moiety comprises an agonist to TLR3, TLR7, TLR8, TLR9, STING, and/or RIG-I.
  • the conjugated moiety comprises a TLR7 agonist selected from the group consisting of imiquimod, gardiquimod, loxoribine, GSK2245035, 852A, GS-9620, RO6864018, RO7020531, CL264, CL307, 852A, BNT411, DSP-0509, LHC165, NJH395, RO7119929 and TQ-A3334, or a TLR8 agonist selected from the group consisting of IRM1, IRM2, IRM3, TL8-506 and the TLR8 agonist moiety of SBT6050, or a TLR7/8 dual agonist selected from the group consisting of resiquimod, MEDI9197, T785, BDB001, BDB018, BDB030, CV8102, NKTR-262, CL097, CL075 and the TLR7/8 agonist moiety of BDC-1001, or a TLR9 agonist selected from the group consisting of MGN1703, SD-101, IMO-21
  • the conjugated moiety comprises an agonist for TLR7 and/or TLR8 that comprises the TLR7/8 agonist moiety of BDC-1001 or the TLR8 agonist moiety of SBT6050, or a TLR9 agonist that comprises the TLR9 agonist moiety of the antibody-drug conjugate TAC-001 or ALTA-002, or a STING agonist that comprises the STING agonist moiety of the antibody-drug conjugate XMT-2056 or CRD-5500.
  • the antigen binding Fab domain of the antibody drug conjugate BDC-1001 comprising a TLR7/8 agonist moiety can be replaced by pH-sensitive CD47-binding Fab domain of the present disclosure to generate a protein drug conjugate targeting CD47.
  • the conjugated moiety comprises photosensitive agents including but not limiting to silicon phthalocyanine dye such as IRDye700DX, that are known in the arts, for example, as described in patent US8524239B2 and the reference (Maczynska et al., Cell Death Dis, 2020) .
  • the conjugated moiety comprises a protein toxin, or an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof.
  • the conjugated moiety comprises an enzymes/pro-drug converting enzyme.
  • the conjugated moiety is covalently conjugated to cystein, lysine, carbohydrate glyco-group or other chemically active group of the protein or Fc through techniques known in the arts.
  • Synthetic receptor for cell therapy comprising a pH-sensitive CD47-binding domain
  • the present disclosure provides a synthetic receptor for engineered cell therapy comprising a pH-sensitive CD47-binding domain of the present disclosure, a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure, or other polypeptide comprising a pH-sensitive CD47-binding domain of the present disclosure.
  • the synthetic receptor comprises a chimeric antigen receptor (CAR) , a synthetic T cell receptor (TCR) , or a T cell-antigen coupler (TAC) , wherein the synthetic receptor comprises an antigen binding domain comprising a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure or other polypeptide comprising a pH-sensitive CD47-binding domain of the present disclosure, a transmembrane domain and an intracellular signaling domain.
  • CAR chimeric antigen receptor
  • TCR synthetic T cell receptor
  • TAC T cell-antigen coupler
  • the synthetic receptor is a chimeric antigen receptor (CAR) comprising from the N-terminal to C-terminal an antigen binding domain comprising a CD47-binding domain of present disclosure, an extracellular spacer domain, a transmembrane domain, a co-stimulatory domainand and an intracellular signaling domain.
  • CAR chimeric antigen receptor
  • the synthetic receptor is a T cell receptor (TCR) fusion protein comprising a CD47-binding domain of present disclosure, wherein the CD47-binding domain is linked directly or through a linker to the N-terminal of a TCR subunit and wherein the TCR fusion protein incorporates into a TCR when expressed in a T cell.
  • TCR subunit is selected from the group consisting of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ and TCR ⁇ .
  • the TCR subunit is preferentially CD3 ⁇ , wherein the T cell receptor complex comprises two CD3 ⁇ units.
  • the synthetic receptor is a T cell receptor (TCR) fusion protein, comprising a CD47-binding domain of present disclosure, wherein the CD47-binding domain is linked to the N-terminal of the constant domain of both partial TCR ⁇ and TCR ⁇ or both partial TCR ⁇ and TCR ⁇ , and wherein the TCR ⁇ and TCR ⁇ fusion protein or the TCR ⁇ and TCR ⁇ fusion protein incorporates into a TCR when expressed in a T cell.
  • TCR T cell receptor
  • the synthetic receptor is a T cell antigen coupler (TAC) , comprising from the N-terminal to C-terminal a CD47-binding domain of present disclosure, a second domain binding to a protein associated with the T cell receptor complex and a third domain comprising a T cell receptor signaling domain.
  • TAC T cell antigen coupler
  • the antigen binding domain of the synthetic receptor comprises an additional antigen binding domain binding to a non-CD47 antigen wherein the additional antigen-binding domain is linked to the C-terminal or N-terminal of a CD47-binding domain of present disclosure. In some embodiments, the additional antigen binding domain binds to CD19 or BCMA.
  • the synthetic receptor is a chimeric antigen receptor (CAR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 407 to 412, a T cell receptor (TCR) fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 413 to 416, or a T cell antigen coupler (TAC) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 421 to 422.
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • TAC T cell antigen coupler
  • the synthetic receptor comprising a pair of TCR ⁇ and TCR ⁇ fusion protein comprising a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure, comprises an amino acid sequence selected from the exemplary group consisting of: SEQ ID NO: 417 x 418, and 419 x 420, wherein “x” symbol indicates a pair of TCR ⁇ and TCR ⁇ fusion protein sequence.
  • a modified cell comprises a CAR, a T cell receptor fusion protein or a TAC comprising a CD47-binding domain of present disclosure. In some embodiments, a modified cell comprises a CAR, a T cell receptor fusion protein or a TAC comprising a CD47-binding domain of present disclosure, wherein the same cell also comprises a second CAR, a second T cell receptor fusion protein or a second TAC targeting against a non-CD47 antigen.
  • a composition comprises a first population of modified T cells comprising a CAR, a T cell receptor or a TAC comprising a CD47-binding domain of present disclosure, and comprises a second population of modified T cells comprising a CAR against CD19 and a polynucleotide comprising a sequence encoding IL-6 and IFN- ⁇ driven by a NFAT promoter, wherein the second population of modified T cells express and secrete IL-6 and IFN- ⁇ in response to activation of the modified T cells.
  • the present disclosure provides a nucleic acid comprising a sequence encoding a CD47-binding domain of the present disclosure, an antibody comprising a CD47-binding domain of the present disclosure, an antibody fusion protein comprising a CD47-binding domain of the present disclosure, a protein comprising a CD47-binding domain of the present disclosure, the protein of a protein drug conjugate of present disclosure, and/or a synthetic receptor of present disclosure.
  • the nucleic acid is selected from the group consisting of a DNA and a RNA.
  • the present disclosure provides an expression vector comprising a nucleic acid or nucleic acids of the present disclosure, wherein the expression vector is selected from the group consisting of plasmids, lentivirus vectors, gamma retrovirus vectors, foamy virus vectors, adeno associated virus vectors, adenovirus vectors, pox virus vectors, herpes virus vectors, engineered hybrid viruses, and transposon mediated vectors.
  • the present disclosure provides a modified cell comprising a nucleic acid, and/or an expression vector of present disclosure.
  • the present disclosure provides a composition of cells comprising a population of modified cells comprising a CAR, a T cell receptor fusion protein or a TAC comprising a CD47-binding domain of present disclosure, wherein the same population of cells and/or a different population of cells comprise 1) a nucleic acid and/or an expression vector comprising a nucleic acid sequence encoding a second CAR, a second T cell receptor fusion protein or a second TAC targeting against CD19, CD20, CD22, CD37, BCMA or GPRCD5, and/or 2) a nucleic acid and/or an expression vector comprising a nucleic acid sequence encoding a therapeutic agent.
  • a second CAR comprises an amino acid sequence of SEQ ID NO: 423.
  • the therapeutic agent is one or more cytokines selected from the group consisting of IL-6, IFN- ⁇ and IL-12.
  • the nucleic acid encoding the therapeutic agent comprises a promoter sequence comprising SEQ ID NO: 427, wherein the therapeutic agent is expressed and secreted in response to activation of the modified cell.
  • the nucleic acid encoding the therapeutic agent encodes an amino acid sequence of SEQ ID NO: 425 and/or 426.
  • the nucleic acid encoding the therapeutic agent comprises a nucleic acid sequence of SEQ ID NO: 427.
  • the modified cell comprises a T cell, NK cell, NKT cell, cytokine-induced killer (CIK) cell, mucosal-associated invariant T (MAIT) cell, monocyte, macrophage, dendritic cell, B cell, granulocyte, neutrophil, innate lymphoid cell (ILC) , mesenchymal stem cell (MSC) and/or induced pluripotent stem cell (iPSC) .
  • the modified cell comprises a T cell, NK cell, NKT cell, CIK cell, macrophage, or iPSC.
  • the T cell comprises ⁇ T cell, ⁇ T cell, double negative T cell and/or Treg cell.
  • the cell is an autologous or allogeneic cell.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprises a pH-sensitive CD47-binding domain of the present disclosure, an antibody comprising a pH-sensitive CD47-binding domain of the present disclosure, an antibody fusion protein comprising a pH-sensitive CD47-binding domain of the present disclosure, a protein comprising a pH-sensitive CD47-binding domain of the present disclosure, a protein drug conjugate of present disclosure, a synthetic receptor of present disclosure, a nucleic acid of present disclosure, a vector of present disclosure, a modified cell of present disclosure, and/or a composition of cells of present disclosure, and the pharmaceutically acceptable carrier.
  • the present disclosure provides a method of treating a CD47-expressing disease in a mammal comprising administrating an effective amount of an antibody comprising a CD47-binding domain of the present disclosure, an antibody fusion protein comprising a CD47-binding domain of the present disclosure, a protein comprising a CD47-binding domain of the present disclosure, a protein drug conjugate of present disclosure, a synthetic receptor of present disclosure, a nucleic acid of present disclosure, a vector of present disclosure, a modified cell of present disclosure, a composition of cells of present disclosure, and/or a pharmaceutical composition of present disclosure, to a subject in need thereof.
  • the CD47-expressing disease is a disease of cancer, fibrosis, atherosclerosis, inflammation or senescence.
  • the CD47-expressing disease is a disease of cancer, comprising ovarian cancer, endometrial cancer, uterine cancer, cervical cancer, breast cancer, gastric cancer, esophageal cancer, lung cancer, head and neck cancer, colorectal cancer, liver cancer, bone cancer, sarcoma, osteosarcoma, brain cancer, multiple myeloma, acute myeloid leukemia (AML) , myelodysplastic syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, basal cell skin cancer, chondrosarcoma, Ewing’s sarcoma, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumor (GIST) , glioma, glioblastoma, hepatocellular cancer, kaposi sarcoma, kidney
  • the CD47-expressing disease is a fibrotic disease of lung, liver, heart, kidney, skin, eye, muscle and/or connective tissues, comprising idiopathic pulmonary fibrosis, liver fibrosis in nonalcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) , scleroderma and Systemic Sclerosis.
  • the mammal is a human.
  • the present disclosure provides a method of combination therapy in human comprising administering a therapeutically effective amount of a pH-sensitive CD47-binding domain of the present disclosure, an antibody comprising a pH-sensitive CD47-binding domain of the present disclosure, an antibody fusion protein comprising a pH-sensitive CD47-binding domain of the present disclosure, a protein comprising a pH-sensitive CD47-binding domain of the present disclosure, a protein drug conjugate of the present disclosure, and/or a cell therapy comprising a synthetic receptor of the present disclosure, and a therapeutically effective amount of an additional therapy.
  • the additional therapy comprises administration of one or more of chemotherapy, molecular targeted therapy, biologics therapy, immunotherapy and radiotherapy.
  • the additional therapy comprises a chemotherapy, molecular targeted therapy, biologics therapy, immunotherapy and/or radiotherapy that enhance pro-phagocytic signal and/or inhibit anti-phagocytic signal.
  • the additional therapy comprises a chemotherapy, molecular targeted therapy, biologics therapy, immunotherapy and/or radiotherapy that comprise cell adhesion among diseased cells and/or between diseased cells and extracellular matrix.
  • FIG. 1 shows illustration of antibody fusion protein comprising an anti-CD47 antibody and a SIRP IgV domain.
  • FIG. 2 shows illustration of antibody fusion protein comprising an anti-CD47 antibody and a non-CD47 binding single chain domain or fragment thereof.
  • FIG. 3 shows illustration of bispecific antibody against CD47 and a non-CD47 target.
  • FIG. 4 shows illustration of symmetric antibody fusion protein comprising an antibody against a non-CD47 target and a non-SIRP single-chain CD47-binding domain or fragment thereof.
  • FIG. 5 shows illustration of asymmetric antibody fusion protein comprising an antibody against a non-CD47 target and a non-SIRP single-chain CD47-binding domain or fragment thereof.
  • FIG. 6 show results of FACS binding on Raji cells of chimeric B6H12 antibody variants and parent wild type antibody at acidic pH and physiological pH.
  • FIG. 7 shows results of FACS binding on Raji cells of humanized B6H12 antibody and its antibody variants with combo mutations at acidic pH and physiological pH.
  • FIG. 8 shows ELISA results of humanized B6H12 antibody and its antibody variants with combo mutations for binding to CD47 protein at acidic pH and physiological pH.
  • CD47 has emerged as a highly attractive therapeutic target for cancer, not only as a functional target for promoting phagocytosis of cancer cells by blocking the CD47’s interaction with SIRP ⁇ , but also as a universal tumor antigen target for directing various therapeutic modalities such as cytotoxic agents and radioligands to cancer cells, given the high and also often uniform expression of CD47 in a wide variety of cancers.
  • CD47 ubiquitous expression of CD47 across normal tissues presents a major obstacle for targeting CD47.
  • anti-CD47 antibody therapeutics stopped clinical development due to safety issues, prominently hemolytic or anemic toxicities due to hemagglutination and/or phagocytic clearance of red blood cells (RBC) induced by the anti-CD47 antibody therapeutics. While a number of anti-CD47 therapeutics for cancer have managed to advance in the clinic through using a priming plus maintenance dosing schedule (e.g. for magrolimab) or using CD47-binders with weak/minimal binding to human RBC (e.g. TTI-621 and TTI-622 using the CD47-binding IgV extracellular domain of SIRP ⁇ with minimal native binding to RBC) , these therapeutics still bind to CD47 on a broad range of other normal cells and tissues with associated safety risks.
  • a priming plus maintenance dosing schedule e.g. for magrolimab
  • CD47-binders with weak/minimal binding to human RBC e.g. TTI-621 and TTI-622 using the CD47-binding Ig
  • thrombocytopenia and neutropenia are commonly reported adverse events for these anti-CD47 therapeutics in clinical development. Consequently, these current anti-CD47 therapeutics are only utilized to functionally block the CD47 “don’t eat me” signal for promoting phagocytosis, but not used for directing other therapeutic modalities such as cytotoxic agents and radioligands to CD47-expressing disease tissue or cells such as cancer cells, due to on-target toxicity risks to the broad CD47-expressing normal tissues. Furthermore, these CD47-blocking protein therapeutics, usually formatted with Fc of inert/weak effector function (e.g.
  • hIgG2, hIgG4 or silent hIgG1 Fc mostly only block the CD47 “don’t eat me” signal, without providing a potent “eat me” signal (e.g. through using hIgG1 Fc with potent ADCP effector function) that’s also critical to promote phagocytosis.
  • TTI-621 uses a wild type hIgG1 Fc (US9969789B2)
  • its dose escalation stopped at a dose of only 2 mg/kg in Phase 1 clinical trial, versus its counterpart TTI-622 with a weak hIgG4 Fc (US10906954B2) has not reached maximal tolerated dose at 18 mg/kg dose.
  • Described herein directs to pH-sensitive CD47-binding protein domains as well as proteins, protein conjugates and synthetic receptor constructs comprising said pH-sensitive CD47-binding domains with differential targeting of CD47 in diseased tissue or cells of interest over normal tissues, and their compositions and methods of use and production related thereto.
  • Acidic extracellular environment is a hallmark of solid tumors and an important determinant of tumor progression, with pH values ranging from 5.8 to 7.4 reported in a variety of solid tumors such as breast, ovarian, sarcoma, brain tumors and melanomas (Erra Diaz et al., Mediators Inflamm, 2018, Jones et al., Mol Imaging Biol, 2017) .
  • Extracellular acidosis is also a frequent feature of infectious and inflammatory processes (Erra Diaz et al., Mediators Inflamm, 2018) .
  • acidic pH values as low as 5.5 associated with the inflammatory responses against bacteria in peripheral tissues have been reported.
  • Acidic extracellular space is also revealed at sites of tissue injury of autoimmune and allergic diseases, such as acidic low pH in the joint synovial fluid of rheumatoid arthritis patients and in the airway surface liquid of patients of asthma and patients with acute lung injury (Erra Diaz et al., Mediators Inflamm, 2018) .
  • pH-sensitive CD47-binding protein domains hold utility to differentially target CD47-expressing disease tissues/cells over normal tissues for cancer, infectious, inflammatory, atherosclerotic and other diseases.
  • the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.
  • the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 15%, 10%, 5%, or 1%.
  • CD47 means CD47 (cluster of differentiation 47) , also known as IAP (integrin-associated protein) .
  • IAP integrated protein
  • CD47 CD47 (cluster of differentiation 47)
  • IAP integrated protein-associated protein
  • four isoforms of the CD47 protein have been reported, with NCBI Accession number of NP_001768.1 (isoform 1) , NP_942088.1 (isoform 2) , NP_001369235.1 (isoform 3) and XP_005247966.1 (isoform X1) , respectively.
  • the amino acid sequences of the four CD47 isoforms only differ in the length of the C-terminal located inside cytosol when CD47 is natively expressed as a transmembrane protein.
  • SIRP means “Signal regulatory protein” , including SIRP ⁇ , SIRP ⁇ , and SIRP ⁇ , which are a family of transmembrane glycoproteins with three extracellular Ig-like domains, including one IgV domain at the N-terminal followed with two IgC domains.
  • the IgV extracellular domain serves as the direct CD47-binding domain of the SIRP proteins (Barclay et al., Nat Rev Immunol, 2006) .
  • SIRP ⁇ comprises 10 members, namely SIRP ⁇ V1 to V10, whose IgV domains all bind to CD47.
  • SIRP IgV means the IgV extracellular domain of the SIRP proteins, including the IgV extracellular domain of SIRP ⁇ , SIRP ⁇ , and SIRP ⁇ .
  • SIRP IgV monomer means one single IgV extracellular domain of a SIRP protein, including one single IgV extracellular domain of SIRP ⁇ , SIRP ⁇ , or SIRP ⁇ .
  • amino acid refers to any organic compound that contains an amino group (-NH2) and a carboxyl group (-COOH) , preferably either as free groups or alternatively after condensation as part of peptide bonds.
  • the "twenty naturally encoded polypeptide-forming alpha-amino acids” are understood in the art and refer to: alanine (ala or A) , arginine (arg or R) , asparagine (asn or N) , aspartic acid (asp or D) , cysteine (cys or C) , gluatamic acid (glu or E) , glutamine (gin or Q) , glycine (gly or G) , histidine (his or H) , isoleucine (ile or I) , leucine (leu or L) , lysine (lys or K) , methionine (met or M) , phenylalanine (phe or F)
  • peptide typically refers to short polypeptides.
  • protein typically refers to longer polypeptides.
  • a “protein” may comprise a single polypeptide, or two or more polypeptides.
  • the left-hand end of a polypeptide sequence is usually described as the amino-terminus (N-terminus) ; and the right-hand end of a polypeptide sequence is usually described as the carboxyl-terminus (C-terminus) .
  • antibody encompasses any immunoglobulin, monoclonal antibody, polyclonal antibody, or multispecific antibody antibody that binds to a specific antigen.
  • a native intact or full antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region ( “VH” ) and a first, second, and third constant region (CH1, CH2 and CH3) , while each light chain consists of a variable region ( “VL” ) and a constant region (CL) .
  • VH variable region
  • CH1, CH2 and CH3 first, second, and third constant region
  • VL variable region
  • Mammalian heavy chains are classified as ⁇ , ⁇ , ⁇ , ⁇ , and ⁇
  • mammalian light chains are classified as ⁇ or ⁇ .
  • the antibody has a “Y” shape, with the stem of the Y consisting of the second and third constant regions of two heavy chains bound together via disulphide bonding.
  • Each arm of the Y includes the variable region and first constant region of a single heavy chain bound to the variable and constant regions of a single light chain.
  • the variable regions of the light and heavy chains are responsible for antigen binding.
  • the variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light (L) chain CDRs including LCDR1, LCDR2, and LCDR3, heavy (H) chain CDRs including HCDR1, HCDR2, HCDR3) .
  • CDRs complementarity determining regions
  • the boundaries of the amino acid sequence of a particular CDR may be defined or identified by the conventions of any of the well-known systems, including Kabat numbering, Chothia numbering and IMGT numering, as described in Kabat et al. (1991) , Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (Kabat numbering system) , (Chothia et al., Nature, 1989) , Al-Lazikani et al., (1997) , JMB 273, 927 -948 (Chothia numbering system) , Lefranc et al., (2003) , Dev. Comp.
  • the three CDRs are interposed between flanking stretches known as framework regions (FRs) , which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops.
  • FRs framework regions
  • Each VH and VL comprises four FRs, and the CDRs and FRs are arranged from amino terminus to carboxy terminus in the order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions.
  • the term “antibody” also includes VHH antibody and heavy-chain only antibody which comprise only a heavy chain without the light chain.
  • antibody also includes VH domain antibody or VL domain antibody which comprises a heavy chain without a light chain or a light chain without a heavy chain respectively.
  • Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain.
  • the five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ heavy chains, respectively.
  • IgG1 ⁇ 1 heavy chain
  • IgG2 ⁇ 2 heavy chain
  • IgG3 ⁇ 3 heavy chain
  • IgG4 ⁇ 4 heavy chain
  • IgA1 ⁇ 1 heavy chain
  • IgA2 ⁇ 2 heavy chain
  • antigen-binding domain refers to polypeptides comprising fragments of a full-length antibody, which retain the ability of specifically binding to an antigen that the full-length antibody specifically binds to, and/or compete with the full-length antibody for binding to the same antigen.
  • antigen-binding domain refers to polypeptides comprising fragments of a full-length antibody, which retain the ability of specifically binding to an antigen that the full-length antibody specifically binds to, and/or compete with the full-length antibody for binding to the same antigen.
  • an antigen-binding domain may comprise an intact variable region (such as VH or VL)
  • an antigen-binding domain may comprise less than an intact variable region yet still retain the capability of binding to an antigen or forming an antigen-binding site.
  • Antigen-binding fragments of an antibody may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of an intact antibody. Under some conditions, antigen binding fragments include Fab, Fab', F (ab') 2, Fd, Fv, dAb and complementary determining region (CDR) fragments, single chain antibody (e.g.
  • Antigen-binding fragments of an antibody may be obtained from a given antibody (e.g., the monoclonal anti-human X antibody provided in the instant application) by conventional techniques known by a person skilled in the art (e.g., recombinant DNA technique or enzymatic or chemical cleavage methods) , and may be screened for specificity in the same manner by which intact antibodies are screened.
  • antibody domain refers to a portion of an antibody, and an antigen-binding antibody domain specifically refers to an antigen-binding domain of an antibody.
  • antibody comprising a pH-sensitive CD47-binding domain specifically refers to an antibody comprising a pH-sensitive CD47-binding domain, wherein the antibody may be an intact antibody or an antibody fragment.
  • monoclonal antibody or “mAb” , as used herein, refer to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody displays a single binding specificity and affinity for a particular epitope.
  • human antibody or “fully human antibody” , as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences.
  • the human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) .
  • chimeric antibody refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.
  • humanized antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
  • a “humanized form” of an antibody, e.g., a non-human antibody refers to an antibody that has undergone humanization.
  • humanized antibody is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.
  • recombinant antibody refers to an antibody that is prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal that is transgenic for another species’ immunoglobulin genes, antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of immunoglobulin gene sequences to other DNA sequences.
  • a Fab fragment consists of a monovalent antigen-binding fragment of an antibody molecule, and can be produced by digestion of a whole antibody molecule with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain.
  • An Fab' fragment of an antibody molecule can be obtained by treating a whole antibody molecule with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain. Two Fab' fragments are obtained per antibody molecule treated in this manner.
  • An (Fab') 2 fragment of an antibody can be obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction.
  • a (Fab') 2 fragment is a dimer of two Fab' fragments, held together by two disulfide bonds.
  • An Fv fragment is defined as a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain expressed as two chains.
  • a Fd chain refers to a fragment of antibody heavy chain comprising VH and CH1.
  • Fc with regard to an antibody refers to that portion of the antibody consisting of the second (CH2) and third (CH3) constant regions of a first heavy chain bound to the second and third constant regions of a second heavy chain via disulphide bonding.
  • the Fc portion of the antibody is responsible for various effector functions such as ADCC, ADCP and CDC, but does not function in antigen binding.
  • CH2 constant region which is also referred to as “CH2 domain” , as used herein refers to the portion of a heavy chain molecule that extends, e.g., from about amino acid 244 to amino acid 360 of an IgG antibody using conventional numbering schemes (amino acids 244 to 360, Kabat numbering system; and amino acids 231-340, EU numbering system; see Kabat, E., et al., U.S. Department of Health and Human Services, (1983) ) .
  • the “CH3 constant region” which is also referred to as “CH3 domain” , extends from the CH2 domain to the C-terminus of the IgG molecule and comprises approximately 108 amino acids.
  • Certain immunoglobulin classes, e.g., IgM further include a CH4 region.
  • “Hinge region” in terms of an antibody includes the portion of a heavy chain molecule that joins the CH1 domain to the CH2 domain. This hinge region is flexible, thus allowing the two N-terminus antigen binding regions to move independently.
  • the hinge region comprises about amino acids 234 to 243 of Kabat numbering system.
  • the hing region comprises about amino acids 226 to 243 of Kabat numbering system.
  • a Fc comprising the hinge region comprising about amino acids 234 to 243 of Kabat numbering system.
  • Single-chain Fv antibody or “scFv” refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to one another directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85: 5879 (1988) ) .
  • fusion protein means a protein comprising at least two fragments that are not naturally fused in the same protein.
  • antibody fusion protein means a “fusion protein” comprising a full antibody or an antibody fragment and at least one additional fragment fused to the antibody or the antibody fragment.
  • the additional fragment may be an antibody fragment or a non-antibody fragment.
  • substitution refers to naturally occurring or induced replacement of one or more amino acids with another in a peptide, polypeptide, or protein. Substitution in a polypeptide may result in diminishment, enhancement, or elimination of the polypeptide’s function.
  • variant refers to a derivative from a parent template wherein the variant has at least one difference from the parent template.
  • a “variant” of a parent peptide, parent polypeptide, parent protein, parent antibody, parent domain or parent CDR means a derivative differing from the parent peptide, parent polypeptide, parent protein, parent antibody, parent domain or parent CDR in one or more amino acid residues of the parent, wherein the original amino acid residues of the parent refer to as the parent residues, and the differing amino acid residues in the variant may be introduced through substitution, insertion and/or deletion.
  • an “antigen” or “Ag” as used herein refers to a compound, composition, peptide, polypeptide, protein, hapten, or substance that can stimulate the production of antibodies or a T cell response in cell culture or in an animal, including compositions (such as one that includes a cancer-specific protein) that are added to a cell culture (such as a hybridoma) , or injected or absorbed into an animal.
  • An antigen reacts with the products of specific humoral or cellular immunity (such as an antibody) , including those induced by heterologous antigens.
  • the term “fusion” or “fused” when used with respect to amino acid sequences e.g.
  • peptide, polypeptide, or protein refers to combination of two or more amino acid sequences, for example by chemical bonding or recombinant means, into a single amino acid sequence that does not exist naturally.
  • a fusion amino acid sequence may be produced by genetic recombination of two encoding polynucleotide sequences, and can be expressed by a method of introducing a construct containing the recombinant polynucleotides into a host cell.
  • binding refers to interaction of a binding domain with an antigen with the interaction depending upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen.
  • a particular structure e.g., an antigenic determinant or epitope
  • an antibody variable region or Fv recognizes and binds to a specific protein structure rather than to proteins generally.
  • the term “specifically binding” or “binding specifically” means that a binding domain binds to or associates with more frequently, more rapidly, with greater duration and/or with greater affinity with a particular antigen than with other proteins.
  • an antibody variable region or Fv specifically binds to its antigen with greater affinity, avidity, more readily, and/or with greater duration than it binds to other antigens.
  • an antibody variable region or Fv binds to a cell surface protein (antigen) with materially greater affinity than it does to related proteins or other cell surface proteins or to antigens commonly recognized by polyreactive natural antibodies (i.e., by naturally occurring antibodies known to bind a variety of antigens naturally found in humans) .
  • polyreactive natural antibodies i.e., by naturally occurring antibodies known to bind a variety of antigens naturally found in humans
  • selective binding does not necessarily require exclusive binding or non-detectable binding of another antigen, this is meant by the term “selective binding” .
  • affinity refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen) .
  • binding affinity refers to intrinsic binding affinity which reflects a 1: 1 interaction between members of a binding pair (e.g., antibody and antigen) .
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd) .
  • pH-sensitive binding or “pH-sensitive” binding refers to the binding of the same antigen-binding domain to the same antigen differing at different pH or pH range.
  • a “pH-sensitive” antigen-binding domain or “pH-sensitive” antigen-binding domain in this disclosure refers to an antigen-binding domain that binds to its antigen with higher affinity at an acidic pH than at physiological pH, wherein an acidic pH is less than 7.0 and physiological pH is 7.2 to 7.5.
  • the term “avidity” refers to the strength of the sum total of noncovalent interactions of two or more antigen binding sites and their binding partner (e.g., an antigen) .
  • valency refers to the number of a specific domain that a molecule consists of in total.
  • “monovalent” “bivalent” , “trivalent” or “tetravalent” refers to respectively one, two, three or four such specific domains comprised by the molecule in total.
  • bivalent binding refers to the binding of a molecule to an antigen wherein the molecule comprises two binding domains to the antigen in total.
  • monovalent binding refers to the binding of a single antigen-binding domain to its antigen.
  • multispecific refers to specificity against two or more different targets.
  • format variant or “FV” in abbreviation, of a protein refers to the structural configuration of the domain components of the protein, as the exemplary format variants (FVs) illustrated in FIG. 1 to 5.
  • effector functions refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype.
  • antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC) ; Fc receptor binding and antibody-dependent cell-mediated cytotoxicity (ADCC) ; Fc receptor binding and antibody-dependent cell-mediated phagocytosis (ADCP) ; down regulation of cell surface receptors (e.g. B cell receptor) ; activation of cells expressing Fc receptor (e.g. B cells, macrophages, dendritic cells) , induction of secretion of cytokines and/or chemokines by cells expressing Fc receptor (e.g. B cells, macrophages, dendritic cells) .
  • phagocytosis refers to a process by which a substance with size (such as a cell, a fragment of cell, a microbe, or a particle) is engulfed and internalized by a cell.
  • phagocyte refers to a cell that is capable of phagocytosis.
  • microenvironment means any portion or region of a tissue, organ or body that has constant or temporal, physical or chemical differences from other regions of the tissue, organ or regions of the body.
  • tumor microenvironment refers to the environment in which a tumor exists, which is the non-cellular area within the tumor and the area directly outside the tumorous tissue but does not pertain to the intracellular compartment of the cancer cell itself.
  • the tumor and the tumor microenvironment are closely related and interact constantly.
  • a tumor can change its microenvironment, and the microenvironment can affect how a tumor grows and spreads.
  • the tumor microenvironment has a low pH in the range of 5.0 to 6.8, or in the range of 5.8 to 6.8, or in the range of 6.2-6.8.
  • the tumor microenvironment has been discussed in (Gillies et al., J Magn Reson Imaging, 2002) , hereby incorporated by reference here.
  • the term “non-tumor microenvironment” refers to a microenvironment at a site other than a tumor.
  • protein drug conjugate refers to a protein comprising a drug payload moiety that is covalently linked to the protein through chemical reaction or recombinant expression.
  • protein drug conjugate refers to a protein comprising at least one CD47-binding protein domain of the present disclosure and a drug payload conjugate.
  • cytotoxic agent refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction.
  • cytostatic agent refers to a compound or composition which arrests growth of a cell either in vitro or in vivo. Thus, a cytostatic agent may be one which significantly reduces the percentage of cells in S phase.
  • chemotherapeutic agent refers to a chemical compound useful in the treatment of cancer.
  • synthetic receptor refers to a non-natural chimeric protein receptor that can be expressed on the membrane of a host cell and the polynucleotide that encodes such a chimeric protein receptor.
  • synthetic receptor include chimeric antigen receptor (CAR) , T cell receptor fusion protein (TFP) and T cell antigen coupler (TAC) .
  • CAR chimeric antigen receptor
  • TCP T cell receptor fusion protein
  • TAC T cell antigen coupler
  • synthetic receptor of the present disclosure it refers to a synthetic receptor comprising at least one CD47-binding protein domain of the present disclosure.
  • vector refers to a nucleic acid vehicle which can have a polynucleotide inserted therein.
  • the vector allows for the expression of the protein encoded by the polynucleotide inserted therein, the vector is called an expression vector.
  • the vector can have the carried genetic material elements expressed in a host cell by transformation, transduction, or transfection into the host cell.
  • Vectors are well known by a person skilled in the art, including, but not limited to plasmids, phages, cosmids, artificial chromosome such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC) ; phage such as ⁇ phage or M13 phage and animal virus.
  • the animal viruses that can be used as vectors include, but are not limited to, retrovirus (including lentivirus) , adenovirus, adeno-associated virus, herpes virus (such as herpes simplex virus) , pox virus, baculovirus, papillomavirus, papova virus (such as SV40) .
  • a vector may comprise multiple elements for controlling expression, including, but not limited to, a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element and a reporter gene.
  • a vector may comprise origin of replication.
  • host cell refers to a cellular system which can be engineered to generate proteins, protein fragments, or peptides of interest.
  • Host cells include, without limitation, cultured cells, e.g., mammalian cultured cells derived from rodents (rats, mice, guinea pigs, or hamsters) such as CHO, BHK, NSO, SP2/0, YB2/0; or human tissues or hybridoma cells, yeast cells, and insect cells, and cells comprised within a transgenic animal or cultured tissue.
  • the term encompasses not only the particular subject cell but also the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term “host cell. ”
  • identity refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent identity” means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) are preferably addressed by a particular mathematical model or computer program (i.e., an “algorithm” ) . Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A. M., ed.
  • the term “subject” is used interchangeably with “patient” and may be a mammal who is in need of prevention or treatment of a disease, such as primates (for example, humans) , companion animals (for example, dogs and cats) , livestock (for example, cows, pigs, horses, sheep, and goats) , and laboratory animals (for example, rats, mice, and guinea pigs) .
  • a disease such as primates (for example, humans) , companion animals (for example, dogs and cats) , livestock (for example, cows, pigs, horses, sheep, and goats) , and laboratory animals (for example, rats, mice, and guinea pigs) .
  • the subject is a human.
  • cancer refers to any or a tumor or a malignant cell growth, proliferation or metastasis-mediated, solid tumors and non-solid tumors such as leukemia and initiate a medical condition.
  • treatment refers generally to treatment and therapy, whether of a human or an animal, in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, regression of the condition, amelioration of the condition, and cure of the condition.
  • Treatment as a prophylactic measure i.e., prophylaxis, prevention
  • treating may refer to dampen or slow the tumor or malignant cell growth, proliferation, or metastasis, or some combination thereof.
  • treatment includes removal of all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of a tumor, or some combination thereof.
  • an effective amount or “a therapeutically-effective amount” , as used herein, pertains to that amount of an active compound, or a material, composition or dosage from comprising an active compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen.
  • the “an effective amount” when used in connection with treatment of target antigen-related diseases or conditions, refers to an antibody or antigen-binding portion thereof in an amount or concentration effective to treat the said diseases or conditions.
  • prevention refers to preventing or delaying the onset of the disease, or preventing the manifestation of clinical or subclinical symptoms thereof.
  • pharmaceutically acceptable means that the vehicle, diluent, excipient and/or salts thereof, are chemically and/or physically is compatible with other ingredients in the formulation, and the physiologically compatible with the recipient.
  • a pharmaceutically acceptable carrier and/or excipient refers to a carrier and/or excipient pharmacologically and/or physiologically compatible with a subject and an active agent, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) , and includes, but is not limited to pH adjuster, surfactant, adjuvant and ionic strength enhancer.
  • the pH adjuster includes, but is not limited to, phosphate buffer;
  • the surfactant includes, but is not limited to, cationic, anionic, or non-ionic surfactant, e.g., Tween-80;
  • the ionic strength enhancer includes, but is not limited to, sodium chloride.
  • the present disclosure provides a CD47-binding domain wherein the domain: a) exhibits higher binding affinity to CD47 at an acidic pH than at physiological pH, wherein the acidic pH is less than 7.0 and the physiological pH is 7.2 to 7.5, and b) is not an IgV extracellular domain of the Signal-regulatory protein (SIRP) family proteins.
  • SIRP Signal-regulatory protein
  • the acidic pH is 4.0-5.0, 5.0-6.0, or 6.0-7.0, In some embodiments, the acidic pH is 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, or 6.9.
  • the CD47-binding domain binds to CD47 with a binding affinity that is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold higher at an acidic pH than at physiological pH.
  • the pH-sensitive CD47-binding domain binds to human wild type CD47 at physiological pH with a monovalent affinity preferably lower than the monovalent affinity of wild type human SIPR ⁇ V2 IgV (SEQ ID NO: 306) to human wild type CD47, wherein the physiological pH is 7.2, 7.3 or 7.4.
  • the pH-sensitive CD47-binding domain binds to human wild type CD47 at physiological pH with a monovalent affinity preferably at least 2-fold lower than the monovalent affinity of wild type human SIPR ⁇ V2 IgV (SEQ ID NO: 306) to human wild type CD47, wherein the physiological pH is 7.2, 7.3 or 7.4.
  • the pH-sensitive CD47-binding domain binds to human wild type CD47 at physiological pH with a monovalent affinity preferably lower than the monovalent affinity of a CD47-binding antibody comprising a heavy chain (HC) sequence of SEQ ID NO: 441 and a light chain (LC) sequence of SEQ ID NO: 442 to CD47, wherein the physiological pH is 7.2, 7.3 or 7.4.
  • the pH-sensitive CD47-binding domain binds to human wild type CD47 at physiological pH preferably with a monovalent affinity weaker than 460 nM in dissociation constant (Kd) measured by surface plasmon resonance (SPR) as applied in (Li et al., J Hematol Oncol, 2023) , wherein the physiological pH is 7.2, 7.3 or 7.4.
  • Kd dissociation constant
  • SPR surface plasmon resonance
  • the pH-sensitive CD47-binding domain binds to human wild type CD47 at physiological pH preferably with a monovalent affinity weaker than 1, 200 nM in dissociation constant (Kd) measured by surface plasmon resonance (SPR) as applied in (Li et al., J Hematol Oncol, 2023) , wherein the physiological pH is 7.2, 7.3 or 7.4.
  • Kd dissociation constant
  • SPR surface plasmon resonance
  • the pH-sensitive CD47-binding domain exhibits negligible binding to human primary platelets at physiological pH, or exhibiting binding to human primary platelets at physiological pH with an EC50 lower than that of monovalent binding of wild type human SIPR ⁇ V2 IgV (SEQ ID NO: 306) as measured by a FACS binding assay, wherein the physiological pH is 7.2, 7.3 or 7.4.
  • the pH-sensitive CD47-binding domain binds to at least one of the following residues: E29, E35, D46, D51, E97, E100, E104 or E106 of SEQ ID NO: 474, wherein the pH-sensitive CD47-binding domain exhibits higher binding affinity to CD47 at an acidic pH than at physiological pH.
  • the pH-sensitive CD47-binding domain binds to at least one of the following residues: E35, D46, D51, E97, or E104 of SEQ ID NO: 474, wherein the pH-sensitive CD47-binding domain exhibits higher binding affinity to CD47 at an acidic pH than at physiological pH.
  • the pH-sensitive CD47-binding domain comprises a Fragment antigen-binding domain (Fab) of antibody, a single chain Fab domain (scFab) , a Fragment variable (Fv) domain of antibody, a single chain Fv (scFv) , a disulfide-stabilized Fv (dsFv) , a VHH antibody or nanobody, a heavy chain variable domain (VH) single chain antibody (VH dAb) or a light chain variable domain (VL) single chain antibody (VL dAb) or other single-chain domain antibody, or an anticalin, DARPIN, affibody, affimer, centryin domain, fibronectin domain, D-Domain or other alternative scaffold known in the arts to function as antigen binding domainthat binds to CD47, or other polypeptide or peptide that binds to CD47.
  • Fab fragment antigen-binding domain
  • scFab single chain Fab domain
  • Fv Fragment variable domain
  • the pH-sensitive CD47-binding domains is a Fab domain, wherein an antibody comprising two said CD47-binding Fab domain binds to human wild type CD47 at physiological pH with a bivalent binding weaker than the monovalent binding of wild type human SIPR ⁇ V2 IgV (SEQ ID NO: 306) to CD47.
  • the pH-sensitive CD47-binding domains is a Fab domain, wherein an antibody comprising two said CD47-binding Fab domain binds to human CD47-expresing Raji cells (ATCC, Cat #CCL-86) at physiological pH weaker than that of monovalent wild type human SIPR ⁇ V2 IgV (SEQ ID NO: 306) , as measured by FACS binding assay set forth in Example 2 and 4.
  • the pH-sensitive CD47-binding domains is a Fab domain, wherein an antibody comprising two said CD47-binding Fab domain binds to monovalent human wild type CD47 at physiological pH weaker than that of monovalent wild type human SIPR ⁇ V2 IgV (SEQ ID NO: 306) , as measured by ELISA assay set forth in Example 5.
  • the pH-sensitive CD47-binding domains is a Fab domain, wherein an antibody comprising two said CD47-binding Fab domain binds to monovalent human wild type CD47 at physiological pH with a bivalent binding dissociation constant weaker than 640 nM as measured by surface plasmon resonance (SPR) as applied in (Li et al., J Hematol Oncol, 2023) , wherein the monovalent human wild type CD47 protein is immobilized and the bivalent antibody comprising two said CD47-binding Fab domain is used as soluble analyte in the SPR, and wherein the physiological pH is 7.2, 7.3 or 7.4.
  • SPR surface plasmon resonance
  • the pH-sensitive CD47-binding domains is a Fab domain, wherein an antibody comprising two said CD47-binding Fab domain binds to monovalent human wild type CD47 at physiological pH with a bivalent binding dissociation constant weaker than 1,000 nM as measured by surface plasmon resonance (SPR) as applied in (Li et al., J Hematol Oncol, 2023) , wherein the monovalent human wild type CD47 protein is immobilized and the bivalent antibody comprising two said CD47-binding Fab domain is used as soluble analyte in the SPR, and wherein the physiological pH is 7.2, 7.3 or 7.4.
  • SPR surface plasmon resonance
  • the pH-sensitive CD47-binding domain comprises an antibody domain comprising a set of variable heavy (VH) chain complementarity determining region (CDR) , and/or variable light (VL) chain complementarity determining region (CDR) sequences.
  • the pH-sensitive CD47-binding domain is a variant of a parent CD47-binding domain or fragment thereof a CD47-binding antibody selected from a group of CD47-binding antibodies as set forth in Table 1, and comprises one or more mutations of residues of the parent CD47-binding domain.
  • the pH-sensitive CD47-binding domain comprises a set of three VH CDR sequences and/or three VL CDR sequences at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, or at least 90%identical to the set of VH and/or VL CDR amino acid sequences of a parent anti-CD47 antibody as set forth in Table 2.
  • the set of VH CDR sequences and/or VL CDR sequences of the pH-sensitive CD47-binding domain comprise one or more substitutions selected from the group of substitutions of the CDR sequences of its parent anti-CD47 antibody as set forth in Table 2.
  • the pH-sensitive CD47-binding domain is a variant derived from the non-pH sensitive parent anti-CD47 antibody B6H12.
  • the antibody B6H12 binds to an epitope on human CD47 extracellular domain (SEQ ID NO: 474) comprising the residues of E29, Q31, N32, T34, E35, V36, Y37, K39, K41, D46, D51, A53, E97, T99, E100, L101, T102, R103 and E104 of SEQ ID NO: 474 (see (Pietsch et al., Blood Cancer J, 2017) ) .
  • the CD47-binding domain exhibits higher binding affinity to CD47 at an acidic pH than at physiological pH, wherein the acidic pH is less than 7.0 and the physiological pH is 7.2 to 7.5, and comprises three VH CDR sequences at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, or at least 80%identical to the three parent VH CDR amino acid sequences of SEQ ID NO: 82, 84 and 89, and/or three VL CDR sequences at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, or at least 80%identical to the three parent VL CDR amino acid sequences of SEQ ID NO: 91, 95 and 99.
  • the CD47-binding domain comprises one or more mutations of parent CDR residues selected from the residues corresponding to the positions 1, 2, 3 and 4 of HCDR1 SEQ ID NO: 82, the positions 1, 2, 3, 4, 6, 7, 8, 9, 10 and 11 of HCDR2 SEQ ID NO: 84, the positions 1, 2, 3, 4, 5, 7 and 9 of HCDR3 SEQ ID NO: 89, the positions 1, 4, 6, 7, 8 and 9 of LCDR1 SEQ ID NO: 91, the positions 1, 2, 4, 5, 6, 7 and 8 of LCDR2 SEQ ID NO: 95, and the positions 2, 3, 5, 6, 7, 8 and 9 of LCDR3 SEQ ID NO: 99.
  • the CD47-binding domain comprises one or more substitutions of the parent CDR residues selected from the group consisting of: G1H, G1E, G1D, Y2H, Y2E, Y2D, G3H, G3E, G3D, M4H, M4E and M4D of HCDR1 SEQ ID NO: 82; T1H, T1E, T1D, I2H, I2E, I2D, T3H, T3E, T3D, S4H, S4E, S4D, G5H, G5E, G5D, G6H, G6E, G6D, T7H, T7E, T7D, Y8H, Y8E, Y8D, T9H, T9E, T9D, Y10H, Y10E, Y10D, Y11H, Y11E and Y11D of HCDR2 SEQ ID NO: 84; S1H, S1D, S1
  • the CD47-binding domain comprises one or more mutations of parent CDR residues selected from the residues corresponding to the positions 2 and 3 of SEQ ID NO: 82, the positions 3, 4, 8 and 10 of SEQ ID NO: 84, the positions 3 and 4 of SEQ ID NO: 89, the positions 7, 8 and 9 of SEQ ID NO: 91, the positions 1, 5, 6, 7 and 8 of SEQ ID NO: 95, and the positions 5, 6 and 8 of SEQ ID NO: 99.
  • the CD47-binding domain comprises one or more substitutions of the parent CDR residues selected from the group consisting of: Y2H, Y2E, Y2D, G3H, G3E and G3D of SEQ ID NO: 82; T3H, T3E, T3D, S4H, S4E, S4D, Y8H, Y8E, Y8D, Y10H, Y10E and Y10D of SEQ ID NO: 84; A3H, A3E, A3D, G4H, G4E and G4D of SEQ ID NO: 89; S7H, S7E, S7D, D8H, D8E , Y9H, Y9E and Y9D of SEQ ID NO: 91; K1H, K1E, K1D, Q5H, Q5E, Q5D, S6H, S6E, S6D, S6R, I7H, I7E, I
  • the CD47-binding domain comprises one or more substitutions of the parent CDR residues selected from the group consisting of: Y2H of SEQ ID NO: 82; T3H, Y8H and Y10H of SEQ ID NO: 84; A3H of SEQ ID NO: 89; S7H, D8H and Y9H of SEQ ID NO: 91; K1H and Q5H of SEQ ID NO: 95; and G5H, F6H and R8H of SEQ ID NO: 99.
  • the CD47-binding domain comprises one or more substitutions of the parent CDR residues selected from the group consisting of: T3H, Y8H and Y10H of SEQ ID NO: 84; A3H of SEQ ID NO: 89; S7H and Y9H of SEQ ID NO: 91; K1H and Q5H of SEQ ID NO: 95; and G5H, F6H and R8H of SEQ ID NO: 99.
  • the CD47-binding domain comprising one or more of the substitutions of parent CDR residues binds to human CD47 through at least one of the CD47 residues of E29, E35, D46, D51, E97, E100 and E104 of SEQ ID NO: 474 and exhibits higher binding affinity to CD47 at an acidic pH than at physiological pH.
  • the CD47-binding domain exhibits higher binding affinity to CD47 at an acidic pH than at physiological pH wherein the acidic pH is less than 7.0 and the physiological pH is 7.2 to 7.5, and comprises one or more histidine substitutions of the CDR residues of a parent antibody comprising a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 82, 84, 89, 91, 95 and 99 respectively, wherein the histidine substitutions of the parent CDR residues are selected from the group consisting of: T3H, Y8H and Y10H of SEQ ID NO: 84; A3H of SEQ ID NO: 89; S7H and Y9H of SEQ ID NO: 91; K1H and Q5H of SEQ ID NO: 95; and G5H, F6H and R8H of SEQ ID NO: 99.
  • the CD47-binding domain comprises a substitution of R8H of SEQ ID NO: 99, and one additional substitution of the parent CDR residues selected from the group consisting of: G1H, G1E, G1D, Y2H, Y2E, Y2D, G3H, G3E, G3D, M4H, M4E and M4D of HCDR1 SEQ ID NO: 82; T1H, T1E, T1D, I2H, I2E, I2D, T3H, T3E, T3D, S4H, S4E, S4D, G5H, G5E, G5D, G6H, G6E, G6D, T7H, T7E, T7D, Y8H, Y8E, Y8D, T9H, T9E, T9D, Y10H, Y10E, Y10D, Y11H, Y11E and Y11D of HCDR2
  • the CD47-binding domain comprises a substitution of K1H of SEQ ID NO: 95, and one additional substitution of the parent CDR residues selected from the group consisting of: G1H, G1E, G1D, Y2H, Y2E, Y2D, G3H, G3E, G3D, M4H, M4E and M4D of HCDR1 SEQ ID NO: 82; T1H, T1E, T1D, I2H, I2E, I2D, T3H, T3E, T3D, S4H, S4E, S4D, G5H, G5E, G5D, G6H, G6E, G6D, T7H, T7E, T7D, Y8H, Y8E, Y8D, T9H, T9E, T9D, Y10H, Y10E, Y10D, Y11H, Y11E and Y11D of HCDR2
  • the CD47-binding domain comprises a substitution of A3H of SEQ ID NO: 89, and one additional substitution of the parent CDR residues selected from the group consisting of: G1H, G1E, G1D, Y2H, Y2E, Y2D, G3H, G3E, G3D, M4H, M4E and M4D of HCDR1 SEQ ID NO: 82; T1H, T1E, T1D, I2H, I2E, I2D, T3H, T3E, T3D, S4H, S4E, S4D, G5H, G5E, G5D, G6H, G6E, G6D, T7H, T7E, T7D, Y8H, Y8E, Y8D, T9H, T9E, T9D, Y10H, Y10E, Y10D, Y11H, Y11E and Y11D of HCDR
  • the CD47-binding domain comprises a substitution of Y8H of SEQ ID NO: 84, and one additional substitution of the parent CDR residues selected from the group consisting of: G1H, G1E, G1D, Y2H, Y2E, Y2D, G3H, G3E, G3D, M4H, M4E and M4D of HCDR1 SEQ ID NO: 82; T1H, T1E, T1D, I2H, I2E, I2D, T3H, T3E, T3D, S4H, S4E, S4D, G5H, G5E, G5D, G6H, G6E, G6D, T7H, T7E, T7D, T9H, T9E, T9D, Y10H, Y10E, Y10D, Y11H, Y11E and Y11D of HCDR2 SEQ ID NO: 84; S1H, SEQ ID NO:
  • the CD47-binding domain comprises a substitution of Y10H of SEQ ID NO: 84, and one additional substitution of the parent CDR residues selected from the group consisting of: G1H, G1E, G1D, Y2H, Y2E, Y2D, G3H, G3E, G3D, M4H, M4E and M4D of HCDR1 SEQ ID NO: 82; T1H, T1E, T1D, I2H, I2E, I2D, T3H, T3E, T3D, S4H, S4E, S4D, G5H, G5E, G5D, G6H, G6E, G6D, T7H, T7E, T7D, Y8H, Y8E, Y8D, T9H, T9E, T9D, Y11H, Y11E and Y11D of HCDR2 SEQ ID NO: 84; S1H, S1D, S
  • the CD47-binding domain comprises a substitution of F6H and R8H of SEQ ID NO: 99, and one additional substitution of the parent CDR residues selected from the group consisting of: G1H, G1E, G1D, Y2H, Y2E, Y2D, G3H, G3E, G3D, M4H, M4E and M4D of HCDR1 SEQ ID NO: 82; T1H, T1E, T1D, I2H, I2E, I2D, T3H, T3E, T3D, S4H, S4E, S4D, G5H, G5E, G5D, G6H, G6E, G6D, T7H, T7E, T7D, Y8H, Y8E, Y8D, T9H, T9E, T9D, Y10H, Y10E, Y10D, Y11H, Y11E and Y11D of
  • the CD47-binding domain comprises a substitution of G5H and R8H of SEQ ID NO: 99, and one additional substitution of the parent CDR residues selected from the group consisting of: G1H, G1E, G1D, Y2H, Y2E, Y2D, G3H, G3E, G3D, M4H, M4E and M4D of HCDR1 SEQ ID NO: 82; T1H, T1E, T1D, I2H, I2E, I2D, T3H, T3E, T3D, S4H, S4E, S4D, G5H, G5E, G5D, G6H, G6E, G6D, T7H, T7E, T7D, Y8H, Y8E, Y8D, T9H, T9E, T9D, Y10H, Y10E, Y10D, Y11H, Y11E and Y11D of
  • the CD47-binding domain comprises a substitution of R8H of SEQ ID NO: 99 and K1H of SEQ ID NO: 95, and one additional substitution of the parent CDR residues selected from the group consisting of: G1H, G1E, G1D, Y2H, Y2E, Y2D, G3H, G3E, G3D, M4H, M4E and M4D of HCDR1 SEQ ID NO: 82; T1H, T1E, T1D, I2H, I2E, I2D, T3H, T3E, T3D, S4H, S4E, S4D, G5H, G5E, G5D, G6H, G6E, G6D, T7H, T7E, T7D, Y8H, Y8E, Y8D, T9H, T9E, T9D, Y10H, Y10E, Y10D, Y11H, Y11
  • the CD47-binding domain comprises a substitution of R8H of SEQ ID NO: 99 and Q5H of SEQ ID NO: 95, and one additional substitution of the parent CDR residues selected from the group consisting of: G1H, G1E, G1D, Y2H, Y2E, Y2D, G3H, G3E, G3D, M4H, M4E and M4D of HCDR1 SEQ ID NO: 82; T1H, T1E, T1D, I2H, I2E, I2D, T3H, T3E, T3D, S4H, S4E, S4D, G5H, G5E, G5D, G6H, G6E, G6D, T7H, T7E, T7D, Y8H, Y8E, Y8D, T9H, T9E, T9D, Y10H, Y10E, Y10D, Y11H, Y11
  • the CD47-binding domain comprises a substitution of R8H of SEQ ID NO: 99 and Y9H of SEQ ID NO: 91, and one additional substitution of the parent CDR residues selected from the group consisting of: G1H, G1E, G1D, Y2H, Y2E, Y2D, G3H, G3E, G3D, M4H, M4E and M4D of HCDR1 SEQ ID NO: 82; T1H, T1E, T1D, I2H, I2E, I2D, T3H, T3E, T3D, S4H, S4E, S4D, G5H, G5E, G5D, G6H, G6E, G6D, T7H, T7E, T7D, Y8H, Y8E, Y8D, T9H, T9E, T9D, Y10H, Y10E, Y10D, Y11H,
  • the CD47-binding domain comprises a substitution of R8H of SEQ ID NO: 99 and S7H of SEQ ID NO: 91, and one additional substitution of the parent CDR residues selected from the group consisting of: G1H, G1E, G1D, Y2H, Y2E, Y2D, G3H, G3E, G3D, M4H, M4E and M4D of HCDR1 SEQ ID NO: 82; T1H, T1E, T1D, I2H, I2E, I2D, T3H, T3E, T3D, S4H, S4E, S4D, G5H, G5E, G5D, G6H, G6E, G6D, T7H, T7E, T7D, Y8H, Y8E, Y8D, T9H, T9E, T9D, Y10H, Y10E, Y10D, Y11H, Y
  • the CD47-binding domain comprises a substitution of R8H of SEQ ID NO: 99 and A3H of SEQ ID NO: 89, and one additional substitution of the parent CDR residues selected from the group consisting of: G1H, G1E, G1D, Y2H, Y2E, Y2D, G3H, G3E, G3D, M4H, M4E and M4D of HCDR1 SEQ ID NO: 82; T1H, T1E, T1D, I2H, I2E, I2D, T3H, T3E, T3D, S4H, S4E, S4D, G5H, G5E, G5D, G6H, G6E, G6D, T7H, T7E, T7D, Y8H, Y8E, Y8D, T9H, T9E, T9D, Y10H, Y10E, Y10D, Y11H, Y
  • the CD47-binding domain comprises a substitution of R8H of SEQ ID NO: 99 and Y8H of SEQ ID NO: 84, and one additional substitution of the parent CDR residues selected from the group consisting of: G1H, G1E, G1D, Y2H, Y2E, Y2D, G3H, G3E, G3D, M4H, M4E and M4D of HCDR1 SEQ ID NO: 82; T1H, T1E, T1D, I2H, I2E, I2D, T3H, T3E, T3D, S4H, S4E, S4D, G5H, G5E, G5D, G6H, G6E, G6D, T7H, T7E, T7D, T9H, T9E, T9D, Y10H, Y10E, Y10D, Y11H, Y11E and Y11D of HCDR2 SEQ ID NO:
  • the CD47-binding domain comprises a substitution of R8H of SEQ ID NO: 99 and Y10H of SEQ ID NO: 84, and one additional substitution of the parent CDR residues selected from the group consisting of: G1H, G1E, G1D, Y2H, Y2E, Y2D, G3H, G3E, G3D, M4H, M4E and M4D of HCDR1 SEQ ID NO: 82; T1H, T1E, T1D, I2H, I2E, I2D, T3H, T3E, T3D, S4H, S4E, S4D, G5H, G5E, G5D, G6H, G6E, G6D, T7H, T7E, T7D, Y8H, Y8E, Y8D, T9H, T9E, T9D, Y11H, Y11E and Y11D of HCDR2 SEQ ID NO:
  • the CD47-binding domain comprises a substitution of K1H of SEQ ID NO: 95 and A3H of SEQ ID NO: 89, and one additional substitution of the parent CDR residues selected from the group consisting of: G1H, G1E, G1D, Y2H, Y2E, Y2D, G3H, G3E, G3D, M4H, M4E and M4D of HCDR1 SEQ ID NO: 82; T1H, T1E, T1D, I2H, I2E, I2D, T3H, T3E, T3D, S4H, S4E, S4D, G5H, G5E, G5D, G6H, G6E, G6D, T7H, T7E, T7D, Y8H, Y8E, Y8D, T9H, T9E, T9D, Y10H, Y10E, Y10D, Y11H, Y
  • the CD47-binding domain exhibits higher binding affinity to CD47 at an acidic pH than at physiological pH, and comprises:
  • VH CDR1 HCDR1 sequence of SEQ ID NO: 76: GX 1 X 2 MS, wherein X 1 is Y, H, E or D, X 2 is G, H, E or D,
  • VH CDR2 (HCDR2) sequence of SEQ ID NO: 77: TIX 1 X 2 GGTX 3 TX 4 YPDSVKG, wherein X 1 is T, H, E or D, X 2 is S, H, E or D, X 3 is Y, H, E or D, X 4 is Y, H, E or D,
  • VH CDR3 HCDR3 sequence of SEQ ID NO: 78: SLX 1 X 2 NAMDY, wherein X 1 is A, H, E or D, X 2 is G, H, E or D,
  • VL CDR1 (LCDR1) sequence of SEQ ID NO: 79: RASQTIX 1 X 2 X 3 , wherein X 1 is S, H, E or D, X 2 is D, H or E, X 3 is Y, H, E or D,
  • LCDR2 VL CDR2 (LCDR2) sequence of SEQ ID NO: 80: X 1 FASX 2 X 3 X 4 X 5 , wherein X 1 is K, H, E or D, X 2 is Q, H, E or D, X 3 is S, H, E, D or R, X 4 is I, H, E, D or A, X 5 is S, H, E, D or T, and
  • VL CDR3 LCDR3 sequence of SEQ ID NO: 81: QNGHX 1 X 2 PX 3 T, wherein X 1 is G, H, E or D, X 2 is F, H, E or D, X 3 is R, H, E or D.
  • the CD47-binding domain exhibits higher binding affinity to CD47 at an acidic pH than at physiological pH, and comprise a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences selected from the group of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences as set forth in Table 3 below.
  • the CD47-binding domain exhibits higher binding affinity to CD47 at an acidic pH than at physiological pH, and comprises:
  • VH CDRs selected from at least one of the group consisting of:
  • a HCDR1 at least 50%, at least 60%, at least 70%, at least 80%or at least 90%identical to a HCDR1 as set forth in one of the sequences selected from the group consisting of SEQ ID NO: 82 to 83 ;
  • a HCDR2 at least 50%, at least 60%, at least 70%, at least 80%or at least 90%identical to a HCDR2 as set forth in one of the sequences selected from the group consisting of SEQ ID NO: 84 to 88 and SEQ ID NO: 111 to 113;
  • a HCDR3 at least 50%, at least 60%, at least 70%, at least 80%or at least 90%identical to a HCDR3 as set forth in one of the sequences selected from the group consisting of SEQ ID NO: 89 to 90;
  • VL CDRs one or more VL CDRs (LCDRs) selected from at least one of the group consisting of:
  • a LCDR1 at least 50%, at least 60%, at least 70%, at least 80%or at least 90%identical to a LCDR1 as set forth in one of the sequences selected from the group consisting of SEQ ID NO: 91 to 94 and SEQ ID NO: 107 to 110;
  • a LCDR2 at least 50%, at least 60%, at least 70%, at least 80%or at least 90%identical to a LCDR2 as set forth in one of the sequences selected from the group consisting of SEQ ID NO: 95 to 98;
  • a LCDR3 at least 50%, at least 60%, at least 70%, at least 80%or at least 90%identical to a LCDR3 as set forth in one of the sequences selected from the group consisting of SEQ ID NO: 99 to 106;
  • the CD47-binding domain comprises a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 82, 85/86/87/88, 89, 91, 95 and 99 respectively; or SEQ ID NO: 82, 84, 90, 91, 95 and 99 respectively; or SEQ ID NO: 82, 84, 89, 92/93/94/107/108/109/110, 95 and 99 respectively; or SEQ ID NO: 82, 84, 89, 91, 96/97/98 and 99 respectively; or SEQ ID NO: 82, 84, 89, 91, 95 and 100/101/102/103/104/105 /106 respectively; or SEQ ID NO: 82, 85/86/87/88, 90, 91, 95 and 99 respectively; or SEQ ID NO: 82, 84, 89, 92/93/94/107/108/109/110, 95 and 102 respectively; or SEQ ID NO: 82
  • the CD47-binding domain comprises a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences as set forth in Table 4 below.
  • the CD47-binding domain comprises a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 82, 85/86/87/88, 89, 91, 95 and 99 respectively; or SEQ ID NO: 82, 84, 90, 91, 95 and 99 respectively; or SEQ ID NO: 82, 84, 89, 92/93/94, 95 and 99 respectively; or SEQ ID NO: 82, 84, 89, 91, 96/97/98 and 99 respectively; or SEQ ID NO: 82, 84, 89, 91, 95 and 100/101/102/103/104/105/106 respectively; or SEQ ID NO: 82, 86/87/88, 90, 91, 95 and 99 respectively; or SEQ ID NO: 82, 86, 89, 93, 96 and 105 respectively; or SEQ ID NO: 82, 87, 89, 93, 97 and 104 respectively;
  • the CD47-binding domain comprises a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 82, 86, 89, 93, 96 and 105 respectively; or SEQ ID NO: 82, 87, 89, 93, 97 and 104 respectively; or SEQ ID NO: 82, 86, 89, 93, 96 and 102 respectively; or SEQ ID NO: 82, 87, 89, 93, 97 and 102 respectively; or SEQ ID NO: 82, 86, 89, 91, 95 and 106 respectively; or SEQ ID NO: 82, 88, 89, 93, 96 and 105 respectively; wherein the “/” indicates alternative sequences for the corresponding CDR.
  • the CD47-binding domain binds to human wild type CD47 at physiological pH with a monovalent affinity preferably lower than the monovalent affinity of wild type human SIPR ⁇ V2 IgV (SEQ ID NO: 306) to human wild type CD47, wherein the physiological pH is 7.2, 7.3 or 7.4. In some embodiments, the CD47-binding domain binds to human wild type CD47 at physiological pH with a monovalent affinity preferably at least 2-fold lower than the monovalent affinity of wild type human SIPR ⁇ V2 IgV (SEQ ID NO: 306) to human wild type CD47, wherein the physiological pH is 7.2, 7.3 or 7.4. In some embodiments, the CD47-binding domain comprises:
  • SEQ ID NO: 121 EIVLTQSPATLSLSPGERATLSCRASQTIX 1 X 2 X 3 LHWYQQKPGQAPRLLIX 4 FASX 5 SISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQNGHX 6 X 7 PX 8 TFGX 9 GTKVEIK, wherein X 1 is S or H, X 2 is D or H, X 3 is Y or H, X 4 is K or H, X 5 is Q or H, X 6 is G or H, X 7 is F or H, X 8 is R or H, X 9 is G or Q; or SEQ ID NO: 122: EIVLTQSPATLSLSPGERATLSCRASQTIX 1 X 2 X 3 LHWYQQKPGQAPRLLIX 4 FASX 5 RATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQX 6 GHX 7 X 8 PX
  • the CD47-binding domain comprises a pair of VH and VL sequences selected from the group of pairs of VH and VL sequences consisting of SEQ ID NO: 124 and 133, SEQ ID NO: 125 and 133, SEQ ID NO: 126 and 133, SEQ ID NO: 127 and 133, SEQ ID NO: 128 and 133, SEQ ID NO: 123 and 134, SEQ ID NO: 123 and 135, SEQ ID NO: 123 and 136, SEQ ID NO: 123 and 137, SEQ ID NO: 123 and 138, SEQ ID NO: 123 and 139, SEQ ID NO: 123 and 140, SEQ ID NO: 123 and 141, SEQ ID NO: 126 and 148, SEQ ID NO: 127 and 144, SEQ ID NO: 126 and 146, SEQ ID NO: 127 and 143, SEQ ID NO: 126 and 153, SEQ ID NO: 126 and 150, and SEQ ID NO: 129
  • the CD47-binding domain comprises a pair of VH and VL sequences selected from the group of pairs of VH and VL sequences consisting of SEQ ID NO: 127 and 133, SEQ ID NO: 128 and 133, SEQ ID NO: 123 and 137, SEQ ID NO: 123 and 141, SEQ ID NO: 126 and 148, SEQ ID NO: 127 and 144, SEQ ID NO: 126 and 146, SEQ ID NO: 127 and 143.
  • the CD47-binding domain is a Fab, a Fab’ , a F (ab’ ) 2, a single chain Fab domain (scFab) , a Fragment variable (Fv) domain, a single chain Fv (scFv) , a disulfide-stabilized Fv (dsFv) , a nanobody, or a single chain antibody.
  • the CD47-binding domain comprises a single chain fragment variable (scFv) comprising a VL-linker-VH or preferably a VH-linker-VL sequence, wherein:
  • (ii) comprises an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%identical to a VH sequence selected from the group consisting of SEQ ID NO: 123 to 132; or
  • (iii) comprises an amino acid sequence with addition, deletion and/or substitution of one or more amino acids compared with SEQ ID NO: 123;
  • (i) comprises the amino acid sequence of SEQ ID NO: 121 or 122;
  • (ii) comprises an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%identical to a VL sequence selected from the group comprising SEQ ID NO: 133 to 174 and 266 to 273; or
  • (iii) comprises an amino acid sequence with addition, deletion and/or substitution of one or more amino acids compared with SEQ ID NO: 133;
  • the linker comprises a sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 276) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 277) .
  • the CD47-binding domain comprises a single chain fragment variable (scFv) comprising a sequence of SEQ ID NO: 175: EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYGMSWVRQAPGKGLEWVATIX 1 SGGTX 2 TX 3 YPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSLX 4 GNAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLS CRASQTIX 5 X 6 X 7 LHWYQQKPGQAPRLLIX 8 FASX 9 SISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQNGHX 10 X 11 PX 12 TFGX 13 GTKVEIK, wherein X 1 is T or H, X 2 is Y or H, X 3 is Y or H, X 4 is A or H, X 5 is S or H, X 6 is D or H
  • the CD47-binding domain comprises a scFv comprising a VH-linker-VL sequence
  • the linker comprises GGGGSGGGGS GGGGS (SEQ ID NO: 276) and the pair of VH and VL sequences comprise SEQ ID NO: 124 and 133, SEQ ID NO: 125 and 133, SEQ ID NO: 126 and 133, SEQ ID NO: 127 and 133, SEQ ID NO: 128 and 133, SEQ ID NO: 123 and 134, SEQ ID NO: 123 and 135, SEQ ID NO: 123 and 136, SEQ ID NO: 123 and 137, SEQ ID NO: 123 and 138, SEQ ID NO: 123 and 139, SEQ ID NO: 123 and 140, SEQ ID NO: 123 and 141, SEQ ID NO: 126 and 148, SEQ ID NO: 127 and 144, SEQ ID NO: 126 and 146, SEQ ID NO: 127 and 143,
  • the CD47-binding domain comprises a scFv comprising a VH-linker-VL sequence, wherein the linker comprises GGGGSGGGGSGGGGS (SEQ ID NO: 276) , and the pair of VH and VL sequences comprise SEQ ID NO: 127 and 133, SEQ ID NO: 128 and 133, SEQ ID NO: 123 and 137, SEQ ID NO: 123 and 141, SEQ ID NO: 126 and 148, SEQ ID NO: 127 and 144, SEQ ID NO: 126 and 146, or SEQ ID NO: 127 and 143.
  • the CD47-binding domain comprises a scFv comprising a sequence of SEQ ID NO: 300 or 301.
  • the pH-sensitive CD47-binding domain is a variant derived from the parent anti-CD47 antibody BC31M4 (see (Li et al., J Hematol Oncol, 2023) ) , wherein the variant exhibits improved pH-sensitive CD47-binding with reduced binding at physiological pH and/or increased binding at acidic pH compared to that of the parent antibody BC31M4.
  • the parent antibody BC31M4 binds to an epitope on human CD47 extracellular domain (SEQ ID NO: 474) comprising the residues of Q1, T34, E35, V36, Y37, K39, D46, D51, A53, L54, E97, L101, T102, R103 and E104 of SEQ ID NO: 474 (see (Li et al., J Hematol Oncol, 2023) ) .
  • BC31M4 exhibits higher binding to CD47 at acidic pH ⁇ 7.0 than at neutral pH but binds to CD47 at physiological pH at a still significant binding affinity with a dissociation constant (KD) of 456 nM, and still binds to platelets and causes platelet depletion (see (Li et al., J Hematol Oncol, 2023) ) .
  • KD dissociation constant
  • the CD47-binding domain comprises three variable VH CDR sequences at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, or at least 80%identical to the three parent VH CDR amino acid sequences of SEQ ID NO: 19, 20 and 21, and/or three VL CDR sequences at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, or at least 80%identical to the three parent VL CDR amino acid sequences of SEQ ID NO: 22, 23 and 24.
  • the CD47-binding domain comprises one or more mutations of parent CDR residues selected from the residues corresponding to the positions 2, 3, 4, 6, 7 and 8 of HCDR1 SEQ ID NO: 19, the positions 1, 3, 5, 6, 8, 9 and 12 of HCDR2 SEQ ID NO: 20, the positions 2, 4, 5, 6, 8 and 9 of HCDR3 SEQ ID NO: 21, the positions 1, 3, 4, 7, 8 and 9 of LCDR1 SEQ ID NO: 22, the positions 2, 4, 5, 6 and 7 of LCDR2 SEQ ID NO: 23, and the positions 4, 5 , 6, 7 and 8 of LCDR3 SEQ ID NO: 24.
  • the CD47-binding domain comprises one or more substitutions of parent CDR residues selected from the group consisting of: Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, Y7H/E/D and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, R2H/E/D, A4H/E/D, V5H/E/D, Y6H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 21, R1H/E/D, S3H/E/D, Q4H/E/D, S7H/E/D, R8H/E/D, and N9H/E/D of SEQ
  • the CD47-binding domain comprising one or more of the substitutions of parent CDR residues binds to human CD47 through at least one of the CD47 residues of E35, D46, D51, E97 and E104 of SEQ ID NO: 474, and exhibits improved pH-sensitive CD47-binding with reduced binding at physiological pH and/or increased binding at acidic pH compared to that of the parent antibody BC31M4.
  • the CD47-binding domain comprises a CDR residue substitution of R8H of SEQ ID NO: 22, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, Y7H/E/D and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, R2H/E/D, A4H/E/D, V5H/E/D, Y6H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 21, R1H/E/D, S3H/E/D, Q4H/E/D, S7H/E/D, and N9H/E
  • the CD47-binding domain comprises a CDR residue substitution of N9H of SEQ ID NO: 22, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, Y7H/E/D and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, R2H/E/D, A4H/E/D, V5H/E/D, Y6H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 21, R1H/E/D, S3H/E/D, Q4H/E/D, S7H/E/D, and R8H/E
  • the CD47-binding domain comprises a CDR residue substitution of S7H of SEQ ID NO: 22, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, Y7H/E/D and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, R2H/E/D, A4H/E/D, V5H/E/D, Y6H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 21, R1H/E/D, S3H/E/D, Q4H/E/D, R8H/E/D, and N9H/E/E of S
  • the CD47-binding domain comprises a CDR residue substitution of W2H of SEQ ID NO: 23, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, Y7H/E/D and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, R2H/E/D, A4H/E/D, V5H/E/D, Y6H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 21, R1H/E/D, S3H/E/D, Q4H/E/D, S7H/E/D, R8H/E/
  • the CD47-binding domain comprises a CDR residue substitution of R8H and N9H of SEQ ID NO: 22, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, Y7H/E/D and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, R2H/E/D, A4H/E/D, V5H/E/D, Y6H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 21, R1H/E/D, S3H/E/D, Q4H/E/D, and S7H/E/D of SEQ ID NO:
  • the CD47-binding domain comprises a CDR residue substitution of R8H and S7H of SEQ ID NO: 22, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, Y7H/E/D and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, R2H/E/D, A4H/E/D, V5H/E/D, Y6H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 21, R1H/E/D, S3H/E/D, Q4H/E/D, and N9H/E/D of SEQ ID NO:
  • the CD47-binding domain comprises a CDR residue substitution of S7H and N9H of SEQ ID NO: 22, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, Y7H/E/D and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, R2H/E/D, A4H/E/D, V5H/E/D, Y6H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 21, R1H/E/D, S3H/E/D, Q4H/E/D, and R8H/E/D of SEQ ID NO:
  • the CD47-binding domain comprises a CDR residue substitution of W8H of SEQ ID NO: 24, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, Y7H/E/D and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, R2H/E/D, A4H/E/D, V5H/E/D, Y6H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 21, R1H/E/D, S3H/E/D, Q4H/E/D, S7H/E/D, R8H/E/
  • the CD47-binding domain comprises a CDR residue substitution of R1H of SEQ ID NO: 22, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, Y7H/E/D and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, R2H/E/D, A4H/E/D, V5H/E/D, Y6H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 21, S3H/E/D, Q4H/E/D, S7H/E/D, R8H/E/D, and N9H/E/E of S
  • the CD47-binding domain comprises a CDR residue substitution of Y7H of SEQ ID NO: 19, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, R2H/E/D, A4H/E/D, V5H/E/D, Y6H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 21, R1H/E/D, S3H/E/D, Q4H/E/D, S7H/E/D, R8H/E/D, and N9H/
  • the CD47-binding domain comprises a CDR residue substitution of R2H of SEQ ID NO: 21, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, Y7H/E/D and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, A4H/E/D, V5H/E/D, Y6H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 21, R1H/E/D, S3H/E/D, Q4H/E/D, S7H/E/D, R8H/E/D, and N9H/E/E of S
  • the CD47-binding domain comprises a CDR residue substitution of V5H of SEQ ID NO: 21, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, Y7H/E/D and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, R2H/E/D, A4H/E/D, Y6H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 21, R1H/E/D, S3H/E/D, Q4H/E/D, S7H/E/D, R8H/E/D, and N9H/E/E of S
  • the CD47-binding domain comprises a CDR residue substitution of Y6H of SEQ ID NO: 21, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, Y7H/E/D and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, R2H/E/D, A4H/E/D, V5H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 21, R1H/E/D, S3H/E/D, Q4H/E/D, S7H/E/D, R8H/E/D, and N9H/E/E of S
  • the CD47-binding domain exhibits improved pH-sensitive CD47-binding with reduced binding at physiological pH and/or increased binding at acidic pH compared to that of the parent antibody BC31M4, and comprise a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences selected from the group of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences as set forth in Table 5 below.
  • the CD47-binding domain comprises a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 177, 20, 21, 22, 23 and 24 respectively; or SEQ ID NO: 178, 20, 21, 22, 23 and 24 respectively; or SEQ ID NO: 179, 20, 21, 22, 23 and 24 respectively; or SEQ ID NO: 19, 20, 180, 22, 23 and 24 respectively; or SEQ ID NO: 19, 20, 181, 22, 23 and 24 respectively; or SEQ ID NO: 19, 20, 182, 22, 23 and 24 respectively; or SEQ ID NO: 19, 20, 21, 183, 23 and 24 respectively; or SEQ ID NO: 19, 20, 21, 184, 23 and 24 respectively; or SEQ ID NO: 19, 20, 21, 185, 23 and 24 respectively; or SEQ ID NO: 19, 20, 21, 186, 23 and 24 respectively; or SEQ ID NO: 19, 20, 21, 187, 23 and 24 respectively; or SEQ ID NO: 19, 20, 21, 188, 23 and 24 respectively; or SEQ ID NO: 19, 20, 21, 189, 23
  • the pH-sensitive CD47-binding domain is a variant derived from the parent anti-CD47 antibody BC27 (see (Li et al., J Hematol Oncol, 2023) ) , wherein the variant exhibits improved pH-sensitive CD47-binding with reduced binding at physiological pH and/or increased binding at acidic pH compared to that of the parent antibody BC27.
  • the antibody BC27 is the parent antibody of the antibody BC31M4 that binds to an epitope on human CD47 extracellular domain (SEQ ID NO: 474) comprising the residues of Q1, T34, E35, V36, Y37, K39, D46, D51, A53, L54, E97, L101, T102, R103 and E104 of SEQ ID NO: 474 (see (Li et al., J Hematol Oncol, 2023) ) .
  • BC27 exhibits higher binding to CD47 at acidic pH ⁇ 7.0 than at neutral pH but binds to CD47 at physiological pH still with an affinity dissociation constant (K D ) of 1169 nM (see (Li et al., J Hematol Oncol, 2023) ) , that is comparable to the binding affinity reported for human SIRP ⁇ V2 binding to CD47 with an affinity dissociation constant (K D ) of about 1-2 ⁇ M ( (Brooke et al., J Immunol, 2004, Hatherley et al., Mol Cell, 2008) ) , while human SIRP ⁇ V2 is known to bind platelets and the SIRP ⁇ V2 IgV-hIgG1 Fc fusion protein therapeutics TTI-621 causes thrombocytopenia in human patients (see (Ansell et al., Clin Cancer Res, 2021) and Blood 138 (2021) 2448-2451) .
  • K D affinity dissociation constant
  • the CD47-binding domain comprises three variable VH CDR sequences at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, or at least 80%identical to the three parent VH CDR amino acid sequences of SEQ ID NO: 19, 20 and 428, and/or three VL CDR sequences at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, or at least 80%identical to the three parent VL CDR amino acid sequences of SEQ ID NO: 22, 23 and 429.
  • the CD47-binding domain comprises one or more mutations of parent CDR residues selected from the residues corresponding to the positions 2, 3, 4, 6, 7 and 8 of HCDR1 SEQ ID NO: 19, the positions 1, 3, 5, 6, 8, 9 and 12 of HCDR2 SEQ ID NO: 20, the positions 2, 4, 5, 6, 8 and 9 of HCDR3 SEQ ID NO: 428, the positions 1, 3, 4, 7, 8 and 9 of LCDR1 SEQ ID NO: 22, the positions 2, 4, 5, 6 and 7 of LCDR2 SEQ ID NO: 23, and the positions 3, 4, 5 , 6, 7 and 8 of LCDR3 SEQ ID NO: 429.
  • the CD47-binding domain comprises one or more substitutions of parent CDR residues selected from the group consisting of: Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, Y7H/E/D and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, R2H/E/D, K4H/E/D/A, V5H/E/D, Y6H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 428, R1H/E/D, S3H/E/D, Q4H/E/D, S7H/E/D, R8H/E/D, and N9
  • the CD47-binding domain comprising one or more of the substitutions of parent CDR residues binds to human CD47 through at least one of the CD47 residues of E35, D46, D51, E97 and E104 of SEQ ID NO: 474, and exhibits improved pH-sensitive CD47-binding with reduced binding at physiological pH and/or increased binding at acidic pH compared to that of the parent antibody BC27.
  • the CD47-binding domain comprises a CDR residue substitution of R8H of SEQ ID NO: 22, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, Y7H/E/D and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, R2H/E/D, K4H/E/D/A, V5H/E/D, Y6H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 428, R1H/E/D, S3H/E/D, Q4H/E/D, S7H/E/D, and N
  • the CD47-binding domain comprises a CDR residue substitution of N9H of SEQ ID NO: 22, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, Y7H/E/D and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, R2H/E/D, K4H/E/D/A, V5H/E/D, Y6H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 428, R1H/E/D, S3H/E/D, Q4H/E/D, S7H/E/D, and R
  • the CD47-binding domain comprises a CDR residue substitution of S7H of SEQ ID NO: 22, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, Y7H/E/D and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, R2H/E/D, K4H/E/D/A, V5H/E/D, Y6H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 428, R1H/E/D, S3H/E/D, Q4H/E/D, R8H/E/D, and N
  • the CD47-binding domain comprises a CDR residue substitution of W2H of SEQ ID NO: 23, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, Y7H/E/D and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, R2H/E/D, K4H/E/D/A, V5H/E/D, Y6H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 428, R1H/E/D, S3H/E/D, Q4H/E/D, S7H/E/D, R8
  • the CD47-binding domain comprises a CDR residue substitution of R8H and N9H of SEQ ID NO: 22, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, Y7H/E/D and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, R2H/E/D, K4H/E/D/A, V5H/E/D, Y6H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 428, R1H/E/D, S3H/E/D, Q4H/E/D, and S7H/E
  • the CD47-binding domain comprises a CDR residue substitution of R8H and S7H of SEQ ID NO: 22, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, Y7H/E/D and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, R2H/E/D, K4H/E/D/A, V5H/E/D, Y6H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 428, R1H/E/D, S3H/E/D, Q4H/E/D, and N9H/E/E of S
  • the CD47-binding domain comprises a CDR residue substitution of S7H and N9H of SEQ ID NO: 22, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, Y7H/E/D and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, R2H/E/D, K4H/E/D/A, V5H/E/D, Y6H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 428, R1H/E/D, S3H/E/D, Q4H/E/D, and R8H/E/E
  • the CD47-binding domain comprises a CDR residue substitution of W8H of SEQ ID NO: 429, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, Y7H/E/D and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, R2H/E/D, K4H/E/D/A, V5H/E/D, Y6H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 428, R1H/E/D, S3H/E/D, Q4H/E/D, S7H/E/D,
  • the CD47-binding domain comprises a CDR residue substitution of R1H of SEQ ID NO: 22, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, Y7H/E/D and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, R2H/E/D, K4H/E/D/A, V5H/E/D, Y6H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 428, S3H/E/D, Q4H/E/D, S7H/E/D, R8H/E/D, and N
  • the CD47-binding domain comprises a CDR residue substitution of Y7H of SEQ ID NO: 19, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, R2H/E/D, K4H/E/D/A, V5H/E/D, Y6H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 428, R1H/E/D, S3H/E/D, Q4H/E/D, S7H/E/D, R8H/E/D, and
  • the CD47-binding domain comprises a CDR residue substitution of R2H of SEQ ID NO: 428, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, Y7H/E/D and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, K4H/E/D/A, V5H/E/D, Y6H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 428, R1H/E/D, S3H/E/D, Q4H/E/D, S7H/E/D, R8H/E/D,
  • the CD47-binding domain comprises a CDR residue substitution of V5H of SEQ ID NO: 428, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, Y7H/E/D and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, R2H/E/D, K4H/E/D/A, Y6H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 428, R1H/E/D, S3H/E/D, Q4H/E/D, S7H/E/D, R8H/E/D,
  • the CD47-binding domain comprises a CDR residue substitution of Y6H of SEQ ID NO: 428, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, F4H/E/D, S6H/E/D, Y7H/E/D and G8H/E/D of SEQ ID NO: 19, W1H/E/D, S3H/E/D, Y5H/E/D, N6H/E/D, N8H/E/D, Y9H/E/D, and K12H/E/D of SEQ ID NO: 20, R2H/E/D, K4H/E/D/A, V5H/E/D, D8H/E, and Y9H/E/D of SEQ ID NO: 428, R1H/E/D, S3H/E/D, Q4H/E/D, S7H/E/D, R8H/E/D,
  • the CD47-binding domain exhibits improved pH-sensitive CD47-binding with reduced binding at physiological pH and/or increased binding at acidic pH compared to that of the parent antibody BC27, and comprise a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences selected from the group of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences as set forth in Table 6 below.
  • the CD47-binding domain comprises a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 177, 20, 428, 22, 23 and 429 respectively; or SEQ ID NO: 178, 20, 428, 22, 23 and 429 respectively; or SEQ ID NO: 179, 20, 428, 22, 23 and 429 respectively; or SEQ ID NO: 19, 20, 180, 22, 23 and 429 respectively; or SEQ ID NO: 19, 20, 181, 22, 23 and 429 respectively; or SEQ ID NO: 19, 20, 182, 22, 23 and 429 respectively; or SEQ ID NO: 19, 20, 428, 183, 23 and 429 respectively; or SEQ ID NO: 19, 20, 428, 184, 23 and 429 respectively; or SEQ ID NO: 19, 20, 428, 185, 23 and 429 respectively; or SEQ ID NO: 19, 20, 428, 186, 23 and 429 respectively; or SEQ ID NO: 19, 20, 428, 187, 23 and 429 respectively; or SEQ ID NO: 19,
  • the pH-sensitive CD47-binding domain is a variant derived from the parent anti-CD47 antibody lemzoparlimab (TJ-C4) (see US20200140565A1) .
  • the antibody lemzoparlimab binds to an epitope on human CD47 extracellular domain (SEQ ID NO: 474) comprising the residues of L3, V25, T26, N27, M28, E29, A30, Q31, T34, E35, Y37, A53, L54, L74, K75, G76, T99, E100, L101, T102 and R103 of SEQ ID NO: 474 (see US20200140565A1) .
  • the CD47-binding domain exhibits higher binding affinity to CD47 at an acidic pH than at physiological pH, and comprises three variable VH CDR sequences at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, or at least 80%identical to the three parent VH CDR amino acid sequences of SEQ ID NO: 37, 38 and 39, and/or three VL CDR sequences at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, or at least 80%identical to the three parent VL CDR amino acid sequences of SEQ ID NO: 40, 41 and 42.
  • the CD47-binding domain comprises one or more mutations of parent CDR residues selected from the residues corresponding to the positions 1, 2, 3 and 5 of HCDR1 SEQ ID NO: 37; 2, 3, 4, 5, 6, 7, 8, 14 and 19 of HCDR2 SEQ ID NO: 38; 2, 3, 4 and 5 of HCDR3 SEQ ID NO: 39; 1, 5, 8, 11, 12, 13, 14 and 15 of LCDR1 SEQ ID NO: 40; 1, 2, 5 and 6 of LCDR2 SEQ ID NO: 41; and 3, 4, 5 and 7 of LCDR3 SEQ ID NO: 42.
  • the CD47-binding domain comprises one or more substitutions of parent CDR residues selected from the group consisting of: R1H/E/D, A2H/E/D, W3H/E/D, and N5H/E/D of SEQ ID NO: 37, R2H/E/D, I3H/E/D, K4H/E/D, R5H/E/D, K6H/E/D, T7H/E/D, D8H/E, Y14H/E/D, and K19H/E/D of SEQ ID NO: 38, N2H/E/D, R3H/E/D, A4H/E/D, and F5H/E/D of SEQ ID NO: 39, K1H/E/D, S5H/E/D, Y8H/E/D, N11H/E/D, N12H/E/D, R13H/E/D, N14H/E/D, and Y15H/E
  • the CD47-binding domain comprising one or more of the substitutions of the parent CDR residues binds to human CD47 through at least one of the CD47 residues of E29, E35 and E100 of SEQ ID NO: 474, and exhibits higher binding affinity to CD47 at an acidic pH than at physiological pH.
  • the CD47-binding domain comprises a CDR residue substitution of R3H of SEQ ID NO: 39, and one additional substitution selected from the group consisting of R1H/E/D, A2H/E/D, W3H/E/D, and N5H/E/D of SEQ ID NO: 37, R2H/E/D, I3H/E/D, K4H/E/D, R5H/E/D, K6H/E/D, T7H/E/D, D8H/E, Y14H/E/D, and K19H/E/D of SEQ ID NO: 38, N2H/E/D, A4H/E/D, and F5H/E/D of SEQ ID NO: 39, K1H/E/D, S5H/E/D, Y8H/E/D, N11H/E/D, N12H/E/D, R13H/E/D, N14H/E/D, and Y15H
  • the CD47-binding domain comprises a CDR residue substitution of R1H of SEQ ID NO: 37, and one additional substitution selected from the group consisting of A2H/E/D, W3H/E/D, and N5H/E/D of SEQ ID NO: 37, R2H/E/D, I3H/E/D, K4H/E/D, R5H/E/D, K6H/E/D, T7H/E/D, D8H/E, Y14H/E/D, and K19H/E/D of SEQ ID NO: 38, N2H/E/D, R3H/E/D, A4H/E/D, and F5H/E/D of SEQ ID NO: 39, K1H/E/D, S5H/E/D, Y8H/E/D, N11H/E/D, N12H/E/D, R13H/E/D, N14H/E/D, and Y15H
  • the CD47-binding domain comprises a CDR residue substitution of R5H of SEQ ID NO: 38, and one additional substitution selected from the group consisting of R1H/E/D, A2H/E/D, W3H/E/D, and N5H/E/D of SEQ ID NO: 37, R2H/E/D, I3H/E/D, K4H/E/D, K6H/E/D, T7H/E/D, D8H/E, Y14H/E/D, and K19H/E/D of SEQ ID NO: 38, N2H/E/D, R3H/E/D, A4H/E/D, and F5H/E/D of SEQ ID NO: 39, K1H/E/D, S5H/E/D, Y8H/E/D, N11H/E/D, N12H/E/D, R13H/E/D, N14H/E/D, and Y15H
  • the CD47-binding domain comprises a CDR residue substitution of R3H of SEQ ID NO: 39 and R5H of SEQ ID NO: 38, and one additional substitution selected from the group consisting of R1H/E/D, A2H/E/D, W3H/E/D, and N5H/E/D of SEQ ID NO: 37, R2H/E/D, I3H/E/D, K4H/E/D, K6H/E/D, T7H/E/D, D8H/E, Y14H/E/D, and K19H/E/D of SEQ ID NO: 38, N2H/E/D, A4H/E/D, and F5H/E/D of SEQ ID NO: 39, K1H/E/D, S5H/E/D, Y8H/E/D, N11H/E/D, N12H/E/D, R13H/E/D, N14H/E/D, and
  • the CD47-binding domain comprises a CDR residue substitution of R3H of SEQ ID NO: 39 and R1H of SEQ ID NO: 37, and one additional substitution selected from the group consisting of A2H/E/D, W3H/E/D, and N5H/E/D of SEQ ID NO: 37, R2H/E/D, I3H/E/D, K4H/E/D, R5H/E/D, K6H/E/D, T7H/E/D, D8H/E, Y14H/E/D, and K19H/E/D of SEQ ID NO: 38, N2H/E/D, A4H/E/D, and F5H/E/D of SEQ ID NO: 39, K1H/E/D, S5H/E/D, Y8H/E/D, N11H/E/D, N12H/E/D, R13H/E/D, N14H/E/D, and
  • the CD47-binding domain comprises a CDR residue substitution of R1H of SEQ ID NO: 37 and R5H of SEQ ID NO: 38, and one additional substitution selected from the group consisting of A2H/E/D, W3H/E/D, and N5H/E/D of SEQ ID NO: 37, R2H/E/D, I3H/E/D, K4H/E/D, K6H/E/D, T7H/E/D, D8H/E, Y14H/E/D, and K19H/E/D of SEQ ID NO: 38, N2H/E/D, R3H/E/D, A4H/E/D, and F5H/E/D of SEQ ID NO: 39, K1H/E/D, S5H/E/D, Y8H/E/D, N11H/E/D, N12H/E/D, R13H/E/D, N14H/E/D, and
  • the CD47-binding domain exhibits higher binding affinity to CD47 at an acidic pH than at physiological pH, and comprise a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences selected from the group of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences as set forth in Table 7 below.
  • the CD47-binding domain comprises a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 222, 38, 39, 40, 41 and 42 respectively; or SEQ ID NO: 37, 223, 39, 40, 41 and 42 respectively; or SEQ ID NO: 37, 38, 224, 40, 41 and 42 respectively; or SEQ ID NO: 37, 38, 39, 225, 41 and 42 respectively; or SEQ ID NO: 37, 38, 39, 40, 41 and 226 respectively; or SEQ ID NO: 37, 38, 39, 40, 41 and 227 respectively; or SEQ ID NO: 37, 38, 39, 40, 41 and 228 respectively; or SEQ ID NO: 37, 223, 224, 40, 41 and 42 respectively; or SEQ ID NO: 222, 38, 224, 40, 41 and 42 respectively; or SEQ ID NO: 222, 223, 39, 40, 41 and 42 respectively; or SEQ ID NO: 37, 38, 224, 40, 41 and 42
  • the pH-sensitive CD47-binding domain is a variant derived from the parent anti-CD47 antibody AO-176 (see Blood (2016) 132 (Supplement 1) : 4180) , wherein the variant exhibits improved pH-sensitive CD47-binding with reduced binding at physiological pH and/or increased binding at acidic pH compared to that of the parent antibody AO-176.
  • the CD47-binding domain comprises three variable VH CDR sequences at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, or at least 80%identical to the three parent VH CDR amino acid sequences of SEQ ID NO: 61, 62 and 63, and/or three VL CDR sequences at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, or at least 80%identical to the three parent VL CDR amino acid sequences of SEQ ID NO: 64, 65 and 66.
  • the CD47-binding domain comprises one or more mutations of parent CDR residues selected from the residues corresponding to the positions 2, 3, 5, 6 and 7 of HCDR1 SEQ ID NO: 61; 1, 2, 5, 6, 8, 9 and 11 of HCDR2 SEQ ID NO: 62; 1, 3, 5, 7 and 8 of HCDR3 SEQ ID NO: 63; 1, 5, 8, 9, 10, 12 and 14 of LCDR1 SEQ ID NO: 64; 1, 2, 5, 6 and 7 of LCDR2 SEQ ID NO: 65; and 2, 4, 8 and 9 of LCDR3 SEQ ID NO: 66.
  • the CD47-binding domain comprises one or more substitutions of parent CDR residues selected from the group consisting of: Y2H/E/D, T3H/E/D, T5H/E/D, N6H/E/D, and Y7H/E/D of SEQ ID NO: 61, Y1H/E/D, T2H/E/D, R5H/E/D, T6H/E/D, Y8H/E/D, T9H/E/D, and Y11H/E/D of SEQ ID NO: 62, G1H/E/D, R3H/E/D, G5H/E/D, G7H/E/D, and Y8H/E/D of SEQ ID NO: 63, R1H/E/D, N5H/E/D, Q8H/E/D, S9H/E/D, N10H/E/D, N12H/E/D, and Y14H/
  • the CD47-binding domain comprises a CDR residue substitution of R5H of SEQ ID NO: 62, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, T5H/E/D, N6H/E/D, and Y7H/E/D of SEQ ID NO: 61, Y1H/E/D, T2H/E/D, T6H/E/D, Y8H/E/D, T9H/E/D, and Y11H/E/D of SEQ ID NO: 62, G1H/E/D, R3H/E/D, G5H/E/D, G7H/E/D, and Y8H/E/D of SEQ ID NO: 63, R1H/E/D, N5H/E/D, Q8H/E/D, S9H/E/D, N10H/E/D, N12H/E/D, and Y
  • the CD47-binding domain comprises a CDR residue substitution of R3H of SEQ ID NO: 63, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, T5H/E/D, N6H/E/D, and Y7H/E/D of SEQ ID NO: 61, Y1H/E/D, T2H/E/D, R5H/E/D, T6H/E/D, Y8H/E/D, T9H/E/D, and Y11H/E/D of SEQ ID NO: 62, G1H/E/D, G5H/E/D, G7H/E/D, and Y8H/E/D of SEQ ID NO: 63, R1H/E/D, N5H/E/D, Q8H/E/D, S9H/E/D, N10H/E/D, N12H/E/D, and Y
  • the CD47-binding domain comprises a CDR residue substitution of K1H of SEQ ID NO: 65, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, T5H/E/D, N6H/E/D, and Y7H/E/D of SEQ ID NO: 61, Y1H/E/D, T2H/E/D, R5H/E/D, T6H/E/D, Y8H/E/D, T9H/E/D, and Y11H/E/D of SEQ ID NO: 62, G1H/E/D, R3H/E/D, G5H/E/D, G7H/E/D, and Y8H/E/D of SEQ ID NO: 63, R1H/E/D, N5H/E/D, Q8H/E/D, S9H/E/D, N10H/E/D, N12H/
  • the CD47-binding domain comprises a CDR residue substitution of R5H of SEQ ID NO: 65, and one additional substitution selected from the group consisting of Y2H/E/D, T3H/E/D, T5H/E/D, N6H/E/D, and Y7H/E/D of SEQ ID NO: 61, Y1H/E/D, T2H/E/D, R5H/E/D, T6H/E/D, Y8H/E/D, T9H/E/D, and Y11H/E/D of SEQ ID NO: 62, G1H/E/D, R3H/E/D, G5H/E/D, G7H/E/D, and Y8H/E/D of SEQ ID NO: 63, R1H/E/D, N5H/E/D, Q8H/E/D, S9H/E/D, N10H/E/D, N12H/
  • Antibody comprising a pH-sensitive CD47-binding domain
  • the present disclosure provides a protein comprising at least one of the pH-sensitive CD47-binding domains of the present disclosure.
  • the protein comprises a Fc or its functional fragment.
  • the Fc is selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and its modification.
  • the Fc is a human IgG1 with wild-type effector function (SEQ ID NO: 291) .
  • the Fc is a human IgG1 with reduced effector function.
  • the Fc comprises a mutation selected from the group consisting of N279A, N297G, N279S, N279Q, L234A+L235A L234A+L235A+G237A, and L234A+L235A+P329G, according to the EU numbering scheme.
  • the Fc comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 288 to 290.
  • the Fc is a human IgG1 Fc with enhanced antibody-dependent cellular phagocytosis (ADCP) function, comprising a mutation selected from the group consisting of G236A, S239D/I332E, G236A/S239D/I332E, S239D/A330L/I332E, G236A/A330L/I332E, G236A/S239D/A330L /I332E, and F243L/R292P/Y300L/V305I/P396L, according to the EU numbering scheme.
  • the Fc is a human IgG4 Fc with a mutation of S228P (SEQ ID NO: 286) .
  • the Fc is a human IgG2 Fc (SEQ ID NO: 287)
  • the Fc comprises a homodimeric Fc, wherein the two Fc chains of the Fc comprise mutations that promote formation of heterodimeric Fc.
  • the Fc comprises a knobs-into-holes heterodimeric Fc, wherein one Fc chain comprises mutation of T336W as the knob Fc half-chain, and the other Fc chain comprises mutation of Y407V or T366S+L368A+Y407V as the hole Fc half-chain, according to the EU numbering scheme.
  • the Fc comprises a knobs-into-holes heterodimeric Fc, wherein one Fc chain comprises mutation of T336W+S354C as the knob Fc half-chain, and the other Fc chain comprises mutation of Y407V+Y349C or T366S+L368A+Y407V+Y349C as the hole Fc half-chain, according to the EU numbering scheme.
  • the knobs-into-holes heterodimeric Fc comprises a pair of Fc amino acid sequence selected from the group consisting of SEQ ID NO: 292: 293, 294: 295, 296: 297 and 298: 299, wherein the “: ” indicates pairing of the two sequences from the left to right of the “:” symbol.
  • the heterodimeric Fc comprises a human IgG1 Fc, comprising a pair of amino acid sequence of SEQ ID NO: 292: 293.
  • the protein is an isolated antibody comprising a full heavy chain and full light chain, wherein the heavy chain and light chain form a pH-sensitive CD47-binding Fab domain of the present disclosure.
  • the present disclosure provides an isolated antibody or the antigen-binding portion thereof, wherein it competes for binding to CD47 with the pH-sensitive CD47-binding domain or the antigen-binding portion thereof the present disclosure.
  • Antibody fusion protein comprising a pH-sensitive CD47-binding domain
  • the present disclosure provides an antibody fusion protein comprising: 1) a full antibody comprising two pH-sensitive CD47-binding Fab domains of the present disclosure, and 2) a CD47-binding SIRP IgV domain, wherein the SIRP IgV domain comprises one, two, three or more SIRP IgV monomers, and is linked preferably through a linker to the N-terminal or C-terminal of the heavy chain or the light chain of the antibody.
  • the antibody fusion protein is configured in a format FV-1, FV-2, FV-3 or FV-4 as set forth in FIG. 1.
  • the SIRP IgV monomer is a SIRP ⁇ , SIRP ⁇ or SIRP ⁇ IgV.
  • the SIRP IgV is a SIRP ⁇ , SIRP ⁇ or SIRP ⁇ IgV comprising one or more mutations selected from the group consisting of K53H, R69H, Q52H, K68H, Q52H+K68H, K53H+K68H, R69H+K68H, K53H+Q52H, R69H+Q52H, K53H+K68H+Q52H, and R69H+K68H+Q52H, wherein the residue is numbered according to SEQ ID NO: 306.
  • the SIRP IgV monomer comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 306 to 311.
  • the CD47-binding Fab domain of the antibody fusion protein comprises a pair of VH and VL sequence selected from the group consisting of SEQ ID NO: 126 and 148, SEQ ID NO: 127 and 144, SEQ ID NO: 126 and 146, and SEQ ID NO: 127 and 143.
  • the antibody fusion protein comprises a heavy chain of SEQ ID NO: 258 and a light chain of SEQ ID NO: 312 or 313.
  • the antibody fusion protein comprises a heavy chain of SEQ ID NO: 259 and a light chain of SEQ ID NO: 314 or 315.
  • the present disclosure provides a fusion protein comprising a CD47-binding domain of the present disclosure, comprises at least one additional domain that binds to a non-CD47 antigen.
  • the additional domain binds to a soluble antigen, an aggregate of antigen, a surface antigen on a cell, a surface antigen on a microbe, or a hapten antigen, wherein the antigen is not CD47.
  • the additional domain comprises a peptide or polypeptide with antigen-binding, a ligand or fragment thereof with receptor binding, a receptor or fragment thereof with ligand binding, a heavy chain variable domain, a light chain variable domain, a single chain fragment variable (scFv) comprising a VH and a VL, a single chain Fab domain (scFab) , a VHH antibody or nanobody, a VH single chain antibody (VH dAb) or a VL single chain antibody (VL dAb) , an immunoglobulin new antigen receptor (IgNAR) , or other single-chain domain antibody, or an anticalin, DARPIN, affibody, affimer, centryin domain, fibronectin domain, D-Domain or other alternative scaffold known in the arts to function as antigen binding domain that binds to the non-CD47 antigen.
  • scFv single chain fragment variable
  • scFab single chain fragment variable
  • VHH antibody or nanobody
  • the fusion protein comprising a CD47-binding domain of the present disclosure and at least one additional non-CD47 binding domain is an antibody fusion protein.
  • the antibody fusion protein comprising a CD47-binding domain of the present disclosure and at least one additional non-CD47 binding domain is configured in a format variant selected from the group consisting of FV-6 to 21 as set forth in FIG. 2 to 5.
  • the additional non-CD47 binding domain of the fusion protein binds to one or more antigens selected from the group consisting of Amyloid Beta, Amyloid fibril, SAP, CD38, SLAMF7, BCMA, GPRC5D, FcRH5, CD138, CD56, CD74, CD26, CD46, CD19, CD20, CD22, CD30, CD33, CD37, CD70, CD117, CD79b, CD123, CD52, CD98, CD205, PD-L1, HER2, EGFR, CD93, TGF ⁇ , IL-6, IL-6R, IL-8, GDF-15, GFRAL, FGFR2, FGFR3, FGFR4, FGFR1, GD2, GD3, B7-H3, B7-H4, phosphatidylserine, DR5, DR4, DR3, TL1A, CD95 (Fas) , TNFR1, TNFR2, CD24, CD31, CD61, CD200, SLAMF3, SLAMF
  • the additional non-CD47 binding domain of the fusion protein comprises an antibody domain or antigen-binding fragment thereof comprising a set of heavy chain variable (VH) and/or light chain variable (VL) complementarity determining region (CDR) sequences at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%or at least 95%identical to the set of VH and/or VL CDR sequences according to the IMGT numbering scheme (The Immunologist, 7, 132-136 (1999) ; Dev Comp Immunol. 2003 Jan; 27 (1) : 55-77) of an antibody selected from a group of exemplary antibodies as set forth in Table 8 to 10.
  • VH heavy chain variable
  • VL light chain variable complementarity determining region
  • the additional non-CD47 binding domain of the fusion protein comprises a peptide, a receptor or fragment thereof, a ligand or fragment thereof, a cytokine or fragment thereof, or a chemokine or fragment thereof, or a growth factor or fragment thereof.
  • the additional non-CD47 binding domain of the antibody fusion protein comprises an amino acid sequence at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%or at least 95%identical to an amino acid sequence selected from a group of exemplary peptides and polypeptides as set forth in Table 11.
  • the additional non-CD47 binding domain of the fusion protein binds to one or more antigens selected from the group consisting of PD-L1, CD38, SLAMF7, CD20, CD19, CD30, CD70, CD117, CA-IX, HER2, EGFR, CD93, FGFR2, GD2, Claudin18.2, Claudin 6, Claudin 1, Claudin 2, Claudin 3, Claudin 4, Claudin 7, B7-H3, DLL3, DR5, DR4, CD95, Phosphatidylserine, Nectin-4, CDH3, CDH6, CDH17, CDH2, integrins, CD44, ICAM-1, EpCAM, CEACAM5, CEACAM1, CEACAM6, CD24, HLA-G, FAP, CTGF and TL1A.
  • one or more antigens selected from the group consisting of PD-L1, CD38, SLAMF7, CD20, CD19, CD30, CD70, CD117, CA-IX, HER
  • the additional non-CD47 binding domain of the fusion protein comprises an antigen binding domain of an antibody comprising a set of VH and/or VL CDR sequences that are at least 70%, 75%, 80%, 85 %, 90%, or 95%identical to the set of VH and/or VL CDR sequences according to the IMGT numbering scheme from an antibody selected from the group of antibodies comprising anti-PD-L1 antibody BMS-936559, atezolizumab, durvalumab, avelumab, cosibelimab, sugemalimab, geptanolimab and envafolimab, anti-CD38 antibody daratumumab, isatuximab, SAR442085, felzartamab, mezagitamab, TAK-169, CID-103 and Y150, anti-SLAMF7 antibody elotuzumab and azintuxizumab, anti-CD20 antibody rituxima
  • anti-CD70 nanobody No. 1 to 14 (CN113292652A) , anti-CD70 antibodies (US11377500B2) , LD70, BR108, MP-0533, and the anti-CD70 antibody moiety of SGN-75, SGN-CD70A, BMS-936561, AMG172, ARX-305, PRO-1160, CTX130, ALLO-316, P-CD70-ALLO1, 4SCAR70, C-4-29 and CAT-248, anti-CA-IX antibody girentuximab and BAY 79-4620, anti-HER2 antibody trastuzumab, pertuzumab, and margetuximab, anti-EGFR antibody cetuximab, panitumumab, necitumumab and nimotuzumab, anti-FGFR2 antibody bemarituzumab and aprutumab, anti-GD2 antibody dinutuximab and n
  • the additional non-CD47 binding domain of the antibody fusion protein binds to one or more antigens selected from the group consisting of Dectin-1, Dectin-2, CLEC5A, MerTK, TREM1, MARCO, CLEVER-1, PSGL-1, VSIG4, CD40, CD205, CD206, CD36, CD91, DC-SIGN, CLEC9A, TLR5, LILRB1 (ILT2) , LILRB2 (ILT4) , LILRB4 (ILT3) , NKG2D, NKp30, NKp46, NKp80, DNAM-1, PD-1, CTLA-4, TIGIT, LAG3, CD3, 4-1BB, OX40, ICOS, CD27 and CD70.
  • one or more antigens selected from the group consisting of Dectin-1, Dectin-2, CLEC5A, MerTK, TREM1, MARCO, CLEVER-1, PSGL-1, VSIG4, CD40, CD205, CD206, CD36, CD91, DC
  • the additional non-CD47 binding domain of the antibody fusion protein comprises an antigen binding domain of an antibody comprising a set of VH and/or VL CDR sequences that are at least 70%, 75%, 80%, 85 %, 90%, or 95%identical to the set of VH and/or VL CDR sequences according to the IMGT numbering scheme from an antibody selected from the group of antibodies comprising anti-Dectin 1 antibody 2M24 and 15E2, anti-Dectin 2 antibody BDC-3042, anti-CLEC5A antibody 1F7, anti-MerTK antibody 18G7 and RGX-019, anti-CD205 antibody 3G9 and OBT076, anti-CD206 antibody CDX-1307, anti-DC-SIGN antibody hD1V1, anti-TREM1 antibody PY159, anti-MARCO antibody PY265, anti-CLEVER1 antibody bexmarilimab, anti-PSGL-1 antibody VTX-0811, neihulizumab, and leiolizum
  • an antibody fusion protein comprising: 1) a full anti-CD47 antibody of the present disclosure comprising two heavy chains and two light chains, wherein the two Fc region of the two heavy chains form a homodimeric Fc, and 2) a single-chain domain binding to a non-CD47 antigen, wherein the single-chain domain is linked preferably through a linker to the N-terminal or C-terminal of the heavy chain or the light chain of the anti-CD47 antibody.
  • the antibody fusion protein is configured in a format of FV-6 or 7 as set forth in FIG. 2, wherein the single-chain domain is linked to the N-terminal of the light chain or heavy chain.
  • the single-chain domain of the antibody fusion protein configured in a format of FV-6 or 7 comprises a sequence of SEQ ID NO: 316, 317, 318 or 319.
  • the antibody fusion protein is configured in a format of FV-8 or 9 as set forth in FIG. 2, wherein the single-chain domain is linked to the C-terminal of the heavy chain or light chain.
  • the single-chain domain of the antibody fusion protein configured in a format of FV-8 or 9 comprises a sequence of SEQ ID NO: 320, 321 or 322.
  • an antibody fusion protein comprising: 1) a first heavy chain comprising VH1-CH1-hinge-CH2-CH3 and a second chimeric heavy chain comprising VH2-CL-hinge-CH2-CH3, wherein the Fc region of the two heavy chains form a heterodimeric Fc, 2) a first light chain comprising VL1-CL, wherein it pairs with the first heavy chain to form the first Fab domain that binds to a non-CD47 antigen, and 3) a second chimeric light chain comprising VL2-CH1, wherein it pairs with the second chimeric heavy chain to form a second anti-CD47 Fab domain of the present disclosure.
  • the antibody fusion protein is configured in a format of FV-10 as set forth in FIG. 3.
  • an antibody fusion protein comprising: 1) a first heavy chain comprising VH1-CH1-hinge-CH2-CH3 and a second chimeric heavy chain comprising VH2-CL-hinge-CH2-CH3, wherein the Fc region of the two heavy chains form a heterodimeric Fc, 2) a first light chain comprising VL1-CL, wherein it pairs with the first heavy chain to form a first anti-CD47 Fab domain of the present disclosure, and 3) a second chimeric light chain comprising VL2-CH1, wherein it pairs with the second chimeric heavy chain to form the second Fab domain that binds to a non-CD47 antigen.
  • the antibody fusion protein is configured in a format of FV-11 as set forth in FIG. 3.
  • an antibody fusion protein comprising: 1) a first heavy chain comprising VH1-CH1-hinge-CH2-CH3 and a second chimeric heavy chain comprising VH2-CL-linker-VH1-CH1-hinge-CH2-CH3, wherein the Fc region of the two heavy chains form a heterodimeric Fc, 2) a first light chain comprising VL1-CL, wherein it pairs with the VH1-CH1 part of the first and second heavy chains to form two Fab domains that bind to a non-CD47 antigen, and 3) a second chimeric light chain comprising VL2-CH1, wherein it pairs with the VH2-CL part of the second chimeric heavy chain to form an anti-CD47 Fab domain of the present disclosure.
  • the antibody fusion protein is configured in a format of FV-13 as set forth in FIG. 3.
  • an antibody fusion protein comprising: 1) a first heavy chain comprising VH1-CH1-hinge-CH2-CH3 and a second chimeric heavy chain comprising VH1-CH1-linker-VH2-CL-hinge-CH2-CH3, wherein the Fc region of the two heavy chains form a heterodimeric Fc, 2) a first light chain comprising VL1-CL, wherein it pairs with the VH1-CH1 part of the first and second heavy chains to form two anti-CD47 Fab domains of the present disclosure, and 3) a second chimeric light chain comprising VL2-CH1, wherein it pairs with the VH2-CL part of the second chimeric heavy chain to form a Fab domain that binds to a non-CD47 antigen.
  • the antibody fusion protein is configured in a format of FV-12 as set forth in FIG. 3.
  • an antibody fusion protein comprising: 1) a full antibody comprising two Fab domains binding to a non-CD47 antigen, and 2) a single-chain CD47-binding domain of the present disclosure, wherein the single-chain CD47-binding domain is linked preferably through a linker to the N-terminal or C-terminal of the heavy chain or the light chain of the antibody.
  • the antibody fusion protein is configured in a format FV-14, FV-15, FV-16 or FV-17 as set forth in FIG. 4.
  • an antibody fusion protein comprising: 1) a first polypeptide comprising a Fc region, and a single-chain CD47-binding domain of the present disclosure, wherein the single-chain CD47-binding domain is linked preferably through a linker to the N-terminal of the Fc region, 2) a full heavy chain of an antibody and a full light chain of an antibody, wherein the heavy chain and the light chain pair to form an half-antibody that binds to a non-CD47 antigen, and 3) the Fc region of the first polypeptide and the antibody heavy chain form a heterodimeric Fc.
  • the antibody fusion protein is configured in a format of FV-18 as set forth in FIG. 5.
  • the linker of an antibody fusion protein configured in a format of FV-18 comprises an amino acid sequence of SEQ ID NO: 274 or 275.
  • the antibody fusion protein further comprises a second single-chain CD47-binding domain of the present disclosure, wherein the second single-chain CD47-binding domain is linked preferably through a linker to the N-terminal of first single-chain CD47-binding domain, the light chain, or the heavy chain of the antibody fusion protein.
  • the antibody fusion protein comprising a second single-chain CD47-binding domain is configured in a format of FV-19, 20 and 21 as set forth in FIG. 5.
  • the CD47-binding domain of the antibody fusion protein configured in a format of FV-1 to 21, comprises a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%or at least 95%identical to the set of CDR sequences of SEQ ID NO: 82 (HCDR1) , SEQ ID NO: 84 (HCDR2) , SEQ ID NO: 89 (HCDR3) , SEQ ID NO: 91 (LCDR1) , SEQ ID NO: 95 (LCDR2) , and SEQ ID NO: 99 (LCDR3) respectively.
  • the CD47-binding domain of the antibody fusion protein configured in a format of FV-1 to 21, comprises a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%or at least 95%identical to the set of CDR sequences of SEQ ID NO: 19 (HCDR1) , SEQ ID NO: 20 (HCDR2) , SEQ ID NO: 21 (HCDR3) , SEQ ID NO: 22 (LCDR1) , SEQ ID NO: 23 (LCDR2) , and SEQ ID NO: 24 (LCDR3) respectively.
  • the CD47-binding domain of the antibody fusion protein configured in a format of FV-1 to 21, comprises a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%or at least 95%identical to the set of CDR sequences of SEQ ID NO: 19 (HCDR1) , SEQ ID NO: 20 (HCDR2) , SEQ ID NO: 428 (HCDR3) , SEQ ID NO: 22 (LCDR1) , SEQ ID NO: 23 (LCDR2) , and SEQ ID NO: 429 (LCDR3) respectively.
  • the CD47-binding domain of the antibody fusion protein configured in a format of FV-1 to 21, comprises a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%or at least 95%identical to the set of CDR sequences of SEQ ID NO: 37 (HCDR1) , SEQ ID NO: 38 (HCDR2) , SEQ ID NO: 39 (HCDR3) , SEQ ID NO: 40 (LCDR1) , SEQ ID NO: 41 (LCDR2) , and SEQ ID NO: 42 (LCDR3) respectively.
  • the CD47-binding domain of the antibody fusion protein configured in a format of FV-1 to 21, comprises a set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%or at least 95%identical to the set of CDR sequences of SEQ ID NO: 61 (HCDR1) , SEQ ID NO: 62 (HCDR2) , SEQ ID NO: 63 (HCDR3) , SEQ ID NO: 64 (LCDR1) , SEQ ID NO: 65 (LCDR2) , and SEQ ID NO: 66 (LCDR3) respectively.
  • the CD47-binding domain of the antibody fusion protein configured in a format of FV-1 to 2121 comprises a VH sequence selected from the group consisting of SEQ ID NO: 123 to 132, and a VL sequence selected from the group consisting of SEQ ID NO: 133 to 174 and 266 to 273.
  • the CD47-binding domain of the antibody fusion protein configured in a format of FV-1 to 21 comprises a VH sequence selected from the group consisting of SEQ ID NO: 196 to 202, and a VL sequence selected from the group consisting of SEQ ID NO: 203 to 221.
  • the CD47-binding domain of the antibody fusion protein configured in a format of FV-1 to 21 comprises a VH sequence selected from the group consisting of SEQ ID NO: 196 to 202 and 433, and a VL sequence selected from the group consisting of SEQ ID NO: 204 to 209, 213 to 218 and 434 to 440.
  • the CD47-binding domain of the antibody fusion protein configured in a format of FV-1 to 21 comprises a VH sequence selected from the group consisting of SEQ ID NO: 229 to 235, and a VL sequence selected from the group consisting of SEQ ID NO: 236 to 240.
  • the CD47-binding domain of the antibody fusion protein configured in a format of FV-1 to 21 comprises a pair of VH and VL sequence of SEQ ID NO: 127 and 133, SEQ ID NO: 128 and 133, SEQ ID NO: 123 and 137, SEQ ID NO: 123 and 141, SEQ ID NO: 126 and 148, SEQ ID NO: 127 and 144, SEQ ID NO: 126 and 146, or SEQ ID NO: 127 and 143.
  • the CD47-binding domain of the antibody fusion protein configured in a format of FV-1 to 21 comprises a pair of VH and VL sequence of SEQ ID NO: 196 and 203.
  • the CD47-binding domain of the antibody fusion protein configured in a format of FV-1 to 21 comprises a pair of VH and VL sequence of SEQ ID NO: 433 and 434. In some embodiments, the CD47-binding domain of the antibody fusion protein configured in a format of FV-1 to 21, comprises a pair of VH and VL sequence of SEQ ID NO: 229 and 236.
  • the single-chain CD47-binding domain of the antibody fusion protein configured in a format of FV-14 to 21, comprises a scFv comprising VH-linker-VL or VL-linker-VH, wherein the linker comprises a sequence of SEQ ID NO: 276 or 277.
  • the VH of the scFv comprises a VH sequence selected from the group consisting of SEQ ID NO: 123 to 132
  • the VL of the scFv comprises a VL sequence selected from the group consisting of SEQ ID NO: 133 to 174 and 266 to 273.
  • the VH of the scFv comprises a VH sequence selected from the group consisting of SEQ ID NO: 196 to 202
  • the VL of the scFv comprises a VL sequence selected from the group consisting of SEQ ID NO: 203 to 221.
  • the VH of the scFv comprises a VH sequence selected from the group consisting of SEQ ID NO: 196 to 202 and 433, and the VL of the scFv comprises a VL sequence selected from the group consisting of SEQ ID NO: 204 to 209, 213 to 218 and 434 to 440.
  • the VH of the scFv comprises a VH sequence selected from the group consisting of SEQ ID NO: 229 to 235
  • the VL of the scFv comprises a VL sequence selected from the group consisting of SEQ ID NO: 236 to 240.
  • the scFv comprises a sequence of SEQ ID NO: 300 or 301.
  • the single-chain non-CD47 binding domain of the antibody fusion protein configured in a format of FV-6 to 9, comprises a scFv comprising VH-linker-VL or VL-linker-VH, wherein the linker comprises a sequence of SEQ ID NO: 276 or 277.
  • the VH and VL sequences of the scFv of the antibody fusion protein configured in a format of FV-6 to 9, comprise a pair of VH and VL sequences selected from the group of VH and VL sequence pairs consisting of SEQ ID NO: 323 and 324, 325 and 326, 327 and 328, 329 and 330, 331 and 332, 333 and 334, 335 and 336, 337 and 338, 339 and 340, 341 and 342, 343 and 344, 345 and 346, 347 and 348, 349 and 350, 351 and 352, 353 and 354, 355 and 356, 357 and 358, 359 and 360, 361 and 362, 363 and 364, 365 and 366, 367 and 368, 369 and 370, 371 and 372, 373 and 374, 375 and 376, 377 and 378, 379 and 380, 381 and 382, 383 and 384, 385 and 386, 387 and 388
  • the VH and VL sequences of the scFv of the antibody fusion protein configured in a format of FV-6 to 7, comprise a pair of VH and VL sequences selected from the group of VH and VL sequence pairs consisting of SEQ ID NO: 323 and 324, 325 and 326, 327 and 328, 329 and 330, 331 and 332, 333 and 334, 335 and 336, 337 and 338, 339 and 340, 341 and 342, 343 and 344, 345 and 346, 347 and 348, 349 and 350, 351 and 352, 353 and 354, 355 and 356, 357 and 358, 359 and 360, 361 and 362, 363 and 364, 365 and 366, 367 and 368, 369 and 370, 371 and 372, 373 and 374, 375 and 376, 377 and 378, 379 and 380, 381 and 382, 383 and 384, 385 and 386, 387 and 388
  • the VH and VL sequences of the scFv of the antibody fusion protein configured in a format of FV-8 to 9, comprise a pair of VH and VL sequences selected from the group of VH and VL sequence pairs consisting of SEQ ID NO: 391 and 392, 393 and 394, 395 and 396, 397 and 398, 399 and 400, 401 and 402, 403 and 404, 405 and 406, 443 and 444, 445 and 446, 447 and 448, 449 and 450, 451 and 452, 453 and 454, 455 and 456, 457 and 458, 459 and 460, 461 and 462, 463 and 464, 465 and 466, 467 and 468, and 469 and 470.
  • the non-CD47 binding Fab domain of the antibody fusion protein configured in a format of FV-10 to 21 comprises a pair of VH and VL sequences selected from the group of VH and VL sequence pairs consisting of SEQ ID NO: 323 and 324, 325 and 326, 327 and 328, 329 and 330, 331 and 332, 333 and 334, 335 and 336, 337 and 338, 339 and 340, 341 and 342, 343 and 344, 345 and 346, 347 and 348, 349 and 350, 351 and 352, 353 and 354, 355 and 356, 357 and 358, 359 and 360, 361 and 362, 363 and 364, 365 and 366, 367 and 368, 369 and 370, 371 and 372, 373 and 374, 375 and 376, 377 and 378, 379 and 380, 381 and 382, 383 and 384, 385 and 386, 387 and 388, 389 and 390
  • the non-CD47 binding Fab domain of the antibody fusion protein configured in a format of FV-13, 14 and 15, comprises a pair of VH and VL sequences selected from the group of VH and VL sequence pairs consisting of SEQ ID NO: 323 and 324, 325 and 326, 327 and 328, 329 and 330, 331 and 332, 333 and 334, 335 and 336, 337 and 338, 339 and 340, 341 and 342, 343 and 344, 345 and 346, 347 and 348, 349 and 350, 351 and 352, 353 and 354, 355 and 356, 357 and 358, 359 and 360, 361 and 362, 363 and 364, 365 and 366, 367 and 368, 369 and 370, 371 and 372, 373 and 374, 375 and 376, 377 and 378, 379 and 380, 381 and 382, 383 and 384, 385 and 386, 387 and 388, 389 and
  • the non-CD47 binding Fab domain of the antibody fusion protein configured in a format of FV-12, 16, 17, 20 and 21, comprises a pair of VH and VL sequences selected from the group of VH and VL sequence pairs consisting of SEQ ID NO: 391 and 392, 393 and 394, 395 and 396, 397 and 398, 399 and 400, 401 and 402, 403 and 404, 405 and 406, 443 and 444, 445 and 446, 447 and 448, 449 and 450, 451 and 452, 453 and 454, 455 and 456, 457 and 458, 459 and 460, 461 and 462, 463 and 464, 465 and 466, 467 and 468, and 469 and 470.
  • an antibody fusion protein configured in a format of FV-10 to 13 and FV-18 to 21, wherein the CD47-binding domain comprising a VH and VL sequence pair of SEQ ID NO: 126 and 148, SEQ ID NO: 127 and 144, SEQ ID NO: 196 and 203, or SEQ ID NO: 433 and 434, and the non-CD47 binding domain comprising a VH and VL sequence pair of SEQ ID NO: 393 and 394.
  • an antibody fusion protein configured in a format of FV-6 to 21, wherein the CD47-binding domain comprising a VH and VL sequence pair of SEQ ID NO: 128 and 133, SEQ ID NO: 123 and 137, SEQ ID NO: 123 and 141, SEQ ID NO: 126 and 148, SEQ ID NO: 127 and 144, SEQ ID NO: 126 and 146, SEQ ID NO: 127 and 143, SEQ ID NO: 196 and 203, or SEQ ID NO: 433 and 434, and the non-CD47 binding domain comprising a VH and VL sequence pair of SEQ ID NO: 337 and 338.
  • an antibody fusion protein configured in a format of FV-6 to 21, wherein the CD47-binding domain comprising a VH and VL sequence pair of SEQ ID NO: 128 and 133, SEQ ID NO: 123 and 137, SEQ ID NO: 123 and 141, SEQ ID NO: 126 and 148, SEQ ID NO: 127 and 144, SEQ ID NO: 126 and 146, SEQ ID NO: 127 and 143, SEQ ID NO: 196 and 203, or SEQ ID NO: 433 and 434, and the non-CD47 binding domain comprising a VH and VL sequence pair of SEQ ID NO: 339 and 340.
  • an antibody fusion protein configured in a format of FV-6 to 21, wherein the CD47-binding domain comprising a VH and VL sequence pair of SEQ ID NO: 128 and 133, and the non-CD47 binding domain comprising a VH and VL sequence at least 80%, at least 90%or at least 100%identical to the VH and VL sequence respectively of the CD70-binding domain from a CD70-targeting agent selected from the group consisting of cusatuzumab, MDX-1411, SEA-CD70, vorsetuzumab, IMM40H, IMM40M, anti-CD70 nanobody No. 1, 2 and 3 to 24 (WO2022262100) , anti-CD70 nanobody No.
  • 1 to 14 (CN113292652A) , anti-CD70 antibodies (US11377500B2) , LD70, BR108, MP-0533, BMS-936561, SGN-CD70A, SGN-75, AMG172, ARX-305, PRO-1160, CTX130, ALLO-316, P-CD70-ALLO1, 4SCAR70, C-4-29 and CAT-248.
  • an antibody fusion protein configured in a format of FV-6 to 21, wherein the CD47-binding domain comprising a VH and VL sequence pair of SEQ ID NO: 123 and 141, and the non-CD47 binding domain comprising a VH and VL sequence at least 80%, at least 90%or at least 100%identical to the VH and VL sequence respectively of the CD70-binding domain from a CD70-targeting agent selected from the group consisting of cusatuzumab, MDX-1411, SEA-CD70, vorsetuzumab, IMM40H, IMM40M, anti-CD70 nanobody No. 1, 2 and 3 to 24 (WO2022262100) , anti-CD70 nanobody No.
  • 1 to 14 (CN113292652A) , anti-CD70 antibodies (US11377500B2) , LD70, BR108, MP-0533, BMS-936561, SGN-CD70A, SGN-75, AMG172, ARX-305, PRO-1160, CTX130, ALLO-316, P-CD70-ALLO1, 4SCAR70, C-4-29 and CAT-248.
  • an antibody fusion protein configured in a format of FV-6 to 21, wherein the CD47-binding domain comprising a VH and VL sequence pair of SEQ ID NO: 126 and 148, and the non-CD47 binding domain comprising a VH and VL sequence at least 80%, at least 90%or at least 100%identical to the VH and VL sequence respectively of the CD70-binding domain from a CD70-targeting agent selected from the group consisting of cusatuzumab, MDX-1411, SEA-CD70, vorsetuzumab, IMM40H, IMM40M, anti-CD70 nanobody No.1, 2 and 3 to 24 (WO2022262100) , anti-CD70 nanobody No.
  • a CD70-targeting agent selected from the group consisting of cusatuzumab, MDX-1411, SEA-CD70, vorsetuzumab, IMM40H, IMM40M, anti-CD70 nanobody No.1, 2 and 3 to 24 (WO2022262100)
  • 1 to 14 (CN113292652A) , anti-CD70 antibodies (US11377500B2) , LD70, BR108, MP-0533, BMS-936561, SGN-CD70A, SGN-75, AMG172, ARX-305, PRO-1160, CTX130, ALLO-316, P-CD70-ALLO1, 4SCAR70, C-4-29 and CAT-248.
  • an antibody fusion protein configured in a format of FV-6 to 21, wherein the CD47-binding domain comprising a VH and VL sequence pair of SEQ ID NO: 127 and 144, and the non-CD47 binding domain comprising a VH and VL sequence at least 80%, at least 90%or at least 100%identical to the VH and VL sequence respectively of the CD70-binding domain from a CD70-targeting agent selected from the group consisting of cusatuzumab, MDX-1411, SEA-CD70, vorsetuzumab, IMM40H, IMM40M, anti-CD70 nanobody No. 1, 2 and 3 to 24 (WO2022262100) , anti-CD70 nanobody No.
  • 1 to 14 (CN113292652A) , anti-CD70 antibodies (US11377500B2) , LD70, BR108, MP-0533, BMS-936561, SGN-CD70A, SGN-75, AMG172, ARX-305, PRO-1160, CTX130, ALLO-316, P-CD70-ALLO1, 4SCAR70, C-4-29 and CAT-248.
  • an antibody fusion protein configured in a format of FV-6 to 21, wherein the CD47-binding domain comprising a VH and VL sequence pair of SEQ ID NO: 126 and 146, and the non-CD47 binding domain comprising a VH and VL sequence at least 80%, at least 90%or at least 100%identical to the VH and VL sequence respectively of the CD70-binding domain from a CD70-targeting agent selected from the group consisting of cusatuzumab, MDX-1411, SEA-CD70, vorsetuzumab, IMM40H, IMM40M, anti-CD70 nanobody No. 1, 2 and 3 to 24 (WO2022262100) , anti-CD70 nanobody No.
  • 1 to 14 (CN113292652A) , anti-CD70 antibodies (US11377500B2) , LD70, BR108, MP-0533, BMS-936561, SGN-CD70A, SGN-75, AMG172, ARX-305, PRO-1160, CTX130, ALLO-316, P-CD70-ALLO1, 4SCAR70, C-4-29 and CAT-248.
  • an antibody fusion protein configured in a format of FV-6 to 21, wherein the CD47-binding domain comprising a VH and VL sequence pair of SEQ ID NO: 127 and 143, and the non-CD47 binding domain comprising a VH and VL sequence at least 80%, at least 90%or at least 100%identical to the VH and VL sequence respectively of the CD70-binding domain from a CD70-targeting agent selected from the group consisting of cusatuzumab, MDX-1411, SEA-CD70, vorsetuzumab, IMM40H, IMM40M, anti-CD70 nanobody No. 1, 2 and 3 to 24 (WO2022262100) , anti-CD70 nanobody No.
  • 1 to 14 (CN113292652A) , anti-CD70 antibodies (US11377500B2) , LD70, BR108, MP-0533, BMS-936561, SGN-CD70A, SGN-75, AMG172, ARX-305, PRO-1160, CTX130, ALLO-316, P-CD70-ALLO1, 4SCAR70, C-4-29 and CAT-248.
  • an antibody fusion protein configured in a format of FV-6 to 21, wherein the CD47-binding domain comprising a VH and VL sequence pair of SEQ ID NO: 196 and 203, and the non-CD47 binding domain comprising a VH and VL sequence at least 80%, at least 90%or at least 100%identical to the VH and VL sequence respectively of the CD70-binding domain from a CD70-targeting agent selected from the group consisting of cusatuzumab, MDX-1411, SEA-CD70, vorsetuzumab, IMM40H, IMM40M, anti-CD70 nanobody No. 1, 2 and 3 to 24 (WO2022262100) , anti-CD70 nanobody No.
  • 1 to 14 (CN113292652A) , anti-CD70 antibodies (US11377500B2) , LD70, BR108, MP-0533, BMS-936561, SGN-CD70A, SGN-75, AMG172, ARX-305, PRO-1160, CTX130, ALLO-316, P-CD70-ALLO1, 4SCAR70, C-4-29 and CAT-248.
  • an antibody fusion protein configured in a format of FV-6 to 21, wherein the CD47-binding domain comprising a VH and VL sequence pair of SEQ ID NO: 433 and 434, and the non-CD47 binding domain comprising a VH and VL sequence at least 80%, at least 90%or at least 100%identical to the VH and VL sequence respectively of the CD70-binding domain from a CD70-targeting agent selected from the group consisting of cusatuzumab, MDX-1411, SEA-CD70, vorsetuzumab, IMM40H, IMM40M, anti-CD70 nanobody No. 1, 2 and 3 to 24 (WO2022262100) , anti-CD70 nanobody No.
  • 1 to 14 (CN113292652A) , anti-CD70 antibodies (US11377500B2) , LD70, BR108, MP-0533, BMS-936561, SGN-CD70A, SGN-75, AMG172, ARX-305, PRO-1160, CTX130, ALLO-316, P-CD70-ALLO1, 4SCAR70, C-4-29 and CAT-248.
  • the homodimeric Fc of the antibody fusion proteins configured in a format of FV-1 to 9 and FV-14 to 17 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 286 to 291. In some embodiments, the homodimeric Fc of the antibody fusion proteins configured in a format of FV-1 to 9 and FV-14 to 17 comprises an amino acid sequence of SEQ ID NO: 291. In some embodiments, the homodimeric Fc of the antibody fusion proteins configured in a format of FV-1 to 9 and FV-14 to 17 comprises an amino acid sequence of SEQ ID NO: 286.
  • the heterodimeric Fc of the antibody fusion proteins configured in a format of FV-10 to 13 and FV-18 to 21 comprises a pair of Fc amino acid sequence selected from the group consisting of SEQ ID NO: 292: 293, 294: 295, 296: 297 and 298: 299, wherein the “: ” indicates pairing of the two Fc sequences from the left to right of the “: ” symbol.
  • the heterodimeric Fc of the antibody fusion proteins configured in a format of FV-10 to 13 and FV-18 to 21 comprises a pair of Fc amino acid sequence of SEQ ID NO: 292: 293.
  • the heterodimeric Fc of the antibody fusion proteins configured in a format of FV-10 to 13 and FV-18 to 21 comprises a pair of Fc amino acid sequence of SEQ ID NO: 294: 295.
  • the homodimeric or heterodimeric Fc comprises a human IgG1 Fc with wild type or enhanced effector fuction.
  • the linker of an antibody fusion protein configured in a format of FV-1 to 9 and FV-12 to 21, comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 274 to 279.
  • the linker of an antibody fusion protein configured in a format of FV-1 to 4, FV-6 to 8, FV-14 to 16, and FV-18 to 21, comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 277.
  • the linker of an antibody fusion protein configured in a format of FV-5, 12, 13, 18, 19, 20 and 21, comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 275.
  • the linker of an antibody fusion protein configured in a format of FV-9 and 17, comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 279.
  • Protein drug conjugate comprising a pH-sensitive CD47-binding domain
  • the present disclosure provides a protein drug conjugate comprising a pH-sensitive CD47-binding domain of the present disclosure, an antibody comprising a pH-sensitive CD47-binding domain of the present disclosure, an antibody fusion protein comprising a pH-sensitive CD47-binding domain of the present disclosure, or a protein comprising a pH-sensitive CD47-binding domain of the present disclosure.
  • the protein of the protein drug conjugate is configured in a format selected from the group consisting of FV-1 to 21 as set forth in FIG. 1 to 5.
  • the protein drug conjugate comprises at least one conjugated moiety selected from the group consisting of a cytotoxic agent, a cytostatic agent, a radioactive isotope or compound, a chelator, a calreticulin-inducing agent, an immune stimulatory adjuvant moiety, a steroid, an immunosuppressor, DNA, RNA, a photosensitizer, a toxin, and an enzyme/pro-drug converting enzyme.
  • a conjugated moiety selected from the group consisting of a cytotoxic agent, a cytostatic agent, a radioactive isotope or compound, a chelator, a calreticulin-inducing agent, an immune stimulatory adjuvant moiety, a steroid, an immunosuppressor, DNA, RNA, a photosensitizer, a toxin, and an enzyme/pro-drug converting enzyme.
  • the therapeutic agent can be conjugated to the protein or antibody by chemical conjugation or enzymatic conjugation in a site-specific or non-spedific manner, using cleavable linkers (e.g. acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker, tumor-enriching linker) or non-cleavable linkers (e.g. cleavable linkers (e.g. acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker, tumor-enriching linker) or non-cleavable linkers (e.g.
  • maleimido-alkylene or maleimide-aryl linker known in the arts, for example, as described in patents such as US7745394, US5208020, US20070092940, EP1968635B1, WO2014140317, EP1370298B1, WO2014202775, US10947317B2, US8685383B2, US20120183566, US9669107B2, US10745488B2 and WO2022170971A1, as well as in journal articles such as (Walsh et al., Chem Soc Rev, 2021, Yao et al., Int J Mol Sci, 2016, Tsuchikama et al., Protein Cell, 2018, Yurkovetskiy et al., Mol Cancer Ther, 2021) , all incorporated herein by reference in its entirety and for all purposes.
  • anti-CD47 antibody-drug conjugates comprising non-pH sensitive anti-CD47 antibody have been successfully generated using different cytotoxic payloads and conjugation technologies known in the arts, as described above, and showed potent anti-tumor activity against CD47-expressing tumor cells in vitro and CD47-expressing tumors in xenograft mouse models in vivo in preclinical studies (see (Si et al., Vaccines (Basel) , 2021) , and (Chiang et al., Front Oncol, 2022) )
  • the therapeutic agent can be conjugated to the protein in a manner that reduces its activity unless it is cleaved off the antibody, for example, by hydrolysis, by reduction, by a cleaving agent or by proteolytic degradation.
  • the therapeutic agent is conjugated to the antibody with a cleavable linker that is sensitive to cleavage within intracellular environment but is not substantially sensitive to the extracellular environment.
  • the conjugate is cleaved off the protein or antibody after it is internalized by the target cell, for example, in the endosomal, lysosomal and/or caveolear environment by virtue of pH sensitivity, protease sensitivity or reduction sensitivity.
  • Protease sensitive linker can be a peptidyl linker (e.g. a linker comprising a Val-Cit or Phe-Leu peptide) that is cleaved by an intracellular peptidase or protease enzyme, including a lysosomal or endosomal protease, such as cathepsins B and D and plasmin that are highly expressed in tumor tissues, e.g. as described in the reference (Dubowchik et al., Pharmacol Ther, 1999) .
  • a peptidyl linker e.g. a linker comprising a Val-Cit or Phe-Leu peptide
  • an intracellular peptidase or protease enzyme including a lysosomal or endosomal protease, such as cathepsins B and D and plasmin that are highly expressed in tumor tissues, e.g. as described in the reference (Dubowchik et al., Pharma
  • pH-sensitive linker for example, an acid-labile linker such as a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like, is hydrolyzable under acidic conditions at usually pH ⁇ 5.5 such as in lyosomes but relatively stable under neutral pH conditions such as in blood, as described in patent such as US5122368, US5824805 and US5622929.
  • Reduction-sensitive linker e.g.
  • Disulfide linkers can be formed using SATA (N-succinimidyl-S-acetylthioacetate) , SPDP (N-succinimidyl-3- (2-pyridyldithio) propionate) , SPDB (N-succinimidyl-3- (2-pyridyldithio) butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha- (2-pyridyl-dithio) toluene) , SPDB and SMPT, as described in patent US4880935A and the reference such as (Thorpe et al., Cancer Res, 1987) .
  • SATA N-succinimidyl-S-acetylthioacetate
  • SPDP N-succinimidyl-3- (2-pyridyldithio) propionate
  • SPDB N-succinimidyl-3
  • the therapeutic agent is conjugated to the antibody with a cleavable linker that is sensitive to cleavage within specific extracellular environment of disease tissues such as tumor, but is not substantially sensitive to the peripheral circulation environment and normal tissue environment, such that the conjugate is cleaved from the antibody after it’s delivered into the extracellular environment of target disease tissue.
  • a cleavable linker that is sensitive to cleavage within specific extracellular environment of disease tissues such as tumor, but is not substantially sensitive to the peripheral circulation environment and normal tissue environment, such that the conjugate is cleaved from the antibody after it’s delivered into the extracellular environment of target disease tissue.
  • a number of protease enzymes such as matriptase (MT-SP1) , matrix metalloproteases (e.g. MMP2, MMP9, MMP7 et al. ) , uPA, ADAMs (e.g. ADAM10, ADAM17) and cathepsins are enriched in the extracellular environment of tumor than peripheral circulation
  • the therapeutic agent can also be conjugated to the antibody with a non-cleavable linker, such as a maleimido-alkylene or maleimide-aryl linker that is directly attached to the therapeutic agent and released by proteolytic degradation of the antibody.
  • a non-cleavable linker such as a maleimido-alkylene or maleimide-aryl linker that is directly attached to the therapeutic agent and released by proteolytic degradation of the antibody.
  • the conjugated moiety comprises a cytotoxic agent selected from the group consisting of tubulin inhibitors, DNA topoisomerase inhibitors, DNA minor groove binders, DNA alkylating agents, DNA intercalating agents, RNA polymerase inhibitors, spliceosome inhibitors and nicotinamide phosphoribosyltransferase inhibitors (NAMPTi) .
  • the foregoing cytotoxic agents comprise cytotoxic agents including but not limiting to auristatins (such as MMAE, MMAF, Auristatin F, Amberstatin, Auristatin W, dolastatin and dolaflexin, see e.g.
  • patents US6884869, US5635483, US5780588, US7498298 and US8685383B2) maytansinoids (such as DM1, DM2, DM3 and DM4, see e.g. patent US5208020, US5416064 and EP0425235Bl) , tubulysins (such as AZ13599185, see e.g. patent US2015141646) , taxanes (such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel, see e.g.
  • patent US7390898 trichothecene, vinca alkaloids (such as vindesine) , methotrexate; camptothecins (such as SN-38, exatecan, DX-8951, Dxd, irrinotecan, topotecan, belotecan and camptothecin, see e.g. patents US10155821B2, US9808537B2) , etoposides (such as etoposide and teniposide) , calicheamicins (such as CM1, see e.g.
  • patents EP2560645A2 and US5475092 benzodiazepines (such as pyrrolo [1, 4] benzodiazepines, indolinobenzodiazepines, and oxazolidinobenzodiazepines, including pyrrolo [1, 4] benzodiazepine dimers, indolinobenzodiazepine dimers, and oxazolidinobenzodiazepine dimmers, see e.g. patents US8765740, EP2766048B1, US2013028919 and US20110256157) , amatoxins (such as ⁇ -amanitin, see e.g. patent EP2436398B1) , thailanstatin A and spliceostatins.
  • benzodiazepines such as pyrrolo [1, 4] benzodiazepines, indolinobenzodiazepines, and oxazolidinobenzodiazepines, including pyrrolo [1, 4] benzodiazepine dimers
  • the conjugated moiety comprises a MMAE.
  • the MMAE payload is conjugated to the protein with a valine-citrulline (VC) dipeptide linker, as described in patents US7745394 and US6884869.
  • the conjugated moiety comprises Dxd.
  • the Dxd payload is conjugated to the protein with a glycyl-glycyl-phenylalanyl-glycine (GGFG) tetrapeptide linker, as described in patents US10155821B2 and US9808537B2.
  • the conjugated moiety comprises PNU-159682.
  • the PNU-159682 payload is conjugated to the protein with a succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker, as described in patent US8389697B2.
  • SMCC succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate
  • DS-8201 comprising a Dxd payload
  • DB-1303 DualityBio
  • SKB264 comprising a toposiomeriase I inhibitor payload
  • the conjugated moiety comprises a radioactive isotope or compound selected from the group consisting of 225 Ac, 211 At, 212 Bi, 14 C, 62 Cu, 64 Cu, 67 Cu, 18 F, 66 Ga, 67 Ga, 68 Ga, 123 I, 125 I, 131 I, 111 In, 177 Lu, 15 O, 212 Pb, 186 Re, 188 Re, 44 Sc, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 90 Y or 89 Zr.
  • a radioactive isotope or compound selected from the group consisting of 225 Ac, 211 At, 212 Bi, 14 C, 62 Cu, 64 Cu, 67 Cu, 18 F, 66 Ga, 67 Ga, 68 Ga, 123 I, 125 I, 131 I, 111 In, 177 Lu, 15 O, 212 Pb, 186 Re, 188 Re, 44 Sc, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 90 Y
  • the conjugated moiety comprises a chelator selected from the group consisting of 1, 4, 7, 10-Tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA) , DOTATATE , DOTA-Bn, DO2A, Diethylenetriamene pentaacetate (DTPA) , 6-amino-1, 4-diazepine-triacetate (DATA) , PCTA, 1, 4, 7-triazacyclononane-N, N', N” -triacetic acid (NOTA) , NOTP, TRAP, 1, 4, 7-Triazacyclononane (TACN) , 6-amino-6-methylperhydro-1, 4-diazepinetetraacetic acid (AAZTA) , H4pypa, H2dedpa, H2CHXhox, H2hox, HBED, and DFO, as described in the reference of chelators (Sneddon et al) ,
  • the chelator preferentially comprises DOTA, DOTATATE, or DOTA-Bn.
  • the foregoing chelator optionally chelates with 177 Lu.
  • anti-CD47 antibody (non-pH sensitive) conjugated with 68 Ga, 89 Zr or 177 Lu through NOTA, DFO or DOTA have all been successfully generated using the technologies known in the arts and reported as effective theranostics for tumor imaging and therapy (see (Zhang et al., Research (Wash D C) , 2023) ) .
  • the conjugated moiety comprises a calreticulin-inducing agent selected from the group consisting of anthracyclin such as doxorubicin, doxorubicin, daunorubicin, epirubicin, idarubicin and mitoxantrone, and a PP1/GADD34 inhibitor such as tautomycin, calyculin A and salubrinal, or fullerenols, that induce translocation of calrecticulin to the cell surface, as described in the reference such as (Obeid et al., Immunol Rev, 2007, Obeid et al., Nat Med, 2007, Kui Chen, Nano Today, 2021) .
  • anthracyclin such as doxorubicin, doxorubicin, daunorubicin, epirubicin, idarubicin and mitoxantrone
  • a PP1/GADD34 inhibitor such as tautomycin, calyculin A and salubrinal,
  • the calreticulin-inducing agent synergizes with CD47-blockade to enhance phagocytosis of the CD47-expressing target cells, as described in the reference such as (Chao et al., Sci Transl Med, 2010, Feng et al., Nat Commun, 2018, Obeid et al., Nat Med, 2007) .
  • the conjugated moiety comprises an agonist to a pattern recognition receptor (PRR) for pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) .
  • PRR pattern recognition receptor
  • the foregoing pattern recognition receptors include but are not limited to, Toll-like receptors (TLRs) , STimulator of INterferon Genes (STING) , C-type lectin receptors (CLRs) , Rig-I-like receptors (RLRs) and NOD-like receptors (NLRs) , as described in reference such as (Li et al., Signal Transduct Target Ther, 2021) .
  • the conjugated moiety comprises TLR2 agonists including but not limiting to lipopeptide, peptide and small molecule agonists CBLB612, SV-283, ISA-201, Pam3Cys, Pam3Cys-Ser- (Lys) 4 (Pam3CSK4) , Triacyl lipid A (OM-174) , Lipoteichoic acid (LTA) , peptidoglycan, and CL419 (S- (2, 3-bis (palmitoyloxy) - (2RS) propyl) - (R) -cysteinyl spermine) , TLR2/6 agonists including but not limiting to Pam2CSK4, and TLR2/7 agonists including but not limiting to CL572, CL413, and CL401, that are known in the arts, for example, as described in patents US10675358B2 and US20170158772A1 and the reference (Anwar et al., Med Res Rev, 2019) .
  • the conjugated moiety comprises TLR4 agonists including but not limiting to lipopolysaccharide (LPS) , monophosphoryl lipid A (MPLA) and small molecule agonists GSK1795091, glycopyranosyl lipid (GLA) -SE/GLA-AF/G-305 (Immune Design) , G100, PEPA-10, PET-lipid A (Cascadian Therapeutics) , and MPL (Allergy Therapeutics) , that are known in the arts, for example, as described in patents US10675358B2 and US20170158772A1 and the reference (Anwar et al., Med Res Rev, 2019) .
  • LPS lipopolysaccharide
  • MPLA monophosphoryl lipid A
  • GSK1795091 glycopyranosyl lipid
  • GLA glycopyranosyl lipid
  • GLA-SE/GLA-AF/G-305 Immune Design
  • G100 glycopyranosyl lipid
  • the conjugated moiety comprises TLR5 agonists including but not limiting to flagellin and recombinant protein agonist thereof including mobilan, entolimod, VAX125 and VAX102, that are known in the arts, for example, as described in patents US10675358B2 and US20170158772A1 and the reference (Anwar et al., Med Res Rev, 2019) .
  • the conjugated moiety comprises TLR3 agonists including but not limiting to dsRNA or polyinosine-polycytidylic acid (poly (I: C) ) , poly-ICLC, Polyadenylic-polyuridylic acid (poly (A: U) , and poly (I) -poly (C12U) , that are known in the arts, for example, as described in patents US10675358B2 and US20170158772A1 and the reference (Anwar et al., Med Res Rev, 2019) .
  • TLR3 agonists including but not limiting to dsRNA or polyinosine-polycytidylic acid (poly (I: C) ) , poly-ICLC, Polyadenylic-polyuridylic acid (poly (A: U) , and poly (I) -poly (C12U) , that are known in the arts, for example, as described in patents US1067
  • the conjugated moiety comprises TLR7 agonists including but not limiting to imidazoquinoline type and other small molecule agonists including imiquimod, Gardiquimod, loxoribine, GSK2245035, 852A, GS-9620, RO6864018, RO7020531, CL264, CL307, 852A, BNT411, DSP-0509, LHC165, NJH395, RO7119929 and TQ-A3334, that are known in the arts, for example, as described in patents US10675358B2 and US20170158772A1 and the reference (Anwar et al., Med Res Rev, 2019, Frega et al., Oncoimmunology, 2020) .
  • TLR7 agonists including but not limiting to imidazoquinoline type and other small molecule agonists including imiquimod, Gardiquimod, loxoribine, GSK2245035, 852A, GS-9620, RO6864018, RO7020531, CL264,
  • the conjugated moiety comprises TLR8 agonists including but not limiting to small molecule agonists motolimod, IRM1, IRM2, IRM3, TL8-506 and the TLR8 agonist of SBT6050, that are known in the arts, for example, as described in patents US10675358B2 and US20170158772A1 and the reference (Anwar et al., Med Res Rev, 2019, Frega et al., Oncoimmunology, 2020) .
  • TLR8 agonists including but not limiting to small molecule agonists motolimod, IRM1, IRM2, IRM3, TL8-506 and the TLR8 agonist of SBT6050, that are known in the arts, for example, as described in patents US10675358B2 and US20170158772A1 and the reference (Anwar et al., Med Res Rev, 2019, Frega et al., Oncoimmunology, 2020) .
  • the conjugated moiety comprises TLR7/8 agonists including but not limiting to small molecule agonists resiquimod, MEDI9197, T785, BDB001, BDB018, BDB030, CV8102, NKTR-262, CL097, CL075 and the TLR7/8 agonist moiety of BDC-1001, that are known in the arts, for example, as described in patents US10675358B2 and US20170158772A1 and the reference (Anwar et al., Med Res Rev, 2019, Frega et al., Oncoimmunology, 2020) .
  • the TLR7/8 agonist comprises BDB001, BDB018, or the TLR7/8 agonist moiety of BDC-1001.
  • the conjugated moiety comprises TLR9 agonists including but not limiting to single strand CpG oligodeoxynucleotides (CpG ODN) and oligonucleotide-based agonists MGN1703, SD-101, CYT003, DUK-CpG-001, CpG-7909, GNKG168, EMD1202081, IMO-2125, CpG10104, and AZD1419, as well as the TLR9 agonist moiety of the antibody-drug conjugate TAC-001 and ALTA-002, that are known in the arts, for example, as described in patents US10675358B2, US20170158772A1, US20220056069A1, WO2018189382A1 and WO2020081744A1 and the reference (Anwar et al., Med Res Rev, 2019) .
  • TLR9 agonists including but not limiting to single strand CpG oligodeoxynucleotides (CpG O
  • the conjugated moiety comprises cytosolic DNA and cyclic dinucleotides (CDN) STING agonists including but not limiting to ADU-S100, Cyclic [G (2', 5') pA (2', 5') p] (2'2'-cGAMP) , cyclic [G (2', 5') pA (3', 5') p] (2'3'-cGAMP) , cyclic [G (3', 5') pA (3', 5') p] (3'3'-cGAMP) , Cyclic di-adenylate monophosphate (c-di-AMP) , 2', 5'-3', 5'-c-diAMP (2'3 '-c-di-AMP) , Cyclic di-guanylate monophosphate (c-di-GMP) , 2', 5'-3', 5'-c-diGMP (2'3 '-c-di-GMP)
  • CDN
  • the conjugated moiety comprises non-CDN small molecule STING agonists including but not limiting to ALG-031048, E7766, JNJ-6196, MK-2118, MSA-1, MSA-2, SNX281, SR-717, TAK676 and TTI-10001, and the STING agonist moiety of the antibody-drug conjugate XMT-2056 and CRD-5500, that are known in the arts, for example, as described in patents US10675358B2, US20170158772A1, WO2021026009A1 and WO2021202984A1 and the reference (Amouzegar et al., Cancers (Basel) , 2021, Yan et al., Vaccines (Basel) , 2021) .
  • STING agonists including but not limiting to ALG-031048, E7766, JNJ-6196, MK-2118, MSA-1, MSA-2, SNX281, SR-717, TAK676 and T
  • the antigen binding Fab domain of the antibody drug conjugate BDC-1001 comprising a TLR7/8 agonist moiety can be replaced by pH-sensitive CD47-binding Fab domain of the present disclosure to generate a protein drug conjugate targeting CD47.
  • the conjugated moiety comprises NLR agonists including but not limiting to acylated derivative of iE-DAP, D-gamma-Glu-mDAP, L-Ala-gamma-D-Glu-mDAP, Muramyldipeptide with a CI8 fatty acid chain, Muramyldipeptide, muramyl tripeptide, and N-glycolylated muramyldipeptide, that are known in the arts, for example, as described in patent US10675358B2.
  • NLR agonists including but not limiting to acylated derivative of iE-DAP, D-gamma-Glu-mDAP, L-Ala-gamma-D-Glu-mDAP, Muramyldipeptide with a CI8 fatty acid chain, Muramyldipeptide, muramyl tripeptide, and N-glycolylated muramyldipeptide, that are known in the arts, for example, as
  • the conjugated moiety comprises RIG-I agonists including but not limiting to 5'ppp-dsRNA (5'-pppGCAUGCGACCUCUGUUUGA -3': 3'-CGUACGCUGGAGACAAACU -5') , 3p-hpRNA, Poly (deoxyadenylic-deoxythymidylic) acid (Poly (dA: dT) ) , Poly (I: C) , MK-4621 (RGT100) , SLR14, SLR20, KIN700, KIN1148, KIN600, KIN500, KIN100, KIN101, KIN400, KIN2000, and SB-9200, that are known in the arts, for example, as described in patents US10675358B2, US20170158772A1 and US8871799B2 and the reference (Iurescia et al., Cancers (Basel) , 2020) .
  • RIG-I agonists including but not limiting to 5'ppp-
  • the conjugated moiety comprises photosensitive agents including but not limiting to silicon phthalocyanine dye such as IRDye700DX, that are known in the arts, for example, as described in patent US8524239B2 and the reference (Maczynska et al., Cell Death Dis, 2020) .
  • the conjugated moiety comprises a protein toxin, or an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof.
  • the toxins include but are not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa ) , Shiga-like toxin A subunit, ricin A chain, abrin A chain, modeccin A chain, alpha-saicm, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S) , momordica charantiain ibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, the tricothecenes, Clos
  • the conjugated moiety comprises an enzymes/pro-drug converting enzyme including but not limiting to alkaline phosphatases, arylsulfatases, cytosine deaminase, proteases such as serratia protease, thermolysis, subtilisin, carboxypeptidases and cathepsins, D-alanylcarboxypeptidases, glycosyltransferase, carbohydrate-cleaving enzymes such as ⁇ -galactosidase, neuraminidase and sialidase, ⁇ -lactamase, ⁇ -glucosidase, ⁇ -glucuronidase, penicillin amidases such as penicillin V amidase and penicillin G amidase, nitroreductase and carboxypeptidase A, that are known in the arts, for example, as described in patent US20170002074A1 and the reference (Sharma et al., Expert Opin Biol Ther, 2017)
  • the conjugated moiety is covalently conjugated to cystein, lysine, carbohydrate glyco-group or other chemically active group of the protein or Fc through techniques known in the arts, for example, as described in patents such as US7745394, US5208020, US20070092940, EP1968635B1, WO2014140317, EP1370298B1, WO2014202775, US10947317B2, US8685383B2, US20120183566, US9669107B2 , US10745488B2, WO2021067776A2, WO2019108733A2, US10155821B2, US9808537B2, WO2021257525A1, WO2017180842A1, WO2018009916A1, WO2020190725A1, WO2021202984A1, US7259249B2 , WO2018189382A1, WO2020081744A1, WO2021174091A1, US10487149B2, US8524239B2,
  • the present disclosure provides a nucleic acid or nucleic acids comprising a sequence encoding a pH-sensitive CD47-binding domain of the present disclosure, an antibody comprising a pH-sensitive CD47-binding domain of the present disclosure, an antibody fusion protein comprising a pH-sensitive CD47-binding domain of the present disclosure, or a protein comprising a pH-sensitive CD47-binding domain of the present disclosure.
  • the nucleic acid is a DNA or RNA.
  • a vector or vectors (e.g. cloning vector, and expression vector) comprise the foregoing nucleic acid or nucleic acids.
  • the vector comprises a plasmid and/or a viral vector.
  • a host cell comprises one or more of the foregoing vectors.
  • a process for production of a polypeptide or protein of the present disclosure comprising culturing the host cell with the foregoing vector or vectors and isolating the polypeptide or protein.
  • a pharmaceutical composition comprises a pH-sensitive CD47-binding domain of the present disclosure, an antibody comprising a pH-sensitive CD47-binding domain of the present disclosure, an antibody fusion protein comprising a pH-sensitive CD47-binding domain of the present disclosure, a protein comprising a pH-sensitive CD47-binding domain of the present disclosure, and/or a protein drug conjugate of the present disclosure, and a pharmaceutically acceptable carrier.
  • a method of treating a CD47-expressing disease in a mammal comprises administering an effective amount of a pH-sensitive CD47-binding domain of the present disclosure, an antibody comprising a pH-sensitive CD47-binding domain of the present disclosure, an antibody fusion protein comprising a pH-sensitive CD47-binding domain of the present disclosure, a protein comprising a pH-sensitive CD47-binding domain of the present disclosure, and/or a protein drug conjugate of the present disclosure to a mammal in need thereof.
  • the disease is a disease of cancer, fibrosis, atherosclerosis, inflammation or senescence.
  • the disease is a disease of cancer, wherein the cancer is ovarian cancer, endometrial cancer, uterine cancer, cervical cancer, breast cancer, gastric cancer, esophageal cancer, lung cancer, head and neck cancer, colorectal cancer, liver cancer, bone cancer, sarcoma, osteosarcoma, brain cancer, multiple myeloma, acute myeloid leukemia (AML) , myelodysplastic syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, basal cell skin cancer, chondrosarcoma, Ewing’s sarcoma, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumor (GIST) , glioma, glioblastoma, hepatocellular cancer, kaposi sarcoma, kidney cancer, laryn
  • the disease is a fibrotic disease, wherein the fibrotic disease comprises comprising fibrosis of lung, liver, heart, kidney, skin, eye, muscle and/or connective tissues, such as idiopathic pulmonary fibrosis, liver fibrosis in nonalcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) , scleroderma and Systemic Sclerosis.
  • the mammal is a human.
  • kits for diagnosis or treatment said kit comprises a pH-sensitive CD47-binding domain of the present disclosure, an antibody comprising a pH-sensitive CD47-binding domain of the present disclosure, an antibody fusion protein comprising a pH-sensitive CD47-binding domain of the present disclosure, or a protein comprising a pH-sensitive CD47-binding domain of the present disclosure, and/or a protein drug conjugate of the present disclosure, or the pharmaceutical composition thereof, and instruction for using it for diagnosis or treatment.
  • Synthetic receptor for cell therapy comprising a pH-sensitive CD47-binding domain
  • the present disclosure provides a synthetic receptor for engineered cell therapy comprising a pH-sensitive CD47-binding domain of the present disclosure, a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure, or other polypeptide comprising a pH-sensitive CD47-binding domain of the present disclosure.
  • the synthetic receptor comprises a chimeric antigen receptor (CAR) , a synthetic T cell receptor (TCR) , or a T cell-antigen coupler (TAC) , wherein the synthetic receptor comprises an antigen binding domain comprising a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure or other polypeptide comprising a pH-sensitive CD47-binding domain of the present disclosure, a transmembrane domain and an intracellular signaling domain.
  • CAR chimeric antigen receptor
  • TCR synthetic T cell receptor
  • TAC T cell-antigen coupler
  • T cells including ⁇ T cells, ⁇ T cells, double negative T cells, Treg) , NK cells, NKT cells, cytokine-induced killer cells (CIK) , mucosal-associated invariant T cells (MAIT) ) , monocytes, macrophages, dendritic cells, B cells, mesenchymal stem cells (MSC) and induced pluripotent stem cells (iPSC) expressing synthetic chimeric antigen receptor (CAR) , namely CAR- ⁇ T, CAR- ⁇ T, CAR-DNT, CAR-Treg, CAR-NK, CAR-NKT, CAR-CIK, CAR-MAIT, CAR-macrophage, CAR-DC, CAR-B, CAR-MSC or CAR-iPSC cells respectively, are well described in the arts such as in the patents WO2005044996, US7446190B2, US20130287748A1, WO2012079000A1, WO
  • T cells expressing a synthetic T cell receptor such as an exogenous T cell receptor (e.g. TCR-T cells as described in (Zhao et al., Front Immunol, 2021) ) , a T cell receptor fusion protein (e.g. TRuC-T as described in the patent WO2016187349A1 and journal article (Baeuerle et al., Nat Commun, 2019) ) , an antibody-T cell recptor chimeric molecule (e.g.
  • a synthetic T cell receptor such as an exogenous T cell receptor (e.g. TCR-T cells as described in (Zhao et al., Front Immunol, 2021) )
  • a T cell receptor fusion protein e.g. TRuC-T as described in the patent WO2016187349A1 and journal article (Baeuerle et al., Nat Commun, 2019)
  • an antibody-T cell recptor chimeric molecule e.g
  • a chimeric antigen receptor comprises an antigen binding domain comprising a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure, or other polypeptide comprising a pH-sensitive CD47-binding domain of the present disclosure.
  • the chimeric antigen receptor (CAR) additionally comprises a transmembrane domain, and an intracellular signaling domain, in addition to the antigen binding domain comprising a CD47-binding domain.
  • the chimeric antigen receptor (CAR) optionally comprises an extracellular spacer domain and/or at least one co-stimulatory domain.
  • the transmembrane domain of the CAR is derived from transmembrane domain of CD28, CD137, CD4, CD8 and/or CD3 ⁇ .
  • the intracellular signaling domain of the CAR is derived from the intracellular signaling domain of CD3 ⁇ .
  • the intracellular signaling domain is derived from the intracellular signaling domain of Fc ⁇ receptors, Megf10, MerTK, Dectin-1, and/or CD147, wherein the CAR comprising the intracellular signaling domain is preferentially expressed in a monocyte, a macrophage, a dendritic cell, or a B cell (see (Sloas et al., Front Immunol, 2021, Wang et al., EBioMedicine, 2022) ) .
  • the co-stimulatory domain of the CAR is derived from the group of proteins comprising CD28, CD137, OX40, CD27, ICOS, GITR, CD40, MyD88, CD86, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, MerTK, Dectin-1, DAP12, DAP10, 2B4, and combination thereof.
  • the co-stimulatory domain of the CAR is derived from CD28 or CD137.
  • the co-stimulatory domain of the CAR is derived from CD40, MyD88, CD86, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, MerTK and/or Dectin-1, wherein the CAR comprising the costimulatory domain is preferentially expressed in a monocyte, a macrophage, a dendritic cell, or a B cell (see (Sloas et al., Front Immunol, 2021, Wang et al., EBioMedicine, 2022) ) .
  • the extracellular spacer domain of the CAR comprises spacer domain selected from the group exemplarily comprising an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an extracellular spacer region of CD8, an extracellular spacer region of CD4, an extracellular spacer region of CD28, an extracellular spacer region of 4-1BB, an artificial spacer sequence and combinations thereof.
  • Spacer domain selected from the group exemplarily comprising an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an extracellular spacer region of CD8, an extracellular spacer region of CD4, an extracellular spacer region of CD28, an extracellular spacer region of 4-1BB, an artificial spacer sequence and combinations thereof.
  • Options of additional transmembrane domain, intracellular signaling domain, co-stimulatory domain and/or spacer domain are described in the arts such as patents WO2005044996, US7446190B2,
  • CAR constructs are well described in the arts such as in the patents WO2005044996, US7446190B2, US20130287748A1, WO2012079000A1, and WO2012129514, WO2013123061A1, WO2021151119A1, US10869888B2, US20210077532A1, US10918667B2, US20210161961A1, WO2019140100A1, US11198729B2, WO2016081518A2 , WO2020072536A1, WO2021108926A1, WO2019178518A1, WO2019213610A1, US20210015859A1, WO2019126748A1, WO2013040371A3 , WO2016049459A1, WO2016071513A1, WO2020127513A1, US11306134B2, WO2021248061A1, US20210252053A1, WO2022051556A1, US20160237407A1, US11246890B2.
  • CD47-targeting CAR-T comprising a non-pH sensitive CD47-binding scFv domain from humanized B6H12 antibody have been generated and reported effective anti-tumor activity in preclinical studies (see (Golubovskaya et al., Cancers (Basel) , 2017, La et al., J Immunol Res, 2021) ) .
  • bispecific CAR-T cells targeting both CD47 and TAG-72 comprising a non-pH sensitive CD47-binding scFv domain from the B6H12 antibody or Hu5F9 antibody, has also been generated and reported as effective anti-tumor activity in preclinical studies (see (Shu et al., Mol Ther Oncolytics, 2021) ) .
  • defective expansion of such non-pH sensitive CD47-targeting CAR-T comprising a fully functional intracellular signaling domain was encountered due to fratricide among the CD47-expressing CAR-T cells (see (Shu et al., Mol Ther Oncolytics, 2021) ) .
  • the antigen-binding domain of an existing CAR known in the arts can be replaced with a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure to generate a new CAR binding to CD47.
  • the antigen-binding domain of an existing CAR known in the arts can also be linked at its N-terminal or C-terminal directly or through a linker to a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure to add binding to CD47 as a bispecific or multispecific CAR.
  • the CAR is a dimeric CAR comprising two separate chimeric antigen receptor polypeptides that homodimerize on the membrane of a cell.
  • dimeric CAR examples include but not limited to, PLoS One, 2016, Fujiwara et al., Cells, 2020, Jayaraman et al., EBioMedicine, 2020) .
  • a modified cell expressing a CAR comprising a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure comprises an ⁇ T cell, a ⁇ T cell, a double negative T cell, a Treg cell, a NK cell, a NKT cell, a cytokine-induced killer cell (CIK) , a mucosal-associated invariant T cell (MAIT) , a monocyte, a macrophage, a dendritic cell, a B cell, a mesenchymal stem cell (MSC) or an induced pluripotent stem cell (iPSC) .
  • the modified cell is a T cell or NK cell.
  • the modified cell is a macrophage.
  • CAR-macrophage is well described in the arts such as in the patents US11306134B2 and WO2021248061A1. Blockade of CD47 is shown to enhance phagocytosis of CAR-macrophages targeting other non-CD47 antigen (see eLife 7: e36688) .
  • a CAR-macrophage comprising a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure can synergistically both block CD47 and self-activate to enhance phagocytosis of CD47-expressing target cells.
  • the chimeric antigen receptor (CAR) comprising a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure, comprises an amino acid sequence selected from the exemplary group consisting of: SEQ ID NO: 407 to 412.
  • a T cell receptor fusion protein comprises an antigen binding domain comprising a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure, or other polypeptide comprising a pH-sensitive CD47-binding domain of the present disclosure.
  • the TCR fusion protein comprises a TCR subunit comprising at least a portion of a TCR extracellular domain sequence, a TCR transmembrane domain and a TCR intracellular domain of a TCR subunit, wherein the pH-sensitive CD47-binding scFv or VHH domain is linked directly or through a linker to the N-terminal of the TCR subunit and wherein the TCR fusion protein incorporates into a TCR when expressed in a T cell.
  • the TCR subunit is selected from the group consisting of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ and TCR ⁇ .
  • the TCR subunit is preferentially CD3 ⁇ , wherein the T cell receptor complex comprises two CD3 ⁇ units.
  • T cell receptor fusion protein comprising a full length TCR subunit fused with scFv-based antigen binding domain is known in the arts, e.g. as described in the patent WO2016187349A1 and journal article (Baeuerle et al., Nat Commun, 2019) .
  • the antigen binding domain of an existing said T cell receptor fusion protein known in the arts can be replaced with a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure to generate a new T cell receptor fusion protein binding to CD47.
  • the antigen-binding domain of an existing said T cell receptor fusion protein known in the arts can also be linked at its N-terminal or C-terminal directly or through a linker to a pH-sensitive CD47-binding scFv or VHH domain to add binding to CD47 as a bispecific or multispecific T cell receptor fusion protein.
  • a modified T cell expressing a T cell receptor fusion protein comprising a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure comprises an ⁇ T cell, a ⁇ T cell, a double negative T cell, a Treg cell, a NKT cell, a cytokine-induced killer cell (CIK) , a mucosal-associated invariant T cell (MAIT) , or an induced pluripotent stem cell (iPSC) .
  • CIK cytokine-induced killer cell
  • MAIT mucosal-associated invariant T cell
  • iPSC induced pluripotent stem cell
  • the T cell receptor fusion protein comprising a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure, comprises an amino acid sequence selected from the exemplary group consisting of: SEQ ID NO: 413 to 416.
  • T cell receptor fusion protein comprising partial TCR chain wherein the variable domain of the TCR ⁇ and TCR ⁇ chain, or the TCR ⁇ and TCR ⁇ chain, is replaced by antibody-derived VH or VL or scFv domain
  • the antigen binding domain of an existing said T cell receptor fusion protein known in the arts can be replaced with a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure to generate a new T cell receptor fusion protein binding to CD47.
  • the antigen-binding domain of an existing said T cell receptor fusion protein known in the arts can also be linked at its N-terminal or C-terminal directly or through a linker to a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure to add binding to CD47 as a bispecific or multispecific T cell receptor fusion protein.
  • a modified T cell expressing a T cell receptor fusion protein comprising a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure comprises an ⁇ T cell, a ⁇ T cell, a double negative T cell, a Treg cell, a NKT cell, a cytokine-induced killer cell (CIK) , a mucosal-associated invariant T cell (MAIT) , or an induced pluripotent stem cell (iPSC) .
  • CIK cytokine-induced killer cell
  • MAIT mucosal-associated invariant T cell
  • iPSC induced pluripotent stem cell
  • the synthetic receptor comprising a pair of TCR ⁇ and TCR ⁇ fusion protein comprising a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure, comprises an amino acid sequence selected from the exemplary group consisting of: SEQ ID NO: 417 x 418, and 419 x 420, wherein “x” symbol indicates a pair of TCR ⁇ and TCR ⁇ fusion protein sequence.
  • a T cell antigen coupler comprises an antigen binding domain comprising a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure, or other polypeptide comprising a pH-sensitive CD47-binding domain of the present disclosure.
  • the T cell antigen coupler additionally comprises a second domain binding to a protein associated with the T cell receptor complex and a third domain comprising a T cell receptor signaling domain.
  • the second domain of the TAC binds to a CD3 subunit associated with a TCR complex on a T cell.
  • the second domain of the TAC comprises a scFv derived from the anti-CD3 antibody UCHT1 or a variant thereof. In another embodiment, the second domain comprises a scFv derived from the anti-CD3 antibody OKT3 or a variant thereof. In another embodiment, the second domain of the TAC comprises a scFv derived from the anti-CD3 antibody SP34 or a variant thereof.
  • the T cell signaling domain of the third domain of the TAC comprises an intracellular cytosolic domain and a transmembrane domain. In one embodiment, the cytosolic domain is a CD4 cytosolic domain and the transmembrane domain is a CD4 transmembrane domain.
  • the T cell signaling domain optionally further comprises a co-stimulatory domain.
  • the co-stimulatory domain is derived from the group of proteins comprising CD28, CD137, OX40, CD27, ICOS and GITR, or combinations thereof.
  • T cell antigen coupler construct linking an antigen-binding domain to a TCR binding domain and a T cell signaling domain is known in the arts, as described in the patent US10435453B2 and journal article (Helsen et al., Nat Commun, 2018) .
  • the antigen-binding domain of an existing TAC known in the arts can be replaced a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure to generate a new TAC binding to CD47.
  • the antigen-binding domain of an existing TAC known in the arts can also be linked at its N-terminal or C-terminal directly or through a linker to a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure to add binding to CD47 as a bispecific or multispecific TAC.
  • a modified cell expressing a TAC comprising a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure, comprises an ⁇ T cell, a ⁇ T cell, a double negative T cell, a Treg cell, a NKT cell, a cytokine-induced killer cell (CIK) , a mucosal-associated invariant T cell (MAIT) , or an induced pluripotent stem cell (iPSC) .
  • CIK cytokine-induced killer cell
  • MAIT mucosal-associated invariant T cell
  • iPSC induced pluripotent stem cell
  • the TAC comprising a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure comprises an amino acid sequence selected from the exemplary group consisting of: SEQ ID NO: 421 to 422.
  • a bispecific CAR, a bispecific T cell receptor fusion protein or a bispecific TAC comprising a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure, and a second antigen binding domain that binds to a non-CD47 antigen, wherein the pH-sensitive CD47-binding scFv or VHH domain is linked through a linker to the N-terminal or C-terminal of the second antigen binding domain.
  • the second antigen binding domain of the bispecific CAR, bispecific T cell receptor fusion protein or bispecific TAC binds to an antigen selected from the group consisting of CD19, CD20, CD22, CD37, BCMA, GPRC5D, CD70, NKG2D ligands, ROR1, MSLN, claudin 18.2, claudin 6, GPC3, HER2, GUCY2C, PAP, TSHR, ALPP, GPC3, EGFR-VIII, GD2, DLL3, IL13Ra2, PSMA, PSCA, MUC1, MUC16, FcRa, CD44v6, Necint-4, CAIX, CEA, B7-H3, HPV16-E6, HPV16-E7, AFP, NY-ESO-1, MAGEA4, MAGEA3, MAGEA8, PRAME, COL6A3 and WT1.
  • an antigen selected from the group consisting of CD19, CD20, CD22, CD37, BCMA, GPRC5D,
  • the second antigen binding domain of the bispecific CAR, bispecific T cell receptor fusion protein or bispecific TAC binds to CD19 or BCMA.
  • bispecific CAR are well described in the arts such as in the patents WO2013123061A1 and US20210077532A1 and the articles (Zah et al., Cancer Immunol Res, 2016, Schneider et al., J Immunother Cancer, 2017) .
  • a modified cell comprises a CAR, a T cell receptor fusion protein or a TAC comprising a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure, wherein the same cell also comprises a second CAR, a second T cell receptor fusion protein or a second TAC targeting against a non-CD47 antigen.
  • a modified cell comprise a CAR, a T cell receptor fusion protein or a TAC comprising a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure, wherein the same cell also comprises a second CAR, a second T cell receptor fusion protein or a second TAC targeting against CD19 or BCMA. Examples of such compound CAR-T cells are well described in the arts such as in the patent WO2019140100A1 and the articles (Petrov et al., Leukemia, 2018, Yan et al., Stem Cell Rev Rep, 2020) .
  • a population of modified cells comprise a CAR, a T cell receptor fusion protein or a TAC comprising a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure, wherein the same population of cells and/or a second population of cells comprises a second CAR, a second T cell receptor fusion protein or a second TAC targeting against a non-CD47 antigen.
  • a population of cells comprise a CAR, a T cell receptor fusion protein or a TAC comprising a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure, wherein the same population of cells and/or a second population of cells comprises a second CAR, a second T cell receptor fusion protein or a second TAC targeting against CD19 or BCMA.
  • Examples of such coupled CAR-T cells are well described in the arts such as in the patents US10869888B2, US20220265708A1, US20220096546A1 and WO2020146743A1.
  • a modified cell comprises a polynucleotide encoding a CAR, a T cell receptor fusion protein or a TAC comprising a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure, wherein the cell comprises a second polynucleotide encoding a therapeutic agent, wherein the modified cell expresses and secretes the therapeutic agent in response to activation of the modified cell.
  • a modified cell comprises a polynucleotide encoding a CAR, a T cell receptor fusion protein or a TAC comprising a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure, and a polynucleotide encoding a second CAR, a second T cell receptor fusion protein or a second TAC that targets against a non-CD47 antigen, wherein the cell comprises another polynucleotide encoding a therapeutic agent, wherein the modified cell expresses and secretes the therapeutic agent in response to activation of the modified cell.
  • a population of modified cells comprise a polynucleotide encoding a CAR, a T cell receptor fusion protein or a TAC comprising a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure, wherein the same population of modified cells and/or a different population of modified cells comprise a second polynucleotide encoding a therapeutic agent, wherein the modified cells express and secrete the therapeutic agent in response to activation of the modified cells.
  • a population of modified cells comprise a polynucleotide encoding a CAR, a T cell receptor fusion protein or a TAC comprising a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure, wherein the same population of modified cells and/or a different population of modified cells comprise a second polynucleotide encoding a second CAR, a second T cell receptor fusion protein or a second TAC that targets against a non-CD47 antigen, and/or a third polynucleotide encoding a therapeutic agent, wherein the modified cells express and secrete the therapeutic agent in response to activation of the modified cells.
  • the non-CD47 antigen targeted by the second CAR, T cell receptor fusion protein or TAC is CD19 or BCMA.
  • the polynucleotide encoding the therapeutic agent comprises a NFAT binding sequence in the promoter sequence regulating the expression of the therapeutic agent.
  • the NFAT promoter sequence of the polynucleotide encoding the therapeutic agent comprises a nucleotide sequence of SEQ ID NO: 424.
  • the therapeutic agent encoded by the polynucleotide comprises a cytokine.
  • the cytokine therapeutic agent comprises one or more cytokine selected from the group consisting of IL-6, IFN- ⁇ , IL-12, IL-7, IL-15, and TNF ⁇ .
  • the cytokine therapeutic agent comprises IL-6 and/or IFN- ⁇ .
  • the polynucleotide encoding therapeutic agents IL-6 and/or IFN- ⁇ is not limited to, but not limited to, IL-6, IFN- ⁇ , IL-12, IL-7, IL-15, and TNF ⁇ .
  • the cytokine therapeutic agent comprises IL-12, comprising an amino acid sequence of SEQ ID NO: 425.
  • the therapeutic cytokine agent comprises IL-6, IFN- ⁇ and/or IL-12.
  • a composition comprises a first population of modified T cells comprising a CAR comprising a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure, and comprises a second population of modified T cells comprising a CAR against CD19 (SEQ ID NO: 423) and a polynucleotide comprising SEQ ID NO: 427 encoding IL-6 and IFN- ⁇ (encoding a polypeptide of IL-6-P2A-IFN- ⁇ with amino acid sequence of SEQ ID NO: 426) driven by a NFAT promoter (SEQ ID NO: 424) , wherein the second population of modified T cells express and secrete IL-6 and IFN- ⁇ in response to activation of their CAR by CD19 antigen.
  • a composition comprises a first population of modified T cells comprising a CAR comprising a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure, and comprises a second population of modified T cells comprising a CAR against CD19 and a polynucleotide encoding IL-12 (with amino acid sequence of SEQ ID NO: 425) driven by a NFAT promoter (SEQ ID NO: 424) , wherein the second population of modified T cells express and secrete IL-12 in response to activation of their CAR by CD19 antigen.
  • a composition comprises a first population of modified T cells comprising a CAR comprising a pH-sensitive CD47-binding scFv or VHH domain of the present disclosure, and comprises a second population of modified T cells comprising a CAR against CD19 and a polynucleotide encoding IL-6, IFN- ⁇ and/or IL-12 driven by a NFAT promoter, wherein the second population of modified T cells express and secrete IL-6, IFN- ⁇ and/or IL-12 in response to activation of their CAR by CD19 antigen.
  • composition of coupled CAR-T cells expressing and secreting cytokines in response to activation of the CAR-T cells are known in the arts, such as described in the patents in the patents US10918667B2, US20210161961A1, US10869888B2, US20220265708A1, WO2020146743A1 and US20220096546A1.
  • Nucleic acid Nucleic acid, vector, host cell for synthetic receptors, and methods of use
  • the present disclosure provides a nucleic acid or nucleic acids comprising a sequence encoding a CAR, a TCR fusion protein, a TAC or other synthetic receptor of the present disclosure.
  • the nucleic acid is a DNA or a RNA.
  • an expression vector comprises a nucleic acid or nucleic acids comprising a sequence encoding a CAR, a TCR fusion protein, a TAC or other synthetic receptor of the present disclosure.
  • the expression vector is selected from the group consisting of lentivirus vectors, gamma retrovirus vectors, foamy virus vectors, adeno associated virus vectors, adenovirus vectors, pox virus vectors, herpes virus vectors, engineered hybrid viruses, and transposon mediated vectors.
  • a cell comprises a CAR, a TCR fusion protein, a TAC or other synthetic receptor of the present disclosure, and/or a nucleic acid or nucleic acids comprising a sequence encoding a CAR, a TCR fusion protein, a TAC or other synthetic receptor of the present disclosure.
  • the cell additionally comprises a nucleic acid sequence and/or a vector encoding a cytokine.
  • the cell comprises a T cell, NK cell, NKT cell, cytokine-induced killer (CIK) cell, mucosal-associated invariant T (MAIT) cell, monocyte, macrophage, dendritic cell, B cell, granulocyte, neutrophil, innate lymphoid cell (ILC) , mesenchymal stem cell (MSC) or induced pluripotent stem cell (iPSC) .
  • the cell comprises a T cell, NK cell, NKT cell, CIK cell, macrophage, B cell, or iPSC.
  • the T cell comprises ⁇ T cell, ⁇ T cell, double negative T cell and/or Treg cell.
  • the cell is an autologous or allogeneic cell.
  • a pharmaceutical composition comprises a nucleic acid, a vector and/or a cell comprising a nucleic acid sequence encoding a CAR, a TCR fusion protein, a TAC or other synthetic receptor of the present disclosure, and a pharmaceutically acceptable carrier.
  • a method of treating a CD47-expressing disease in a mammal comprises administering an effective amount of a nucleic acid, a vector and/or a cell comprising a nucleic acid sequence encoding a CAR, a TCR fusion protein, a TAC or other synthetic receptor of the present disclosure, to a mammal in need thereof.
  • the disease is a disease of cancer, fibrosis, atherosclerosis, inflammation or senescence.
  • the disease is a disease of cancer, wherein the cancer is ovarian cancer, endometrial cancer, uterine cancer, cervical cancer, breast cancer, gastric cancer, esophageal cancer, lung cancer, head and neck cancer, colorectal cancer, liver cancer, bone cancer, sarcoma, osteosarcoma, brain cancer, multiple myeloma, acute myeloid leukemia (AML) , myelodysplastic syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, basal cell skin cancer, chondrosarcoma, Ewing’s sarcoma, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumor (GIST) , glioma, glioblastoma, hepatocellular cancer, kaposi sarcoma, kidney cancer, laryn
  • the present disclosure provides a method of combination therapy in human comprising administering a therapeutically effective amount of a pH-sensitive CD47-binding domain of the present disclosure, an antibody comprising a pH-sensitive CD47-binding domain of the present disclosure, an antibody fusion protein comprising a pH-sensitive CD47-binding domain of the present disclosure, a protein comprising a pH-sensitive CD47-binding domain of the present disclosure, a protein drug conjugate of the present disclosure, and/or a cell therapy comprising a synthetic receptor of the present disclosure, and a therapeutically effective amount of an additional therapy.
  • the additional therapy comprises administration of one or more of chemotherapy, molecular targeted therapy, biologics therapy, immunotherapy and radiotherapy.
  • the additional therapy comprises a chemotherapy, molecular targeted therapy, biologics therapy, immunotherapy and/or radiotherapy that enhance pro-phagocytic signal and/or inhibit anti-phagocytic signal.
  • the additional therapy comprises a chemotherapy, molecular targeted therapy, biologics therapy, immunotherapy and/or radiotherapy that comprise cell adhesion among diseased cells and/or between diseased cells and extracellular matrix.
  • the additional therapy comprises a molecular targeted therapgy targeting CD117.
  • the additional therapy comprises a molecular targeted therapgy targeting CD117 selected from the group consisting of CDX-0158, CDX-0159, briquilimab (JSP191) , eglatoprutug and MGTA-117.
  • a pH-sensitive CD47-binding domain with differential binding to CD47 at acidic pH than at physiological pH is generated or improved through mutating one or more amino acid residues within and/or surrounding the CD47-binding site or paratope of a parent CD47-binding protein domain to preferentially histidine, aspartate or glutamate.
  • the candidate residues for substitution are selected based on insight through analysis of the amino acid sequence, crystal structure and/or interaction of the parent CD47-binding domain and CD47.
  • substitution mutants of the parent CD47-binding domain are then selected for pH-sensitive binding to CD47 through ELISA (enzyme-linked immunosorbent assay) assay against CD47 antigen and/or FACS (fluorescence-activated cell sorting) binding assay against CD47-expressing cells at an acidic pH versus at physiological pH, wherein the acidic pH is lower than pH 7.0 and the physiological pH is between 7.2 and 7.5.
  • the acidic pH and physiological pH for the initial selection are pH 6.0 and 7.3 respectively. Mutants with higher binding affinity at an acidic pH than at physiological pH are selected. Alternatively, mutants with lower binding affinity at an acidic pH than at physiological pH are selected.
  • substitution mutants of the parent CD47-binding domain are measured for affinity constant (KA) or dissociation constant (KD) at an acidic pH and physiological pH (e.g. pH 5, 5.5, 6.0, 6.5, 6.8, 7.0, 7.2, 7.3, 7.5) by various techniques known in the arts such as preferably surface plasmon resonance (e.g., using Biacore T200, Biacore 3000 or Biacore 8K TM surface plasmon resonance (SPR) system, Cytiva, Marlborough, MA. ) , to then select mutants with lower dissociation constant at an acidic pH than at physiological pH. Alternatively, mutants with higher dissociation constant at an acidic pH than at physiological pH are selected.
  • KA affinity constant
  • KD dissociation constant
  • a CD47-binding domain derived from a CD47-binding antibody selected from a group of CD47-binding antibodies known in the arts are used as the parent CD47-binding domain.
  • CD47-binding antibodies known in the arts include B6H12, C47B161, BC31M4, BC31M5, BC27, C47B222, C47B227, magrolimab (Hu5F9) , lemzoparlimab (TJC4) , letaplimab (IBI188) , ligufalimab (AK117) , ZL-1201, SHR-1603, CC-90002, SRF231, AO-176, AO-104, SGN-CD47M, PT240, IMC-002, TQB2928, AMMS4, ZF1, HLX24, CNTO-7108, Vx-1000, Vx1000R, Vx-1004, HuNb1, LQ001, PT240, BCD-106, BAT6004, LB1, LYN-00301, BSI-082, ADG-153, SCT-113, MIL-95/CM-312, kintuzumab, STI-6643, 2D3, MA
  • the amino acid sequence and available crystal structure and/or interaction of the parent CD47-antibody binding domain with CD47 are analyzed to guide rational selection of candidate residues for mutation to generate or improve pH-sensitive binding to CD47.
  • One or more amino acid residues within and/or surrounding the CD47-binding paratope of the parent CD47-binding domain are then mutated to preferentially histidine, aspartate or glutamate.
  • Such mutations of a number of exemplary parent anti-CD47 antibodies are provided as set forth in Table 2.
  • Antibody variants comprising one or more CDR mutations of a parent CD47-binding antibody, as set forth in Table 1 and 2, are then constructed as isolated antibodies comprising a human IgG1 Fc or IgG4 Fc with S228P mutation.
  • DNAs encoding the full heavy chain and/or light chain of the antibody variants are cloned into pcDNA3.4 mammalian expression vector through gene synthesis and molecular cloning as the heavy chain expression vector and/or light chain expression vector respectively.
  • the CD47-binding antibody variants and the parent antibody are then produced through transient co-expression of the heavy chain and light chain vectors at 2: 3 ratio in HEK293 cells and single step purification of Protein A chromatography purification.
  • the purified antibodies are analyzed by SDS-PAGE and SEC-HPLC.
  • the CD47-binding antibody variants generated above are analyzed for binding to CD47-expressing Raji cells by FACS.
  • Raji cells are reported to express the similar copy number of surface CD47 receptors per cell as primary human epithelial cells which are among the highest CD47-expressing human tissue cells tested (see (Puro et al., Mol Cancer Ther, 2020) ) .
  • Raji cells serve as a good surrogate for selecting pH-sensitive CD47-binding antibody variants with favorable low binding to even the highest CD47-expressing human tissues at physiological pH through FACS binding assay.
  • Raji cells are plated at 1E5 cells/well in 96-well plate and cultured in RPMI 1640 medium with 10%FBS.
  • the FACS binding results of the antibody variants at the acidic pH 6.0 and physiological pH 7.3 are compared to each other and also compared to that of the parent antibody to identify positive antibody variants showing de novo or improved pH-sensitive binding to CD47.
  • the antibody variants show higher binding to the CD47-expressing Raji cells at pH 6.0 than at pH 7.3 are identified as de novo pH-sensitive CD47-binding antibody variants.
  • the antibody variants that show one of the following binding profiles are identified as the positive antibody variants with improved pH-sensitive CD47-binding compared to the parent antibody: 1) antibody variants showing favorably reduced CD47-binding than that of the parent antibody at physiological pH, and also favorably increased CD47-binding than that of the parent antibody at acidic pH; 2) antibody variants showing favorably reduced CD47-binding than that of the parent antibody at physiological pH, and meanwhile comparable CD47-binding to that of the parent antibody at acidic pH; 3) antibody variants showing reduced CD47-binding than that of the parent antibody at both acidic pH and physiological pH, but favorably more reduction in binding at physiological pH than at acidic pH; 4) antibody variants showing favorably reduced CD47-binding than that of the parent antibody at physiological pH, although also with similar reduction in CD47-binding than that of the parent antibody at acidic pH; 5) antibody variants showing favorably increased CD47-
  • the antibody variants are also tested for pH-sensitive binding to CD47 through ELISA assay at pH 7.3 and at pH 6.0.
  • ELISA assay at pH 7.3 and at pH 6.0.
  • 96-well ELISA plate is coated with His-tagged CD47 extracellular domain (ECD) protein at 3 ⁇ g/mL in 50 ⁇ l 1x PBS/well at 4°C overnight.
  • ECD extracellular domain
  • PBST 1x PBS + 0.05%Tween 20
  • the plate is blocked with 1x PBS + 1%BSA for 1 hour at room temperature (RT) .
  • serially diluted anti-CD47 antibody variants and the parent antibody in 1x PBS+0.2%BSA at pH 7.3 or 6.0 are added to the coated wells (30 ⁇ l/well) and incubated for 1 hour at RT.
  • diluted anti-hIgG-Fc-HRP in 1x PBS + 0.2%BSA at pH 7.3 or 6.0 is added (30 ⁇ l/well) and incubated for 1 hour at RT.
  • 100 ⁇ l/well tetramethylbenzidine (TMB) substrate solution is added and incubated in dark at room temperature for 20 minutes.
  • pH-sensitive CD47-binding antibody variants of a number of exemplary parent anti-CD47 antibodies, as set forth in Table 2, are provided in following embodiments using the foregoing experimental methods.
  • Embodiment Set 1 is a diagrammatic representation of Embodiment Set 1:
  • de novo pH-sensitive CD47-binding antibody variants are generated using the non-pH sensitive anti-CD47 antibody B6H12 as the parent CD47-binding domain.
  • top VH CDR and VL CDR residues of B6H12 for pH-sensitive histidine substitution are identified, including Y2H of HCDR1 SEQ ID NO: 82, T3H, Y8H and Y10H of HCDR2 SEQ ID NO: 84, A3H of HCDR3 SEQ ID NO: 89, S7H, D8H and Y9H of LCDR1 SEQ ID NO: 91, K1H and Q5H of LCDR2 SEQ ID NO: 95, and G5H, F6H and R8H of LCDR3 SEQ ID NO: 99.
  • Various B6H12 antibody variants comprising one, two, three or more of these histidine substitutions with expected pH-sensitive binding to CD47 are selected and constructed as isolated hIgG1 antibody variants.
  • DNAs encoding the full heavy chain and light chain of the antibody variants and the parent antibody AV-1 with the same wild type CDR sequences as B6H12 are cloned into pcDNA3.4 mammalian expression vector through gene synthesis and molecular cloning as the heavy chain expression vector and light chain expression vector respectively.
  • the antibody variants and the parent antibody are then produced through transient co-expression of the heavy chain and light chain vectors at 2: 3 ratio in HEK293 cells and single step purification of Protein A chromatography purification.
  • the purified antibodies are analyzed by SDS-PAGE and SEC-HPLC.
  • the purified B6H12 antibody variants generated above are then analyzed for binding to CD47-expressing Raji cells by FACS assay at acidic pH 6.0 and physiological pH 7.3 to reveal pH-sensitive binding to CD47.
  • Raji cells are plated at 1E5 cells/well in 96-well plate and cultured in RPMI 1640 medium with 10%FBS.
  • the Raji cells are then analyzed by FACS in 1x PBS+2%FBS at pH 7.3 or 6.0. While the parent antibody (AV-1) does not have pH-sensitive CD47-binding, the FACS assay is expected to reveal de novo pH-sensitive binding to CD47 from a list of antibody variants comprising one, two, three or more of the histidine substitutions, named as AV-2 to AV-163 in Table 4 and 12, with higher binding to CD47-expressing Raji cells at acidic pH 6.0 than at physiological pH 7.3 in several ways compared to the parent antibody AV-1, including: 1) antibody variants such as AV-3, AV-6, AV-7, AV-10, AV-14, AV-97, AV-98, AV-99, AV-100 and AV-101 showing reduced binding to Raji cells at both physiological pH 7.3 and acidic pH 6.0 compared to the parent antibody AV-1, but favorably more reduction in binding at physiological pH 7.3 than at acidic pH 6.0, with thus higher binding to CD47 at
  • the B6H12 antibody variants are further analyzed for CD47-binding at pH 7.3 and at pH 6.0 by ELISA assay using recombinant human CD47 protein as immobilized ligand and the B6H12 antibody variants as detecting analyte.
  • 96-well ELISA plate is coated with His-tagged CD47 extracellular domain (ECD) protein at 3 ⁇ g/mL in 50 ⁇ l 1x PBS/well at 4°C overnight. After washing with 1x PBS + 0.05%Tween 20 (PBST) , the plate is blocked with 1x PBS + 1%BSA for 1 hour at room temperature (RT) .
  • ECD extracellular domain
  • TMB tetramethylbenzidine
  • H2SO4 50 ⁇ l/well stop solution
  • Embodiment Set 2 is a diagrammatic representation of Embodiment Set 2:
  • antibody variants with improved pH-sensitive binding to CD47 with higher binding at acidic pH than at physiological pH are generated using the pH-sensitive antibody BC31M4 (see (Li et al., J Hematol Oncol, 2023) ) as the parent CD47-binding domain.
  • top VH CDR and VL CDR residues of BC31M4 for improving pH-sensitive binding are identified for histidine substitution, including T3H, S6H and Y7H of HCDR1 SEQ ID NO: 19; R2H, A4H and V5H of HCDR3 SEQ ID NO: 21; R1H, S7H, R8H and N9H of LCDR1 SEQ ID NO: 22; W2H and T5H of LCDR2 SEQ ID NO: 23, and Q5H, N6H and W8H of LCDR3 SEQ ID NO: 24.
  • BC31M4 antibody variants comprising one, two, or more of these histidine substitutions with expected improved pH-sensitive binding to CD47 are selected and constructed as isolated hIgG1 antibody variants.
  • DNAs encoding the full heavy chain and light chain of the antibody variants and the parent antibody AV-164 with the same wild type sequences CDR as BC31M4 are cloned into pcDNA3.4 mammalian expression vector through gene synthesis and molecular cloning as the heavy chain expression vector and light chain expression vector respectively.
  • the antibody variants and the parent antibody are then produced through transient co-expression of the heavy chain and light chain vectors at 2: 3 ratio in HEK293 cells and single step of Protein A chromatography purification, as similarly done in the Embodiment Set 1 above.
  • the purified antibodies are analyzed by SDS-PAGE and SEC-HPLC.
  • the purified BC31M4 antibody variants generated above are analyzed for binding to CD47-expressing Raji cells by FACS assay and to CD47 protein by ELISA assay, as similarly done in the Embodiment Set 1 above. While the parent antibody (AV-164) itself shows higher binding at acidic pH 6.0 than at physiological pH 7.3, the assays are expected to reveal improved pH-sensitive binding to CD47 from a list of antibody variants, named as AC-165 to 218 in Table 13 below, compared to the parent antibody AV-164 in several ways in the assay, including: 1) antibody variants showing favorably reduced CD47-binding than that of the parent antibody at physiological pH, and also favorably increased CD47-binding than that of the parent antibody at acidic pH; 2) antibody variants showing favorably reduced CD47-binding than that of the parent antibody at physiological pH, and meanwhile comparable CD47-binding to that of the parent antibody at acidic pH; 3) antibody variants showing reduced CD47-binding than that of the parent antibody at both acidic pH and physiological pH, but favor
  • Embodiment Set 3 is a diagrammatic representation of Embodiment Set 3:
  • antibody variants with improved pH-sensitive binding to CD47 with higher binding at acidic pH than at physiological pH are generated using the pH-sensitive antibody BC27 (see (Li et al., J Hematol Oncol, 2023) ) as the parent CD47-binding domain.
  • top VH CDR and VL CDR residues of BC27 for improving pH-sensitive binding are identified for histidine substituion, including T3H, S6H and Y7H of HCDR1 SEQ ID NO: 19; R2H, K4H and V5H of HCDR3 SEQ ID NO: 428; R1H, S7H, R8H and N9H of LCDR1 SEQ ID NO: 22; W2H and T5H of LCDR2 SEQ ID NO: 23, and Y3H, T5H and A6H of LCDR3 SEQ ID NO: 429.
  • BC27 antibody variants comprising one, two, or more histidine substitutions of these histidine substitutions with expected improved pH-sensitive binding to CD47 are selected and constructed as isolated hIgG1 antibody variants.
  • DNAs encoding the full heavy chain and light chain of the antibody variants and the parent antibody AV-219 with the same wild type CDR sequences as BC27 are cloned into pcDNA3.4 mammalian expression vector through gene synthesis and molecular cloning as the heavy chain expression vector and light chain expression vector respectively.
  • the antibody variants and the parent antibody are then produced through transient co-expression of the heavy chain and light chain vectors at 2: 3 ratio in HEK293 cells and single step of Protein A chromatography purification, as similarly done in the Embodiment Set 1 above.
  • the purified antibodies are analyzed by SDS-PAGE and SEC-HPLC.
  • the purified BC27 antibody variants generated above are analyzed for binding to CD47-expressing Raji cells by FACS assay and to CD47 protein by ELISA assay, as similarly done in the Embodiment Set 1 above. While the parent antibody (AV-219) itself shows higher binding at acidic pH 6.0 than at physiological pH 7.3, the assays are expected to reveal improved pH-sensitive binding to CD47 from a list of antibody variants, named as AV-220 to 273 in Table 14 below, compared to the parent antibody AV-219 in several ways in the assay, including: 1) antibody variants showing favorably reduced CD47-binding than that of the parent antibody at physiological pH, and also favorably increased CD47-binding than that of the parent antibody at acidic pH; 2) antibody variants showing favorably reduced CD47-binding than that of the parent antibody at physiological pH, and meanwhile comparable CD47-binding to that of the parent antibody at acidic pH; 3) antibody variants showing reduced CD47-binding than that of the parent antibody at both acidic pH and physiological pH, but favorably
  • Embodiment Set 4 is a diagrammatic representation of Embodiment Set 4:
  • de novo pH-sensitive CD47-binding antibody variants are generated using the non-pH sensitive anti-CD47 antibody lemzoparlimab (TJ-C4) as the parent CD47-binding domain.
  • TJ-C4 non-pH sensitive anti-CD47 antibody lemzoparlimab
  • top VH CDR and VL CDR residues of lemzoparlimab for pH-sensitive histidine substitution are identified, including R1H of HCDR1 SEQ ID NO: 37; R5H of HCDR2 SEQ ID NO: 38; R3H of HCDR3 SEQ ID NO: 39; R13H of LCDR1 SEQ ID NO: 40; and Y3H and Y4H of LCDR3 SEQ ID NO: 42.
  • Lemzoparlimab antibody variants comprising one, two or more of these histidine substitutions are selected and constructed as isolated hIgG1 antibody variants. Specifically, DNAs encoding the full heavy chain and light chain of the antibody variants and the parent antibody AV-274 with the same wild type CDR sequences as lemzoparlimab are cloned into pcDNA3.4 mammalian expression vector through gene synthesis and molecular cloning as the heavy chain expression vector and light chain expression vector respectively.
  • the antibody variants and the parent antibody are then produced through transient co-expression of the heavy chain and light chain vectors at 2: 3 ratio in HEK293 cells and single step of Protein A chromatography purification, as similarly done in the Embodiment Set 1 above.
  • the purified antibodies are analyzed by SDS-PAGE and SEC-HPLC.
  • the purified lemzoparlimab antibody variants generated above are analyzed for binding to CD47-expressing Raji cells by FACS assay and to CD47 protein by ELISA assay, as similarly done in the Embodiment Set 1 above. While the parent antibody (AV-274) does not have pH-sensitive CD47-binding, the assays are expected to reveal de novo pH-sensitive binding to CD47 from a list of antibody variants comprising one, two or more of the histidine substitutions, named AV-275 to 296 in Table 15 below, with higher binding at acidic pH 6.0 than at physiological pH 7.3 compared to the parent antibody (AV-274) in several ways, including: 1) antibody variants showing reduced CD47-binding at both physiological pH and acidic pH compared to the parent antibody, but favorably more reduction in binding at physiological pH than at acidic pH, with thus higher binding to CD47 at acidic pH than at physiological pH; 2) antibody variants showing favorably reduced CD47-binding at physiological pH but no significant change at acidic pH compared to the parent antibody
  • Embodiment Set 5 multispecific antibody fusion proteins comprising a pH-sensitive CD47-binding domain
  • multispecific antibody fusion proteins comprising a pH-sensitive CD47-binding domain and at least one non-CD47 binding domain can be configured in exemplary format variants FV-6 to 21 as illustrated in FIG. 2 to 5 and constructed in exemplary compositions as depicted in Table 16, 17 and 18 below.
  • the DNA sequence encoding each polypeptide of a specific antibody fusion protein as included in Table 16 to 18 is optimized for expression in Homo sapiens, synthesized and cloned into the pcDNA3.4 mammalian expression vector using standard molecular cloning techniques.
  • the plasmids encoding the polypeptides of a given antibody fusion protein is co-transfected into HEK293 cells.
  • a 3: 2 ratio of the light chain : the heavy chain are usually used for the plasmids co-transfection.
  • a 1: 1 ratio of knob : hole heavy chain are usually used for the plasmids co-transfection.
  • the antibody fusion proteins produced through the transient expression in HEK293 cells are then collected and purified through single step purification of Protein A chromatography purification, as similarly done in Embodiment Set 1 to 3 above.
  • the purified antibody fusion proteins are analyzed by SDS-PAGE and SEC-HPLC, as similarly done in Embodiment Set 1 to 4 above.
  • the purified antibody fusion proteins are analyzed for binding to CD47 protein by ELISA assay at both acidic pH 6.0 and physiological pH 7.3 expected to reveal that the purified antibody fusion proteins, as constructed in Table 16 to 18, exhibit pH-sensitive binding to CD47with higher binding at acidic pH than at physiological pH, consistent with that of the monospecific counterpart antibody protein comprising the same pH-sensitive CD47-binding domain.
  • the purified antibody fusion proteins are further analyzed by FACS assay at both acidic pH 6.0 and physiological pH 7.3 for binding to cells that express both CD47 and the antigen targeted by the non-CD47 binding domain of the antibody fusion protein on the cell surface.
  • the FACS assay is expected to reveal that the purified antibody fusion proteins, as constructed in Table 16 to 18, exhibit higher binding to the cells at acidic pH than at physiological pH consistent with higher binding to CD47 on the cell surface at acidic pH by the pH-sensitive CD47-binding domain of the antibody fusion protein, and that the purified antibody fusion proteins exhibit binding to the cells higher than the binding of the monospecific counterpart antibody protein comprising either the same pH-sensitive CD47-binding domain or the same non-CD47 binding domain at an acidic pH, indicating simultaneous binding of CD47 and the non-CD47 cell surface antigen by the antibody fusion proteins at an acidic pH.
  • the mouse anti-CD47 antibody B6H12 a well characterized functional blocking antibody against human CD47, was used as an example anti-CD47 parent antibody for generating antibody variants with differential binding to human CD47 at acidic pH than at physiological pH. Insight gained through proprietary analysis of the amino acid sequence, crystal structure and interaction of B6H12 and CD47 was used to identify residues of B6H12 VH CDRs (SEQ ID NO: 82, 84 and 89) and VL CDRs (SEQ ID NO: 91, 95 and 99) for histidine substitution to generate pH-sensitive binding to CD47.
  • Top VH and VL CDR candidate residues including G3 of SEQ ID NO: 82, T3, S4, Y8 and Y10 of SEQ ID NO: 84, A3 and G4 of SEQ ID NO: 89, S7 and Y9 of SEQ ID NO: 91, K1 and Q5 of SEQ ID NO: 95, and G5, F6 and R8 of SEQ ID NO: 99 were selected to generate single histidine substitution for higher binding to CD47 at acidic pH than at physiological pH.
  • B6H12 antibody variants comprising single histidine substitutions at these CDR residues were constructed as isolated chimeric antibody variants AV-298 to 311 comprising hIgG4 Fc with S228P mutation, as set forth in Table 19 below.
  • DNAs encoding the full chimeric heavy chain and chimeric light chain of the antibody variants AV-298 to 311 and the parent antibody AV-297 with the same wild type CDR sequences as B6H12 were cloned into pcDNA3.4 mammalian expression vector through gene synthesis and molecular cloning as the heavy chain expression vector and light chain expression vector respectively.
  • the antibody variants and the parent antibody are then produced through transient co-expression of the heavy chain and light chain vectors at 2: 3 ratio in HEK293 cells and single step of Protein A chromatography purification.
  • the purified antibodies are analyzed by SDS-PAGE and SEC-HPLC. As shown in Table 19 below, all the B6H12 antibody variants were expressed and purified with yield and purity similar to the parent antibody.
  • the B6H12 antibody variants generated in Example 1 were analyzed for binding to CD47-expressing Raji cells by FACS at acidic pH 6.0 and physiological pH 7.3. Specifically, Raji cells were plated at 1E5 cells/well in 96-well plate and cultured in RPMI 1640 medium with 10%FBS.
  • FITC-labeled anti-human IgG Fc secondary antibody (Abcam, CAT#ab6854) was added and incubated at 4°C for 30 minutes at pH 7.3 or 6.0. After washing, the Raji cells were then analyzed by FACS in 1x PBS+2%FBS at pH 7.3 or 6.0.
  • FIG. 6A-C show five types of binding profiles among the B6H12 antibody variants: 1) the antibody variant AV-303 and AV-309 showed favorably reduced binding to Raji cells at physiological pH 7.3 and favorably increased binding at acidic pH 6.0 compared to the binding of the parent wild type antibody AV-297, with thus higher binding to CD47 at acidic pH than at physiological pH (FIG.
  • the antibody variants AV-298, AV-300, AV-304, AV-306 and AV-310 showed reduced binding to Raji cells at both physiological pH 7.3 and acidic pH 6.0 compared to the binding of the parent wild type antibody AV-297, but favorably more reduction in binding at physiological pH 7.3 than at acidic pH 6.0, with thus higher binding to CD47 at acidic pH than at physiological pH (FIG. 6A-C) .
  • AV-310, AV-304 and AV-300 exhibited particularly large reduction of binding at physiological pH 7.3 compared to the parent wild type antibody AV-297, while still retain significant binding at acidic pH 6.0 (FIG.
  • the antibody variants AV-299 and AV-302 showed favorably reduced binding to Raji cells at physiological pH 7.3 but no significant change at acidic pH 6.0 compared to the binding of the parent wild type antibody AV-297, with thus higher binding to CD47 at acidic pH than at physiological Ph (FIG. 6B) ;
  • the antibody variants AV-301 and AV-308 showed favorably increased binding to Raji cells at acidic pH 6.0 but no significant change at physiological pH 7.3 compared to the binding of the parent wild type antibody AV-297, with thus higher binding to CD47 at acidic pH than at physiological pH (FIG.
  • Human IGHV3-7_IGHJ4 was selected as the VH template and IGKV3-11_IGKJ4 was selected as the VL template with also cross reference to IGKV6D-21 for CDR grafting and humanization of B6H12 VH and VL sequences according to the methods described in (Almagro et al., Front Biosci, 2008, Gilliland et al., Methods Mol Biol, 2012, Golubovskaya et al., Cancers (Basel) , 2017, La et al., J Immunol Res, 2021) and US9382320B2.
  • Exemplary humanized B6H12 VH sequences with one or more pH-sensitive CDR mutations are shown in SEQ ID NO: 124 to 132, and Exemplary humanized B6H12 VL sequences with one or more pH-sensitive CDR mutations are shown in SEQ ID NO: 134 to 174 and 266 to 273.
  • Humanized B6H12 antibody variants comprising two or more of the 11 pH-sensitive individual CDR residue mutations can be generated by pairing a VH sequence selected from SEQ ID NO: 123 to 132 and a VL sequence selected from SEQ ID NO: 133 to 174 and 266 to 273.
  • a number of exemplary humanized B6H12 antibody variants comprising combination of four, five or six of the 11 pH-sensitive histidine substitutions of parent CDR residues and human IgG1 Fc, as set forth in Table 20 below, were produced similarly as done in Example 1.
  • These humanized B6H12 antibody variants comprising combo mutations of four or more of the pH-sensitive histidine substitutions were still expressed with good yield and purity as the parent wild type humanized B6H12 antibody (AV-1) , indicating the various histidine substitutions of parent CDR residues of B6H12 are generally compatible for combination.
  • the exemplary humanized B6H12 antibody variants comprising the combo mutations produced in Example 3 were then analyzed for binding to CD47-expressing Raji cells by FACS assay, as similarly done in Example 2, to identify combo mutations that exhibit higher CD47-binding at acidic pH than at physiological pH with preferably further reduced CD47-binding at physiological pH compared to the corresponding single mutations that a combo mutation comprises.
  • the result in FIG. 7 shows that all these humanized B6H12 antibody variants with combo mutations (AV-97 to 101) continued to exhibit higher binding to the CD47-expressing Raji cells at acidic pH 6.0 than at physiological pH 7.3.
  • FIG. 7 shows the minimal or weak binding of the bivalent humanized B6H12 antibody variants at physiological pH 7.3 was favorably even much less than the binding of a benchmark protein comprising a monovalent human wild type SIPR ⁇ V2 IgV (SEQ ID NO: 306) .
  • AV-97 exhibited significant CD47 binding similar to the binding of a benchmark protein comprising bivalent human wild type SIRP ⁇ V2 IgV-IgG1 Fc fusion protein that’s an analog to TTI-621, aclinically active anti-CD47 therapeutics (Ansell et al., Clin Cancer Res, 2021) (FIG. 7) .
  • AV-98, AV-100 and AV-101 also exhibited significant binding at acidic pH 6.0 that’s higher than the benchmark protein comprising monovalent human wild type SIPR ⁇ V2 IgV, but less than that of AV-97 (FIG. 7) .
  • the humanized B6H12 antibody variants with combo mutations showing pH-sensitive binding to CD47 by FACS assay in Example 4 above was further measured for CD47 binding at acidic pH 6.0 and at physiological pH 7.3 by ELISA, using recombinant human CD47 protein as immobilized ligand and the humanized B6H12 antibody variants as detecting analyte.
  • 96-well ELISA plate was coated with His-tagged CD47 extracellular domain (ECD) protein (Abclonal, CAT #RP01306) at 3 ⁇ g/mL in 50 ⁇ l 1x PBS/well at 4°Covernight.
  • ECD extracellular domain
  • the ELISA results in FIG. 8 also show that all these humanized B6H12 antibody variants with combo mutations exhibited higher CD47-binding at acidic pH 6.0 than at physiological pH.
  • the antibody variants AV-97, AV-98 and AV-101 exhibited favorably high CD47-binding at acidic pH 6.0 that’s comparable to that of the parent wild type antibody AV-1 and the benchmark TTI-621 analog in the ELISA assay, but favorably exihibited substantially reduced CD47-binding at physiological pH 7.3 (FIG. 8) .
  • a novel antibody-TCR (AbTCR) platform combines Fab-based antigen recognition with gamma/delta-TCR signaling to facilitate T-cell cytotoxicity with low cytokine release.

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Abstract

L'invention concerne des domaines de liaison à CD47 sensibles au pH, ainsi que des anticorps, des protéines de fusion d'anticorps, des conjugués de protéine-médicament et des constructions de récepteur synthétique comprenant lesdits domaines de liaison à CD47 pour cibler de manière différentielle des tissus/cellules malades exprimant CD47, et des procédés d'utilisation et de production associés à ceux-ci.
PCT/CN2024/087509 2023-04-14 2024-04-12 Domaines de liaison à cd47 sensibles au ph et leurs utilisations Pending WO2024213119A1 (fr)

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WO2016179399A1 (fr) * 2015-05-06 2016-11-10 The Board Of Trustees Of The Leland Stanford Junior University Analogues du cd47 à haute affinité
US20180142019A1 (en) * 2016-10-21 2018-05-24 Tioma Therapeutics, Inc. Therapeutic cd47 antibodies
WO2018201056A1 (fr) * 2017-04-28 2018-11-01 Novartis Ag Cellules exprimant un récepteur antigénique chimérique ciblant le bcma, et polythérapie comprenant un inhibiteur de gamma sécrétase
US20180346605A1 (en) * 2017-06-05 2018-12-06 Janssen Biotech, Inc. Engineered Multispecific Antibodies and Other Multimeric Proteins with Asymmetrical CH2-CH3 Region Mutations
CN110713547A (zh) * 2018-11-30 2020-01-21 北京泽勤生物医药有限公司 CD47-低pH插入肽在标记或治疗肿瘤中的应用
CN113621075A (zh) * 2014-08-08 2021-11-09 Alx肿瘤生物技术公司 SIRP-α变体构建体及其用途
WO2023235522A1 (fr) * 2022-06-02 2023-12-07 Blaze Bioscience, Inc. Compositions et procédés d'épuisement sélectif de molécules cibles d'egfr

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113621075A (zh) * 2014-08-08 2021-11-09 Alx肿瘤生物技术公司 SIRP-α变体构建体及其用途
WO2016179399A1 (fr) * 2015-05-06 2016-11-10 The Board Of Trustees Of The Leland Stanford Junior University Analogues du cd47 à haute affinité
US20180142019A1 (en) * 2016-10-21 2018-05-24 Tioma Therapeutics, Inc. Therapeutic cd47 antibodies
WO2018201056A1 (fr) * 2017-04-28 2018-11-01 Novartis Ag Cellules exprimant un récepteur antigénique chimérique ciblant le bcma, et polythérapie comprenant un inhibiteur de gamma sécrétase
US20180346605A1 (en) * 2017-06-05 2018-12-06 Janssen Biotech, Inc. Engineered Multispecific Antibodies and Other Multimeric Proteins with Asymmetrical CH2-CH3 Region Mutations
CN110713547A (zh) * 2018-11-30 2020-01-21 北京泽勤生物医药有限公司 CD47-低pH插入肽在标记或治疗肿瘤中的应用
WO2023235522A1 (fr) * 2022-06-02 2023-12-07 Blaze Bioscience, Inc. Compositions et procédés d'épuisement sélectif de molécules cibles d'egfr

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