WO2020198370A2 - Therapeutic cd47 antibodies - Google Patents

Therapeutic cd47 antibodies Download PDF

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Publication number
WO2020198370A2
WO2020198370A2 PCT/US2020/024730 US2020024730W WO2020198370A2 WO 2020198370 A2 WO2020198370 A2 WO 2020198370A2 US 2020024730 W US2020024730 W US 2020024730W WO 2020198370 A2 WO2020198370 A2 WO 2020198370A2
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seq
amino acid
acid sequence
human
binding fragment
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PCT/US2020/024730
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French (fr)
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WO2020198370A3 (en
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Pamela Manning
Robyn PURO
Juan Almagro
Robert Karr
Prabir CHAKRABORTY
Daniel Pereira
W. Casey WILSON
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Arch Oncology, Inc.
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Publication of WO2020198370A2 publication Critical patent/WO2020198370A2/en
Publication of WO2020198370A3 publication Critical patent/WO2020198370A3/en

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    • 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
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/524—CH2 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • 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

  • This disclosure is related generally to anti-CD47 monoclonal antibodies (anti-CD47 mAbs) with distinct functional profiles as described herein, methods to generate anti-CD47 mAbs, and methods of using these anti-CD47 mAbs as therapeutics for the prevention and treatment of solid and hematological cancers, ischemia-reperfusion injury, cardiovascular diseases, autoimmune diseases, or inflammatory diseases, or as diagnostics for determining the level of CD47 in tissue samples.
  • CD47 is a cell surface receptor comprised of an extracellular IgV set domain, a 5 transmembrane domain, and a cytoplasmic tail that is alternatively spliced.
  • Two ligands bind CD47: signal-regulatory protein alpha (SIRPoc) and thrombospondin- 1 (TSP1).
  • SIRPoc signal-regulatory protein alpha
  • TSP1 thrombospondin- 1
  • SIRPa is an innate immune checkpoint receptor primarily expressed on macrophages, granulocytes, dendritic cells and monocytes. SIRPa suppresses innate immunity upon interaction with its ligand, CD47, which is widely expressed on normal tissues and is upregulated in most human tumors as a means to escape macrophage recognition and programmed cell removal.
  • CD47 expression and/or activity has been implicated in a number of diseases and disorders. Accordingly, there exists a need for therapeutic compositions and methods for treating diseases and conditions associated with CD47 in humans and animals, including the prevention and treatment of solid and hematological cancers, ischemia-reperfusion injury (IRI), cardiovascular diseases, or an autoimmune or inflammatory disease.
  • IRI ischemia-reperfusion injury
  • cardiovascular diseases or an autoimmune or inflammatory disease.
  • the present disclosure describes anti-CD47 m Ahs with distinct functional profiles. These antibodies possess distinct combinations of properties selected from the following: 1) exhibit cross-reactivity with one or more species homologs of CD47; 2) block the interaction between CD47 and its ligand SIRPoc; 3) increase phagocytosis of human tumor cells; 4) induce death of susceptible human tumor cells; 5) do not induce cell death of human tumor cells; 6) do not have reduced or minimal binding to human red blood cells (hRBCs); 7) have reduced binding to hRBCs; 8) have minimal binding to hRBCs; 9) cause reduced agglutination of hRBCs; 10) cause no detectable agglutination of hRBCs; 11) reverse TSP1 inhibition of the nitric oxide (NO) pathway; 12) do not reverse TSP1 inhibition of the NO pathway; 13) cause loss of mitochondrial membrane potential; 14) do not cause cause cause loss of mitochondrial membrane potential; 15) cause an
  • the anti-CD47 mAbs of the disclosure are useful in various therapeutic methods for treating diseases and conditions associated with CD47 in humans and animals, including the prevention and treatment of solid and hematological cancers, autoimmune diseases, inflammatory diseases, IRI, and cardiovascular diseases.
  • the antibodies of the disclosure are also useful as diagnostics to determine the level of CD47 expression in tissue samples.
  • Embodiments of the disclosure include isolated antibodies and immunologically active binding fragments thereof; pharmaceutical compositions comprising one or more of the anti-CD47 mAbs, preferably chimeric or humanized forms of said anti-CD47 mAbs; methods of therapeutic use of such anti-CD47 monoclonal antibodies; and cell lines that produce these anti-CD47 mAbs.
  • the embodiments of the disclosure include the mAbs, or antigen-binding fragments thereof, which are defined herein by reference to specific structural characteristics, i.e. , specified amino acid sequences of either the CDRs or entire heavy chain or light chain variable domains. All antibodies of the disclosure bind to CD47.
  • the monoclonal antibodies, or antigen binding fragments thereof may comprise at least one, usually at least three, CDR sequences as provided herein, usually in combination with framework sequences from a human variable region or as an isolated CDR peptide.
  • an antibody comprises at least one light chain comprising the three light chain CDR sequences provided herein situated in a variable region framework, which may be, without limitation, a murine or human variable region framework, and at least one heavy chain comprising the three heavy chain CDR sequences provided herein situated in a variable region framework, which may be, without limitation, a human or murine variable region framework.
  • variable heavy chain CDR1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: l, SEQ ID NO:2, and SEQ ID NO:3
  • said variable heavy chain CDR2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6
  • said variable heavy chain CDR3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO: 10.
  • the heavy chain variable (VH) domain may comprise any one of the listed variable heavy chain CDR1 sequences (HCDR1) in combination with any one of the variable heavy chain CDR2 sequences (HCDR2) and any one of the variable heavy chain CDR3 sequences (HCDR3).
  • HCDR1 and HCDR2 and HCDR3 are provided that derive from a single common VH domain, examples of which are described herein.
  • the antibody or antigen binding fragment thereof may additionally comprise a light chain variable (VL) domain, which is paired with the VH domain to form an antigen binding domain.
  • VL light chain variable domains
  • light chain variable domains are those comprising a variable light chain CDR1, variable light chain CDR2, and a variable light chain CDR3, wherein said variable light chain CDR1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, and SEQ ID NO:14; said variable light chain CDR2 optionally comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:15, SEQ ID NO: 16, and SEQ ID NO: 17; and said variable light chain CDR3 optionally comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO:20.
  • the light chain variable domain may comprise any one of the listed variable light chain CDR1 sequences (LCDR1) in combination with any one of the variable light chain CDR2 sequences (LCDR2) and any one of the variable light chain CDR3 sequences (LCDR3).
  • LCDR1 and LCDR2 and LCDR3 are provided that derive from a single common VL domain, examples of which are described herein.
  • any given CD47 antibody or antigen binding fragment thereof comprising a VH domain paired with a VL domain will comprise a combination of 6 CDRs: variable heavy chain CDR1 (HCDR1), variable heavy chain CDR2 (HCDR2), variable heavy chain CDR3 (HCDR3), variable light chain CDR1 (LCDR1), variable light chain CDR2 (LCDR2), and variable light chain CDR3 (LCDR3).
  • HCDR1 variable heavy chain CDR1
  • HCDR2 variable heavy chain CDR2
  • HCDR3 variable heavy chain CDR3
  • LCDR1 variable light chain CDR1
  • LCDR2 variable light chain CDR2
  • LCDR3 variable light chain CDR3
  • combinations of 6 CDRs include, but are not limited to, the combinations of variable heavy chain CDR1 (HCDR1), variable heavy chain CDR2 (HCDR2), variable heavy chain CDR3 (HCDR3), variable light chain CDR1 (LCDR1), variable light chain CDR2 (LCDR2), and variable light chain CDR3 (LCDR3) selected from the group consisting of:
  • HCDR1 comprising SEQ ID NO: 1
  • HCDR2 comprising SEQ ID NO:4
  • HCDR3 comprising SEQ ID NO:7
  • LCDR1 comprising SEQ ID NO:l l
  • LCDR2 comprising SEQ ID NO: 15, LCDR3 comprising SEQ ID NO: 18;
  • HCDR1 comprising SEQ ID NO: 1
  • HCDR2 comprising SEQ ID NO:4
  • LCDR2 comprising SEQ ID NO: 15, LCDR3 comprising SEQ ID NO: 18;
  • HCDR1 comprising SEQ ID NO:2, HCDR2 comprising SEQ ID NO:5, HCDR3 comprising SEQ ID NO:9, LCDR1 comprising SEQ ID NO: 12, LCDR2 comprising SEQ ID NO: 16, LCDR3 comprising SEQ ID NO: 19;
  • HCDR1 comprising SEQ ID NO:2, HCDR2 comprising SEQ ID NO:5, HCDR3 comprising SEQ ID NO:9, LCDR1 comprising SEQ ID NO:13, LCDR2 comprising SEQ ID NO: 16, LCDR3 comprising SEQ ID NO: 19;
  • HCDR1 comprising SEQ ID NO:3, HCDR2 comprising SEQ ID NO:6, HCDR3 comprising SEQ ID NO: 10, LCDR1 comprising SEQ ID NO: 14, LCDR2 comprising SEQ ID NO: 17, LCDR3 comprising SEQ ID NO: 18.
  • anti-CD47 m Ahs include antibodies or antigen binding fragments thereof, comprising a heavy chain variable domain having an amino acid sequence selected from the group consisting of: the amino acid sequences of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, and amino acid sequences exhibiting at least 90%, 95%, 97%, 98%, or 99% sequence identity to one of the recited sequences.
  • anti-CD47 mAbs including antibodies or antigen binding fragments thereof may comprise a light chain variable domain having an amino acid sequence selected from the group consisting of: the amino acid sequences of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, and SEQ ID NO:52, and amino acid sequences exhibiting at least 90%, 95%, 97%, 98%, or 99% sequence identity to one of the recited sequences.
  • anti-CD47 mAbs, or antigen binding fragments thereof are those comprising a combination of a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the combination is selected from the group consisting of:
  • anti-CD47 antibodies or antigen binding fragments thereof may also comprise a combination of a heavy chain variable domain and a light chain variable domain wherein the heavy chain variable domain comprises a VH sequence with at least 85% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 97%, 98% or 99% sequence identity, to the heavy chain amino acid sequences shown above in (i) to (xxxiv) and/or the light chain variable domain comprises a VL sequence with at least 85% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 97%, 98% or 99% sequence identity, to the light chain amino acid sequences shown above in (i) to (xxxiv).
  • the specific VH and VL pairings or combinations in parts (i) through (xxxiv) may be preserved for anti-CD47 antibodies having VH and VL domain sequences with a particular percentage sequence identity to these reference sequences.
  • the VH and/or VL domains may retain identical CDR sequences to those present in the reference sequence such that the variation is present only within the framework regions.
  • the preferred CD47 antibodies, or antigen binding fragments thereof are those comprising a combination of a heavy chain (HC) and a light chain (LC), wherein the combination is selected from the group consisting of:
  • VH amino acid sequence is at least 90%, 95%, 97%, 98% or 99% identical thereto and the a VL amino acid sequence is at least 90%, 95%, 97%, 98% or 99% identical thereto.
  • anti-CD47 antibodies as described herein may also be characterized by combinations of properties which are not exhibited by prior art anti-CD47 antibodies proposed for human therapeutic use. Accordingly, in some embodiments, anti-CD47 antibodies described herein are characterized by:
  • a. binds to human CD47 ; b. blocks SIRPoc binding to human CD47;
  • the anti-CD47 antibodies are characterized by:
  • a. binds to human CD47 ;
  • hRBCs human red blood cells
  • the anti-CD47 antibodies are characterized by:
  • a. binds to human CD47 ;
  • hRBCs human red blood cells
  • the anti-CD47 antibodies are characterized by:
  • a. binds to human CD47 ;
  • the anti-CD47 antibodies are characterized by:
  • hRBCs human red blood cells
  • the anti-CD47 antibodies are characterized by: a. binds to human CD47 ;
  • hRBCs human red blood cells
  • anti-CD47 antibodies described herein are also characterized by combinations of properties which are not exhibited by prior art anti-CD47 antibodies proposed for human therapeutic use. Accordingly, anti-CD47 antibodies as described herein may be further characterized by one or more among the following characteristics:
  • a causes an increase in cell surface calreticulin expression on human tumor cells
  • b causes an increase in adenosine triphosphate (ATP) release by human tumor cells
  • c. causes an increase in high mobility group box 1 (HMGB1) release by human tumor cells
  • d. causes an increase in annexin A1 release by human tumor cells
  • CXCL10 C-X-C Motif Chemokine Ligand 10
  • g. causes an increase in cell surface protein disulfide-isomerase A3 (PDIA3) expression on human tumor cells;
  • HSP70 cell surface heat shock protein 70
  • HSP90 cell surface heat shock protein 90
  • the monoclonal antibody, or antigen binding fragment thereof binds to human, non-human primate, mouse, rabbit, and rat CD47.
  • the monoclonal antibody, or antigen binding fragment thereof specifically also binds to non-human primate CD47, wherein non human primate may include, but is not limited to, cynomolgus monkey, green monkey, rhesus monkey, and squirrel monkey.
  • the monoclonal antibody, or antigen binding fragment thereof has reduced binding to normal human cells, which includes, but is not limited to, endothelial cells, skeletal muscle cells, epithelial cells, and peripheral blood mononuclear cells (e.g., human aortic endothelial cells, human skeletal muscle cells, human micro vascular endothelial cells, human renal tubular epithelial cells, human peripherial blood CD3+ cells, and human peripheral blood mononuclear cells).
  • endothelial cells e.g., endothelial cells, skeletal muscle cells, epithelial cells, and peripheral blood mononuclear cells
  • peripheral blood mononuclear cells e.g., human aortic endothelial cells, human skeletal muscle cells, human micro vascular endothelial cells, human renal tubular epithelial cells, human peripherial blood CD3+ cells, and human peripheral blood mononuclear cells.
  • the monoclonal antibody, or antigen binding fragment thereof has a greater have a greater affinity for human CD47 at an acidic pH compared to physiological pH.
  • the monoclonal antibody, or antigen binding fragment thereof may additionally possess one or more of the following characteristics: 1) exhibit cross reactivity with one or more species homologs of CD47 ; 2) block the interaction between CD47 and its ligand SIRPoc; 3) increase phagocytosis of human tumor cells; 4) induce death of susceptible human tumor cells; 5) do not induce cell death of human tumor cells; 6) do not have reduced or minimal binding to human red blood cells (hRBCs); 7) have reduced binding to hRBCs; 8) have minimal binding to hRBCs; 9) cause reduced agglutination of hRBCs; 10) cause no detectable agglutination of hRBCs; 11) reverse TSP1 inhibition of the nitric oxide (NO) pathway; 12) do not reverse TSP1 inhibition of the NO pathway; 13) cause loss of mitochondrial membrane potential; 14) do not cause cause cause loss of mitochondrial membrane potential; 15) cause an increase in cell surface calreticulin
  • the anti-CD47 mAbs can be full-length humanized antibodies with human frameworks and constant regions of the isotypes, IgA, IgD, IgE, IgG, and IgM, more particularly, IgGl, IgG2, IgG3, IgG4, and in some cases with various mutations to alter Fc receptor function or prevent Fab arm exchange or an antibody fragment, e.g., a F(ab’)2 fragment, a F(ab) fragment, a single chain Fv fragment (scFv), etc., as disclosed herein.
  • compositions comprise one or more of the anti-CD47 mAbs or fragments disclosed herein, optionally chimeric or humanized forms, and a pharmaceutically acceptable carrier, diluent, or excipient.
  • anti-CD47 mAbs Prior to the present disclosure, there was a need to identify anti-CD47 mAbs that possess the functional profiles as described herein.
  • the anti-CD47 mAbs of the present disclosure exhibit distinct combinations of properties, particularly combinations of properties that render the m Ahs particularly advantageous or suitable for use in human therapy, particularly in the prevention and/or treatment of solid and hematological cancers, ischemia- reperfusion injury, autoimmune and/or inflammatory diseases.
  • the disclosure provides a monoclonal antibody, or an antigen binding fragment thereof, which: binds to human CD47; blocks SIRPoc binding to human CD47; increases phagocytosis of human tumor cells; and induces death of human tumor cells; wherein said monoclonal antibody, or an antigen binding fragment thereof, exhibits pH- dependent binding to CD47 present on a cell.
  • the disclosure provides a monoclonal antibody, or an antigen binding fragment thereof, which: binds to human CD47; blocks SIRPoc binding to human CD47; increases phagocytosis of human tumor cells; wherein said monoclonal antibody, or an antigen binding fragment thereof, exhibits pH-dependent binding to CD47 present on a cell.
  • the disclosure provides a monoclonal antibody, or an antigen binding fragment thereof, which: binds to human CD47; blocks SIRPoc binding to human CD47; increases phagocytosis of human tumor cells; and induces death of human tumor cells; wherein said monoclonal antibody, or an antigen binding fragment thereof, exhibits reduced binding to normal cells.
  • these cells may be an endothelial cell, a skeletal muscle cell, an epithelial cell, a PBMC or a RBC (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubular epithelial cells, human peripherial blood CD3+ cells, human peripheral blood mononuclear cells or human RBC).
  • the disclosure provides a monoclonal antibody, or an antigen binding fragment thereof, which: binds to human CD47; blocks SIRPoc binding to human CD47; increases phagocytosis of human tumor cells; wherein said monoclonal antibody, or an antigen binding fragment thereof, exhibits reduced binding to normal cells.
  • these cells may be an endothelial cell, a skeletal muscle cell, an epithelial cell, a PBMC or a RBC (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubular epithelial cells, human peripherial blood CD3+ cells, human peripheral blood mononuclear cells or human RBC).
  • the monoclonal antibody, or an antigen binding fragment thereof exhibits both pH dependent binding and reduced binding to a cell.
  • FIG. 1A Binding of VLX4 Humanized m Ahs to Human OVIO Cells Expressing Human CD47.
  • VLX4 humanized m Ahs VLX4hum_01 IgGl, VLX4hum_02 IgGl, VLX4hum_01 IgG4PE, and VLX4hum_02 IgG4PE
  • OVIO hCD47 OVIO cell line expressing human CD47
  • Various concentrations of m Ahs were added to the cells for 1 hr. Cells were washed and then incubated with HRP-labelled secondary antibody for 1 hr followed by addition of peroxidase substrate.
  • FIG. IB Binding of VLX4 Humanized m Ahs to Human OVIO Cells Expressing Human CD47. Binding of VLX4 humanized mAbs (VLX4hum_06 IgG4PE, VLX4hum_07 IgG4PE, VLX4hum_12 IgG4PE, and VLX4hum_13 IgG4PE) to human CD47 was determined using an OVIO CD47 cell-based ELISA. OVIO hCD47 cells were plated into 96 well plates and were confluent at the time of assay. Various concentrations of VLX4 representative m Ahs were added to the cells for 1 hr. Cells were washed and then incubated with HRP-labelled secondary antibody for 1 hr followed by addition of peroxidase substrate.
  • VLX4hum_06 IgG4PE VLX4hum_07 IgG4PE
  • VLX4hum_12 IgG4PE VLX4hum_13 I
  • FIG. 2A Binding of VLX4 Humanized m Ahs to Human RBCs (hRBCs). Binding of VLX4 humanized mAbs (VLX4hum_01 IgGl, VLX4hum_02 IgGl, VLX4hum_01 IgG4PE, and VLX4hum_02 IgG4PE) to human CD47 was determined using freshly isolated hRBCs. hRBCs were incubated for 60 minutes at 37°C with various concentrations of VLX4 mAbs, washed and incubated for lhr with FITC-labelled donkey anti-human antibody. Cells were washed and antibody binding measured using flow cytometry.
  • FIG. 2B Binding of VLX4 Humanized mAbs to Human RBCs. Binding of VLX4 humanized mAbs (VLX4hum_07 IgG4PE, VLX4hum_12 IgG4PE, and VLX4hum_13 IgG4PE) to human CD47 was determined using freshly isolated hRBCs. hRBCs were incubated for 60 minutes at 37°C with various concentrations of VLX4 mAbs, washed and incubated for lhr with FITC-labelled donkey anti-human antibody. Cells were washed and antibody binding measured using flow cytometry.
  • FIG. 3A Binding of VLX8 Humanized m Ahs to Human OVIO hCD47 Cells. Binding of VLX8 IgG4PE chimera (xi) or humanized m Ahs (VLX8hum_01 IgG4PE, VLX8hum_04 IgG4PE, VLX8hum_07 IgG4PE, and VLX8hum_09 IgG4PE) to human CD47 was determined using an OVIO hCD47 cell-based ELISA. OVIO hCD47 cells were plated into 96 well plates and were confluent at the time of assay. Various concentrations of VLX8 representative m Ahs were added to the cells for 1 hr.
  • FIG. 3B Binding of VLX8 Humanized mAbs to Human OVIO hCD47 Cells. Binding of VLX8 chimera or humanized mAbs (VLX8hum_06 IgG2, VLX8hum_07 IgG2, VLX8hum_08 IgG2, and VLX8hum_09 IgG2) to human CD47 was determined using an OVIO hCD47 cell-based ELISA. OVIO hCD47 cells were plated into 96 well plates and were confluent at the time of assay. Various concentrations of VLX8 representative mAbs were added to the cells for 1 hr. Cells were washed and then incubated with HRP-labelled secondary antibody for 1 hr followed by addition of peroxidase substrate.
  • FIG. 4A Binding of VLX8 Humanized mAbs to Human RBCs. Binding of VLX8
  • IgG4PE xi
  • humanized mAbs VLX8hum_01 IgG4PE, VLX8hum_03 IgG4PE, VLX8hum_07 IgG4PE, and VLX8hum_10 IgG4PE
  • VLX8hum_01 IgG4PE VLX8hum_03 IgG4PE
  • VLX8hum_07 IgG4PE VLX8hum_10 IgG4PE
  • FIG. 4B Binding of VLX8 Humanized mAbs to Human RBCs. Binding of VLX8 IgG4PE xi or humanized mAbs (VLX8hum_06 IgG2, VLX8hum_07 IgG2, VLX8hum_08 IgG2 and VLX8hum_09 IgG2) to human CD47 was determined using freshly isolated human RBCs. RBCs were incubated for 1 hr at 37°C with various concentrations of VLX8 mAbs, washed and incubated for lhr with FITC-labelled donkey anti-human antibody. Cells were washed and antibody binding measured using flow cytometry.
  • FIG. 5A Binding of VLX9 Humanized mAbs to Human OVIO hCD47 Cells. Binding of VLX9 IgG2 (xi) or humanized mAbs (VLX9hum_01 IgG2, VLX9hum_02 IgG2, VLX9hum_03 IgG2, VLX9hum_04 IgG2 and VLX9hum_05 IgG2) to human CD47 was determined using an OVIO human CD47 cell-based ELISA. OVIO hCD47 cells were plated into 96 well plates and were confluent at the time of assay. Various concentrations of mAbs were added to the cells for 1 hr. Cells were washed and then incubated with HRP-labelled secondary antibody for 1 hr followed by addition of peroxidase substrate.
  • FIG. 5B Binding of VLX9 Humanized mAbs to Human OVIO hCD47 Cells. Binding of VLX9 IgG2 (xi) or humanized mAbs (VLX9hum_06 IgG2, VLX9hum_07 IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2 and VLX9hum_10 IgG2) to human CD47 was determined using a OVIO hCD47 cell-based ELISA. OVIO hCD47 cells were plated into 96 well plates and were confluent at the time of assay. Various concentrations of mAbs were added to the cells for 1 hr.
  • FIG. 6A Specific Binding of VLX Humanized m Ahs to CD47. Binding of VLX humanized mAh VLX4hum_07 IgG4PE to wildtype and CD47 knockout Jurkat cells was determined by flow cytometry. Various concentrations of m Ahs were added to 1 X 10 4 cells for 1 hr. The cells were washed and then incubated with FITC-labelled secondary antibody for 1 hr. Cells were washed and antibody binding measured using flow cytometry.
  • FIG. 6B Specific Binding of VLX Humanized mAhs to CD47. Binding of VLX humanized mAh VLX9hum_04 IgG2 to wildtype and CD47 knockout Jurkat cells was determined by flow cytometry. Various concentrations of mAhs were added to 1 X 10 4 cells for 1 hr. The cells were washed and then incubated with FITC-labelled secondary antibody for 1 hr. Cells were washed and antibody binding measured using flow cytometry.
  • FIG. 7 Binding of VLX9 Humanized mAhs to Human RBCs. Binding of VLX9 IgG2 xi or humanized VLX9 mAhs to human CD47 (VLX9hum_01 IgG2, VLX9hum_02 IgG2 and VLX9hum_07 IgG2) was determined using freshly isolated human hRBCs. RBCs were incubated for 60 minutes at 37°C with various concentrations of VLX9 mAbs, washed and incubated for lhr with FITC-labelled donkey anti-human antibody. Cells were washed and antibody binding measured using flow cytometry.
  • FIG. 8A Binding of VLX Humanized mAhs to Human Aortic Endothelial Cells (HAEC). Binding of VLX humanized mAbs (VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_l 1 IgG4PE, VLX4hum_01 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2, VLX9hum_03 IgG2 and VLX9hum_04 IgG2) to HAEC was determined by flow cytometry. HAEC were removed from the flask using acutase. Various concentrations of mAhs were added to 1 X 10 4 cells for 1 hr. The cells were washed and then incubated with FITC-labelled secondary antibody for 1 hr followed by measurement of FITC label by flow cytometry.
  • FIG. 8B Binding of VLX Humanized mAhs to Skeletal Human Muscle Cells (SkMC). Binding of VLX humanized mAhs (VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_l 1 IgG4PE, VLX4hum_01 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2, VLX9hum_03 IgG2 and VLX9hum_04 IgG2) to SkMc was determined by flow cytometry. SkMC were removed from the flask using acutase.
  • FIG. 8C Binding of VLX Humanized m Ahs to Human Lung Microvascular Endothelial Cells (HMVEC-L).
  • VLX humanized m Ahs (VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_l l IgG4PE, VLX4hum_01 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2, VLX9hum_03 IgG2 and VLX9hum_04 IgG2) to HMVEC-L was determined by flow cytometry. HMVEC-L were removed from the flask using acutase. Various concentrations of m Ahs were added to 1 X 10 4 cells for 1 hr. The cells were washed and then incubated with FITC-labelled secondary antibody for 1 hr followed by measurement of FITC label by flow cytometry.
  • FIG. 8D Binding of VLX Humanized mAhs to Human Renal Tubular Epithelial Cells (RTEC). Binding of VLX humanized mAhs (VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_l 1 IgG4PE, VLX4hum_01 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2, VLX9hum_03 IgG2 and VLX9hum_04 IgG2) to RTEC by flow cytometry. RTEC were removed from the flask using acutase. Various concentrations of mAhs were added to 1 X 10 4 cells for 1 hr. The cells were washed and then incubated with FITC- labelled secondary antibody for 1 hr followed by measurement of FITC label by flow cytometry.
  • FIG. 8E Binding of VLX Humanized mAhs to Human Peripheral Blood CD3 + Cells. Binding of VLX humanized mAhs (VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_l 1 IgG4PE, VLX4hum_01 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2, VLX9hum_03 IgG2 and VLX9hum_04 IgG2) to CD3 + cells was determined by flow cytometry. PBMC were plated into 96 well plates.
  • FIG. 8F Binding of VLX Humanized mAhs to Human Peripheral Blood Mononuclear Cells (PBMC). Binding of VLX humanized mAhs (VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_l l IgG4PE, VLX4hum_01 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2, VLX9hum_03 IgG2 and VLX9hum_04 IgG2) to PBMC was determined by flow cytometry. PBMCs were plated into 96 well plates. Various concentrations of mAhs were added to the cells for 1 hr. Cells were washed and then incubated with FITC-labelled secondary antibody for 1 hr followed by measurement of FITC label by flow cytometry.
  • FIG. 9A pH Dependent and pH Independent Binding of Humanized mAh to His- CD47. Binding of VLX9hum_09 IgG2 to human CD47 was determined using a solid-phase CD47 ELISA assay. His-CD47 was adsorbed to microtiter wells, washed and various concentrations of humanized m Ahs were added to the wells for lhr at pH 6 or 8. The wells were washed and then incubated with HRP-labelled secondary antibody for 1 hour followed by addition of peroxidase substrate.
  • FIG. 9B pH Dependent and pH Independent Binding of Humanized mAh to His- CD47. Binding of VLX9hum_04 IgG2 to human CD47 was determined using a solid-phase CD47 ELISA assay. His-CD47 was adsorbed to microtiter wells, washed and various concentrations of humanized m Ahs were added to the wells for lhr at pH 6 or 8. The wells were washed and then incubated with HRP-labelled secondary antibody for 1 hour followed by addition of peroxidase substrate.
  • FIG. 9C pH Dependent and pH Independent Binding of Humanized mAh to His- CD47. Binding of VLX4hum_07 IgG4PE to human CD47 was determined using a solid-phase CD47 ELISA assay. His-CD47 was adsorbed to microtiter wells, washed and various concentrations of humanized m Ahs were added to the wells for lhr at pH 6 or 8. The wells were washed and then incubated with HRP-labelled secondary antibody for 1 hour followed by addition of peroxidase substrate.
  • FIG. 9D pH Dependent and pH Independent Binding of Humanized mAh to His- CD47. Binding of VLX8hum_10 IgG4PE to human CD47 was determined using a solid-phase CD47 ELISA assay. His-CD47 was adsorbed to microtiter wells, washed and various concentrations of humanized m Ahs were added to the wells for lhr at pH 6 or 8. The wells were washed and then incubated with HRP-labelled secondary antibody for 1 hour followed by addition of peroxidase substrate.
  • FIG. 10 VLX4, VLX8, and VLX9 Humanized m Ahs Block SIRPoc binding to CD47 on Human Jurkat Cells.
  • 1.5 x 10 6 Jurkat cells were incubated with 5pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE, VLX4hum_l l IgG4PE, VLX9hum_03 IgG2, VLX9hum_06 IgG2, and VLX9hum_08 IgG2) or a control antibody or no antibody in RPMI containing 10% FBS for 30 min at 37°C. An equal volume of media containing fluorescently labelled SIRPoc-Fc fusion protein was added and incubated for an additional 30 min at 37°C. Cells were washed and binding was assessed using flow cytometry.
  • FIG. 11 VLX4 CD47 Chimeric mAbs Increase Phagocytosis of Human Jurkat Cells by Human Macrophages.
  • Human macrophages were plated at a concentration of lxlO 4 cells per well in a 96 well plate and allowed to adhere for 24 hrs.
  • 5xl0 4 CFSE-labehed human Jurkat cells and 1 mg/ml of the VLX4 chimeric m Ahs (VLX4 IgGl xi, VLX4 IgGl N297Q xi, VLX4 IgG4PE xi, VLX4 IgG4 S228P xi) were added to the macrophage cultures and incubated at 37°C for 2 hrs.
  • Non-phagocytosed Jurkat cells were removed and macrophage cultures were washed extensively. Macrophages were trypsinized and stained for CD 14. Flow cytometry was used to determine the percentage of CD14 + /CFSE + cells in the total CD14 + population.
  • FIG. 12A VEX4 Humanized mAbs Increase Phagocytosis of Human Jurkat Cells by Human Macrophages.
  • Human macrophages were plated at a concentration of lxlO 4 cells per well in a 96 well plate and allowed to adhere for 24 hrs.
  • 5xl0 4 CFSE-labelled human Jurkat cells and 1 pg/ml of antibody (VEX4hum_01 IgGl and VEX4hum_01 IgG4PE) were added to the macrophage cultures and incubated at 37°C for 2 hrs.
  • Non-phagocytosed Jurkat cells were removed and macrophage cultures were washed extensively.
  • Macrophages were trypsinized and stained for CD14. Flow cytometry was used to determine the percentage of CD14 + /CFSE + cells in the total CD14 + population.
  • FIG. 12B VEX4 Humanized mAbs Increase Phagocytosis of Human Jurkat Cells by Human Macrophages.
  • Human macrophages were plated at a concentration of lxlO 4 cells per well in a 96 well plate and allowed to adhere for 24 hrs.
  • 5xl0 4 CFSE-labelled human Jurkat cells and 1 pg/ml of antibody (VEX4 IgG4PE xi, VEX4hum_06 IgG4PE, VEX4hum_07 IgG4PE, VEX4hum_012 IgG4PE and VEX4hum_13 IgG4PE) were added to the macrophage cultures and incubated at 37°C for 2 hrs.
  • Non-phagocytosed Jurkat cells were removed and macrophage cultures were washed extensively. Macrophages were trypsinized and stained for CD14. Flow cytometry was used to determine the percentage of CD14 + /CFSE + cells in the total CD14 + population.
  • FIG. 13A VFX8 CD47 Chimeric m Ahs Increase Phagocytosis of Human Jurkat Cells by Human Macrophages.
  • Human macrophages were plated at a concentration of lxlO 4 cells per well in a 96 well plate and allowed to adhere for 24 hrs.
  • 5xl0 4 CFSE- labelled human Jurkat cells and 1 pg/ml of the VFX8 chimeric mAbs (VFX8 IgGl N297Q xi and VFX8 Ig4PE xi) were added to the macrophage cultures and incubated at 37°C for 2 hrs.
  • Non-phagocytosed Jurkat cells were removed and macrophage cultures were washed extensively.
  • Macrophages were trypsinized and stained for CD 14. Flow cytometry was used to determine the percentage of CD14 + /CFSE + cells in the total CD14 + population.
  • FIG. 13B VFX8 Humanized mAbs Increase Phagocytosis of Human Jurkat Cells by Human Macrophages. Human macrophages were plated at a concentration of lxlO 4 cells per well in a 96 well plate and allowed to adhere for 24 hrs.
  • VFX8 IgG4PE xi 5xl0 4 CFSE-labelled human Jurkat cells and 1 pg/ml of antibody (VFX8 IgG4PE xi, VFX8hum_01 IgG4PE, VFX8hum_03 IgG4PE, VLX8hum_07 IgG4PE, VLX8hum_08 IgG4PE and VLX8hum_09 IgG4PE) were added to the macrophage cultures and incubated at 37°C for 2 hrs. Non-phagocytosed Jurkat cells were removed and macrophage cultures were washed extensively. Macrophages were trypsinized and stained for CD 14. Flow cytometry was used to determine the percentage of CD14 + /CFSE + cells in the total CD14 + population.
  • FIG. 14A VLX9 CD47 Chimeric m Ahs Increase Phagocytosis of Human Jurkat Cells by Human Macrophages.
  • Human macrophages were plated at a concentration of lxlO 4 cells per well in a 96 well plate and allowed to adhere for 24 hours.
  • 5xl0 4 CFSE-labelled human Jurkat cells and 1 pg/ml of the VLX9 chimeric m Ahs (VLX9 IgGl N297 xi, VLX9 IgG2 xi and VLX9 IgG4PE xi) were added to the macrophage cultures and incubated at 37°C for two hours.
  • Non-phagocytosed Jurkat cells were removed and macrophage cultures were washed extensively. Macrophages were trypsinized and stained for CD 14. Flow cytometry was used to determine the percentage of CD14+/CFSE+ cells in the total CD 14+ population.
  • FIG. 14B VEX9 Humanized mAbs Increase Phagocytosis of Human Jurkat Cells by Human Macrophages. Human macrophages were plated at a concentration of lxlO 4 cells per well in a 96 well plate and allowed to adhere for 24 hours.
  • VEX9 IgG2 xi 5xl0 4 CFSE-labelled human Jurkat cells and 1 pg/ml of antibody (VEX9 IgG2 xi, VEX9hum_01 IgG2, VEX9hum_02 IgG2, VEX9hum_03 IgG2, VEX9hum_04 IgG2, VEX9hum_05 IgG2, VEX9hum_06 IgG2, VEX9hum_07 IgG2, VEX9hum_08 IgG2, VEX9hum_09 IgG2 and VEX9hum_10 IgG2) were added to the macrophage cultures and incubated at 37°C for two hours. Non-phagocytosed Jurkat cells were removed and macrophage cultures were washed extensively. Macrophages were trypsinized and stained for CD 14. Flow cytometry was used to determine the percentage of CD14+/CFSE+ cells in the total CD 14+ population.
  • FIG. 15A Induction of Cell Death in Human Jurkat Cells by Soluble VFX4 Humanized m Ahs.
  • Jurkat cells (lxlO 4 ) were incubated with 1 pg/ml VFX4 humanized m Ahs (VFX4hum_01 IgGl, VFX4hum_01 IgG4PE, VFX4hum_02 IgGl, VFX4hum_02 IgG4PE) in RPMI media for 24 hours at 37°C. Cells were then stained with annexin V and the signal was detected by flow cytometry. The data are shown as % of cells that are annexin V positive (annexin V + ).
  • FIG. 15B Induction of Cell Death in Human Jurkat Cells by Soluble VFX4 Humanized m Ahs.
  • Jurkat cells (lxlO 4 ) were incubated with 1 pg/ml VFX4 humanized m Ahs (VFX4hum_01 IgGl, VFX4hum_01 IgG4PE, VFX4hum_02 IgGl, VFX4hum_02 IgG4PE) in RPMI media for 24 hours at 37°C.
  • Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. The data are shown as % of the cells that are annexin V positive/7 - AAD negative (annexin VV7-AAD ).
  • FIG. 15C Induction of Cell Death in Human Jurkat Cells by Soluble VLX4 Humanized m Ahs.
  • Jurkat cells (lxlO 4 ) were incubated with 1 pg/ml VLX4 humanized m Ahs (VLX4hum_01 IgGl, VLX4hum_01 IgG4PE, VLX4hum_02 IgGl, VLX4hum_02 IgG4PE) in RPMI media for 24 hours at 37°C.
  • Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. The data are shown as % of cells that are annexin V positive/7- AAD positive (annexin V + /7-AAD + ).
  • FIG. 15D Induction of Cell Death in Human Jurkat Cells by Soluble VLX4 Humanized m Ahs.
  • Jurkat cells (lxlO 4 ) were incubated with 1 pg/ml VLX4 humanized m Ahs (VLX4hum_06 IgG4PE, VLX4hum_07 IgG4PE, VLX4hum_08 IgG4PE, VLX4hum_l l IgG4PE, VLX4hum_12 IgG4PE, VLX4hum_13 IgG4PE) in RPMI media for 24 hours at 37°C. Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. The data are shown as the % of cells that are annexin V positive (annexin V + ).
  • FIG. 15E Induction of Cell Death in Human Jurkat Cells by Soluble VLX4 Humanized m Ahs.
  • Jurkat cells (lxlO 4 ) were incubated with 1 pg/ml VLX4 humanized m Ahs (VLX4hum_06 IgG4PE, VLX4hum_07 IgG4PE, VLX4hum_08 IgG4PE, VLX4hum_l l IgG4PE, VLX4hum_12 IgG4PE, VLX4hum_13 IgG4PE) in RPMI media for 24 hours at 37°C. Cells were then stained with annexin V and 7-AAD by flow cytometry. The data are shown as the % of cells that are annexin V positive/7-AAD negative (annexin V77-AAD ).
  • FIG. 15F Induction of Cell Death in Human Jurkat Cells by Soluble VLX4 Humanized m Ahs.
  • Jurkat cells (lxlO 4 ) were incubated with 1 pg/ml VLX4 humanized m Ahs (VLX4hum_06 IgG4PE, VLX4hum_07 IgG4PE, VLX4hum_08 IgG4PE, VLX4hum_l l IgG4PE, VLX4hum_12 IgG4PE, VLX4hum_13 IgG4PE) in RPMI media for 24 hours at 37°C. Cells were then stained with annexin V and and 7-AAD and analyzed by flow cytometry. The data are shown as the % of cells that are annexin V positive/7- A AD positive (annexin + /7- AAD + ).
  • FIG. 16A Induction of Cell Death in Human Jurkat Cells by Soluble VLX8 CD47 Chimeric mAbs.
  • Jurkat cells (lxlO 4 ) were incubated with 1 pg/ml VLX8 chimeric m Ahs (VLX8 IgGl N297Q xi and VLX8 IgG4PE xi) in RPMI media for 24 hrs at 37°C. Cells were then stained with annexin V and analyzed by flow cytometry. The data are presented as % of cells that are annexin V positive (annexin V + ).
  • FIG. 16B Induction of Cell Death in Human Jurkat Cells by Soluble VLX8 Chimeric m Ahs.
  • Jurkat cells (lxlO 4 ) were incubated with 1 pg/ml VLX8 chimeric m Ahs (VLX8 IgGl N297Q xi and VLX8 IgG4PE xi) in RPMI media for 24 hrs at 37°C.
  • Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. The data are presented as the % of cells that are annexin V positive/7- A AD negative (annexin VV7-AAD ).
  • FIG. 16C Induction of Cell Death in Human Jurkat Cells by Soluble VLX8 Chimeric m Ahs.
  • Jurkat cells (lxlO 4 ) were incubated with 1 pg/ml VLX8 chimeric m Ahs (VLX8 IgGl N297Q xi and VLX8 IgG4PE (xi) in RPMI media for 24 hrs at 37°C.
  • Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. The data are presented as the % of cells that are annexin V positive/7-AAD positive (annexin V + /7-AAD + ).
  • FIG. 16D Induction of Cell Death in Human Jurkat Cells by Soluble VLX8 Humanized mAbs.
  • Jurkat cells (lxlO 4 ) were incubated with 1 pg/ml VLX8 humanized m Ahs (VLX8hum_02 IgG4PE, VLX8hum_04 IgG4PE, VLX8hum_07 IgG4PE and VLX8hum_08 IgG4PE) and chimeric mAh VLX8 IgG4PE in RPMI media for 24 hrs at 37°C.
  • Cells were then stained with annexin V and analyzed by flow cytometry. The data are presented as the % of cells that are annexin V positive (annexin V + ).
  • FIG. 16E Induction of Cell Death in Human Jurkat Cells by Soluble VLX8 Humanized mAbs.
  • Jurkat cells (lxlO 4 ) were incubated with 1 pg/ml VLX8 humanized m Ahs (VLX8hum_02 IgG4PE, VLX8hum_04 IgG4PE, VLX8hum_07 IgG4PE and VLX8hum_08 IgG4PE) and chimeric mAh VLX8 IgG4PE in RPMI media for 24 hrs at 37°C.
  • Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. The data are shown as the % of cells that are annexin V positive/7- A AD negative (annexin V77-AAD ).
  • FIG. 16F Induction of Cell Death in Human Jurkat Cells by Soluble VLX8 Humanized mAbs.
  • Jurkat cells (lxlO 4 ) were incubated with 1 pg/ml VLX8 humanized m Ahs (VLX8hum_02 IgG4PE, VLX8hum_04 IgG4PE, VLX8hum_07 IgG4PE and VLX8hum_08 IgG4PE) and chimeric mAh VLX8 IgG4PE in RPMI media for 24 hrs at 37°C.
  • Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. The data are shown as the % of cells that are annexin V positive/7- A AD positive (annexin V + /7-AAD + ).
  • FIG. 17A Induction of Cell Death of Human Jurkat Cells by Soluble VLX9 Chimeric mAbs.
  • lxlO 4 Jurkat cells were incubated with 1 pg/ml of the VLX9 CD47 chimeric mAbs (VLX9 IgGl N297Q xi, VLX9 IgG2 xi and VLX9 IgG4PE xi) in RPMI media for 24 hours 37°C. Cells were then stained with annexin V and the signal analyzed by flow cytometry. The data are shown as % of cells that are annexin V positive (annexin V + ).
  • FIG. 17B Induction of Cell Death of Human Jurkat Cells by Soluble VLX9 Chimeric mAbs.
  • lxlO 4 Jurkat cells were incubated with 1 pg/ml of the VLX9 CD47 chimeric mAbs (VLX9 IgGl N297Q xi, VLX9 IgG2 xi and VLX9 IgG4PE xi) in RPMI media for 24 hours 37°C. Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. The data are shown as % of cells that are annexin V positive/7- A AD negative (annexin VV7-AAD ).
  • FIG. 17C Induction of Cell Death of Human Jurkat Cells by Soluble VLX9 Chimeric mAbs.
  • lxlO 4 Jurkat cells were incubated with lpg/ml of the VLX9 CD47 chimeric mAbs (VLX9 IgGl N297Q xi, VLX9 IgG2 xi and VLX9 IgG4PE xi) in RPMI media for 24 hours 37°C. Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. The data are shown as % of cells that are annexin V positive/7- A AD positive (annexin V + /7-AAD + ).
  • FIG. 17D Induction of Cell Death in Human Jurkat Cells by Soluble VLX9 Humanized mAbs.
  • Jurkat cells (lxlO 4 ) were incubated with 1 pg/ml VLX9 humanized mAbs (VLX9hum_01 to 10 IgG2) and chimeric mAh VLX9 IgG2 xi in RPMI media for 24 hours at 37°C. Cells were then stained with annexin V and the signal was detected by flow cytometry.
  • VLX9 IgG2 (xi) is a murine/human chimera. The data are shown as % of cells that are annexin
  • FIG. 17E Induction of Cell Death in Human Jurkat Cells by Soluble VLX9 Humanized mAbs.
  • Jurkat cells (lxlO 4 ) were incubated with 1 pg/ml VLX9 humanized mAbs (VLX9hum_01 to 10 IgG2) and chimeric mAh VLX9 IgG2 (xi) in RPMI media for 24 hours at 37°C.
  • Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry.
  • VLX9 IgG2 (xi) is a murine/human chimera. The data are shown as % of cells that are annexin
  • FIG. 17F Induction of Cell Death in Human Jurkat Cells by Soluble VLX9 Humanized mAbs.
  • Jurkat cells (lxlO 4 ) were incubated with 1 pg/ml VLX9 humanized mAbs (VLX9hum_01 to 10 IgG2) and chimeric mAh VLX9 IgG2 xi in RPMI media for 24 hours at 37°C.
  • Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry.
  • VLX9 IgG2 (xi) is a murine/human chimera. The data are shown as the % of cells that are annexin V positive/7- A AD positive (annexin V + /7-AAD + ).
  • FIG. 18 Induction of Mitochondrial Depolarization in Human Raji Cells by Soluble VLX4, VLX8 and VLX9 Humanized mAbs.
  • lxlO 4 Raji cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l l IgG4PE, VLX9hum_03 IgG2, VLX9hum_06 IgG2 and VLX9hum_08 IgG2), a negative IgG control antibody or 1 mM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours. Cells were washed and the change in JC-1 dye fluorescence was assessed using flow cytometry. The data are expressed as % of cells with mitochondrial depolarization.
  • FIG. 19 Soluble VLX4, VLX8 and VLX9 Humanized m Ahs cause an Increase in Cell Surface Calreticulin Expression on Human Raji Cells.
  • lxlO 4 Raji cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l l IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 mM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours. Cells were washed and calreticulin expression was assessed using flow cytometry. The data are expressed as % of cells that are calreticulin positive.
  • FIG. 20 Soluble VLX4, VLX8 and VLX9 Humanized m Ahs cause an Increase in Cell Surface Protein Disulfide-Isomerase A3 (PDIA3) Expression on Human Raji Cells.
  • lxlO 4 Raji cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l l IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 pM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours. Cells were washed and PDIA3 expression was assessed using flow cytometry. The data are expressed as % of cells that are PDIA3 positive.
  • FIG. 21 Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase Cell Surface HSP70 Expression on Human Raji Cells. lxlO 4 Raji cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l 1 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 pM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours. Cells were washed and HSP70 expression was assessed using flow cytometry. The data are expressed as % of cells that are HSP70 positive.
  • FIG. 22 Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase Cell Surface HSP90 Expression on Human Raji Cells. lxlO 4 Raji cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l 1 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 pM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours.
  • VLX4hum_01 IgG4PE VLX4hum_07 IgG4PE
  • VLX8hum_l 1 IgG4PE VLX9hum_06 IgG2
  • FIG. 23 Soluble VLX4, VLX8 and VLX9 Humanized m Ahs Increase Release of Adenosine Triphosphate (ATP) by Human Raji Cells.
  • FIG. 24 Soluble VLX4, VLX8 and VLX9 Humanized m Ahs cause an Increase in Release of High Mobility Group Box 1 (HMGB 1) by Human Raji Cells.
  • HMGB 1 High Mobility Group Box 1
  • lxlO 4 Raji cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l l IgG4PE, VLX9hum_03 IgG2, VLX9hum_06 IgG2 and VLX9hum_08 IgG2), a negative IgG control antibody or 1 pM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours.
  • Cell-free supernatant was collected and analyzed using an HMGB 1 immunoassay. The data are expressed as ng/ml of
  • FIG. 25 Soluble VLX4, VLX8 and VLX9 Humanized m Ahs Increase CXCL10 Release by Human Raji Cells.
  • lxlO 4 Raji cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l 1 IgG4PE, VLX9hum_03 IgG2, VLX9hum_06 IgG2 and VLX9hum_08 IgG2), a negative IgG control antibody or 1 pM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours.
  • Cell-free supernatant was collected and analyzed using an CXCL10 immunoassay. The data are expressed as pg/ml of CXCL10 in the supernatant.
  • FIG.26 Induction Mitochondrial Depolarization in Human Jurkat Cells by Soluble VLX4, VLX8 and VLX9 Humanized m Ahs.
  • lxlO 4 Jurkat cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l 1 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 pM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours. Cells were washed and the change in JC-1 dye fluorescence was assessed using flow cytometry. The data are expressed as % of cells with mitochondrial depolarization.
  • FIG. 27 Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase Cell Surface Calreticulin Expression on Human Jurkat Cells.
  • lxlO 4 Jurkat cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l l IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 mM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours. Cells were washed and calreticulin expression was assessed using flow cytometry. The data are expressed as % of cells that are calreticulin positive.
  • FIG. 28 Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase Cell Surface PDIA3 Expression on Human Jurkat Cells.
  • lxlO 4 Jurkat cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l 1 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 mM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours. Cells were washed and PDIA3 expression was assessed using flow cytometry. The data are expressed as % of cells that are PDIA3 positive.
  • FIG. 29 Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase Cell Surface HSP70 Expression on Human Jurkat Cells.
  • lxlO 4 Jurkat cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l 1 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 mM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours. Cells were washed and HSP70 expression was assessed using flow cytometry. The data are expressed as % of cells that are HSP70 positive.
  • FIG. 30 Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase Cell Surface HSP90 Expression on Human Jurkat Cells.
  • lxlO 4 Jurkat cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l 1 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 mM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours. Cells were washed and HSP90 expression was assessed using flow cytometry. The data are expressed as % of cells that are HSP90 positive.
  • FIG.31 Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase ATP Release by Human Jurkat Cells.
  • lxlO 4 Jurkat cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l 1 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 mM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours. Cell-free supernatant was collected and analyzed using an ATP determination kit. The data are expressed as pM ATP in the supernatant.
  • FIG. 32 Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase HMGB 1 Release by Human Jurkat Cells.
  • lxlO 4 Jurkat cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l 1 IgG4PE, VLX9hum_03 IgG2, VLX9hum_06 IgG2 and VLX9hum_08 IgG2), a negative IgG control antibody or 1 mM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours. Cell-free supernatant was collected and analyzed using an HMGB 1 immunoassay. The data are expressed as ng/ml of HMGB 1 in the supernatant.
  • FIG. 33A Agglutination of hRBCs by VLX4 Humanized mAbs. Hemagglutination was assessed following incubation of hRBCs with various concentrations of humanized VLX4 mAbs (VLX4hum_01 IgGl and VLX4hum_01 IgG4PE). Blood was diluted (1 :50) and washed 3 times with PBS/EDTA/BSA. hRBCs were added to U-bottomed 96 well plates with equal volumes of the antibodies (75 m ⁇ ) and incubated for 3 hrs at 37°C and overnight at 4°C.
  • FIG. 33B Agglutination of hRBCs by VLX8 Chimeric and Humanized mAbs. Hemagglutination was assessed following incubation of hRBCs with various concentrations of humanized VLX8 mAbs (VLX8hum_01 IgG4PE, VLX8hum_02 IgG4PE VLX8hum_03 IgG4PE, VLX8hum_08 IgG4PE, VLX8hum_09 IgG4PE, VLX8hum_10 IgG4PE and VLX8hum_ll IgG4PE) and the chimeric mAh VLX8 IgG4PE xi.
  • VLX8hum_01 IgG4PE VLX8hum_02 IgG4PE
  • VLX8hum_03 IgG4PE VLX8hum_08 IgG4PE
  • VLX8hum_09 IgG4PE VLX8hum_10 IgG4PE and V
  • Blood was diluted (1 :50) and washed 3 times with PBS/EDTA/BSA.
  • hRBCs were added to U-bottomed 96 well plates with equal volumes of the antibodies (75 m ⁇ ) and incubated for 3 hrs at 37°C and overnight at 4°C.
  • FIG. 34A Agglutination of Human RBCs by VLX9 Humanized mAbs. Hemagglutination was assessed following incubation of human RBCs with various concentrations of VLX9 IgG2 chimera (xi) and humanized VLX9 mAbs (VLX9hum_01 to _05 IgG2). Blood was diluted (1 :50) and washed 3 times with PBS/EDTA/BSA. RBCs were added to U-bottomed 96 well plates with equal volumes of the antibodies (75 m ⁇ ) and incubated for 3 hrs at 37°C and overnight at 4°C.
  • FIG. 34B Agglutination of Human RBCs by VLX9 Humanized mAbs. Hemagglutination was assessed following incubation of human RBCs with various concentrations of VLX9 IgG2 chimera (xi) and humanized VLX9 mAbs (VLX9hum_06 to _10 IgG2). Blood was diluted (1 :50) and washed 3 times with PBS/EDTA/BSA. RBCs were added to U-bottomed 96 well plates with equal volumes of the antibodies (75 m ⁇ ) and incubated for 3 hrs at 37°C and overnight at 4°C.
  • FIG.35 VLX4 Humanized mAh Reduces Tumor Growth in Raji Xenograft Model.
  • Female NSG mice were inoculated subcutaneously in the right flank with 0.1 mL of a 30% RPMI / 70% MatrigelTM mixture containing a suspension of 5xl0 6 Raji tumor cells.
  • Five days following inoculation tumor volumes were measured and mice with palpable tumor volumes of 31-74 mm 3 were randomized into 8-10/group.
  • VLX4hum_07 IgG4PE or PBS (control) administration was initiated at this time.
  • Mice were treated with 5 mg/kg of antibody 5X/week for 4 weeks by intraperitoneal injection. Tumor volumes and body weights were recorded twice weekly.
  • FIG.36 VLX8 Humanized mAh Reduces Tumor Growth in Raji Xenograft Model.
  • Female NSG mice were inoculated subcutaneously in the right flank with 0.1 mL of a 30% RPMI / 70% MatrigelTM mixture containing a suspension of 5xl0 6 Raji tumor cells.
  • Five days following inoculation tumor volumes were measured and mice with palpable tumor volumes of 31-74 mm 3 were randomized into 8-10/group.
  • VFX8hum_10 IgG4PE or PBS (control) administration was initiated at this time.
  • Mice were treated with 5 mg/kg of antibody 5X/week for 4 weeks by intraperitoneal injection. Tumor volumes and body weights were recorded twice weekly.
  • FIG.37 VEX9 Humanized mAh Reduces Tumor Growth in Raji Xenograft Model.
  • Female NSG mice were inoculated subcutaneously in the right flank with 0.1 mL of a 30% RPMI / 70% MatrigelTM mixture containing a suspension of 5xl0 6 Raji tumor cells.
  • Five days following inoculation tumor volumes were measured and mice with palpable tumor volumes of 31-74 mm 3 were randomized into 8-10/group.
  • VFX9hum_08 IgG2 or PBS (control) administration was initiated at this time.
  • Mice were treated with 5 mg/kg of antibody 5X/week for 4 weeks by intraperitoneal injection. Tumor volumes and body weights were recorded twice weekly.
  • FIG. 38A Hemoglobin Fevels in Blood Following Administration of a Humanized VFX9 mAh to Cynomolgus Monkeys by Intravenous Infusion.
  • VFX9hum_08 IgG2 or vehicle were administered as a one hour intravenous infusion a dose of 5mg/kg on day 1 and a dose of 15mg/kg on day 18.
  • Hemoglobin levels were monitored throughout the study and normali ed to control values.
  • FIG. 38B RBC Fevels in Blood Following Administration of Humanized VFX9 m Ahs to Cynomolgus Monkeys by Intravenous Infusion.
  • VFX9hum_08 IgG2 or vehicle was administered as a one hour intraveneous infusion a dose of 5mg/kg on day 1 and a dose of 15mg/kg on day 18.
  • RBC levels were monitored throughout the study and normalized to control values.
  • FIG. 39A Confocal microscopy of CD47 staining pattern on human red blood cells with CD47 antibodies VFX9_04, VFX9_06, VFX9_08 or VLX4_01. RBCs were incubated with primary antibody for 1 hour, washed and stained with fluorophore labeled secondary antibody. After staining, cells were washed and imaged (Top: immunofluorescence, Bottom: brightfield)
  • FIG. 39B Confocal microscopy of CD47 (red) and b ⁇ Integrin (green) staining pattern on Jurkat T-ALL cells with CD47 antibodies.
  • Jurkats were co-stained with CD47 antibodies VLX9_04 IgG2, VLX9_06 IgG2, VLX9_08 IgG2, or VLX4_01 IgG4PE and b ⁇ Integrin antibodies for 1 hour, washed, and stained with flurorophore labeled secondary antibody. After staining, cells were washed and imaged.
  • FIG. 40 Summary of Anti-CD47 Antibody Properties.
  • FIG. 41 Confocal microscopy of CD47 (yellow), b ⁇ Integrin (blue), GFP (purple) staining on Jurkat T-ALL b ⁇ Integrin CRISPR/Cas9 knockout cells with CD47 antibody VLX9_04 IgG2, VLX9_06 IgG2, VLX_08 IgG2 or another anti-CD47 mAh (Hu5L9-G4, PCT International Publication WO 2011/143624). Arrows indicate b ⁇ Integrin knocked-out cells that do not bind with VLX9_06 IgG2, but do bind with the Hu5F9-G4 CD47 antibody.
  • FIG. 42 Confocal microscopy staining pattern on Jurkat T-ALL cells with SIRPa variant vl or v2 (red) for 1 hour, and then incubated with b ⁇ Integrin antibodies for another 1 hour and stained with fluorophore labeled secondary antibodies.
  • FIG. 43 VLX9_06 IgG2 Humanized mAh blocks SIRPa variant 1 and 2 binding to CD47 on Human Jurkat Cells.
  • FIG. 44A Immunoprecipitation of CD47 from Jurkat cells with the anti-CD47 antibodies VLX9_06 IgG2 or Hu5F9-G4, or a negative control IgG2.
  • the immunoprecipitated CD47 was determined by immunoblotting using the anti-CD47 antibody BRIC126.
  • Control lysate shows endogenous levels of CD47 in Jurkat cells.
  • FIG. 44B Ability of b ⁇ -integrin to be co-immunoprecipitated by the anti-CD47 mAbs, VLX9_06 and Hu5F9-G4 and IgG2 control antibodies. Control lysate shows endogenous levels of b ⁇ -integrin in Jurkat cells.
  • FIG. 45 Co-localization of CD47 and b ⁇ -integrin on gastric carcinoma cells. Localization of anti-CD47 antibodies on tumor cells expressing b ⁇ -integrin was determined by confocal microscopy. SNU-1 gastric carcinoma cells were seeded on glass and incubated with either VLX9hum_06 IgG2 or Hu5L9-G4 and an integrin beta 1 antibody [mouse anti-Integrin beta 1 antibody for 1 hour at 37°C, then washed and stained with both anti-human IgG (H+L) antibody and anti-mouse IgG (H+L) antibody for 1 hour. After fixation, cells were imaged using a confocal microscope.
  • FIG. 46A - FIG. 46C Decreased binding of humanized VLX9hum_06 IgG2 antibody to normal hematological cells compared to tumor by confocal microcopy. Co localization of CD47 and b ⁇ -integrin on tumor cells and normal hematological cells was determined by confocal microscopy (FIG. 46 A).
  • FIG. 47A - FIG. 47B Comparison of the binding of VLX9hum_06 IgG2 and b ⁇ - integrin antibodies to normal hematological cells to tumor by flow cytometry. Binding of anti- CD47 and anti-b ⁇ -integrin antibodies to tumor cells and normal hematological cells was determined by flow cytometry. Cultured Jurkat T-ALL cells or healthy normal human blood derived hRBCs, neutrophils, monocytes, platelets and T cells were incubated for 60 min at 37°C with increasing concentrations of either VLX9hum_06 IgG2 or anti-b ⁇ -integrin antibodies diluted in PBS.
  • FIG. 48A - FIG. 48C Binding of VLX9hum_06 IgG2 involves b ⁇ -integrin expression. Binding of CD47 antibodies to K562 erythroblast-like cells and to mature hRBCs was determined by flow cytometry b ⁇ -integrin levels in these cell lines was determined by western blot. K562 or freshly isolated hRBCs were incubated for 60 min at 37°C with increasing concentrations of VLX9hum_06 IgG2 or Hu5F9-G4 diluted in PBS. Antibody binding was determined using flow cytometry. Quantification of b ⁇ -integrin levels was determined by Western blot on protein lysates taken from K562 cells and hRBCs.
  • FIG. 49A - FIG. 49B Binding of VLX9hum_06 IgG2 interferes with b ⁇ -integrin signaling pathways in Jurkat cells.
  • the effect of CD47 antibodies on downstream mediators of b ⁇ -integrin signaling in tumor cells was determined by phosphoblot analysis of treated Jurkat cells.
  • FIG. 49A wildtype Jurkat cells were treated either with control IgG2 antibody, various concentrations of VLX9hum_06 IgG2 or Hu5F9-G4 for 90 minutes then the cells were lysed in RIPA buffer with HALT protease-phosphatase inhibitor. Lysates were blotted with antibodies to FAK, pFAK, SRC, and pSRC.
  • FIG. 49A wildtype Jurkat cells were treated either with control IgG2 antibody, various concentrations of VLX9hum_06 IgG2 or Hu5F9-G4 for 90 minutes then the cells were lysed in RIPA buffer with HALT protease-phosphatase inhibitor
  • b ⁇ -integrin knockout Jurkat cells were generated by CRISPR/Cas9 along with control lines and cell lysates were harvested and blotted with antibodies to b ⁇ -integrin, FAK, pFAK, SRC, and pSRC.
  • FIG. 50A - FIG. 50B VLX9hum_06 IgG2 interferes with b ⁇ -integrin signaling pathways in SNU-1 gastric carcinoma cells.
  • the effect of CD47 antibodies on downstream mediators of b ⁇ -integrin signaling in tumor cells was determined by phosphoblot analysis of treated SNU-lcells.
  • SNU-1 cells were treated either with control IgG2 antibody, VLX9hum_06 IgG2 or Hu5F9-G4 for 1, 2 or 3 hours. Lysates were blotted with antibodies to SRC, and pSRC (FIG. 50A). Quantification of pSrc/total Src levels was determined using ImageJ software (FIG. 50B).
  • FIG. 51A - FIG. 51H Induction of Cell Death in Gastric Carcinoma Cells by Soluble VLX9hum_06 IgG2.
  • Gastric carcinoma cell lines SNU-1, KATOIII, SNU-16, Hs746T, SNU-5, NCI-N87, AGS or OE19 cells (lxlO 4 ) were incubated with 100 pg/ml VLX9hum_06 IgG2 or control for 24 hours at 37°C. Cells were then stained with annexin V and the signal was detected by flow cytometry. The data are shown as % of cells that are annexin V positive (annexin V + ).
  • CD47 “integrin-associated protein (IAP),”“ovarian cancer antigen OA3,”“Rh-related antigen,” and“MERG” are synonymous and may be used interchangeably.
  • anti-CD47 antibody refers to an antibody of the disclosure which is intended for use as a therapeutic or diagnostic agent, and therefore will typically possess the binding affinity required to be useful as a therapeutic and/or diagnostic agent.
  • the term“antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e. , molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • immunoglobulin immunoglobulin
  • By “specifically binds” or“immunoreacts with” or“directed against” is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides or binds at a much lower affinity (Kd > 10 6 ).
  • Antibodies include but are not limited to, polyclonal, monoclonal, chimeric, Fab fragments, Fab’ fragments, F(ab’)2 fragments, single chain Fv fragments, and one-armed antibodies.
  • mAb monoclonal antibody
  • mAb monoclonal antibody
  • the term“monoclonal antibody” (mAb) as applied to the present antibody compounds refers to an antibody that is derived from a single copy or clone including, for example, any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced m Ahs of the present disclosure preferably exist in a homogeneous or substantially homogeneous population.
  • Complete mAbs contain 2 heavy chains and 2 light chains.
  • an“antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include but are not limited to Fv, Fab, Fab’, Fab’ -SH, F(ab’)2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.
  • antibody compounds refers to mAbs and antigen-binding fragments thereof. Additional antibody compounds exhibiting similar functional properties according to the present disclosure can be generated by conventional methods. For example, mice can be immunized with human CD47 or fragments thereof, the resulting antibodies can be recovered and purified, and determination of whether they possess binding and functional properties similar to or the same as the antibody compounds disclosed herein can be assessed by the methods described in Examples 3-17 below. Antigen-binding fragments can also be prepared by conventional methods.
  • the monoclonal antibodies encompass antibodies in which a portion of the heavy and/or light chain is identical with, or homologous to, corresponding sequences in murine antibodies, in particular the murine CDRs, while the remainder of the chain(s) is (are) identical with, or homologous to, corresponding sequences in human antibodies.
  • Other embodiments of the disclosure include antigen-binding fragments of these monoclonal antibodies that exhibit binding and biological properties similar or identical to the monoclonal antibodies.
  • the antibodies of the present disclosure can comprise kappa or lambda light chain constant regions, and heavy chain IgA, IgD, IgE, IgG, or IgM constant regions, including those of IgG subclasses IgGl, IgG2, IgG3, and IgG4 and in some cases with various mutations to alter Fc receptor function.
  • the monoclonal antibodies containing the presently disclosed murine CDRs can be prepared by any of the various methods known to those skilled in the art, including recombinant DNA methods.
  • a full-length antibody as it exists naturally is a“Y” shaped immunoglobulin (Ig) molecule comprising four polypeptide chains: two identical heavy (H) chains and two identical light (L) chains, interconnected by disulfide bonds.
  • the amino terminal portion of each chain termed the fragment antigen binding region (FAB), includes a variable region of about 100- 110 or more amino acids primarily responsible for antigen recognition via the complementarity determining regions (CDRs) contained therein.
  • CDRs complementarity determining regions
  • the carboxy-terminal portion of each chain defines a constant region (the“Fc” region) primarily responsible for effector function.
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of 3 CDRs and 4 FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the 3 CDRs of the light chain are referred to as “LCDR1, LCDR2, and LCDR3” and the 3 CDRs of the heavy chain are referred to as“HCDRl, HCDR2, and HCDR3.”
  • the CDRs contain most of the residues which form specific interactions with the antigen.
  • the “antigen-binding site” can also be defined as the “hypervariable regions,”“HVRs,” or“HVs,” and refer to the structurally hypervariable regions of antibody variable domains as defined by Chothia and Lesk (Chothia and Lesk, Mol. Biol. 196:901-917, 1987). There are six HVRs, three in VH (HI, H2, H3) and three in VL (LI, L2, L3).
  • the CDRs used herein are as defined by Kabat except in H-CDR1, which is extended to include HI.
  • Ig heavy chains There are five types of mammalian immunoglobulin (Ig) heavy chains, denoted by the Greek letters a (alpha), d (delta), e (epsilon), g (gamma), and m (mu), which define the class or isotype of an antibody as IgA, IgD, IgE, IgG, or IgM, respectively.
  • IgG antibodies can be further divided into subclasses, for example, IgGl, IgG2, IgG3, and IgG4.
  • Each heavy chain type is characterized by a particular constant region with a sequence well known in the art.
  • the constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes.
  • Heavy chains g, a, and d have a constant region composed of three tandem immunoglobulin (Ig) domains, and a hinge region for added flexibility.
  • Heavy chains m and e have a constant region composed of four Ig domains.
  • the hinge region is a flexible amino acid stretch that links the Fc and Fab portions of an antibody. This regions contains cysteine residues that can form disulfide bonds, connecting two heavy chains together.
  • variable region of the heavy chain differs in antibodies produced by different B cells, but is the same for all antibodies produced by a single B cell or B cell clone.
  • the variable region of each heavy chain is approximately 110 amino acids long and is composed of a single Ig domain.
  • light chains are classified as kappa (K) or lambda (l), and are characterized by a particular constant region as known in the art.
  • a light chain has two successive domains: one variable domain at the amino-terminal end, and one constant domain at the carboxy-terminal end.
  • Each antibody contains two light chains that are always identical; only one type of light chain, k or l, is present per antibody in mammals.
  • the Fc region composed of two heavy chains that contribute three or four constant domains depending on the class of the antibody, plays a role in modulating immune cell activity. By binding to specific proteins, the Fc region ensures that each antibody generates an appropriate immune response for a given antigen.
  • the Fc region also binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins. By doing this, it mediates different physiological effects, including opsonization, cell lysis, and degranulation of mast cells, basophils and eosinophils.
  • epitope refers to a specific arrangement of amino acids located on a peptide or protein to which an antibody or antibody fragment binds.
  • Epitopes often consist of a chemically active surface grouping of molecules such as amino acids or sugar side chains, and have specific three dimensional structural characteristics as well as specific charge characteristics.
  • Epitopes can be linear, i.e. , involving binding to a single sequence of amino acids, or conformational, i.e., involving binding to two or more sequences of amino acids in various regions of the antigen that may not necessarily be contiguous in the linear sequence.
  • the terms “specifically binds,” “bind specifically,” “specific binding,” and the like as applied to the present antibody compounds refer to the ability of a specific binding agent (such as an antibody) to bind to a target molecular species in preference to binding to other molecular species with which the specific binding agent and target molecular species are admixed.
  • a specific binding agent is said specifically to recognize a target molecular species when it can bind specifically to that target.
  • binding affinity refers to the strength of binding of one molecule to another at a site on the molecule. If a particular molecule will bind to or specifically associate with another particular molecule, these two molecules are said to exhibit binding affinity for each other. Binding affinity is related to the association constant and dissociation constant for a pair of molecules as measured in a 1 : 1 interaction. Affinities as used herein to describe interactions between molecules of the described methods which can be used to compare the relative strength with which one molecule (e.g . , an antibody or other specific binding partner) will bind two other molecules (e.g., two versions or variants of a peptide) in a univalent interaction. The concepts of binding affinity, association constant, and dissociation constant are well known.
  • the term“apparent binding affinity” refers to the apparent strength of binding of one molecule to another at a site on the molecule. If a particular molecule will bind to or specifically associate with another particular molecule, these two molecules are said to exhibit binding affinity for each other.
  • Apparent binding affinity is related to the association constant and dissociation constant for a pair of molecules, and relates to a non 1 :1 or multivalent association between the pair of molecules.
  • Apparent affinities as used herein to describe interactions between molecules of the described methods are observed in empirical studies, which can be used to compare the relative strength with which one molecule (e.g., an antibody or other specific binding partner) will bind two other molecules (e.g. , two versions or variants of a peptide).
  • the concept of binding affinity may be described as apparent Kd, apparent binding constant, EC50 or other measurements of binding.
  • sequence identity means the percentage of identical nucleotide or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e. , taking into account gaps and insertions. Identity can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H.
  • Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, by the homology alignment algorithms, by the search for similarity method or, by computerized implementations of these algorithms (GAP, BESTFIT, PASTA, and TFASTA in the GCG Wisconsin Package, available from Accelrys, Inc., San Diego, California, United States of America), or by visual inspection. See generally, Altschul, S. F. et al , J. Mol. Biol. 215: 403-410 (1990) and Altschul et al. Nucl. Acids Res. 25: 3389-3402 (1997).
  • One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BEAST algorithm, which is described in (Altschul, S. et al, NCBI NEM NIH Bethesda, Md. 20894; and Altschul, S. et al, J. Mol. Biol. 215: 403-410 (1990).
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold.
  • HSPs high scoring sequence pairs
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix.
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences.
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is in one embodiment less than about 0.1, in another embodiment less than about 0.01, and in still another embodiment less than about 0.001.
  • the terms“humanized,”“humanization,” and the like refer to grafting of the murine monoclonal antibody CDRs disclosed herein to human FRs and constant regions. Also encompassed by these terms are possible further modifications to the murine CDRs, and human FRs, by the methods disclosed in, for example, Kashmiri et al. (2005) Methods 36(l):25-34 and Hou et al. (2008) J. Biochem. 144(1): 115- 120, respectively, to improve various antibody properties, as discussed below.
  • humanized antibodies refers to mAbs and antigen binding fragments thereof, including antibody compounds, that have binding and functional properties similar to those disclosed herein, and that have FRs and constant regions that are substantially human or fully human surrounding CDRs derived from a non-human antibody.
  • FR or“framework sequence” refers to any one of FRs 1 to 4.
  • Humanized antibodies and antigen binding fragments encompassed by the present disclosure include molecules wherein any one or more of FRs 1 to 4 is substantially or fully human, i.e., wherein any of the possible combinations of individual substantially or fully human FRs 1 to 4, is present. For example, this includes molecules in which FR1 and FR2, FR1 and FR3, FR1, FR2, and FR3, etc., are substantially or fully human.
  • Substantially human frameworks are those that have at least 80% sequence identity to a known human germline framework sequence.
  • the substantially human frameworks have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, to a framework sequence disclosed herein, or to a known human germline framework sequence.
  • Fully human frameworks are those that are identical to a known human germline framework sequence.
  • Human FR germline sequences can be obtained from the international ImMunoGeneTics (IMGT) database and from The Immunoglobulin FactsBook by Marie-Paule Lefranc and Gerard Lefranc, Academic Press, 2001, the contents of which are herein incorporated by reference in their entirety.
  • IMGT ImMunoGeneTics
  • the Immunoglobulin Facts Book is a compendium of the human germline immunoglobulin genes that are used to create the human antibody repertoire, and includes entries for 203 genes and 459 alleles, with a total of 837 displayed sequences.
  • the individual entries comprise all the human immunoglobulin constant genes, and germline variable, diversity, and joining genes that have at least one functional or open reading frame allele, and which are localized in the three major loci.
  • germline light chain FRs can be selected from the group consisting of: IGKV3D-20, IGKV2-30, IGKV2-29, IGKV2-28, IGKV1-27, IGKV3-20, IGKV1-17, IGKV1-16, 1-6, IGKV1-5, IGKV1-12, IGKV1D-16, IGKV2D-28, IGKV2D-29, IGKV3-11, IGKV1-9, IGKV1-39, IGKV1D-39, IGKV1D-33, and IGKJ1-5; and germline heavy chain FRs can be selected from the group consisting of: IGHV1- 2, IGHV1-18, IGHV1-46, IGHV1-69, IGHV2-5, IGHV2-26, IGHV2-70, IGHV1-3, IGHV1- 8, IGHV3-9, IGHV3-11, IGHV3-15, IGHV
  • Substantially human FRs are those that have at least 80% sequence identity to a known human germline FR sequence.
  • the substantially human frameworks have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, to a framework sequences disclosed herein, or to a known human germline framework sequence.
  • CDRs encompassed by the present disclosure include not only those specifically disclosed herein, but also CDR sequences having sequence identities of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a CDR sequence disclosed herein.
  • CDRs encompassed by the present disclosure include not only those specifically disclosed herein, but also CDR sequences having 1, 2, 3, 4, or 5 amino acid changes at corresponding positions compared to CDR sequences disclosed herein.
  • Such sequence identical, or amino acid modified, CDRs preferably bind to the antigen recognized by the intact antibody.
  • Humanized antibodies in addition to those disclosed herein exhibiting similar functional properties according to the present disclosure can be generated using several different methods, including those disclosed by Almagro et al. (Frontiers in Biosciences. Humanization of antibodies. (2008) Jan 1; 13: 1619-33).
  • the parent antibody compound CDRs are grafted into a human framework that has a high sequence identity with the parent antibody compound framework.
  • the sequence identity of the new framework will generally be at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identical to the sequence of the corresponding framework in the parent antibody compound.
  • frameworks having fewer than 100 amino acid residues one, two, three, four, five, six, seven, eight, nine, or ten amino acid residues can be changed.
  • the parent antibody compound CDRs are grafted into a human FR that has a high sequence identity with the parent antibody compound framework.
  • the sequence identity of the new FR will generally be at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of the corresponding FR in the parent antibody compound.
  • FRs having fewer than 100 amino acid residues one, two, three, four, five, or more amino acid residues can be changed.
  • any side chain atom of a framework amino acid is within about 5-6 angstroms
  • Another approach to generating humanized antibodies exhibiting similar functional properties to the antibody compounds disclosed herein involves randomly mutating amino acids within the grafted CDRs without changing the framework, and screening the resultant molecules for binding affinity and other functional properties that are as good as, or better than, those of the parent antibody compounds.
  • Single mutations can also be introduced at each amino acid position within each CDR, followed by assessing the effects of such mutations on binding affinity and other functional properties. Single mutations producing improved properties can be combined to assess their effects in combination with one another.
  • amino acid substitution within the frameworks is restricted to one, two, three, four, or five positions within any one or more of the four light chain and/or heavy chain FRs disclosed herein.
  • amino acid substitution within the CDRs is restricted to one, two, three, four, or five positions within any one or more of the three light chain and/or heavy chain CDRs. Combinations of the various changes within these FRs and CDRs described above are also possible.
  • murine antibodies have been genetically manipulated to progressively replace their murine content with the amino acid residues present in their human counterparts by grafting their complementarity determining regions (CDRs) onto the variable light (VL) and variable heavy (VH) frameworks of human immunoglobulin molecules, while retaining those murine framework residues deemed essential for the integrity of the antigen-combining site.
  • CDRs complementarity determining regions
  • VL variable light
  • VH variable heavy
  • the xenogeneic CDRs of the humanized antibodies may evoke anti-idiotypic (anti-id) response in patients.
  • SDR grafting a procedure to humanize xenogeneic antibodies by grafting onto the human frameworks only the CDR residues most crucial in the antibody- ligand interaction, called“SDR grafting”, has been developed, wherein only the crucial specificity determining residues (SDRs) of CDRS are grafted onto the human frameworks.
  • This procedure described inskyi et al. (2005, Methods 36(l):25-34), involves identification of SDRs through the help of a database of the three-dimensional structures of the antigen- antibody complexes of known structures, or by mutational analysis of the antibody-combining site.
  • Embodiments of the present disclosure encompass antibodies created to avoid recognition by the human immune system containing CDRs disclosed herein in any combinatorial form such that contemplated m Ahs can contain the set of CDRs from a single murine mAh disclosed herein, or light and heavy chains containing sets of CDRs comprising individual CDRs derived from two or three of the disclosed murine mAbs.
  • Such mAbs can be created by standard techniques of molecular biology and screened for desired activities using assays described herein. In this way, the disclosure provides a“mix and match” approach to create novel m Ahs comprising a mixture of CDRs from the disclosed murine m Ahs to achieve new, or improved, therapeutic activities.
  • Monoclonal antibodies or antigen-binding fragments thereof encompassed by the present disclosure that“compete” with the molecules disclosed herein are those that bind human CD47 at site(s) that are identical to, or overlapping with, the site(s) at which the present molecules bind. Competing monoclonal antibodies or antigen-binding fragments thereof can be identified, for example, via an antibody competition assay. For example, a sample of purified or partially purified human CD47 extracellular domain can be bound to a solid support. Then, an antibody compound, or antigen binding fragment thereof, of the present disclosure and a monoclonal antibody or antigen-binding fragment thereof suspected of being able to compete with such disclosure antibody compound are added. One of the two molecules is labelled.
  • the labelled compound and the unlabelled compound bind to separate and discrete sites on CD47, the labelled compound will bind to the same level whether or not the suspected competing compound is present. However, if the sites of interaction are identical or overlapping, the unlabelled compound will compete, and the amount of labelled compound bound to the antigen will be lowered. If the unlabelled compound is present in excess, very little, if any, labelled compound will bind.
  • competing monoclonal antibodies or antigen-binding fragments thereof are those that decrease the binding of the present antibody compounds to CD47 by about 50%, about 60%, about 70%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%.
  • Details of procedures for carrying out such competition assays are well known in the art and can be found, for example, in Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Such assays can be made quantitative by using purified antibodies.
  • a standard curve is established by titrating one antibody against itself, i.e., the same antibody is used for both the label and the competitor.
  • the capacity of an unlabelled competing monoclonal antibody or antigen-binding fragment thereof to inhibit the binding of the labelled molecule to the plate is titrated. The results are plotted, and the concentrations necessary to achieve the desired degree of binding inhibition are compared.
  • m Ahs or antigen-binding fragments thereof that compete with antibody compounds of the present disclosure in such competition assays possess the same or similar functional properties of the present antibody compounds can be determined via these methods in conjunction with the methods described in Examples below.
  • competing antibodies possess about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or identical biological activity compared to that of the antibody compounds disclosed herein as determined by the methods disclosed in the Examples presented below.
  • the m Ahs or antigen-binding fragments thereof, or competing antibodies useful in the compositions and methods can be any of the isotypes described herein. Furthermore, any of these isotypes can comprise further amino acid modifications as follows.
  • the monoclonal antibody or antigen-binding fragment thereof, or competing antibody described herein can be of the human IgGl isotype.
  • the human IgGl constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to alter antibody half-life.
  • Antibody half-life is regulated in large part by Fc-dependent interactions with the neonatal Fc receptor (Roopenian and Alikesh, 2007).
  • the human IgGl constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody can be modified to increase half-life include, but are not limited to amino acid modifications N434A, T307A/E380A/N434A (Petkova et al, 2006, Yeung et al , 2009); M252Y/S254T/T256E (Dall’Acqua et al. , 2006); T250Q/M428L (Hinton et al., 2006); and M428L/N434S (Zalevsky et al. , 2010).
  • ADCC Antibody-Dependent Cellular Cytotoxicity
  • CDC Complement-Dependent Cytotoxicity
  • the human IgGl constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease half-life and/or decrease endogenous IgG include, but are not limited to amino acid modifications I253A (Petkova et al, 2006); P257I/N434H, D376V/N434H (Datta-Mannan et al, 2007); and M252Y/S254T/T256E/H433K/N434F (Vaccaro et al., 2005).
  • the human IgGl constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to increase or decrease antibody effector functions.
  • These antibody effector functions include, but are not limited to, Antibody-Dependent Cellular Cytotoxicity (ADCC), Complement-Dependent Cytotoxicity (CDC), Antibody-Dependent Cellular Phagocytosis (ADCP), Clq binding, and altered binding to Fc receptors.
  • the human IgGl constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to increase antibody effector function include, but are not limited to amino acid modifications S298A/E333A/K334 (Shields et al, 2001); S239D/I332E and S239D/A330L/I332E (Lazar et al , 2006); F234L/R292P/Y300L, F234L/R292P/Y300L/P393L, and F243L/R292P/Y300L/V305I/P396L (Stevenhagen et al, 2007); G236A, G236A/S239D/I332E, and G236A/S239D/A330L/I332E (Richards et al, 2008); K326A/E333A, K326A/E333S and K326W/E333S
  • the human IgGl constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease antibody effector function include, but are not limited to amino acid modifications N297A and N297Q (Bolt et al , 1993, Walker et al , 1989); L234A/L235A (Xu et al, 2000); K214T/E233P/L234V/L235A/G236-deleted/A327G/P331A/D356E/L358M (Ghevaert et al, 2008); C226S/C229S/E233P/L234V/L235A (McEarchern et al, 2007); S267E/L328F (Chu et al, 2008).
  • the human IgGl constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease antibody effector function include, but are not limited to amino acid modifications V234A/G237A (Cole et al, 1999); E233D, G237D, P238D, H268Q, H268D, P271G, V309L, A330S, A330R, P331S, H268 Q/A330S/V309L/P331 S , H268D/A330S/V309L/P331S,
  • H268Q/A330R/V309L/P331S H268D/A330R/V309L/P331S, E233D/A330R,
  • E233D/A330S E233D/P271G/A330R, E233D/P271G/A330S, G237D/H268D/P271G,
  • P238D/G237D/H268 Q/P271 G P238D/G237D/ P271G/A330R, P238D/G237D/ P271G/A330S, P238D/E233D/H268D/P271G/A330R,
  • the monoclonal antibody or antigen-binding fragment thereof, or competing antibody described herein can be of the human IgG2 isotype.
  • the human IgG2 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to increase or decrease antibody effector functions.
  • antibody effector functions include, but are not limited to, Antibody-Dependent Cellular Cytotoxicity (ADCC), Complement-Dependent Cytotoxicity (CDC), Antibody-Dependent Cellular Phagocytosis (ADCP), and Clq binding, and altered binding to Fc receptors.
  • the human IgG2 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to increase antibody effector function include, but are not limited to the amino acid modification K326A/E333S
  • the human IgG2 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease antibody effector function include, but are not limited to amino acid modifications V234A/G237A (Cole et al, 1999); V234A, G237A, P238S, H268A, E233D, G237D, P238D, H268Q, H268D,
  • H268D/A330S/V309L/P331S H268Q/A330R/V309L/P331S, H268D/A330R/V309L/P331S, E233D/A330R, E233D/A330S, E233D/P271G/A330R, E233D/P271G/A330S,
  • G237D/H268D/P271G G237D/H268Q/P271G, G237D/ P271G/A330R, G237D/
  • P238D/G237D/H268 Q/P271 G P238D/G237D/ P271G/A330R, P238D/G237D/ P271G/A330S, P238D/E233D/H268D/P271G/A330R, P238D/E233D/H268 Q/P271 G/A330R, P238D/E233D/H268D/P271G/A330S,
  • the Fc region of a human IgG2 of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to alter isoform and/or agonistic activity, include, but are not limited to amino acid modifications C127S (CHI domain), C232S, C233S, C232S/C233S, C236S, and C239S (White et al, 2015, Lightle et al , 2010).
  • the monoclonal antibody or antigen-binding fragment thereof, or competing antibody described herein can be of the human IgG3 isotype.
  • the human IgG3 constant region of the monoclonal antibody, or antigen binding fragment thereof wherein said human IgG3 constant region of the monoclonal antibody, or antigen-binding fragment thereof can be modified at one or more amino acid(s) to increase antibody half-life, Antibody-Dependent Cellular Cytotoxicity (ADCC), Complement- Dependent Cytotoxicity (CDC), or apoptosis activity.
  • ADCC Antibody-Dependent Cellular Cytotoxicity
  • CDC Complement- Dependent Cytotoxicity
  • apoptosis activity apoptosis activity.
  • the human IgG3 constant region of the monoclonal antibody, or antigen-binding fragment thereof wherein said human IgG3 constant region of the monoclonal antibody, or antigen-binding fragment thereof can be modified at amino acid R435H to increase antibody half-life.
  • the monoclonal antibody or antigen-binding fragment thereof, or competing antibody described herein can be of the human IgG4 isotype.
  • the human IgG4 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease antibody effector functions.
  • These antibody effector functions include, but are not limited to, Antibody- Dependent Cellular Cytotoxicity (ADCC) and Antibody-Dependent Cellular Phagocytosis (ADCP).
  • the human IgG4 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to prevent Fab arm exchange and/or decrease antibody effector function include, but are not limited to amino acid modifications F234A/L235A (Alegre et ah , 1994); S228P, L235E and S228P/L235E (Reddy et ah , 2000).
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer refers to or describe the physiological condition in mammals that is typically characterized by aberrant cell growth/proliferation.
  • cancers include, but are not limited to, carcinoma, lymphoma (i.e., Hodgkin’s and non- Hodgkin’s lymphoma), blastoma, sarcoma, and leukemia.
  • cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.
  • the term“susceptible cancer” as used herein refers to a cancer, cells of which express CD47, and are responsive to treatment with an anti-CD47 antibody or antigen binding fragment thereof, or competing antibody or antigen binding fragment thereof, of the present disclosure.
  • Nitric oxide (NO) donor, precursor, or nitric oxide generating topical agent refers to a compound or agent that either delivers NO, or that can be converted to NO through enzymatic or non-enzymatic processes. Examples include, but are not limited to, NO gas, isosorbide dinitrite, nitrite, nitroprusside, nitroglycerin, 3-Morpholinosydnonimine (SIN-1), S- nitroso-N-acetyl-penicillamine (SNAP), Diethylenetriamine/NO (DETA/NO), S-nitrosothiols, Bidil®, and arginine.
  • NO gas isosorbide dinitrite, nitrite, nitroprusside, nitroglycerin, 3-Morpholinosydnonimine (SIN-1), S- nitroso-N-acetyl-penicillamine (SNAP), Diethylenetriamine/NO (DETA/NO), S-nitrosothio
  • Soluble guanylyl cyclase is the receptor for nitric oxide in vascular smooth muscle.
  • nitric oxide is endogenously generated by endothelial nitric oxide synthase from L-arginine, and activates soluble guanylyl cyclase in adjacent vascular smooth muscle cells to increase cGMP levels, inducing vascular relaxation.
  • Nitric oxide binds to the normally reduced heme moiety of soluble guanylyl cyclase, and increases the formation of cGMP from GTP, leading to a decrease in intracellular calcium, vasodilation, and anti-inflammatory effects.
  • Oxidation of the heme iron on sGC decreases responsiveness of the enzyme to nitric oxide, and promotes vasoconstriction.
  • the nitric oxide-sGC-cGMP pathway therefore plays an important role in cardiovascular diseases.
  • Nitrogen-containing compounds such as sodium azide, sodium nitrite, hydroxylamine, nitroglycerin, and sodium nitroprusside have been shown to stimulate sGC, causing an increase in cGMP, and vascular relaxation.
  • activators of sGC activate the oxidized or heme-deficient sGC enzyme that is not responsive to nitric oxide, i.e., they stimulate sGC independent of redox state.
  • stimulators of of sGC can enhance the sensitivity of reduced sGC to nitric oxide
  • activators of sGC can increase sGC enzyme activity even when the enzyme is oxidized and is therefore less, or unresponsive, to nitric oxide.
  • sGC activators are non-nitric oxide based.
  • An agent that activates soluble guanylyl cyclase refers, for example, to organic nitrates (Artz et al. (2002) J. Biol. Chem. 277: 18253-18256); protoporphyrin IX (Ignarro et al. (1982) Proc. Natl. Acad. Sci. USA 79:2870-2873); YC-1 (Ko et al. (1994) Blood 84:4226- 4233); BAY 41-2272 and BAY 41-8543 (Stasch et al.
  • cGMP can also be increased by inhibiting degradation using phosphodiesterase inhibitors.
  • an agent that inhibits cyclic nucleotide phosphodiesterases include, tadalafil, vardenafil, udenafil, and sildenafil avanafil.
  • term “treating” or “treat” or “treatment” means slowing, interrupting, arresting, controlling, stopping, reducing, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily involve a total elimination of all disease-related signs, symptoms, conditions, or disorders.
  • the term“treating” and the like refer to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop.
  • term“effective amount” refers to the amount or dose of an antibody compound of the present disclosure which, upon single or multiple dose administration to a patient or organ, provides the desired treatment or prevention.
  • Therapeutically effective amounts of the present antibody compounds can also comprise an amount in the range of from about 0.1 mg/kg to about 150 mg/kg, from about 0.1 mg/kg to about 100 mg/kg, from about 0.1 mg/kg to about 50 mg/kg, or from about 0.05 mg/kg to about 10 mg/kg per single dose administered to a harvested organ or to a patient.
  • Known antibody-based pharmaceuticals provide guidance in this respect.
  • HerceptinTM is administered by intravenous infusion of a 21 mg/ml solution, with an initial loading dose of 4 mg/kg body weight and a weekly maintenance dose of 2 mg/kg body weight; RituxanTM is administered weekly at 375 mg/m2; for example.
  • a therapeutically effective amount for any individual patient can be determined by the health care provider by monitoring the effect of the antibody compounds on tumor regression, circulating tumor cells, tumor stem cells or anti-tumor responses. Analysis of the data obtained by these methods permits modification of the treatment regimen during therapy so that optimal amounts of antibody compounds of the present disclosure, whether employed alone or in combination with one another, or in combination with another therapeutic agent, or both, are administered, and so that the duration of treatment can be determined as well. In this way, the dosing/treatment regimen can be modified over the course of therapy so that the lowest amounts of antibody compounds used alone or in combination that exhibit satisfactory efficacy are administered, and so that administration of such compounds is continued only so long as is necessary to successfully treat the patient.
  • Known antibody-based pharmaceuticals provide guidance relating to frequency of administration e.g., whether a pharmaceutical should be delivered daily, weekly, monthly, etc. Frequency and dosage may also depend on the severity of symptoms.
  • antibody compounds of the present disclosure can be used as medicaments in human and veterinary medicine, administered by a variety of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intraperitoneal, intrathecal, intraventricular, transdermal, transcutaneous, topical, subcutaneous, intratumoral, intranasal, enteral, sublingual, intravaginal, intravesiciular or rectal routes.
  • the compositions can also be administered directly into a lesion such as a tumor. Dosage treatment may be a single dose schedule or a multiple dose schedule. Hypo sprays may also be used to administer the pharmaceutical compositions.
  • the therapeutic compositions can be prepared as injectables, either as liquid solutions or suspensions.
  • Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
  • Veterinary applications include the treatment of companion/pet animals, such as cats and dogs; working animals, such as guide or service dogs, and horses; sport animals, such as horses and dogs; zoo animals, such as primates, cats such as lions and tigers, bears, etc. ⁇ , and other valuable animals kept in captivity.
  • compositions can be prepared by methods well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy, 21st Edition (2005), Lippincott Williams & Wilkins, Philadelphia, PA, and comprise one or more antibody compounds disclosed herein, and a pharmaceutically or veterinarily acceptable, for example, physiologically acceptable, carrier, diluent, or excipient.
  • the present disclosure describes anti-CD47 m Ahs with distinct functional profiles. These antibodies possess distinct combinations of properties selected from the following: 1) exhibit cross-reactivity with one or more species homologs of CD47; 2) block the interaction between CD47 and its ligand SIRPoc; 3) increase phagocytosis of human tumor cells; 4) induce death of susceptible human tumor cells; 5) do not induce cell death of human tumor cells; 6) do not have reduced or minimal binding to human red blood cells (hRBCs); 7) have reduced binding to hRBCs; 8) have minimal binding to hRBCs; 9) cause reduced agglutination of hRBCs; 10) cause no detectable agglutination of hRBCs; 11) reverse TSP1 inhibition of the nitric oxide (NO) pathway; 12) do not reverse TSP1 inhibition of the NO pathway; 13) cause loss of mitochondrial membrane potential; 14) do not cause cause cause loss of mitochondrial membrane potential; 15) cause
  • anti-CD47 antibodies and antigen binding fragments thereof of the present disclosure possess combinations of properties that are distinct from the anti-CD47 antibodies of the prior art. These properties and characteristics will now be described in further detail.
  • the term“binds to human CD47” refers to binding with an apparent Kd greater than 50 nM, for example, in a solid phase ELISA assay or cell based assay.
  • the terms “apparent binding affinity and apparent Kd” are determined by non-linear fit (Prism GraphPad software) of the binding data at the various antibody concentrations.
  • the anti-CD47 antibodies, and antigen binding fragments thereof, of the present disclosure bind human CD47.
  • the anti-CD47 antibodies exhibit cross reactivity with one or more species homologs of CD47, for example CD47 homologs of non human primate origin.
  • the anti-CD47 antibodies and antigen binding fragments thereof of the present disclosure bind to human CD47 and to CD47 of non-human primate, mouse, rat, and/or rabbit origin. The cross-reactivity with other species homologs can be particularly advantageous in the development and testing of therapeutic antibodies.
  • pre-clinical toxicology testing of therapeutic antibodies is frequently carried out in non-human primate species including, but not limited to, cynomolgus monkey, green monkey, rhesus monkey and squirrel monkey.
  • Cross-reactivity with these species homologs can therefore be particularly advantageous for the development of antibodies as clinical candidates.
  • cross-reacts with one or more species homologs of CD47 refers to binding with an apparent Kd greater than 50 nM.
  • CD47 also known as integrin associated protein (IAP) is a 50 kDa cell surface receptor that is comprised of an extracellular N-terminal IgV domain, a five membrane- spanning transmembrane domain, and a short C-terminal intracellular tail that is alternatively spliced.
  • IAP integrin associated protein
  • TSP1 Signal Regulatory Protein alpha
  • SIRPa Signal Regulatory Protein alpha
  • TSP1 Thrombospondin- 1
  • SIRPa on a phagocyte engages CD47 on a target cell
  • this interaction prevents phagocytosis of the target cell.
  • the interaction of CD47 and SIRPa effectively sends a“don’t eat me” signal to the phagocyte (Oldenborg et al. Science 288: 2051-2054, 2000).
  • Blocking the interaction of SIRPa and CD47 with an anti-CD47 mAh in a therapeutic context can provide an effective anti-cancer treatment by promoting the uptake and clearance of cancer cells by the host’s immune system.
  • an important functional characteristic of some anti-CD47 mAbs is the ability to block the interaction of CD47 and SIRPa, resulting in phagocytosis of CD47 expressing tumor cells by phagocytes including macrophages.
  • Several anti-CD47 mAbs have been shown to block the interaction of CD47 and SIRPa, including B6H12 (Seiffert et al. Blood 94:3633-3643,1999; Latour et al. J. Immunol. 167: 2547-2554, 2001; Subramanian et al. Blood 107: 2548-2556, 2006; Liu et al. J Biol. Chem. Ill: 10028-10036, 2002; Rebres et al. J.
  • SIRPa contains 3 Ig-like domains in its extracellular region, including 1 NH2- terminal V-set domain and 2 Cl-set domains.
  • the NH2-terminal V-set domain of SIRPa recognizes the Ig-like domain of CD47.
  • CD47 is highly conserved, extensive polymorphisms localized to the CD47-binding IgV domain of SIRPa occur in numerous ethnic groups.
  • Ten allelic variants of human SIRPa have been identified with two prevalent alleles, vl and v2.
  • SIRPa variants, vl and v2 is known to be most widespread among the Caucasian population.
  • the term“blocks SIRPa binding to human CD47” refers to a greater than 50% reduction of SIRPa-Fc binding to CD47 on cells by an anti-CD47 mAh compared to either untreated cells or cells treated with a negative antibody.
  • the anti-CD47 mAbs of the disclosure described herein block the interaction of CD47 and SIRPa and increase phagocytosis of human tumor cells.
  • “Phagocytosis” of cancer cells refers to the engulfment and digestion of such cells by phagocytes including, but not limited to, macrophages and dendritic cells, and the eventual digestion or degradation of these cancer cells and the release of digested or degraded cellular components extracellularly, or intracellularly to undergo further processing.
  • Anti-CD47 monoclonal antibodies that block SIRPa binding to CD47 increase phagocytosis of cancer cells. SIRPa binding to CD47 on cancer cells would otherwise allow these cells to escape phagocytosis.
  • the cancer cell may be viable or living cancer cells.
  • the term“increases phagocytosis of human tumor cells” refers to a greater than 2-fold increase in phagocytosis of human tumor cells by human macrophages in the presence of an anti-CD47 mAh compared to either untreated cells or cells treated with a negative control antibody. Inducing Death of Tumor Cells
  • soluble anti-CD47 mAbs initiate a cell death program on binding to CD47 on tumor cells, resulting in collapse of mitochrondrial membrane potential, loss of ATP generating capacity, increased cell surface expression of phosphatidylserine (detected by increased staining for annexin V) and cell death without the participation of caspases or fragmentation of DNA.
  • Such soluble anti-CD47 mAbs have the potential to treat a variety of solid and hematological cancers.
  • soluble anti-CD47 mAbs which have been shown to induce tumor cell death, including MABL-1, MABL-2 and fragments thereof (US Patent 8,101,719; Uno et al. Oncol Rep.
  • Induction of cell death refers to the ability of certain of the soluble anti-CD47 antibodies, murine antibodies, chimeric antibodies, humanized antibodies, or antigen-binding fragments thereof (and competing antibodies and antigen-binding fragments thereof) disclosed herein to kill cancer cells via a cell autonomous mechanism without participation of complement or other cells including, but not limited to, T cells, neutrophils, natural killer cells, macrophages, or dendritic cells.
  • the term“induces death of human tumor cells” refers to increased binding of annexin V (in the presence of calcium) and increased 7-aminoactinomycin D (7- AAD) or propidium iodide uptake in response to treatement with an anti-CD47 mAh. These features may be quantitated in three cell populations: annexin V positive (annexin V + ), annexin V positive/7-AAD negative (annexin VV7-AAD ) and annexin V positive/7- A AD positive (annexin VV7-AAD + ) by flow cytometry.
  • Induction of cell death may be defined by a greater than 2-fold increase in each of the above cell populations in human tumor cells caused by soluble anti-CD47 mAh compared to the background obtained with the negative control antibody, (humanized, isotype-matched antibody) or untreated cells.
  • Another indicator of cell death is loss of mitochondrial function and membrane potential by the tumor cells as assayed by one of several available measures (potentiometric fluorescent dyes such as DiO-C6 or JC1 or formazan-based assays such as MTT or WST-1).
  • potentiometric fluorescent dyes such as DiO-C6 or JC1 or formazan-based assays such as MTT or WST-1).
  • the term“causes loss of mitochondrial membrane potential” refers to a statistically significant (p ⁇ 0.05) decrease in mitochondrial membrane potential by a soluble anti-CD47 mAh compared to the background obtained with a negative control, humanized isotype-matched antibody or no treatment.
  • Cell viability assays are described in NCI/NIFl guidance manual that describes numerous types of cell based assays that can be used to assess induction of cell death caused by CD47 antibodies:“Cell Viability Assays”, Terry L Riss, PhD, Richard A Moravec, BS, Andrew L Niles, MS, Flelene A Benink, PhD, Tracy J Worzella, MS, and Lisa Minor, PhD. Contributor Information, published May 1, 2013.
  • CD47 is expressed on human erythrocytes (hRBCs) (Brown. J Cell Biol. I l l : 2785- 2794, 1990; Avent. Biochem J., (1988) 251: 499-505; Knapp. Blood, (1989) Vol. 74, No. 4, 1448-1450; Oliveira et al. Biochimica et Biophysica Acta 1818: 481-490, 2012; Petrova P. et al. Cancer Res 2015; 75(15 Suppl): Abstract no. 4271). It has been shown that anti-CD47 mAbs bind to RBCs, including B6F112 (Brown et al. J.
  • Binding to RBCs can be reduced by generation of bi-specific antibodies with only one CD47 binding arm (Masternak et al. Cancer Res 2015; 75(15 Suppl): Abstract no. 2482). Because some anti-CD47 m Ahs have been shown to result in reduction of RBCs when administered to cynomolgus monkeys (Mounho-Zamora B. et al. The Toxicologist, Supplement to Toxicological Sciences, 2015; 144 (1): Abstract 596: 127, Liu et al. PLoS One.
  • the anti-CD47 mAbs have reduced, minimal, or no binding to human RBCs .
  • some of the anti-CD47 mAbs require ITOb I for maximal binding to human CD47.
  • some of the anti-CD47 mAbs do not bind RBCs because RBCs do not express ITOb I .
  • red blood cell(s) and“erythrocyte(s)” are synonymous and used interchangeably herein.
  • the term“reduced binding to hRBCs” refers to an apparent Kd of an anti-CD47 mAh binding to a hRBC which is 8 -fold or greater than the apparent Kd on a human tumor cell, wherein the tumor cell is an OV10 hCD47 cell (human OV10 ovarian cancer cell line expressing human CD47).
  • minimal binding or“MB“ refers to no measurable binding to hRBCs at an anti-CD47 mAh concentration up to 5,000 pM.
  • Some of the anti-CD47 mAbs, disclosed herein, have reduced or minimal binding to human RBCs.
  • CD47 is also expressed, by many but not all, normal cell types, including, but not limited to RBCs (see previous section), lymphocytes and mononuclear cells, endothelial cells, and brain, liver, muscle cells and/or tissues (Brown et al, J Cell Biol 1990; Reinhold et al , J Cell Sci. 1995; Matozaki et al , Cell 2009; Stefanidakis et al , Blood 2008; Xiao et al, Cancer Letters 2015).
  • anti-CD47 m Ahs Because of this expression, it is expected that some anti-CD47 m Ahs would bind to these normal cell types/tissues in addition to the cancer cells which are the therapeutic target. It is therefore desirable to identify anti-CD47 m Ahs that either have reduced or minimal binding to some of these normal cells to both reduce potential non-desired effects on these normal cells and also allow more available antibody for binding to the tumor cells. Anti-CD47 m Ahs with such reduced or minimal binding to normal cells have not been described.
  • the terms“reduced binding to normal human cells which includes, but is not limited to, endothelial cells, skeletal muscle cells, epithelial cells, and peripheral blood mononuclear cells (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubular epithelial cells, human peripherial blood CD3+ cells, and human peripheral blood mononuclear cells) refers to the apparent Kd of an anti-CD47 mAh binding to these cells which is 8-fold or greater than the apparent Kd of the anti-CD47 mAh binding to a human tumor cell, wherein the tumor cell is OVIO hCD47.
  • the term“mimimal binding” or“MB” refers to no measurable binding of an antibody or other molecule as described herein to normal human cells which includes, but is not limited to, endothelial cells, skeletal muscle cells, epithelial cells, and peripheral blood mononuclear cells (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubular epithelial cells, human peripherial blood CD3+ cells, and human peripheral blood mononuclear cells) at an anti-CD47 mAh concentration up to 5,000 pM.
  • endothelial cells e.g., endothelial cells, skeletal muscle cells, epithelial cells, and peripheral blood mononuclear cells
  • peripheral blood mononuclear cells e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubular epithelial cells, human peripherial blood
  • Red blood cell (RBC) agglutination or hemagglutination is a homotypic interaction that occurs when RBCs aggregate or clump together following incubation with various agents, including antibodies to RBC antigens and cell surface proteins such as CD47.
  • RBC antigens and cell surface proteins such as CD47.
  • Many anti-CD47 antibodies have been reported to cause hemagglutination of washed human RBCs in vitro, in a concentration dependent manner, including B6H12, BRIC126, MABL-1, MABL-2, CC2C6, and 5F9 (Uger R. et al. Cancer Res 2014; 74(19 Suppl): Abstract no. 5011, US Patent 9,045,541, Uno et al. Oncol Rep.
  • mouse antibody 2D3 is an example of an anti-CD47 antibody that binds to CD47 on red blood cells but does not cause hemagglutination (US Patent 9,045,541, Petrova et al. Cancer Res 2015; 75(15 Suppl): Abstract no. 4271).
  • the term “reduced hemagglutination” refers to detectable agglutination activity of hRBCs at anti-CD47 mAh concentrations greater than or equal to 1.85 mg/ml, and no measurable activity at concentrations less than 1.85 mg/mI in a washed RBC assay, as visualized by discrete punctate dot compared to a diffuse pattern that represents hemagglutination.
  • the term“no detectable hemagglutination” refers to no visible or detectable agglutination activity of hRBCs at anti-CD47 mAh concentrations greater or equal to 0.3 pg/ml to a concentration less than or equal to 10 mg/mI in a washed RBC assay, as visualized by discrete punctate dot compared to a diffuse pattern that represents hemagglutination.
  • a method for treating a human subject having cancer, wherein an antibody or antigen binding fragment thereof is administered to a human subject that prevents the binding of human CD47 with SIRPoc without agglutination of normal cells, and wherein the human CD47 is associated with an integrin on a cancer cell, and wherein said integrin allows for the binding of said antibody or antigen binding fragment thereof to human CD47.
  • a method is disclosed of preventing agglutination of normal cells in human subject having cancer treatment with a CD47 antibody or antigen binding fragment thereof, wherein a CD47 antibody or antigen binding fragment thereof is admini tered to a human subject an antibody or antigen binding fragment thereof that prevents the binding of human CD47 with SIRPoc, wherein the human CD47 is associated with an integrin on a cancer cell, and wherein said integrin allows for the binding of said antibody or antigen binding fragment thereof to human CD47.
  • Integrins are transmembrane adhesion receptors localized at cell-matrix contact sites where they link extracellular matrix (ECM) components to the actin cytoskeleton and interact with multiple downstream structural and signaling molecules, such as Src and FAK (Berrier and Yamada, 2007).
  • ECM extracellular matrix
  • the integrin family of proteins consists of an alpha (a) and a beta (b) subunit, associated by noncovalent interactions, forming an extracellular ligand-binding head, two multi-domain Tegs’, two single-pass transmembrane helices and two short cytoplasmic tails.
  • the a and b subunits show no homology to each other, however, conserved regions are found among each a and b subtype.
  • Src is a non-receptor cytoplasmic tyrosine kinase which becomes activated following the stimulation of plasma membrane receptors, including receptor tyrosine kinases and integrins, and is involved in multiple physiological homeostatic pathways. Once activated, Src is the starting point for several biochemical cascades that thereby propagate signals generated extracellularly along intracellular interconnected transduction pathways. Src transmits signals promoting cell survival, and the reorganization of the cytoskeleton, and adhesion systems that underpin cell migration and invasion.
  • Src activation appears to be pleiotropic as numerous Src substrates are phosphorylated in tumor cells with activated forms of Src. Many of these phosphorylated Src substrates have been linked to processes inducing tumorigenicity and metastasis (Brown and Cooper, 1996). Furthermore, these alterations in signal transduction likely influence multiple downstream transcriptional events which include alterations in homotypic adhesion, angiogenesis, tumor cell invasivity, tumor growth, and apoptosis, all of which play a significant role in the development of the metastatic phenotype.
  • the tyrosine kinase FAK acts both as a signaling molecule and a scaffold able to recruit Src and the Src substrates to sites of integrin engagement, and has an important role in cell cycle progression and survival as well as in adhesion and migration (Berrier and Yamada, 2007).
  • FAK-Src complex in which activated Src phosphorylates FAK at several tyrosine residues, further enhances the kinase activity of FAK (Playford and Schaller, 2004). Finally, the resulting highly active FAK-Src complex promotes the phosphorylation of many FAK associated Src substrates including CAS, paxillin, and pl90RhoGAP which have a central role in the reorganization of the actin cytoskeleton and migration (Playford and Schaller, 2004).
  • phosphorylated FAK can recruit Grb2 and the p85 regulatory subunit of PI3K, thus leading respectively to stimulation of Grb2/Ras/MAPK and PI3K/Akt pathways which enhance cell survival and proliferation and contribute to cytoskeleton remodeling.
  • CD47 has been shown to be associated with integrins, including anb3, anb ⁇ , aI3 ⁇ 4b3, a2b1 and a4b1, and may interact with other integrin complexes (Lindberg FP et al., 1996, Ticchioni et al., 1997, Reinhold MI et al., 1997, Wu et al., 1999).
  • the interaction of CD47 with integrins is important for mechano-transduction, cell adhesion, signaling and cell migration (Lindberg FP et al., 2008, Schwartz MA et al., 1998).
  • the monoclonal antibody or antigen-binding fragment thereof binds to human CD47 which is associated with an integrin.
  • the integrin comprises ab integrin heterodimeric complex with an alpha (a) and a beta (b) subunit.
  • the b subunit is selected from P ⁇ bI, ITOb2, PT ⁇ b3, ITOb4, P ⁇ b5, P ⁇ b6, P ⁇ b7, and IT ⁇ b8.
  • the a subunit is selected from ITGocl, ITGoc2, ITGoc3, ITGoc4, ITGoc5, ITGoc6, ITGoc7, ITGoc8, ITGoc9, ITGoclO, ITGocl 1, and ITGocv .
  • the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with ITOb I .
  • the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with an ab integrin heterodimeric complex selected from P ⁇ bIaI, IT ⁇ b1a2, IT ⁇ b1a3, IT ⁇ b1a4, IT ⁇ b1a5, P ⁇ b1a6, IT ⁇ b1a7, P ⁇ b1a8, P ⁇ b1a9, IT ⁇ bIaIO, P ⁇ bIaI I, and ITGav.
  • an ab integrin heterodimeric complex selected from P ⁇ bIaI, IT ⁇ b1a2, IT ⁇ b1a3, IT ⁇ b1a4, IT ⁇ b1a5, P ⁇ b1a6, IT ⁇ b1a7, P ⁇ b1a8, P ⁇ b1a9, IT ⁇ bIaIO, P ⁇ bIaI I, and ITGav.
  • the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with an integrin and at least a second protein.
  • the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with an integrin and at least a second protein, wherein the at least second protein can be selected from an integrin or a non-integrin protein, wherein the non-integrin protein can be a receptor tyrosine kinase, a checkpoint inhibitor protein, etc.
  • the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with ITOb I and at least a second protein.
  • the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with ITOb 1 and at least a second protein, wherein the second protein is selected from ACTN1, CD46, CD9, FHL2, Filamin, FLNB, CD81, GNB2L1, IT ⁇ bIBRI, LGALS8, MAP4K4, NME1, PKCoc, TLN1, TSPAN4, and YWHAB.
  • the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with a beta (b) subunit of an integrin selected from IT ⁇ bI, IT ⁇ b2, PCTb3, PCTb4, IT ⁇ b5, IT ⁇ b6, IT ⁇ b7, and PCTb8, and at least a second protein, wherein the at least a second protein is a receptor tyrosine kinase (RTK), i.e., Src and FAK, wherein the receptor tyrosine kinase is not known to bind to a beta (b) subunit of an integrin.
  • RTK receptor tyrosine kinase
  • the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with a beta (b) subunit of an integrin selected from IT ⁇ bI, IT ⁇ b2, PCTb3, PCTb4, IT ⁇ b5, IT ⁇ b6, IT ⁇ b7, and PCTb8, and at least a second protein, wherein the at least a second protein is a checkpoint inhibitor (CTLA-4, PD-1, PD-L1, etc.), wherein the receptor tyrosine kinase is not known to bind to a beta (b) subunit of an integrin.
  • a checkpoint inhibitor CTL-4, PD-1, PD-L1, etc.
  • administering the monoclonal antibody or antigen-binding fragment thereof binds to human CD47 which is associated with an integrin decreases Src phosphorylation (e.g., de-phosphorylation or inhibition of phosphorylation at Tyr416), reversing a pro-survival effect of the cancer cell.
  • Src phosphorylation e.g., de-phosphorylation or inhibition of phosphorylation at Tyr416
  • the autophosphorylation of Src is driven by conformation changes mediated by integrins and receptor tyrosine kinases (e.g., epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR)).
  • EGFR epidermal growth factor receptor
  • PDGFR platelet-derived growth factor receptor
  • the ability of Src to phosphorylate or autophosphorylate Y416 is decreased.
  • Src phosphorylation (e.g., in a test sample) is decreased by at least about 1.1-, 1.2-, 1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, 2-, 2.1-, 2.2-, 2.3-, 2.4-, 2.5-, 2.6-, 2.7-, 2.8-, 2.9-, 3-, 3.1-, 3.2-, 3.3-, 3.4-, 3.5-, 3.6-, 3.7-, 3.8-, 3.9-, 4-, 4.1-, 4.2-, 4.3-, 4.4-, 4.5-, 4.6-, 4.7- , 4.8-, 4.9-, 5-, 5.5-, 6-, 6.5-, 7-, 7.5-, 8-, 8.5-, 9-, 9.5-, 10-, 10.5-, 11-, 11.5-, 12-, 12.5-, 13-, 13.5-, 14-, 14.5-, 15-, 15.5-, 16-,
  • the phosphorylation of a downstream Src target is increased or decreased and is selected from STAT3, PI3K/Akt, ERK/MAPK and / or a combination thereof.
  • administering a monoclonal antibody or antigen-binding fragment thereof that binds to human CD47 which is associated with an integrin decreases FAK phosphorylation (de-phosphorylation or inhibition of phosphorylation at Tyr397), reversing a pro-survival effect of the cancer cell.
  • the phosphorylation or autophosphorylation of FAK is driven by conformation changes mediated by integrins, G-protein coupled receptor (GPCR) occupancy by ligands such as bombesin or lysophosphatidic acid, or via FDF receptor occupancy.
  • GPCR G-protein coupled receptor
  • the ability of FAK to autophosphorylate FAK at Y397 is decreased.
  • FAK phosphorylation (e.g., in a test sample) is decreased by at least about 1.1-, 1.2-, 1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, 2-, 2.1-, 2.2-, 2.3-, 2.4-, 2.5-, 2.6-, 2.7-, 2.8-, 2.9-, 3-, 3.1-, 3.2-, 3.3-, 3.4-, 3.5-, 3.6-, 3.7-, 3.8-, 3.9-, 4-, 4.1-, 4.2-, 4.3-, 4.4- , 4.5-, 4.6-, 4.7-, 4.8-, 4.9-, 5-, 5.5-, 6-, 6.5-, 7-, 7.5-, 8-, 8.5-, 9-, 9.5-, 10-, 10.5-, 11-, 11.5-, 12-, 12.5-, 13-, 13.5-, 14-, 14.5-, 15-, 15.5-, 16-, 1
  • the phosphorylation of a downstreatm FAK target is increased or decreased and is selected from paxillin, ERK/MAPK, and Rho/Rac, and / or a combination thereof.
  • the anti-CD47 mAbs or antigen binding fragments thereof cause release of or exposure on tumor cell surfaces of one or more of the DAMPs listed in the above section, characteristics of Immunogenic Cell Death (ICD). These DAMPS are expected to promote a therapeutically beneficial adaptive anti-tumor immune response.
  • the binding of the anti-CD47 mAbs disclosed herein to human CD47 which is associated with an integrin, particularly ITOb I is linked to the release of DAMPs that cause maturation, activation and homing of DCs and attraction of T cells connects anti-CD47 mAh treatment to the development of the therapeutically desirable anti-tumor immune response.
  • TSP1 is also a ligand for CD47.
  • the TSP1/CD47 pathway opposes the beneficial effects of the NO pathway in many cell types, including, but not limited to, vascular cells.
  • the NO pathway consists of any of three enzymes (nitric oxide synthases, NOS I, NOS II and NOS III) that generate bioactive gas NO using arginine as a substrate. NO can act within the cell in which it is produced, or in neighboring cells, to activate the enzyme soluble guanylyl cyclase that produces the messenger molecule cyclic GMP (cGMP).
  • cGMP messenger molecule cyclic GMP
  • the proper functioning of the NO/cGMP pathway is essential for protecting the cardiovascular system against stresses including, but not limited to, those resulting from wounding, inflammation, hypertension, metabolic syndrome, ischemia, and IRI.
  • stresses including, but not limited to, those resulting from wounding, inflammation, hypertension, metabolic syndrome, ischemia, and IRI.
  • the inhibition of the NO/cGMP pathway by the TSP1/CD47 system exacerbates the effects of stress. This is a particular problem in the cardiovascular system where both cGMP and cAMP play important protective roles.
  • ischemia and reperfusion injury cause or contribute to disease, trauma, and poor outcomes of surgical procedures.
  • one of more of the chimeric or humanized anti-CD47 antibodies will reverse TSP1 inhibition of cGMP production. Reversal will be complete (>80 %) or intermediate (20% -80%). This reversal of TSP1 inhibition of cGMP production will demonstrate that the anti-CD47 m Ahs have the ability to increase NO signaling and suggest utility in protecting the cardiovascular system against stresses including, but not limited to, those resulting from wounding, inflammation, hypertension, metabolic syndrome, ischemia, and ischemia-reperfusion injury (IRI). Additional assay systems, for example smooth muscle cell contraction, will also be expected to show that some of the chimeric or humanized antibodies reverse the inhibitory actions of TSP1 on downstream effects resulting from the activation of NO signaling.
  • IRI ischemia-reperfusion injury
  • “complete reversal of NO pathway inhibition” refers to greater than 80% reversal of TSP1 inhibition of NO signaling by an anti-CD47 mAh compared to a negative control, humanized isotype-matched antibody or no treatment.
  • “intermediate reversal of NO pathway inhibition” refers to 20- 80% reversal of TSP1 inhibition of NO signaling by an anti-CD47 mAh compared to a negative control, humanized isotype-matched antibody or no treatment.
  • no reversal of NO pathway inhibition refers to less than 20% reversal of TSP1 inhibition of NO signaling by an anti-CD47 mAh compared to a negative control, humanized isotype-matched antibody or no treatment.
  • ICD immunogenic cell death
  • ICD of tumor cells are the release from or exposure on tumor cell surfaces of specific ligands: 1) the pre-apoptotic cell surface exposure of calreticulin, 2) the secretion of adenosine triphosphate (ATP), 3) release of high mobility group box 1 (HMGB 1), 4) annexin Al release, 5) type I interferon release and 6) C-X-C motif chemokine ligand 10 (CXCL10) release.
  • DAMPs endogenous damage- associated molecular patterns
  • CD AMs cell death-associated molecules
  • each of these ligands induced during ICD binds to specific receptors, referred to as pattern recognition receptors (PRRs), that contribute to an anti-tumor immune response.
  • PRRs pattern recognition receptors
  • ATP binds the purinergic receptors PY2, G-protein coupled, 2 (P2RY2) and PX2, ligand-gated ion channel, 7 (P2RX7) on dendritic cells causing dendritic cell recruitment and activation, respectively.
  • Annexin Al binds to formyl peptide receptor 1 (FPR1) on dendritic cells causing dendritic cell homing.
  • Calreticulin expressed on the surface of tumor cells binds to LRP1 (CD91) on dendritic cells promoting antigen uptake by dendritic cells.
  • HMGB 1 binds to toll-like receptor 4 (TLR4) on dendritic cells to cause dendritic cell maturation.
  • TLR4 toll-like receptor 4
  • tumor cells release type I interferon leading to signaling via the type I interferon receptor and the release of the CXCL10 which favors the recruitment of effector CXCR3+ T cells
  • the actions of these ligands on their receptors facilitate recruitment of DCs into the tumor, the engulfment of tumor antigens by DCs and optimal antigen presentation to T cells.
  • Calreticulin is one of the most abundant proteins in the endoplasmic reticulum (ER). Calreticulin was shown to rapidly translocate preapoptotically from the ER lumen to the surface of cancer cells in response to multiple ICD inducers, including anthracyclines (Obeid et al. Nat Med 13: 54-61, 2007; Kroemer et al. Annu Rev Immunol 31 : 51-72, 2013). Blockade or knockdown of calretiulin suppressed the phagocytosis of anthracycline-treated tumor cells by dendritic cells and abolished their immunogenicity in mice.
  • calreticulin caused by anthracyclines or oxaliplatin is activated by an ER stress response that involves the phosphorylation of the eukaryotic translation initiation factor eIF2oc by the PKR-like ER kinase.
  • Calretiulin which has a prominent function as an“eat-me” signal (Gardai et al. Cell 123: 321-334, 2005) binds to LRP1 (CD91) on dendritic cells and macrophages resulting in phagocytosis of the calreticulin expressing cell, unless the calreticulin-expressing cell expresses a don’t eat me signal, such as CD47.
  • Calreticulin also signals through CD91 on antigen presenting cells to cause the release of proinflammatory cytokines and to program Thl7 cell responses.
  • calreticulin expressed as part of immunogenic cell death stimulates antigen presenting cells to engulf dying cells, process their antigens and prime an immune response.
  • PDIA3 protein disulfide-isomerase A3
  • Erp57 protein disulfide-isomerase A3
  • HSP70 and HSP90 bind to the PRR LRP1 on antigen presenting cells; the PRR to which PDIA3 binds has not been identified (Galluzi et al. Nat Rev Immunol 17: 97-111, 2016).
  • TLR4 was shown to be required for cross-presentation of dying tumor cells and to control tumor antigen processing and presentation.
  • HMGB1 was uniquely released by mouse tumor cells in which ICD was induced by irradiation or doxorubicin (Apetoh et al. Nat Med 13: 1050-1059, 2007).
  • ICD was induced by irradiation or doxorubicin
  • the highly efficient induction of an in vivo anti-tumor immune by doxorubicin treatment of mouse tumor cells required the presence of HMGB 1 and TLR4, as demonstrated by abrogation of the immune response by inhibition of HMGB 1 and knock-out TLR4.
  • ATP-elicited production of IL-Ib by DCs appears to be one of the critical factors for the immune system to perceive cell death induced by certain chemotherapy drugs as immunogenic.
  • Ma et al. addressed the question of how chemotherapy-induced cell death leads to efficient antigen presentation to T cells (Ma et al. Immunity 38: 729-741, 2013). They found that at specific kind of tumor infiltrating lymphocyte, CDl lc + CDl lb + Ly6C hl cells, are particularly important for the induction of anticancer immune responses by anthracy dines. ATP released by dying cancer cells recruited myeloid cells into tumors and stimulated the local differentiation of CDl lc + CDl lb + Ly6C hl cells. These cells were shown to be particularly efficient in capturing and presenting tumor cell antigens and, after adoptive transfer into naive mice, conferring protection to challenge with living tumor cells of the same cell line.
  • Type I interferons bind to IFN-oc and IFN-b receptors on cancer cells and trigger autocrine and paracrine signaling pathways that result in release of CXCL10. Tumors lacking Tlr3 or Ifnar failed to respond to chemotherapy unless type I IFN or CXCL10, respectively, was supplied. These preclinical findings have clinical relevance.
  • a type I IFN-related gene expression signature predicted clinical responses to anthracycline-based chemotherapy in independent cohorts of breast cancer patients.
  • Vacchelli et al. Another receptor on dendritic cells that is involved in chemotherapy-induced anti cancer immune response was recently identified: formyl peptide receptor- 1, which binds annexin Al (Vacchelli et al. Science 350: 972-978, 2015).
  • Vacchelli et al. designed a screen to identify candidate genetic defects that negatively affect responses to chemotherapy. They identified a loss-of-function allele of the gene encoding formyl peptide receptor 1 (FPR1) that was associated with poor metastatis-free survival and overall survival in breast and colorectal cancer patients receiving adjuvant chemotherapy.
  • FPR1 formyl peptide receptor 1
  • the therapeutic effects of anthracyclines were abrogated in tumor-bearing Fprl -/- mice due to impaired antitumor immunity.
  • FPR1- deficient DCs did not approach dying tumor cells and, therefore, could not elicit antitumor T cell immunity.
  • FPR1 and annexin A1 promoted stable interactions between dying cancer cells and human or mouse leukocytes.
  • the anti-CD20 monoclonal antibody rituximab has improved outcomes in multiple B-cell malignancies.
  • Cheadle et al investigated the induction of immunogenic cell death by anti-CD20 mAbs (Cheadle et al. Brit J Haematol 162: 842-862, 2013).
  • the cell death induced by obinutuzumab and tositumomab is a form of immunogenic cell death characterized by the release of F1MGB 1, F1SP90 and ATP.
  • a type I anti-CD20 mAh did not cause release of F1MGB 1 , F1SP90 and ATP.
  • Incubation of supernatants from a human tumor cell line treated with obinutuzumab caused maturation of human dendritic cells, consistent with the previously described effects of F1MGB 1 and ATP on dendritic cells. In contrast to the results reported by Cheadle et al. , Zhao et al.
  • “causes an increase in cell surface calreticulin expression on human tumor cells” refers to a statistically significant increase (p ⁇ 0.05) in calreticulin expression by a soluble anti-CD47 mAh compared to the background obtained with a negative control, humanized isotype-matched antibody or no treatment.
  • the term“the release of’ is synonymous with secretion and is defined as the extracellular appearance of ATP, HMGB1, annexin Al, type I interferon and CXCL10.
  • “cause an increase in the release of adenosine triphosphate by human tumor cells” refers to a statistically significant increase (p ⁇ 0.05) in ATP in the supernatant caused by a soluble anti-CD47 mAh compared to the background obtained with a negative control, humanized isotype-matched antibody or no treatment.
  • “cause an increase in the release of high mobility group box 1 by human tumor cells” refers to a statistically significant increase (p ⁇ 0.05) in HMGB1 in the supernatant caused by a soluble anti-CD47 mAh compared to the background obtained with a negative control, humanized isotype-matched antibody or no treatment.
  • “causes an increase in the release of type I interferon by human tumor cells” refers to a statistically significant increase (p ⁇ 0.05) in type I interferon in the supernatant or type I interferon mRNA caused by a soluble anti-CD47 mAh compared to the background obtained with a negative control, humanized isotype-matched antibody or no treatment.
  • CXCL10 C-X-C Motif Chemokine Ligand 10
  • “causes an increase in cell surface HSP70 expression on human tumor cells” refers to a statistically significant increase (p ⁇ 0.05) in HSP70 expression by a soluble anti-CD47 mAh compared to the background obtained with a negative control, humanized isotype-matched antibody or no treatment.
  • “causes an increase in cell surface HSP90 expression on human tumor cells” refers to statistically significant increase (p ⁇ 0.05) in HSP90 expression by a soluble anti-CD47 mAh compared to the background obtained with a negative control, humanized isotype-matched antibody or no treatment.
  • TME tumor microenvironment
  • the acidic pH may provide an advantage to the tumor by promoting invasiveness, metastatic behavior, chronic autophagy, resistance to chemotherapies and reduced efficacy of immune cells in the tumor microenvironment (Estrella et al. Cancer Res 2013; Wojtkowiak et al, Cancer Res 2012; Song et al, Cancer Drug Discovery and Development 2006; Barar, Bioimpacts, 2012).
  • the identification of anti-CD47 antibodies with the property of increased binding affinity at acidic pH would confer a therapeutic advantage with higher binding to CD47 on tumor cells within the acidic TME compared to cells at physiological pH.
  • Antibodies with pH-dependent properties have been generated with the goal of recycling antibodies.
  • anti-CD47 antibodies described herein are also characterized by combinations of properties which are not exhibited by prior art anti-CD47 antibodies proposed for human therapeutic use. Accordingly, in some embodiments, anti-CD47 antibodies described herein may be characterized by:
  • a. binds to human CD47 ;
  • the anti-CD47 antibodies are characterized by:
  • a. binds to human CD47 ;
  • hRBCs human red blood cells
  • the anti-CD47 antibodies are characterized by:
  • a. binds to human CD47 ;
  • hRBCs human red blood cells
  • the anti-CD47 antibodies are characterized by:
  • a. binds to human CD47 ;
  • the anti-CD47 antibodies are characterized by:
  • a. binds to human CD47 ;
  • hRBCs human red blood cells
  • the anti-CD47 antibodies are characterized by:
  • a. binds to human CD47 ; b. blocks SIRPoc binding to human CD47;
  • hRBCs human red blood cells
  • the monoclonal antibody, or antigen binding fragment thereof binds to human, non-human primate, mouse, rabbit, and rat CD47.
  • the monoclonal antibody, or antigen binding fragment thereof specifically also binds to non-human primate CD47, wherein non human primate may include, but is not limited to, cynomolgus monkey, green monkey, rhesus monkey and squirrel monkey.
  • the anti-CD47 monoclonal antibody, or antigen binding fragment thereof may additionally possess one or more of the following characteristics: 1) exhibit cross-reactivity with one or more species homologs of CD47; 2) block the interaction between CD47 and its ligand SIRPoc; 3) increase phagocytosis of human tumor cells; 4) induce death of susceptible human tumor cells; 5) do not induce cell death of human tumor cells; 6) do not have reduced or minimal binding to human red blood cells (hRBCs); 7) have reduced binding to hRBCs; 8) have minimal binding to hRBCs; 9) cause reduced agglutination of hRBCs; 10) cause no detectable agglutination of hRBCs; 11) reverse TSP1 inhibition of the nitric oxide (NO) pathway; 12) do not reverse TSP1 inhibition of the NO pathway; 13) cause loss of mitochondrial membrane potential; 14) do not cause cause cause loss of mitochondrial membrane potential; 15) cause an increase in cell surface ca reti
  • a monoclonal antibody, or an antigen binding fragment thereof which: binds to human CD47; blocks SIRPoc binding to human CD47; increases phagocytosis of human tumor cells; and induces death of human tumor cells; wherein said monoclonal antibody, or an antigen binding fragment thereof, exhibits pH-dependent binding to CD47 present on a cell.
  • the disclosure provides a monoclonal antibody, or an antigen binding fragment thereof, which: binds to human CD47; blocks SIRPoc binding to human CD47; increases phagocytosis of human tumor cells; and induces death of human tumor cells; wherein said monoclonal antibody, or an antigen binding fragment thereof, exhibits reduced binding to normal cells.
  • a cell to which such an antibody may bind may be of any cell type as described herein.
  • a monoclonal antibody as described herein, or an antigen binding fragment thereof may exhibit any combination of characteristics provided in the present disclosure. For example, a monoclonal antibody may beneficially exhibit both pH dependent binding and reduced binding to a cell.
  • These cells may be an endothelial cell, a skeletal muscle cell, an epithelial cell, a PBMC or a RBC (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubular epithelial cells, human peripherial blood CD3+ cells, human peripheral blood mononuclear cells or human RBC).
  • aortic endothelial cells e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubular epithelial cells, human peripherial blood CD3+ cells, human peripheral blood mononuclear cells or human RBC.
  • pH dependent binding of an antibody of the disclosure may refer to altered binding of the antibody at a particular pH, for example an antibody that exhibits increased binding affinity at acidic pH.
  • CD47 expression is thought to protect cancer cells from phagocytic clearance by sending a“don’t eat me” signal to macrophages via SIRPoc, an inhibitory receptor that prevents phagocytosis of CD47-bearing cells (Oldenborg et al. Science 288: 2051-2054, 2000; Jaiswal et al. (2009) Cell 138(2):271-851; Chao et al. (2010) Science Translational Medicine 2(63):63ra94).
  • SIRPoc an inhibitory receptor that prevents phagocytosis of CD47-bearing cells
  • Antibodies that block CD47 and prevent its binding to SIRPoc have shown efficacy in human tumor in murine (xenograft) tumor models.
  • Such blocking anti-CD47 mAbs exhibiting this property increase the phagocytosis of cancer cells by macrophages, which can reduce tumor burden (Majeti et al. (2009) Cell 138 (2): 286-99; US 9,045,541 ; Willingham et al. (2012) Proc Natl Acad. Sci. USA 109(17):6662-6667; Xiao et al. (2015) Cancer Letters 360:302-309; Chao et al. (2012) Cell 142:699-713; Kim et al. (2012) Leukemia 26:2538-2545).
  • Anti-CD47 mAbs have also been shown to promote an adaptive immune response to tumors in vivo (Tseng et al. (2013) PNAS 110 (27): 11103-11108; Soto-Pantoja et al. (2014) Cancer Res. 74 (23): 6771-6783; Liu et al. (2015) Nat. Med. 21 (10): 1209-1215; Xu et al. (2017) Immunity 47: 363-373).
  • anti-CD47 mAbs can attack transformed cells that have not yet been exploited in the treatment of cancer.
  • Multiple groups have shown that particular anti-human CD47 mAbs induce cell death of human tumor cells.
  • Anti-CD47 mAh Ad22 induces cell death of multiple human tumor cells lines (Pettersen et al. J. Immunol. 166: 4931-4942, 2001 ; Lamy et al. J. Biol. Chem. 278: 23915-23921, 2003).
  • AD22 was shown to indice rapid mitochondrial dysfunction and rapid cell death with early phosphatidylserine exposure and a drop in mitochondrial membrane potential (Lamy et al. J. Biol. Chem.
  • Anti-CD47 mAh MABL-2 and fragments thereof induce cell death of human leukemia cell lines, but not normal cells in vitro and had an anti-tumor effect in in vivo xenograft models. (Uno et al. (2007) Oncol. Rep. 17 (5): 1189-94).
  • Anti-human CD47 mAh 1F7 induces cell death of human T cell leukemias (Manna and Frazier (2003) J. Immunol. 170: 3544-53) and several breast cancers (Manna and Frazier (2004) Cancer Research 64 (3): 1026- 36). 1F7 kills CD47-bearing tumor cells without the action of complement or cell mediated killing by NK cells, T cells, or macrophages.
  • anti-CD47 mAh 1F7 acts via a non- apoptotic mechanism that involves a direct CD47-dependent attack on mitochondria, discharging their membrane potential and destroying the ATP-generating capacity of the cell leading to rapid cell death. It is noteworthy that anti-CD47 mAh 1F7 does not kill resting leukocytes, which also express CD47, but only those cells that are “activated” by transformation. Thus, normal circulating cells, many of which express CD47, are spared while cancer cells are selectively killed by the tumor-toxic CD47 mAh (Manna and Frazier (2003) J. Immunol. 170: 3544-53).
  • This mechanism can be thought of as a proactive, selective and direct attack on tumor cells in contrast to the passive mechanism of causing phagocytosis by simply blocking CD47/SIRP0C binding.
  • mAh 1F7 also blocks binding of SIRPoc to CD47 (Rebres et al., J. Cellular Physiol. 205: 182-193, 2005) and thus it can act via two mechanisms: (1) direct tumor toxicity, and (2) causing phagocytosis of cancer cells.
  • a single mAh that can accomplish both functions may be superior to one that only blocks CD47/SIRP0C binding.
  • anti-CD47 m Ahs An additional mechanism by which anti-CD47 m Ahs can be exploited in the treatment of cancer is through the promotion of an anti-tumor immune response.
  • Anti-CD47 mAbs have not been previously shown to cause tumor cell release of ATP, HMGB1, annexin Al, type I interferons and CXCL10 and tumor cell expression of calreticulin, PDIA3, HSP70 and HSP90.
  • IRI ischemia-reperfusion injury
  • TSP1-CD47 The pathways that are limited by the TSP1-CD47 system are precisely those that would be of most benefit in combating the damage of IRI, including the NO pathway. Thus, blocking the TSP1-CD47 pathway, as with the antibodies disclosed herein, will provide more robust functioning of these endogenous protective pathways.
  • Anti-CD47 m Ahs have been shown to reduce organ damage in rodent models of renal warm ishchemia (Rogers et al. J Am Soc Nephrol. 23: 1538-1550, 2012), liver ischemia-reperfusion injury (Isenberg et al. Surgery. 144: 752-761, 2008), renal transplantation (Lin et al. Transplantation. 98: 394-401, 2014; Rogers et al. Kidney Interantional.
  • liver transplantation including steatotic livers (Xiao et al. Liver Transpl. 21 : 468-477, 2015; Xiao et al. Transplantation. 100: 1480-1489, 2016).
  • anti-CD47 mAh caused significant reductions of right ventricular systolic pressure and right ventricular hypertrophy in the monocrotaline model of pulmonary arterial hypertension (Bauer et al. Cardiovasc Res. 93: 682-693, 2012).
  • Studies in skin flap models have shown that modulation of CD47, including with anti-CD47 mAbs, inhibits TSPl-mediated CD47 signaling.
  • Anti-CD47 m Ahs have also been shown to be efficacious in models of other cardiovascular diseases.
  • anti-CD47 mAh mitigated cardiac myocyte hypertrophy, decreased left ventricular fibrosis, prevented an increase in left ventricular weight, decreased ventricular stiffness, and normalized changes in the pressure volume loop profile (Sharifi- Sanjani et al. J Am Heart Assoc., 2014).
  • An anti-CD47 mAh ameliorated atherosclerosis in multiple mouse models (Kojima et al. Nature., 2016).
  • anti-CD47 m Ahs and antigen binding fragments thereof effective as cancer therapeutics which can be administered to patients who exhibit anemia.
  • Anemia is a condition in which a patient lacks enough healthy red blood cells to carry adequate oxygen to the tissues of the body. Anemia can be temporary or long term, and it can range from mild to severe.
  • anemia can be caused by the cancer itself, chemotherapy treatments, or both. Cancers that involve the marrow space, such as leukemia or lymphoma, compete with the marrow’s function and interfere with normal red blood cell production.
  • Grade 1 is considered mild anemia, is Hb from 10 g/dL to the lower limit of normal; Grade 2 anemia, or moderate anemia, is Hb from 8 to less than 10 g/dL; Grade 3, or severe anemia, is below 8 g/dL; Grade 4, is life-threatening anemia; and Grade 5 is death.
  • the disclosed anti-CD47 mAbs can be administered to treat cancer in a patient with anemia, wherein the disclosed anti-CD47 mAbs bind to human CD47 on the surface of a tumor cell with minimal binding to human red blood cells (hRBCs), and wherein the human red blood cells (hRBCs) lack b ⁇ -integrin expression.
  • hRBCs human red blood cells
  • the disclosed anti-CD47 mAbs can be administered to treat cancer in a patient with anemia, wherein the disclosed anti-CD47 mAbs bind to human CD47 in association with an integrin on the surface of a tumor cell with minimal binding to human red blood cells (hRBCs), and wherein the human red blood cells (hRBCs) lack b ⁇ -integrin expression.
  • hRBCs human red blood cells
  • anti-CD47 mAbs and antigen binding fragments thereof effective as cancer therapeutics which can be administered to patients, preferably parenterally, with susceptible hematologic cancers and solid tumors including, but not limited to, leukemias, including systemic mastocytosis, acute lymphocytic (lymphoblastic) leukemia (ALL), T cell - ALL, acute myeloid leukemia (AML), myelogenous leukemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), myeloproliferative disorder / neoplasm, monocytic cell leukemia, and plasma cell leukemia; multiple myeloma (MM); Waldenstrom’s Macroglobulinemia; lymphomas, including histiocytic lymphoma and T cell lymphoma, B cell lymphomas, including Hodgkin’s lymphoma and non-Hodgkin’s lymphoma, such as low
  • the disclosed anti-CD47 m Ahs can be used in a method to treat gastric cancer in a human subject, wherein said monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with an integrin on the surface of a tumor cell, thereby causing an increase in cell death of tumor cells.
  • the gastric cancer can be selected from adenocarcinoma, lymphoma, gastrointentinal stromal tumor, or GIST, and carcinoid tumor (Type I and II ECL- cell carcinoids, Type III ECL-cell carcinoids).
  • combination therapies are often employed in cancer treatment as single-agent therapies or procedures may not be sufficient to treat or cure the disease or condition.
  • Conventional cancer treatments often involve surgery, radiation treatment, the administration of a combination of cytotoxic drugs to achieve additive or synergistic effects, and combinations of any or all of these approaches.
  • chemotherapeutic and biologic therapy combinations employ drugs that work via different mechanisms of action, increasing cancer cell control or killing, increasing the ability of the immune system to control cancer cell growth, reducing the likelihood of drug resistance during therapy, and minimizing possible overlapping toxicities by permitting the use of reduced doses of individual drugs.
  • Classes of conventional anti-tumor/anti-neoplastic agents useful in the combination therapies encompassed by the present methods are disclosed, for example, in Goodman & Gilman’s The Pharmacological Basis of Therapeutics, Twelfth Edition (2010) L.L. Brunton, B.A. Chabner, and B. C. Knollmann Eds., Section VIII, “Chemotherapy of Neoplastic Diseases”, Chapters 60-63, pp. 1665-1770, McGraw-Hill, NY, and include, for example, alkylating agents, antimetabolites, natural products, a variety of miscellaneous agents, hormones and antagonists, targeted drugs, monoclonal antibodies and other protein therapeutics.
  • the methods of the present disclosure are related to treatment of cancer indications and further comprises treating the patient via surgery, radiation, and/or administering to a patient in need thereof an effective amount of a chemical small molecule or biologic drug including, but not limited to, a peptide, polypeptide, protein, nucleic acid therapeutic, conventionally used or currently being developed, to treat tumorous conditions.
  • a chemical small molecule or biologic drug including, but not limited to, a peptide, polypeptide, protein, nucleic acid therapeutic, conventionally used or currently being developed, to treat tumorous conditions.
  • the therapeutic methods disclosed and claimed herein include the use of the antibodies disclosed herein alone, and/or in combinations with one another, and/or with antigen-binding fragments thereof of the present disclosure that bind to CD47, and/or with competing antibodies exhibiting appropriate biological/therapeutic activity, as well, for example, all possible combinations of these antibody compounds to achieve the greatest treatment efficacy.
  • the present therapeutic methods also encompass the use of these antibodies, antigen-binding fragments thereof, competing antibodies and combinations thereof further in combination with: (1) any one or more anti-tumor therapeutic treatments selected from surgery, radiation, anti-tumor, anti-neoplastic agents, and combinations of any of these, or (2) any one or more of anti-tumor biological agents, or (3) equivalents of any of the foregoing of (1) or (2) as would be apparent to one of ordinary skill in the art, in appropriate combination(s) to achieve the desired therapeutic treatment effect for the particular indication.
  • Antibody and small molecule drugs that increase the immune response to cancer by modulating co-stimulatory or inhibitory interactions that influence the T cell response to tumor antigens are also of particular interest in the context of the combination therapeutic methods encompassed herein and include, but are not limited to, other anti-CD47 antibodies.
  • Administration of therapeutic agents that bind to the CD47 protein for example, antibodies or small molecules that bind to CD47 and prevent interaction between CD47 and SIRPa, are administered to a patient, causing the clearance of cancer cells via phagocytosis.
  • the therapeutic agent that binds to the CD47 protein is combined with a therapeutic agent such as an antibody, a chemical small molecule or biologic drug disclosed herein, directed against one or more additional cellular targets of CD70 (Cluster of Differentiation 70), CD200 (OX-2 membrane glycoprotein, Cluster of Differentiation 200), CD 154 (Cluster of Differentiation 154, CD40L, CD40 ligand, Cluster of Differentiation 40 ligand), CD223 (Lymphocyte- activation gene 3, LAG3, Cluster of Differentiation 223), KIR (Killer-cell immunoglobulin like receptors), GITR (TNFRSF18, glucocorticoid-induced TNFR-related protein, activation- inducible TNFR family receptor, AITR, Tumor necrosis factor receptor superfamily member 18), CD28 (Cluster of Differentiation 28), CD40 (Cluster of Differentiation 40, Bp50, CDW40, TNFRSF5, Tumor necrosis factor receptor superfamily member 5, p50), CD86 (B
  • Carcinoembryonic antigen-related cell adhesion molecule 1 Carcinoembryonic antigen-related cell adhesion molecule 1, biliary glycoprotein, BGP, BGP1, BGPI, Cluster of Differentiation 66a), CD80 (B7-1, Cluster of Differentiation 80), CD94 (Cluster of Differentiation 94), NKG2A (Natural killer group 2A, killer cell lectin-like receptor subfamily D member 1, KLRDl), CD96 (Cluster of Differentiation 96, TActILE, T cell activation increased late expression), CD112 (PVRL2, nectin, Poliovirus receptor-related
  • CD115 CSF1R, Colony stimulating factor 1 receptor, macrophage colony-stimulating factor receptor, M-CSFR, Cluster of Differentiation 115
  • CD205 DEC-205, LY75, Lymphocyte antigen 75, Cluster of Differentiation 205
  • CD226 DNAM1, Cluster of Differentiation 226, DNAX Accessory Molecule-1, PTA1, platelet and T cell activation antigen 1
  • CD244 Cluster of Differentiation 244, Natural killer cell receptor 2B4
  • CD262 D5, TrailR2, TRAIL-R2, Tumor necrosis factor receptor superfamily member 10b, TNFRSF10B, Cluster of Differentiation 262, KILLER, TRICK2, TRICKB, ZTNFR9, TRICK2A, TRICK2B
  • CD284 Toll-like Receptor-4, TLR4, Cluster of Differentiation 284)
  • CD288 Toll-like Receptor-4, TLR4, Cluster of Differentiation 284)
  • CD288 Toll-like Receptor-4,
  • YERVOY® ipilimumab; Bristol-Meyers Squibb
  • YERVOY® is an example of an approved anti-CTLA-4 antibody.
  • KEYTRUDA® pembrolizumab; Merck
  • OPDIVO® nivolumab; Bristol- Meyers Squibb Company
  • TECENTRIQ® atezolizumab; Roche
  • SPRYCEL® dasatinib; Bristol-Meyers Squibb Company
  • ATP- competitive protein tyrosine kinase inhibitor The main targets of dasatinib are BCR/Abl (the "Philadelphia chromosome"), Src, c-Kit, ephrin receptors, and several other tyrosine kinases.
  • Defactinib (Verastem Oncology) is an orally available small molecule inhibitor of FAK and PYK2 that is currently in clinical development with potential anti-angiogeneic and anti-neoplastic activities.
  • IRI Ischemia-Reperfusion Injury
  • a CD47 mAh or antigen binding fragment thereof disclosed herein can be used to treat a number of diseases and conditions in which IRI is a contributing feature, and to treat various autoimmune, autoinflammatory, inflammatory and cardiovascular diseases.
  • diseases and conditions in which IRI is a contributing feature include: organ transplantation in which a mAh or antigen binding fragment thereof of the present disclosure is administered to the donor prior to organ harvest, to the harvested donor organ in the organ preservation solution, to the recipient patient, or to any combination thereof; skin grafting; surgical resections or tissue reconstruction in which such mAh or fragment is administered either locally by injection to the affected tissue or parenterally to the patient; reattachment of body parts; treatment of traumatic injury; pulmonary hypertension; pulmonary arterial hypertension; sickle cell disease (crisis); myocardial infarction; cerebrovascular disease; stroke; surgically-induced ischemia; acute kidney disease/kidney failure; any other condition in which IRI occurs and contributes to the pathogenesis of disease; autoimmune and inflammatory diseases, including arthritis,
  • Anti-CD47 m Ahs and antigen binding fragments thereof of the present disclosure can also be used to increase tissue perfusion in a subject in need of such treatment.
  • Such subjects can be identified by diagnostic procedures indicating a need for increased tissue perfusion.
  • the need for increased tissue perfusion may arise because the subject has had, is having, or will have, a surgery selected from integument surgery, soft tissue surgery, composite tissue surgery, skin graft surgery, resection of a solid organ, organ transplant surgery, or reattachment or an appendage or other body part.
  • IRI Ischemia-Reperfusion Injury
  • the methods of the present disclosure can further comprise administering to a patient in need thereof an effective amount of therapeutic agent that binds to the CD47 protein and a nitric oxide donor, precursor, or both; a nitric oxide generating topical agent; an agent that activates soluble guanylyl cyclase; an agent that inhibits cyclic nucleotide phosphodiesterases; or any combination of any of the foregoing.
  • the nitric oxide donor or precursor can be selected from NO gas, isosorbide dinitrate, nitrite, nitroprusside, nitroglycerin, 3-Morpholinosydnonimine (SIN-1), S- nitroso-N-acetylpenicillamine (SNAP), Diethylenetriamine/NO (DETA/NO), S-nitrosothiols, Bidil ® , and arginine.
  • NO gas isosorbide dinitrate, nitrite, nitroprusside, nitroglycerin, 3-Morpholinosydnonimine (SIN-1), S- nitroso-N-acetylpenicillamine (SNAP), Diethylenetriamine/NO (DETA/NO), S-nitrosothiols, Bidil ® , and arginine.
  • the agent that activates soluble guanylyl cyclase can be a non-NO (nitric oxide)- based chemical activator of soluble guanylyl cyclase that increases cGMP levels in vascular cells.
  • Such agents bind soluble guanylyl cyclase in a region other than the NO binding motif, and activate the enzyme regardless of local NO or reactive oxygen stress (ROS).
  • ROS reactive oxygen stress
  • Non-limiting examples of chemical activators of soluble guanylyl cyclase include organic nitrates (Artz et al. (2002) J. Biol. Chem. 277:18253-18256); protoporphyrin IX (Ignarro et al. (1982) Proc. Natl.
  • the agent that inhibits cyclic nucleotide phosphodiesterases can be selected from, tadalafil, vardenafil, udenafil, sildenafil and avanafil. Treatment of Autoimmune, Autoinflammatory, Inflammatory, and Cardiovascular Diseases
  • a therapeutic agent that binds to the CD47 protein for the treatment of an autoimmune, autoinflammatory, inflammatory disease and / or cardiovascular disease can be combined with one or more therapeutic agent(s) such as an antibody, a chemical small molecule, or biologic or a medical or surgical procedure which include, but are not limited to the following.
  • the combined therapeutic agents are: hydroxychloroquine, leflunomide, methotrexate, minocycline, sulfasalazine, abatacept, rituximab, tocilizumab, anti-TNF inhibitors or blockers (adalimumab, etanercept, infliximab, certolizumab pegol, golimumab), non-steroidal anti inflammatory drugs, glucocorticoids, corticosteroids, intravenous immunoglobulin, anakinra, canakinumab, rilonacept, cyclophosphamide, mycophenolate mofetil, azathioprine, 6- mercaptopurine, belimumab, beta interferons, glatiramer acetate, dimethyl fumarate, fingolimod, teriflunomide, natalizumab, 5 -aminosalicy
  • the combined therapeutic agents or procedures are: medical procedures and/or surgery, including percutaneous coronary intervention (coronary angioplasty and stenting), coronary artery bypass grafting, and carotid endarterectomy; therapeutic agents, including angiotensin-converting enzyme (ACE) inhibitors (including ramipril, quinapril, captopril, and enalapril), calcium channel blockers (including amiodipine, nifedipine, verapamil, felodipine and diltiazem), angiotensin-receptor blockers (including eposartan, olmesarten, azilsartan, valsartan, telmisartan, losartan, candesartan, and irbesartan), the combination of ezetimibe and simvastatin, PCSK9 inhibitors (including alirocumab and evolocumab), anacetrapib, and HMG-CoA
  • ACE angiotens
  • the combined therapeutic agents are: ACE inhibitors, angiotensin receptor blockers, angiotensin receptor neprilsyn inhibitors (including the combination of sacubitril and valsartan), diuretics, digoxin, inotropes, beta blockers and aldosterone antagonists.
  • the combined therapeutic agents are: sildenafil, tadalafil, ambrisentan, bosentan, macitentan, riociguat, treprostinil, epoprostenol, iloprost, and selexipag.
  • the anti-CD47 mAh is administered before, at the same time or after the combined therapeutic agents or medical or surgical procedures.
  • Another useful class of compounds for the combination therapies contemplated herein includes modulators of SIRPoc/CD47 binding such as antibodies to SIRPa, as well as soluble protein fragments of this ligand, or CD47 itself, inhibiting binding of, or interfering with binding of, SIRPa to CD47.
  • the therapeutic methods encompassed herein include the use of the antibodies disclosed herein alone, in combination with one another, and/or with antigen-binding fragments thereof as well, for example, a l possible combinations of these antibody compounds.
  • Diagnostics have been an area of focus in the field of oncology.
  • a number of diagnostic assays have been developed for targeted therapeutics such as Herceptin (Genentech), Tarceva (OSI Pharmaceuticals/Genentech), Iressa (Astra Zeneca), and Erbitux (Imclone / Bristol Myers Squibb).
  • the anti-CD47 mAbs antibodies of the disclosure are particularly well-suited to use in diagnostic applications. Accordingly, the disclosure provides a method to measure CD47 expression in tumor and / or immune cells, using an anti-CD47 mAh of the disclosure.
  • the anti-CD47 mAbs of the disclosure may be used in a diagnostic assay and / or in vitro method to measure CD47 expression in tumor and / or immune cells present in a patient’s tumor sample.
  • the anti-CD47 mAbs of the disclosure may bind CD47 on approximately 1% or more of tumor and / or immune cells present in a patient’s sample as compared to a reference level.
  • the anti-CD47 mAbs may bind CD47 on approximately 5% or more of tumor and/ or immune cells in a patient’s sample as compared to a reference level, for example, or binding at least 10%, or at least 20%, or at least 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or between 10-100% as compared to a reference level.
  • the anti-CD47 mAbs may bind CD47 on tumor and / or immune cells in a patient’s sample to at least about a 2-fold increase as compared to a reference level, or at least about 3 -fold, or at least about a 4-fold, or at least about a 5 -fold or at least about a 8- fold increase, or between 2-fold and 8-fold, or about 10-fold or greater as compared to a reference level.
  • the measurement of CD47 expression in a patient’s sample provides biological and/or clinical information that enables decision making about the development and use of a potential drug therapy, notably the use of anti-CD47 antibodies for treating solid and hematological cancers, autoimmune disease, inflammatory disease, atherosclerosis, heart failure, in which the CD47 receptor plays a role.
  • the in vitro method comprises, obtaining a patient sample, contacting the patient sample with a monoclonal antibody, or antigen-binding fragment thereof, which specifically binds to an epitope within the sequence of SEQ ID NO:66, and assaying for binding of the antibody to the patient sample, wherein binding of the antibody to the patient sample is diagnostic of CD47 expression in a patient sample.
  • a diagnostic assay in accordance with the disclosure may comprise contacting tumor and/or immune cells in a patient’s sample with an anti-CD47 mAh, or an antigen binding fragment thereof, and assaying for binding of the anti-CD47 mAh to a patient’s tumor sample, wherein binding of the anti-CD47 mAh to the patient sample is diagnostic of CD47 expression.
  • the patient’s sample is a sample containing tumor cells.
  • binding of the anti-CD47 mAh of the disclosure, or antigen binding fragment thereof, to the tumor cells may be assessed for CD47 expression.
  • the levels of CD47 expression by tumor cells and/or immune cells of a patient’s tumor sample may be predictive of clinical outcome in a patient.
  • Increased binding of anti-CD47 m Ahs binding to cells in a patient’s sample is associated with increased CD47 expression.
  • the anti-CD47 m Ahs of the disclosure may bind to approximately 5% or more of tumor cells in a patient’s sample and this may indicate that the patient would benefit from rapid intervention to a solid and hematological cancer.
  • a diagnostic assay of this sort may be used to determine suitable therapeutic regimes for solid and hematological cancers with relatively high binding of anti-CD47 m Ahs of the disclosure, i.e. , increased CD47 expression.
  • the diagnostic assay disclosed herein has a number of advantages. The most important of these advantages is that the diagnostic assay of the disclosure may allow the user a greater deal of confidence in the CD47 expression in tumor and / or immune cells. The increased sensitivity of the diagnostic assay of the disclosure allows detection of CD47 in a patient’s sample at lower levels than has previously been the case.
  • the anti-CD47 mAbs of the disclosure may be used as a diagnostic assay in relation to many forms of cancer.
  • Particular forms of cancer that may advantageously be investigated for CD47 expression include susceptible hematologic cancers and solid tumors including, but not limited to, leukemias, lymphomas, and solid tumors.
  • the diagnostic assays of the disclosure may utilize any suitable means for detecting binding of an anti-CD47 mAh to measure CD47 expression. Suitable methods may be selected with reference to the nature of any reporter moiety used to label the anti-CD47 m Ahs of the disclosure. Suitable techniques include, but are by no means limited to, flow cytometry, and enzyme linked immunosorbent assays (ELISA) and assays utilizing nanoparticles.
  • Suitable techniques include, but are by no means limited to, flow cytometry, and enzyme linked immunosorbent assays (ELISA) and assays utilizing nanoparticles.
  • Chimeric antibodies disclosed herein comprise a mouse heavy chain variable domain and a light chain variable domain combined with a human kappa or human Fc IgGl , IgGl-N297Q, IgG2, IgG4, IgG4 S228P, and IgG4 PE constant domains, respectively. These were designed to incorporate a secretion signal and cloned into a mammalian expression system, and transfected into CHO cells to generate chimeric (murine -human) antibodies. The chimeric variants were expressed as full length IgG molecules, secreted into the medium, and purified using protein A.
  • CDRs complementarity determining regions
  • the humanized variable domains were then combined with a secretion signal and human kappa and human Fc IgGl, IgGl-N297Q, IgG2, IgG3, IgG4 S228P and IgG4 PE constant domains, cloned into a mammalian expression system, and transfected into CHO cells to generate humanized m Ahs.
  • the humanized variants were expressed as full length IgG molecules, secreted into the medium and purified using protein A.
  • a non-glycosylated version (IgGl-N297Q) was created by site directed mutagenesis of heavy chain position 297 to change the asparagine to glutamine (Human Fc IgGl-N297Q, SEQ ID NO:54).
  • An IgG4 variant was created by site-directed mutagenesis at position 228 to change the serine to proline thereby preventing in vivo Fab arm exchange.
  • An IgG4 double mutant was created by site-directed mutagenesis at positions 228 (serine to proline) and 235 (leucine to glutamate) to prevent Fab arm exchange and to further reduce Fc effector function.
  • IgG2, IgG3, IgG4 S228P, and IgG4PE isotypes were constructed by cloning the heavy chain variable domain in frame with the human IgG2, IgG3, IgG4 S228P, and IgG4PE constant domains (Human Fc-IgG2, SEQ ID NO:56 Human Fc-IgG3, SEQ ID NO:57; Human Fc-IgG4 S228P, SEQ ID NO:59; and Human Fc-IgG4PE, SEQ ID NO:60).
  • chimeric (murine-human) and humanized antibodies of the present disclosure was determined by ELISA using OV10 cells transfected with human CD47 (OV10 hCD47) or using freshly isolated human red blood cells (hRBCs), which display CD47 on their surface (Kamel et al. (2010) Blood. Transfus. 8(4):260-266).
  • Binding activities of VLX4, VLX8, and VLX9 chimeric (xi) and humanized mAbs were determined using a cell-based ELISA assay with human OV10 hCD47cells expressing cell surface human CD47.
  • OV10 hCD47 cells were grown in IMDM medium containing 10% heat inactivated fetal bovine serum (BioWest; S01520).
  • 3xl0 4 cells were plated in 96 well cell bind plates (Corning #3300, VWR #66025-626) and were 95-100% confluent at the time of assay.
  • Binding activities of chimeric and humanized VLX4, VLX8, and VLX9 mAbs to human CD47 on hRBCs were also determined using flow cytometry. Blood was obtained from normal volunteers and RBCs were washed 3 times with phosphate buffered saline, pH 7.2 containing 2.5 mM EDTA (PBS+E). hRBCs were incubated for 60 min at 37°C with various concentrations of the chimeric or humanized antibodies in a PBS+E.

Abstract

Provided are anti-CD47 monoclonal antibodies (anti-CD47 mAbs) with distinct functional profiles as described herein, methods to generate anti-CD47 mAbs, and to methods of using these anti-CD47 mAbs as therapeutics for the prevention and treatment of solid and hematological cancers, ischemia-reperfusion injury, cardiovascular diseases, autoimmune diseases, inflammatory diseases or as diagnostics for determining the level of CD47 in tissue samples.

Description

THERAPEUTIC CD47 ANTIBODIES
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application cl aim the benefit of U.S. Provisional Application No. 62/823,303, filed March 25, 2019, U.S. Provisional Application No. 62/877,069 filed July 22, 2019, and U.S. Provisional Application No. 62/909,603 filed October 2, 2019 the disclosures of which are incorporated herein in their entirety.
FIELD OF THE DISCLOSURE
[0002] This disclosure is related generally to anti-CD47 monoclonal antibodies (anti-CD47 mAbs) with distinct functional profiles as described herein, methods to generate anti-CD47 mAbs, and methods of using these anti-CD47 mAbs as therapeutics for the prevention and treatment of solid and hematological cancers, ischemia-reperfusion injury, cardiovascular diseases, autoimmune diseases, or inflammatory diseases, or as diagnostics for determining the level of CD47 in tissue samples.
BACKGROUND OF THE DISCLOSURE
[0003] CD47 is a cell surface receptor comprised of an extracellular IgV set domain, a 5 transmembrane domain, and a cytoplasmic tail that is alternatively spliced. Two ligands bind CD47: signal-regulatory protein alpha (SIRPoc) and thrombospondin- 1 (TSP1).
[0004] SIRPa is an innate immune checkpoint receptor primarily expressed on macrophages, granulocytes, dendritic cells and monocytes. SIRPa suppresses innate immunity upon interaction with its ligand, CD47, which is widely expressed on normal tissues and is upregulated in most human tumors as a means to escape macrophage recognition and programmed cell removal.
[0005] CD47 expression and/or activity has been implicated in a number of diseases and disorders. Accordingly, there exists a need for therapeutic compositions and methods for treating diseases and conditions associated with CD47 in humans and animals, including the prevention and treatment of solid and hematological cancers, ischemia-reperfusion injury (IRI), cardiovascular diseases, or an autoimmune or inflammatory disease.
SUMMARY OF THE DISCLOSURE [0006] The present disclosure describes anti-CD47 m Ahs with distinct functional profiles. These antibodies possess distinct combinations of properties selected from the following: 1) exhibit cross-reactivity with one or more species homologs of CD47; 2) block the interaction between CD47 and its ligand SIRPoc; 3) increase phagocytosis of human tumor cells; 4) induce death of susceptible human tumor cells; 5) do not induce cell death of human tumor cells; 6) do not have reduced or minimal binding to human red blood cells (hRBCs); 7) have reduced binding to hRBCs; 8) have minimal binding to hRBCs; 9) cause reduced agglutination of hRBCs; 10) cause no detectable agglutination of hRBCs; 11) reverse TSP1 inhibition of the nitric oxide (NO) pathway; 12) do not reverse TSP1 inhibition of the NO pathway; 13) cause loss of mitochondrial membrane potential; 14) do not cause cause loss of mitochondrial membrane potential; 15) cause an increase in cell surface calreticulin expression on human tumor cells; 16) do not cause an increase in cell surface calreticulin expression on human tumor cells; 17) cause an increase in adenosine triphosphate (ATP) release by human tumor cells; 18) do not cause an increase in adenosine triphosphate (ATP) release by human tumor cells; 19) cause an increase in high mobility group box 1 (HMGB 1) release by human tumor cells; 20) do not cause an increase in high mobility group box 1 (HMGB 1) release by human tumor cells; 21) cause an increase in type I interferon release by human tumor cells; 22) do not cause an increase in type I interferon release by human tumor cells; 23) cause an increase in C-X-C Motif Chemokine Ligand 10 (CXCL10) release by human tumor cells; 24) do not cause an increase in C-X-C Motif Chemokine Ligand 10 (CXCL10) release by human tumor cells; 25) cause an increase in cell surface protein disulfide-isomerase A3 (PDIA3) expression on human tumor cells; 26) do not cause an increase in cell surface protein disulfide-isomerase A3 (PDIA3) expression on human tumor cells; 27) cause an increase in cell surface heat shock protein 70 (HSP70) expression on human tumor cells; 28) do not cause an increase in cell surface heat shock protein 70 (HSP70) expression on human tumor cells; 29) cause an increase in cell surface heat shock protein 90 (HSP90) expression on human tumor cells; 30) do not cause an increase in cell surface heat shock protein 90 (HSP90) expression on human tumor cells; 31) have reduced binding to normal human cells, which includes, but is not limited to, endothelial cells, skeletal muscle cells, epithelial cells, and peripheral blood mononuclear cells (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubular epithelial cells, human peripherial blood CD3+ cells, and human peripheral blood mononuclear cells); 32) do not have reduced binding to normal human cells, which includes, but is not limited to, endothelial cells, skeletal muscle cells, epithelial cells, and peripheral blood mononuclear cells (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubular epithelial cells, human peripherial blood CD3+ cells, and human peripheral blood mononuclear cells); 33) have a greater affinity for human CD47 at an acidic pH compared to physiological pH; 34) do not have a greater affinity for human CD47 at an acidic pH compared to physiological pH; and 35) cause an increase in annexin A1 release by human tumor cells. The anti-CD47 mAbs of the disclosure are useful in various therapeutic methods for treating diseases and conditions associated with CD47 in humans and animals, including the prevention and treatment of solid and hematological cancers, autoimmune diseases, inflammatory diseases, IRI, and cardiovascular diseases. The antibodies of the disclosure are also useful as diagnostics to determine the level of CD47 expression in tissue samples. Embodiments of the disclosure include isolated antibodies and immunologically active binding fragments thereof; pharmaceutical compositions comprising one or more of the anti-CD47 mAbs, preferably chimeric or humanized forms of said anti-CD47 mAbs; methods of therapeutic use of such anti-CD47 monoclonal antibodies; and cell lines that produce these anti-CD47 mAbs.
[0007] The embodiments of the disclosure include the mAbs, or antigen-binding fragments thereof, which are defined herein by reference to specific structural characteristics, i.e. , specified amino acid sequences of either the CDRs or entire heavy chain or light chain variable domains. All antibodies of the disclosure bind to CD47.
[0008] The monoclonal antibodies, or antigen binding fragments thereof, may comprise at least one, usually at least three, CDR sequences as provided herein, usually in combination with framework sequences from a human variable region or as an isolated CDR peptide. In some embodiments, an antibody comprises at least one light chain comprising the three light chain CDR sequences provided herein situated in a variable region framework, which may be, without limitation, a murine or human variable region framework, and at least one heavy chain comprising the three heavy chain CDR sequences provided herein situated in a variable region framework, which may be, without limitation, a human or murine variable region framework.
[0009] Some embodiments of the disclosure are anti-CD47 mAbs, or antigen binding fragments thereof, comprising a heavy chain variable domain comprising a variable heavy chain CDR1, variable heavy chain CDR2, and a variable heavy chain CDR3, wherein said variable heavy chain CDR1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: l, SEQ ID NO:2, and SEQ ID NO:3; said variable heavy chain CDR2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; and said variable heavy chain CDR3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO: 10.
[0010] The heavy chain variable (VH) domain may comprise any one of the listed variable heavy chain CDR1 sequences (HCDR1) in combination with any one of the variable heavy chain CDR2 sequences (HCDR2) and any one of the variable heavy chain CDR3 sequences (HCDR3). However, certain embodiments of HCDR1 and HCDR2 and HCDR3 are are provided that derive from a single common VH domain, examples of which are described herein.
[0011] The antibody or antigen binding fragment thereof may additionally comprise a light chain variable (VL) domain, which is paired with the VH domain to form an antigen binding domain. In some embodiments, light chain variable domains are those comprising a variable light chain CDR1, variable light chain CDR2, and a variable light chain CDR3, wherein said variable light chain CDR1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, and SEQ ID NO:14; said variable light chain CDR2 optionally comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:15, SEQ ID NO: 16, and SEQ ID NO: 17; and said variable light chain CDR3 optionally comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO:20.
[0012] The light chain variable domain may comprise any one of the listed variable light chain CDR1 sequences (LCDR1) in combination with any one of the variable light chain CDR2 sequences (LCDR2) and any one of the variable light chain CDR3 sequences (LCDR3). However, certain embodiments of LCDR1 and LCDR2 and LCDR3 are provided that derive from a single common VL domain, examples of which are described herein.
[0013] Any given CD47 antibody or antigen binding fragment thereof comprising a VH domain paired with a VL domain will comprise a combination of 6 CDRs: variable heavy chain CDR1 (HCDR1), variable heavy chain CDR2 (HCDR2), variable heavy chain CDR3 (HCDR3), variable light chain CDR1 (LCDR1), variable light chain CDR2 (LCDR2), and variable light chain CDR3 (LCDR3). Although all combinations of 6 CDRs selected from the CDR sequence groups listed above are permissible, and within the scope of the disclosure, certain combinations of 6 CDRs are provided.
[0014] In some embodiments, combinations of 6 CDRs include, but are not limited to, the combinations of variable heavy chain CDR1 (HCDR1), variable heavy chain CDR2 (HCDR2), variable heavy chain CDR3 (HCDR3), variable light chain CDR1 (LCDR1), variable light chain CDR2 (LCDR2), and variable light chain CDR3 (LCDR3) selected from the group consisting of:
(i) HCDR1 comprising SEQ ID NO: 1 , HCDR2 comprising SEQ ID NO:4, HCDR3 comprising SEQ ID NO:7, LCDR1 comprising SEQ ID NO:l l, LCDR2 comprising SEQ ID NO: 15, LCDR3 comprising SEQ ID NO: 18;
(ii) HCDR1 comprising SEQ ID NO: 1 , HCDR2 comprising SEQ ID NO:4, HCDR3 comprising SEQ ID NO:8, LCDR1 comprising SEQ ID NO:l l, LCDR2 comprising SEQ ID NO: 15, LCDR3 comprising SEQ ID NO: 18;
(iii) HCDR1 comprising SEQ ID NO:2, HCDR2 comprising SEQ ID NO:5, HCDR3 comprising SEQ ID NO:9, LCDR1 comprising SEQ ID NO: 12, LCDR2 comprising SEQ ID NO: 16, LCDR3 comprising SEQ ID NO: 19;
(iv) HCDR1 comprising SEQ ID NO:2, HCDR2 comprising SEQ ID NO:5, HCDR3 comprising SEQ ID NO:9, LCDR1 comprising SEQ ID NO:13, LCDR2 comprising SEQ ID NO: 16, LCDR3 comprising SEQ ID NO: 19; and
(v) HCDR1 comprising SEQ ID NO:3, HCDR2 comprising SEQ ID NO:6, HCDR3 comprising SEQ ID NO: 10, LCDR1 comprising SEQ ID NO: 14, LCDR2 comprising SEQ ID NO: 17, LCDR3 comprising SEQ ID NO: 18.
[0015] In some embodiments, anti-CD47 m Ahs include antibodies or antigen binding fragments thereof, comprising a heavy chain variable domain having an amino acid sequence selected from the group consisting of: the amino acid sequences of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, and amino acid sequences exhibiting at least 90%, 95%, 97%, 98%, or 99% sequence identity to one of the recited sequences. Alternatively, or in addition, anti-CD47 mAbs including antibodies or antigen binding fragments thereof, may comprise a light chain variable domain having an amino acid sequence selected from the group consisting of: the amino acid sequences of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, and SEQ ID NO:52, and amino acid sequences exhibiting at least 90%, 95%, 97%, 98%, or 99% sequence identity to one of the recited sequences.
[0016] Although all possible pairing of VH domains and VL domains selected from the VH and VL domain sequence groups listed above are permissible, and within the scope of the disclosure, some embodiments provide certain combinations of VH and VL domains. Accordingly, in some embodiments, anti-CD47 mAbs, or antigen binding fragments thereof, are those comprising a combination of a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the combination is selected from the group consisting of:
(i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:21 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:41;
(ii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:23 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:43;
(iii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:34 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:49;
(iv) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:36 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:52;
(v) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:38 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:52;
(vi) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:39 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:52;
(vii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:24 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:43;
(viii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:37 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:52;
(ix) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:33 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:48;
(x) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:26 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:44; (xi) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:27 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:44; and
(xii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:38 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:51;
(xiii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:39 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:51;
(xiv) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:40 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:52;
(xv) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:36 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:51;
(xvi) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:29 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:47;
(xvii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:30 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:47;
(xviii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:31 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:47;
(xix) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:32 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:47;
(xx) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:33 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:47;
(xxi) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:29 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:48; (xxii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:30 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:48;
(xxiii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:31 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:48;
(xxiv) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:32 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:48;
(xxv) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:26 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:43;
(xxvi) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:27 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:43;
(xxvii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:28 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:46;
(xxviii)a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:35 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:50;
(xxix) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:29 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:48;
(xxx) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:30 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:48;
(xxxi) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:31 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:48;
(xxxii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:32 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:48; (xxxiii)a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:37 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:51 ; and
(xxxiv)a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:40 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:51.
[0017] In some embodiments, anti-CD47 antibodies or antigen binding fragments thereof may also comprise a combination of a heavy chain variable domain and a light chain variable domain wherein the heavy chain variable domain comprises a VH sequence with at least 85% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 97%, 98% or 99% sequence identity, to the heavy chain amino acid sequences shown above in (i) to (xxxiv) and/or the light chain variable domain comprises a VL sequence with at least 85% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 97%, 98% or 99% sequence identity, to the light chain amino acid sequences shown above in (i) to (xxxiv). The specific VH and VL pairings or combinations in parts (i) through (xxxiv) may be preserved for anti-CD47 antibodies having VH and VL domain sequences with a particular percentage sequence identity to these reference sequences.
[0018] For all embodiments wherein the heavy chain and/or light chain variable domains of the antibodies or antigen binding fragments are defined by a particular percentage sequence identity to a reference sequence, the VH and/or VL domains may retain identical CDR sequences to those present in the reference sequence such that the variation is present only within the framework regions.
[0019] In another embodiment, the preferred CD47 antibodies, or antigen binding fragments thereof, are those comprising a combination of a heavy chain (HC) and a light chain (LC), wherein the combination is selected from the group consisting of:
(i) a heavy chain comprising the amino acid sequence of SEQ ID NO:76 and a light chain comprising the amino acid sequence SEQ ID NO:66;
(ii) a heavy chain comprising the amino acid sequence of SEQ ID NO:77 and a light chain comprising the amino acid sequence SEQ ID NO: 68;
(iii) a heavy chain comprising the amino acid sequence of SEQ ID NO:78 and a light chain comprising the amino acid sequence SEQ ID NO: 69;
(iv) a heavy chain comprising the amino acid sequence of SEQ ID NO:79 and a light chain comprising the amino acid sequence SEQ ID NO: 70; (v) a heavy chain comprising the amino acid sequence of SEQ ID NO: 80 and a light chain comprising the amino acid sequence SEQ ID NO: 70;
(vi) a heavy chain comprising the amino acid sequence of SEQ ID NO: 81 and a light chain comprising the amino acid sequence SEQ ID NO: 70;
(vii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 82 and a light chain comprising the amino acid sequence SEQ ID NO: 68;
(viii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 83 and a light chain comprising the amino acid sequence SEQ ID NO: 70;
(ix) a heavy chain comprising the amino acid sequence of SEQ ID NO: 84 and a light chain comprising the amino acid sequence SEQ ID NO:71 ;
(x) a heavy chain comprising the amino acid sequence of SEQ ID NO: 85 and a light chain comprising the amino acid sequence SEQ ID NO: 72;
(xi) a heavy chain comprising the amino acid sequence of SEQ ID NO: 86 and a light chain comprising the amino acid sequence SEQ ID NO: 72;
(xii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 80 and a light chain comprising the amino acid sequence SEQ ID NO: 73;
(xiii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 81 and a light chain comprising the amino acid sequence SEQ ID NO: 73;
(xiv) a heavy chain comprising the amino acid sequence of SEQ ID NO: 87 and a light chain comprising the amino acid sequence SEQ ID NO: 70;
(xv) a heavy chain comprising the amino acid sequence of SEQ ID NO:79 and a light chain comprising the amino acid sequence SEQ ID NO: 73;
(xvi) a heavy chain comprising the amino acid sequence of SEQ ID NO:88 and a light chain comprising the amino acid sequence SEQ ID NO: 74;
(xvii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 89 and a light chain comprising the amino acid sequence SEQ ID NO: 74;
(xviii) a heavy chain comprising the amino acid sequence of SEQ ID NO:90 and a light chain comprising the amino acid sequence SEQ ID NO: 74;
(xix) a heavy chain comprising the amino acid sequence of SEQ ID NO:91 and a light chain comprising the amino acid sequence SEQ ID NO: 74;
(xx) a heavy chain comprising the amino acid sequence of SEQ ID NO: 84 and a light chain comprising the amino acid sequence SEQ ID NO: 74;
(xxi) a heavy chain comprising the amino acid sequence of SEQ ID NO:92 and a light chain comprising the amino acid sequence SEQ ID NO:71 ; (xxii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 89 and a light chain comprising the amino acid sequence SEQ ID NO:71 ;
(xxiii) a heavy chain comprising the amino acid sequence of SEQ ID NO:90 and a light chain comprising the amino acid sequence SEQ ID NO:71 ;
(xxiv) a heavy chain comprising the amino acid sequence of SEQ ID NO:91 and a light chain comprising the amino acid sequence SEQ ID NO:71 ;
(xxv) a heavy chain comprising the amino acid sequence of SEQ ID NO: 85 and a light chain comprising the amino acid sequence SEQ ID NO: 68;
(xxvi) a heavy chain comprising the amino acid sequence of SEQ ID NO: 86 and a light chain comprising the amino acid sequence SEQ ID NO: 68;
(xxvii) a heavy chain comprising the amino acid sequence of SEQ ID NO:93 and a light chain comprising the amino acid sequence SEQ ID NO: 100;
(xxviii)a heavy chain comprising the amino acid sequence of SEQ ID NO:94 and a light chain comprising the amino acid sequence SEQ ID NO: 75;
(xxix) a heavy chain comprising the amino acid sequence of SEQ ID NO:95 and a light chain comprising the amino acid sequence SEQ ID NO:71 ;
(xxx) a heavy chain comprising the amino acid sequence of SEQ ID NO:96 and a light chain comprising the amino acid sequence SEQ ID NO:71 ;
(xxxi) a heavy chain comprising the amino acid sequence of SEQ ID NO:97 and a light chain comprising the amino acid sequence SEQ ID NO:71 ;
(xxxii) a heavy chain comprising the amino acid sequence of SEQ ID NO:98 and a light chain comprising the amino acid sequence SEQ ID NO:71 ;
(xxxiii)a heavy chain comprising the amino acid sequence of SEQ ID NO: 83 and a light chain comprising the amino acid sequence SEQ ID NO: 73; and (xxxiv)a heavy chain comprising the amino acid sequence of SEQ ID NO: 87 and a light chain comprising the amino acid sequence SEQ ID NO: 73;
wherein the VH amino acid sequence is at least 90%, 95%, 97%, 98% or 99% identical thereto and the a VL amino acid sequence is at least 90%, 95%, 97%, 98% or 99% identical thereto.
[0020] In some embodiments, anti-CD47 antibodies as described herein may also be characterized by combinations of properties which are not exhibited by prior art anti-CD47 antibodies proposed for human therapeutic use. Accordingly, in some embodiments, anti-CD47 antibodies described herein are characterized by:
a. binds to human CD47 ; b. blocks SIRPoc binding to human CD47;
c. increases phagocytosis of human tumor cells; and
d. induces death of susceptible human tumor cells.
[0021] In another embodiment described herein, the anti-CD47 antibodies are characterized by:
a. binds to human CD47 ;
b. blocks SIRPoc binding to human CD47;
c. increases phagocytosis of human tumor cells;
d. induces death of susceptible human tumor cells; and
e. causes no detectable agglutination of human red blood cells (hRBCs).
[0022] In yet another embodiment described herein, the anti-CD47 antibodies are characterized by:
a. binds to human CD47 ;
b. blocks SIRPoc binding to human CD47;
c. increases phagocytosis of human tumor cells;
d. induces death of susceptible human tumor cells; and
e. causes reduced agglutination of human red blood cells (hRBCs).
[0023] In another embodiment described herein, the anti-CD47 antibodies are characterized by:
a. binds to human CD47 ;
b. blocks SIRPoc binding to human CD47;
c. increases phagocytosis of human tumor cells;
d. induces death of susceptible human tumor cells; and
e. has reduced hRBC binding.
[0024] In another embodiment described herein, the anti-CD47 antibodies are characterized by:
a. binds to human CD47,
b. blocks SIRPoc binding to human CD47,
c. increases phagocytosis of human tumor cells,
d. causes no detectable agglutination of human red blood cells (hRBCs); and e. has minimal binding to hRBCs.
[0025] In another embodiment described herein, the anti-CD47 antibodies are characterized by: a. binds to human CD47 ;
b. blocks SIRPoc binding to human CD47;
c. increases phagocytosis of human tumor cells;
d. causes no detectable agglutination of human red blood cells (hRBCs); and e. has reduced hRBC binding.
[0026] Additional embodiments of the anti-CD47 antibodies described herein, are also characterized by combinations of properties which are not exhibited by prior art anti-CD47 antibodies proposed for human therapeutic use. Accordingly, anti-CD47 antibodies as described herein may be further characterized by one or more among the following characteristics:
a. causes an increase in cell surface calreticulin expression on human tumor cells; b. causes an increase in adenosine triphosphate (ATP) release by human tumor cells;
c. causes an increase in high mobility group box 1 (HMGB1) release by human tumor cells;
d. causes an increase in annexin A1 release by human tumor cells;
e. causes an increase in Type I Interferon release by human tumor cells;
f. causes an increase in C-X-C Motif Chemokine Ligand 10 (CXCL10) release by human tumor cells;
g. causes an increase in cell surface protein disulfide-isomerase A3 (PDIA3) expression on human tumor cells;
h. causes an increase in cell surface heat shock protein 70 (HSP70) expression on human tumor cells; and
i. causes an increase in cell surface heat shock protein 90 (HSP90) expression on human tumor cells.
[0027] In another embodiment described herein, the monoclonal antibody, or antigen binding fragment thereof binds to human, non-human primate, mouse, rabbit, and rat CD47.
[0028] In yet another embodiment described herein, the monoclonal antibody, or antigen binding fragment thereof specifically also binds to non-human primate CD47, wherein non human primate may include, but is not limited to, cynomolgus monkey, green monkey, rhesus monkey, and squirrel monkey.
[0029] In yet another embodiment described herein, the monoclonal antibody, or antigen binding fragment thereof, has reduced binding to normal human cells, which includes, but is not limited to, endothelial cells, skeletal muscle cells, epithelial cells, and peripheral blood mononuclear cells (e.g., human aortic endothelial cells, human skeletal muscle cells, human micro vascular endothelial cells, human renal tubular epithelial cells, human peripherial blood CD3+ cells, and human peripheral blood mononuclear cells).
[0030] In yet another embodiment described herein, the monoclonal antibody, or antigen binding fragment thereof, has a greater have a greater affinity for human CD47 at an acidic pH compared to physiological pH.
[0031] In some embodiments, the monoclonal antibody, or antigen binding fragment thereof, may additionally possess one or more of the following characteristics: 1) exhibit cross reactivity with one or more species homologs of CD47 ; 2) block the interaction between CD47 and its ligand SIRPoc; 3) increase phagocytosis of human tumor cells; 4) induce death of susceptible human tumor cells; 5) do not induce cell death of human tumor cells; 6) do not have reduced or minimal binding to human red blood cells (hRBCs); 7) have reduced binding to hRBCs; 8) have minimal binding to hRBCs; 9) cause reduced agglutination of hRBCs; 10) cause no detectable agglutination of hRBCs; 11) reverse TSP1 inhibition of the nitric oxide (NO) pathway; 12) do not reverse TSP1 inhibition of the NO pathway; 13) cause loss of mitochondrial membrane potential; 14) do not cause cause loss of mitochondrial membrane potential; 15) cause an increase in cell surface calreticulin expression on human tumor cells; 16) do not cause an increase in cell surface calreticulin expression on human tumor cells; 17) cause an increase in adenosine triphosphate (ATP) release by human tumor cells; 18) do not cause an increase in adenosine triphosphate (ATP) release by human tumor cells; 19) cause an increase in high mobility group box 1 (HMGB1) release by human tumor cells; 20) do not cause an increase in high mobility group box 1 (HMGB 1) release by human tumor cells; 21) cause an increase in type I interferon release by human tumor cells; 22) do not cause an increase in type I interferon release by human tumor cells; 23) cause an increase in C-X-C Motif Chemokine Ligand 10 (CXCL10) release by human tumor cells; 24) do not cause an increase in C-X-C Motif Chemokine Ligand 10 (CXCL10) release by human tumor cells; 25) cause an increase in cell surface protein disulfide-isomerase A3 (PDIA3) expression on human tumor cells; 26) do not cause an increase in cell surface protein disulfide-isomerase A3 (PDIA3) expression on human tumor cells; 27) cause an increase in cell surface heat shock protein 70 (HSP70) expression on human tumor cells; 28) do not cause an increase in cell surface heat shock protein 70 (HSP70) expression on human tumor cells; 29) cause an increase in cell surface heat shock protein 90 (HSP90) expression on human tumor cells; 30) do not cause an increase in cell surface heat shock protein 90 (HSP90) expression on human tumor cells; 31) have reduced binding to normal human cells, which includes, but is not limited to, endothelial cells, skeletal muscle cells, epithelial cells, and peripheral blood mononuclear cells (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubular epithelial cells, human peripherial blood CD3+ cells, and human peripheral blood mononuclear cells); 32) do not have reduced binding to normal human cells, which includes, but is not limited to, endothelial cells, skeletal muscle cells, epithelial cells, and peripheral blood mononuclear cells (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubular epithelial cells, human peripherial blood CD3+ cells, and human peripheral blood mononuclear cells); 33) have a greater affinity for human CD47 at an acidic pH compared to physiological pH; 34) do not have a greater affinity for human CD47 at an acidic pH compared to physiological pH; and 35) cause an increase in annexin A1 release by human tumor cells.
[0032] Various forms of the anti-CD47 m Ahs disclosed are contemplated herein. For example, the anti-CD47 mAbs can be full-length humanized antibodies with human frameworks and constant regions of the isotypes, IgA, IgD, IgE, IgG, and IgM, more particularly, IgGl, IgG2, IgG3, IgG4, and in some cases with various mutations to alter Fc receptor function or prevent Fab arm exchange or an antibody fragment, e.g., a F(ab’)2 fragment, a F(ab) fragment, a single chain Fv fragment (scFv), etc., as disclosed herein.
[0033] In some embodiments, pharmaceutical or veterinary compositions are provided that comprise one or more of the anti-CD47 mAbs or fragments disclosed herein, optionally chimeric or humanized forms, and a pharmaceutically acceptable carrier, diluent, or excipient.
[0034] Prior to the present disclosure, there was a need to identify anti-CD47 mAbs that possess the functional profiles as described herein. The anti-CD47 mAbs of the present disclosure exhibit distinct combinations of properties, particularly combinations of properties that render the m Ahs particularly advantageous or suitable for use in human therapy, particularly in the prevention and/or treatment of solid and hematological cancers, ischemia- reperfusion injury, autoimmune and/or inflammatory diseases.
[0035] In some embodiments, the disclosure provides a monoclonal antibody, or an antigen binding fragment thereof, which: binds to human CD47; blocks SIRPoc binding to human CD47; increases phagocytosis of human tumor cells; and induces death of human tumor cells; wherein said monoclonal antibody, or an antigen binding fragment thereof, exhibits pH- dependent binding to CD47 present on a cell. In other embodiments, the disclosure provides a monoclonal antibody, or an antigen binding fragment thereof, which: binds to human CD47; blocks SIRPoc binding to human CD47; increases phagocytosis of human tumor cells; wherein said monoclonal antibody, or an antigen binding fragment thereof, exhibits pH-dependent binding to CD47 present on a cell. In other embodiments, the disclosure provides a monoclonal antibody, or an antigen binding fragment thereof, which: binds to human CD47; blocks SIRPoc binding to human CD47; increases phagocytosis of human tumor cells; and induces death of human tumor cells; wherein said monoclonal antibody, or an antigen binding fragment thereof, exhibits reduced binding to normal cells. In one embodiment, these cells may be an endothelial cell, a skeletal muscle cell, an epithelial cell, a PBMC or a RBC (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubular epithelial cells, human peripherial blood CD3+ cells, human peripheral blood mononuclear cells or human RBC). In other embodiments, the disclosure provides a monoclonal antibody, or an antigen binding fragment thereof, which: binds to human CD47; blocks SIRPoc binding to human CD47; increases phagocytosis of human tumor cells; wherein said monoclonal antibody, or an antigen binding fragment thereof, exhibits reduced binding to normal cells. In one embodiment, these cells may be an endothelial cell, a skeletal muscle cell, an epithelial cell, a PBMC or a RBC (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubular epithelial cells, human peripherial blood CD3+ cells, human peripheral blood mononuclear cells or human RBC). In another embodiment, the monoclonal antibody, or an antigen binding fragment thereof, exhibits both pH dependent binding and reduced binding to a cell.
[0036] Further scope of the applicability of the present disclosure will become apparent from the detailed description provided below. However, it should be understood that the detailed description and specific examples, while indicating some embodiments of the disclosure, are given by way of illustration only since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] The above and other aspects, features, and advantages of the present disclosure will be better understood from the following detailed descriptions taken in conjunction with the accompanying drawing(s), all of which are given by way of illustration only, and are not limitative of the present disclosure. [0038] FIG. 1A. Binding of VLX4 Humanized m Ahs to Human OVIO Cells Expressing Human CD47. Binding of VLX4 humanized m Ahs (VLX4hum_01 IgGl, VLX4hum_02 IgGl, VLX4hum_01 IgG4PE, and VLX4hum_02 IgG4PE) to human CD47 was determined using a OVIO cell line expressing human CD47 (OVIO hCD47) cell-based ELISA. OVIO hCD47 cells were plated into 96 well plates and were confluent at the time of assay. Various concentrations of m Ahs were added to the cells for 1 hr. Cells were washed and then incubated with HRP-labelled secondary antibody for 1 hr followed by addition of peroxidase substrate.
[0039] FIG. IB. Binding of VLX4 Humanized m Ahs to Human OVIO Cells Expressing Human CD47. Binding of VLX4 humanized mAbs (VLX4hum_06 IgG4PE, VLX4hum_07 IgG4PE, VLX4hum_12 IgG4PE, and VLX4hum_13 IgG4PE) to human CD47 was determined using an OVIO CD47 cell-based ELISA. OVIO hCD47 cells were plated into 96 well plates and were confluent at the time of assay. Various concentrations of VLX4 representative m Ahs were added to the cells for 1 hr. Cells were washed and then incubated with HRP-labelled secondary antibody for 1 hr followed by addition of peroxidase substrate.
[0040] FIG. 2A. Binding of VLX4 Humanized m Ahs to Human RBCs (hRBCs). Binding of VLX4 humanized mAbs (VLX4hum_01 IgGl, VLX4hum_02 IgGl, VLX4hum_01 IgG4PE, and VLX4hum_02 IgG4PE) to human CD47 was determined using freshly isolated hRBCs. hRBCs were incubated for 60 minutes at 37°C with various concentrations of VLX4 mAbs, washed and incubated for lhr with FITC-labelled donkey anti-human antibody. Cells were washed and antibody binding measured using flow cytometry.
[0041] FIG. 2B. Binding of VLX4 Humanized mAbs to Human RBCs. Binding of VLX4 humanized mAbs (VLX4hum_07 IgG4PE, VLX4hum_12 IgG4PE, and VLX4hum_13 IgG4PE) to human CD47 was determined using freshly isolated hRBCs. hRBCs were incubated for 60 minutes at 37°C with various concentrations of VLX4 mAbs, washed and incubated for lhr with FITC-labelled donkey anti-human antibody. Cells were washed and antibody binding measured using flow cytometry.
[0042] FIG. 3A. Binding of VLX8 Humanized m Ahs to Human OVIO hCD47 Cells. Binding of VLX8 IgG4PE chimera (xi) or humanized m Ahs (VLX8hum_01 IgG4PE, VLX8hum_04 IgG4PE, VLX8hum_07 IgG4PE, and VLX8hum_09 IgG4PE) to human CD47 was determined using an OVIO hCD47 cell-based ELISA. OVIO hCD47 cells were plated into 96 well plates and were confluent at the time of assay. Various concentrations of VLX8 representative m Ahs were added to the cells for 1 hr. Cells were washed and then incubated with HRP-labelled secondary antibody for 1 hr followed by addition of peroxidase substrate. [0043] FIG. 3B. Binding of VLX8 Humanized mAbs to Human OVIO hCD47 Cells. Binding of VLX8 chimera or humanized mAbs (VLX8hum_06 IgG2, VLX8hum_07 IgG2, VLX8hum_08 IgG2, and VLX8hum_09 IgG2) to human CD47 was determined using an OVIO hCD47 cell-based ELISA. OVIO hCD47 cells were plated into 96 well plates and were confluent at the time of assay. Various concentrations of VLX8 representative mAbs were added to the cells for 1 hr. Cells were washed and then incubated with HRP-labelled secondary antibody for 1 hr followed by addition of peroxidase substrate.
[0044] FIG. 4A. Binding of VLX8 Humanized mAbs to Human RBCs. Binding of VLX8
IgG4PE (xi) or humanized mAbs (VLX8hum_01 IgG4PE, VLX8hum_03 IgG4PE, VLX8hum_07 IgG4PE, and VLX8hum_10 IgG4PE) to human CD47 was determined using freshly isolated human RBCs. RBCs were incubated for 1 hr at 37°C with various concentrations of VLX8 mAbs, washed and incubated for lhr with FITC-labelled donkey anti human antibody. Cells were washed and antibody binding measured using flow cytometry.
[0045] FIG. 4B. Binding of VLX8 Humanized mAbs to Human RBCs. Binding of VLX8 IgG4PE xi or humanized mAbs (VLX8hum_06 IgG2, VLX8hum_07 IgG2, VLX8hum_08 IgG2 and VLX8hum_09 IgG2) to human CD47 was determined using freshly isolated human RBCs. RBCs were incubated for 1 hr at 37°C with various concentrations of VLX8 mAbs, washed and incubated for lhr with FITC-labelled donkey anti-human antibody. Cells were washed and antibody binding measured using flow cytometry.
[0046] FIG. 5A. Binding of VLX9 Humanized mAbs to Human OVIO hCD47 Cells. Binding of VLX9 IgG2 (xi) or humanized mAbs (VLX9hum_01 IgG2, VLX9hum_02 IgG2, VLX9hum_03 IgG2, VLX9hum_04 IgG2 and VLX9hum_05 IgG2) to human CD47 was determined using an OVIO human CD47 cell-based ELISA. OVIO hCD47 cells were plated into 96 well plates and were confluent at the time of assay. Various concentrations of mAbs were added to the cells for 1 hr. Cells were washed and then incubated with HRP-labelled secondary antibody for 1 hr followed by addition of peroxidase substrate.
[0047] FIG. 5B. Binding of VLX9 Humanized mAbs to Human OVIO hCD47 Cells. Binding of VLX9 IgG2 (xi) or humanized mAbs (VLX9hum_06 IgG2, VLX9hum_07 IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2 and VLX9hum_10 IgG2) to human CD47 was determined using a OVIO hCD47 cell-based ELISA. OVIO hCD47 cells were plated into 96 well plates and were confluent at the time of assay. Various concentrations of mAbs were added to the cells for 1 hr. Cells were washed and then incubated with HRP-labelled secondary antibody for 1 hr followed by addition of peroxidase substrate. [0048] FIG. 6A. Specific Binding of VLX Humanized m Ahs to CD47. Binding of VLX humanized mAh VLX4hum_07 IgG4PE to wildtype and CD47 knockout Jurkat cells was determined by flow cytometry. Various concentrations of m Ahs were added to 1 X 104 cells for 1 hr. The cells were washed and then incubated with FITC-labelled secondary antibody for 1 hr. Cells were washed and antibody binding measured using flow cytometry.
[0049] FIG. 6B. Specific Binding of VLX Humanized mAhs to CD47. Binding of VLX humanized mAh VLX9hum_04 IgG2 to wildtype and CD47 knockout Jurkat cells was determined by flow cytometry. Various concentrations of mAhs were added to 1 X 104 cells for 1 hr. The cells were washed and then incubated with FITC-labelled secondary antibody for 1 hr. Cells were washed and antibody binding measured using flow cytometry.
[0050] FIG. 7. Binding of VLX9 Humanized mAhs to Human RBCs. Binding of VLX9 IgG2 xi or humanized VLX9 mAhs to human CD47 (VLX9hum_01 IgG2, VLX9hum_02 IgG2 and VLX9hum_07 IgG2) was determined using freshly isolated human hRBCs. RBCs were incubated for 60 minutes at 37°C with various concentrations of VLX9 mAbs, washed and incubated for lhr with FITC-labelled donkey anti-human antibody. Cells were washed and antibody binding measured using flow cytometry.
[0051] FIG. 8A. Binding of VLX Humanized mAhs to Human Aortic Endothelial Cells (HAEC). Binding of VLX humanized mAbs (VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_l 1 IgG4PE, VLX4hum_01 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2, VLX9hum_03 IgG2 and VLX9hum_04 IgG2) to HAEC was determined by flow cytometry. HAEC were removed from the flask using acutase. Various concentrations of mAhs were added to 1 X 104 cells for 1 hr. The cells were washed and then incubated with FITC-labelled secondary antibody for 1 hr followed by measurement of FITC label by flow cytometry.
[0052] FIG. 8B. Binding of VLX Humanized mAhs to Skeletal Human Muscle Cells (SkMC). Binding of VLX humanized mAhs (VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_l 1 IgG4PE, VLX4hum_01 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2, VLX9hum_03 IgG2 and VLX9hum_04 IgG2) to SkMc was determined by flow cytometry. SkMC were removed from the flask using acutase. Various concentrations of mAhs were added to 1 X 104 cells for 1 hr. The cells were washed and then incubated with FITC-labelled secondary antibody for 1 hr followed by measurement of FITC label by flow cytometry. [0053] FIG. 8C. Binding of VLX Humanized m Ahs to Human Lung Microvascular Endothelial Cells (HMVEC-L). Binding of VLX humanized m Ahs (VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_l l IgG4PE, VLX4hum_01 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2, VLX9hum_03 IgG2 and VLX9hum_04 IgG2) to HMVEC-L was determined by flow cytometry. HMVEC-L were removed from the flask using acutase. Various concentrations of m Ahs were added to 1 X 104 cells for 1 hr. The cells were washed and then incubated with FITC-labelled secondary antibody for 1 hr followed by measurement of FITC label by flow cytometry.
[0054] FIG. 8D. Binding of VLX Humanized mAhs to Human Renal Tubular Epithelial Cells (RTEC). Binding of VLX humanized mAhs (VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_l 1 IgG4PE, VLX4hum_01 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2, VLX9hum_03 IgG2 and VLX9hum_04 IgG2) to RTEC by flow cytometry. RTEC were removed from the flask using acutase. Various concentrations of mAhs were added to 1 X 104 cells for 1 hr. The cells were washed and then incubated with FITC- labelled secondary antibody for 1 hr followed by measurement of FITC label by flow cytometry.
[0055] FIG. 8E. Binding of VLX Humanized mAhs to Human Peripheral Blood CD3+ Cells. Binding of VLX humanized mAhs (VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_l 1 IgG4PE, VLX4hum_01 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2, VLX9hum_03 IgG2 and VLX9hum_04 IgG2) to CD3+ cells was determined by flow cytometry. PBMC were plated into 96 well plates. Various concentrations of mAhs were added to the cells for 1 hr. Cells were washed and then incubated with FITC- labelled secondary antibody and (APC)-labelled anti-CD3 antibody for 1 hr followed by measurement of FITC-labelled APC -positive cells by flow cytometry.
[0056] FIG. 8F. Binding of VLX Humanized mAhs to Human Peripheral Blood Mononuclear Cells (PBMC). Binding of VLX humanized mAhs (VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_l l IgG4PE, VLX4hum_01 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2, VLX9hum_03 IgG2 and VLX9hum_04 IgG2) to PBMC was determined by flow cytometry. PBMCs were plated into 96 well plates. Various concentrations of mAhs were added to the cells for 1 hr. Cells were washed and then incubated with FITC-labelled secondary antibody for 1 hr followed by measurement of FITC label by flow cytometry.
[0057] FIG. 9A. pH Dependent and pH Independent Binding of Humanized mAh to His- CD47. Binding of VLX9hum_09 IgG2 to human CD47 was determined using a solid-phase CD47 ELISA assay. His-CD47 was adsorbed to microtiter wells, washed and various concentrations of humanized m Ahs were added to the wells for lhr at pH 6 or 8. The wells were washed and then incubated with HRP-labelled secondary antibody for 1 hour followed by addition of peroxidase substrate.
[0058] FIG. 9B. pH Dependent and pH Independent Binding of Humanized mAh to His- CD47. Binding of VLX9hum_04 IgG2 to human CD47 was determined using a solid-phase CD47 ELISA assay. His-CD47 was adsorbed to microtiter wells, washed and various concentrations of humanized m Ahs were added to the wells for lhr at pH 6 or 8. The wells were washed and then incubated with HRP-labelled secondary antibody for 1 hour followed by addition of peroxidase substrate.
[0059] FIG. 9C. pH Dependent and pH Independent Binding of Humanized mAh to His- CD47. Binding of VLX4hum_07 IgG4PE to human CD47 was determined using a solid-phase CD47 ELISA assay. His-CD47 was adsorbed to microtiter wells, washed and various concentrations of humanized m Ahs were added to the wells for lhr at pH 6 or 8. The wells were washed and then incubated with HRP-labelled secondary antibody for 1 hour followed by addition of peroxidase substrate.
[0060] FIG. 9D. pH Dependent and pH Independent Binding of Humanized mAh to His- CD47. Binding of VLX8hum_10 IgG4PE to human CD47 was determined using a solid-phase CD47 ELISA assay. His-CD47 was adsorbed to microtiter wells, washed and various concentrations of humanized m Ahs were added to the wells for lhr at pH 6 or 8. The wells were washed and then incubated with HRP-labelled secondary antibody for 1 hour followed by addition of peroxidase substrate.
[0061] FIG. 10. VLX4, VLX8, and VLX9 Humanized m Ahs Block SIRPoc binding to CD47 on Human Jurkat Cells. 1.5 x 106 Jurkat cells were incubated with 5pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE, VLX4hum_l l IgG4PE, VLX9hum_03 IgG2, VLX9hum_06 IgG2, and VLX9hum_08 IgG2) or a control antibody or no antibody in RPMI containing 10% FBS for 30 min at 37°C. An equal volume of media containing fluorescently labelled SIRPoc-Fc fusion protein was added and incubated for an additional 30 min at 37°C. Cells were washed and binding was assessed using flow cytometry.
[0062] FIG. 11. VLX4 CD47 Chimeric mAbs Increase Phagocytosis of Human Jurkat Cells by Human Macrophages. Human macrophages were plated at a concentration of lxlO4 cells per well in a 96 well plate and allowed to adhere for 24 hrs. 5xl04 CFSE-labehed human Jurkat cells and 1 mg/ml of the VLX4 chimeric m Ahs (VLX4 IgGl xi, VLX4 IgGl N297Q xi, VLX4 IgG4PE xi, VLX4 IgG4 S228P xi) were added to the macrophage cultures and incubated at 37°C for 2 hrs. Non-phagocytosed Jurkat cells were removed and macrophage cultures were washed extensively. Macrophages were trypsinized and stained for CD 14. Flow cytometry was used to determine the percentage of CD14+/CFSE+ cells in the total CD14+ population.
[0063] FIG. 12A. VEX4 Humanized mAbs Increase Phagocytosis of Human Jurkat Cells by Human Macrophages. Human macrophages were plated at a concentration of lxlO4 cells per well in a 96 well plate and allowed to adhere for 24 hrs. 5xl04 CFSE-labelled human Jurkat cells and 1 pg/ml of antibody (VEX4hum_01 IgGl and VEX4hum_01 IgG4PE) were added to the macrophage cultures and incubated at 37°C for 2 hrs. Non-phagocytosed Jurkat cells were removed and macrophage cultures were washed extensively. Macrophages were trypsinized and stained for CD14. Flow cytometry was used to determine the percentage of CD14+/CFSE+ cells in the total CD14+ population.
[0064] FIG. 12B. VEX4 Humanized mAbs Increase Phagocytosis of Human Jurkat Cells by Human Macrophages. Human macrophages were plated at a concentration of lxlO4 cells per well in a 96 well plate and allowed to adhere for 24 hrs. 5xl04 CFSE-labelled human Jurkat cells and 1 pg/ml of antibody (VEX4 IgG4PE xi, VEX4hum_06 IgG4PE, VEX4hum_07 IgG4PE, VEX4hum_012 IgG4PE and VEX4hum_13 IgG4PE) were added to the macrophage cultures and incubated at 37°C for 2 hrs. Non-phagocytosed Jurkat cells were removed and macrophage cultures were washed extensively. Macrophages were trypsinized and stained for CD14. Flow cytometry was used to determine the percentage of CD14+/CFSE+ cells in the total CD14+ population.
[0065] FIG. 13A. VFX8 CD47 Chimeric m Ahs Increase Phagocytosis of Human Jurkat Cells by Human Macrophages. Human macrophages were plated at a concentration of lxlO4 cells per well in a 96 well plate and allowed to adhere for 24 hrs. 5xl04 CFSE- labelled human Jurkat cells and 1 pg/ml of the VFX8 chimeric mAbs (VFX8 IgGl N297Q xi and VFX8 Ig4PE xi) were added to the macrophage cultures and incubated at 37°C for 2 hrs. Non-phagocytosed Jurkat cells were removed and macrophage cultures were washed extensively. Macrophages were trypsinized and stained for CD 14. Flow cytometry was used to determine the percentage of CD14+/CFSE+ cells in the total CD14+ population.
[0066] FIG. 13B. VFX8 Humanized mAbs Increase Phagocytosis of Human Jurkat Cells by Human Macrophages. Human macrophages were plated at a concentration of lxlO4 cells per well in a 96 well plate and allowed to adhere for 24 hrs. 5xl04 CFSE-labelled human Jurkat cells and 1 pg/ml of antibody (VFX8 IgG4PE xi, VFX8hum_01 IgG4PE, VFX8hum_03 IgG4PE, VLX8hum_07 IgG4PE, VLX8hum_08 IgG4PE and VLX8hum_09 IgG4PE) were added to the macrophage cultures and incubated at 37°C for 2 hrs. Non-phagocytosed Jurkat cells were removed and macrophage cultures were washed extensively. Macrophages were trypsinized and stained for CD 14. Flow cytometry was used to determine the percentage of CD14+/CFSE+ cells in the total CD14+ population.
[0067] FIG. 14A. VLX9 CD47 Chimeric m Ahs Increase Phagocytosis of Human Jurkat Cells by Human Macrophages. Human macrophages were plated at a concentration of lxlO4 cells per well in a 96 well plate and allowed to adhere for 24 hours. 5xl04 CFSE-labelled human Jurkat cells and 1 pg/ml of the VLX9 chimeric m Ahs (VLX9 IgGl N297 xi, VLX9 IgG2 xi and VLX9 IgG4PE xi) were added to the macrophage cultures and incubated at 37°C for two hours. Non-phagocytosed Jurkat cells were removed and macrophage cultures were washed extensively. Macrophages were trypsinized and stained for CD 14. Flow cytometry was used to determine the percentage of CD14+/CFSE+ cells in the total CD 14+ population.
[0068] FIG. 14B. VEX9 Humanized mAbs Increase Phagocytosis of Human Jurkat Cells by Human Macrophages. Human macrophages were plated at a concentration of lxlO4 cells per well in a 96 well plate and allowed to adhere for 24 hours. 5xl04 CFSE-labelled human Jurkat cells and 1 pg/ml of antibody (VEX9 IgG2 xi, VEX9hum_01 IgG2, VEX9hum_02 IgG2, VEX9hum_03 IgG2, VEX9hum_04 IgG2, VEX9hum_05 IgG2, VEX9hum_06 IgG2, VEX9hum_07 IgG2, VEX9hum_08 IgG2, VEX9hum_09 IgG2 and VEX9hum_10 IgG2) were added to the macrophage cultures and incubated at 37°C for two hours. Non-phagocytosed Jurkat cells were removed and macrophage cultures were washed extensively. Macrophages were trypsinized and stained for CD 14. Flow cytometry was used to determine the percentage of CD14+/CFSE+ cells in the total CD 14+ population.
[0069] FIG. 15A. Induction of Cell Death in Human Jurkat Cells by Soluble VFX4 Humanized m Ahs. Jurkat cells (lxlO4) were incubated with 1 pg/ml VFX4 humanized m Ahs (VFX4hum_01 IgGl, VFX4hum_01 IgG4PE, VFX4hum_02 IgGl, VFX4hum_02 IgG4PE) in RPMI media for 24 hours at 37°C. Cells were then stained with annexin V and the signal was detected by flow cytometry. The data are shown as % of cells that are annexin V positive (annexin V+).
[0070] FIG. 15B. Induction of Cell Death in Human Jurkat Cells by Soluble VFX4 Humanized m Ahs. Jurkat cells (lxlO4) were incubated with 1 pg/ml VFX4 humanized m Ahs (VFX4hum_01 IgGl, VFX4hum_01 IgG4PE, VFX4hum_02 IgGl, VFX4hum_02 IgG4PE) in RPMI media for 24 hours at 37°C. Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. The data are shown as % of the cells that are annexin V positive/7 - AAD negative (annexin VV7-AAD ).
[0071] FIG. 15C. Induction of Cell Death in Human Jurkat Cells by Soluble VLX4 Humanized m Ahs. Jurkat cells (lxlO4) were incubated with 1 pg/ml VLX4 humanized m Ahs (VLX4hum_01 IgGl, VLX4hum_01 IgG4PE, VLX4hum_02 IgGl, VLX4hum_02 IgG4PE) in RPMI media for 24 hours at 37°C. Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. The data are shown as % of cells that are annexin V positive/7- AAD positive (annexin V+/7-AAD+).
[0072] FIG. 15D. Induction of Cell Death in Human Jurkat Cells by Soluble VLX4 Humanized m Ahs. Jurkat cells (lxlO4) were incubated with 1 pg/ml VLX4 humanized m Ahs (VLX4hum_06 IgG4PE, VLX4hum_07 IgG4PE, VLX4hum_08 IgG4PE, VLX4hum_l l IgG4PE, VLX4hum_12 IgG4PE, VLX4hum_13 IgG4PE) in RPMI media for 24 hours at 37°C. Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. The data are shown as the % of cells that are annexin V positive (annexin V+).
[0073] FIG. 15E. Induction of Cell Death in Human Jurkat Cells by Soluble VLX4 Humanized m Ahs. Jurkat cells (lxlO4) were incubated with 1 pg/ml VLX4 humanized m Ahs (VLX4hum_06 IgG4PE, VLX4hum_07 IgG4PE, VLX4hum_08 IgG4PE, VLX4hum_l l IgG4PE, VLX4hum_12 IgG4PE, VLX4hum_13 IgG4PE) in RPMI media for 24 hours at 37°C. Cells were then stained with annexin V and 7-AAD by flow cytometry. The data are shown as the % of cells that are annexin V positive/7-AAD negative (annexin V77-AAD ).
[0074] FIG. 15F. Induction of Cell Death in Human Jurkat Cells by Soluble VLX4 Humanized m Ahs. Jurkat cells (lxlO4) were incubated with 1 pg/ml VLX4 humanized m Ahs (VLX4hum_06 IgG4PE, VLX4hum_07 IgG4PE, VLX4hum_08 IgG4PE, VLX4hum_l l IgG4PE, VLX4hum_12 IgG4PE, VLX4hum_13 IgG4PE) in RPMI media for 24 hours at 37°C. Cells were then stained with annexin V and and 7-AAD and analyzed by flow cytometry. The data are shown as the % of cells that are annexin V positive/7- A AD positive (annexin+/7- AAD+).
[0075] FIG. 16A. Induction of Cell Death in Human Jurkat Cells by Soluble VLX8 CD47 Chimeric mAbs. Jurkat cells (lxlO4) were incubated with 1 pg/ml VLX8 chimeric m Ahs (VLX8 IgGl N297Q xi and VLX8 IgG4PE xi) in RPMI media for 24 hrs at 37°C. Cells were then stained with annexin V and analyzed by flow cytometry. The data are presented as % of cells that are annexin V positive (annexin V+).
[0076] FIG. 16B. Induction of Cell Death in Human Jurkat Cells by Soluble VLX8 Chimeric m Ahs. Jurkat cells (lxlO4) were incubated with 1 pg/ml VLX8 chimeric m Ahs (VLX8 IgGl N297Q xi and VLX8 IgG4PE xi) in RPMI media for 24 hrs at 37°C. Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. The data are presented as the % of cells that are annexin V positive/7- A AD negative (annexin VV7-AAD ).
[0077] FIG. 16C. Induction of Cell Death in Human Jurkat Cells by Soluble VLX8 Chimeric m Ahs. Jurkat cells (lxlO4) were incubated with 1 pg/ml VLX8 chimeric m Ahs (VLX8 IgGl N297Q xi and VLX8 IgG4PE (xi) in RPMI media for 24 hrs at 37°C. Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. The data are presented as the % of cells that are annexin V positive/7-AAD positive (annexin V+/7-AAD+).
[0078] FIG. 16D. Induction of Cell Death in Human Jurkat Cells by Soluble VLX8 Humanized mAbs. Jurkat cells (lxlO4) were incubated with 1 pg/ml VLX8 humanized m Ahs (VLX8hum_02 IgG4PE, VLX8hum_04 IgG4PE, VLX8hum_07 IgG4PE and VLX8hum_08 IgG4PE) and chimeric mAh VLX8 IgG4PE in RPMI media for 24 hrs at 37°C. Cells were then stained with annexin V and analyzed by flow cytometry. The data are presented as the % of cells that are annexin V positive (annexin V+).
[0079] FIG. 16E. Induction of Cell Death in Human Jurkat Cells by Soluble VLX8 Humanized mAbs. Jurkat cells (lxlO4) were incubated with 1 pg/ml VLX8 humanized m Ahs (VLX8hum_02 IgG4PE, VLX8hum_04 IgG4PE, VLX8hum_07 IgG4PE and VLX8hum_08 IgG4PE) and chimeric mAh VLX8 IgG4PE in RPMI media for 24 hrs at 37°C. Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. The data are shown as the % of cells that are annexin V positive/7- A AD negative (annexin V77-AAD ).
[0080] FIG. 16F. Induction of Cell Death in Human Jurkat Cells by Soluble VLX8 Humanized mAbs. Jurkat cells (lxlO4) were incubated with 1 pg/ml VLX8 humanized m Ahs (VLX8hum_02 IgG4PE, VLX8hum_04 IgG4PE, VLX8hum_07 IgG4PE and VLX8hum_08 IgG4PE) and chimeric mAh VLX8 IgG4PE in RPMI media for 24 hrs at 37°C. Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. The data are shown as the % of cells that are annexin V positive/7- A AD positive (annexin V+/7-AAD+).
[0081] FIG. 17A. Induction of Cell Death of Human Jurkat Cells by Soluble VLX9 Chimeric mAbs. lxlO4 Jurkat cells were incubated with 1 pg/ml of the VLX9 CD47 chimeric mAbs (VLX9 IgGl N297Q xi, VLX9 IgG2 xi and VLX9 IgG4PE xi) in RPMI media for 24 hours 37°C. Cells were then stained with annexin V and the signal analyzed by flow cytometry. The data are shown as % of cells that are annexin V positive (annexin V+).
[0082] FIG. 17B. Induction of Cell Death of Human Jurkat Cells by Soluble VLX9 Chimeric mAbs. lxlO4 Jurkat cells were incubated with 1 pg/ml of the VLX9 CD47 chimeric mAbs (VLX9 IgGl N297Q xi, VLX9 IgG2 xi and VLX9 IgG4PE xi) in RPMI media for 24 hours 37°C. Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. The data are shown as % of cells that are annexin V positive/7- A AD negative (annexin VV7-AAD ).
[0083] FIG. 17C. Induction of Cell Death of Human Jurkat Cells by Soluble VLX9 Chimeric mAbs. lxlO4 Jurkat cells were incubated with lpg/ml of the VLX9 CD47 chimeric mAbs (VLX9 IgGl N297Q xi, VLX9 IgG2 xi and VLX9 IgG4PE xi) in RPMI media for 24 hours 37°C. Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. The data are shown as % of cells that are annexin V positive/7- A AD positive (annexin V+/7-AAD+).
[0084] FIG. 17D. Induction of Cell Death in Human Jurkat Cells by Soluble VLX9 Humanized mAbs. Jurkat cells (lxlO4) were incubated with 1 pg/ml VLX9 humanized mAbs (VLX9hum_01 to 10 IgG2) and chimeric mAh VLX9 IgG2 xi in RPMI media for 24 hours at 37°C. Cells were then stained with annexin V and the signal was detected by flow cytometry. VLX9 IgG2 (xi) is a murine/human chimera. The data are shown as % of cells that are annexin
V positive (annexin V+).
[0085] FIG. 17E. Induction of Cell Death in Human Jurkat Cells by Soluble VLX9 Humanized mAbs. Jurkat cells (lxlO4) were incubated with 1 pg/ml VLX9 humanized mAbs (VLX9hum_01 to 10 IgG2) and chimeric mAh VLX9 IgG2 (xi) in RPMI media for 24 hours at 37°C. Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. VLX9 IgG2 (xi) is a murine/human chimera. The data are shown as % of cells that are annexin
V positive/7- A AD negative (annexin V77-AAD ).
[0086] FIG. 17F. Induction of Cell Death in Human Jurkat Cells by Soluble VLX9 Humanized mAbs. Jurkat cells (lxlO4) were incubated with 1 pg/ml VLX9 humanized mAbs (VLX9hum_01 to 10 IgG2) and chimeric mAh VLX9 IgG2 xi in RPMI media for 24 hours at 37°C. Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. VLX9 IgG2 (xi) is a murine/human chimera. The data are shown as the % of cells that are annexin V positive/7- A AD positive (annexin V+/7-AAD+).
[0087] FIG. 18. Induction of Mitochondrial Depolarization in Human Raji Cells by Soluble VLX4, VLX8 and VLX9 Humanized mAbs. lxlO4 Raji cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l l IgG4PE, VLX9hum_03 IgG2, VLX9hum_06 IgG2 and VLX9hum_08 IgG2), a negative IgG control antibody or 1 mM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours. Cells were washed and the change in JC-1 dye fluorescence was assessed using flow cytometry. The data are expressed as % of cells with mitochondrial depolarization.
[0088] FIG. 19. Soluble VLX4, VLX8 and VLX9 Humanized m Ahs Cause an Increase in Cell Surface Calreticulin Expression on Human Raji Cells. lxlO4 Raji cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l l IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 mM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours. Cells were washed and calreticulin expression was assessed using flow cytometry. The data are expressed as % of cells that are calreticulin positive.
[0089] FIG. 20. Soluble VLX4, VLX8 and VLX9 Humanized m Ahs Cause an Increase in Cell Surface Protein Disulfide-Isomerase A3 (PDIA3) Expression on Human Raji Cells. lxlO4 Raji cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l l IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 pM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours. Cells were washed and PDIA3 expression was assessed using flow cytometry. The data are expressed as % of cells that are PDIA3 positive.
[0090] FIG. 21. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase Cell Surface HSP70 Expression on Human Raji Cells. lxlO4 Raji cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l 1 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 pM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours. Cells were washed and HSP70 expression was assessed using flow cytometry. The data are expressed as % of cells that are HSP70 positive.
[0091] FIG. 22. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase Cell Surface HSP90 Expression on Human Raji Cells. lxlO4 Raji cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l 1 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 pM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours. Cells were washed and HSP90 expression was assessed using flow cytometry. The data are expressed as % of cells that are HSP90 positive. [0092] FIG. 23. Soluble VLX4, VLX8 and VLX9 Humanized m Ahs Increase Release of Adenosine Triphosphate (ATP) by Human Raji Cells. lxlO4 Raji cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l l IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 mM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours. Cell-free supernatant was collected and analyzed using an ATP determination kit. The data are expressed as pM ATP in the supernatant.
[0093] FIG. 24. Soluble VLX4, VLX8 and VLX9 Humanized m Ahs Cause an Increase in Release of High Mobility Group Box 1 (HMGB 1) by Human Raji Cells. lxlO4 Raji cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l l IgG4PE, VLX9hum_03 IgG2, VLX9hum_06 IgG2 and VLX9hum_08 IgG2), a negative IgG control antibody or 1 pM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours. Cell-free supernatant was collected and analyzed using an HMGB 1 immunoassay. The data are expressed as ng/ml of HMGB 1 in the supernatant.
[0094] FIG. 25. Soluble VLX4, VLX8 and VLX9 Humanized m Ahs Increase CXCL10 Release by Human Raji Cells. lxlO4 Raji cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l 1 IgG4PE, VLX9hum_03 IgG2, VLX9hum_06 IgG2 and VLX9hum_08 IgG2), a negative IgG control antibody or 1 pM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours. Cell-free supernatant was collected and analyzed using an CXCL10 immunoassay. The data are expressed as pg/ml of CXCL10 in the supernatant.
[0095] FIG.26. Induction Mitochondrial Depolarization in Human Jurkat Cells by Soluble VLX4, VLX8 and VLX9 Humanized m Ahs. lxlO4 Jurkat cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l 1 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 pM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours. Cells were washed and the change in JC-1 dye fluorescence was assessed using flow cytometry. The data are expressed as % of cells with mitochondrial depolarization.
[0096] FIG. 27. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase Cell Surface Calreticulin Expression on Human Jurkat Cells. lxlO4 Jurkat cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l l IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 mM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours. Cells were washed and calreticulin expression was assessed using flow cytometry. The data are expressed as % of cells that are calreticulin positive.
[0097] FIG. 28. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase Cell Surface PDIA3 Expression on Human Jurkat Cells. lxlO4 Jurkat cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l 1 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 mM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours. Cells were washed and PDIA3 expression was assessed using flow cytometry. The data are expressed as % of cells that are PDIA3 positive.
[0098] FIG. 29. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase Cell Surface HSP70 Expression on Human Jurkat Cells. lxlO4 Jurkat cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l 1 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 mM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours. Cells were washed and HSP70 expression was assessed using flow cytometry. The data are expressed as % of cells that are HSP70 positive.
[0099] FIG. 30. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase Cell Surface HSP90 Expression on Human Jurkat Cells. lxlO4 Jurkat cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l 1 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 mM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours. Cells were washed and HSP90 expression was assessed using flow cytometry. The data are expressed as % of cells that are HSP90 positive.
[0100] FIG.31. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase ATP Release by Human Jurkat Cells. lxlO4 Jurkat cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l 1 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 mM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours. Cell-free supernatant was collected and analyzed using an ATP determination kit. The data are expressed as pM ATP in the supernatant.
[0101] FIG. 32. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase HMGB 1 Release by Human Jurkat Cells. lxlO4 Jurkat cells were incubated with 10 pg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l 1 IgG4PE, VLX9hum_03 IgG2, VLX9hum_06 IgG2 and VLX9hum_08 IgG2), a negative IgG control antibody or 1 mM of mitoxantrone as a positive control in RPMI media at 37°C for 24 hours. Cell-free supernatant was collected and analyzed using an HMGB 1 immunoassay. The data are expressed as ng/ml of HMGB 1 in the supernatant.
[0102] FIG. 33A. Agglutination of hRBCs by VLX4 Humanized mAbs. Hemagglutination was assessed following incubation of hRBCs with various concentrations of humanized VLX4 mAbs (VLX4hum_01 IgGl and VLX4hum_01 IgG4PE). Blood was diluted (1 :50) and washed 3 times with PBS/EDTA/BSA. hRBCs were added to U-bottomed 96 well plates with equal volumes of the antibodies (75 mΐ) and incubated for 3 hrs at 37°C and overnight at 4°C.
[0103] FIG. 33B. Agglutination of hRBCs by VLX8 Chimeric and Humanized mAbs. Hemagglutination was assessed following incubation of hRBCs with various concentrations of humanized VLX8 mAbs (VLX8hum_01 IgG4PE, VLX8hum_02 IgG4PE VLX8hum_03 IgG4PE, VLX8hum_08 IgG4PE, VLX8hum_09 IgG4PE, VLX8hum_10 IgG4PE and VLX8hum_ll IgG4PE) and the chimeric mAh VLX8 IgG4PE xi. Blood was diluted (1 :50) and washed 3 times with PBS/EDTA/BSA. hRBCs were added to U-bottomed 96 well plates with equal volumes of the antibodies (75 mΐ) and incubated for 3 hrs at 37°C and overnight at 4°C.
[0104] FIG. 34A. Agglutination of Human RBCs by VLX9 Humanized mAbs. Hemagglutination was assessed following incubation of human RBCs with various concentrations of VLX9 IgG2 chimera (xi) and humanized VLX9 mAbs (VLX9hum_01 to _05 IgG2). Blood was diluted (1 :50) and washed 3 times with PBS/EDTA/BSA. RBCs were added to U-bottomed 96 well plates with equal volumes of the antibodies (75 mΐ) and incubated for 3 hrs at 37°C and overnight at 4°C.
[0105] FIG. 34B. Agglutination of Human RBCs by VLX9 Humanized mAbs. Hemagglutination was assessed following incubation of human RBCs with various concentrations of VLX9 IgG2 chimera (xi) and humanized VLX9 mAbs (VLX9hum_06 to _10 IgG2). Blood was diluted (1 :50) and washed 3 times with PBS/EDTA/BSA. RBCs were added to U-bottomed 96 well plates with equal volumes of the antibodies (75 mΐ) and incubated for 3 hrs at 37°C and overnight at 4°C.
[0106] FIG.35. VLX4 Humanized mAh Reduces Tumor Growth in Raji Xenograft Model. Female NSG mice were inoculated subcutaneously in the right flank with 0.1 mL of a 30% RPMI / 70% Matrigel™ mixture containing a suspension of 5xl06 Raji tumor cells. Five days following inoculation, tumor volumes were measured and mice with palpable tumor volumes of 31-74 mm3 were randomized into 8-10/group. VLX4hum_07 IgG4PE or PBS (control) administration was initiated at this time. Mice were treated with 5 mg/kg of antibody 5X/week for 4 weeks by intraperitoneal injection. Tumor volumes and body weights were recorded twice weekly.
[0107] FIG.36. VLX8 Humanized mAh Reduces Tumor Growth in Raji Xenograft Model. Female NSG mice were inoculated subcutaneously in the right flank with 0.1 mL of a 30% RPMI / 70% Matrigel™ mixture containing a suspension of 5xl06 Raji tumor cells. Five days following inoculation, tumor volumes were measured and mice with palpable tumor volumes of 31-74 mm3 were randomized into 8-10/group. VFX8hum_10 IgG4PE or PBS (control) administration was initiated at this time. Mice were treated with 5 mg/kg of antibody 5X/week for 4 weeks by intraperitoneal injection. Tumor volumes and body weights were recorded twice weekly.
[0108] FIG.37. VEX9 Humanized mAh Reduces Tumor Growth in Raji Xenograft Model. Female NSG mice were inoculated subcutaneously in the right flank with 0.1 mL of a 30% RPMI / 70% Matrigel™ mixture containing a suspension of 5xl06 Raji tumor cells. Five days following inoculation, tumor volumes were measured and mice with palpable tumor volumes of 31-74 mm3 were randomized into 8-10/group. VFX9hum_08 IgG2 or PBS (control) administration was initiated at this time. Mice were treated with 5 mg/kg of antibody 5X/week for 4 weeks by intraperitoneal injection. Tumor volumes and body weights were recorded twice weekly.
[0109] FIG. 38A. Hemoglobin Fevels in Blood Following Administration of a Humanized VFX9 mAh to Cynomolgus Monkeys by Intravenous Infusion. VFX9hum_08 IgG2 or vehicle were administered as a one hour intravenous infusion a dose of 5mg/kg on day 1 and a dose of 15mg/kg on day 18. Hemoglobin levels were monitored throughout the study and normali ed to control values.
[0110] FIG. 38B. RBC Fevels in Blood Following Administration of Humanized VFX9 m Ahs to Cynomolgus Monkeys by Intravenous Infusion. VFX9hum_08 IgG2 or vehicle was administered as a one hour intraveneous infusion a dose of 5mg/kg on day 1 and a dose of 15mg/kg on day 18. RBC levels were monitored throughout the study and normalized to control values.
[0111] FIG. 39A. Confocal microscopy of CD47 staining pattern on human red blood cells with CD47 antibodies VFX9_04, VFX9_06, VFX9_08 or VLX4_01. RBCs were incubated with primary antibody for 1 hour, washed and stained with fluorophore labeled secondary antibody. After staining, cells were washed and imaged (Top: immunofluorescence, Bottom: brightfield)
[0112] FIG. 39B. Confocal microscopy of CD47 (red) and bΐ Integrin (green) staining pattern on Jurkat T-ALL cells with CD47 antibodies. Jurkats were co-stained with CD47 antibodies VLX9_04 IgG2, VLX9_06 IgG2, VLX9_08 IgG2, or VLX4_01 IgG4PE and bΐ Integrin antibodies for 1 hour, washed, and stained with flurorophore labeled secondary antibody. After staining, cells were washed and imaged.
[0113] FIG. 40. Summary of Anti-CD47 Antibody Properties.
[0114] FIG. 41. Confocal microscopy of CD47 (yellow), bΐ Integrin (blue), GFP (purple) staining on Jurkat T-ALL bΐ Integrin CRISPR/Cas9 knockout cells with CD47 antibody VLX9_04 IgG2, VLX9_06 IgG2, VLX_08 IgG2 or another anti-CD47 mAh (Hu5L9-G4, PCT International Publication WO 2011/143624). Arrows indicate bΐ Integrin knocked-out cells that do not bind with VLX9_06 IgG2, but do bind with the Hu5F9-G4 CD47 antibody.
[0115] FIG. 42. Confocal microscopy staining pattern on Jurkat T-ALL cells with SIRPa variant vl or v2 (red) for 1 hour, and then incubated with bΐ Integrin antibodies for another 1 hour and stained with fluorophore labeled secondary antibodies.
[0116] FIG. 43. VLX9_06 IgG2 Humanized mAh blocks SIRPa variant 1 and 2 binding to CD47 on Human Jurkat Cells.
[0117] FIG. 44A. Immunoprecipitation of CD47 from Jurkat cells with the anti-CD47 antibodies VLX9_06 IgG2 or Hu5F9-G4, or a negative control IgG2. The immunoprecipitated CD47 was determined by immunoblotting using the anti-CD47 antibody BRIC126. Control lysate shows endogenous levels of CD47 in Jurkat cells.
[0118] FIG. 44B. Ability of bΐ-integrin to be co-immunoprecipitated by the anti-CD47 mAbs, VLX9_06 and Hu5F9-G4 and IgG2 control antibodies. Control lysate shows endogenous levels of bΐ -integrin in Jurkat cells.
[0119] FIG. 45. Co-localization of CD47 and bΐ-integrin on gastric carcinoma cells. Localization of anti-CD47 antibodies on tumor cells expressing bΐ -integrin was determined by confocal microscopy. SNU-1 gastric carcinoma cells were seeded on glass and incubated with either VLX9hum_06 IgG2 or Hu5L9-G4 and an integrin beta 1 antibody [mouse anti-Integrin beta 1 antibody for 1 hour at 37°C, then washed and stained with both anti-human IgG (H+L) antibody and anti-mouse IgG (H+L) antibody for 1 hour. After fixation, cells were imaged using a confocal microscope. Arrows indicate staining with VLX9hum_06 IgG2 (top panel), anti- bΐ-integrin antibody (middle panel), or the colocalization of both antibodies. [0120] FIG. 46A - FIG. 46C. Decreased binding of humanized VLX9hum_06 IgG2 antibody to normal hematological cells compared to tumor by confocal microcopy. Co localization of CD47 and bΐ-integrin on tumor cells and normal hematological cells was determined by confocal microscopy (FIG. 46 A). Jurkat T-ALL cells, neutrophils, T-cells, and monocytes were seeded on glass slides and incubated with either VLX9hum_06 IgG2 or Hu5F9-G4 and integrin beta 1 antibody [mouse anti-Integrin bΐ antibody] for 1 hour at 37°C, then washed and stained with both anti-human IgG (H+L) antibody and anti-mouse IgG (H+L) antibody for 1 hour. After fixation, cells were imaged using a confocal microscope. Quantification of the intensity of binding determined using ImageJ software (FIG. 46B). Normalization of VLX9hum_06 IgG2 binding to Hu5F9-G4 binding on normal cells (FIG. 46C).
[0121] FIG. 47A - FIG. 47B. Comparison of the binding of VLX9hum_06 IgG2 and bΐ- integrin antibodies to normal hematological cells to tumor by flow cytometry. Binding of anti- CD47 and anti-bΐ -integrin antibodies to tumor cells and normal hematological cells was determined by flow cytometry. Cultured Jurkat T-ALL cells or healthy normal human blood derived hRBCs, neutrophils, monocytes, platelets and T cells were incubated for 60 min at 37°C with increasing concentrations of either VLX9hum_06 IgG2 or anti-bΐ -integrin antibodies diluted in PBS. Cells were washed and incubated for an additional hour with FITC labelled donkey anti-human IgG antibody or PE-labeled anti-mouse IgG antibody. Antibody binding was determined using flow cytometry and binding curves of normal cell types were normalized to binding to tumor (Jurkat) cells.
[0122] FIG. 48A - FIG. 48C. Binding of VLX9hum_06 IgG2 involves bΐ -integrin expression. Binding of CD47 antibodies to K562 erythroblast-like cells and to mature hRBCs was determined by flow cytometry bΐ -integrin levels in these cell lines was determined by western blot. K562 or freshly isolated hRBCs were incubated for 60 min at 37°C with increasing concentrations of VLX9hum_06 IgG2 or Hu5F9-G4 diluted in PBS. Antibody binding was determined using flow cytometry. Quantification of bΐ -integrin levels was determined by Western blot on protein lysates taken from K562 cells and hRBCs.
[0123] FIG. 49A - FIG. 49B. Binding of VLX9hum_06 IgG2 interferes with bΐ-integrin signaling pathways in Jurkat cells. The effect of CD47 antibodies on downstream mediators of bΐ -integrin signaling in tumor cells was determined by phosphoblot analysis of treated Jurkat cells. In FIG. 49A, wildtype Jurkat cells were treated either with control IgG2 antibody, various concentrations of VLX9hum_06 IgG2 or Hu5F9-G4 for 90 minutes then the cells were lysed in RIPA buffer with HALT protease-phosphatase inhibitor. Lysates were blotted with antibodies to FAK, pFAK, SRC, and pSRC. In FIG. 49B, bΐ-integrin knockout Jurkat cells were generated by CRISPR/Cas9 along with control lines and cell lysates were harvested and blotted with antibodies to bΐ-integrin, FAK, pFAK, SRC, and pSRC.
[0124] FIG. 50A - FIG. 50B. VLX9hum_06 IgG2 interferes with bΐ-integrin signaling pathways in SNU-1 gastric carcinoma cells. The effect of CD47 antibodies on downstream mediators of bΐ-integrin signaling in tumor cells was determined by phosphoblot analysis of treated SNU-lcells. SNU-1 cells were treated either with control IgG2 antibody, VLX9hum_06 IgG2 or Hu5F9-G4 for 1, 2 or 3 hours. Lysates were blotted with antibodies to SRC, and pSRC (FIG. 50A). Quantification of pSrc/total Src levels was determined using ImageJ software (FIG. 50B).
[0125] FIG. 51A - FIG. 51H. Induction of Cell Death in Gastric Carcinoma Cells by Soluble VLX9hum_06 IgG2. Gastric carcinoma cell lines SNU-1, KATOIII, SNU-16, Hs746T, SNU-5, NCI-N87, AGS or OE19 cells (lxlO4) were incubated with 100 pg/ml VLX9hum_06 IgG2 or control for 24 hours at 37°C. Cells were then stained with annexin V and the signal was detected by flow cytometry. The data are shown as % of cells that are annexin V positive (annexin V+).
DETAILED DESCRIPTION
Definitions
[0126] Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art.
[0127] As used herein, the term“CD47,”“integrin-associated protein (IAP),”“ovarian cancer antigen OA3,”“Rh-related antigen,” and“MERG” are synonymous and may be used interchangeably.
[0128] The term“anti-CD47 antibody” refer to an antibody of the disclosure which is intended for use as a therapeutic or diagnostic agent, and therefore will typically possess the binding affinity required to be useful as a therapeutic and/or diagnostic agent.
[0129] As used herein, the term“antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e. , molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. By “specifically binds” or“immunoreacts with” or“directed against” is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides or binds at a much lower affinity (Kd > 10 6). Antibodies include but are not limited to, polyclonal, monoclonal, chimeric, Fab fragments, Fab’ fragments, F(ab’)2 fragments, single chain Fv fragments, and one-armed antibodies.
[0130] As used herein, the term“monoclonal antibody” (mAb) as applied to the present antibody compounds refers to an antibody that is derived from a single copy or clone including, for example, any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced m Ahs of the present disclosure preferably exist in a homogeneous or substantially homogeneous population. Complete mAbs contain 2 heavy chains and 2 light chains.
[0131] An“antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab’, Fab’ -SH, F(ab’)2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.
[0132] As disclosed herein,“antibody compounds” refers to mAbs and antigen-binding fragments thereof. Additional antibody compounds exhibiting similar functional properties according to the present disclosure can be generated by conventional methods. For example, mice can be immunized with human CD47 or fragments thereof, the resulting antibodies can be recovered and purified, and determination of whether they possess binding and functional properties similar to or the same as the antibody compounds disclosed herein can be assessed by the methods described in Examples 3-17 below. Antigen-binding fragments can also be prepared by conventional methods. Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art and can be found, for example, in Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, chapters 5-8 and 15.
[0133] The monoclonal antibodies encompass antibodies in which a portion of the heavy and/or light chain is identical with, or homologous to, corresponding sequences in murine antibodies, in particular the murine CDRs, while the remainder of the chain(s) is (are) identical with, or homologous to, corresponding sequences in human antibodies. Other embodiments of the disclosure include antigen-binding fragments of these monoclonal antibodies that exhibit binding and biological properties similar or identical to the monoclonal antibodies. The antibodies of the present disclosure can comprise kappa or lambda light chain constant regions, and heavy chain IgA, IgD, IgE, IgG, or IgM constant regions, including those of IgG subclasses IgGl, IgG2, IgG3, and IgG4 and in some cases with various mutations to alter Fc receptor function.
[0134] The monoclonal antibodies containing the presently disclosed murine CDRs can be prepared by any of the various methods known to those skilled in the art, including recombinant DNA methods.
[0135] Reviews of current methods for antibody engineering and improvement can be found, for example, in P. Chames, Ed., (2012) Antibody Engineering: Methods and Protocols, Second Edition (Methods in Molecular Biology, Book 907), Humana Press, ISBN-10: 1617799734; C. R. Wood, Ed., (2011) Antibody Drug Discovery (Molecular Medicine and Medicinal Chemistry, Book 4), Imperial College Press; R. Kontermann and S. Dubel, Eds., (2010) Antibody Engineering Volumes 1 and 2 (Springer Protocols), Second Edition; and W. Strohl and L. Strohl (2012) Therapeutic antibody engineering: Current and future advances driving the strongest growth area in the pharmaceutical industry, Woodhead Publishing.
[0136] Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art and can be found, for example, in Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, chapters 5-8 and 15.
[0137] A full-length antibody as it exists naturally is a“Y” shaped immunoglobulin (Ig) molecule comprising four polypeptide chains: two identical heavy (H) chains and two identical light (L) chains, interconnected by disulfide bonds. The amino terminal portion of each chain, termed the fragment antigen binding region (FAB), includes a variable region of about 100- 110 or more amino acids primarily responsible for antigen recognition via the complementarity determining regions (CDRs) contained therein. The carboxy-terminal portion of each chain defines a constant region (the“Fc” region) primarily responsible for effector function.
[0138] The CDRs are interspersed with regions that are more conserved, termed frameworks (“FRs”). Amino acid sequences of many FRs are well known in the art. Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of 3 CDRs and 4 FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDRs of the light chain are referred to as “LCDR1, LCDR2, and LCDR3” and the 3 CDRs of the heavy chain are referred to as“HCDRl, HCDR2, and HCDR3.” The CDRs contain most of the residues which form specific interactions with the antigen. The numbering and positioning of CDR amino acid residues within the LCVR and HCVR regions are in accordance with the well-known Rabat numbering convention Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition. NIH Publication No. 91-3242.
[0139] As described herein, the “antigen-binding site” can also be defined as the “hypervariable regions,”“HVRs,” or“HVs,” and refer to the structurally hypervariable regions of antibody variable domains as defined by Chothia and Lesk (Chothia and Lesk, Mol. Biol. 196:901-917, 1987). There are six HVRs, three in VH (HI, H2, H3) and three in VL (LI, L2, L3). The CDRs used herein are as defined by Kabat except in H-CDR1, which is extended to include HI.
[0140] There are five types of mammalian immunoglobulin (Ig) heavy chains, denoted by the Greek letters a (alpha), d (delta), e (epsilon), g (gamma), and m (mu), which define the class or isotype of an antibody as IgA, IgD, IgE, IgG, or IgM, respectively. IgG antibodies can be further divided into subclasses, for example, IgGl, IgG2, IgG3, and IgG4.
[0141] Each heavy chain type is characterized by a particular constant region with a sequence well known in the art. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. Heavy chains g, a, and d have a constant region composed of three tandem immunoglobulin (Ig) domains, and a hinge region for added flexibility. Heavy chains m and e have a constant region composed of four Ig domains.
[0142] The hinge region is a flexible amino acid stretch that links the Fc and Fab portions of an antibody. This regions contains cysteine residues that can form disulfide bonds, connecting two heavy chains together.
[0143] The variable region of the heavy chain differs in antibodies produced by different B cells, but is the same for all antibodies produced by a single B cell or B cell clone. The variable region of each heavy chain is approximately 110 amino acids long and is composed of a single Ig domain.
[0144] In mammals, light chains are classified as kappa (K) or lambda (l), and are characterized by a particular constant region as known in the art. A light chain has two successive domains: one variable domain at the amino-terminal end, and one constant domain at the carboxy-terminal end. Each antibody contains two light chains that are always identical; only one type of light chain, k or l, is present per antibody in mammals.
[0145] The Fc region, composed of two heavy chains that contribute three or four constant domains depending on the class of the antibody, plays a role in modulating immune cell activity. By binding to specific proteins, the Fc region ensures that each antibody generates an appropriate immune response for a given antigen. The Fc region also binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins. By doing this, it mediates different physiological effects, including opsonization, cell lysis, and degranulation of mast cells, basophils and eosinophils.
[0146] As used herein, the term“epitope” refers to a specific arrangement of amino acids located on a peptide or protein to which an antibody or antibody fragment binds. Epitopes often consist of a chemically active surface grouping of molecules such as amino acids or sugar side chains, and have specific three dimensional structural characteristics as well as specific charge characteristics. Epitopes can be linear, i.e. , involving binding to a single sequence of amino acids, or conformational, i.e., involving binding to two or more sequences of amino acids in various regions of the antigen that may not necessarily be contiguous in the linear sequence.
[0147] As used herein, the terms “specifically binds,” “bind specifically,” “specific binding,” and the like as applied to the present antibody compounds refer to the ability of a specific binding agent (such as an antibody) to bind to a target molecular species in preference to binding to other molecular species with which the specific binding agent and target molecular species are admixed. A specific binding agent is said specifically to recognize a target molecular species when it can bind specifically to that target.
[0148] As used herein, the term“binding affinity” refers to the strength of binding of one molecule to another at a site on the molecule. If a particular molecule will bind to or specifically associate with another particular molecule, these two molecules are said to exhibit binding affinity for each other. Binding affinity is related to the association constant and dissociation constant for a pair of molecules as measured in a 1 : 1 interaction. Affinities as used herein to describe interactions between molecules of the described methods which can be used to compare the relative strength with which one molecule ( e.g . , an antibody or other specific binding partner) will bind two other molecules (e.g., two versions or variants of a peptide) in a univalent interaction. The concepts of binding affinity, association constant, and dissociation constant are well known.
[0149] As used herein, the term“apparent binding affinity” refers to the apparent strength of binding of one molecule to another at a site on the molecule. If a particular molecule will bind to or specifically associate with another particular molecule, these two molecules are said to exhibit binding affinity for each other. Apparent binding affinity is related to the association constant and dissociation constant for a pair of molecules, and relates to a non 1 :1 or multivalent association between the pair of molecules. Apparent affinities as used herein to describe interactions between molecules of the described methods are observed in empirical studies, which can be used to compare the relative strength with which one molecule (e.g., an antibody or other specific binding partner) will bind two other molecules (e.g. , two versions or variants of a peptide). The concept of binding affinity may be described as apparent Kd, apparent binding constant, EC50 or other measurements of binding.
[0150] As used herein, the term“sequence identity” means the percentage of identical nucleotide or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e. , taking into account gaps and insertions. Identity can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs.
[0151] Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, by the homology alignment algorithms, by the search for similarity method or, by computerized implementations of these algorithms (GAP, BESTFIT, PASTA, and TFASTA in the GCG Wisconsin Package, available from Accelrys, Inc., San Diego, California, United States of America), or by visual inspection. See generally, Altschul, S. F. et al , J. Mol. Biol. 215: 403-410 (1990) and Altschul et al. Nucl. Acids Res. 25: 3389-3402 (1997).
[0152] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BEAST algorithm, which is described in (Altschul, S. et al, NCBI NEM NIH Bethesda, Md. 20894; and Altschul, S. et al, J. Mol. Biol. 215: 403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold.
[0153] These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always; 0) and N (penalty score for mismatching residues; always; 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, a cutoff of 100, M = 5, N = -4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix.
[0154] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is in one embodiment less than about 0.1, in another embodiment less than about 0.01, and in still another embodiment less than about 0.001.
[0155] As used herein, the terms“humanized,”“humanization,” and the like, refer to grafting of the murine monoclonal antibody CDRs disclosed herein to human FRs and constant regions. Also encompassed by these terms are possible further modifications to the murine CDRs, and human FRs, by the methods disclosed in, for example, Kashmiri et al. (2005) Methods 36(l):25-34 and Hou et al. (2008) J. Biochem. 144(1): 115- 120, respectively, to improve various antibody properties, as discussed below.
[0156] As used herein, the term“humanized antibodies” refers to mAbs and antigen binding fragments thereof, including antibody compounds, that have binding and functional properties similar to those disclosed herein, and that have FRs and constant regions that are substantially human or fully human surrounding CDRs derived from a non-human antibody.
[0157] As used herein, the term“FR” or“framework sequence” refers to any one of FRs 1 to 4. Humanized antibodies and antigen binding fragments encompassed by the present disclosure include molecules wherein any one or more of FRs 1 to 4 is substantially or fully human, i.e., wherein any of the possible combinations of individual substantially or fully human FRs 1 to 4, is present. For example, this includes molecules in which FR1 and FR2, FR1 and FR3, FR1, FR2, and FR3, etc., are substantially or fully human. Substantially human frameworks are those that have at least 80% sequence identity to a known human germline framework sequence. Preferably, the substantially human frameworks have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, to a framework sequence disclosed herein, or to a known human germline framework sequence.
[0158] Fully human frameworks are those that are identical to a known human germline framework sequence. Human FR germline sequences can be obtained from the international ImMunoGeneTics (IMGT) database and from The Immunoglobulin FactsBook by Marie-Paule Lefranc and Gerard Lefranc, Academic Press, 2001, the contents of which are herein incorporated by reference in their entirety.
[0159] The Immunoglobulin Facts Book is a compendium of the human germline immunoglobulin genes that are used to create the human antibody repertoire, and includes entries for 203 genes and 459 alleles, with a total of 837 displayed sequences. The individual entries comprise all the human immunoglobulin constant genes, and germline variable, diversity, and joining genes that have at least one functional or open reading frame allele, and which are localized in the three major loci. For example, germline light chain FRs can be selected from the group consisting of: IGKV3D-20, IGKV2-30, IGKV2-29, IGKV2-28, IGKV1-27, IGKV3-20, IGKV1-17, IGKV1-16, 1-6, IGKV1-5, IGKV1-12, IGKV1D-16, IGKV2D-28, IGKV2D-29, IGKV3-11, IGKV1-9, IGKV1-39, IGKV1D-39, IGKV1D-33, and IGKJ1-5; and germline heavy chain FRs can be selected from the group consisting of: IGHV1- 2, IGHV1-18, IGHV1-46, IGHV1-69, IGHV2-5, IGHV2-26, IGHV2-70, IGHV1-3, IGHV1- 8, IGHV3-9, IGHV3-11, IGHV3-15, IGHV3-20, IGHV3-66, IGHV3-72, IGHV3-74, IGHV4- 31, IGHV3-21, IGHV3-23, IGHV3-30, IGHV3-48, IGHV4-39, IGHV4-59, IGHV5-51, and IGHJ1-6.
[0160] Substantially human FRs are those that have at least 80% sequence identity to a known human germline FR sequence. Preferably, the substantially human frameworks have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, to a framework sequences disclosed herein, or to a known human germline framework sequence.
[0161] CDRs encompassed by the present disclosure include not only those specifically disclosed herein, but also CDR sequences having sequence identities of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a CDR sequence disclosed herein. Alternatively, CDRs encompassed by the present disclosure include not only those specifically disclosed herein, but also CDR sequences having 1, 2, 3, 4, or 5 amino acid changes at corresponding positions compared to CDR sequences disclosed herein. Such sequence identical, or amino acid modified, CDRs preferably bind to the antigen recognized by the intact antibody.
[0162] Humanized antibodies in addition to those disclosed herein exhibiting similar functional properties according to the present disclosure can be generated using several different methods, including those disclosed by Almagro et al. (Frontiers in Biosciences. Humanization of antibodies. (2008) Jan 1; 13: 1619-33).
[0163] In one approach, the parent antibody compound CDRs are grafted into a human framework that has a high sequence identity with the parent antibody compound framework. The sequence identity of the new framework will generally be at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identical to the sequence of the corresponding framework in the parent antibody compound. In the case of frameworks having fewer than 100 amino acid residues, one, two, three, four, five, six, seven, eight, nine, or ten amino acid residues can be changed. This grafting may result in a reduction in binding affinity compared to that of the parent antibody. If this is the case, the framework can be back-mutated to the parent framework at certain positions based on specific criteria disclosed by Queen et al. (1991) Proc. Natl. Acad. Sci. USA 88:2869. Additional references describing methods useful to generate humanized variants based on homology and back mutations include as described in Olimpieri et al. ( Bioinformatics 2015 Feb l ;31(3):434-435) and U.S. Patents 4,816,397, 5,225,539, and 5,693,761 ; and the method of Winter and co-workers (Jones et al. (1986) Nature 321 :522-525; Riechmann et al. (1988) Nature 332:323-327; and Verhoeyen et al. (1988) Science 239: 1534-1536).
[0164] Humanization began with chimerization, a method developed during the first half of the 1980’s (Morrison, S. L., M. J. Johnson, L. A. Herzenberg & V. T. Oi: Chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains. Proc. Natl. Acad. Sci. USA 81, 6851-5 (1984)), consisting of combining the variable (V) domains of murine antibodies with human constant (C) domains to generate molecules with -70% of human content.
[0165] Several different methods can be used to generate humanized antibodies, which are described herein. In one approach, the parent antibody compound CDRs are grafted into a human FR that has a high sequence identity with the parent antibody compound framework. The sequence identity of the new FR will generally be at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of the corresponding FR in the parent antibody compound. In the case of FRs having fewer than 100 amino acid residues, one, two, three, four, five, or more amino acid residues can be changed. This grafting may result in a reduction in binding affinity compared to that of the parent antibody. If this is the case, the FR can be back-mutated to the parent framework at certain positions based on specific criteria disclosed by Queen et al. (1991) Proc. Natl. Acad. Sci. USA 88:2869. Additional references describing methods useful to generate humanized variants based on homology and back mutations include as described in Olimpieri et al. ( Bioinformatics . 2015 Feb l ;31(3):434-435) and U.S. Patents 4,816,397, 5,225,539, and 5,693,761 ; and the method of Winter and co workers (Jones et al. (1986) Nature 321 :522-525; Riechmann et al. (1988) Nature 332:323- 327; and Verhoeyen et al. (1988) Science 239: 1534-1536).
[0166] The identification of residues to consider for back-mutation can be carried out as described below. When an amino acid falls under the following category, the framework amino acid of the human germ-line sequence that is being used (the“acceptor FR”) is replaced by a framework amino acid from a framework of the parent antibody compound (the“donor FR”):
[0167] (a) the amino acid in the human FR of the acceptor framework is unusual for human frameworks at that position, whereas the corresponding amino acid in the donor immunoglobulin is typical for human frameworks at that position;
[0168] (b) the position of the amino acid is immediately adjacent to one of the CDRs; or
[0169] (c) any side chain atom of a framework amino acid is within about 5-6 angstroms
(center-to-center) of any atom of a CDR amino acid in a three dimensional immunoglobulin model.
[0170] When each of the amino acids in the human FR of the acceptor framework and a corresponding amino acid in the donor framework is generally unusual for human frameworks at that position, such amino acid can be replaced by an amino acid typical for human frameworks at that position. This back-mutation criterion enables one to recover the activity of the parent antibody compound.
[0171] Another approach to generating humanized antibodies exhibiting similar functional properties to the antibody compounds disclosed herein involves randomly mutating amino acids within the grafted CDRs without changing the framework, and screening the resultant molecules for binding affinity and other functional properties that are as good as, or better than, those of the parent antibody compounds. Single mutations can also be introduced at each amino acid position within each CDR, followed by assessing the effects of such mutations on binding affinity and other functional properties. Single mutations producing improved properties can be combined to assess their effects in combination with one another.
[0172] Further, a combination of both of the foregoing approaches is possible. After CDR grafting, one can back-mutate specific FRs in addition to introducing amino acid changes in the CDRs. This methodology is described in Wu et al. (1999, J. Mol. Biol. 294: 151-162).
[0173] Applying the teachings of the present disclosure, a person skilled in the art can use common techniques, e.g., site-directed mutagenesis, to substitute amino acids within the presently disclosed CDR and FR sequences and thereby generate further variable region amino acid sequences derived from the present sequences. Up to all naturally occurring amino acids can be introduced at a specific substitution site. The methods disclosed herein can then be used to screen these additional variable region amino acid sequences to identify sequences having the indicated in vivo functions. In this way, further sequences suitable for preparing humanized antibodies and antigen-binding portions thereof in accordance with the present disclosure can be identified. Preferably, amino acid substitution within the frameworks is restricted to one, two, three, four, or five positions within any one or more of the four light chain and/or heavy chain FRs disclosed herein. Preferably, amino acid substitution within the CDRs is restricted to one, two, three, four, or five positions within any one or more of the three light chain and/or heavy chain CDRs. Combinations of the various changes within these FRs and CDRs described above are also possible.
[0174] That the functional properties of the antibody compounds generated by introducing the amino acid modifications discussed above conform to those exhibited by the specific molecules disclosed herein can be confirmed by the methods in Examples disclosed herein.
[0175] As described above, to circumvent the problem of eliciting human anti-murine antibody (HAMA) response in patients, murine antibodies have been genetically manipulated to progressively replace their murine content with the amino acid residues present in their human counterparts by grafting their complementarity determining regions (CDRs) onto the variable light (VL) and variable heavy (VH) frameworks of human immunoglobulin molecules, while retaining those murine framework residues deemed essential for the integrity of the antigen-combining site. However, the xenogeneic CDRs of the humanized antibodies may evoke anti-idiotypic (anti-id) response in patients.
[0176] To minimize the anti-id response, a procedure to humanize xenogeneic antibodies by grafting onto the human frameworks only the CDR residues most crucial in the antibody- ligand interaction, called“SDR grafting”, has been developed, wherein only the crucial specificity determining residues (SDRs) of CDRS are grafted onto the human frameworks. This procedure, described in Kashmiri et al. (2005, Methods 36(l):25-34), involves identification of SDRs through the help of a database of the three-dimensional structures of the antigen- antibody complexes of known structures, or by mutational analysis of the antibody-combining site. An alternative approach to humanization involving retention of more CDR residues is based on grafting of the‘abbreviated’ CDRs, the stretches of CDR residues that include all the SDRs. Kashmiri et al. also discloses a procedure to assess the reactivity of humanized antibodies to sera from patients who had been administered the murine antibody.
[0177] Another strategy for constructing human antibody variants with improved immunogenic properties is disclosed in Hou et al. (2008, J. Biochem. 144(1): 115-120). These authors developed a humanized antibody from 4C8, a murine anti-human CD34 monoclonal antibody, by CDR grafting using a molecular model of 4C8 built by computer-assisted homology modelling. Using this molecular model, the authors identified FR residues of potential importance in antigen binding. A humanized version of 4C8 was generated by transferring these key murine FR residues onto a human antibody framework that was selected based on homology to the murine antibody FR, together with the murine CDR residues. The resulting humanized antibody was shown to possess antigen-binding affinity and specificity similar to that of the original murine antibody, suggesting that it might be an alternative to murine anti-CD34 antibodies routinely used clinically.
[0178] Embodiments of the present disclosure encompass antibodies created to avoid recognition by the human immune system containing CDRs disclosed herein in any combinatorial form such that contemplated m Ahs can contain the set of CDRs from a single murine mAh disclosed herein, or light and heavy chains containing sets of CDRs comprising individual CDRs derived from two or three of the disclosed murine mAbs. Such mAbs can be created by standard techniques of molecular biology and screened for desired activities using assays described herein. In this way, the disclosure provides a“mix and match” approach to create novel m Ahs comprising a mixture of CDRs from the disclosed murine m Ahs to achieve new, or improved, therapeutic activities.
[0179] Monoclonal antibodies or antigen-binding fragments thereof encompassed by the present disclosure that“compete” with the molecules disclosed herein are those that bind human CD47 at site(s) that are identical to, or overlapping with, the site(s) at which the present molecules bind. Competing monoclonal antibodies or antigen-binding fragments thereof can be identified, for example, via an antibody competition assay. For example, a sample of purified or partially purified human CD47 extracellular domain can be bound to a solid support. Then, an antibody compound, or antigen binding fragment thereof, of the present disclosure and a monoclonal antibody or antigen-binding fragment thereof suspected of being able to compete with such disclosure antibody compound are added. One of the two molecules is labelled. If the labelled compound and the unlabelled compound bind to separate and discrete sites on CD47, the labelled compound will bind to the same level whether or not the suspected competing compound is present. However, if the sites of interaction are identical or overlapping, the unlabelled compound will compete, and the amount of labelled compound bound to the antigen will be lowered. If the unlabelled compound is present in excess, very little, if any, labelled compound will bind. For purposes of the present disclosure, competing monoclonal antibodies or antigen-binding fragments thereof are those that decrease the binding of the present antibody compounds to CD47 by about 50%, about 60%, about 70%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%. Details of procedures for carrying out such competition assays are well known in the art and can be found, for example, in Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Such assays can be made quantitative by using purified antibodies. A standard curve is established by titrating one antibody against itself, i.e., the same antibody is used for both the label and the competitor. The capacity of an unlabelled competing monoclonal antibody or antigen-binding fragment thereof to inhibit the binding of the labelled molecule to the plate is titrated. The results are plotted, and the concentrations necessary to achieve the desired degree of binding inhibition are compared.
[0180] Whether m Ahs or antigen-binding fragments thereof that compete with antibody compounds of the present disclosure in such competition assays possess the same or similar functional properties of the present antibody compounds can be determined via these methods in conjunction with the methods described in Examples below. In various embodiments, competing antibodies for use in the therapeutic methods encompassed herein possess biological activities as described herein in the range of from about 50% to about 100% or about 125%, or more, compared to that of the antibody compounds disclosed herein. In some embodiments, competing antibodies possess about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or identical biological activity compared to that of the antibody compounds disclosed herein as determined by the methods disclosed in the Examples presented below. [0181] The m Ahs or antigen-binding fragments thereof, or competing antibodies useful in the compositions and methods can be any of the isotypes described herein. Furthermore, any of these isotypes can comprise further amino acid modifications as follows.
[0182] The monoclonal antibody or antigen-binding fragment thereof, or competing antibody described herein can be of the human IgGl isotype.
[0183] The human IgGl constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to alter antibody half-life. Antibody half-life is regulated in large part by Fc-dependent interactions with the neonatal Fc receptor (Roopenian and Alikesh, 2007). The human IgGl constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody can be modified to increase half-life include, but are not limited to amino acid modifications N434A, T307A/E380A/N434A (Petkova et al, 2006, Yeung et al , 2009); M252Y/S254T/T256E (Dall’Acqua et al. , 2006); T250Q/M428L (Hinton et al., 2006); and M428L/N434S (Zalevsky et al. , 2010).
[0184] As opposed to increasing half-life, there are some circumstances where decreased half-life would be desired, such as to reduce the possibility of adverse events associated with high Antibody-Dependent Cellular Cytotoxicity (ADCC) and Complement-Dependent Cytotoxicity (CDC) antibodies (Presta 2008). The human IgGl constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease half-life and/or decrease endogenous IgG include, but are not limited to amino acid modifications I253A (Petkova et al, 2006); P257I/N434H, D376V/N434H (Datta-Mannan et al, 2007); and M252Y/S254T/T256E/H433K/N434F (Vaccaro et al., 2005).
[0185] The human IgGl constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to increase or decrease antibody effector functions. These antibody effector functions include, but are not limited to, Antibody-Dependent Cellular Cytotoxicity (ADCC), Complement-Dependent Cytotoxicity (CDC), Antibody-Dependent Cellular Phagocytosis (ADCP), Clq binding, and altered binding to Fc receptors.
[0186] The human IgGl constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to increase antibody effector function include, but are not limited to amino acid modifications S298A/E333A/K334 (Shields et al, 2001); S239D/I332E and S239D/A330L/I332E (Lazar et al , 2006); F234L/R292P/Y300L, F234L/R292P/Y300L/P393L, and F243L/R292P/Y300L/V305I/P396L (Stevenhagen et al, 2007); G236A, G236A/S239D/I332E, and G236A/S239D/A330L/I332E (Richards et al, 2008); K326A/E333A, K326A/E333S and K326W/E333S (Idusogie et al , 2001); S267E and S267E/L328F (Smith et al, 2012); H268F/S324T, S267E/H268F, S267E/S234T, and S267E/H268F/S324T (Moore et al, 2010); S298G/T299A (Sazinsky et al, 2008); E382V/M428I (Jung et al , 2010).
[0187] The human IgGl constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease antibody effector function include, but are not limited to amino acid modifications N297A and N297Q (Bolt et al , 1993, Walker et al , 1989); L234A/L235A (Xu et al, 2000); K214T/E233P/L234V/L235A/G236-deleted/A327G/P331A/D356E/L358M (Ghevaert et al, 2008); C226S/C229S/E233P/L234V/L235A (McEarchern et al, 2007); S267E/L328F (Chu et al, 2008).
[0188] The human IgGl constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease antibody effector function include, but are not limited to amino acid modifications V234A/G237A (Cole et al, 1999); E233D, G237D, P238D, H268Q, H268D, P271G, V309L, A330S, A330R, P331S, H268 Q/A330S/V309L/P331 S , H268D/A330S/V309L/P331S,
H268Q/A330R/V309L/P331S, H268D/A330R/V309L/P331S, E233D/A330R,
E233D/A330S, E233D/P271G/A330R, E233D/P271G/A330S, G237D/H268D/P271G,
G237D/H268 Q/P271 G, G237D/ P271G/A330R, G237D/ P271G/A330S,
E233D/H268D/P271G/A330R, E233D/H268Q/P271G/A330R,
E233D/H268D/P271G/A330S, E233D/H268 Q/P271 G/A330S ,
G237D/H268D/P271G/A330R, G237D/H268Q/P271G/A330R,
G237D/H268D/P271G/A330S, G237D/H268Q/P271G/A330S,
E233D/G237D/H268D/P271G/A330R, E233D/G237D/H268Q/P271G/A330R,
E233D/G237D/H268D/P271G/A330S, E233D/G237D/H268Q/P271G/A330S,
P238D/E233D/A330R, P238D/E233D/A330S, P238D/E233D/P271G/A330R,
P238D/E233D/P271G/A330S , P238D/G237D/H268D/P271 G,
P238D/G237D/H268 Q/P271 G, P238D/G237D/ P271G/A330R, P238D/G237D/ P271G/A330S, P238D/E233D/H268D/P271G/A330R,
P238D/E233D/H268 Q/P271 G/A330R, P238D/E233D/H268D/P271G/A330S,
P238D/E233D/H268 Q/P271 G/A330S , P238D/G237D/H268D/P271G/A330R,
P238D/G237D/H268 Q/P271 G/A330R, P238D/G237D/H268D/P271G/A330S,
P238D/G237D/H268 Q/P271 G/A330S , P238D/E233D/G237D/H268D/P271G/A330R, P238D/E233D/G237D/H268Q/P271 G/A330R,
P238D/E233D/G237D/H268D/P271 G/A330S , P238D/E233D/G237D/H268Q/P271G/A330S (An et al , 2009, Mimoto, 2013).
[0189] The monoclonal antibody or antigen-binding fragment thereof, or competing antibody described herein can be of the human IgG2 isotype.
[0190] The human IgG2 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to increase or decrease antibody effector functions. These antibody effector functions include, but are not limited to, Antibody-Dependent Cellular Cytotoxicity (ADCC), Complement-Dependent Cytotoxicity (CDC), Antibody-Dependent Cellular Phagocytosis (ADCP), and Clq binding, and altered binding to Fc receptors.
[0191] The human IgG2 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to increase antibody effector function include, but are not limited to the amino acid modification K326A/E333S
(Idusogie et al, 2001).
[0192] The human IgG2 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease antibody effector function include, but are not limited to amino acid modifications V234A/G237A (Cole et al, 1999); V234A, G237A, P238S, H268A, E233D, G237D, P238D, H268Q, H268D,
P271G, V309L, A330S, A330R, P331S, P238S/H268A,
V 234 A/G237 A/P238 S/H268 A/V309L/A330S/P331 S , H268Q/A330S/V309L/P331S,
H268D/A330S/V309L/P331S, H268Q/A330R/V309L/P331S, H268D/A330R/V309L/P331S, E233D/A330R, E233D/A330S, E233D/P271G/A330R, E233D/P271G/A330S,
G237D/H268D/P271G, G237D/H268Q/P271G, G237D/ P271G/A330R, G237D/
P271G/A330S, E233D/H268D/P271G/A330R, E233D/H268Q/P271G/A330R,
E233D/H268D/P271G/A330S, E233D/H268Q/P271G/A330S,
G237D/H268D/P271G/A330R, G237D/H268Q/P271G/A330R,
G237D/H268D/P271G/A330S, G237D/H268Q/P271G/A330S,
E233D/G237D/H268D/P271G/A330R, E233D/G237D/H268Q/P271G/A330R,
E233D/G237D/H268D/P271G/A330S, E233D/G237D/H268Q/P271G/A330S,
P238D/E233D/A330R, P238D/E233D/A330S, P238D/E233D/P271G/A330R,
P238D/E233D/P271G/A330S , P238D/G237D/H268D/P271 G,
P238D/G237D/H268 Q/P271 G, P238D/G237D/ P271G/A330R, P238D/G237D/ P271G/A330S, P238D/E233D/H268D/P271G/A330R, P238D/E233D/H268 Q/P271 G/A330R, P238D/E233D/H268D/P271G/A330S,
P238D/E233D/H268 Q/P271 G/A330S , P238D/G237D/H268D/P271G/A330R,
P238D/G237D/H268 Q/P271 G/A330R, P238D/G237D/H268D/P271G/A330S,
P238D/G237D/H268 Q/P271 G/A330S , P238D/E233D/G237D/H268D/P271G/A330R,
R238D/E233D/G237D/H268Q/P271 G/A330R,
R238D/E233D/G237D/H268D/P271 G/A330S , P238D/E233D/G237D/H268Q/P271G/A330S (An et al , 2009, Mimoto, 2013).
[0193] The Fc region of a human IgG2 of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to alter isoform and/or agonistic activity, include, but are not limited to amino acid modifications C127S (CHI domain), C232S, C233S, C232S/C233S, C236S, and C239S (White et al, 2015, Lightle et al , 2010).
[0194] The monoclonal antibody or antigen-binding fragment thereof, or competing antibody described herein can be of the human IgG3 isotype.
[0195] The human IgG3 constant region of the monoclonal antibody, or antigen binding fragment thereof, wherein said human IgG3 constant region of the monoclonal antibody, or antigen-binding fragment thereof can be modified at one or more amino acid(s) to increase antibody half-life, Antibody-Dependent Cellular Cytotoxicity (ADCC), Complement- Dependent Cytotoxicity (CDC), or apoptosis activity.
[0196] The human IgG3 constant region of the monoclonal antibody, or antigen-binding fragment thereof, wherein said human IgG3 constant region of the monoclonal antibody, or antigen-binding fragment thereof can be modified at amino acid R435H to increase antibody half-life.
[0197] The monoclonal antibody or antigen-binding fragment thereof, or competing antibody described herein can be of the human IgG4 isotype.
[0198] The human IgG4 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease antibody effector functions. These antibody effector functions include, but are not limited to, Antibody- Dependent Cellular Cytotoxicity (ADCC) and Antibody-Dependent Cellular Phagocytosis (ADCP).
[0199] The human IgG4 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to prevent Fab arm exchange and/or decrease antibody effector function include, but are not limited to amino acid modifications F234A/L235A (Alegre et ah , 1994); S228P, L235E and S228P/L235E (Reddy et ah , 2000).
[0200] As used herein, the term“tumor” refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
[0201] The terms“cancer,”“cancerous,” and“tumor” are not mutually exclusive as used herein.
[0202] The terms“cancer” and“cancerous” refer to or describe the physiological condition in mammals that is typically characterized by aberrant cell growth/proliferation. Examples of cancers include, but are not limited to, carcinoma, lymphoma (i.e., Hodgkin’s and non- Hodgkin’s lymphoma), blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.
[0203] The term“susceptible cancer” as used herein refers to a cancer, cells of which express CD47, and are responsive to treatment with an anti-CD47 antibody or antigen binding fragment thereof, or competing antibody or antigen binding fragment thereof, of the present disclosure.
[0204] “Nitric oxide (NO) donor, precursor, or nitric oxide generating topical agent” refers to a compound or agent that either delivers NO, or that can be converted to NO through enzymatic or non-enzymatic processes. Examples include, but are not limited to, NO gas, isosorbide dinitrite, nitrite, nitroprusside, nitroglycerin, 3-Morpholinosydnonimine (SIN-1), S- nitroso-N-acetyl-penicillamine (SNAP), Diethylenetriamine/NO (DETA/NO), S-nitrosothiols, Bidil®, and arginine.
[0205] “Soluble guanylyl cyclase (sGC)” is the receptor for nitric oxide in vascular smooth muscle. In the cardiovascular system, nitric oxide is endogenously generated by endothelial nitric oxide synthase from L-arginine, and activates soluble guanylyl cyclase in adjacent vascular smooth muscle cells to increase cGMP levels, inducing vascular relaxation. Nitric oxide binds to the normally reduced heme moiety of soluble guanylyl cyclase, and increases the formation of cGMP from GTP, leading to a decrease in intracellular calcium, vasodilation, and anti-inflammatory effects. Oxidation of the heme iron on sGC decreases responsiveness of the enzyme to nitric oxide, and promotes vasoconstriction. The nitric oxide-sGC-cGMP pathway therefore plays an important role in cardiovascular diseases. Nitrogen-containing compounds such as sodium azide, sodium nitrite, hydroxylamine, nitroglycerin, and sodium nitroprusside have been shown to stimulate sGC, causing an increase in cGMP, and vascular relaxation. In contrast to stimulators of sGC, which bind to reduced sGC, activators of sGC activate the oxidized or heme-deficient sGC enzyme that is not responsive to nitric oxide, i.e., they stimulate sGC independent of redox state. While stimulators of of sGC can enhance the sensitivity of reduced sGC to nitric oxide, activators of sGC can increase sGC enzyme activity even when the enzyme is oxidized and is therefore less, or unresponsive, to nitric oxide. Thus, sGC activators are non-nitric oxide based. Note the reviews of Nossaman et al. (2012) Critical Care Research and Practice, Volume 2012, article 290805, and Derbyshire and Marietta (2012 ) Ann. Rev. Biochem. 81:533-559.
[0206] “An agent that activates soluble guanylyl cyclase” refers, for example, to organic nitrates (Artz et al. (2002) J. Biol. Chem. 277: 18253-18256); protoporphyrin IX (Ignarro et al. (1982) Proc. Natl. Acad. Sci. USA 79:2870-2873); YC-1 (Ko et al. (1994) Blood 84:4226- 4233); BAY 41-2272 and BAY 41-8543 (Stasch et al. (2001 Nature 410 (6825): 212-5), CMF- 1571, and A-350619 (reviewed in Evgenov et al. (2006) Nat. Rev. Drug. Discov. 5:755-768); BAY 58-2667 (Cinaciguat; Frey etal. (2008) Journal of Clinical Pharmacology 48 (12): 1400- 10); BAY 63-2521 (Riociguat; Mittendorf et al. (2009) Chemmedchem 4 (5): 853-65). Additional soluble guanylyl cyclase activators are disclosed in Stasch et al. (2011) Circulation 123:2263-2273; Derbyshire and Marietta (2012) Ann. Rev. Biochem. 81 :533-559, and Nossaman et al. (2012) Critical Care Research and Practice, Volume 2012, Article ID 290805, pages 1-12.
[0207] cGMP can also be increased by inhibiting degradation using phosphodiesterase inhibitors. Examples of“an agent that inhibits cyclic nucleotide phosphodiesterases” include, tadalafil, vardenafil, udenafil, and sildenafil avanafil.
[0208] As used herein, term “treating” or “treat” or “treatment” means slowing, interrupting, arresting, controlling, stopping, reducing, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily involve a total elimination of all disease-related signs, symptoms, conditions, or disorders. The term“treating” and the like refer to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. [0209] As used herein, term“effective amount” refers to the amount or dose of an antibody compound of the present disclosure which, upon single or multiple dose administration to a patient or organ, provides the desired treatment or prevention.
[0210] The precise effective amount for any particular subject will depend upon their size and health, the nature and extent of their condition, and the therapeutics or combination of therapeutics selected for administration. The effective amount for a given patient is determined by routine experimentation and is within the judgment of a clinician. Therapeutically effective amounts of the present antibody compounds can also comprise an amount in the range of from about 0.1 mg/kg to about 150 mg/kg, from about 0.1 mg/kg to about 100 mg/kg, from about 0.1 mg/kg to about 50 mg/kg, or from about 0.05 mg/kg to about 10 mg/kg per single dose administered to a harvested organ or to a patient. Known antibody-based pharmaceuticals provide guidance in this respect. For example, Herceptin™ is administered by intravenous infusion of a 21 mg/ml solution, with an initial loading dose of 4 mg/kg body weight and a weekly maintenance dose of 2 mg/kg body weight; Rituxan™ is administered weekly at 375 mg/m2; for example.
[0211] A therapeutically effective amount for any individual patient can be determined by the health care provider by monitoring the effect of the antibody compounds on tumor regression, circulating tumor cells, tumor stem cells or anti-tumor responses. Analysis of the data obtained by these methods permits modification of the treatment regimen during therapy so that optimal amounts of antibody compounds of the present disclosure, whether employed alone or in combination with one another, or in combination with another therapeutic agent, or both, are administered, and so that the duration of treatment can be determined as well. In this way, the dosing/treatment regimen can be modified over the course of therapy so that the lowest amounts of antibody compounds used alone or in combination that exhibit satisfactory efficacy are administered, and so that administration of such compounds is continued only so long as is necessary to successfully treat the patient. Known antibody-based pharmaceuticals provide guidance relating to frequency of administration e.g., whether a pharmaceutical should be delivered daily, weekly, monthly, etc. Frequency and dosage may also depend on the severity of symptoms.
[0212] In some embodiments antibody compounds of the present disclosure can be used as medicaments in human and veterinary medicine, administered by a variety of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intraperitoneal, intrathecal, intraventricular, transdermal, transcutaneous, topical, subcutaneous, intratumoral, intranasal, enteral, sublingual, intravaginal, intravesiciular or rectal routes. The compositions can also be administered directly into a lesion such as a tumor. Dosage treatment may be a single dose schedule or a multiple dose schedule. Hypo sprays may also be used to administer the pharmaceutical compositions. Typically, the therapeutic compositions can be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. Veterinary applications include the treatment of companion/pet animals, such as cats and dogs; working animals, such as guide or service dogs, and horses; sport animals, such as horses and dogs; zoo animals, such as primates, cats such as lions and tigers, bears, etc. ·, and other valuable animals kept in captivity.
[0213] Such pharmaceutical compositions can be prepared by methods well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy, 21st Edition (2005), Lippincott Williams & Wilkins, Philadelphia, PA, and comprise one or more antibody compounds disclosed herein, and a pharmaceutically or veterinarily acceptable, for example, physiologically acceptable, carrier, diluent, or excipient.
[0214] The present disclosure describes anti-CD47 m Ahs with distinct functional profiles. These antibodies possess distinct combinations of properties selected from the following: These antibodies possess distinct combinations of properties selected from the following: 1) exhibit cross-reactivity with one or more species homologs of CD47; 2) block the interaction between CD47 and its ligand SIRPoc; 3) increase phagocytosis of human tumor cells; 4) induce death of susceptible human tumor cells; 5) do not induce cell death of human tumor cells; 6) do not have reduced or minimal binding to human red blood cells (hRBCs); 7) have reduced binding to hRBCs; 8) have minimal binding to hRBCs; 9) cause reduced agglutination of hRBCs; 10) cause no detectable agglutination of hRBCs; 11) reverse TSP1 inhibition of the nitric oxide (NO) pathway; 12) do not reverse TSP1 inhibition of the NO pathway; 13) cause loss of mitochondrial membrane potential; 14) do not cause cause loss of mitochondrial membrane potential; 15) cause an increase in cell surface calreticulin expression on human tumor cells; 16) do not cause an increase in cell surface calreticulin expression on human tumor cells; 17) cause an increase in adenosine triphosphate (ATP) release by human tumor cells; 18) do not cause an increase in adenosine triphosphate (ATP) release by human tumor cells; 19) cause an increase in high mobility group box 1 (HMGB 1) release by human tumor cells; 20) do not cause an increase in high mobility group box 1 (HMGB 1) release by human tumor cells; 21) cause an increase in type I interferon release by human tumor cells; 22) do not cause an increase in type I interferon release by human tumor cells; 23) cause an increase in C-X-C Motif Chemokine Ligand 10 (CXCL10) release by human tumor cells; 24) do not cause an increase in C-X-C Motif Chemokine Ligand 10 (CXCL10) release by human tumor cells; 25) cause an increase in cell surface protein disulfide-isomerase A3 (PDIA3) expression on human tumor cells; 26) do not cause an increase in cell surface protein disulfide-isomerase A3 (PDIA3) expression on human tumor cells; 27) cause an increase in cell surface heat shock protein 70 (HSP70) expression on human tumor cells; 28) do not cause an increase in cell surface heat shock protein 70 (HSP70) expression on human tumor cells; 29) cause an increase in cell surface heat shock protein 90 (HSP90) expression on human tumor cells; 30) do not cause an increase in cell surface heat shock protein 90 (HSP90) expression on human tumor cells; 31) have reduced binding to normal human cells, which includes, but is not limited to, endothelial cells, skeletal muscle cells, epithelial cells, and peripheral blood mononuclear cells (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubular epithelial cells, human peripherial blood CD3+ cells, and human peripheral blood mononuclear cells); 32) do not have reduced binding to normal human cells, which includes, but is not limited to, endothelial cells, skeletal muscle cells, epithelial cells, and peripheral blood mononuclear cells (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubular epithelial cells, human peripherial blood CD3+ cells, and human peripheral blood mononuclear cells); 33) have a greater affinity for human CD47 at an acidic pH compared to physiological pH; 34) do not have a greater affinity for human CD47 at an acidic pH compared to physiological pH; and 35) cause an increase in annexin A1 release by human tumor cells.
[0215] The anti-CD47 antibodies and antigen binding fragments thereof of the present disclosure possess combinations of properties that are distinct from the anti-CD47 antibodies of the prior art. These properties and characteristics will now be described in further detail.
[0216] As used herein, the term“binds to human CD47” refers to binding with an apparent Kd greater than 50 nM, for example, in a solid phase ELISA assay or cell based assay.
[0217] As used herein, the terms “apparent binding affinity and apparent Kd” are determined by non-linear fit (Prism GraphPad software) of the binding data at the various antibody concentrations.
Binding to CD47 of Different Species
[0218] The anti-CD47 antibodies, and antigen binding fragments thereof, of the present disclosure bind human CD47. In certain embodiments, the anti-CD47 antibodies exhibit cross reactivity with one or more species homologs of CD47, for example CD47 homologs of non human primate origin. In certain embodiments, the anti-CD47 antibodies and antigen binding fragments thereof of the present disclosure bind to human CD47 and to CD47 of non-human primate, mouse, rat, and/or rabbit origin. The cross-reactivity with other species homologs can be particularly advantageous in the development and testing of therapeutic antibodies. For example, pre-clinical toxicology testing of therapeutic antibodies is frequently carried out in non-human primate species including, but not limited to, cynomolgus monkey, green monkey, rhesus monkey and squirrel monkey. Cross-reactivity with these species homologs can therefore be particularly advantageous for the development of antibodies as clinical candidates.
[0219] As used herein, the term“cross-reacts with one or more species homologs of CD47” refers to binding with an apparent Kd greater than 50 nM.
Blocking the Interaction Between CD47 and SIRPa and Promoting Phagocytosis
[0220] CD47, also known as integrin associated protein (IAP), is a 50 kDa cell surface receptor that is comprised of an extracellular N-terminal IgV domain, a five membrane- spanning transmembrane domain, and a short C-terminal intracellular tail that is alternatively spliced.
[0221] Two ligands bind to CD47: Signal Regulatory Protein alpha (SIRPa) and Thrombospondin- 1 (TSP1). TSP1 is present in plasma and synthesized by many cells, including platelets. SIRPa is expressed on hematopoietic cells, which include macrophages and dendritic cells.
[0222] When SIRPa on a phagocyte engages CD47 on a target cell, this interaction prevents phagocytosis of the target cell. The interaction of CD47 and SIRPa effectively sends a“don’t eat me” signal to the phagocyte (Oldenborg et al. Science 288: 2051-2054, 2000). Blocking the interaction of SIRPa and CD47 with an anti-CD47 mAh in a therapeutic context can provide an effective anti-cancer treatment by promoting the uptake and clearance of cancer cells by the host’s immune system. Thus, an important functional characteristic of some anti-CD47 mAbs is the ability to block the interaction of CD47 and SIRPa, resulting in phagocytosis of CD47 expressing tumor cells by phagocytes including macrophages. Several anti-CD47 mAbs have been shown to block the interaction of CD47 and SIRPa, including B6H12 (Seiffert et al. Blood 94:3633-3643,1999; Latour et al. J. Immunol. 167: 2547-2554, 2001; Subramanian et al. Blood 107: 2548-2556, 2006; Liu et al. J Biol. Chem. Ill: 10028-10036, 2002; Rebres et al. J. Cellular Physiol. 205: 182-193, 2005), BRIC126 (Vernon-Wilson et al. Eur J Immunol. 30: 2130-2137, 2000; Subramanian et al. Blood 107: 2548-2556, 2006), CC2C6 (Seiffert et al. Blood 94:3633-3643,1999), 1F7 (Rebres et al. J. Cellular Phy iol. 205: 182-193, 2005), 5F9 (Liu et al. PLoS One. 2015 Sep 21 ;10(9): e0137345) and CC-90002 (Narla et al. Proc Am Assoc Cancer Res 58: 1200, 2017; abst 469)4. B6H12 and BRIC126 have also been shown to cause phagocytosis of human tumor cells by human and mouse macrophages (Willingham et al. Proc Natl Acad Sci USA 109(17):6662-6667, 2012; Chao et al. Cell 142:699-713, 2012; EP 2 242 512 Bl). Other existing anti-CD47 mAbs, such as 2D3, does not block the interaction of CD47 and SIRPoc (Seiffert et al. Blood 94:3633-3643,1999; Latour et al. J. Immunol. 167: 2547-2554, 2001 ; Rebres et al. J. Cellular Physiol. 205: 182-193, 2005), and does not cause phagocytosis of tumor cells (Willingham et al. Proc Natl Acad Sci USA 109(17):6662-6667, 2012; Chao et al. Cell 142:699-713, 2012; EP 2 242 512 B l).
[0223] SIRPa contains 3 Ig-like domains in its extracellular region, including 1 NH2- terminal V-set domain and 2 Cl-set domains. The NH2-terminal V-set domain of SIRPa recognizes the Ig-like domain of CD47. Although CD47 is highly conserved, extensive polymorphisms localized to the CD47-binding IgV domain of SIRPa occur in numerous ethnic groups. Ten allelic variants of human SIRPa have been identified with two prevalent alleles, vl and v2. SIRPa variants, vl and v2, is known to be most widespread among the Caucasian population.
[0224] As used herein, the term“blocks SIRPa binding to human CD47” refers to a greater than 50% reduction of SIRPa-Fc binding to CD47 on cells by an anti-CD47 mAh compared to either untreated cells or cells treated with a negative antibody.
[0225] The anti-CD47 mAbs of the disclosure described herein, block the interaction of CD47 and SIRPa and increase phagocytosis of human tumor cells.
[0226] “Phagocytosis” of cancer cells refers to the engulfment and digestion of such cells by phagocytes including, but not limited to, macrophages and dendritic cells, and the eventual digestion or degradation of these cancer cells and the release of digested or degraded cellular components extracellularly, or intracellularly to undergo further processing. Anti-CD47 monoclonal antibodies that block SIRPa binding to CD47 increase phagocytosis of cancer cells. SIRPa binding to CD47 on cancer cells would otherwise allow these cells to escape phagocytosis. The cancer cell may be viable or living cancer cells.
[0227] As used herein, the term“increases phagocytosis of human tumor cells” refers to a greater than 2-fold increase in phagocytosis of human tumor cells by human macrophages in the presence of an anti-CD47 mAh compared to either untreated cells or cells treated with a negative control antibody. Inducing Death of Tumor Cells
[0228] Some soluble anti-CD47 mAbs initiate a cell death program on binding to CD47 on tumor cells, resulting in collapse of mitochrondrial membrane potential, loss of ATP generating capacity, increased cell surface expression of phosphatidylserine (detected by increased staining for annexin V) and cell death without the participation of caspases or fragmentation of DNA. Such soluble anti-CD47 mAbs have the potential to treat a variety of solid and hematological cancers. Several soluble anti-CD47 mAbs which have been shown to induce tumor cell death, including MABL-1, MABL-2 and fragments thereof (US Patent 8,101,719; Uno et al. Oncol Rep. 17: 1189-94, 2007; Kikuchi et al. Biochem Biophys Res. Commun. 315: 912-8, 2004), Ad22 (Pettersen et al. J. Immuno. 166: 4931-4942, 2001 ; Lamy et al. J. Biol. Chem. 278: 23915-23921, 2003), and 1F7 (Manna et al. J. Immunol. 170: 3544-3553, 2003; Manna et al. Cancer Research, 64: 1026-1036, 2004). Some of the anti-CD47 mAbs of the disclosure described herein induce cell death of human tumor cells.
[0229] Induction of cell death refers to the ability of certain of the soluble anti-CD47 antibodies, murine antibodies, chimeric antibodies, humanized antibodies, or antigen-binding fragments thereof (and competing antibodies and antigen-binding fragments thereof) disclosed herein to kill cancer cells via a cell autonomous mechanism without participation of complement or other cells including, but not limited to, T cells, neutrophils, natural killer cells, macrophages, or dendritic cells.
[0230] The terms“inducing cell death” or“kills” and the like, are used interchangeably herein.
[0231] As used herein, the term“induces death of human tumor cells” refers to increased binding of annexin V (in the presence of calcium) and increased 7-aminoactinomycin D (7- AAD) or propidium iodide uptake in response to treatement with an anti-CD47 mAh. These features may be quantitated in three cell populations: annexin V positive (annexin V+), annexin V positive/7-AAD negative (annexin VV7-AAD ) and annexin V positive/7- A AD positive (annexin VV7-AAD+) by flow cytometry. Induction of cell death may be defined by a greater than 2-fold increase in each of the above cell populations in human tumor cells caused by soluble anti-CD47 mAh compared to the background obtained with the negative control antibody, (humanized, isotype-matched antibody) or untreated cells.
[0232] Another indicator of cell death is loss of mitochondrial function and membrane potential by the tumor cells as assayed by one of several available measures (potentiometric fluorescent dyes such as DiO-C6 or JC1 or formazan-based assays such as MTT or WST-1). [0233] As used herein, the term“causes loss of mitochondrial membrane potential” refers to a statistically significant (p < 0.05) decrease in mitochondrial membrane potential by a soluble anti-CD47 mAh compared to the background obtained with a negative control, humanized isotype-matched antibody or no treatment.
[0234] Among the present humanized or chimeric mAbs, those that induce cell death of human tumor cells cause increased annexin V binding similar to the findings reported for anti- CD47 mAbs Ad22 (Pettersen et al. J. Immunol. 166: 4931-4942, 2001 ; Lamy et al. J. Biol. Chem. 278: 23915-23921, 2003); 1F7 (Manna and Frazier J. Immunol. 170:3544-3553, 2003; Manna and Frazier Cancer Res. 64: 1026-1036, 2004); and MABL-1 and 2 (US Patent 7,531,643 B2; US Patent 7,696,325 B2; US Patent 8,101719 B2).
[0235] Cell viability assays are described in NCI/NIFl guidance manual that describes numerous types of cell based assays that can be used to assess induction of cell death caused by CD47 antibodies:“Cell Viability Assays”, Terry L Riss, PhD, Richard A Moravec, BS, Andrew L Niles, MS, Flelene A Benink, PhD, Tracy J Worzella, MS, and Lisa Minor, PhD. Contributor Information, published May 1, 2013.
Binding to hRBCs
[0236] CD47 is expressed on human erythrocytes (hRBCs) (Brown. J Cell Biol. I l l : 2785- 2794, 1990; Avent. Biochem J., (1988) 251: 499-505; Knapp. Blood, (1989) Vol. 74, No. 4, 1448-1450; Oliveira et al. Biochimica et Biophysica Acta 1818: 481-490, 2012; Petrova P. et al. Cancer Res 2015; 75(15 Suppl): Abstract no. 4271). It has been shown that anti-CD47 mAbs bind to RBCs, including B6F112 (Brown et al. J. Cell Biol., 1990, Oliveira et al. Biochimica et Biophysica Acta 1818: 481-490, 2012, Petrova P. et al. Cancer Res 2015; 75(15 Suppl): Abstract no. 4271), BRIC125 (Avent. Biochem J., (1988) 251 : 499-505), BRIC126 (Avent. Biochem J., (1988) 251 : 499-505; Petrova P. et al. Cancer Res 2015; 75(15 Suppl): Abstract no. 4271), 5F9 (Uger R. et al. Cancer Res 2014; 74(19 Suppl): Abstract no. 5011, Liu et al. PLoS One. 2015 Sep 21 ;10(9): e0137345; Sikic B. et al. J Clin Oncol 2016;34 (suppl; abstract 3019)), anti-CD47 antibodies disclosed in US Patent Publication 2014/0161799, WO Publication 2014/093678, US Patent Publication 2014/0363442, and CC2C6 (Petrova P. et al. Cancer Res 2015; 75(15 Suppl): Abstract no. 4271, Uger R. et al. Cancer Res 2014; 74(19 Suppl): Abstract no. 5011). It has also been shown that a SIRPa-Fc fusion protein, which binds to human CD47, has reduced binding to human RBCs compared to other human cells (Uger R. et al. Cancer Res 2014; 74(19 Suppl: Abstract no. 5011 ; Petrova et al. Clin Cancer Res 23: 1068-1079, 2017). Binding to RBCs can be reduced by generation of bi-specific antibodies with only one CD47 binding arm (Masternak et al. Cancer Res 2015; 75(15 Suppl): Abstract no. 2482). Because some anti-CD47 m Ahs have been shown to result in reduction of RBCs when administered to cynomolgus monkeys (Mounho-Zamora B. et al. The Toxicologist, Supplement to Toxicological Sciences, 2015; 144 (1): Abstract 596: 127, Liu et al. PLoS One. 2015 Sep 21 ; 10(9): e0137345; Pietsch et al. Cancer Res 2015; 75(15 Suppl): Abstract nr 2470), it is highly desirable to identify anti-CD47 m Ahs that have reduced or minimal binding to CD47-expressing RBCs.
[0237] As disclosed herein, the anti-CD47 mAbs have reduced, minimal, or no binding to human RBCs . As disclosure herein, some of the anti-CD47 mAbs require ITOb I for maximal binding to human CD47. As a result, some of the anti-CD47 mAbs do not bind RBCs because RBCs do not express ITOb I .
[0238] As used herein, the terms“red blood cell(s)” and“erythrocyte(s)” are synonymous and used interchangeably herein.
[0239] As used herein, the term“reduced binding to hRBCs” refers to an apparent Kd of an anti-CD47 mAh binding to a hRBC which is 8 -fold or greater than the apparent Kd on a human tumor cell, wherein the tumor cell is an OV10 hCD47 cell (human OV10 ovarian cancer cell line expressing human CD47).
[0240] As used herein, the term“minimal binding” or“MB“ refers to no measurable binding to hRBCs at an anti-CD47 mAh concentration up to 5,000 pM.
[0241] Prior to the disclosure described herein, no monospecific anti-CD47 m Ahs have been reported that have minimal binding to human RBCs expressing CD47.
[0242] Some of the anti-CD47 mAbs, disclosed herein, have reduced or minimal binding to human RBCs.
Binding to Human Endothelial Cells and Other Normal Human Cells
[0243] In addition to expression/overexpression on most hematological malignancies and solid tumors (Willingham et al , Proc Natl Acad Sci 2012), CD47 is also expressed, by many but not all, normal cell types, including, but not limited to RBCs (see previous section), lymphocytes and mononuclear cells, endothelial cells, and brain, liver, muscle cells and/or tissues (Brown et al, J Cell Biol 1990; Reinhold et al , J Cell Sci. 1995; Matozaki et al , Cell 2009; Stefanidakis et al , Blood 2008; Xiao et al, Cancer Letters 2015). Because of this expression, it is expected that some anti-CD47 m Ahs would bind to these normal cell types/tissues in addition to the cancer cells which are the therapeutic target. It is therefore desirable to identify anti-CD47 m Ahs that either have reduced or minimal binding to some of these normal cells to both reduce potential non-desired effects on these normal cells and also allow more available antibody for binding to the tumor cells. Anti-CD47 m Ahs with such reduced or minimal binding to normal cells have not been described.
[0244] As used herein, the terms“reduced binding to normal human cells which includes, but is not limited to, endothelial cells, skeletal muscle cells, epithelial cells, and peripheral blood mononuclear cells (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubular epithelial cells, human peripherial blood CD3+ cells, and human peripheral blood mononuclear cells) refers to the apparent Kd of an anti-CD47 mAh binding to these cells which is 8-fold or greater than the apparent Kd of the anti-CD47 mAh binding to a human tumor cell, wherein the tumor cell is OVIO hCD47.
[0245] As used herein, the term“mimimal binding” or“MB” refers to no measurable binding of an antibody or other molecule as described herein to normal human cells which includes, but is not limited to, endothelial cells, skeletal muscle cells, epithelial cells, and peripheral blood mononuclear cells (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubular epithelial cells, human peripherial blood CD3+ cells, and human peripheral blood mononuclear cells) at an anti-CD47 mAh concentration up to 5,000 pM.
Agglutination of RBCs
[0246] Red blood cell (RBC) agglutination or hemagglutination is a homotypic interaction that occurs when RBCs aggregate or clump together following incubation with various agents, including antibodies to RBC antigens and cell surface proteins such as CD47. Many anti-CD47 antibodies have been reported to cause hemagglutination of washed human RBCs in vitro, in a concentration dependent manner, including B6H12, BRIC126, MABL-1, MABL-2, CC2C6, and 5F9 (Uger R. et al. Cancer Res 2014; 74(19 Suppl): Abstract no. 5011, US Patent 9,045,541, Uno et al. Oncol Rep. 17: 1189-94, 2007; Kikuchi et al. Biochem Biophys Res. Commun. 315: 912-8, 2004; Sikic B. et al. J Clin Oncol 2016;34 (suppl; abstract 3019)). This functional effect requires binding to RBCs by an intact, bivalent antibody and can be reduced or eliminated by generating antibody fragments, either a F(ab’) or svFv (Uno et al. Oncol Rep. 17: 1189-94, 2007; Kikuchi et al. Biochem Biophys Res. Commun. 315: 912-8, 2004) or bi specific antibodies with only one CD47 binding arm (Masternak et al. Cancer Res 2015; 75(15 Suppl): Abstract no. 2482). Other functional properties of these fragments, including cell killing, were shown to be either reduced or retained in these fragments (Uno et al. Oncol Rep. 17: 1189-94, 2007; Kikuchi et al. Biochem Biophys Res. Commun. 315: 912-8, 2004). The mouse antibody 2D3 is an example of an anti-CD47 antibody that binds to CD47 on red blood cells but does not cause hemagglutination (US Patent 9,045,541, Petrova et al. Cancer Res 2015; 75(15 Suppl): Abstract no. 4271).
[0247] Hemagglutination has been shown to be reduced/eliminated by reducing the binding selectively to human RBCs, but not other cells, using a SIRPoc-Fc fusion protein (Uger R. et al. Blood 2013; 122(21): 3935). In addition, mouse anti-CD47 mAh 2A1 and humanized versions of 2A1 have been reported to block CD47/SIRP0C but do not exhibit hemagglutination activity in a washed RBC assay (US Patent 9,045,541 ; Narla et al. Proc Am Assoc Cancer Res 58: 1200, 2017; abst 4694). A small number of a panel of mouse anti-human CD47 antibodies (8 of 23) were reported to not cause hemagglutination of human RBCs (Pietsch E et al. Blood Cancer Journal (2017) 7, e536; doi: 10.1038/bcj.2017.7). Therefore, prior to the disclosure described herein, there was a need to identify CD47 m Ahs that block SIRPoc/CD47 binding, have reduced or minimal binding to RBCs and/or cause no detectable hemagglutination. The term“agglutination” refers to cellular clumping, while the term“hemagglutination” refers to clumping of a specific subset of cells, i.e., RBCs. Thus, hemagglutination is a type of agglutination.
[0248] As used herein, the term “reduced hemagglutination” refers to detectable agglutination activity of hRBCs at anti-CD47 mAh concentrations greater than or equal to 1.85 mg/ml, and no measurable activity at concentrations less than 1.85 mg/mI in a washed RBC assay, as visualized by discrete punctate dot compared to a diffuse pattern that represents hemagglutination.
[0249] As used herein, the term“no detectable hemagglutination” refers to no visible or detectable agglutination activity of hRBCs at anti-CD47 mAh concentrations greater or equal to 0.3 pg/ml to a concentration less than or equal to 10 mg/mI in a washed RBC assay, as visualized by discrete punctate dot compared to a diffuse pattern that represents hemagglutination.
[0250] In some embodiments a method is disclosed for treating a human subject having cancer, wherein an antibody or antigen binding fragment thereof is administered to a human subject that prevents the binding of human CD47 with SIRPoc without agglutination of normal cells, and wherein the human CD47 is associated with an integrin on a cancer cell, and wherein said integrin allows for the binding of said antibody or antigen binding fragment thereof to human CD47.
[0251] In another embodiment, a method is disclosed of preventing agglutination of normal cells in human subject having cancer treatment with a CD47 antibody or antigen binding fragment thereof, wherein a CD47 antibody or antigen binding fragment thereof is admini tered to a human subject an antibody or antigen binding fragment thereof that prevents the binding of human CD47 with SIRPoc, wherein the human CD47 is associated with an integrin on a cancer cell, and wherein said integrin allows for the binding of said antibody or antigen binding fragment thereof to human CD47.
[0252] Some of the anti-CD47 antibodies described herein, cause reduced or no detectable hemagglutination of human RBCs.
Integrins, Src, and FAK
[0253] Integrins are transmembrane adhesion receptors localized at cell-matrix contact sites where they link extracellular matrix (ECM) components to the actin cytoskeleton and interact with multiple downstream structural and signaling molecules, such as Src and FAK (Berrier and Yamada, 2007). The integrin family of proteins consists of an alpha (a) and a beta (b) subunit, associated by noncovalent interactions, forming an extracellular ligand-binding head, two multi-domain Tegs’, two single-pass transmembrane helices and two short cytoplasmic tails. The a and b subunits show no homology to each other, however, conserved regions are found among each a and b subtype.
[0254] Src is a non-receptor cytoplasmic tyrosine kinase which becomes activated following the stimulation of plasma membrane receptors, including receptor tyrosine kinases and integrins, and is involved in multiple physiological homeostatic pathways. Once activated, Src is the starting point for several biochemical cascades that thereby propagate signals generated extracellularly along intracellular interconnected transduction pathways. Src transmits signals promoting cell survival, and the reorganization of the cytoskeleton, and adhesion systems that underpin cell migration and invasion.
[0255] The effect of Src activation appears to be pleiotropic as numerous Src substrates are phosphorylated in tumor cells with activated forms of Src. Many of these phosphorylated Src substrates have been linked to processes inducing tumorigenicity and metastasis (Brown and Cooper, 1996). Furthermore, these alterations in signal transduction likely influence multiple downstream transcriptional events which include alterations in homotypic adhesion, angiogenesis, tumor cell invasivity, tumor growth, and apoptosis, all of which play a significant role in the development of the metastatic phenotype.
[0256] The tyrosine kinase FAK acts both as a signaling molecule and a scaffold able to recruit Src and the Src substrates to sites of integrin engagement, and has an important role in cell cycle progression and survival as well as in adhesion and migration (Berrier and Yamada, 2007).
[0257] It is known that clustering of integrins subsequent to cell-matrix contact results in FAK transient dimerization, intermolecular autophosphorylation, and activation. Tyrosine phosphorylation of FAK creates a high-affinity binding site for Src, specifically the SFI2 domain, leading to recruitment and activation of Src. The formation of a stable
FAK-Src complex, in which activated Src phosphorylates FAK at several tyrosine residues, further enhances the kinase activity of FAK (Playford and Schaller, 2004). Finally, the resulting highly active FAK-Src complex promotes the phosphorylation of many FAK associated Src substrates including CAS, paxillin, and pl90RhoGAP which have a central role in the reorganization of the actin cytoskeleton and migration (Playford and Schaller, 2004). Furthermore, phosphorylated FAK can recruit Grb2 and the p85 regulatory subunit of PI3K, thus leading respectively to stimulation of Grb2/Ras/MAPK and PI3K/Akt pathways which enhance cell survival and proliferation and contribute to cytoskeleton remodeling.
CD47 and Integrins
[0258] CD47 has been shown to be associated with integrins, including anb3, anbΐ, aI¾b3, a2b1 and a4b1, and may interact with other integrin complexes (Lindberg FP et al., 1996, Ticchioni et al., 1997, Reinhold MI et al., 1997, Wu et al., 1999). The interaction of CD47 with integrins is important for mechano-transduction, cell adhesion, signaling and cell migration (Lindberg FP et al., 2008, Schwartz MA et al., 1998).
[0259] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof binds to human CD47 which is associated with an integrin.
[0260] In some embodiments, the integrin comprises ab integrin heterodimeric complex with an alpha (a) and a beta (b) subunit.
[0261] In some embodiments, the b subunit is selected from PΌbI, ITOb2, PT}b3, ITOb4, PΌb5, PΌb6, PΌb7, and ITϋb8.
[0262] In some embodiments, the a subunit is selected from ITGocl, ITGoc2, ITGoc3, ITGoc4, ITGoc5, ITGoc6, ITGoc7, ITGoc8, ITGoc9, ITGoclO, ITGocl 1, and ITGocv.
[0263] In some embodiments, the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with ITOb I .
[0264] In some embodiments, the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with an ab integrin heterodimeric complex selected from PΌbIaI, ITϋb1a2, ITϋb1a3, ITϋb1a4, ITϋb1a5, PΌb1a6, ITϋb1a7, PΌb1a8, PΌb1a9, ITϋbIaIO, PΌbIaI I, and ITGav.
[0265] In some embodiments, the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with an integrin and at least a second protein.
[0266] In some embodiments, the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with an integrin and at least a second protein, wherein the at least second protein can be selected from an integrin or a non-integrin protein, wherein the non-integrin protein can be a receptor tyrosine kinase, a checkpoint inhibitor protein, etc.
[0267] In some embodiments the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with ITOb I and at least a second protein.
[0268] In some embodiments, the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with ITOb 1 and at least a second protein, wherein the second protein is selected from ACTN1, CD46, CD9, FHL2, Filamin, FLNB, CD81, GNB2L1, ITϋbIBRI, LGALS8, MAP4K4, NME1, PKCoc, TLN1, TSPAN4, and YWHAB.
[0269] In some embodiments, the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with a beta (b) subunit of an integrin selected from ITϋbI, ITϋb2, PCTb3, PCTb4, ITϋb5, ITϋb6, ITϋb7, and PCTb8, and at least a second protein, wherein the at least a second protein is a receptor tyrosine kinase (RTK), i.e., Src and FAK, wherein the receptor tyrosine kinase is not known to bind to a beta (b) subunit of an integrin.
[0270] In some embodiments, the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with a beta (b) subunit of an integrin selected from ITϋbI, ITϋb2, PCTb3, PCTb4, ITϋb5, ITϋb6, ITϋb7, and PCTb8, and at least a second protein, wherein the at least a second protein is a checkpoint inhibitor (CTLA-4, PD-1, PD-L1, etc.), wherein the receptor tyrosine kinase is not known to bind to a beta (b) subunit of an integrin.
[0271] In some embodiments, administering the monoclonal antibody or antigen-binding fragment thereof binds to human CD47 which is associated with an integrin decreases Src phosphorylation (e.g., de-phosphorylation or inhibition of phosphorylation at Tyr416), reversing a pro-survival effect of the cancer cell. The autophosphorylation of Src is driven by conformation changes mediated by integrins and receptor tyrosine kinases (e.g., epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR)). In some embodiments, the ability of Src to phosphorylate or autophosphorylate Y416 is decreased. In some embodiments, Src phosphorylation (e.g., in a test sample) is decreased by at least about 1.1-, 1.2-, 1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, 2-, 2.1-, 2.2-, 2.3-, 2.4-, 2.5-, 2.6-, 2.7-, 2.8-, 2.9-, 3-, 3.1-, 3.2-, 3.3-, 3.4-, 3.5-, 3.6-, 3.7-, 3.8-, 3.9-, 4-, 4.1-, 4.2-, 4.3-, 4.4-, 4.5-, 4.6-, 4.7- , 4.8-, 4.9-, 5-, 5.5-, 6-, 6.5-, 7-, 7.5-, 8-, 8.5-, 9-, 9.5-, 10-, 10.5-, 11-, 11.5-, 12-, 12.5-, 13-, 13.5-, 14-, 14.5-, 15-, 15.5-, 16-, 16.5-, 17-, 17.5-, 18-, 18.5-, 19-, 19.5-, or 20-fold (e.g., compared to when the monoclonal antibody or antigen-binding fragment thereof is not administered).
[0272] In some embodiments, the phosphorylation of a downstream Src target is increased or decreased and is selected from STAT3, PI3K/Akt, ERK/MAPK and / or a combination thereof.
[0273] In some embodiments, administering a monoclonal antibody or antigen-binding fragment thereof that binds to human CD47 which is associated with an integrin decreases FAK phosphorylation (de-phosphorylation or inhibition of phosphorylation at Tyr397), reversing a pro-survival effect of the cancer cell. The phosphorylation or autophosphorylation of FAK is driven by conformation changes mediated by integrins, G-protein coupled receptor (GPCR) occupancy by ligands such as bombesin or lysophosphatidic acid, or via FDF receptor occupancy. In some embodiments, the ability of FAK to autophosphorylate FAK at Y397 is decreased. In some embodiments, FAK phosphorylation (e.g., in a test sample) is decreased by at least about 1.1-, 1.2-, 1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, 2-, 2.1-, 2.2-, 2.3-, 2.4-, 2.5-, 2.6-, 2.7-, 2.8-, 2.9-, 3-, 3.1-, 3.2-, 3.3-, 3.4-, 3.5-, 3.6-, 3.7-, 3.8-, 3.9-, 4-, 4.1-, 4.2-, 4.3-, 4.4- , 4.5-, 4.6-, 4.7-, 4.8-, 4.9-, 5-, 5.5-, 6-, 6.5-, 7-, 7.5-, 8-, 8.5-, 9-, 9.5-, 10-, 10.5-, 11-, 11.5-, 12-, 12.5-, 13-, 13.5-, 14-, 14.5-, 15-, 15.5-, 16-, 16.5-, 17-, 17.5-, 18-, 18.5-, 19-, 19.5-, or 20- fold (e.g., compared to when the monoclonal antibody or antigen-binding fragment thereof is not administered).
[0274] In some embodiments, the phosphorylation of a downstreatm FAK target is increased or decreased and is selected from paxillin, ERK/MAPK, and Rho/Rac, and / or a combination thereof.
[0275] As disclosed herein, the anti-CD47 mAbs or antigen binding fragments thereof cause release of or exposure on tumor cell surfaces of one or more of the DAMPs listed in the above section, characteristics of Immunogenic Cell Death (ICD). These DAMPS are expected to promote a therapeutically beneficial adaptive anti-tumor immune response. The binding of the anti-CD47 mAbs disclosed herein to human CD47 which is associated with an integrin, particularly ITOb I , is linked to the release of DAMPs that cause maturation, activation and homing of DCs and attraction of T cells connects anti-CD47 mAh treatment to the development of the therapeutically desirable anti-tumor immune response.
Modulation of the NO Pathway
[0276] As noted above, TSP1 is also a ligand for CD47. The TSP1/CD47 pathway opposes the beneficial effects of the NO pathway in many cell types, including, but not limited to, vascular cells. The NO pathway consists of any of three enzymes (nitric oxide synthases, NOS I, NOS II and NOS III) that generate bioactive gas NO using arginine as a substrate. NO can act within the cell in which it is produced, or in neighboring cells, to activate the enzyme soluble guanylyl cyclase that produces the messenger molecule cyclic GMP (cGMP). The proper functioning of the NO/cGMP pathway is essential for protecting the cardiovascular system against stresses including, but not limited to, those resulting from wounding, inflammation, hypertension, metabolic syndrome, ischemia, and IRI. In the context of these cellular stresses the inhibition of the NO/cGMP pathway by the TSP1/CD47 system exacerbates the effects of stress. This is a particular problem in the cardiovascular system where both cGMP and cAMP play important protective roles. There are many cases in which ischemia and reperfusion injury cause or contribute to disease, trauma, and poor outcomes of surgical procedures.
[0277] As disclosed herein, one of more of the chimeric or humanized anti-CD47 antibodies will reverse TSP1 inhibition of cGMP production. Reversal will be complete (>80 %) or intermediate (20% -80%). This reversal of TSP1 inhibition of cGMP production will demonstrate that the anti-CD47 m Ahs have the ability to increase NO signaling and suggest utility in protecting the cardiovascular system against stresses including, but not limited to, those resulting from wounding, inflammation, hypertension, metabolic syndrome, ischemia, and ischemia-reperfusion injury (IRI). Additional assay systems, for example smooth muscle cell contraction, will also be expected to show that some of the chimeric or humanized antibodies reverse the inhibitory actions of TSP1 on downstream effects resulting from the activation of NO signaling.
[0278] As disclosed herein,“complete reversal of NO pathway inhibition” refers to greater than 80% reversal of TSP1 inhibition of NO signaling by an anti-CD47 mAh compared to a negative control, humanized isotype-matched antibody or no treatment. [0279] As disclosed herein,“intermediate reversal of NO pathway inhibition” refers to 20- 80% reversal of TSP1 inhibition of NO signaling by an anti-CD47 mAh compared to a negative control, humanized isotype-matched antibody or no treatment.
[0280] As disclosed herein,“no reversal of NO pathway inhibition” refers to less than 20% reversal of TSP1 inhibition of NO signaling by an anti-CD47 mAh compared to a negative control, humanized isotype-matched antibody or no treatment.
Immunogenic Cell Death
[0281] The concept of immunogenic cell death (ICD) has emerged in recent years. This form of cell death, unlike non-immunogenic cell death, stimulates an immune response against antigens from cancer cells. ICD is induced by specific chemotherapy drugs, including anthracyclines (doxorubicin, daurorubicin and mitoxantrone) and oxaliplatin, but not by cisplatin and other chemotherapy drugs. ICD is also induced by bortezomib, cardiac glycosides, photodynamic therapy and radiation Galluzi et al. Nat Rev Immunol 17: 97-111, 2016). The distinctive characteristics of ICD of tumor cells are the release from or exposure on tumor cell surfaces of specific ligands: 1) the pre-apoptotic cell surface exposure of calreticulin, 2) the secretion of adenosine triphosphate (ATP), 3) release of high mobility group box 1 (HMGB 1), 4) annexin Al release, 5) type I interferon release and 6) C-X-C motif chemokine ligand 10 (CXCL10) release. These ligands are endogenous damage- associated molecular patterns (DAMPs), which include the cell death-associated molecules (CD AMs) (Kroemer et al. Annu Rev Immunol 31: 51-72, 2013). Importantly, each of these ligands induced during ICD binds to specific receptors, referred to as pattern recognition receptors (PRRs), that contribute to an anti-tumor immune response. ATP binds the purinergic receptors PY2, G-protein coupled, 2 (P2RY2) and PX2, ligand-gated ion channel, 7 (P2RX7) on dendritic cells causing dendritic cell recruitment and activation, respectively. Annexin Al binds to formyl peptide receptor 1 (FPR1) on dendritic cells causing dendritic cell homing. Calreticulin expressed on the surface of tumor cells binds to LRP1 (CD91) on dendritic cells promoting antigen uptake by dendritic cells. HMGB 1 binds to toll-like receptor 4 (TLR4) on dendritic cells to cause dendritic cell maturation. As a component of ICD, tumor cells release type I interferon leading to signaling via the type I interferon receptor and the release of the CXCL10 which favors the recruitment of effector CXCR3+ T cells Together, the actions of these ligands on their receptors facilitate recruitment of DCs into the tumor, the engulfment of tumor antigens by DCs and optimal antigen presentation to T cells. Kroemer et al. have proposed that a precise combination of the CD AMs mentioned above elicited by ICD can overcome the mechanisms that normally prevent the activation of anti-tumor immune responses (Kroemer et al. Annu Rev Immunol 31 : 51-72, 2013; Galluzi et al. Nat Rev Immunol 17: 97-111, 2016). When mouse tumor cells treated in vitro with ICD-inducing modalities are administered in vivo to syngeneic mice, they provide effective vaccination that leads to an anti-tumor adaptive immune response, including memory. This vaccination effect cannot be tested in xenograft tumor models because the mice used in these studies lack a complete immune system. The available data indicate that ICD effects induced by chemotherapy or radiation will promote an adaptive anti-tumor immune response in cancer patients. The components of ICD are described in more detail below.
[0282] In 2005, it was reported that tumor cells which were dying in response to anthracycline chemotherapy in vitro caused an effective anti-tumor immune response when administered in vivo in the absence of adjuvant (Casares et al. J Exp Med 202 : 16911701, 2005). This immune response protected mice from subsequent re-challenge with viable cells of the same tumor and caused regression of established tumors. Anthracyclines (doxorubicin, daunorubicin and idarubicin) and mitomycin C induced tumor cell apoptosis with caspase activation, but only apoptosis induced by anthracyclines resulted in immunogenic cell death. Caspase inhibition did not inhibit cell death induced by doxorubicin but did suppress the immunogenicity of tumor cells dying in response to doxorubicin. The central roles of dendritic cells and CD8+ T cells in the immune response elicited by doxorubicin-treated apoptotic tumor cells were established by the demonstration that depletion of these cells abolished the immune response in vivo.
[0283] Calreticulin is one of the most abundant proteins in the endoplasmic reticulum (ER). Calreticulin was shown to rapidly translocate preapoptotically from the ER lumen to the surface of cancer cells in response to multiple ICD inducers, including anthracyclines (Obeid et al. Nat Med 13: 54-61, 2007; Kroemer et al. Annu Rev Immunol 31 : 51-72, 2013). Blockade or knockdown of calretiulin suppressed the phagocytosis of anthracycline-treated tumor cells by dendritic cells and abolished their immunogenicity in mice. The exposure of calreticulin caused by anthracyclines or oxaliplatin is activated by an ER stress response that involves the phosphorylation of the eukaryotic translation initiation factor eIF2oc by the PKR-like ER kinase. Calretiulin, which has a prominent function as an“eat-me” signal (Gardai et al. Cell 123: 321-334, 2005) binds to LRP1 (CD91) on dendritic cells and macrophages resulting in phagocytosis of the calreticulin expressing cell, unless the calreticulin-expressing cell expresses a don’t eat me signal, such as CD47. Calreticulin also signals through CD91 on antigen presenting cells to cause the release of proinflammatory cytokines and to program Thl7 cell responses. In summary, calreticulin expressed as part of immunogenic cell death stimulates antigen presenting cells to engulf dying cells, process their antigens and prime an immune response.
[0284] In addition to calreticulin, protein disulfide-isomerase A3 (PDIA3), also called Erp57, was shown to translocate from the ER to the surface of tumor cells following treatment with mitoxantrone, oxaliplatin and irradiation with UVC light (Panaretakis et al. Cell Death Differ 15: 1499-1509, 2008; Panaretakis et al. EMBL J 28: 578-590, 2009). A human ovarian cancer cell line, primary ovarian cancer cells and a human prostate cancer cell line expressed cell-surface calreticulin, HSP70 and HSP90 following treatment with the anthracyclines doxorubicin and idarubicin (Fucikova et al. Cancer Res 71 : 4821-4833, 2011). HSP70 and HSP90 bind to the PRR LRP1 on antigen presenting cells; the PRR to which PDIA3 binds has not been identified (Galluzi et al. Nat Rev Immunol 17: 97-111, 2016).
[0285] TLR4 was shown to be required for cross-presentation of dying tumor cells and to control tumor antigen processing and presentation. Among proteins that were known to bind to and stimulate TLR4, HMGB1 was uniquely released by mouse tumor cells in which ICD was induced by irradiation or doxorubicin (Apetoh et al. Nat Med 13: 1050-1059, 2007). The highly efficient induction of an in vivo anti-tumor immune by doxorubicin treatment of mouse tumor cells required the presence of HMGB 1 and TLR4, as demonstrated by abrogation of the immune response by inhibition of HMGB 1 and knock-out TLR4. These preclinical findings are clinically relevant. Patients with breast cancer who carry a TLR4 loss-of-function allele relapse more quickly after radiotherapy and chemotherapy than those carrying the normal TLR4 allele.
[0286] Ghiringhelli et al. showed that mouse tumor cells treated with oxaliplatin, doxorubicin and mitoxanthrone in vitro released ATP and that the ATP binds to the purinergic receptors PY2, G-protein coupled, 2 (P2RY2) and PX2, ligand-gated ion channel, 7 (P2RX7) on dendritic cells (Ghiringhelli et al. Nat Med 15: 1170-1178, 2009). Binding of ATP to P2RX7 on DCs triggers the NOD-like receptor family, pyrin domain containing-3 protein (NLRP3)- dependent caspase-1 activation complex (inflammasome), allowing for the secretion of interleukin- 1b (IL-Ib), which is essential for the priming of interferon-gamma-producing CD8+ T cells by dying tumor cells. Therefore, the ATP-elicited production of IL-Ib by DCs appears to be one of the critical factors for the immune system to perceive cell death induced by certain chemotherapy drugs as immunogenic. This paper also reports that HMGB 1, a TLR4 agonist, also contributes to the stimulation of the NLRP3 inflammasome in DCs and the secretion of IL-Ib. These preclinical results have been shown to have clinical relevance; in a breast cancer cohort, the presence of the P2RX7 loss-of-function allele had a significant negative prognostic impact of metastatic disease-free survival. ATP binding to P2RY2 causes the recruitment of myeloid cells into the tumor microenvironment (Vacchelli et al. Oncoimmunology 5: el 118600, 2016)
[0287] Michaud et al. demonstrated that autophagy is required for the immunogenicity of chemotherapy-induced cell death (Michaud et al. Science 334: 1573-1577, 2011). Release of ATP from dying tumor cells required autophagy and autophagy-competent, but not autophagy- deficient, mouse tumors attracted dendritic cells and T lymphocytes into the tumor microenvironment in response to chemotherapy that induces ICD.
[0288] Ma et al. addressed the question of how chemotherapy-induced cell death leads to efficient antigen presentation to T cells (Ma et al. Immunity 38: 729-741, 2013). They found that at specific kind of tumor infiltrating lymphocyte, CDl lc+CDl lb+Ly6Chl cells, are particularly important for the induction of anticancer immune responses by anthracy dines. ATP released by dying cancer cells recruited myeloid cells into tumors and stimulated the local differentiation of CDl lc+CDl lb+Ly6Chl cells. These cells were shown to be particularly efficient in capturing and presenting tumor cell antigens and, after adoptive transfer into naive mice, conferring protection to challenge with living tumor cells of the same cell line.
[0289] It has been shown that anthracyclines stimulate the rapid production of type I interferons by tumor cells after activation of TLR3 (Sistugu et al. Nat Med 20: 1301-1309, 2014). Type I interferons (IFN) bind to IFN-oc and IFN-b receptors on cancer cells and trigger autocrine and paracrine signaling pathways that result in release of CXCL10. Tumors lacking Tlr3 or Ifnar failed to respond to chemotherapy unless type I IFN or CXCL10, respectively, was supplied. These preclinical findings have clinical relevance. A type I IFN-related gene expression signature predicted clinical responses to anthracycline-based chemotherapy in independent cohorts of breast cancer patients.
[0290] Another receptor on dendritic cells that is involved in chemotherapy-induced anti cancer immune response was recently identified: formyl peptide receptor- 1, which binds annexin Al (Vacchelli et al. Science 350: 972-978, 2015). Vacchelli et al. designed a screen to identify candidate genetic defects that negatively affect responses to chemotherapy. They identified a loss-of-function allele of the gene encoding formyl peptide receptor 1 (FPR1) that was associated with poor metastatis-free survival and overall survival in breast and colorectal cancer patients receiving adjuvant chemotherapy. The therapeutic effects of anthracyclines were abrogated in tumor-bearing Fprl -/- mice due to impaired antitumor immunity. FPR1- deficient DCs did not approach dying tumor cells and, therefore, could not elicit antitumor T cell immunity. Two anthracyclines, doxorubicin and mitoxantrone, stimulated the secretion of annexin Al, one of four known ligands of FPR1. FPR1 and annexin A1 promoted stable interactions between dying cancer cells and human or mouse leukocytes.
[0291] In addition to anthracyclines and oxaliplatin, other drugs have been shown to induce immunogenic cell death. Cardiac glycosides, including clinically used digoxin and digitoxin, were also shown to be efficient inducers of immunogenic cell death of tumor cells (Menger et al. Sci Transl Med 4: 143ra99, 2012). Other chemotherapy agents and cancer drugs that have been reported to induce DAMP expression or release are bleomycin, bortezomib, cyclophosphamide, paclitaxel, vorinistat and cisplatin (Garg et al. Front Immunol 588: 1-24, 2015; Menger et al. Sci Transl Med 4: 143ra99, 2012; Martins et al. Oncogene 30: 1147-1158, 2011). Importantly, these results have clinical relevance. Administration of digoxin during chemotherapy had a significant positive impact on the overall survival of patients with breast, colorectal, head and neck, and hepatocellular cancers, but failed to improve overall survival of lung and prostate cancer patients.
[0292] The anti-CD20 monoclonal antibody rituximab has improved outcomes in multiple B-cell malignancies. The success of rituximab, referred to as a type I anti-CD20 mAh, led to the development of type II anti-CD20 mAbs, including obinutuzumab and tositumomab. Cheadle et al, investigated the induction of immunogenic cell death by anti-CD20 mAbs (Cheadle et al. Brit J Haematol 162: 842-862, 2013). They found that the cell death induced by obinutuzumab and tositumomab is a form of immunogenic cell death characterized by the release of F1MGB 1, F1SP90 and ATP. A type I anti-CD20 mAh did not cause release of F1MGB 1 , F1SP90 and ATP. Incubation of supernatants from a human tumor cell line treated with obinutuzumab caused maturation of human dendritic cells, consistent with the previously described effects of F1MGB 1 and ATP on dendritic cells. In contrast to the results reported by Cheadle et al. , Zhao et al. reported that both type I and II anti-CD20 mAbs increased F1MGB 1 release from human diffuse large B cell lymphoma cell lines, but did not cause ATP release or cell surface expression of calreticulin (Zhao et al. Oncotarget 6: 27817-27831, 2015).
[0293] The release from or exposure on tumor cell surfaces of the DAMPs calreticulin, ATP, F1MGB 1, annexin Al, type I interferon release, CXCL10, PDIA3, F1SP70 and/or F1SP90 in response to anti-CD47 mAbs has not been reported. As disclosed herein, anti-CD47 mAbs cause release from or exposure on tumor cell surfaces of one or more of the DAMPs listed above (characteristics of ICD), an unexpected result. These DAMPS are expected to promote a therapeutically beneficial adaptive anti-tumor immune response. [0294] As disclosed herein,“causes an increase in cell surface calreticulin expression on human tumor cells” refers to a statistically significant increase (p < 0.05) in calreticulin expression by a soluble anti-CD47 mAh compared to the background obtained with a negative control, humanized isotype-matched antibody or no treatment.
[0295] As disclosed herein, the term“the release of’ is synonymous with secretion and is defined as the extracellular appearance of ATP, HMGB1, annexin Al, type I interferon and CXCL10.
[0296] As disclosed herein,“cause an increase in the release of adenosine triphosphate by human tumor cells” refers to a statistically significant increase (p < 0.05) in ATP in the supernatant caused by a soluble anti-CD47 mAh compared to the background obtained with a negative control, humanized isotype-matched antibody or no treatment.
[0297] As disclosed herein,“cause an increase in the release of high mobility group box 1 by human tumor cells” refers to a statistically significant increase (p < 0.05) in HMGB1 in the supernatant caused by a soluble anti-CD47 mAh compared to the background obtained with a negative control, humanized isotype-matched antibody or no treatment.
[0298] As disclosed herein,“causes an increase in the release of type I interferon by human tumor cells” refers to a statistically significant increase (p < 0.05) in type I interferon in the supernatant or type I interferon mRNA caused by a soluble anti-CD47 mAh compared to the background obtained with a negative control, humanized isotype-matched antibody or no treatment.
[0299] As disclosed herein,“causes an increase in the release of C-X-C Motif Chemokine Ligand 10 (CXCL10) by human tumor cells” refers to a statistically significant increase (p < 0.05) in CXCL10 in the supernatant or CXCL10 mRNA caused by a soluble anti-CD47 mAh compared to the background obtained with a negative control, humanized isotype-matched antibody or no treatment.
[0300] As disclosed herein,“causes an increase in cell surface PDIA3 expression on human tumor cells” refers to a statistically significant increase (p < 0.05) in PDIA3 expression by a soluble anti-CD47 mAh compared to the background obtained with a negative control, humanized isotype-matched antibody or no treatment.
[0301] As disclosed herein,“causes an increase in cell surface HSP70 expression on human tumor cells” refers to a statistically significant increase (p < 0.05) in HSP70 expression by a soluble anti-CD47 mAh compared to the background obtained with a negative control, humanized isotype-matched antibody or no treatment. [0302] As disclosed herein,“causes an increase in cell surface HSP90 expression on human tumor cells” refers to statistically significant increase (p < 0.05) in HSP90 expression by a soluble anti-CD47 mAh compared to the background obtained with a negative control, humanized isotype-matched antibody or no treatment.
pH Dependence of Anti-CD47 mAb Binding
[0303] Most antibody binding, particularly in the blood compartment and to normal cells occurs at physiological pH (approximately 7.4). Therefore, the binding affinity of therapeutic m Ahs is normally assessed in vitro at physiological pH. However, the tumor microenvironment (TME) is more acidic in nature, with pH values below 7.4. This appears to be due to a number of differences including hypoxia, anaerobic glycolysis leading to the production of lactic acid and hydrolysis of ATP (Tannock and Rotin, Cancer Res 1989; Song et al, Cancer Drug Discovery and Development 2006; Chen and Pagel, Advan Radiol 2015). The acidic pH may provide an advantage to the tumor by promoting invasiveness, metastatic behavior, chronic autophagy, resistance to chemotherapies and reduced efficacy of immune cells in the tumor microenvironment (Estrella et al. Cancer Res 2013; Wojtkowiak et al, Cancer Res 2012; Song et al, Cancer Drug Discovery and Development 2006; Barar, Bioimpacts, 2012). The identification of anti-CD47 antibodies with the property of increased binding affinity at acidic pH would confer a therapeutic advantage with higher binding to CD47 on tumor cells within the acidic TME compared to cells at physiological pH. Antibodies with pH-dependent properties have been generated with the goal of recycling antibodies. However, in contrast to exhibiting the properties of enhanced binding at acidic pH, these bind with high affinity to their target antigen at physiological pH, but release their target at acidic pH (Bonvin et al, mAbs 2015; Igawa and Hattori, Biochem Biophys Acta 2014).
[0304] As disclosed herein,“has a greater affinity for CD47 at an acidic pH compared to physiological pH” refers to an apparent Kd that is increased 5-fold or more at acidic pH (< 7.4) compared to physiological pH (7.4).
Combinations of Functional Properties
[0305] In some embodiments, the anti-CD47 antibodies described herein, are also characterized by combinations of properties which are not exhibited by prior art anti-CD47 antibodies proposed for human therapeutic use. Accordingly, in some embodiments, anti-CD47 antibodies described herein may be characterized by:
a. binds to human CD47 ;
b. blocks SIRPoc binding to human CD47; c. increases phagocytosis of human tumor cells; and
d. induces death of susceptible human tumor cells.
[0306] In another embodiment described herein, the anti-CD47 antibodies are characterized by:
a. binds to human CD47 ;
b. blocks SIRPoc binding to human CD47;
c. increases phagocytosis of human tumor cells;
d. induces death of susceptible human tumor cells; and
e. causes no detectable agglutination of human red blood cells (hRBCs).
[0307] In yet another embodiment described herein, the anti-CD47 antibodies are characterized by:
a. binds to human CD47 ;
b. blocks SIRPoc binding to human CD47;
c. increases phagocytosis of human tumor cells;
d. induces death of susceptible human tumor cells; and
e. causes reduced agglutination of human red blood cells (hRBCs).
[0308] In another embodiment described herein, the anti-CD47 antibodies are characterized by:
a. binds to human CD47 ;
b. blocks SIRPoc binding to human CD47;
c. increases phagocytosis of human tumor cells;
d. induces death of susceptible human tumor cells; and
e. has reduced hRBC binding.
[0309] In another embodiment described herein, the anti-CD47 antibodies are characterized by:
a. binds to human CD47 ;
b. blocks SIRPoc binding to human CD47;
c. increases phagocytosis of human tumor cells;
d. causes no detectable agglutination of human red blood cells (hRBCs); and e. has minimal binding to hRBCs.
[0310] In another embodiment described herein, the anti-CD47 antibodies are characterized by:
a. binds to human CD47 ; b. blocks SIRPoc binding to human CD47;
c. increases phagocytosis of human tumor cells;
d. causes no detectable agglutination of human red blood cells (hRBCs); and e. has reduced hRBC binding.
[0311] In another embodiment described herein, the monoclonal antibody, or antigen binding fragment thereof binds to human, non-human primate, mouse, rabbit, and rat CD47.
[0312] In yet another embodiment described herein, the monoclonal antibody, or antigen binding fragment thereof specifically also binds to non-human primate CD47, wherein non human primate may include, but is not limited to, cynomolgus monkey, green monkey, rhesus monkey and squirrel monkey.
[0313] In another embodiment, the anti-CD47 monoclonal antibody, or antigen binding fragment thereof, may additionally possess one or more of the following characteristics: 1) exhibit cross-reactivity with one or more species homologs of CD47; 2) block the interaction between CD47 and its ligand SIRPoc; 3) increase phagocytosis of human tumor cells; 4) induce death of susceptible human tumor cells; 5) do not induce cell death of human tumor cells; 6) do not have reduced or minimal binding to human red blood cells (hRBCs); 7) have reduced binding to hRBCs; 8) have minimal binding to hRBCs; 9) cause reduced agglutination of hRBCs; 10) cause no detectable agglutination of hRBCs; 11) reverse TSP1 inhibition of the nitric oxide (NO) pathway; 12) do not reverse TSP1 inhibition of the NO pathway; 13) cause loss of mitochondrial membrane potential; 14) do not cause cause loss of mitochondrial membrane potential; 15) cause an increase in cell surface ca reticulin expression on human tumor cells; 16) do not cause an increase in cell surface caireticulin expression on human tumor cells; 17) cause an increase in adenosine triphosphate (ATP) release by human tumor cells; 18) do not cause an increase in adenosine triphosphate (ATP) release by human tumor cells; 19) cause an increase in high mobility group box 1 (HMGB 1) release by human tumor cells; 20) do not cause an increase in high mobility group box 1 (HMGB 1) release by human tumor cells; 21) cause an increase in type I interferon release by human tumor cells; 22) do not cause an increase in type I interferon release by human tumor cells; 23) cause an increase in C-X-C Motif Chemokine Ligand 10 (CXCL10) release by human tumor cells; 24) do not cause an increase in C-X-C Motif Chemokine Ligand 10 (CXCL10) release by human tumor cells; 25) cause an increase in cell surface protein disulfide-isomerase A3 (PDIA3) expression on human tumor cells; 26) do not cause an increase in cell surface protein disulfide-isomerase A3 (PDIA3) expression on human tumor cells; 27) cause an increase in cell surface heat shock protein 70 (HSP70) expression on human tumor cells; 28) do not cause an increase in cell surface heat shock protein 70 (HSP70) expression on human tumor cells; 29) cause an increase in cell surface heat shock protein 90 (HSP90) expression on human tumor cells; 30) do not cause an increase in cell surface heat shock protein 90 (HSP90) expression on human tumor cells; 31) have reduced binding to normal human cells, which includes, but is not limited to, endothelial cells, skeletal muscle cells, epithelial cells, and peripheral blood mononuclear cells (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubular epithelial cells, human peripherial blood CD3+ cells, and human peripheral blood mononuclear cells); 32) do not have reduced binding to normal human cells, which includes, but is not limited to, endothelial cells, skeletal muscle cells, epithelial cells, and peripheral blood mononuclear cells (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubular epithelial cells, human peripherial blood CD3+ cells, and human peripheral blood mononuclear cells); 33) have a greater affinity for human CD47 at an acidic pH compared to physiological pH; 34) do not have a greater affinity for human CD47 at an acidic pH compared to physiological pH; and 35) cause an increase in annexin A1 release by human tumor cells.
[0314] In some embodiments, a monoclonal antibody, or an antigen binding fragment thereof, is provided, which: binds to human CD47; blocks SIRPoc binding to human CD47; increases phagocytosis of human tumor cells; and induces death of human tumor cells; wherein said monoclonal antibody, or an antigen binding fragment thereof, exhibits pH-dependent binding to CD47 present on a cell. In other embodiments, the disclosure provides a monoclonal antibody, or an antigen binding fragment thereof, which: binds to human CD47; blocks SIRPoc binding to human CD47; increases phagocytosis of human tumor cells; and induces death of human tumor cells; wherein said monoclonal antibody, or an antigen binding fragment thereof, exhibits reduced binding to normal cells. In some embodiments, a cell to which such an antibody may bind may be of any cell type as described herein. In other embodiments, a monoclonal antibody as described herein, or an antigen binding fragment thereof, may exhibit any combination of characteristics provided in the present disclosure. For example, a monoclonal antibody may beneficially exhibit both pH dependent binding and reduced binding to a cell. These cells may be an endothelial cell, a skeletal muscle cell, an epithelial cell, a PBMC or a RBC (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubular epithelial cells, human peripherial blood CD3+ cells, human peripheral blood mononuclear cells or human RBC). Such characteristics may be exhibited individually or in any combination as described herein. As used herein, pH dependent binding of an antibody of the disclosure may refer to altered binding of the antibody at a particular pH, for example an antibody that exhibits increased binding affinity at acidic pH.
CD47 Antibodies
[0315] Many human cancers up-regulate cell surface expression of CD47 and those expressing the highest levels of CD47 appear to be the most aggressive and the most lethal for patients. Increased CD47 expression is thought to protect cancer cells from phagocytic clearance by sending a“don’t eat me” signal to macrophages via SIRPoc, an inhibitory receptor that prevents phagocytosis of CD47-bearing cells (Oldenborg et al. Science 288: 2051-2054, 2000; Jaiswal et al. (2009) Cell 138(2):271-851; Chao et al. (2010) Science Translational Medicine 2(63):63ra94). Thus, the increase of CD47 expression by many cancers provides them with a cloak of “selfness” that slows their phagocytic clearance by macrophages and dendritic cells.
[0316] Antibodies that block CD47 and prevent its binding to SIRPoc have shown efficacy in human tumor in murine (xenograft) tumor models. Such blocking anti-CD47 mAbs exhibiting this property increase the phagocytosis of cancer cells by macrophages, which can reduce tumor burden (Majeti et al. (2009) Cell 138 (2): 286-99; US 9,045,541 ; Willingham et al. (2012) Proc Natl Acad. Sci. USA 109(17):6662-6667; Xiao et al. (2015) Cancer Letters 360:302-309; Chao et al. (2012) Cell 142:699-713; Kim et al. (2012) Leukemia 26:2538-2545).
[0317] Anti-CD47 mAbs have also been shown to promote an adaptive immune response to tumors in vivo (Tseng et al. (2013) PNAS 110 (27): 11103-11108; Soto-Pantoja et al. (2014) Cancer Res. 74 (23): 6771-6783; Liu et al. (2015) Nat. Med. 21 (10): 1209-1215; Xu et al. (2017) Immunity 47: 363-373).
[0318] However, there are mechanisms by which anti-CD47 mAbs can attack transformed cells that have not yet been exploited in the treatment of cancer. Multiple groups have shown that particular anti-human CD47 mAbs induce cell death of human tumor cells. Anti-CD47 mAh Ad22 induces cell death of multiple human tumor cells lines (Pettersen et al. J. Immunol. 166: 4931-4942, 2001 ; Lamy et al. J. Biol. Chem. 278: 23915-23921, 2003). AD22 was shown to indice rapid mitochondrial dysfunction and rapid cell death with early phosphatidylserine exposure and a drop in mitochondrial membrane potential (Lamy et al. J. Biol. Chem. 278: 23915-23921, 2003). Anti-CD47 mAh MABL-2 and fragments thereof induce cell death of human leukemia cell lines, but not normal cells in vitro and had an anti-tumor effect in in vivo xenograft models. (Uno et al. (2007) Oncol. Rep. 17 (5): 1189-94). Anti-human CD47 mAh 1F7 induces cell death of human T cell leukemias (Manna and Frazier (2003) J. Immunol. 170: 3544-53) and several breast cancers (Manna and Frazier (2004) Cancer Research 64 (3): 1026- 36). 1F7 kills CD47-bearing tumor cells without the action of complement or cell mediated killing by NK cells, T cells, or macrophages. Instead, anti-CD47 mAh 1F7 acts via a non- apoptotic mechanism that involves a direct CD47-dependent attack on mitochondria, discharging their membrane potential and destroying the ATP-generating capacity of the cell leading to rapid cell death. It is noteworthy that anti-CD47 mAh 1F7 does not kill resting leukocytes, which also express CD47, but only those cells that are “activated” by transformation. Thus, normal circulating cells, many of which express CD47, are spared while cancer cells are selectively killed by the tumor-toxic CD47 mAh (Manna and Frazier (2003) J. Immunol. 170: 3544-53). This mechanism can be thought of as a proactive, selective and direct attack on tumor cells in contrast to the passive mechanism of causing phagocytosis by simply blocking CD47/SIRP0C binding. Importantly, mAh 1F7 also blocks binding of SIRPoc to CD47 (Rebres et al., J. Cellular Physiol. 205: 182-193, 2005) and thus it can act via two mechanisms: (1) direct tumor toxicity, and (2) causing phagocytosis of cancer cells. A single mAh that can accomplish both functions may be superior to one that only blocks CD47/SIRP0C binding.
[0319] An additional mechanism by which anti-CD47 m Ahs can be exploited in the treatment of cancer is through the promotion of an anti-tumor immune response. The discovery that anti-CD47 mAbs cause tumor cells to release DAMPs that cause maturation, activation and homing of DCs and attraction of T cells connects anti-CD47 mAh treatment to the development of the therapeutically desirable anti-tumor immune response. Anti-CD47 mAbs have not been previously shown to cause tumor cell release of ATP, HMGB1, annexin Al, type I interferons and CXCL10 and tumor cell expression of calreticulin, PDIA3, HSP70 and HSP90.
[0320] Following periods of tissue ischemia, the initiation of blood flow causes damage referred to as“ischemia-reperfusion injury” or IRI. IRI contributes to poor outcomes in many surgical procedures where IRI occurs due to the necessity to stop blood flow for a period of time, in many forms/causes of trauma in which blood flow is interrupted and later restored by therapeutic intervention and in procedures required for organ transplantation, cardio/pulmonary bypass procedures, reattachment of severed body parts, reconstructive and cosmetic surgeries and other situations involving stopping and restarting blood flow. Ischemia itself causes many physiological changes that, by themselves would eventually lead to cell and tissue necrosis and death. Reperfusion poses its own set of damaging events including generation of reactive oxygen species, thrombosis, inflammation and cytokine mediated damage. The pathways that are limited by the TSP1-CD47 system are precisely those that would be of most benefit in combating the damage of IRI, including the NO pathway. Thus, blocking the TSP1-CD47 pathway, as with the antibodies disclosed herein, will provide more robust functioning of these endogenous protective pathways. Anti-CD47 m Ahs have been shown to reduce organ damage in rodent models of renal warm ishchemia (Rogers et al. J Am Soc Nephrol. 23: 1538-1550, 2012), liver ischemia-reperfusion injury (Isenberg et al. Surgery. 144: 752-761, 2008), renal transplantation (Lin et al. Transplantation. 98: 394-401, 2014; Rogers et al. Kidney Interantional. 90: 334-347, 2016)) and liver transplantation, including steatotic livers (Xiao et al. Liver Transpl. 21 : 468-477, 2015; Xiao et al. Transplantation. 100: 1480-1489, 2016). In addition, anti-CD47 mAh caused significant reductions of right ventricular systolic pressure and right ventricular hypertrophy in the monocrotaline model of pulmonary arterial hypertension (Bauer et al. Cardiovasc Res. 93: 682-693, 2012). Studies in skin flap models have shown that modulation of CD47, including with anti-CD47 mAbs, inhibits TSPl-mediated CD47 signaling. This results in inceased activity of the NO pathway, resulting in reduced IRI (Maxhimer et al. Plast Reconstr Surg. 124: 1880-1889, 2009; Isenberg et al. Arterioscler Throm Vase Biol. 27: 2582-2588, 2007; Isenberg et al. Curr Drug Targets. 9: 833-841, 2008; Isenberg et al. Ann Surg. 247: 180-190, 2008)
[0321] Anti-CD47 m Ahs have also been shown to be efficacious in models of other cardiovascular diseases. In the mouse transverse aortic constriction model of pressure overload left ventricular heart failure, anti-CD47 mAh mitigated cardiac myocyte hypertrophy, decreased left ventricular fibrosis, prevented an increase in left ventricular weight, decreased ventricular stiffness, and normalized changes in the pressure volume loop profile (Sharifi- Sanjani et al. J Am Heart Assoc., 2014). An anti-CD47 mAh ameliorated atherosclerosis in multiple mouse models (Kojima et al. Nature., 2016).
Anemia
[0322] Presently disclosed are anti-CD47 m Ahs and antigen binding fragments thereof effective as cancer therapeutics which can be administered to patients who exhibit anemia. Anemia is a condition in which a patient lacks enough healthy red blood cells to carry adequate oxygen to the tissues of the body. Anemia can be temporary or long term, and it can range from mild to severe.
[0323] In the context of a diagnosis of cancer, anemia can be caused by the cancer itself, chemotherapy treatments, or both. Cancers that involve the marrow space, such as leukemia or lymphoma, compete with the marrow’s function and interfere with normal red blood cell production.
[0324] There are different types of anemia common to the oncology practice. Grade 1 is considered mild anemia, is Hb from 10 g/dL to the lower limit of normal; Grade 2 anemia, or moderate anemia, is Hb from 8 to less than 10 g/dL; Grade 3, or severe anemia, is below 8 g/dL; Grade 4, is life-threatening anemia; and Grade 5 is death.
[0325] In one embodiment, the disclosed anti-CD47 mAbs can be administered to treat cancer in a patient with anemia, wherein the disclosed anti-CD47 mAbs bind to human CD47 on the surface of a tumor cell with minimal binding to human red blood cells (hRBCs), and wherein the human red blood cells (hRBCs) lack bΐ-integrin expression.
[0326] In another embodiment, the disclosed anti-CD47 mAbs can be administered to treat cancer in a patient with anemia, wherein the disclosed anti-CD47 mAbs bind to human CD47 in association with an integrin on the surface of a tumor cell with minimal binding to human red blood cells (hRBCs), and wherein the human red blood cells (hRBCs) lack bΐ-integrin expression.
Cancer Indications
[0327] Presently disclosed are anti-CD47 mAbs and antigen binding fragments thereof effective as cancer therapeutics which can be administered to patients, preferably parenterally, with susceptible hematologic cancers and solid tumors including, but not limited to, leukemias, including systemic mastocytosis, acute lymphocytic (lymphoblastic) leukemia (ALL), T cell - ALL, acute myeloid leukemia (AML), myelogenous leukemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), myeloproliferative disorder / neoplasm, monocytic cell leukemia, and plasma cell leukemia; multiple myeloma (MM); Waldenstrom’s Macroglobulinemia; lymphomas, including histiocytic lymphoma and T cell lymphoma, B cell lymphomas, including Hodgkin’s lymphoma and non-Hodgkin’s lymphoma, such as low grade/follicular non-Hodgkin’s lymphoma (NHL), cell lymphoma (FCC), mantle cell lymphoma (MCL), diffuse large cell lymphoma (DLCL), small lymphocytic (SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL; solid tumors, including ovarian cancer, breast cancer, endometrial cancer, colon cancer (colorectal cancer), rectal cancer, bladder cancer, urothelial cancer, lung cancer (non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung), bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, gastric cancer, hepatocellular carcinoma (liver cancer, hepatoma), gall bladder cancer, bile duct cancer, esophageal cancer, renal cell carcinoma, thyroid cancer, squamous cell carcinoma of the head and neck (head and neck cancer), testicular cancer, cancer of the endocrine gland, cancer of the adrenal gland, cancer of the pituitary gland, cancer of the skin, cancer of soft tissues, cancer of blood vessels, cancer of brain, cancer of nerves, cancer of eyes, cancer of meninges, cancer of oropharynx, cancer of hypopharynx, cancer of cervix, and cancer of uterus, glioblastoma, meduloblastoma, astrocytoma, glioma, meningioma, gastrinoma, neuroblastoma, myelodysplastic syndrome, and sarcomas including, but not limited to, osteosarcoma, Ewing’s sarcoma, leiomyosarcoma, synovial sarcoma, alveolar soft part sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, chrondrosarcoma, and melanoma.
[0328] In some embodiments, the disclosed anti-CD47 m Ahs can be used in a method to treat gastric cancer in a human subject, wherein said monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with an integrin on the surface of a tumor cell, thereby causing an increase in cell death of tumor cells.
[0329] In some embodiments, the gastric cancer can be selected from adenocarcinoma, lymphoma, gastrointentinal stromal tumor, or GIST, and carcinoid tumor (Type I and II ECL- cell carcinoids, Type III ECL-cell carcinoids).
Treatment of Cancer
[0330] As is well known to those of ordinary skill in the art, combination therapies are often employed in cancer treatment as single-agent therapies or procedures may not be sufficient to treat or cure the disease or condition. Conventional cancer treatments often involve surgery, radiation treatment, the administration of a combination of cytotoxic drugs to achieve additive or synergistic effects, and combinations of any or all of these approaches. Especially useful chemotherapeutic and biologic therapy combinations employ drugs that work via different mechanisms of action, increasing cancer cell control or killing, increasing the ability of the immune system to control cancer cell growth, reducing the likelihood of drug resistance during therapy, and minimizing possible overlapping toxicities by permitting the use of reduced doses of individual drugs.
[0331] Classes of conventional anti-tumor/anti-neoplastic agents useful in the combination therapies encompassed by the present methods are disclosed, for example, in Goodman & Gilman’s The Pharmacological Basis of Therapeutics, Twelfth Edition (2010) L.L. Brunton, B.A. Chabner, and B. C. Knollmann Eds., Section VIII, “Chemotherapy of Neoplastic Diseases”, Chapters 60-63, pp. 1665-1770, McGraw-Hill, NY, and include, for example, alkylating agents, antimetabolites, natural products, a variety of miscellaneous agents, hormones and antagonists, targeted drugs, monoclonal antibodies and other protein therapeutics.
[0332] In addition to the foregoing, the methods of the present disclosure are related to treatment of cancer indications and further comprises treating the patient via surgery, radiation, and/or administering to a patient in need thereof an effective amount of a chemical small molecule or biologic drug including, but not limited to, a peptide, polypeptide, protein, nucleic acid therapeutic, conventionally used or currently being developed, to treat tumorous conditions. This includes antibodies and antigen-binding fragments, other than those disclosed herein, cytokines, antisense oligonucleotides, siRNAs, and miRNAs.
[0333] The therapeutic methods disclosed and claimed herein include the use of the antibodies disclosed herein alone, and/or in combinations with one another, and/or with antigen-binding fragments thereof of the present disclosure that bind to CD47, and/or with competing antibodies exhibiting appropriate biological/therapeutic activity, as well, for example, all possible combinations of these antibody compounds to achieve the greatest treatment efficacy.
[0334] In addition, the present therapeutic methods also encompass the use of these antibodies, antigen-binding fragments thereof, competing antibodies and combinations thereof further in combination with: (1) any one or more anti-tumor therapeutic treatments selected from surgery, radiation, anti-tumor, anti-neoplastic agents, and combinations of any of these, or (2) any one or more of anti-tumor biological agents, or (3) equivalents of any of the foregoing of (1) or (2) as would be apparent to one of ordinary skill in the art, in appropriate combination(s) to achieve the desired therapeutic treatment effect for the particular indication.
[0335] Antibody and small molecule drugs that increase the immune response to cancer by modulating co-stimulatory or inhibitory interactions that influence the T cell response to tumor antigens, including inhibitors of immune checkpoints and modulators of co-stimulatory molecules, are also of particular interest in the context of the combination therapeutic methods encompassed herein and include, but are not limited to, other anti-CD47 antibodies. Administration of therapeutic agents that bind to the CD47 protein, for example, antibodies or small molecules that bind to CD47 and prevent interaction between CD47 and SIRPa, are administered to a patient, causing the clearance of cancer cells via phagocytosis. The therapeutic agent that binds to the CD47 protein is combined with a therapeutic agent such as an antibody, a chemical small molecule or biologic drug disclosed herein, directed against one or more additional cellular targets of CD70 (Cluster of Differentiation 70), CD200 (OX-2 membrane glycoprotein, Cluster of Differentiation 200), CD 154 (Cluster of Differentiation 154, CD40L, CD40 ligand, Cluster of Differentiation 40 ligand), CD223 (Lymphocyte- activation gene 3, LAG3, Cluster of Differentiation 223), KIR (Killer-cell immunoglobulin like receptors), GITR (TNFRSF18, glucocorticoid-induced TNFR-related protein, activation- inducible TNFR family receptor, AITR, Tumor necrosis factor receptor superfamily member 18), CD28 (Cluster of Differentiation 28), CD40 (Cluster of Differentiation 40, Bp50, CDW40, TNFRSF5, Tumor necrosis factor receptor superfamily member 5, p50), CD86 (B7-2, Cluster of Differentiation 86), CD160 (Cluster of Differentiation 160, BY55, NK1, NK28), CD258 (LIGHT, Cluster of Differentiation 258, Tumor necrosis factor ligand superfamily member 14, TNFSF14, HVEML, HVEM ligand, herpesvirus entry mediator ligand, LTg), CD270 (HVEM, Tumor necrosis factor receptor superfamily member 14, herpesvirus entry mediator, Cluster of Differentiation 270, LIGHTR, HVEA), CD275 (ICOSL, ICOS ligand, Inducible T-cell co stimulator ligand, Cluster of Differentiation 275), CD276 (B7-H3, B7 homolog 3, Cluster of Differentiation 276), OX40L (0X40 Ligand), B7-H4 (B7 homolog 4, VTCN1, V-set domain- containing T-cell activation inhibitor 1), GITRL (Glucocorticoid-induced tumor necrosis factor receptor-ligand, glucocorticoid-induced TNFR-ligand), 4-1BBL (4-1BB ligand), CD3 (Cluster of Differentiation 3, T3D), CD25 (IL2Ra, Cluster of Differentiation 25, Interleukin-2 Receptor a chain, IL-2 Receptor a chain), CD48 (Cluster of Differentiation 48, B-lymphocyte activation marker, BLAST-1, signaling lymphocytic activation molecule 2, SLAMF2), CD66a (Ceacam-
1, Carcinoembryonic antigen-related cell adhesion molecule 1, biliary glycoprotein, BGP, BGP1, BGPI, Cluster of Differentiation 66a), CD80 (B7-1, Cluster of Differentiation 80), CD94 (Cluster of Differentiation 94), NKG2A (Natural killer group 2A, killer cell lectin-like receptor subfamily D member 1, KLRDl), CD96 (Cluster of Differentiation 96, TActILE, T cell activation increased late expression), CD112 (PVRL2, nectin, Poliovirus receptor-related
2, herpesvirus entry mediator B, HVEB, nectin-2, Cluster of Differentiation 112), CD115 (CSF1R, Colony stimulating factor 1 receptor, macrophage colony-stimulating factor receptor, M-CSFR, Cluster of Differentiation 115), CD205 (DEC-205, LY75, Lymphocyte antigen 75, Cluster of Differentiation 205), CD226 (DNAM1, Cluster of Differentiation 226, DNAX Accessory Molecule-1, PTA1, platelet and T cell activation antigen 1), CD244 (Cluster of Differentiation 244, Natural killer cell receptor 2B4), CD262 (DR5, TrailR2, TRAIL-R2, Tumor necrosis factor receptor superfamily member 10b, TNFRSF10B, Cluster of Differentiation 262, KILLER, TRICK2, TRICKB, ZTNFR9, TRICK2A, TRICK2B), CD284 (Toll-like Receptor-4, TLR4, Cluster of Differentiation 284), CD288 (Toll-like Receptor-8, TLR8, Cluster of Differentiation 288), TNFSF15 (Tumor necrosis factor superfamily member 15, Vascular endothelial growth inhibitor, VEGI, TL1 A), TD02 (Tryptophan 2,3-dioxygenase, TPH2, TRPO), IGF-1R (Type I Insulin-like Growth Factor), GD2 (Disialoganglioside 2), TMIGD2 (Transmembrane and immunoglobulin domain-containing protein 2), RGMB (RGM domain family, member B), VISTA (V-domain immunoglobulin-containing suppressor of T- cell activation, B7-H5, B7 homolog 5), BTNL2 (Butyrophilin-like protein 2), Btn (Butyrophilin family), TIGIT (T cell Immunoreceptor with Ig and ITIM domains, Vstm3, WUCAM), Siglecs (Sialic acid binding Ig-like lectins), Neurophilin, VEGFR (Vascular endothelial growth factor receptor), ILT family (LIRs, immunoglobulin-like transcript family, leukocyte immunoglobulin-like receptors), NKG families (Natural killer group families, C- type lectin transmembrane receptors), MICA (MHC class I polypeptide-related sequence A), TGF (Transforming growth factor b), STING pathway (Stimulator of interferon gene pathway), Leukemia inhibitory factor (LIF), Arginase (Arginine amidinase, canavanase, L- arginase, arginine transamidinase), EGFRvIII (Epidermal growth factor receptor variant III), and HHLA2 (B7-H7, B7y, HERV-H LTR-associating protein 2, B7 homolog 7), inhibitors of PD-1 (Programmed cell death protein 1, PD-1, CD279, Cluster of Differentiation 279), PD-L1 (B7-H1, B7 homolog 1, Programmed death-ligand 1, CD274, cluster of Differentiation 274), PD-L2 (B7-DC, Programmed cell death 1 ligand 2, PDCD1LG2, CD273, Cluster of Differentiation 273), CTLA-4 (Cytotoxic T-lymphocyte-associated protein 4, CD 152, Cluster of Differentiation 152), BTLA (B- and T-lymphocyte attenuator, CD272, Cluster of Differentiation 272), Indoleamine 2,3-dioxygenase (IDO, IDOl), TIM3 (HAVCR2, Hepatitis A virus cellular receptor 2, Angiopoietin-like 4 (Angptl4), T cell immunoglobulin mucin-3, KIM-3, Kidney injury molecule 3, TIMD-3, T cell immunoglobulin mucin-domain 3), A2A adenosine receptor (ADO receptor), CD39 (ectonucleoside triphosphate diphosphohydrolase- 1, Cluster of Differentiation 39, ENTPD1), and CD73 (Ecto-5’-nucleotidase, 5’-nucleotidase, 5’-NT, Cluster of Differentiation 73), CD27 (Cluster of Differentiation 27), ICOS (CD278, Cluster of Differentiation 278, Inducible T-cell Co- stimulator), CD137 (4-1BB, Cluster of Differentiation 137, Thrombospondin- 1 (TSP1), tumor necrosis factor receptor superfamily member 9, TNFRSF9), 0X40 (CD 134, Cluster of Differentiation 134), and TNFSF25 (Tumor necrosis factor receptor superfamily member 25), including antibodies, small molecules, and agonists, are also specifically contemplated herein. Additional agents include IL-10 (Interleukin- 10, human cytokine synthesis inhibitory factor, CSIF), and Galectins.
[0336] YERVOY® (ipilimumab; Bristol-Meyers Squibb) is an example of an approved anti-CTLA-4 antibody.
[0337] KEYTRUDA® (pembrolizumab; Merck) and OPDIVO® (nivolumab; Bristol- Meyers Squibb Company) are examples of approved anti-PD-1 antibodies. [0338] TECENTRIQ® (atezolizumab; Roche) is an example of an approved anti-PD-Ll antibody.
[0339] SPRYCEL® (dasatinib; Bristol-Meyers Squibb Company) is an example of ATP- competitive protein tyrosine kinase inhibitor. The main targets of dasatinib are BCR/Abl (the "Philadelphia chromosome"), Src, c-Kit, ephrin receptors, and several other tyrosine kinases.
[0340] Defactinib (Verastem Oncology) is an orally available small molecule inhibitor of FAK and PYK2 that is currently in clinical development with potential anti-angiogeneic and anti-neoplastic activities.
[0341] Ischemia-Reperfusion Injury (IRI)-Related, Autoimmune, Autoinflammatory, Inflammatory, Cardiovascular Conditions and Diseases
[0342] Administration of a CD47 mAh or antigen binding fragment thereof disclosed herein can be used to treat a number of diseases and conditions in which IRI is a contributing feature, and to treat various autoimmune, autoinflammatory, inflammatory and cardiovascular diseases. These include: organ transplantation in which a mAh or antigen binding fragment thereof of the present disclosure is administered to the donor prior to organ harvest, to the harvested donor organ in the organ preservation solution, to the recipient patient, or to any combination thereof; skin grafting; surgical resections or tissue reconstruction in which such mAh or fragment is administered either locally by injection to the affected tissue or parenterally to the patient; reattachment of body parts; treatment of traumatic injury; pulmonary hypertension; pulmonary arterial hypertension; sickle cell disease (crisis); myocardial infarction; cerebrovascular disease; stroke; surgically-induced ischemia; acute kidney disease/kidney failure; any other condition in which IRI occurs and contributes to the pathogenesis of disease; autoimmune and inflammatory diseases, including arthritis, rheumatoid arthritis, multiple sclerosis, psoriasis, psoriatic arthritis, Crohn’s disease, inflammatory bowel disease, ulcerative colitis, lupus, systemic lupus erythematous, juvenile rheumatoid arthritis, juvenile idiopathic arthritis, Grave’s disease, Hashimoto’s thyroiditis, Addison’s disease, celiac disease, dermatomyositis, multiple sclerosis, myasthenia gravis, pernicious anemia, Sjogren syndrome, type I diabetes, vasculitis, uveitis, and ankylosing spondylitis; autoinflammatory diseases, including familial Mediterranean fever, neonatal onset multisystem inflammatory disease, tumor necrosis factor (TNF) receptor-associated periodic syndrome, deficiency of the interleukin- 1 receptor antagonist, Behcet’s disease; cardiovascular diseases, including coronary heart disease, coronary artery disease, atherosclerosis, myocardial infarction, heart failure, and left ventricular heart failure. [0343] Anti-CD47 m Ahs and antigen binding fragments thereof of the present disclosure can also be used to increase tissue perfusion in a subject in need of such treatment. Such subjects can be identified by diagnostic procedures indicating a need for increased tissue perfusion. In addition, the need for increased tissue perfusion may arise because the subject has had, is having, or will have, a surgery selected from integument surgery, soft tissue surgery, composite tissue surgery, skin graft surgery, resection of a solid organ, organ transplant surgery, or reattachment or an appendage or other body part.
Treatment of Ischemia-Reperfusion Injury (IRI)-Related Indications
[0344] The methods of the present disclosure, for example those related to treatment of IRI-related indications, can further comprise administering to a patient in need thereof an effective amount of therapeutic agent that binds to the CD47 protein and a nitric oxide donor, precursor, or both; a nitric oxide generating topical agent; an agent that activates soluble guanylyl cyclase; an agent that inhibits cyclic nucleotide phosphodiesterases; or any combination of any of the foregoing.
[0345] In these methods, the nitric oxide donor or precursor can be selected from NO gas, isosorbide dinitrate, nitrite, nitroprusside, nitroglycerin, 3-Morpholinosydnonimine (SIN-1), S- nitroso-N-acetylpenicillamine (SNAP), Diethylenetriamine/NO (DETA/NO), S-nitrosothiols, Bidil®, and arginine.
[0346] The agent that activates soluble guanylyl cyclase can be a non-NO (nitric oxide)- based chemical activator of soluble guanylyl cyclase that increases cGMP levels in vascular cells. Such agents bind soluble guanylyl cyclase in a region other than the NO binding motif, and activate the enzyme regardless of local NO or reactive oxygen stress (ROS). Non-limiting examples of chemical activators of soluble guanylyl cyclase include organic nitrates (Artz et al. (2002) J. Biol. Chem. 277:18253-18256); protoporphyrin IX (Ignarro et al. (1982) Proc. Natl. Acad. Sci. USA 79:2870-2873); YC-1 (Ko et al. (1994) Blood 84:4226-4233); BAY 41- 2272 and BAY 41-8543 (Stasch et al. (2001 Nature 410 (6825): 212-5), CMF-1571, and A- 350619 (reviewed in Evgenov et al. (2006) Nat. Rev. Drug. Discov. 5:755-768); BAY 58-2667 (Cinaciguat; Frey et al. (2008) Journal of Clinical Pharmacology 48 (12): 1400-10); BAY 63- 2521 (Riociguat; Mittendorf et al. (2009) Chemmedchem 4 (5): 853-65). Additional soluble guanylyl cyclase activators are disclosed in Stasch et al. (2011) Circulation 123:2263-2273; Derbyshire and Marietta (2012) Ann. Rev. Biochem. 81 :533-559, and Nossaman et al. (2012) Critical Care Research and Practice, Volume 2012, Article ID 290805, pages 1-12.
[0347] The agent that inhibits cyclic nucleotide phosphodiesterases can be selected from, tadalafil, vardenafil, udenafil, sildenafil and avanafil. Treatment of Autoimmune, Autoinflammatory, Inflammatory, and Cardiovascular Diseases
[0348] A therapeutic agent that binds to the CD47 protein for the treatment of an autoimmune, autoinflammatory, inflammatory disease and / or cardiovascular disease can be combined with one or more therapeutic agent(s) such as an antibody, a chemical small molecule, or biologic or a medical or surgical procedure which include, but are not limited to the following. For the treatment of autoimmune, autoinflammatory and inflammatory diseases, the combined therapeutic agents are: hydroxychloroquine, leflunomide, methotrexate, minocycline, sulfasalazine, abatacept, rituximab, tocilizumab, anti-TNF inhibitors or blockers (adalimumab, etanercept, infliximab, certolizumab pegol, golimumab), non-steroidal anti inflammatory drugs, glucocorticoids, corticosteroids, intravenous immunoglobulin, anakinra, canakinumab, rilonacept, cyclophosphamide, mycophenolate mofetil, azathioprine, 6- mercaptopurine, belimumab, beta interferons, glatiramer acetate, dimethyl fumarate, fingolimod, teriflunomide, natalizumab, 5 -aminosalicylic acid, mesalamine, cyclosporine, tacrolimus, pimecrolimus, vedolizumab, ustekinumab, secukinumab, ixekizumab, apremilast, budesonide and tofacitinib. For the treatment of atherosclerosis, the combined therapeutic agents or procedures are: medical procedures and/or surgery, including percutaneous coronary intervention (coronary angioplasty and stenting), coronary artery bypass grafting, and carotid endarterectomy; therapeutic agents, including angiotensin-converting enzyme (ACE) inhibitors (including ramipril, quinapril, captopril, and enalapril), calcium channel blockers (including amiodipine, nifedipine, verapamil, felodipine and diltiazem), angiotensin-receptor blockers (including eposartan, olmesarten, azilsartan, valsartan, telmisartan, losartan, candesartan, and irbesartan), the combination of ezetimibe and simvastatin, PCSK9 inhibitors (including alirocumab and evolocumab), anacetrapib, and HMG-CoA inhibitors (including atorvastatin, pravastatin, simvastatin, rosuvastatin, pitavastatin, lovastatin and fluvastatin). For the treatment of heart failure, the combined therapeutic agents are: ACE inhibitors, angiotensin receptor blockers, angiotensin receptor neprilsyn inhibitors (including the combination of sacubitril and valsartan), diuretics, digoxin, inotropes, beta blockers and aldosterone antagonists. For the treatment of pumonary hypertension the combined therapeutic agents are: sildenafil, tadalafil, ambrisentan, bosentan, macitentan, riociguat, treprostinil, epoprostenol, iloprost, and selexipag.
[0349] As disclosed herein, the anti-CD47 mAh is administered before, at the same time or after the combined therapeutic agents or medical or surgical procedures. [0350] Another useful class of compounds for the combination therapies contemplated herein includes modulators of SIRPoc/CD47 binding such as antibodies to SIRPa, as well as soluble protein fragments of this ligand, or CD47 itself, inhibiting binding of, or interfering with binding of, SIRPa to CD47. It should be noted that the therapeutic methods encompassed herein include the use of the antibodies disclosed herein alone, in combination with one another, and/or with antigen-binding fragments thereof as well, for example, a l possible combinations of these antibody compounds.
[0351] The examples illustrate various embodiments of the present disclosure, but should not be considered as limiting the disclosure to only these particularly disclosed embodiments.
Diagnostics for CD47 Expression
[0352] Diagnostics (including complementary and companion) have been an area of focus in the field of oncology. A number of diagnostic assays have been developed for targeted therapeutics such as Herceptin (Genentech), Tarceva (OSI Pharmaceuticals/Genentech), Iressa (Astra Zeneca), and Erbitux (Imclone / Bristol Myers Squibb). The anti-CD47 mAbs antibodies of the disclosure are particularly well-suited to use in diagnostic applications. Accordingly, the disclosure provides a method to measure CD47 expression in tumor and / or immune cells, using an anti-CD47 mAh of the disclosure.
[0353] The anti-CD47 mAbs of the disclosure may be used in a diagnostic assay and / or in vitro method to measure CD47 expression in tumor and / or immune cells present in a patient’s tumor sample. In particular, the anti-CD47 mAbs of the disclosure may bind CD47 on approximately 1% or more of tumor and / or immune cells present in a patient’s sample as compared to a reference level. In another embodiment, the anti-CD47 mAbs may bind CD47 on approximately 5% or more of tumor and/ or immune cells in a patient’s sample as compared to a reference level, for example, or binding at least 10%, or at least 20%, or at least 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or between 10-100% as compared to a reference level. In yet another embodiment, the anti-CD47 mAbs may bind CD47 on tumor and / or immune cells in a patient’s sample to at least about a 2-fold increase as compared to a reference level, or at least about 3 -fold, or at least about a 4-fold, or at least about a 5 -fold or at least about a 8- fold increase, or between 2-fold and 8-fold, or about 10-fold or greater as compared to a reference level. As described herein, the measurement of CD47 expression in a patient’s sample provides biological and/or clinical information that enables decision making about the development and use of a potential drug therapy, notably the use of anti-CD47 antibodies for treating solid and hematological cancers, autoimmune disease, inflammatory disease, atherosclerosis, heart failure, in which the CD47 receptor plays a role.
[0354] In one embodiment, the in vitro method comprises, obtaining a patient sample, contacting the patient sample with a monoclonal antibody, or antigen-binding fragment thereof, which specifically binds to an epitope within the sequence of SEQ ID NO:66, and assaying for binding of the antibody to the patient sample, wherein binding of the antibody to the patient sample is diagnostic of CD47 expression in a patient sample.
[0355] Accordingly, a diagnostic assay in accordance with the disclosure may comprise contacting tumor and/or immune cells in a patient’s sample with an anti-CD47 mAh, or an antigen binding fragment thereof, and assaying for binding of the anti-CD47 mAh to a patient’s tumor sample, wherein binding of the anti-CD47 mAh to the patient sample is diagnostic of CD47 expression. Preferably, the patient’s sample is a sample containing tumor cells. In this case, binding of the anti-CD47 mAh of the disclosure, or antigen binding fragment thereof, to the tumor cells may be assessed for CD47 expression. The levels of CD47 expression by tumor cells and/or immune cells of a patient’s tumor sample may be predictive of clinical outcome in a patient.
[0356] Increased binding of anti-CD47 m Ahs binding to cells in a patient’s sample is associated with increased CD47 expression. In one embodiment, the anti-CD47 m Ahs of the disclosure may bind to approximately 5% or more of tumor cells in a patient’s sample and this may indicate that the patient would benefit from rapid intervention to a solid and hematological cancer. A diagnostic assay of this sort may be used to determine suitable therapeutic regimes for solid and hematological cancers with relatively high binding of anti-CD47 m Ahs of the disclosure, i.e. , increased CD47 expression.
[0357] It will be appreciated that the diagnostic assay disclosed herein has a number of advantages. The most important of these advantages is that the diagnostic assay of the disclosure may allow the user a greater deal of confidence in the CD47 expression in tumor and / or immune cells. The increased sensitivity of the diagnostic assay of the disclosure allows detection of CD47 in a patient’s sample at lower levels than has previously been the case.
[0358] The anti-CD47 mAbs of the disclosure may be used as a diagnostic assay in relation to many forms of cancer. Particular forms of cancer that may advantageously be investigated for CD47 expression include susceptible hematologic cancers and solid tumors including, but not limited to, leukemias, lymphomas, and solid tumors.
[0359] The diagnostic assays of the disclosure may utilize any suitable means for detecting binding of an anti-CD47 mAh to measure CD47 expression. Suitable methods may be selected with reference to the nature of any reporter moiety used to label the anti-CD47 m Ahs of the disclosure. Suitable techniques include, but are by no means limited to, flow cytometry, and enzyme linked immunosorbent assays (ELISA) and assays utilizing nanoparticles.
EXAMPLES
Example 1
Amino Acid Sequences
Light Chain CDRs
Figure imgf000092_0001
Heavy Chain CDRs
Figure imgf000092_0002
Figure imgf000093_0001
Murine Light Chain Variable Domains
>Vx4murL01
DVLMTQTPLSLPVNLGDQASISCRSRQSIVHTNGNTYLGWFLQKPGQSPKLLIYKVS NRFSGVPDRFSGSGSGTDFTLTISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK (SEQ ID NO:41).
>Vx4murL02
DVLMTQTPLSLPVNLGDQASISCRSRQSIVHTNGNTYLGWFLQKPGQSPKLLIYKVS NRFSGVPDRFSGSGSGTDFTLTISRVEAEDLGVYYCFQGSHVPYTFGQGTKVEIK
(SEQ ID NO:42).
>Vx8murL03
DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLYSGV PSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIK (SEQ ID NO:46).
>Vx9murL04
DVFMTQTPLSLPVSLGDQASISCRSSQNIVQSNGNTYLEWYLQKPGQSPKLLIYKVFH RFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKVEIK(SEQ ID NO:50)
Murine Heavy Chain Variable Domains
>Vx4murH01 EVQLQQSGPELVKPGASVKMSCKASGYTFTNYVIHWVKRRPGQGLEWIGYIYPYND GILYNEKFKGKATVTSDKSSSTAYMDLSSLTSEDSAVYYCTRGGYYVPDYWGQGTTL TVSS (SEQ ID NO:21).
> Vx4mur-H02
EVQLQQSGPELVKPGASVKMSCKASGYTFTNYVIHWVKRRPGQGLEWIGYIYPYND GILYNEKFKGKATVTSDKSSSTAYMDLSSLTSEDSAVYYCTRGGYYVPDYWGQGTL VTVSS (SEQ ID NO:22).
> Vx8murH03
EVQLQQSGPELMKPGASVKISCKASGYSFTNYYIHWVNQSHGKSLEWIGYIDPLNGD TTYNQKFKGKATLTVDKSSSTAYMRLSSLTSADSAVYYCARGGKRAMDYWGQGTSV TVSS (SEQ ID NO:28).
>Vx9murH04
QV QLQQFGAELAKPGAS V QMSCKASGYTFTNYWIHWVKQRPGQGLEWIGYTDPRT DYTEYNQKFKDKATLAADRSSSTAYMRLSSLTSEDSAVYYCAGGGRVGLGYWGHGS SVTVSS (SEQ ID NO:35)
Human Light Chain Variable Domains
>Vx4humL01
DIVMTQSPLSLPVTPGEPASISCRSRQSIVHTNGNTYLGWYLQKPGQSPRLLIYKVSNR FSGVPDRFSGSGSGTDFTLKISRVEADDVGIYYCFQGSHVPYTFGQGTKLEIK (SEQ ID NO:43)
>Vx4humF02
DVVMTQSPFSFPVTFGQPASISCRSRQSIVHTNGNTYFGWFQQRPGQSPRRFIYKVSN RFSGVPDRFSGSGSGTDFTFKISRVEAEDVGVYYCFQGSHVPYTFGQGTKFEIK (SEQ ID NO:44)
>Vx4humE03
DIVMTQSPDSEAVSEGERATINCRSRQSIVHTNGNTYEGWYQQKPGQPPKEEIYKVSN RFSGVPDRFSGSGSGTDFTETISSEQAEDVAVYYCFQGSHVPYTFGQGTKEEIK (SEQ ID NO:45)
> Vx8humL04 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLYSGV PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPWTFGQGTKVEIK (SEQ ID
NO:47).
> Vx8humL05
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIYYTSRLYSGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK (SEQ ID
NO:48).
> Vx8humL06
DIVMTQSPLSLPVTPGEPASISCRASQDISNYLNWYLQKPGQSPRLLIYYTSRLYSGVP DRFSGSGSGTDFTLKISRVEADDVGIYYCQQGNTLPWTFGQGTKLEIK (SEQ ID
NO:49)
>Vx9humL07
DVVMTQSPLSLPVTLGQPASISCRSSQNIVQSNGNTYLEWFQQRPGQSPRRLIYKVFH RFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIK (SEQ ID N0:51).
>Vx9humL08
DIVMTQSPDSLAVSLGERATINCRSSQNIVQSNGNTYLEWYQQKPGQPPKLLIYKVFH RFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGSHVPYTFGQGTKLEIK (SEQ ID NO:52).
Human Heavy Chain Variable Domains >Vx4humH01
QV QLV QSGAEVKKPGAS V QVSCKASGYTFTNYVIHWLRQAPGQGLEWMGYIYPYN DGILYNEKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGGYYVPDYWGQATL VTVSS (SEQ ID NO:23).
>Vx4humH02
QV QLV QSGAEVKKPGAS V QVSCKASGYTFTNYVIHWLRQAPGQGLEWMGYIYPYN DGILYNEKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGGYYVYDYWGQATL VTVSS (SEQ ID NO:24).
>Vx4humH03 EVQLVQSGAEVKKPGATVKISCKVSGYTFTNYVIHWVQQAPGKGLEWMGYIYPYND GILYNEKFKGRVTITADTSTDTAYMELSSLRSEDTAVYYCATGGYYVPDYWGQGTTV TVSS (SEQ ID NO:25)
>Vx4humH04
EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYVIHWVRQMPGKGLEWMGYIYPYND GILYNEKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGYYVPDYWGQGTT VTVSS (SEQ ID NO:26)
>Vx4humH05
QV QLV QSGAEVKKPGAS VKVSCKASGYTFTNYVIHWVRQAPGQGLEWMGYIYPYN DGILYNEKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGGYYVPDYWGQGT TVTVSS (SEQ ID NO:27)
>Vx8humH06
QV QLV QSGAEVKKPGAS VKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLN GDTTYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGKRAMDYWGQG TLVTVSS (SEQ ID NO:29).
>Vx8humH07
QV QLV QSGAEVKKPGSS VKV SCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLN GDTTYNQKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGKRAMDYWGQGT LVTVSS (SEQ ID NO:30).
>Vx8humH08
EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIHWVRQMPGKGLEWMGYIDPLNG DTTYNQKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGKRAMDYWGQGT LVTVSS (SEQ ID NO:31).
>Vx8humH09
QV QLV QSGAEVKKPGSS VKV SCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLN GDTTYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGKRAMDYWGQGT LVTVSS (SEQ ID NO:32).
>Vx8humH10 EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIHWVRQMPGKGLEWMGYIDPLNG DTTYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGKRAMDYWGRGTL VTVSS (SEQ ID NO:33).
>Vx8humHl 1
QV QLV QSGAEVKKPGAS V QVSCKASGYSFTNYYIHWLRQAPGQGLEWMGYIDPLN GDTTYNQKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGGKRAMDYWGQAT LVTVSS (SEQ ID NO:34)
>Vx9humH12
QV QLV QSGAEVKKPGAS VKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGYTDPR TDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGRVGLGYWGQ GTLVTVSS (SEQ ID NO:36).
>Vx9humH13
QV QLV QSGAEVKKPGSS VKV SCKASGYTFTNYWIHWVRQAPGQGLEWMGYTDPRT DYTEYNQKFKDRVTITADESTSTAYMELSSLRSEDTAVYYCARGGRVGLGYWGQGTL VTVSS (SEQ ID NO: 37).
>Vx9humH14
EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYWIHWVRQMPGKGLEWMGYTDPRT DYTEYNQKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARGGRVGLGYWGQG TLVTVSS (SEQ ID NO:38).
>Vx9humH15
QV QLV QSGAEVKKPGSS VKV SCKASGYTFTNYWIHWVRQAPGQGLEWMGYTDPRT DYTEYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGRVGLGYWGQGTL VTVSS (SEQ ID NO:39).
>Vx9humH16
EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYWIHWVRQMPGKGLEWMGYTDPRT DYTEYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGRVGLGYWGQGT LVTVSS (SEQ ID NO:40).
Human IgG-Fc >Human Fc IgGl
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:53).
>Human Fc IgGl-N297Q
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:54).
>Human Fc-IgG2
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVA
GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPR
EEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVY
TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:55).
>Human Fc-IgG3
ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRC
PEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEM
TKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKS
RWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO:56)
>Human Fc-IgG4 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGG PS VFLFPPKPKDTLMISRTPEVTC VVVD V SQEDPEV QFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO:57).
>Human Fc-IgG4 S228P
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGG PS VFLFPPKPKDTLMISRTPEVTC VVVD V SQEDPEV QFNWYVDGVEVFINAKTKPREE QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO:58).
>Human Fc-IgG4 PE
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEG
GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR
EEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY
TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 59)
>Human Fc-IgG4 PE’
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEG
GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR
EEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY
TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO:99)
>Human kappa LC
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:60). >Rat Fc-IgG2c
ARTTAPSVYPLVPGCSGTSGSLVTLGCLVKGYFPEPVTVKWNSGALSSGVHTFPAVL
QSGLYTLSSSVTVPSSTWSSQTVTCSVAHPATKSNLIKRIEPRRPKPRPPTDICSCDDN
LGRPSVFIFPPKPKDILMITLTPKVTCVVVDVSEEEPDVQFSWFVDNVRVFTAQTQPH
EEQLNGTFRVVSTLHIQHQDWMSGKEFKCKVNNKDLPSPIEKTISKPRGKARTPQVY
TIPPPREQMSKNKVSLTCMVTSFYPASISVEWERNGELEQDYKNTLPVLDSDESYFLY
SKLSVDTDSWMRGDIYTCSVVHEALHNHHTQKNLSRSPGK (SEQ ID NO:61).
>Rat kappa LC
RADAAPTVSIFPPSMEQLTSGGATVVCFVNNFYPRDISVKWKIDGSEQRDGVLDSVT DQDSKDSTYSMSSTLSLTKVEYERHNLYTCEVVHKTSSSPVVKSFNRNEC (SEQ ID NO:62).
Rabbit IgG-Fc
>Rabbit IgG
GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVR QSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPS VFIFPPKPKDTLMISRTPE VTC V V VD V S QDDPEV QFTWYINNEQ VRT ARPPLREQQFN STIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPR EELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVP TSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO:63).
>Rabbit kappa LC
RDPV APT VLIFPP AAD Q V ATGT VTI V C V ANKYFPD VTVTWE VDGTTQTTGIEN S KTP QNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO:64).
>CD47
MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYV
KWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNY
TCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGM
DEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIG
LTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVE
(SEQ ID NO:65). Chimera and Fluman Light Chains
>Vx4murL01 Full length
DVLMTQTPLSLPVNLGDQASISCRSRQSIVHTNGNTYLGWFLQKPGQSPKLLIYKVS NRFSGVPDRFSGSGSGTDFTLTISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:66).
>Vx4murL01 Full length
DVLMTQTPLSLPVNLGDQASISCRSRQSIVHTNGNTYLGWFLQKPGQSPKLLIYKVS NRFSGVPDRFSGSGSGTDFTLTISRVEAEDLGVYYCFQGSHVPYTFGQGTKVEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:67).
>Vx4humL01 Full length LC
DIVMTQSPLSLPVTPGEPASISCRSRQSIVHTNGNTYLGWYLQKPGQSPRLLIYKVSN RFSGVPDRFSGSGSGTDFTLKISRVEADDVGIYYCFQGSHVPYTFGQGTKLEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:68).
>Vx8humL03 Full length LC
DIVMTQSPLSLPVTPGEPASISCRASQDISNYLNWYLQKPGQSPRLLIYYTSRLYSGVP DRFSGSGSGTDFTLKISRVEADDVGIYYCQQGNTLPWTFGQGTKLEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:69).
>Vx9humL02 Full length LC
DIVMTQSPDSLAVSLGERATINCRSSQNIVQSNGNTYLEWYQQKPGQPPKLLIYKVF HRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGSHVPYTFGQGTKLEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:70). >Vx8humL02 Full length LC
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIYYTSRLYSGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:71).
>Vx4humL02 Full length LC
DVVMTQSPLSLPVTLGQPASISCRSRQSIVHTNGNTYLGWFQQRPGQSPRRLIYKVSN RFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:72).
>Vx9humL07 Full length LC
DVVMTQSPLSLPVTLGQPASISCRSSQNIVQSNGNTYLEWFQQRPGQSPRRLIYKVFH RFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:73).
>Vx8humL01 Full length LC
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLYSGV PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPWTFGQGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:74).
>Vx8murL03 Full length LC
DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLYSGV PSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 100).
>Vx9mur_L04 Full length LC
DVFMTQTPLSLPVSLGDQASISCRSSQNIVQSNGNTYLEWYLQKPGQSPKLLIYKVFH
RFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKVEIKRTV
AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:75).
Chimera and Fluman Fleavv Chains
>Vx4murH01 Full length HC
EVQLQQSGPELVKPGASVKMSCKASGYTFTNYVIHWVKRRPGQGLEWIGYIYPYND
GILYNEKFKGKATVTSDKSSSTAYMDLSSLTSEDSAVYYCTRGGYYVPDYWGQGTTL
TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP
EFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT
KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP
QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:76).
>Vx4humF101 Full length F1C
QV QLV QSGAEVKKPGAS VQV SCKASGYTFTNYVIF1WLRQAPGQGLEWMGYIYPYN
DGILYNEKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGGYYVPDYWGQAT
LVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP
APEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA
KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR
EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID
NO:77).
> Vx8humFll 1 Full length F1C
QV QLV QSGAEVKKPGAS V QVSCKASGYSFTNYYIFIWLRQAPGQGLEWMGYIDPLN
GDTTYNQKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGGKRAMDYWGQAT
LVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP
APEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA
KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR
EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:78).
>Vx9humH12 Full length HC
QV QLV QSGAEVKKPGAS VKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGYTDPR
TDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGRVGLGYWGQ
GTLVTV SS ASTKGPS VFPLAPCSRSTSESTAALGCLVKDYFPEPVTV SWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECP
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVH
NAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPML
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID
NO:79).
>Vx9humH14 Full length HC
EVQFVQSGAEVKKPGESFKISCKGSGYTFTNYWIHWVRQMPGKGFEWMGYTDPRT
DYTEYNQKFKDQVTISADKSISTAYFQWSSFKASDTAMYYCARGGRVGFGYWGQG
TFVTVSSASTKGPSVFPFAPCSRSTSESTAAFGCFVKDYFPEPVTVSWNSGAFTSGVH
TFPAVFQSSGFYSFSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPP
CPAPPVAGPS VFFFPPKPKDTFMISRTPEVTC VVVDVSHEDPEV QFNWYVDGVEVHN
AKTKPREEQFNSTFRVVSVFTVVHQDWFNGKEYKCKVSNKGFPAPIEKTISKTKGQP
REPQVYTFPPSREEMTKNQVSFTCFVKGFYPSDIAVEWESNGQPENNYKTTPPMFDS
DGSFFFYSKFTVDKSRWQQGNVFSCSVMHEAFHNHYTQKSFSFSPGK (SEQ ID
NO:80).
>Vx9humH15 Full length HC
QV QFV QSGAEVKKPGSS VKV SCKASGYTFTNYWIHWVRQAPGQGFEWMGYTDPRT
DYTEYAQKFQGRVTITADESTSTAYMEFSSFRSEDTAVYYCARGGRVGFGYWGQGTF
VTVSS ASTKGPS VFPFAPCSRSTSESTAAFGCFVKDYFPEPVTV S WN SGAFTSGVHTF
PAVFQSSGFYSFSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCP
APPVAGPSVFFFPPKPKDTFMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNA
KTKPREEQFNSTFRVVSVFTVVHQDWFNGKEYKCKVSNKGFPAPIEKTISKTKGQPR
EPQVYTFPPSREEMTKNQVSFTCFVKGFYPSDIAVEWESNGQPENNYKTTPPMFDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:81).
>Vx4humH02 Full length HC
QV QLV QSGAEVKKPGAS V QVSCKASGYTFTNYVIHWLRQAPGQGLEWMGYIYPYN DGILYNEKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGGYYVYDYWGQATL VTVSS ASTKGPS VFPLAPCSRSTSESTAALGCLVKDYFPEPVTV S WN SGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA PEFEGGPS VFLFPPKPKDTLMISRTPEVTC VVVD V SQEDPEV QFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:82).
>Vx9humH13 Full length HC
QV QLV QSGAEVKKPGSS VKV SCKASGYTFTNYWIHWVRQAPGQGLEWMGYTDPRT
DYTEYNQKFKDRVTITADESTSTAYMELSSLRSEDTAVYYCARGGRVGLGYWGQGTL
VTVSS ASTKGPS VFPLAPCSRSTSESTAALGCLVKDYFPEPVTV S WN SGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCP
APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNA
KTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPR
EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID
NO:83).
>Vx8humH10 Full length HC
EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIHWVRQMPGKGLEWMGYIDPLNG DTTYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGKRAMDYWGRGTL VTVSS ASTKGPS VFPLAPCSRSTSESTAALGCLVKDYFPEPVTV S WN SGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA PEFEGGPS VFLFPPKPKDTLMISRTPEVTC VVVD V SQEDPEV QFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:84).
>Vx4humH04 Full length HC EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYVIHWVRQMPGKGLEWMGYIYPYND GILYNEKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGYYVPDYWGQGTT VTVSS ASTKGPS VFPLAPCSRSTSESTAALGCLVKDYFPEPVTV S WN SGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA PEFEGGPS VFLFPPKPKDTLMISRTPEVTC VVVD V SQEDPEV QFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:85).
>Vx4humH05 Full length HC
QV QLV QSGAEVKKPGAS VKVSCKASGYTFTNYVIHWVRQAPGQGLEWMGYIYPYN
DGILYNEKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGGYYVPDYWGQGT
TVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP
APEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA
KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR
EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID
NO:86).
>Vx9humH16 Full length HC
EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYWIHWVRQMPGKGLEWMGYTDPRT
DYTEYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGRVGLGYWGQGT
LVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPC
PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNA
KTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPR
EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID
NO:87).
>Vx8humH06 Full length HC
QV QLV QSGAEVKKPGAS VKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLN GDTTYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGKRAMDYWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC
PAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN
AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP
REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID
NO:88).
>Vx8humH07 Full length HC
QV QLV QSGAEVKKPGSS VKV SCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLN
GDTTYNQKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGKRAMDYWGQGT
LVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP
APEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA
KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR
EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID
NO:89).
>Vx8humH08 Full length HC
EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIHWVRQMPGKGLEWMGYIDPLNG
DTTYNQKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGKRAMDYWGQGT
LVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP
APEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA
KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR
EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID
NO:90).
>Vx8humH09 Full length HC
QV QLV QSGAEVKKPGSS VKV SCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLN GDTTYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGKRAMDYWGQGT LVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP
APEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA
KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR
EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID
NO:91).
>Vx8humH06 Full length HC
QV QLV QSGAEVKKPGAS VKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLN
GDTTYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGKRAMDYWGQG
TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC
PAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN
AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP
REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID
NO:92).
>Vx8mur-H03 Full length HC
EVQLQQSGPELMKPGASVKISCKASGYSFTNYYIHWVNQSHGKSLEWIGYIDPLNGD
TTYNQKFKGKATLTVDKSSSTAYMRLSSLTSADSAVYYCARGGKRAMDYWGQGTSV
TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP
EFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT
KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP
QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:93).
>Vx9mur-H04 Full length HC
QV QLQQFGAELAKPGAS V QMSCKASGYTFTNYWIHWVKQRPGQGLEWIGYTDPRT
DYTEYNQKFKDKATLAADRSSSTAYMRLSSLTSEDSAVYYCAGGGRVGLGYWGHGS
SVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPC
PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:94).
>Vx8humH06 Full length HC
QV QLV QSGAEVKKPGAS VKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLN
GDTTYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGKRAMDYWGQG
TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPP
CPAPPVAGPS VFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEV QFNWYVDGVEVHN
AKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQP
REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID
NO:95).
>Vx8humH07 Full length HC
QV QLV QSGAEVKKPGSS VKV SCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLN
GDTTYNQKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGKRAMDYWGQGT
LVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPC
PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNA
KTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPR
EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID
NO:96).
>Vx8humH08 Full length HC
EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIHWVRQMPGKGLEWMGYIDPLNG
DTTYNQKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGKRAMDYWGQGT
LVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPC
PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNA
KTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:97).
>Vx8humH09 Full length HC
QV QLV QSGAEVKKPGSS VKV SCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLN
GDTTYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGKRAMDYWGQGT
LVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPC
PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNA
KTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPR
EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID
NO:98).
>Vx4mur-ratL01 Full length LC
DVLMTQTPLSLPVNLGDQASISCRSRQSIVHTNGNTYLGWFLQKPGQSPKLLIYKVS NRFSGVPDRFSGSGSGTDFTLTISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK RADAAPTVSIFPPSMEQLTSGGATVVCFVNNFYPRDISVKWKIDGSEQRDGVLDSVT DQDSKDSTYSMSSTLSLTKVEYERHNLYTCEVVHKTSSSPVVKSFNRNEC (SEQ ID NO: 101).
>Vx4mur-ratH01 Full length HC
EVQLQQSGPELVKPGASVKMSCKASGYTFTNYVIHWVKRRPGQGLEWIGYIYPYND
GILYNEKFKGKATVTSDKSSSTAYMDLSSLTSEDSAVYYCTRGGYYVPDYWGQGTTL
TVSS
ARTTAPSVYPLVPGCSGTSGSLVTLGCLVKGYFPEPVTVKWNSGALSSGVHTFPAVL
QSGLYTLSSSVTVPSSTWSSQTVTCSVAHPATKSNLIKRIEPRRPKPRPPTDICSCDDN
LGRPSVFIFPPKPKDILMITLTPKVTCVVVDVSEEEPDVQFSWFVDNVRVFTAQTQPH
EEQLNGTFRVVSTLHIQHQDWMSGKEFKCKVNNKDLPSPIEKTISKPRGKARTPQVY
TIPPPREQMSKNKVSLTCMVTSFYPASISVEWERNGELEQDYKNTLPVLDSDESYFLY
SKLSVDTDSWMRGDIYTCSVVHEALHNHHTQKNLSRSPGK (SEQ ID NO: 102).
>Vx4mur-rabL01 Full length LC DVLMTQTPLSLPVNLGDQASISCRSRQSIVHTNGNTYLGWFLQKPGQSPKLLIYKVS NRFSGVPDRFSGSGSGTDFTLTISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK RDPV APT VLIFPP AAD Q V ATGT VTI V C V ANKYFPD VTVTWE VDGTTQTTGIEN S KTP QNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO: 103).
>Vx4mur-rabH01 Full length HC
EVQLQQSGPELVKPGASVKMSCKASGYTFTNYVIHWVKRRPGQGLEWIGYIYPYND
GILYNEKFKGKATVTSDKSSSTAYMDLSSLTSEDSAVYYCTRGGYYVPDYWGQGTTL
TVSS
GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVR QSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPS VFIFPPKPKDTLMISRTPE VTC V V VD V S QDDPEV QFTWYINNEQ VRT ARPPLREQQFN STIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPR EELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVP TSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 104).
Example 2
Production of CD47 Antibodies
[0360] Chimeric antibodies disclosed herein comprise a mouse heavy chain variable domain and a light chain variable domain combined with a human kappa or human Fc IgGl , IgGl-N297Q, IgG2, IgG4, IgG4 S228P, and IgG4 PE constant domains, respectively. These were designed to incorporate a secretion signal and cloned into a mammalian expression system, and transfected into CHO cells to generate chimeric (murine -human) antibodies. The chimeric variants were expressed as full length IgG molecules, secreted into the medium, and purified using protein A.
[0361] Multiple methods for humanizing antibodies are well-known to those of ordinary skill in the art. One such method, as used herein, has previously been described (Making and Using Antibodies a Practical Handbook, Second Edition, Ed. Matthew R. Kase, Chapter 15: Humanization of Antibodies, Juan Carlos Almagro et al., CRC Press 2013). As such, the humanized antibodies disclosed herein comprise frameworks derived from the human genome. The collection covers the diversity found in the human germ line sequences, yielding functionally expressed antibodies in vivo. The complementarity determining regions (CDRs) in the light and heavy chain variable regions of the murine and chimeric (murine -human) are described herein and were determined by following commonly accepted rules disclosed in “Protein Sequence and Structure Analysis of Antibody Variable Domains,” In: Antibody Engineering Lab Manual, eds. S. Duebel and R. Kontermann, Springer-Verlag, Heidelberg (2001)). The human light chain variable domains were then designed. The humanized variable domains were then combined with a secretion signal and human kappa and human Fc IgGl, IgGl-N297Q, IgG2, IgG3, IgG4 S228P and IgG4 PE constant domains, cloned into a mammalian expression system, and transfected into CHO cells to generate humanized m Ahs. The humanized variants were expressed as full length IgG molecules, secreted into the medium and purified using protein A.
[0362] A non-glycosylated version (IgGl-N297Q) was created by site directed mutagenesis of heavy chain position 297 to change the asparagine to glutamine (Human Fc IgGl-N297Q, SEQ ID NO:54). An IgG4 variant was created by site-directed mutagenesis at position 228 to change the serine to proline thereby preventing in vivo Fab arm exchange. An IgG4 double mutant was created by site-directed mutagenesis at positions 228 (serine to proline) and 235 (leucine to glutamate) to prevent Fab arm exchange and to further reduce Fc effector function. IgG2, IgG3, IgG4 S228P, and IgG4PE isotypes were constructed by cloning the heavy chain variable domain in frame with the human IgG2, IgG3, IgG4 S228P, and IgG4PE constant domains (Human Fc-IgG2, SEQ ID NO:56 Human Fc-IgG3, SEQ ID NO:57; Human Fc-IgG4 S228P, SEQ ID NO:59; and Human Fc-IgG4PE, SEQ ID NO:60).
Example 3
Binding of CD47 Monoclonal Antibodies im
Figure imgf000112_0001
[0363] The binding of chimeric (murine-human) and humanized antibodies of the present disclosure was determined by ELISA using OV10 cells transfected with human CD47 (OV10 hCD47) or using freshly isolated human red blood cells (hRBCs), which display CD47 on their surface (Kamel et al. (2010) Blood. Transfus. 8(4):260-266).
[0364] Binding activities of VLX4, VLX8, and VLX9 chimeric (xi) and humanized mAbs were determined using a cell-based ELISA assay with human OV10 hCD47cells expressing cell surface human CD47. OV10 hCD47 cells were grown in IMDM medium containing 10% heat inactivated fetal bovine serum (BioWest; S01520). One day before assay, 3xl04 cells were plated in 96 well cell bind plates (Corning #3300, VWR #66025-626) and were 95-100% confluent at the time of assay. Cells were washed, various concentrations of purified antibodies added in IMDM and incubated at 37°C for 1 hr in 95%02 / 5%C02- Cells were then washed with media and incubated for an additional hour at 37°C with HRP labelled secondary anti human antibody (Promega) diluted 1/2500 in media. Cells were washed three times with PBS, and the peroxidase substrate 3,3’, 5,5’-tetramethylbenzidine was added (Sigma; Catalog #T4444). Reactions were terminated by the addition of HC1 to 0.7N, and absorbance at 450nM determined using a Tecan model Infinite M200 plate reader. The apparent binding affinities of these clones to human OVIO hCD47 cells was determined by non-linear fit (Prism GraphPad software).
[0365] Binding activities of chimeric and humanized VLX4, VLX8, and VLX9 mAbs to human CD47 on hRBCs were also determined using flow cytometry. Blood was obtained from normal volunteers and RBCs were washed 3 times with phosphate buffered saline, pH 7.2 containing 2.5 mM EDTA (PBS+E). hRBCs were incubated for 60 min at 37°C with various concentrations of the chimeric or humanized antibodies in a PBS+E. Cells were then washed with cold PBS+E and incubated for an additional hour on ice with FITC labelled donkey anti human antibody (Jackson Tmmiino Research Labs, West Grove, PA; Catalogue # 709-096-149) in PBS +E. Cells were washed with PBS+E, antibody binding was analyzed using a C6 Accuri Flow Cytometer (Becton Dickinson) and apparent binding affinities determined by non-linear fit (Prism GraphPad software) of the median fluorescence intensities at the various antibody concentrations.
[0366] All of the VLX4 chimeric (murine -human) mAbs bound to human OVIO hCD47 tumor cells with apparent affinities in the picomolar (pM) range (Table 1).
[0367] Similarly, the humanized VLX4 mAbs bound to human OVIO hCD47 tumor cells in a concentration-dependent manner (FIG. 1A and FIG. IB) with apparent binding affinities ranging from the picomolar to low nanomolar range (Table 2).
[0368] All of the chimeric VLX4 mAbs bound to human RBCs with apparent Kd values in the picomolar range and these were similar to the Kd values obtained for OVIO hCD47 tumor cells by ELISA (Table 1).
[0369] The humanized VLX4 mAbs VLX4hum_01 IgGl N297Q, VLX4hum_02 IgGl N297Q, VLX4hum_01 IgG4PE, VLX4hum_02 IgG4PE, VLX4hum_12 IgG4PE, and VLX4hum_13 IgG4PE bound to human RBCs with Kd values similar to those obtained for OVIO hCD47 tumor cells whereas VLX4hum_06 IgG4PE and VLX4hum_07 IgG4 PE exhibited reduced binding to hRBCs (FIG.2A, FIG.2B, and Table 2). This differential binding of the humanized mAbs to tumor cells and RBCs was unexpected as the VLX4 IgG4PE chimeric mAh bound with similar apparent Kd values to both tumor and RBC CD47 (Table 1). [0370] As shown in Table 1, all the VLX8 chimeric m Ahs bound to human OVIO hCD47 tumor cells in a concentration-dependent manner with apparent affinities in the picomolar (pM) range.
[0371] Similarly, the humanized VLX8 mAbs bound to human OVIO hCD47 tumor cells in a concentration-dependent manner (FIG. 3A and FIG. 3B) with apparent affinities in the picomolar range (Table 2).
[0372] All the VLX8 chimeric m Ahs bound to hRBCs with apparent Kd values in the picomolar range and these were similar to the apparent K values obtained for OVIO hCD47 tumor cells by ELISA (Table 1).
[0373] The VLX8 humanized m Ahs VLX8hum_01 IgG4PE, VLX8hum_02 IgG4PE, VLX8hum_03 IgG4PE, VLX8hum_04 IgG4PE, VLX8hum_05 IgG4 PE, and VLX8hum_06 IgG4PE, VLX8hum_07 IgG4PE, VLX8hum_08 IgG4 PE, VLX8hum_09 IgG4 PE, VLX8hum_l l IgG4 PE, VLX8hum_06 IgG2, VLX8hum_07 IgG2, VLX8hum_08 and VLX8hum_09 IgG2 IgG2 bound to human RBCs with Kd values similar to the values obtained for OVIO hCD47 tumor cells whereas VLX8hum_10 IgG4PE exhibited reduced to hRBCs (FIG. 4A, FIG. 4B, and Table 2). This differential binding of the humanized m Ahs to tumor cells and RBCs was unexpected as the VLX8 IgG4PE chimeric mAh bound with similar apparent Kd values to both tumor and RBC CD47 (Table 1).
[0374] Table 1 shows the apparent binding affinities of VLX9 chimeric m Ahs to human OVIO hCD47 cells and to human RBCs. All of the chimeric m Ahs bound to OVIO hCD47 tumor cells with apparent binding constants in the picomolar range. Similarly, the humanized VLX9 m Ahs bound to human OVIO hCD47 tumor cells in a concentration-dependent manner (FIG. 5A and FIG. 5B) with apparent affinities in the picomolar to nanomolar range (Table 2).
[0375] All the VLX9 chimeric m Ahs bound to hRBCs with apparent Kd values in the picomolar range and these were similar to the apparent Kd values obtained for OVIO hCD47 tumor cells by ELISA (Table 1). In contrast to the chimeric mAbs, the VLX9 humanized m Ahs VLX9hum_01 IgG2, VLX9hum_02 IgG2 and VLX9hum_07 IgG2 exhibited reduced binding to human RBCs (FIG. 7, Table 2). By contrast, the humanized mAbs VLX9hum_03 IgG2, VLX9hum_04 IgG2, VLX9hum_05 IgG2, VLX9hum_06 IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2 and VLX9hum_10 IgG2 exhibited no measureable binding to RBCs up to 5,000 pM (Table 2). This differential binding of the humanized m Ahs to tumor cells and RBCs was unexpected as the VLX9 IgG2 chimeric m Ahs all bound with similar apparent Kd values to both tumor and RBC CD47 (Table 1). [0376] Specific binding of CD47 humanized mAbs was demonstrated using Jurkat wildtype and Jurkat CD47 knockout (KO) cells. Jurkat wildtype and Jurkat CD47 KO cells were grown in RPMI medium containing 10% heat inactivated fetal bovine serum (BioWest; S01520). The cells were washed and lxlO4 cells were resuspended media and incubated with various antibody concentrations for one hour at 37° in 5% CO2. Cells were then washed twice with lx PBS and then resuspended 1: 1000 in secondary antibody (goat anti-human IgG (H+L) FITC-labelled, Jackson Labs, 109-095-003) for one hour at 37°in 5% CO2. Cells were then washed twice with lx PBS and resuspended in lx PBS. Median fluorescence intensity was determined by flow cytometry and the apparent binding affinities determined by non-linear fit (Prism GraphPad software).
[0377] As shown in FIG. 6, VLX4hum_07 IgG4PE (FIG. 6A) and VLX9hum_09 IgG2 (FIG. 6B) bound to Jurkat cells expressing CD47, whereas no binding is observed to Jurkat
CD47KO cells.
Table 1. Binding of VLX4, VLX8, and VLX9 Chimeric (xi) mAbs to OV10 hCD47 Cells and Human Red Blood Cells (hRBCs).
Figure imgf000115_0001
Table 2. Binding of VLX4, VLX8, and VLX9 Humanized mAbs to Human OVIO hCD47 and
Human Red Blood Cells (hRBCs).
Figure imgf000116_0001
Figure imgf000117_0001
*MB - Minimal biniding; no measurable binding detected at mAb concentration up to 5,000 pM. ** - Reduced RBC binding.
***/? - Reduced hemagglutination.
[0378] Cross-species binding of humanized VLX4, VLX8, and VLX9 m Ahs was determined using flow cytometry. Mouse, rat, rabbit or cynomolgus monkey RBCs were incubated for 60 min on at 37°C with various concentrations of the humanized antibodies in a solution of phosphate buffered saline, pH 7.2, 2.5 mM EDTA (PBS+E). Cells were then washed with cold PBS+E, and incubated for an additional hr on ice with FITC labelled donkey anti human antibody (Jackson Immuno Research Labs, West Grove, PA; Catalogue # 709-096-149) in PBS +E. Cells were washed with PBS+E, and antibody binding analyzed using a C6 Accuri Flow Cytometer (Becton Dickinson).
[0379] Table 3 shows the apparent binding affinities of the humanized VLX4 and VLX8 m Ahs to RBCs from mouse, rat, and cynomolgus monkey determined by non-linear fit (Prism GraphPad software) of the median fluorescence intensities at various antibody concentrations. This data demonstrates that humanized VLX4 and VLX8 m Ahs bind to mouse, rat, rabbit (data not shown) and cynomolgus monkey RBCs with apparent Kd values in the picomolar to nanomolar range.
Table 3. Binding of VLX4 and VLX8 Humanized m Ahs to Mouse, Rat and Cynomolgus Monkey RBCs.
Figure imgf000118_0001
Example 4
Binding of Humanized Anti-CD47 mAhs Determined by Surface Plasmon Resonance
[0380] Binding of soluble anti-CD47 mAhs to recombinant human His-CD47 was measured in vitro by surface plasmon resonance on a Biacore 2000. An Anti-Human IgG (GE Lifesciences) was amine coupled to a CM5 chip on flow cells 1 and 2. The humanized mAhs VLX4hum_07 IgG4PE, VLX8hum_l l IgG4PE, VLX9hum_08 IgG2 or VLX9hum_03 IgG2 diluted in HBS-EP+ running buffer (pH 7.2) were captured onto flow cell 2. Multi-cycle kinetics were determined using 0 to lOOOnM His-tagged human CD47 (Aero Biosystems) diluted in HBS-EP+ running buffer (pH 7.2) with contact time of 180 seconds and dissociation time of 300 seconds. A 1 : 1 binding model was employed for kinetic analysis of binding curves. The on-rate, off-rate and Dissociation constants for VLX4hum_07 IgG4PE, VLX8hum_l l IgG4PE, VLX9hum_08 IgG2 and VLX9hum_03 IgG2 are shown in Table 4.
Table 4. Binding of VLX4, VLX8 and VLX9 Humanized mAhs to Human Recombinant His-
CD47 by Surface Plasmon Resonance at pH 7.2.
Figure imgf000118_0002
Example 5
Differential Binding of Anti-GD47 mAhs
[0381] Some soluble CD47 antibodies described herein have been shown to differentially bind to normal cells. This additional property of selective binding is expected to have advantages compared to mAbs that bind with equal affinity to normal and tumor cells. Anti- CD47 mAbs with such reduced binding have not been described.
[0382] Binding by soluble anti-CD47 mAbs is measured in vitro. Binding activities of VLX4, VLX8, and VLX9 humanized mAbs were determined using a flow cytometry based binding assay with human aortic endothelial cells (HAEC), skeletal muscle cells (SkMC), human lung microvascular endothelial cells (HMVEC-L), renal tubular epithelial cells (RTEC), CD3+ cells or peripheral blood mononuclear cells (PBMC). HAEC, SkMC, HMVEC- L and RTEC cells were purchased from Lonza and cultured according to the manufacturer’s recommendations. Adherent cells were removed from the culture flask with accutase, resuspended in the recommended media and lxlO4 cells were incubated with various antibody concentrations for one hour at 37°, 5% CO2. For non-adherent cells, lxlO4 cells were resuspended in the recommended media and incubated with various antibody concentrations for one hour at 37°, 5% CO2. Cells were then washed twice with lx PBS and then resuspended 1 : 1000 in secondary antibody (goat anti-human IgG (H+L) - FITC, Jackson Labs, 109-095- 003) for one hour at 37°C, 5% CO2.
[0383] PBMC were isolated by ficoll gradient and were incubated with an FcR blocking reagent (Miltenyi Biotec) for 10 min at 4°C per manufacturer’s recommendation immediately preceding the addition of various concentrations of antibodies diluted in PBS. CD3 cells were detected using an ahophycocyanin (APC) -labelled anti-CD3 antibody (BD BioSciences) which was added at the same time as the FITC-labehed goat anti-human IgG (H+L) antibody. Cells were washed twice with lxPBS and antibody binding was assessed by flow cytometry analysis.
[0384] As shown in FIG. 8A, VLX4 and VLX8 humanized mAbs bound to HAEC cells whereas VLX9 humanized mAbs had reduced or minimal binding to HAEC cells as compared to tumor cells (Table 5). VLX9 humanized mAbs also showed reduced binding to SkMC cells (FIG. 8B), reduced or minimal binding to HMVEC-L cells (FIG. 8C), reduced binding to RPTEC cells (FIG. 8D) as compared to binding to tumor cells (Table 5). Reduced binding of VLX9 humanized mAbs was also observed to CD3+ cells (FIG. 8E) and PBMC (FIG. 8F) as compared to tumor cells (Table 5). This selective binding imparts an additional desirable antibody property and potential therapeutic benefit in the treatment of cancer. Table 5. VLX4, VLX8 and VLX9 Humanized mAbs Binding to Normal Cells.
Figure imgf000120_0001
*MB - Minimal binding, no measureable binding detected at mAb concentration up to 5,000pM.
Reduced binding.
Example 6
pH Dependent and Independent Binding of Humanized Anti-CD47 mAbs
[0385] Some soluble anti-CD47 mAbs described herein have been shown to bind tumor cells at acidic pH with greater affinity compared to physiologic pH. This additional property is expected to have advantages compared to mAbs that bind at similar affinities to CD47 at both acidic and physiologic pH, in part due to the acidic nature of the tumor microenvironment (Tannock and Rotin, Cancer Res 1989; Song et al. Cancer Drug Discovery and Development 2006; Chen and Pagel, Advan Radiol 2015).
[0386] Binding by soluble anti-CD47 mAbs to immobilized recombinant human CD47 and to human CD47 expressed on cells was measured in vitro. For the in vitro binding to recombinant CD47, His-CD47 (AcroBiosy stems) was adsorbed to high-binding microtiter plates overnight at 4°C. The wells were washed and varying concentrations of anti-CD47 mAbs were added to the wells in buffers with a of either pH 6 or pH 8 for 1 hour. The wells were washed and then incubated with HRP-labelled secondary antibody for 1 hour at pH 6 or pH 8 followed by addition of peroxidase substrate. The apparent affinities were calculated using non-linear fit model (Graphpad Prism).
[0387] For analysis of pH dependent binding by surface plasmon resonance using a Biacore 2000, an Anti-Human IgG (GE Lifesciences) was amine coupled to a CM5 chip on flow cells 1 and 2. An Fc-tagged human CD47 (Aero Biosystems) was diluted in PBS-EP+ running buffer (pH 7.5, 6.5 or 6.0) and captured onto flow cell 2. Multi-cycle kinetics were determined using 0 to lOOnM VLX8hum_l l Fab or VLX9hum_08 Fab diluted in PBS-EP+ running buffer (pH 7.5, 6.5 or 6.0) with contact time of 180 seconds and dissociation time of 300 seconds. A 1: 1 binding model was employed for kinetic analysis of binding curves.
[0388] For the in vitro binding to cells expressing CD47, Jurkat cells were grown in RPMI medium containing 10% heat inactivated fetal bovine serum (BioWest; S01520). The cells were washed and lxlO4 cells were resuspended in PBS supplementated with 2% FBS at either pH 7.4 or 6.5 and incubated with various antibody concentrations for 1 hour at 37°C. Cells were then washed twice and resuspended with 1 : 1000 of secondary antibody (goat anti-human IgG (H+L) labelled with AIexa488, Jacksonlmmunoresearch) for 1 hour at 37°C at pH 6 or pH 8. Cells were then washed twice and the median fluorescence intensity was determined by flow cytometry. The apparent binding affinities were determined by non-linear fit (Prism GraphPad software).
[0389] As shown in FIG. 9A and FIG. 9B, the soluble VLX9 humanized mAbs (VLX9hum_09 IgG2 and VLX9hum_04 IgG2) bound to His-CD47 with greater affinity at the more acidic pH 6.0 than at pH 8.0. Neither VLX4hum_07 IgG4PE (FIG. 9C) nor VLX8hum_10 IgG4PE (FIG. 9D) displayed pH dependent binding. In addition, the murine VLX9 antibody and VLX9 chimeric antibodies containing human Fc from isoytpes IgGl, IgG2 and IgG4PE did not display pH dependence (Table 6) whereas VLX9hum_04 as either an IgGl , IgG2 or an IgG4PE demonstrated greater binding to His-CD47 at acidic pH (Table 7). The apparent binding affinities for additional humanized mAbs to recombinant human CD47 are shown in Table 8. All humanized VLX9 mAbs exhibited pH dependent binding whereas the VLX4 and VLX8 humanized mAbs did not. To determine the effect of pH on on-rates, off- rates and dissociation constants, Biacore analysis was performed for humanized mAbs VLX8hum_l l Fab fragment and VLX9hum_08 Fab at pH 6, pH 6.5 and pH 7.5. The VLX9hum_08 Fab exhibited pH dependent binding that increased with decreasing pH wheras the VLX8hum_l l Fab did not. The on-rate, off-rate and dissociation constants for VLX8hum_l l Fab and VLX9hum_08 Fab are shown in Table 9. Table 10 illustrates the pFl dependent binding exhibited by VLX9hum_04 IgG2 to CD47 expressed on Jurkat cells. No pFl dependent binding was exhibited by VLX4hum_07 IgG4PE. This pFl dependence of the VLX9 humanized mAbs imparts an additional desirable antibody property and therapeutic benefit in the treatment of cancer.
Table 6. Murine VLX9 and mouse-human chimeric VLX9 Binding to CD47 is not pFl
Figure imgf000122_0003
Table 7. VLX9hum_04 Flumanized mAbs Bind to CD47 in a pH Dependent Manner and
Figure imgf000122_0001
Table 8. pH Dependent and Independent Binding of VLX4, VLX8 and VLX9 Humanized mAbs.
Figure imgf000122_0002
Figure imgf000123_0001
Table 9. pH Independent and Dependent Binding of VLX8hum_l 1 Fab and VLX9hum_08 Fab to Recombinant Human CD47.
Figure imgf000123_0002
Table 10. pH Dependent and Independent Binding of VLX4 and VLX9 Humanized m Ahs to
Jurkat Cells.
Figure imgf000123_0003
Example 7
CD47 Antibodies Block CD47/SIRP0C binding
[0390] To assess the effect of humanized CD47 m Ahs on binding of CD47 to SIRPoc in vitro the following method is employed using the binding of fluorescently-labelled SIRPoc-Fc fusion protein to CD47 expressing Jurkat cells.
[0391] SIRPoc-Fc fusion protein (R&D Systems, cat #4546-SA) was labelled using an Alexa Fluor® antibody labelling kit (Invitrogen Cat No. A20186) according to the manufacturers specifications. 1.5 x 106 Jurkat cells were incubated with humanized mAbs (5pg/ml), a human control antibody in RPMI containing 10% media or media alone for 30 min at 37°C. An equal volume of fluorescently labelled SIRPoc-Fc fusion protein was added and incubated for an additional 30 min at 37°C. Cells were washed once with PBS and the amount of labelled SIRPoc-Fc bound to the Jurkat cells analyzed by flow cytometry.
[0392] As shown in FIG. 10, the humanized VLX4, VLX8 and VLX9 m Ahs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_l l IgG4PE, VLX9hum_03 IgG2, VLX9hum_06 IgG2 and VLX9hum_08 IgG2) blocked the interaction of CD47 expressed on the Jurkat cells with soluble SIPRoc, while the human control antibody (which does not bind to CD47) or media alone, did not block the CD47/SIRP0C interaction.
Example 8
CD47 Antibodies Increase Phagocytosis
[0393] To assess the effect of chimeric (murine-human) and humanized VLX4, VLX8, and VLX9 CD47 mAbs on phagocytosis of tumor cells by macrophages in vitro the following method is employed using flow cytometry (Willingham et al. (2012) Proc Natl Acad Sci USA 109(17):6662-7 and Tseng et al. (2013) Proc Natl Acad Sci U S A 110(27): 11103-8).
[0394] Human derived macrophages were derived from leukapheresis of healthy human peripheral blood and incubated in AIM-V media (Life Technologies) for 7-10 days. For the in vitro phagocytosis assay, macrophages were re -plated at a concentration of lxlO4 cells per well in 100 ul of AIM-V media in a 96-well plate and allowed to adhere for 24 hrs. Once the effector macrophages adhered to the culture dish, the target human cancer cells (Jurkat) were labelled with ImM 5(6)-Carboxyfluorescein diacetate /V-succinimidyl ester (CFSE; Sigma Aldrich) and added to the macrophage cultures at a concentration of 5xl04 cells in 1ml of AIM-V media (5: 1 target to effector ratio). VLX4, VLX8, and VLX9 CD47 mAbs (1 pg/ml) were added immediately upon mixture of target and effector cells and allowed to incubate at 37°C for 2-3 hours. After 2-3 hrs, all non-phagocytosed cells were removed and the remaining cells washed three times with phosphate buffered saline (PBS; Sigma Aldrich). Cells were then trypsinized, collected into microcentrifuge tubes, and incubated in lOOng of allophycocyanin (APC) labelled CD14 antibodies (BD Biosciences) for 30 minutes, washed once, and analyzed by flow cytometry (Accuri C6; BD Biosciences) for the percentage of CD14+ cells that were also CFSE+ indicating complete phagocytosis.
[0395] As shown in FIG. 11, the VLX4 chimeric mAbs VLX4 IgGl xi, VLX4 IgGl N297Q xi, VLX4 IgG4PE xi, and VLX4 IgG4 S228P xi increased phagocytosis of Jurkat cells by human macrophages by blocking the CD47/SIRP0C interaction. This enhanced phagocytosis is independent of Fc function.
[0396] Similarly, as shown in FIG. 12A and FIG. 12B, humanized mAbs VLX4hum_01 IgGl, VLX4hum_01 IgG4PE, VLX4hum_06 IgG4PE, VLX4hum_07 IgG4PE, VLX4hum_12 IgG4PE, and VLX4hum_13 IgG4PE increased phagocytosis of Jurkat cells by human macrophages by blocking the CD47/SIRP0C interaction. This enhanced phagocytosis is independent of Fc function.
[0397] As shown in FIG. 13A, the VLX8 chimeric mAbs VLX8 IgGl N297Q xi and VLX8 IgG4PE xi increase phagocytosis of Jurkat cells by human macrophages by blocking the CD47/SIRP0C interaction. This enhanced phagocytosis is independent of Fc function.
[0398] Similarly, as shown in FIG. 13B, humanized mAbs VLX8hum_01 IgG4PE, VLX8hum_03 IgG4PE, VLX8hum_07 IgG4PE, VLX8hum_08 IgG4PE, and VLX8hum_09 IgG4PE and chimeric mAh VLX8 IgG4PE xi increased phagocytosis of Jurkat cells by human macrophage by blocking the CD47/SIRP0C interaction.
[0399] As shown in FIG. 14A, the VLX9 IgGl N297Q xi, VLX9 IgG2 xi and VLX9 IgG4PE xi chimeric mAbs all increased phagocytosis of Jurkat cells by human macrophages by blocking the CD47/SIRP0C interaction. This enhanced phagocytosis is independent of Fc effector function. Similarly as shown in FIG. 14B, all of the humanized VLX9 IgG2 mAbs (VLX9hum_01 to _10 IgG2) increased phagocytosis of Jurkat cells.
Example 9
Induction of Cell Death by Soluble CD47 Antibodies
[0400] Some soluble CD47 antibodies have been shown to induce selective cell death of tumor cells. This additional property of selective toxicity to cancer cells is expected to have advantages compared to mAbs that only block SIRPoc binding to CD47. [0401] Induction of cell death by soluble anti-CD47 mAbs is measured in vitro (Manna et al. (2003) J. Immunol. 107 (7): 3544-53). For the in vitro cell death assay, lxlO5 transformed human T cells (Jurkat cells) were incubated with soluble humanized VLX4, VLX8, and VLX9 CD47 mAbs ( 1 pg/ml ) for 24 hrs at 37°C. As cell death occurs, mitochondrial membrane potential is decreased, the inner leaflet of the cell membrane is inverted, exposing phosphatidylserines (PS), and propidium iodide (PI) or 7-aminoactinomycin D (7-AAD) is able to incorporate into nuclear DNA. In order to detect these cellular changes, cells were then stained with fluorescently labelled annexin V and PI or 7-aminoactinomycin D (7-AAD) (BD Biosciences) and the signal detected using an Accuri C6 flow cytometer (BD Biosciences). The increase in PS exposure is determined by measuring the percent increase in annexin V signal and the percent of dead cells by measuring the percent increase in PI or 7-AAD signal. Annexin V positive (annexin V+) or annexin V positive/7- AAD negative (annexin VV7-AAD ) cells are observed in early stages of cell death and annexin V positive/7- A AD positive (annexin VV7- AAD+) cells are dead cells. Importantly for therapeutic purposes, these mAbs induce cell death of tumor cells directly and do not require complement or the intervention of other cells (e.g. , NK cells, T cells, or macrophages) to kill. Thus, the mechanism is independent of both other cells and of Fc effector function. Therefore, therapeutic antibodies developed from these mAbs can be engineered to reduce Fc effector functions such as ADCC and CDC and thereby limit the potential for side effects common to humanized mAbs with intact Fc effector functions.
[0402] As shown in FIG. 15A - FIG. 15F, the soluble VLX4 humanized mAbs induced increased PS exposure and cell death of Jurkat cells as measured by increased % of the cells that are annexin V+ (FIG. 15A and FIG. 15D), annexin VV7-AAD (FIG. 15B and FIG. 15E), or annexin V77-AAD+ (FIG. 15C and FIG. 15F). The humanized mAbs VLX4hum_01 IgGl, VLX4hum_01 IgG4PE, VLX4hum_02 IgGl, VLX4hum_02 IgG4PE, VLX4hum_06 IgG4 PE, VLX4hum_07 IgG4PE, VLX4hum_12 IgG4PE, and VLX4hum_13 IgG4PE caused increased PS exposure and cell death. In contrast, the humanized mAbs VLX4hum_08 IgG4PE and VLX4hum_l 1 IgG4PE did not cause increased PS exposure and cell death of Jurkat cells. Induction of cell death and the promotion of phagocytosis of susceptible cancer cells imparts an additional desirable antibody property and potential therapeutic benefit in the treatment of cancer.
[0403] As shown in FIG. 16A - FIG. 16F, the soluble VLX8 chimeric and humani ed mAbs induced increased PS exposure and cell death of Jurkat cells as measured by the % of the cells that are annexin V+ (FIGs. 16A, 16D), annexin VV7-AAD (FIGs. 16B, 16E), or annexin VV7-AAD+ (FIGs. 16C, 16F). The chimeric mAbs, VLX8 IgGl N297Q xi and VLX8 IgG4PE xi, and the humanized mAbs, VLX8hum_07 IgG4PE and VLX8hum_08 IgG4PE, induced increased PS exposure and cell death of Jurkat cells. In contrast, the humanized mAbs VLX8hum_02 IgG4PE and VLX8hum_04 IgG4PE did not cause increased PS exposure and cell death of Jurkat cells. Induction of cell death and the promotion of phagocytosis of susceptible cancer cells imparts an additional desirable antibody property and potential therapeutic benefit in the treatment of cancer.
[0404] As shown in FIG. 17A - FIG. 17F, the soluble VLX9 chimeric and humani ed antibodies induced increased PS exposure and cell death of Jurkat cells as measured by % of the cells that are annexin V+ (FIG. 17A and FIG. 17D), annexin V77-AAD (FIG. 17B and FIG. 17E), or annexin V+/7-AAD+ (FIG. 17C and FIG. 17F). The chimeric VLX9 IgG2xi mAh and the humanized mAbs VLX9hum_06 IgG2, VLX9hum_07 IgG2, VLX9hum_08 IgG2, and VLX9hum_09 IgG2 induced increased PS exposure and cell death of Jurkat cells. In contrast, the humanized mAbs VLX9hum_01 IgG2, VLX9hum_02 IgG2, VLX9hum_03 IgG2, VLX9hum_04 IgG2, VLX9hum_05 IgG2 and VLX9hum_010 IgG2 did not cause increased PS exposure and cell death of Jurkat cells. Induction of cell death and the promotion of phagocytosis of susceptible cancer cells imparts an additional desirable antibody property and potential therapeutic benefit in the treatment of cancer. Importantly, chimeric and humanized mAbs that cause cell death of tumor cells do not cause cell death of normal cells.
Example 10
Damage- Associated Molecular Pattern (DAMP) Expression and Release, Mitochondrial Depolarization and Cell Death Caused by Humanized Anti-CD47 mAh Humanized Anti-CD47 mAbs Cause Loss of Mitochondrial Membrane Potential
[0405] These experiments demonstrate that humanized anti-CD47 mAbs of the present disclosure exhibit the ability to induce the loss of mitochondrial membrane potential in tumor cell as described previously (Manna and Frazier, 2014 Journal of Immunology 170(7):3544- 3553).
[0406] Loss of mitochondrial membrane potential in the tumor cell was determined using JC-1 dye (Thermo; Catalogue #M34152). Human Raji lymphoma cells (ATCC, Manassas, VA; Catalog # CCL-86) or other cells types that express sufficient levels of CD47 will be used. Cells were grown in RPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovine serum (BioWest; Catalogue # S01520), 100 units/mL penicillin, 100 pg mL streptomycin (Sigma; Catalogue # P4222) at densities less than 1 x 106 cells/mL. For this assay, Raji cells were plated in 96 well tissue culture plates at a density of lxlO5 cells/ml RPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovine serum (BioWest; Catalog # S01520), 100 units/mL penicillin, 100 mg/mL streptomycin (Sigma; #P4222).
[0407] The humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l 1 IgG4PE, VLX9hum_06 IgG2 VLX9hum_08 IgG2 and VLX9hum_03 IgG2) as disclosed herein, purified from transient transfections in CHO cells as described above, as well as the control chimeric antibody, were added at a final concentration of 10 pg/ml. As a positive control for loss of mitochondrial membrane potential, cells were treated with 1 mM of chemotherapeutic anthracycline mitoxantrone. The cells were incubated at 37°C for 24 hours, after which the cells were harvested, washed twice with PBS, and incubated for 30 minutes with JC-1 dye as described above, diluted 1 :2000 in PBS. After 30 minutes the cells were washed twice with PBS, resuspended in 100 pi of PBS, and analyzed for the percent of cells that shift their fluorescence emission from red to green by flow cytometry (Accuri C6, Becton Dickinson, Franklin Lakes, NJ). Results are presented as means ± SEM and analyzed for statistical significance using ANOVA in GraphPad Prism 6.
[0408] Some of the chimeric or humanized antibodies induce the loss of mitochondrial membrane potential in the tumor cell. As shown in FIG. 18, the percent of cells with mitochondrial membrane depolarization in all anti-CD47 mAh treated cultures was significantly increased (p < 0.05) compared to an isotype control. This increase in the amount of mitochondrial membrane depolarization demonstrates that anti-CD47 chimeric or humanized antibodies induce mitochondrial depolarization that leads to cell death in human tumor cells.
Humanized Anti-CD47 mAbs Cause Increase in Cell Surface Calreticulin Expression
[0409] These experiments demonstrate that humanized anti-CD47 mAbs of the present disclosure exhibit the ability to expose the endoplasmic reticulum resident chaperone calreticulin on the surface of the tumor cell as, for example, described previously using chemotherapeutic anthracyclines such as doxorubicin and mitoxantrone, as disclosed by Obeid et al. (2007) Nat. Med. 13(1):54-61.
[0410] Cell surface exposure of calreticulin was determined using a rabbit monoclonal antibody against calreticulin conjugated to Alexa Fluor 647 (Abeam; Catalogue #abl96159). Human Raji lymphoma cells (ATCC, Manassas, VA; Catalog # CCL-86) or other cells types that express sufficient levels of CD47 will be used. Cells were grown in RPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovine serum (BioWest; Catalogue # S01520), 100 units/mL penicillin, 100 pg mL streptomycin (Sigma; Catalogue # P4222) at densities less than 1 x 106 cells/mL. For this assay, cells were plated in 96 well tissue culture plates at a density of lxlO5 cells/ml RPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovine serum (BioWest; Catalog # S01520), 100 units/mL penicillin, 100 mg/mL streptomycin (Sigma; #P4222).
[0411] The humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l 1 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 VLX9hum_03 IgG2) as disclosed herein, purified from transient transfections in CHO cells as described above, as well as the control chimeric antibody, were added at a final concentration of 10 pg/ml. As a positive control for calreticulin exposure, cells were treated with 1 mM of chemotherapeutic anthracycline mitoxantrone. The cells were incubated at 37°C for 24 hours, after which the cells were harvested, washed twice with PBS, and incubated for 30 minutes with anti- calreticulin antibody as described above, diluted 1 :200 in PBS. After 30 minutes the cells were washed twice with PBS, resuspended in 100 pi of PBS, and analyzed for the mean fluorescence intensity of the anti -calreticulin antibody signal as well as the percent of cells that stain positive for cell surface calreticulin by flow cytometry (Accuri C6, Becton Dickinson, Franklin Lakes, NJ). Results are presented as means ± SEM and analyzed for statistical significance using ANOVA in GraphPad Prism 6.
[0412] As shown in FIG. 19, the humanized antibodies induced the preapoptotic exposure of calreticulin on the tumor cell surface. The percent of calreticulin positive cells in all anti- CD47 mAh treated cultures was significantly increased (p < 0.05) compared to an isotype control. This increase in the exposure of calreticulin on the cell surface demonstrates that some of the humanized antibodies induce DAMPs from tumor cells that can lead to phagocytosis of tumor cells and processing of tumor antigen by innate immune cells.
Humanized Anti-CD47 mAbs Cause Increased Protein Disulfide-Isomerase 3 (PDIA3) Expression
[0413] These experiments demonstrate that humanized anti-CD47 mAbs of the present disclosure exhibit the ability to expose the endoplasmic reticulum resident chaperone PDIA3 on the surface of the tumor cell as, for example, described previously using chemotherapeutic anthracyclines such as doxorubicin and mitoxantrone, as disclosed by Panaretakis et al. (2008) Cell Death & Differentiation 15: 1499-1509.
[0414] Cell surface exposure of PDIA3 was determined using a mouse monoclonal antibody against PDIA3 conjugated to FITC (Abeam; Catalogue #ab 183396). Human Raji lymphoma cells (ATCC, Manassas, VA; Catalog # CCL-86) or other cells types that express sufficient levels of CD47 will be used. Cells were grown in RPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovine serum (BioWest; Catalogue # S01520), 100 units/mL penicillin, 100 pg mL streptomycin (Sigma; Catalogue # P4222) at densities less than 1 x 106 cells/mL. For this assay, cells were plated in 96 well tissue culture plates at a density of lxlO5 cells/ml RPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovine serum (BioWest; Catalog # S01520), 100 units/mL penicillin, 100 pg/mL streptomycin (Sigma; #P4222).
[0415] The humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l 1 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2) as disclosed herein, purified from transient transfections in CHO cells as described above, as well as the control chimeric antibody, were added at a final concentration of 10 pg/ml. As a positive control for PDIA3 exposure, cells were treated with 1 pM of chemotherapeutic anthracycline mitoxantrone. The Raji cells were incubated at 37°C for 24 hours, after which the cells were harvested, washed twice with PBS, and incubated for 30 minutes with anti- PDIA3 antibody as described above, diluted 1 :200 in PBS. After 30 minutes the cells were washed twice with PBS, resuspended in 100 pi of PBS, and analyzed for the mean fluorescence intensity of the anti-PDIA3 antibody signal as well as the percent of cells that stain positive for cell surface calreticulin by flow cytometry (Accuri C6, Becton Dickinson, Franklin Lakes, NJ). Results are presented as means ± SEM and analyzed for statistical significance using ANOVA in GraphPad Prism 6.
[0416] Some of the chimeric or humanized antibodies induce the preapoptotic exposure of PDIA3 on the tumor cell surface. As shown in FIG. 20, the percent of PDIA3 positive cells in all the soluble anti-CD47 mAh treated cultures was significantly increased (p < 0.05) compared to the background obtained with a negative control, humanized isotype-matched antibody. This increase in the exposure of PDIA3 on the cell surface demonstrates that some of the chimeric or humanized antibodies induce DAMPs from tumor cells that can lead to phagocytosis of tumor cells and processing of tumor antigen by innate immune cells.
Humanized Anti-CD47 mAbs Cause Increased Cell Surface HSP70 Expression
[0417] These experiments demonstrate that humanized anti-CD47 mAbs of the present disclosure exhibit the ability to expose the endoplasmic reticulum resident chaperone HSP70 on the surface of the tumor cell as, for example, described previously using chemotherapeutic anthracyclines such as doxorubicin and mitoxantrone, as disclosed by Fucikova et al. (2011) Cancer Research 71(14):4821-4833. [0418] Cell surface exposure of HSP70 was determined using a mouse monoclonal antibody against HSP70 conjugated to Phycoerythrin (Abeam; Catalogue #ab65174). Human Raji lymphoma cells (ATCC, Manassas, VA; Catalog # CCL-86) or other cells types that express sufficient levels of CD47 were used. Cells were grown in RPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovine serum (BioWest; Catalogue # S01520), 100 units/mL penicillin, 100 pg mL streptomycin (Sigma; Catalogue # P4222) at densities less than 1 x 106 cells/mL. For this assay, cells were plated in 96 well tissue culture plates at a density of lxlO5 cells/ml RPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovine serum (BioWest; Catalog # S01520), 100 units/mL penicillin, 100 pg/mL streptomycin (Sigma; #P4222).
[0419] The humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l 1 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2) as disclosed herein, purified from transient transfections in CHO cells as described above, as well as the control chimeric antibody, were added at a final concentration of 10 pg/ml. As a positive control for HSP70 exposure, Raji cells were treated with 1 pM of chemotherapeutic anthracycline mitoxantrone. The cells were incubated at 37°C for 24 hours, after which the cells were harvested, washed twice with PBS, and incubated for 30 minutes with anti-HSP70 antibody as described above, diluted 1:200 in PBS. After 30 minutes the cells were washed twice with PBS, resuspended in 100 pi of PBS, and analyzed for the mean fluorescence intensity of the anti-HSP70 antibody signal as well as the percent of cells that stain positive for cell surface calreticulin by flow cytometry (Accuri C6, Becton Dickinson, Franklin Lakes, NJ). Results are presented as means ± SEM and analyzed for statistical significance using ANOVA in GraphPad Prism 6.
[0420] Some of the chimeric or humanized antibodies induce the preapoptotic exposure of HSP70 on the tumor cell surface. As shown in FIG. 21, the percent of HSP70 positive cells in all anti-CD47 mAh treated cultures was significantly increased (p < 0.05) compared to those seen in isotype control treated cultures. This increase in the exposure of HSP70 on the cell surface demonstrates that some of the chimeric or humanized antibodies induce DAMPs from tumor cells and can lead to phagocytosis of tumor cells and processing of tumor antigen by innate immune cells.
Humanized Anti-CD47 mAbs Cause Increased Cell Surface HSP90 Expression
[0421] These experiments demonstrate that humanized anti-CD47 mAbs of the present disclosure expose the endoplasmic reticulum resident chaperone HSP70 on the surface of the tumor cell as, for example, described previously using chemotherapeutic anthracyclines such as doxorubicin and mitoxantrone, as disclosed by Fucikova et al. (2011) Cancer Research 71(14):4821-4833.
[0422] Cell surface exposure of HSP90 was determined using a mouse monoclonal antibody against HSP70 conjugated to Phycoerythrin (Abeam; Catalogue #ab65174). Human Raji lymphoma cells (ATCC, Manassas, VA; Catalog # CCL-86) or other cells types that express sufficient levels of CD47 were used. Cells are grown in RPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovine serum (BioWest; Catalogue # S01520), 100 units/mL penicillin, 100 pg mL streptomycin (Sigma; Catalogue # P4222) at densities less than 1 x 106 cells/mL. For this assay, cells were plated in 96 well tissue culture plates at a density of lxlO5 cells/ml RPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovine serum (BioWest; Catalog # S01520), 100 units/mL penicillin, 100 pg/iuL streptomycin (Sigma; #P4222).
[0423] The humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l 1 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2) as disclosed herein, purified from transient transfections in CHO cells as described above, as well as the control chimeric antibody, were added at a final concentration of 10 pg/ml. As a positive control for HSP90 exposure, cells were treated with 1 mM of chemotherapeutic anthracycline mitoxantrone. The Raji cells were incubated at 37°C for 24 hours, after which the cells were harvested, washed twice with PBS, and incubated for 30 minutes with anti- HSP70 antibody as described above, diluted 1 :200 in PBS. After 30 minutes the cells were washed twice with PBS, resuspended in 100 pi of PBS, and analyzed for the mean fluorescence intensity of the anti-HSP70 antibody signal as well as the percent of cells that stain positive for cell surface calreticulin by flow cytometry (Accuri C6, Becton Dickinson, Franklin Lakes, NJ). Results are presented as means ± SEM and analyzed for statistical significance using ANOVA in GraphPad Prism 6.
[0424] Some of the chimeric or humanized antibodies induce the preapoptotic exposure of HSP90 on the tumor cell surface. As shown in FIG. 22, the percent of HSP90 positive cells in soluble anti-CD47 m Ah-treated cultures was significantly increased (p < 0.05) compared to the background obtained with a negative control, humanized isotype-matched antibody, except for VLXhum_06 IgG2 and VLX4hum_01 IgG4PE (ns, not significant). This increase in the exposure of HSP90 on the cell surface demonstrates that some of the chimeric or humani ed antibodies induce DAMPs from tumor cells and can lead to phagocytosis of tumor cells and processing of tumor antigen by innate immune cells.
Humanized Anti-CD47 mAbs Cause Increased ATP release
[0425] These experiments demonstrate that humanized anti-CD47 mAbs of the present disclosure induce increased release of adenosine triphosphate (ATP) from the tumor cell as described previously using anthracy cline chemotherapy drugs (Martins et al. , 2014 Cell Death and Differentiation 21 :79-91).
[0426] Release of ATP from the tumor cell is determined by quantitative bioluminescence assay as described by the manufacturer (Molecular Probes; Catalogue #A22066). Human Raji lymphoma cells (ATCC, Manassas, VA; Catalog # CCL-86) or other cells types that express sufficient levels of CD47 were used. Cells were grown in RPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovine serum (BioWest; Catalogue # S01520), 100 units/ml , penicillin, 100 pg mL streptomycin (Sigma; Catalogue # P4222) at densities less than 1 x 106 cells/mL. For this assay, cells were plated in 96 well tissue culture plates at a density of lxlO5 cells/ml RPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovine serum (BioWest; Catalog # S01520), 100 units/ml , penicillin, 100 pg/mL streptomycin (Sigma; #P4222).
[0427] The humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l 1 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03) as disclosed herein, purified from transient transfections in CHO cells as described above, as well as the control chimeric antibody, were added at a final concentration of 10 pg/ml. As a positive control for ATP release, cells were treated with 1 pM of chemotherapeutic anthracycline mitoxantrone. The cells were incubated at 37°C for 24 hours, after which the cell-free supernatant was collected and stored at -80°C. After all samples have been collected, lOpl of each sample was tested by the ATP determination kit as described above. Final concentrations were determined by comparing experimental values to a standard curve and displayed as the concentration of ATP (pM) released by tumor cells in response to antibody treatment. Results are presented as means ± SEM and analyzed for statistical significance using ANOVA in GraphPad Prism 6.
[0428] The humanized antibodies increased the release of ATP from the tumor cells. As shown in FIG. 23, the amount of released ATP in all anti-CD47 mAh treated cultures was significantly increased (p < 0.05) compared to an isotype control. This increase in the release of ATP demonstrates that some of the chimeric or humanized antibodies induce the release of ATP from tumor cells and can lead to dendritic cell migration through its cognate purinergic receptors.
Humanized Anti-CD47 mAbs Cause HMGB1 Release
[0429] These experiments demonstrate that humanized anti-CD47 mAbs of the present disclosure increase the release of the non-histone chromatin protein high-mobility group box 1 (HMGB1) from the tumor cell as described previously using chemotherapy agents, such as oxaliplatin (Tesniere et al , 2010 Oncogene, 29:482-491) and mitoxantrone (Michaud et al , 2011 Science 334: 1573-1577).
[0430] Release of HMGB 1 protein from the tumor cell was determined by enzyme immunoassay as described by the manufacturer (IBL International; Hamburg, Germany, Catalogue #ST51011). Human Raji lymphoma cells (ATCC, Manassas, VA; Catalog # CCL- 86) or other cells types that express sufficient levels of CD47 were used. Cells will be grown in RPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovine serum (BioWest; Catalogue # S01520), 100 units/mL penicillin, 100 pg mL streptomycin (Sigma; Catalogue # P4222) at densities less than 1 x 106 cells/mL. For this assay, cells were plated in 96 well tissue culture plates at a density of lxlO5 cells/ml RPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovine serum (BioWest; Catalog # S01520), 100 units/ml , penicillin, 100 pg/mL streptomycin (Sigma; #P4222).
[0431] The humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l 1 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2) as disclosed herein, purified from transient transfections in CHO cells as described above, as well as the control chimeric antibody, will then be added at a final concentration of 10 pg/ml. As a positive control for HMGB 1 release, Raji cells were treated with 1 pM of chemotherapeutic anthracycline mitoxantrone. The cells were incubated at 37°C for 24 hours, after which the cell-free supernatant was collected and stored at -80°C. After all samples have been collected, lOpl of each sample was tested by HMGB1 ELISA as described above. Final concentrations were determined by comparing experimental values to a standard curve and reported as the concentration of HMGB1 (ng/ml) released by tumor cells in response to antibody treatment. Results are presented as means ± SEM and analyzed for statistical significance using ANOVA in GraphPad Prism 6.
[0432] As shown in FIG. 24, the humanized antibodies increased the release of HMGB 1 protein from the tumor cells. The amount of released HMGB1 protein in all anti-CD47 mAh treated cultures was significantly increased (p < 0.05) compared to an isotype control, except for VLX9hum_06 IgG2 (ns, not significant). This increase in the release of HMGB 1 demonstrates that some of the chimeric or humanized antibodies induce release of DAMPs from tumor cells and can lead to dendritic cell activation.
Humanized Anti-CD47 mAbs Cause CXCL10 Release
[0433] These experiments demonstrate that humanized anti-CD47 mAbs of the present disclosure increase the production and release of the chemokine CXCL10 from the human tumor cells as described previously using anthracycline chemotherapy drugs (Sistigu et al , 2014 Nat. Med. 20(11): 1301 - 1309).
[0434] Release of the CXCL10 from the tumor cell was determined by enzyme immunoassay as described by the manufacturer (R&D Systems; Catalogue #DIP100). Human Raji lymphoma cells (ATCC, Manassas, VA; Catalog # CCL-86) or other cells types that express sufficient levels of CD47 will be used. Cells were grown in RPMI-1640 medium containing 5% (v/v) heat inactivated fetal bovine serum (BioWest; Catalogue # S01520), 100 units/mL penicillin, 100 pg mL streptomycin (Sigma; Catalogue # P4222) at densities less than 1 x 106 cells/mL. For this assay, cells were plated in 96 well tissue culture plates at a density of lxlO5 cells/ml RPMI-1640 medium containing 5% (v/v) heat inactivated fetal bovine serum (BioWest; Catalog # S01520), 100 units/ml , penicillin, 100 pg/mL streptomycin (Sigma; #P4222).
[0435] The humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l 1 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2) as disclosed herein, purified from transient transfections in CHO cells as described above, as well as the control chimeric antibody, were added at a final concentration of 10 pg/ml. As a positive control for CXCL 10 release, Raji cells were treated with 1 pM of the chemotherapeutic anthracycline mitoxantrone. The cells were incubated at 37°C for 24 hours, after which the cell-free supernatant was collected and stored at -80°C. After all samples have been collected, lOpl of each sample was tested by the CXCL10 ELISA as described above. Final concentrations were determined by comparing experimental values to a standard curve and displayed as the concentration of CXCL 10 (pg/ml) released by tumor cells in response to antibody treatment.
[0436] Some of the chimeric or humanized antibodies induce release of CXCL10 by human tumor cells. As shown in FIG. 25, the amount of released CXCL 10 in all anti-CD47 mAh treated cultures significantly increased (p < 0.05) compared to an isotype control. This increase in the release of CXCL 10 demonstrates that some of the chimeric or humanized antibodies induce the release of CXCL10 from tumor cells and suggest a role in the recruitment of immune cells to the tumor.
Example 11
Damage- Associated Molecular Pattern (DAMP) Expression and Release. Mitochondrial Depolarization and Cell Death Caused by Humanized Anti-CD47 m Ahs
[0437] These studies were conducted as described in Example 10, except that the human Jurkat T ALL cell line (ATCC, Manassas, VA; Catalog # TIB- 152) was used.
Humanized Anti-CD47 mAbs Cause Loss of Mitochondrial Membrane Potential
[0438] As shown in FIG. 26, the humanized m Ahs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l l IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2) caused a significant increase in the percent of cells with mitochondrial membrane depolarization (p < 0.05) compared to an isotype control. This increase in the amount of mitochondrial membrane depolarization demonstrates that some of the chimeric or humanized antibodies induce cell death in human tumor cells.
Humanized Anti-CD47 mAbs Cause Increase in Cell Surface Calreticulin Expression
[0439] As shown in FIG. 27, the humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l l IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2) induced the preapoptotic exposure of calreticulin on the tumor cell surface. The percent of calreticulin positive cells in all anti-CD47 mAh treated cultures were significantly increased (p < 0.05) compared to an isotype control, except VLX9hum_03 IgG2 (ns). This increase in the exposure of calreticulin on the cell surface demonstrated that some of the humanized antibodies induce DAMPs from tumor cells and can lead to phagocytosis of tumor cells and processing of tumor antigen by innate immune cells.
Humanized Anti-CD47 mAbs Cause Increase in Cell Surface PDIA3 Expression
[0440] As shown in FIG. 28, the percent of PDIA3 positive cells in soluble anti-CD47 mAh (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l 1 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2) treated cultures were significantly increased (p < 0.05) compared to the background obtained with a negative control, humanized isotype-matched antibody. This increase in the exposure of PDIA3 on the cell surface demonstrates that some of the chimeric or humanized antibodies induce DAMPs from tumor cells and can lead to phagocytosis of tumor cells and processing of tumor antigen by innate immune cells.
Humanized Anti-CD47 mAbs Cause Increase in Cell Surface HSP70 Expression [0441] As shown in FIG. 29, the percent of HSP70 positive cells in anti-CD47 mAh (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l l IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2) treated cultures were significantly increased (p < 0.05) compared to those seen in isotype control treated cultures. Although each of the anti-CD47 m Ahs caused a statistically significant increase in HSP70 expression, mitoxantrone did not. This increase in the exposure of HSP70 on the cell surface demonstrates that some of the chimeric or humanized antibodies induce DAMPs from tumor cells and can lead to phagocytosis of tumor cells and processing of tumor antigen by innate immune cells. Humanized Anti-CD47 mAbs Cause Increase in Cell Surface HSP90 Expression
[0442] As shown in FIG. 30, the percent of HSP90 positive cells in soluble anti-CD47 mAh (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l 1 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2) treated cultures were significantly increased (p < 0.05) compared to the background obtained with a negative control, humanized isotype-matched antibody. This increase in the exposure of HSP90 on the cell surface demonstrates that some of the chimeric or humanized antibodies induce DAMPs from tumor cells and can lead to phagocytosis of tumor cells and processing of tumor antigen by innate immune cells.
Humanized Anti-CD47 mAbs Cause Increase in ATP Release
[0443] As shown in FIG. 31, the amount of released ATP in humanized anti-CD47 mAh (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l l IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2) treated cultures was significantly increased (p < 0.05) compared to an isotype control. Although each of the anti-CD47 m Ahs caused a statistically significant increase in HSP70 expression, mitoxantrone did not (ns). This increase in the release of ATP will demonstrates that some of the chimeric or humani ed antibodies induce the release of ATP from tumor cells and can lead to dendritic cell migration through its cognate purinergic receptors.
Humanized Anti-CD47 mAbs Cause Increase in HMGB1 Release
[0444] As shown in FIG. 32, the amount of released HMGB 1 protein in anti-CD47 mAh (VLX4hum_01 IgG4PE,VLX4hum_07 IgG4PE, VLX8hum_l l IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2) treated cultures was significantly increased (p < 0.05) compared to an isotype control, except for VLX4hum_01 IgG4PE (ns). This increase in the release of HMGB 1 demonstrates that some of the chimeric or humanized antibodies induce DAMPs from tumor cells and can lead to dendritic cell activation. Example 12
Hemagglutination of Human Red Blood Cells (hRBCs )
[0445] Many CD47 antibodies, including B6H12, BRIC126, MABL1, MABL2, CC2C6, 5F9, have been shown to cause hemagglutination (HA) of washed RBCs in vitro or in vivo (Petrova P. et al. Cancer Res 2015; 75(15 Suppl): Abstract nr 4271 ; US Patent 9,045,541 ; Uno et al. Oncol Rep. 17: 1189-94, 2007; Kikuchi et al. Biochem Biophys Res. Commun. 315: 912- 8, 2004; Sikic B. et al. J Clin Oncol 2016;34 (suppl; abstract 3019)). Hemagglutination of hRBCs was assessed following incubation of hRBCs with various concentrations of chimeric and humanized VLX4, VLX8, and VLX9 mAbs in vitro essentially as described by Kikuchi et al. Biochem Biophys Res. Commun (2004) 315:912-918. Blood was obtained from healthy donors, diluted (1 :50) in PBS/1 mM EDTA/BSA and washed 3 times with PBS/EDTA/BSA. hRBCs were added to U-bottomed 96 well plates with equal volumes of the antibodies (75 mΐ of each) and incubated for 3 hrs at 37°C and overnight at 4°C. A tight RBC pellet is observed with antibodies that do not cause hemagglutination, and a diffuse, hazy pattern is observed with antibodies that cause hemagglutination.
[0446] As shown in FIG. 33A and Tables 1 and 2, The VLX4hum_01 IgGl caused visible hemagglutination of hRBCs, whereas the humanized VLX4hum_01 IgG4PE mAh did not (mAb concentrations 50 mg/ml to 0.3 ng/ml). The lack of detectable hemagglutination by VLX4hum_01 IgG4 PE imparts an additional desirable antibody property and potential therapeutic benefit in the treatment of cancer.
[0447] As shown in FIG. 33B and Tables 1 and 2, the chimeric antibody VLX8 IgG4PE (xi) and the humanized antibodies VLX8hum_08 IgG4PE, VLX8hum_09 IgG4PE, and VLX8hum_10 IgG4PE caused visible hemagglutination of hRBCs, whereas the VLX8 humanized Abs VLX8hum_01 IgG4PE, VLX8hum_02 IgG4 PE, VLX8hum_03 IgG4 PE and VLX8hum_l 1 IgG4PE did not (mAb concentrations 50 mg/ml to 0.3 ng/ml).
[0448] The lack of detectable hemagglutination by humanized antibodies VLX4hum_01 IgG4PE, VLX8hum_01 IgG4PE, VLX8hum_02 IgG4 PE, VLX8hum_03 IgG4 PE and VLX8hum_l l IgG4 PE imparts an additional desirable antibody property and a potential therapeutic benefit in the treatment of cancer.
[0449] As shown in FIG. 34A and FIG. 34B, the chimeric antibody VLX9 IgG2 xi caused visible hemagglutination of hRBCs, whereas all of the humanized VLX9 mAbs except for VLX9hum_07 IgG2, did not cause detectable hemagglutination (at concentrations from 50 ug/ml to 0.3 pg/ml). However, the amount of detectable hemagglutination caused by VLX9hum_07 was reduced compared to the VLX9 IgG2 chimeric mAh. Again, the reduced or lack of detectable hemagglutination by the VLX9 humanized m Ahs imparts an additional desirable antibody property and a potential therapeutic benefit in the treatment of cancer.
Example 13
Anti-Tumor Activity in vivo
[0450] The purpose of this experiment was to demonstrate that VLX4, VLX8 and VLX9 humanized antibodies, exemplified by VLX4_07 IgG4PE, VLX8_10 IgG4PE and VLX9hum_08 IgG2, reduce tumor burden in vivo in a mouse xenograft model of lymphoma.
[0451] Raji human Burkitt’s lymphoma cells (ATCC #CCL-86, Manassas, VA) were maintained in RPMI-1640 (Lonza; Walkersville, MD) supplemented with 10% Fetal Bovine Serum (FBS; Omega Scientific; Tarzana, CA) within a 5% C02 atmosphere. Cultures were expanded in tissue culture flasks.
[0452] Female NSG (NOD-Cg-Prkdes l I 12rglm 1 Wjl/Sz.l) were obtained from Jackson Faboratory (Bar Harbor, ME) at 5-6 weeks of age. Mice were acclimated prior to handling and housed in microisolator cages (Fab Products, Seaford, DE) under specific pathogen-free conditions. Mice were fed Teklad Global Diet® 2920x irradiated laboratory animal diet (Envigo, Formerly Harlan; Indianapolis, IN) and provided autoclaved water ad libitum. All procedures were carried out under Institutional Animal Care and Use guidelines.
[0453] Female NSG mice were inoculated subcutaneously in the right flank with 0.1 mL of a 30% RPMI / 70% Matrigel™ (BD Biosciences; Bedford, MA) mixture containing a suspension of 5xl06 Raji tumor cells. Five days following inoculation, digital calipers were used to measure width and length diameters of the tumor. Tumor volumes were calculated utilizing the formula: tumor volume (mm3) = (a x b2/2) where‘b’ is the smallest diameter and ‘a’ is the largest diameter. Mice with palpable tumor volumes of 31-74 mm3 were randomized into 8-10/group and VFX9hum_08 or PBS (control) administration was initiated at this time. Mice were treated with 5 mg/kg of antibody 5X/week for 4 weeks by intraperitoneal injection. Tumor volumes and body weights were recorded twice weekly.
[0454] As shown in FIG. 35, treatment with the humanized VFX4hum_07 IgG4PE significantly reduced tumor growth of the Raji tumors (p < 0.05, two-way ANOVA), demonstrating anti-tumor efficacy in vivo.
[0455] As shown in FIG. 36, treatment with the humanized anti-CD47 mAh, VFX8hum_10 IgG4PE significantly reduced (p < 0.0001, two-way ANOVA) tumor growth of the Raji tumors, demonstrating anti-tumor efficacy in vivo. [0456] As shown in FIG. 37, treatment with the humanized anti-CD47 mAh, VLX9hum_08 IgG2 significantly reduced (p < 0.05, two-way ANOVA) tumor growth of the Raji tumors, demonstrating anti-tumor efficacy in vivo.
Example 14
Effect on Circulating Red Blood Cell Parameters
[0457] The purpose of this experiment is to demonstrate that VLX9 humanized antibodies that do not bind to human RBC in vitro (Table 2), exemplified by huml017_08 IgG2, do not cause a reduction in either hemoglobin (Hg) or circulating RBCs following administration to cynomolgus monkeys.
[0458] Female Chinese cynomolgus monkeys (Charles River Laboratories, Houston, TX) 2.5 - 3 kg were used in accordance with the Institutional Animal Care and Use guidelines. VLX9hum_08 IgG2 or vehicle (PBS) was administered as a 1 hour intravenous infusion on day 1 at a dose of 5 mg/kg and on day 18 at a dose of 15 mg/kg (3 animals/group). Hematological parameters were measured throughout the study on days -7, -3 (not shown), pre dose, 3, 8, 12, 18 (pre-dose), 20, 25, 29, 35 and 41 and compared/normalized to the means values of control animals. The pre-treatment RBC and Hg values on day 0 in the VLX9hum_08 IgG2 group were lower than the control group. Following treatment with either dose of VLX9hum_08 IgG2, there were minimal changes (< 10%) in Hg (FIG. 38A) or RBC counts (FIG. 38B) compared to the control group demonstrating that VLX9hum_08 IgG2 causes minimal reductions in RBC hematological parameters when administered to cynomolgus monkeys.
Example 15
Antibodies to CD47 Regulate Nitric Oxide Signaling
[0459] TSP1 binding to CD47 activates the heterotrimeric G protein Gi, which leads to suppression of intracellular cyclic AMP (cAMP) levels. In addition, the TSP1/CD47 pathway opposes the beneficial effects of the nitric oxide (NO) pathway in all vascular cells. The NO pathway consists of any of three nitric oxide synthase enzymes (NOS I, NOS II and NOS III) that generate bioactive gas NO using arginine as a substrate. NO can act within the cell in which it is produced or in neighboring cells, to activate the enzyme soluble guanylyl cyclase that produces the messenger molecule cyclic GMP (cGMP). The proper functioning of the NO/cGMP pathway is essential for protecting the cardiovascular system against stresses including, but not limited to, those resulting from wounding, inflammation, hypertension, metabolic syndrome, ischemia, and ischemia-reperfusion injury (IRI). In the context of these cellular stresses, the inhibition of the NO/cGMP pathway by the TSP1/CD47 system exacerbates the effects of stress. This is a particular problem in the cardiovascular system where both cGMP and cAMP play important protective roles. There are many cases in which ischemia and reperfusion injury cause or contribute to disease, trauma, and poor outcomes of surgical procedures.
[0460] The purpose of these experiment will be to demonstrate that humanized anti-CD47 m Ahs of the present disclosure exhibit the ability to reverse TSP1 -mediated inhibition of NO- stimulated cGMP synthesis as, for example, described previously using mouse monoclonal antibodies to CD47 as disclosed by Isenberg et al. (2006) J. Biol. Chem. 281 :26069-80, or alternatively other downstream markers of or effects resulting from NO signaling, for example smooth muscle cell relaxation or platelet aggregation as described previously by Miller et al. (2010) Br J. Pharmacol. 159: 1542-1547.
[0461] The method employed that will be to measure cGMP as described by the manufacturer (CatchPoint Cyclic-GMP Fluorescent Assay Kit, Molecular Devices, Sunnyvale, CA). Jurkat JE6.1 cells (ATCC, Manassas, VA; Catalog # TIB- 152) or other cells types that retain the NO/cGMP signaling pathway when grown in culture and exhibit a robust and reproducible inhibitory response to TSP1 ligation of CD47 will be used. Cells will be grown in Iscove’s modified Dulbeccco’s medium containing 5% (v/v) heat inactivated fetal bovine serum (BioWest; Catalogue # S01520), 100 units/mL penicillin, 100 pg mL streptomycin (Sigma; Catalogue # P4222) at densities less than 1 x 106 cells/mL. For the cGMP assay, cells will be plated in 96 well tissue culture plates at a density of lxlO5 cells/ml in Iscoves modified Dulbecco’s medium containing 5% (v/v) heat inactivated fetal bovine serum (BioWest; Catalog # S01520), 100 units/ml , penicillin, 100 pg/mL streptomycin (Sigma; #P4222) for 24 hours and then transferred to serum free medium overnight.
[0462] The humanized antibodies as disclosed herein, purified from transient transfections in CFIO cells as described above in Example 3, as well as the control chimeric antibody, will then be added at a final concentration of 20 ng/ml, followed 15 minutes later by 0 or 1 pg/ml human TSP1 (Athens Research and Technology, Athens, GA, Catalogue # 16-20-201319). After an additional 15 minutes, the NO donor, diethylamine (DEA) NONOate (Cayman Chemical, Ann Arbor, MI, Catalog # 82100), will be added to half the wells at a final concentration of 1 pM. Five minutes later, the cells will be lysed with buffer supplied in the cGMP kit, and aliquots of each well assayed for cGMP content.
[0463] It is anticipated that some of the chimeric or humanized antibodies will reverse TSP1 inhibition of cGMP. Reversal will be complete (>80 %) or intermediate (20% -80%). This reversal of TSP1 inhibition of cGMP will demonstrate that they have the ability to increase NO signaling and suggest utility in protecting the cardiovascular system against stresses including, but not limited to, those resulting from wounding, inflammation, hypertension, metabolic syndrome, ischemia, and ischemia-reperfusion injury (IRI). Additional assay systems, for example smooth muscle cell contraction, will also be expected to show that some of the chimeric or humanized antibody clones reverse the inhibitory actions of TSP1 on downstream effects resulting from the activation of NO signaling.
Example 16
Co-localization of Humanized VLX Antibodies bound to CD47 with Bl-integrin
[0464] The localization of anti-CD47 antibodies of the present disclosure to cell surface CD47 on tumor and normal cells was determined by confocal microscopy. Co-localization with bΐ-integrin protein was also performed by confocal microscopy.
[0465] For staining of CD47 on human red blood cells, RBCs were collected in microcentrifuge tubes and incubated with VLX9hum_04 IgG2, VLX9hum_06 IgG2, VLX9hum _08 IgG2, or VLX4hum _01 IgG4PE for 1 hour at 37°C. The cells were washed three times and then stained with a fluorophore labeled anti-human IgG (H+L) antibody antibody for 1 hour followed by three washes and stained cells were imaged using a confocal microscope (Leica SP8) at 100X magnification.
[0466] For co- localization studies with bΐ-integrin, Jurkat T-ALL tumor cells were collected in microcentrifuge tubes and incubated simultaneously with VLX9hum _04 IgG2, VLX9hum _06 IgG2, VLX9hum _08 IgG2, VLX4hum _01 IgG4PE and bΐ-integrin antibody [Anti^l-integrin antibody [12G10] (ab30394)] for 1 hour at 37°C. The fixed cells were washed thrice and then stained with a fluorophore labeled anti-human IgG (FI+L) antibody or anti-mouse IgG (FI+L) antibody for 1 hour followed by three washes and fixation using 1 % paraformaldehyde. The cells were again washed, and stained cells were imaged using a confocal microscope (Leica SP8) at 100X magnification.
[0467] As shown in FIG. 39A, VLX9hum_04 IgG2, VLX9hum_06 IgG2, VLX9hum_08 IgG2 do not bind to human RBCs as indicated by lack of staining to red blood cells in immunofluorescence imaging shown in the upper panel. The lower panel captures the brighfield image of the cells. In contrast, VLX4hum _01 IgG4PE uniformly binds to the surface of red blood cells.
[0468] As shown in FIG. 39B, VLX9hum_04 IgG2, VLX9hum_06 IgG2, VLX9hum_08 IgG2 displayed a distinct capped pattern on the surface of Jurkat T-ALL cells. A uniform staining pattern with VLX4hum_01 IgG4PE is shown over the surface of the cell in FIG. 39B. In FIG. 39B, exclusive co-localization of bΐ-integrin with VLX4_01 IgG4PE was not evident, whereas VLX9hum_04 IgG2, VLX9hum_06 IgG2, VLX9hum_08 IgG2 bound CD47 specifically and co-localized with bΐ-integrin.
Example 17
Humanized CD47 Antibodies Specifically Bind to CD47 that is Associated with bΐ -integrin
[0469] The localization of anti-CD47 antibodies of the present disclosure to cell surface CD47 on tumor cells expressing bΐ-integrin was determined by confocal microscopy. Co localization of CD47 with integrin bΐ protein was also performed by confocal microscopy.
[0470] bΐ -integrin knock-out (DITϋbI) cells were generated using a CRISPR/Cas9 targeting approach. Jurkat cells were generated to express Cas9 nuclease using a lentiviral based expression system (. Nature Methods. 10 (10): 957-63). Next, ITGB1 targeted sgRNAs in recombinant lentiviral vectors were used to infect the Cas9-expressing Jurkat cells. Successfully transduced cells were selected by monitoring co-expression of fluorescent proteins (purple staining) and antibiotic resistance.
[0471] For staining of CD47 with anti-CD47 antibodies alone, Jurkat T-ALL DPΌbI cells were collected in microcentrifuge tubes and incubated with VXL9hum_04 IgG2,
VLX9hum_06 IgG2, or VLX9hum_08 IgG2 which exhibit differential binding or a CD47 antibody that lacks differential binding (Hu5F9-G4) for 1 hour at 37°C. The cells were washed thrice and then stained with a fluorophore labeled anti-human IgG (H+L) antibody for 1 hour followed by three washes and fixation using 1% paraformaldehyde. The cells were again washed, and stained cells were imaged using a confocal microscope (Leica SP8) at 100X magnification.
[0472] For co-localization studies of CD47 and bΐ-integrin, Jurkat T-AFF DITObI cells were collected in microcentrifuge tubes and incubated with VFX9hum_04 IgG2,
VFX9hum_06 IgG2, VFX9hum_08 IgG2, or Hu5F9-G4 mAh for 1 hour at 37 °C and an integrin beta 1 antibody [mouse anti-Integrin bΐ antibody [12G10] (ab30394)] for 1 hour at 37°C. The cells were washed three times and then stained with a fluorophore labeled anti human IgG (H+F) antibody or anti-mouse IgG (H+F) antibody for 1 hour followed by three washes and fixation using 1% paraformaldehyde. The cells were again washed, and CD47 (yellow), bΐ integrin (blue) or GFP (purple indicating virally transduced ITGB 1KO cells) were imaged using a confocal microscope (Feica SP8) at 100X magnification. [0473] As shown in FIG. 41 (shown in row 2, row 3, and row 4), neither VLX9hum_04 IgG2, VLX9hum_06 IgG2, VLX9hum_08 IgG2, or the anti-integrin bΐ antibody bound to the surface of Jurkat T-ALL cells that did not express bΐ-integrin (arrows indicate DITOb I cells). In contrast, control non-transduced cells that still expressed bΐ-integrin were still efficiently stained by VLX9hum_04 IgG2, VLX9hum_06 IgG2, VLX9hum_08 IgG2, and the integrin bΐ antibody . In contrast, (FIG. 41, top row), Hu5F9-G4, a non-differentially binding anti-CD47 mAh stained both DITOb 1 cells ( arrow , purple cells ) and control non-transduced cells.
[0474] Interestingly, RBCs which also do not express bΐ -integrin, are negligibly bound by VLX9hum_04 IgG2, VLX9hum_06 IgG2, or VLX9hum_08 IgG2, yet are bound highly by non-differentially binding CD47 antibodies (as shown in FIG. 39 A).
Example 18
Co-localization of SIRPa variants bound CD47 with bΐ -integrin
[0475] The localization of recombinant SIRPa variants vl and v2 to cell surface CD47 on tumor cells were determined by confocal microscopy. These recombinant SIRPa variants, vl and v2, possessed the NH2-terminal V-set domain of SIRPa required for CD47 binding. Co localization with integrin beta 1 protein (bΐ -integrin) was also performed by confocal microscopy.
[0476] To demonstrate the SIRPa variants binding/localization to the surface of tumor cells, Jurkat T-ALL cells (hematologic tumor cells) were collected in microcentrifuge tubes and incubated simultaneously with recombinant SIRPa variant vl (Aero Biosystems) or SIRPa variant v2 (developed by Arch Oncology, Inc.) and integrin bΐ antibody [Anti-integrin bΐ antibody [12G10] (ab30394)] for 1 hour at 37°C. The cells were washed thrice and then stained with a fluorophore labeled anti -human IgG (H+L) antibody or anti-mouse IgG (H+L) antibody for 1 hour, followed by three washes and fixation using 1% paraformaldehyde. The cells were washed again, and stained cells were imaged using a confocal microscope (Leica SP8) at 100X magnification.
[0477] As shown in FIG. 42, top, left panel, SIRPa variant vl displayed a distinct clustered/capped pattern on the surface of Jurkat T-ALL cells while SIRPa variant v2 demonstrated a uniform staining pattern over the surface of the cell (FIG. 42, bottom left panel). Exclusive co-localization of bΐ-integrin with SIRPa variant v2 (FIG. 42, bottom middle and right panels) was not evident whereas SIRPa variant vl bound CD47 specifically co-localized with bΐ-integrin (FIG. 42, top middle and right panels). [0478] In summary, the binding pattern of SIRPa variant vl and its colocalization with bΐ- integrin was found to be similar to VLX9hum_04 IgG2, VLX9hum_06 IgG2 and VLX9hum_08 IgG2 binding of CD47 on cancer cell surfaces (as shown in FIG. 39B).
Example 19
VLX9hum 06 IgG2 Blocks CD47/SIRPa Variant 1 and 2 Binding
[0479] To assess the effect of humanized CD47 mAh (VLX9hum_06 IgG2) on the binding of CD47 to SIRPa variants 1 and 2, the in vitro binding of fluorescently labeled SIRPa-Fc fusion proteins to CD47 expressed on Jurkat T cells is measured in the presence or absence of VLX9hum_06 IgG2.
[0480] PE-labeled SIRPa-Fc variant 1 fusion protein was purchased from Aero Biosystems (Cat. No. SIA-HP252). SIRPa-Fc variant 2 fusion protein (made in-house, concentration 18 pg/ml) was labeled with Alexa Fluor 647 using a microscale protein labeling kit (ThermoFisher, Cat No: A30009) according to the manufacture’s specifications. Jurkat T-ALL cells were seeded at lxlO5 cells/well and incubated with increasing concentrations (0- 40 pg/ml) of VLX9hum_06 IgG2 for 30 min at 37°C. An equal volume of fluorescently labeled SIRPa-Fc variant 1 or 2 was added and incubated for an additional 30 min at 37°C. Cells were washed once with PBS and the amount of labeled SIRPa-Fc variants 1 and 2 bound to Jurkat T cells was assessed by flow cytometry. Percent inhibition was determined using the following equation [100 x (averaged median well fluorescence - negative control average median fluorescence)/ (positive control average median fluorescence - negative control average median fluorescence)] and graphed versus concentration of VLX9hum_06 IgG2.
[0481] As shown in FIG. 43, VLX9hum_06 IgG2 mAh blocked the interaction of CD47 expressed on the Jurkat T cells with SIRPa variant 1 and SIRPa variant 2 in a concentration- dependent manner.
Example 20
VLX9hum 06 IgG2 Binds to CD47 in a Protein Complex that Contains bΐ-integrin
[0482] The present disclosure shows the interaction of cell surface CD47 and bΐ-integrin on tumor cells as determined by co-immunoprecipitation with certain anti-CD47 antibodies.
[0483] Hematologic tumor cells Jurkat T-ALL were incubated with 50 ug/ml biotinylated VLX9hum_06 IgG2 or 1 ug/ml biotinylated Hu5F9-G4-IgG4 for 1 h at 37°C, then cross-linked with a water-soluble and cell impermeable crosslinker BS3 (bisfsulfosuccinimidyl] suberate) [Themo Scientific # 21586] for 30 min at room temperature, followed by quenching with 20mM Tris. Cells were lysed and incubated with Streptavidin Mag beads for 1 h followed by elution with elution buffer (0.1 M glycine, ph=2.5). The protein complexes were disrupted and resolved by SDS-PAGE and proteins transferred to PVDF (Biorad #1620177). Immunoprecipitation efficiency of CD47 by VLX9_06, Hu-5F9-G4 and IgG2 control antibody was determined by immunoblot using anti-CD47 antibody BRIC126 (SCBT #sc-59079). Endogenous levels of CD47 in the control lysate are shown as a positive control. Co-immunoprecipitation of bΐ-integrin was determined by immunoblot using anti-Integrin bΐ (D2E5) (cell signaling technology #9699). Endogenous levels of bΐ-integrin in the control lysate are shown as a positive control.
[0484] FIG. 44 (left panel) shows the amount of CD47 immunoprecipitated with the anti- CD47 antibodies VLX9hum_06 IgG2 or Hu5F9-G4. IgG2 served as a negative control. FIG. 44 (right panel) shows the western blot for bΐ-integrin co-immunoprecipitated in the same samples. VLX9hum_06 IgG2 co-immunoprecipitated cell surface CD47 bound to bΐ-integrin whereas Hu5F9-G4 did not.
Example 21
Humanized VLX9hum 06 IgG2 Antibody specifically binds to CD47 that is associated with bΐ-integrin in SNU-1 Cells
[0485] The localization of anti-CD47 antibodies of the present disclosure to cell surface CD47 on tumor cells expressing bΐ-integrin was determined by confocal microscopy. Co localization with integrin beta 1 protein was also performed by confocal microscopy.
[0486] For co-localization studies of CD47 and bΐ -integrin, SNU-1 gastric carcinoma cells were seeded on glass slides and incubated with VLX9hum_06 IgG2 or Hu5F9-G4 for 1 hour at 37°C and bΐ-integrin antibody [mouse anti-Integrin beta 1 antibody [12G10] (ab30394)] for 1 hour at 37°C. The fixed cells were washed thrice and then stained with a fluorophore labeled anti-human IgG (H+L) antibody or anti-mouse IgG (H+L) antibody for 1 hour, followed by three washes and fixation using 1% paraformaldehyde. The cells were again washed, and stained cells were imaged using a confocal microscope (Leica SP8) at 100X magnification.
[0487] As shown in FIG. 45, VLX9hum_06 IgG2 bound to SNU-1 cells in distinct capped pattern (upper left panel, indicated by arrow) co-localizing with bΐ -integrin staining (middle and lower left panels, arrow denotes area where VLX9hum_06 IgG2 strongly colocalizes with bΐ-integrin). In contrast, Hu5F9-G4 stained in a diffuse pattern around the cell (upper right panel) and did not specifically co-localize with bΐ -integrin staining (middle and lower right panels).
Example 22
Decreased Binding of Humanized VLX9hum 06 IgG2 Antibody to Normal Hematological
Cells [0488] The localization of anti-CD47 antibodies of the present disclosure to cell surface CD47 on tumor cells and normal hematological cells was determined by confocal microscopy. Localization of integrin bΐ -protein was also performed by confocal microscopy.
[0489] For localization studies of CD47 and bΐ -integrin, Jurkat T-ALL were seeded on glass slides and incubated with VLX9hum_06 IgG2 or Hu5F9-G4 for 1 hour at 37°C and integrin bΐ -antibody [mouse anti-Integrin beta 1 antibody [12G10] (ab30394)] for 1 hour at 37°C. The cells were washed thrice and then stained with a fluorophore labeled anti-human IgG (Fi+L) antibody or anti-mouse IgG (H+L) antibody for 1 hour, followed by three washes and fixation using 1 % paraformaldehyde. Fiuman normal neutrophils, monocytes and T cells (Astarte Biologies, Bothell, WA) were and incubated in suspension with VLX9hum_06 IgG2 or Fiu5F9-G4 for 1 hour at 37°C and integrin beta 1 antibody [mouse anti-Integrin beta 1 antibody [12G10] (ab30394)] for 1 hour at 37°C. The cells were washed thrice and then stained with a fluorophore labeled anti -human IgG (H+L) antibody or anti-mouse IgG (H+L) antibody for 1 hour, followed by three washes and fixation using 1 % paraformaldehyde. The cells were again washed, and stained cells were imaged using a confocal microscope (Leica SP8) at 100X magnification. Intensity of binding was determined using ImageJ image processing program.
[0490] As shown in FIG. 46A, binding of VLX9hum_06 IgG2 antibody to normal hematological cells was observed to be greatly reduced in comparison to tumor cell binding, reflective in part because of the lower overall amount of CD47 on normal cells compared to tumor cells. Similarly, bΐ-integrin staining was less intense on the normal hematological cells as compared to tumor cells. In contrast, Hu5F9-G4 strongly stained in a diffuse pattern around all the normal hematological cells to a greater level than VLX9hum_06 IgG2. Unlike other hematological cells, neutrophils had an inherent diffuse low intensity staining pattern of bΐ- integrin, which was mirrored by VLX9hum_06 IgG2 binding intensity and staining pattern. Quantification of the staining to normal hematological cells normali ed to Jurkat T-ALL tumor cell binding shows the reduced binding of VLX9hum_06 IgG2 to normal cells (FIG. 46B, left panel). A similar reduction in bΐ -integrin staining by confocal imaging in normal hematological cells normalized to Jurkat T-ALL tumor cell is graphically shown FIG. 46B, right panel. The quantified staining intensity of VLX9hum_06 IgG2 on normal hematological cells normalized to the binding of Hu5F9-G4 on normal cells, shows the reduced VLX9hum_06 IgG2 binding to normal cells compared to Hu5F9-G4 (FIG. 46C).
Example 23
Comparison of VLX9hum 06 IgG2 Antibody Binding to Tumor versus Normal
Hematological Cells [0491] The binding of VLX9hum_06 IgG2 in the present disclosure was determined by flow cytometry using Jurkat T-ALL cells, freshly isolated human red blood cells (hRBCs), human monocytes, human neutrophils, human T-cells and human platelets all of which express CD47 on their cell surface bΐ-integrin staining/levels were also determined by flow cytometry.
[0492] Binding activities of VLX9hum_06 IgG2 on Jurkats, hRBCs, neutrophils, monocytes, platelets and T-cells was determined using flow cytometry. For hRBCs, blood was obtained from normal volunteers and RBCs were washed 3 times with phosphate buffered saline, pH 7.2 containing 2.5 mM EDTA (PBS+E). Monocytes, T cells neutrophils and platelets were obtained from Astarte Biologies (Bothell, WA). Cells were incubated for 60 min at 37°C with increasing concentrations of VLX9hum_06 IgG2 diluted in PBS. Cells were then washed with PBS and incubated for an additional hour with FITC labelled donkey anti human IgG antibody (Jackson Tmmuno Research Labs, West Grove, PA; Catalogue # 709-096- 149). Cells were washed with PBS and antibody binding was analyzed using an Attune Nxt Flow Cytometer (ThermoFisher). Binding to bΐ-integrin was also determined by staining cells with increasing concentrations of Integrin beta 1 antibody [12G10]. Cells were then washed with PBS and incubated for an additional hour with PE labeled donkey anti-mouse IgG antibody (Jackson Tmmuno Research Labs, West Grove, PA). Binding curves of normal cell types were normalized to binding to tumor (Jurkat) cells.
[0493] As shown in FIG. 47A, VLX9hum_06 IgG2 binds with lower intensity to normal hematologic cells and with little to no binding to hRBCs, as compared to Jurkat cells. VLX9hum_06 IgG2 also binds with lower affinity to normal hemtatolgoical cells as compared to Jurkat cells observed as a right shift in binding. Overall, VLX9hum_06 IgG2 binds strongly to tumor cells with little binding to hRBCs and significantly reduced binding to other normal cells. The binding of Integrin beta 1 antibody on Jurkat and normal cells demonstrates that the overall levels of bΐ -integrin are either absent (RBCs) or significantly reduced on normal hematological cells compared to tumor cells (FIG. 47B).
Example 24
VLX9hum 06 IgG2 Binding Involves Expression of B1 -Integrin
[0494] The binding of VLX9hum_06 IgG2 in the present disclosure to K562 ery throblast like cells and to mature hRBCs was determined by flow cytometry bΐ-integrin levels were determined by western blot.
[0495] Binding of VLX9hum_06 IgG2 or Hu5F9-G4 on the erythroblast-like cell line K562, a model cell line used to study erythropoiesis, was determined using flow cytometry. For hRBCs, blood was obtained from normal volunteers and RBCs were washed 3 times with phosphate buffered saline, pH 7.2 containing 2.5 mM EDTA (PBS+E). K562 cells were obtained from ATCC. Cells were incubated for 60 min at 37°C with increasing concentrations of VLX9hum_06 IgG2 or Hu5F9-G4 diluted in PBS. Cells were then washed with PBS and incubated for an additional hour with FITC labelled donkey anti-human IgG antibody (Jackson Tmmuno Research Labs, West Grove, PA; Catalogue # 709-096-149). Cells were washed with PBS and antibody binding was analyzed using an Attune Nxt Flow Cytometer (ThermoFisher).
[0496] As shown in FIG. 48A - FIG. 48B, VLX9hum_06 IgG2 displayed little to no binding to hRBCs, compared to greater binding to K562 cells. This decreased binding to RBCs is consistent with the fact that during erythropoiesis, cell surface expression of bΐ-integrin is lost. Western blot analysis of bΐ-integrin confirmed undetectable levels of the bΐ-integrin in mature RBCs, in contrast to high levels in K562 cells (FIG. 48C). In contrast, Hu5F9-G4 bound with high affinity to both K562 and hRBCs. Overall, VLX9hum_06 IgG2 binds to cells that express bΐ-integrin but exhibits little to no binding to mature hRBCs which lack bΐ- integrin expression.
Example 25
VLX9hum 06 IgG2 Interferes with bΐ-lntegrin Signaling Pathways
[0497] The effect of VLX9hum_06 IgG2 in the present disclosure on downstream mediators of bΐ-integrin signaling in tumor cells was determined by phosphoblot analysis of treated Jurkat cells.
[0498] Wildtype Jurkat cells were treated either with control 100 pg/mL IgG2 antibody, 10, 30 or 100 pg/mL VLX9hum_06 IgG2 or 10 or 100 pg/mL Hu5F9-G4 for 90 minutes bΐ- integrin knock-out cells were also harvested for phosphoblot analysis. Cells were lysed in RIP A buffer with HALT protease-phosphatase inhibitor (Roche). A total of 30pg protein of each sample was prepared by boiling 10 minutes at 95 °C in 4X lamellae buffer with b- mercaptoethanol. The protein samples were electrophoretically separated by SDS-PAGE run at 100V in IX SDS running buffer (BioRad). Proteins were transferred to PVDF at lOOv for 65 minutes with ice pack. The blot was blocked using 5%-BSA in TBS solution then incubated with primary antibody (Src 1 :1000 dilution, pSrc 1 :1000 dilution, FAK 1 : 1000 dilution, pFAK 1 :500 dilution and GAPDH 1 :3000 dilution). Following primary antibody incubation, the blots were washed using TBS-T and incubated with a 1 :2500 dilution of the secondary antibodies (anti-rabbit HRP for pSrc, Src, FAK and GAPDH; anti-mouse for pFAK) and incubated with rocking for 1 hr at room temperature. The blots were washed and developed using ECL substrate mix. Total FAK and Src and GAPDH were also assessed by western blot. [0499] As shown in FIG. 49A, VLX9hum_06 IgG2 interferes with known bΐ-integrin signaling pathways as seen with the decrease in pSrc and pFAK levels in treated samples. In contrast, Hu5F9-G4 had no discernable effect on pFAK or pSrc. In addition, bΐ-integrin knockout cells had lower overall levels in pFAK and pSrc, consistent with the fact that bΐ- integrin signals through the Src and FAK family pathways (FIG.49B). Overall, VLX9hum_06 IgG2 alters known bΐ-integrin signaling pathways to decrease pSrc and pFAK in Jurkat cells in contrast to Flu5F9-G4 which does not.
Example 26
VLX9hum 06 IgG2 Interferes with Bl-Integrin Signaling Pathways in SNU-1 Gastric
Carcinoma Cells
[0500] The effect of VLX9hum_06 IgG2 in the present disclosure on downstream mediators of bΐ-integrin signaling in tumor cells was also determined by phosphoblot analysis of treated SNU-1 cells.
[0501] Wildtype SNU-1 cells were treated either with control IgG2 antibody, VLX9hum_06 IgG2 or Hu5F9-G4 for 1, 2 or 3 hours. Cells were lysed in RIPA buffer with HALT protease-phosphatase inhibitor (Roche). A total of 30pg protein of each sample was prepared by boiling 10 minutes at 95 °C in 4X lamellae buffer with b-mercaptoethanol. The protein samples were electrophoretically separated by SDS-PAGE run at 100V in IX SDS running buffer (BioRad). Proteins were transferred to PVDF at lOOv for 65 minutes with ice pack. The blot was blocked using 5%-BSA in TBS solution then incubated with primary antibodies (pSrc 1 :1000 dilution, Src 1 : 1000 dilution). Following primary antibody incubation, the blots were washed using TBS-T and incubated with a 1 :2500 dilution of the secondary antibodies (anti-rabbit HRP for pSrc and Src) and incubated with rocking for 1 hr at room temperature. The blots were washed and developed using ECL substrate mix. Intensity of western blot signal was determined using ImageJ image processing program.
[0502] As shown in FIG. 50A, VLX9hum_06 IgG2 interferes with known bΐ-integrin signaling pathways as can be seen with the decrease in pSrc levels in treated samples. In contrast, Hu5F9-G4 had no discernable effect on Src signaling. Quantification of the pSrc levels normalized to total Src levels is graphically depicted in FIG. 50B. Overall, VLX9hum_06 IgG2 alters known bΐ-integrin signaling pathways to decrease pSrc in SNU-1 cells.
Example 27
VLX9hum 06 IgG2 Interference in bΐ-lntegrin Signaling Pathways and Killing of Gastric
Carcinoma Cells [0503] The effect of VLX9hum_06 IgG2 in the present disclosure on interference in bΐ- integrin signaling and direct killing of gastric carcinoma cell lines.
[0504] Gastric carcinoma cell lines SNU-l, KATO III, SNU-16, Hs746T, NCI-N87, AGS, SNU-5 and OE-19 were treated either with media, IgG2 control antibody or VLX9hum_06 IgG2 for 24 hours. Cells were harvested and stained with were then stained with fluorescently labelled annexin V and PI or 7-aminoactinomycin D (7-AAD) (BD Biosciences) and the signal detected using an Attune Nxt flow cytometer (ThermoFisher).
[0505] Gastric carcinoma cell lines SNU-l, KATO III, SNU-16, Hs746T, NCI-N87, AGS, SNU-5 and OE-19 were treated either with control IgG2 antibody, VLX9hum_06 IgG2 or Hu5F9-G4 for 1, 2 or 3 hours. Cells were lysed in RIPA buffer with HALT protease- phosphatase inhibitor (Roche). A total of 30pg protein of each sample was prepared by boiling 10 minutes at 95 °C in 4X lamellae buffer with b-mercaptoethanol. The protein samples were electrophoretically separated by SDS-PAGE run at 100V in IX SDS running buffer (BioRad). Proteins were transferred to PVDF at lOOv for 65 minutes with ice pack. The blot was blocked using 5%-BSA in TBS solution then incubated with primary antibody (pSrc 1 : 1000 dilution, Src 1: 1000 dilution). Following the primary antibody incubation, the blots were washed using TBS-T and incubated with a 1 :2500 dilution of the secondary antibodies (anti-rabbit HRP for pSrc and Src) and incubated with rocking for 1 hr at room temperature. The blots were washed and developed using ECL substrate mix.
[0506] As shown in FIG. 51A - FIG. 51D, VLX9hum_06 IgG2 induced an increase in PS exposure and cell death of SNU-l, KATO III, SNU-16 and Hs746T cells as measured by an increase in Annexin V positivity. In contrast, the NCI-N87, AGS, SNU-5 and OE19 cells were not killed by VLX9hum_06 IgG2 (FIG. 51E - FIG. 51H). Table 11 provides the percent and fold change increase in Annexin V positivity and the % pSrc decrease in eight gastric carcinoma cell lines. Annexin V+ correlates with decreased pSrc signaling in SNU-l, KATO III, and SNU-16 gastric carcinoma cell lines.
Table 11. Annexin V+ and pSrc signaling in gastric carcinoma cell lines.
Figure imgf000151_0001
Figure imgf000152_0001

Claims

CLAIMS What is claimed is:
1. A monoclonal antibody or an antigen binding fragment thereof, which:
a. binds to human CD47 ;
b. blocks SIRPoc binding to human CD47;
c. increases phagocytosis of human tumor cells; and
d. induces death of human tumor cells;
wherein said monoclonal antibody or an antigen binding fragment thereof, possesses one or more among the following characteristics:
e. causes an increase in cell surface calreticulin expression on human tumor cells; f. causes an increase in adenosine triphosphate (ATP) release by human tumor cells;
g. causes an increase in high mobility group box 1 (HMGB1) release by human tumor cells;
h. causes an increase in annexin A1 release by human tumor cells;
i. causes an increase in Type I Interferon release by human tumor cells; j. causes an increase in C-X-C Motif Chemokine Ligand 10 (CXCL10) release by human tumor cells;
k. causes an increase in cell surface protein disulfide-isomerase A3 (PDIA3) expression on human tumor cells;
l. causes an increase in cell surface heat shock protein 70 (HSP70) expression on human tumor cells; and
m. causes an increase in cell surface heat shock protein 90 (HSP90) expression on human tumor cells.
2. A monoclonal antibody or an antigen binding fragment thereof, which:
a. binds to human CD47 ;
b. blocks SIRPoc binding to human CD47;
c. increases phagocytosis of human tumor cells;
d. induces death of human tumor cells; and
e. causes no detectable agglutination of human red blood cells (hRBCs); wherein said monoclonal antibody, or an antigen binding fragment thereof, possesses one or more among the following characteristics:
f. causes an increase in cell surface calreticulin expression on human tumor cells; g. causes an increase in adenosine triphosphate (ATP) release by human tumor cells;
h. causes an increase in high mobility group box 1 (HMGB1) release by human tumor cells;
i. causes an increase in annexin A1 release by human tumor cells;
j. causes an increase in Type I Interferon release by human tumor cells; k. causes an increase in C-X-C Motif Chemokine Ligand 10 (CXCL10) release by human tumor cells;
l. causes an increase in cell surface protein disulfide-isomerase A3 (PDIA3) expression on human tumor cells;
m. causes an increase in cell surface heat shock protein 70 (HSP70) expression on human tumor cells; and
n. causes an increase in cell surface heat shock protein 90 (HSP90) expression on human tumor cells.
3. The monoclonal antibody, or antigen binding fragment thereof, of any one of the preceding claims, which is a chimeric or humanized antibody.
4. The monoclonal antibody, or antigen binding fragment thereof, of any one of the preceding claims, wherein the monoclonal antibody, or antigen binding fragment thereof cross-reacts with one or more species homologs of CD47.
5. The monoclonal antibody or antigen binding fragment thereof, of any one of the preceding claims, wherein the monoclonal antibody or antigen binding fragment thereof has minimal binding to hRBCs.
6. The monoclonal antibody or antigen binding fragment thereof, of any one of the preceding claims, wherein the monoclonal antibody or antigen binding fragment thereof has reduced binding to normal human cells, wherein the normal human cells include endothelial cells, skeletal muscle cells, epithelial cells, and peripheral blood mononuclear cells (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubular epithelial cells, and human peripheral blood CD3+ cells).
7. The monoclonal antibody or antigen binding fragment thereof, of any of the preceding claims, wherein the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with an integrin.
8. The monoclonal antibody or antigen binding fragment thereof claim 7, wherein the integrin comprises a heterodimer of an a and b subunit.
9. The monoclonal antibody or antigen binding fragment thereof claim 8, wherein the b subunit is selected from ITOb 1 , ITOb2, ITOb3, ITOb4, ITOb5, ITObό, ITOb7, and PΌb8.
10. The monoclonal antibody or antigen binding fragment thereof of claim 8, wherein the a subunit is selected from ITGal, ITGa2, ITGa3, ITGa4, ITGa5, ITGa6, ITGa7, ITGa8, ITGa9, ITGalO, ITGal 1, and ITGav.
11. The monoclonal antibody or antigen binding fragment thereof of claims 1-6, wherein the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with ITObI .
12. The monoclonal antibody or antigen binding fragment thereof of claims 1-6, wherein the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with an ab integral complex selected from PΌbIaI, ITObI «2, PΌb1a3, PΌb1a4, PΌb1a5, PΌb1a6, PΌb1a7, PΌbIad, PΌb1a9, PΌbIaIO, PΌbIaϋ, and ITGav-
13. The monoclonal antibody or antigen binding fragment thereof of claims 1-6, wherein the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with ITObI and at least a second protein.
14. The monoclonal antibody or antigen binding fragment thereof of claim 13, wherein the second protein is selected from a receptor tyrosine kinase (RTK), a checkpoint inhibitor, ACTN1, CD46, CD9, FHL2, Filamin, FLNB, CD81, GNB2L1, ITϋbIBRI, LGALS8, MAP4K4, NME1, PKCa, TLN1, TSPAN4, and YWHAB.
15. The monoclonal antibody or antigen binding fragment thereof of any one of the preceding claims, wherein the monoclonal antibody or antigen binding fragment thereof has a greater affinity for CD47 at an acidic pFi compared to physiological pH.
16. The monoclonal antibody or antigen binding fragment thereof according to any one of the preceding claims, comprising a heavy chain variable domain comprising a variable heavy chain CDR1, variable heavy chain CDR2, and a variable heavy chain CDR3, wherein said variable chain CDR1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:l, SEQ ID NO:2, SEQ ID NOG;
wherein said variable heavy chain CDR2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:4, SEQ ID NOG, SEQ ID NOG; and wherein said variable heavy chain CDR3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO: 10.
17. The monoclonal antibody or antigen binding fragment thereof of claim 16, further comprising a light chain variable domain comprising a variable light chain CDR1, variable light chain CDR2, and a variable light chain CDR3, wherein said variable chain CDR1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:l l, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14;
wherein said variable light chain CDR2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and
wherein said variable light chain CDR3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20.
18. The monoclonal antibody or antigen binding fragment thereof, of claim 17, comprising a combination of variable heavy chain CDR1 (HCDR1), variable heavy chain CDR2 (HCDR2), and a variable heavy chain CDR3 (HCDR3), variable light chain CDR1 (LCDR1), variable light chain CDR2 (LCDR2), and a variable light chain CDR3 (LCDR3), wherein the combination is selected from the group consisting of:
(i) HCDR1 comprising SEQ ID NO: 1 , HCDR2 comprising SEQ ID NO:4, HCDR3 comprising SEQ ID NO:7, LCDR1 comprising SEQ ID NO:l l, LCDR2 comprising SEQ ID NO: 15, LCDR3 comprising SEQ ID NO: 18;
(ii) HCDR1 comprising SEQ ID NO: 1 , HCDR2 comprising SEQ ID NO:4, HCDR3 comprising SEQ ID NO:8, LCDR1 comprising SEQ ID NO:l l, LCDR2 comprising SEQ ID NO: 15, LCDR3 comprising SEQ ID NO: 18;
(iii) HCDR1 comprising SEQ ID NO:2, HCDR2 comprising SEQ ID NO:5, HCDR3 comprising SEQ ID NO:9, LCDR1 comprising SEQ ID NO: 12, LCDR2 comprising SEQ ID NO: 16, LCDR3 comprising SEQ ID NO: 19;
(iv) HCDR1 comprising SEQ ID NO:3, HCDR2 comprising SEQ ID NO:6, HCDR3 comprising SEQ ID NO: 10, LCDR1 comprising SEQ ID NO: 14, LCDR2 comprising SEQ ID NO: 17, LCDR3 comprising SEQ ID NO: 18.
19. The monoclonal antibody or antigen binding fragment thereof of claim 18, comprising a heavy chain variable domain (VH) having an amino acid sequence selected from the group consisting of: the amino acid sequences of: SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, and optionally comprising a light chain variable domain (VL) having an amino acid sequence selected from the group consisting of: SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:51, and SEQ ID NO:52.
20. The monoclonal antibody or antigen binding fragment thereof of claim 19, comprising a combination of a heavy chain variable domain (VH) and light chain variable domain (VL), wherein the combination is selected from the group consisting of:
(i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:27 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:44;
(ii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:23 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:43;
(iii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:33 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:48;
(iv) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:34 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:49;
(v) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:36 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:52;
(vi) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:38 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:52; and
(vii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:39 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:52.
21. A monoclonal antibody or an antigen binding fragment thereof, which:
a. binds to human CD47,
b. blocks SIRPoc binding to human CD47,
c. increases phagocytosis of human tumor cells,
d. induces death of human tumor cells; and
e. causes reduced agglutination of human red blood cells (hRBCs). wherein said monoclonal antibody or an antigen binding fragment thereof, possesses one or more among the following characteristics:
f. causes an increase in cell surface calreticulin expression on human tumor cells; g. causes an increase in adenosine triphosphate (ATP) release by human tumor cells;
h. causes an increase in high mobility group box 1 (HMGB1) release by human tumor cells;
i. causes an increase in annexin A1 release by human tumor cells;
j. causes an increase in Type I Interferon release by human tumor cells; k. causes an increase in C-X-C Motif Chemokine Ligand 10 (CXCL10) release by human tumor cells;
l. causes an increase in cell surface protein disulfide-isomerase A3 (PDIA3) expression on human tumor cells;
m. causes an increase in cell surface heat shock protein 70 (HSP70) expression on human tumor cells; and
n. causes an increase in cell surface heat shock protein 90 (HSP90) expression on human tumor cells.
22. The monoclonal antibody or antigen binding fragment thereof of claim 21, which is a chimeric or humanized antibody.
23. The monoclonal antibody or antigen binding fragment thereof of claim 21 or claim 22, wherein the monoclonal antibody, or antigen binding fragment thereof cross-reacts with one or more species homologs of CD47.
24. The monoclonal antibody or antigen binding fragment thereof according to any one of claims 21-23, wherein the monoclonal antibody or antigen binding fragment thereof has reduced hRBC binding.
25. The monoclonal antibody or antigen binding fragment thereof according to any one of claims 21-24, wherein the monoclonal antibody or antigen binding fragment thereof has reduced binding to normal human cells, wherein the normal human cells include endothelial cells, skeletal muscle cells, epithelial cells, and peripheral blood mononuclear cells (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubular epithelial cells, and human peripheral blood CD3+ cells).
26. The monoclonal antibody or antigen binding fragment thereof according to any one of claims 21-25, wherein the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with an integrin.
27. The monoclonal antibody or antigen binding fragment thereof claim 26, wherein the integrin comprises a heterodimer of an a and b subunit.
28. The monoclonal antibody or antigen binding fragment thereof claim 27, wherein the b subunit is selected from ITOb 1 , ITOb2, ITOb3, ITOb4, ITOb5, ITOb6, ITOb7, and ITϋbd.
29. The monoclonal antibody or antigen binding fragment thereof of claim 27, wherein the a subunit is selected from ITGal, ITGa2, ITGa3, ITGa4, ITGa5, ITGa6, ITGa7, ITGa8, ITGa9, ITGalO, ITGal 1, and ITGav.
30. The monoclonal antibody or antigen binding fragment thereof of claims 21-25, wherein the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with ITObI .
31. The monoclonal antibody or antigen binding fragment thereof of claims 21-25, wherein the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with an ab integrin complex selected from PΌbIaI, ITObI «2, PΌb1a3, PΌb1a4, PΌb1a5, PΌb1a6, PΌb1a7, PΌbIad, PΌb1a9, PΌbIaIO, PΌbIaϋ, and ITGav.
32. The monoclonal antibody or antigen binding fragment thereof of claims 21-25, wherein the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with ITObI and at least a second protein selected from ACTN1, CD46, CD9, FHL2, Filamin, FLNB, CD81, GNB2L1, PΌbIBRI, LGALS8, MAP4K4, NME1, PKCa, TLN1, TSPAN4, and YWHAB.
33. The monoclonal antibody or antigen binding fragment thereof according to any one of claims 21-32, wherein the monoclonal antibody, or antigen binding fragment thereof has a greater affinity for human CD47 at an acidic pFi compared to physiological pH.
34. The monoclonal antibody or antigen binding fragment thereof, according to any one of claims 21-33, comprising a heavy chain variable domain comprising a variable heavy chain CDR1, variable heavy chain CDR2, and a variable heavy chain CDR3, wherein said variable chain CDR1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:l and SEQ ID NOG, wherein said variable heavy chain CDR2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 4 and SEQ ID NO:6; and
wherein said variable heavy chain CDR3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:7, SEQ ID:8, and SEQ ID:10.
35. The monoclonal antibody or antigen binding fragment thereof of claim 34, further comprising a light chain variable domain comprising a variable light chain CDR1, variable light chain CDR2, and a variable light chain CDR3, wherein said variable chain CDR1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 11 and SEQ ID NO: 14;
wherein said variable light chain CDR2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:15 and SEQ ID NO:17; and
wherein said variable light chain CDR3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 18 and SEQ ID NO:20.
36. The monoclonal antibody or antigen binding fragment thereof of claim 35, comprising a combination of variable heavy chain CDR1 (HCDR1), variable heavy chain CDR2 (HCDR2), and a variable heavy chain CDR3 (HCDR3), variable light chain CDR1 (LCDR1), variable light chain CDR2 (LCDR2), and a variable light chain CDR3 (LCDR3), wherein the combination is selected from the group consisting of:
(i) HCDR1 comprising SEQ ID NO:l, HCDR2 comprising SEQ ID NO:4, HCDR3 comprising SEQ ID NO:8, LCDR1 comprising SEQ ID NO: 11 , LCDR2 comprising SEQ ID NO: 15, and LCDR3 comprising SEQ ID NO:18;
(ii) HCDR1 comprising SEQ ID NO: 3, HCDR2 comprising SEQ ID NO: 6, HCDR3 comprising SEQ ID NO: 10, LCDR1 comprising SEQ ID NO: 14, LCDR2 comprising SEQ ID NO: 17, and LCDR3 comprising SEQ ID NO:20.
37. The monoclonal antibody or antigen binding fragment thereof of claim 36, comprising a heavy chain variable domain (VH) having an amino acid sequence selected from the group consisting of: the amino acid sequences of: SEQ ID NO:24 and SEQ ID NO:37, and optionally comprising a light chain variable domain (VL) having an amino acid sequence selected from the group consisting of: SEQ ID NO:43 and SEQ ID NO:52.
38. The monoclonal antibody or antigen binding fragment thereof of claim 37, comprising a combination of a heavy chain variable domain (VH) and light chain variable domain (VL), wherein the combination is selected from the group consisting of: (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:24 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:43; and
(ii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:37 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:52.
39. A monoclonal antibody or an antigen binding fragment thereof, which:
a. binds to human CD47,
b. blocks SIRPoc binding to human CD47,
c. increases phagocytosis of human tumor cells
d. induces death of human tumor cells; and
e. has reduced hRBC binding.
wherein said monoclonal antibody or an antigen binding fragment thereof, possesses one or more among the following characteristics:
f. causes an increase in cell surface calreticulin expression on human tumor cells; g. causes an increase in adenosine triphosphate (ATP) release by human tumor cells;
h. causes an increase in high mobility group box 1 (HMGB1) release by human tumor cells;
i. causes an increase in annexin A1 release by human tumor cells;
j. causes an increase in Type I Interferon release by human tumor cells; k. causes an increase in C-X-C Motif Chemokine Ligand 10 (CXCL10) release by human tumor cells;
l. causes an increase in cell surface protein disulfide-isomerase A3 (PDIA3) expression on human tumor cells;
m. causes an increase in cell surface heat shock protein 70 (HSP70) expression on human tumor cells; and
n. causes an increase in cell surface heat shock protein 90 (HSP90) expression on human tumor cells.
40. The monoclonal antibody, or antigen binding fragment thereof of claim 39, which is a chimeric or humanized antibody.
41. The monoclonal antibody or antigen binding fragment thereof of claim 39 or claim 40, wherein the monoclonal antibody, or antigen binding fragment thereof cross-reacts with one or more species homologs of CD47.
42. The monoclonal antibody or antigen binding fragment thereof according to any one of claims 39-41, wherein the monoclonal antibody or antigen binding fragment thereof has reduced binding to normal human cells, wherein the normal human cells include endothelial cells, skeletal muscle cells, epithelial cells, and peripheral blood mononuclear cells (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubular epithelial cells, and human peripheral blood CD3+ cells).
43. The monoclonal antibody or antigen binding fragment thereof according to any one of claims 39-42, wherein the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with an integrin.
44. The monoclonal antibody or antigen binding fragment thereof of claim 43, wherein the integrin comprises a heterodimer of an a and b subunit.
45. The monoclonal antibody or antigen binding fragment thereof of claim 44, wherein the b subunit is selected from PΌbI, PΌb2, PΌb3, PΌb4, PΌb5, ITObό, PΌb7, and PΌb8.
46. The monoclonal antibody or antigen binding fragment thereof of claim 44, wherein the a subunit is selected from ITGal, ITGa2, ITGa3, ITGa4, ITGa5, ITGa6, ITGa7, ITGa8, ITGa9, ITGalO, ITGal 1, and ITGav.
47. The monoclonal antibody or antigen binding fragment thereof of claims 39-42, wherein the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with ITObI .
48. The monoclonal antibody or antigen binding fragment thereof of claims 39-42, wherein the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with an ab integrin complex selected from PΌbIaI, ITObI «2, PΌb1a3, PΌb1a4, PΌb1a5, PΌb1a6, PΌb1a7, PΌbIad, PΌb1a9, PΌbIaIO, PΌbIaI I, and ITGav.
49. The monoclonal antibody or antigen binding fragment thereof of claims 39-42, wherein the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with ITObI and at least a second protein selected from ACTN1, CD46, CD9, FHL2, Filamin, FLNB, CD81, GNB2L1, PΌbIBRI, LGALS8, MAP4K4, NME1, PKCoc, TLN1, TSPAN4, and YWHAB.
50. The monoclonal antibody or antigen binding fragment thereof, according to any one of claims 39-49, wherein the monoclonal antibody, or antigen binding fragment thereof has a greater affinity for human CD47 at an acidic pFi compared to physiological pH.
51. The monoclonal antibody or antigen binding fragment thereof, according to any one of claims 39-50, comprising a heavy chain variable domain comprising a variable heavy chain CDR1, variable heavy chain CDR2, and a variable heavy chain CDR3, wherein said variable chain CDR1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:l and SEQ ID NO:2;
wherein said variable heavy chain CDR2 comprises an amino acid sequence selected from the group consisting of: SEQ ID:4 and SEQ ID:5, and
wherein said variable heavy chain CDR3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:7, SEQ ID NO:8, and SEQ ID:9.
52. The monoclonal antibody or antigen binding fragment thereof, of claim 51, further comprising a light chain variable domain comprising a variable light chain CDR1, variable light chain CDR2, and a variable light chain CDR3, wherein said variable chain CDR1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:l l, SEQ ID NO:12, and SEQ ID NO: 13;
wherein said variable light chain CDR2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:15 and SEQ ID NO:16; and
wherein said variable light chain CDR3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 18 and SEQ ID NO: 19.
53. The monoclonal antibody or antigen binding fragment thereof, of claim 52, comprising a combination of variable heavy chain CDR1 (HCDR1), variable heavy chain CDR2 (HCDR2), and a variable heavy chain CDR3 (HCDR3), variable light chain CDR1 (LCDR1), variable light chain CDR2 (LCDR2), and a variable light chain CDR3 (LCDR3), wherein the combination is selected from the group consisting of:
(i) HCDR1 comprising SEQ ID NO:l, HCDR2 comprising SEQ ID NO:4, HCDR3 comprising SEQ ID NO:7, LCDR1 comprising SEQ ID NO: 11 , LCDR2 comprising SEQ ID NO: 15, LCDR3 comprising SEQ ID NO: 18; and (ii) HCDR1 comprising SEQ ID NO:2, HCDR2 comprising SEQ ID NO:5, HCDR3 comprising SEQ ID NO:9, LCDR1 comprising SEQ ID NO: 13, LCDR2 comprising SEQ ID NO: 16, LCDR3 comprising SEQ ID NO:19.
54. The monoclonal antibody or antigen binding fragment thereof, of claim 53, comprising a heavy chain variable domain (VH) having an amino acid sequence selected from the group consisting of: the amino acid sequences of: SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:33, and optionally comprising a light chain variable domain (VL) having an amino acid sequence selected from the group consisting of: SEQ ID NO:44 and SEQ ID NO:48.
55. The monoclonal antibody or antigen binding fragment thereof, of claim 54, comprising a combination of a heavy chain variable domain (VH) and light chain variable domain (VL), wherein the combination is selected from the group consisting of:
(i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:26 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:44;
(ii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:27 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:44; and
(iii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:33 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:48.
56. A monoclonal antibody or an antigen binding fragment thereof, which:
a. binds to human CD47,
b. blocks SIRPoc binding to human CD47,
c. increases phagocytosis of human tumor cells,
d. causes no detectable agglutination of human red blood cells (hRBCs); and e. has minimal hRBC binding.
wherein said monoclonal antibody, or an antigen binding fragment thereof, possesses one or more among the following characteristics:
f. causes an increase in cell surface calreticulin expression on human tumor cells; g. causes an increase in adenosine triphosphate (ATP) release by human tumor cells; h. causes an increase in high mobility group box 1 (HMGB1) release by human tumor cells;
i. causes an increase in annexin A1 release by human tumor cells;
j. causes an increase in Type I Interferon release by human tumor cells; k. causes an increase in C-X-C Motif Chemokine Ligand 10 (CXCL10) release by human tumor cells;
l. causes an increase in cell surface protein disulfide-isomerase A3 (PDIA3) expression on human tumor cells;
m. causes an increase in cell surface heat shock protein 70 (HSP70) expression on human tumor cells; and
n. causes an increase in cell surface heat shock protein 90 (HSP90) expression on human tumor cells.
57. The monoclonal antibody or antigen binding fragment thereof of claim 56, which is a chimeric or humanized antibody.
58. The monoclonal antibody, or antigen binding fragment thereof of claim 56 or claim 57, wherein the monoclonal antibody, or antigen binding fragment thereof cross-reacts with one or more species homologs of CD47.
59. The monoclonal antibody or antigen binding fragment thereof according to any one of claims 56-58, wherein the monoclonal antibody or antigen binding fragment thereof has reduced binding to normal human cells, wherein the normal human cells include endothelial cells, skeletal muscle cells, epithelial cells, and peripheral blood mononuclear cells (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubular epithelial cells, and human peripheral blood CD3+ cells).
60. The monoclonal antibody or antigen binding fragment thereof according to any one of claims 56-59, wherein the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with an integrin.
61. The monoclonal antibody or antigen binding fragment thereof of claim 60, wherein the integrin comprises a heterodimer of an a and b subunit.
62. The monoclonal antibody or antigen binding fragment thereof of claim 61, wherein the b subunit is selected from ITObI , ITOb2, ITOb3, ITOb4, ITOb5, ITObό, ITOb7, and ITϋbd.
63. The monoclonal antibody or antigen binding fragment thereof of claim 61, wherein the a subunit is selected from ITGal, ITGa2, ITGa3, ITGa4, ITGa5, ITGa6, ITGa7, ITGa8, ITGa9, ITGalO, ITGal 1, and ITGav.
64. The monoclonal antibody or antigen binding fragment thereof of claims 56-59, wherein the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with ITϋbI.
65. The monoclonal antibody or antigen binding fragment thereof of claims 56-59, wherein the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with an ab integral complex selected from ITϋbIaI, ITOb I «2, ITϋb1a3, ITϋb1a4, ITϋb1a5, ITϋbIaό, ITϋb1a7, ITϋb1a8, ITϋb1a9, ITϋbIaIO, ITϋbIaϋ, and ITGav-
66. The monoclonal antibody or antigen binding fragment thereof according to any one of claims 56-65, wherein the monoclonal antibody or antigen binding fragment thereof has a greater affinity for human CD47 at an acidic pH compared to physiological pH.
67. The monoclonal antibody or antigen binding fragment thereof, according to any one of claims 56-65, comprising a combination of variable heavy chain CDR1 (HCDR1), variable heavy chain CDR2 (HCDR2), and a variable heavy chain CDR3 (HCDR3), variable light chain CDR1 (LCDR1), variable light chain CDR2 (LCDR2), and a variable light chain CDR3 (LCDR3), wherein the combination is:
(i) HCDR1 comprising SEQ ID NOG, HCDR2 comprising SEQ ID NO: 6, HCDR3 comprising SEQ ID NO: 10, LCDR1 comprising SEQ ID NO: 14, LCDR2 comprising SEQ ID NO: 17, LCDR3 comprising SEQ ID NO:20.
68. The monoclonal antibody or antigen binding fragment thereof of claim 67, comprising a heavy chain variable domain (VH) having an amino acid sequence selected from the group consisting of: the amino acid sequences of: SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40 and optionally comprising a light chain variable domain (VL) having an amino acid sequence selected from the group consisting of: SEQ ID NO:51 and SEQ ID NO:52.
69. The monoclonal antibody or antigen binding fragment thereof of claim 68, comprising a combination of a heavy chain variable domain (VH) and light chain variable domain (VL), wherein the combination is selected from the group consisting of: (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:38 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:51;
(ii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:39 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:51; and
(iii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:40 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:52.
70. An monoclonal antibody or an antigen binding fragment thereof, which:
a. binds to human CD47 ;
b. blocks SIRPoc binding to human CD47;
c. increases phagocytosis of human tumor cells;
d. causes no detectable agglutination of human red blood cells (hRBCs); and e. has reduced hRBC binding.
wherein said monoclonal antibody, or an antigen binding fragment thereof, possesses one or more among the following characteristics:
f. causes an increase in cell surface calreticulin expression on human tumor cells; g. causes an increase in adenosine triphosphate (ATP) release by human tumor cells;
h. causes an increase in high mobility group box 1 (HMGB1) release by human tumor cells;
i. causes an increase in annexin A1 release by human tumor cells;
j. causes an increase in Type I Interferon release by human tumor cells; k. causes an increase in C-X-C Motif Chemokine Ligand 10 (CXCL10) release by human tumor cells;
l. causes an increase in cell surface protein disulfide-isomerase A3 (PDIA3) expression on human tumor cells;
m. causes an increase in cell surface heat shock protein 70 (HSP70) expression on human tumor cells; and
n. causes an increase in cell surface heat shock protein 90 (HSP90) expression on human tumor cells.
71. The monoclonal antibody or antigen binding fragment thereof of claim 70, which is a chimeric or humanized antibody.
72. The monoclonal antibody or antigen binding fragment thereof of claim 70 or claim 71, wherein the monoclonal antibody, or antigen binding fragment thereof cross-reacts with one or more species homologs of CD47.
73. The monoclonal antibody or antigen binding fragment thereof according to any one of claims 70-72, wherein the monoclonal antibody or antigen binding fragment thereof has reduced binding to normal human cells, wherein the normal human cells include endothelial cells, skeletal muscle cells, epithelial cells, and peripheral blood mononuclear cells (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubular epithelial cells, and human peripheral blood CD3+ cells).
74. The monoclonal antibody or antigen binding fragment thereof according to any one of claims 70-73, wherein the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with an integrin.
75. The monoclonal antibody or antigen binding fragment thereof of claim 74, wherein the integrin comprises a heterodimer of an a and b subunit.
76. The monoclonal antibody or antigen binding fragment thereof of claim 75, wherein the b subunit is selected from PΌbI, PΌb2, PΌb3, PΌb4, PΌb5, PΌb6, PΌb7, and PΌb8.
77. The monoclonal antibody or antigen binding fragment thereof of claim 75, wherein the a subunit is selected from ITGal, ITGa2, ITGa3, ITGa4, ITGa5, ITGa6, ITGa7, ITGa8, ITGa9, ITGalO, ITGal 1, and ITGav.
78. The monoclonal antibody or antigen binding fragment thereof of claims 70-73, wherein the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with ITObI .
79. The monoclonal antibody or antigen binding fragment thereof of claims 70-73, wherein the monoclonal antibody or antigen binding fragment thereof binds to human CD47 which is associated with an ab integrin complex selected from PΌbIaI, ITObI «2, PΌb1a3, PΌb1a4, PΌb1a5, PΌb1a6, PΌb1a7, PΌbIad, PΌb1a9, PΌbIaIO, PΌbIaI I, and ITGav.
80. The monoclonal antibody or antigen binding fragment thereof according to any one of claims 70-79, wherein the monoclonal antibody or antigen binding fragment thereof has a greater affinity for human CD47 at an acidic pH compared to physiological pH.
81. The monoclonal antibody or antigen binding fragment thereof according to any one of claims 70-80, comprising a combination of variable heavy chain CDR1 (HCDR1), variable heavy chain CDR2 (HCDR2), and a variable heavy chain CDR3 (HCDR3), variable light chain CDR1 (LCDR1), variable light chain CDR2 (LCDR2), and a variable light chain CDR3 (LCDR3), wherein the combination is:
(i) HCDR1 comprising SEQ ID NO: 3, HCDR2 comprising SEQ ID NO: 6, HCDR3 comprising SEQ ID NO: 10, LCDR1 comprising SEQ ID NO: 14, LCDR2 comprising SEQ ID NO: 17, LCDR3 comprising SEQ ID NO:20.
82. The monoclonal antibody or antigen binding fragment thereof of claim 81, comprising a combination of a heavy chain variable domain (VH) and light chain variable domain (VL), wherein the combination is:
(i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:36 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:51.
83. An monoclonal antibody or an antigen binding fragment thereof which binds to CD47, wherein the antibody or antigen binding fragment comprises a combination of and variable heavy chain CDR1 (HCDR1), variable heavy chain CDR2 (HCDR2), and variable heavy chain CDR3 (HCDR3), wherein the combination is selected from the group consisting of:
(i) HCDR1 comprising SEQ ID NO:l, HCDR2 comprising SEQ ID NO:4, HCDR3 comprising SEQ ID NO:7;
(ii) HCDR1 comprising SEQ ID NO:l, HCDR2 comprising SEQ ID NO:4, HCDR3 comprising SEQ ID NO: 8;
(iii) HCDR1 comprising SEQ ID NO:2, HCDR2 comprising SEQ ID NO:5, HCDR3 comprising SEQ ID NO:9; and
(iv) HCDR1 comprising SEQ ID NO: 3, HCDR2 comprising SEQ ID NO: 6, HCDR3 comprising SEQ ID NO: 10.
84. The monoclonal antibody or antigen-binding fragment of claim 83, comprising a light chain variable domain comprising a combination of variable light chain CDR3 (LCDR3), variable light chain CDR2 (LCDR2) and variable light chain CDR1 (LCDR1) selected from the group consisting of:
(i) LCDR1 comprising SEQ ID NO: 11, LCDR2 comprising SEQ ID NO: 15, LCDR3 comprising SEQ ID NO: 18;
(ii) LCDR1 comprising SEQ ID NO: 12, LCDR2 comprising SEQ ID NO: 16, LCDR3 comprising SEQ ID NO: 19;
(iii) LCDR1 comprising SEQ ID NO: 13, LCDR2 comprising SEQ ID NO: 16, LCDR3 comprising SEQ ID NO: 19; and
(iv) LCDR1 comprising SEQ ID NO: 14, LCDR2 comprising SEQ ID NO: 17, LCDR3 comprising SEQ ID NO:20.
85. The monoclonal antibody or antigen-binding fragment of claim 84 comprising a combination of variable heavy chain CDR sequences and a combination of variable light chain CDR sequences selected from the group:
(i) HCDR1 comprising SEQ ID NO:l, HCDR2 comprising SEQ ID NO:4, HCDR3 comprising SEQ ID NO:7, LCDR1 comprising SEQ ID NO: 11 , LCDR2 comprising SEQ ID NO: 15, LCDR3 comprising SEQ ID NO:18;
(ii) HCDR1 comprising SEQ ID NO:l, HCDR2 comprising SEQ ID NO:4, HCDR3 comprising SEQ ID NO:8, LCDR1 comprising SEQ ID NO: 11 , LCDR2 comprising SEQ ID NO: 15, LCDR3 comprising SEQ ID NO:18;
(iii) HCDR1 comprising SEQ ID NO:2, HCDR2 comprising SEQ ID NO:5, HCDR3 comprising SEQ ID NO:9, LCDR1 comprising SEQ ID NO: 12, LCDR2 comprising SEQ ID NO: 16, LCDR3 comprising SEQ ID NO:19;
(iv) HCDR1 comprising SEQ ID NO:2, HCDR2 comprising SEQ ID NO:5, HCDR3 comprising SEQ ID NO:9, LCDR1 comprising SEQ ID NO: 13, LCDR2 comprising SEQ ID NO: 16, LCDR3 comprising SEQ ID NO: 19; and
(v) HCDR1 comprising SEQ ID NO: 3, HCDR2 comprising SEQ ID NO: 6, HCDR3 comprising SEQ ID NO: 10, LCDR1 comprising SEQ ID NO: 14, LCDR2 comprising SEQ ID NO: 17, LCDR3 comprising SEQ ID NO:20.
86. The monoclonal antibody or antigen binding fragment of claim 85, wherein the antibody or antigen binding fragment thereof comprises a combination of a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of:
(i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:21 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:41 ;
(ii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:23 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:43;
(iii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:34 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:49;
(iv) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:36 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:52;
(v) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:38 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:52;
(vi) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:39 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:52;
(vii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:24 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:43;
(viii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:37 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:52;
(ix) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:33 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:48;
(x) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:26 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:44; (xi) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:27 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:44;
(xii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:38 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:51 ;
(xiii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:39 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:51 ;
(xiv) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:40 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:52;
(xv) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:36 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:51 ;
(xvi) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:29 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:47;
(xvii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:30 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:47;
(xviii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:31 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:47;
(xix) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:32 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:47;
(xx) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:33 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:47;
(xxi) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:29 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:48; (xxii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:30 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:48;
(xxiii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:31 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:48;
(xxiv) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:32 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:48;
(xxv) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:26 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:43;
(xxvi) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:27 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:43;
(xxvii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:28 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:46;
(xxviii)a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:35 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:50;
(xxix) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:29 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:48;
(xxx) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:30 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:48;
(xxxi) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:31 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:48;
(xxxii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:32 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:48; (xxxiii)a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:37 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:51; and
(xxxiv)a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:40 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:51;
wherein the VH amino acid sequence is at least 90%, 95%, 97%, 98% or 99% identical thereto and the a VL amino acid sequence is at least 90%, 95%, 97%, 98% or 99% identical thereto.
87. An monoclonal antibody according to any one of claims 83-86, comprising at least one heavy chain and at least one light chain selected from the selected from the group consisting of:
(i) a heavy chain comprising the amino acid sequence of SEQ ID NO:76 and a light chain comprising the amino acid sequence SEQ ID NO:66;
(ii) a heavy chain comprising the amino acid sequence of SEQ ID NO:77 and a light chain comprising the amino acid sequence SEQ ID NO: 68;
(iii) a heavy chain comprising the amino acid sequence of SEQ ID NO:78 and a light chain comprising the amino acid sequence SEQ ID NO: 69;
(iv) a heavy chain comprising the amino acid sequence of SEQ ID NO:79 and a light chain comprising the amino acid sequence SEQ ID NO: 70;
(v) a heavy chain comprising the amino acid sequence of SEQ ID NO: 80 and a light chain comprising the amino acid sequence SEQ ID NO: 70;
(vi) a heavy chain comprising the amino acid sequence of SEQ ID NO: 81 and a light chain comprising the amino acid sequence SEQ ID NO: 70;
(vii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 82 and a light chain comprising the amino acid sequence SEQ ID NO: 68;
(viii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 83 and a light chain comprising the amino acid sequence SEQ ID NO: 70;
(ix) a heavy chain comprising the amino acid sequence of SEQ ID NO: 84 and a light chain comprising the amino acid sequence SEQ ID NO:71;
(x) a heavy chain comprising the amino acid sequence of SEQ ID NO: 85 and a light chain comprising the amino acid sequence SEQ ID NO: 72; (xi) a heavy chain comprising the amino acid sequence of SEQ ID NO: 86 and a light chain comprising the amino acid sequence SEQ ID NO: 72;
(xii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 80 and a light chain comprising the amino acid sequence SEQ ID NO: 73;
(xiii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 81 and a light chain comprising the amino acid sequence SEQ ID NO: 73;
(xiv) a heavy chain comprising the amino acid sequence of SEQ ID NO: 87 and a light chain comprising the amino acid sequence SEQ ID NO: 70;
(xv) a heavy chain comprising the amino acid sequence of SEQ ID NO:79 and a light chain comprising the amino acid sequence SEQ ID NO: 73;
(xvi) a heavy chain comprising the amino acid sequence of SEQ ID NO:88 and a light chain comprising the amino acid sequence SEQ ID NO: 74;
(xvii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 89 and a light chain comprising the amino acid sequence SEQ ID NO: 74;
(xviii) a heavy chain comprising the amino acid sequence of SEQ ID NO:90 and a light chain comprising the amino acid sequence SEQ ID NO: 74;
(xix) a heavy chain comprising the amino acid sequence of SEQ ID NO:91 and a light chain comprising the amino acid sequence SEQ ID NO: 74;
(xx) a heavy chain comprising the amino acid sequence of SEQ ID NO: 84 and a light chain comprising the amino acid sequence SEQ ID NO: 74;
(xxi) a heavy chain comprising the amino acid sequence of SEQ ID NO:88 and a light chain comprising the amino acid sequence SEQ ID NO:71 ;
(xxii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 89 and a light chain comprising the amino acid sequence SEQ ID NO:71 ;
(xxiii) a heavy chain comprising the amino acid sequence of SEQ ID NO:90 and a light chain comprising the amino acid sequence SEQ ID NO:71 ;
(xxiv) a heavy chain comprising the amino acid sequence of SEQ ID NO:91 and a light chain comprising the amino acid sequence SEQ ID NO:71 ;
(xxv) a heavy chain comprising the amino acid sequence of SEQ ID NO: 85 and a light chain comprising the amino acid sequence SEQ ID NO: 68;
(xxvi) a heavy chain comprising the amino acid sequence of SEQ ID NO: 86 and a light chain comprising the amino acid sequence SEQ ID NO: 68;
(xxvii) a heavy chain comprising the amino acid sequence of SEQ ID NO:93 and a light chain comprising the amino acid sequence SEQ ID NO: 100; (xxviii)a heavy chain comprising the amino acid sequence of SEQ ID NO:94 and a light chain comprising the amino acid sequence SEQ ID NO: 75;
(xxix) a heavy chain comprising the amino acid sequence of SEQ ID NO:95 and a light chain comprising the amino acid sequence SEQ ID NO:71;
(xxx) a heavy chain comprising the amino acid sequence of SEQ ID NO:96 and a light chain comprising the amino acid sequence SEQ ID NO:71;
(xxxi) a heavy chain comprising the amino acid sequence of SEQ ID NO:97 and a light chain comprising the amino acid sequence SEQ ID NO:71;
(xxxii) a heavy chain comprising the amino acid sequence of SEQ ID NO:98 and a light chain comprising the amino acid sequence SEQ ID NO:71;
(xxxiii)a heavy chain comprising the amino acid sequence of SEQ ID NO: 83 and a light chain comprising the amino acid sequence SEQ ID NO: 73;
(xxxiv)a heavy chain comprising the amino acid sequence of SEQ ID NO: 87 and a light chain comprising the amino acid sequence SEQ ID NO: 73;
(xxxv) a heavy chain comprising the amino acid sequence of SEQ ID NO: 102 and a light chain comprising the amino acid sequence SEQ ID NO: 101;
(xxxvi)a heavy chain comprising the amino acid sequence of SEQ ID NO: 104 and a light chain comprising the amino acid sequence SEQ ID NO: 103;
wherein the VH amino acid sequence is at least 90%, 95%, 97%, 98% or 99% identical thereto and the a VL amino acid sequence is at least 90%, 95%, 97%, 98% or 99% identical thereto.
88. The monoclonal antibody or antigen binding fragment thereof for use according to any one the preceding claims, wherein the monoclonal antibody or antigen binding fragment thereof causes complete, intermediate, or no reversal of NO pathway inhibition.
89. The monoclonal antibody or antigen binding fragment thereof for use according to any one of the preceding claims, which displays one or more effector functions selected from antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and Clq binding against CD47-expressing cancer cells.
90. The monoclonal antibody or antigen binding fragment thereof for use according to any one of claims 1-89, wherein the monoclonal antibody or antigen binding fragment thereof is formulated as a pharmaceutical composition comprising a pharmaceutically or physiologically acceptable carrier, diluent, or excipient.
91. The monoclonal antibody or antigen binding fragment thereof or pharmaceutical composition for use according to claim 90, wherein said cancer is selected from leukemia, lymphoma, multiple myeloma, ovarian cancer, breast cancer, endometrial cancer, colon cancer (colorectal cancer), rectal cancer, bladder cancer, urothelial cancer, lung cancer (non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung), bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, gastric cancer, hepatocellular carcinoma, gall bladder cancer, bile duct cancer, esophageal cancer, renal cell carcinoma, thyroid cancer, squamous cell carcinoma of the head and neck (head and neck cancer), testicular cancer, cancer of the endocrine gland, cancer of the adrenal gland, cancer of the pituitary gland, cancer of the skin, cancer of soft tissues, cancer of blood vessels, cancer of brain, cancer of nerves, cancer of eyes, cancer of meninges, cancer of oropharynx, cancer of hypopharynx, cancer of cervix, and cancer of uterus, glioblastoma, meduloblastoma, astrocytoma, glioma, meningioma, gastrinoma, neuroblastoma, melanoma, myelodysplastic syndrome, and a sarcoma.
92. The monoclonal antibody or antigen binding fragment thereof or pharmaceutical composition for use according to claim 91, wherein said leukemia is selected from systemic mastocytosis, acute lymphocytic (lymphoblastic) leukemia (ALL), T cell - ALL, acute myeloid leukemia (AML), myelogenous leukemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), myeloproliferative disorder / neoplasm, myelodysplastic syndrome, monocytic cell leukemia, and plasma cell leukemia; wherein said lymphoma is selected from the group consisting of histiocytic lymphoma and T cell lymphoma, B cell lymphomas, including Hodgkin’s lymphoma and non-Hodgkin’s lymphoma, such as low grade/follicular non-Hodgkin’s lymphoma (NHL), cell lymphoma (FCC), mantle cell lymphoma (MCL), diffuse large cell lymphoma (DLCL), small lymphocytic (SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL, and Waldenstrom’s Macroglobulinemia; and wherein said sarcoma is selected from the group consisting of osteosarcoma, Ewing’s sarcoma, leiomyosarcoma, synovial sarcoma, alveolar soft part sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chrondrosarcoma.
93. The monoclonal antibody or antigen binding fragment thereof for use according to any one of claims 1-92 wherein the antibody or antigen binding fragment thereof is administered intravenously or subcutaneously.
94. A monoclonal antibody or antigen binding fragment thereof for use in a method of treating cancer in a human subject with anemia, wherein said monoclonal antibody or antigen binding fragment thereof binds to human CD47 on the surface of a tumor cell with minimal binding to human red blood cells (hRBCs), wherein the human red blood cells (hRBCs) lack bΐ-integrin expression.
95. The monoclonal antibody or antigen binding fragment thereof for use according to claim 94, wherein the monoclonal antibody or antigen binding fragment thereof binds human CD47 in association with an integrin on the surface of a tumor cell.
96. A monoclonal antibody or antigen binding fragment thereof for use in a method of enhancing the effect of a therapeutic agent in a human subject receiving said therapeutic agent, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 90.
97. The monoclonal antibody or antigen binding fragment thereof for use according to claim 96, wherein the human subject is being treated for cancer.
98. The monoclonal antibody or antigen binding fragment thereof for use according to claim 96, wherein the therapeutic agent is an inhibitor of a cell signaling pathway.
99. The monoclonal antibody or antigen binding fragment thereof for use according to claim 98, wherein the therapeutic agent is selected from the Src family kinase inhibitor dasatinib (BMS-354825) and the focal adhesion kinase (FAK) inhibitor defactinib (VS- 6063).
100. A monoclonal antibody or antigen binding fragment thereof for use in a method to decrease Src activity in a human subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 90.
101. The monoclonal antibody or antigen binding fragment thereof for use according to claim 100, wherein the activity of a downstream Src target is increased or decreased.
102. The method of claim 101, wherein the downstream Src target is selected from STAT3, PI3K/Akt, ERK/MAPK and / or a combination thereof.
103. A monoclonal antibody or antigen binding fragment thereof for use in a method of decreasing and / or inhibiting Src phosphorylation or auto-phosphorylation in a human subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 90.
104. The monoclonal antibody or antigen binding fragment thereof for use according to claim 103, wherein the ability of Src to phosphorylate or auto-phosphorylate is decreased.
105. The monoclonal antibody or antigen binding fragment thereof for use according to claim 103, wherein the phosphorylation or autophosphorylation of Src at one or more tyrosine amino acid residues is decreased.
106. A monoclonal antibody or antigen binding fragment thereof for use in a method to decrease FAK activity in a human subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 90.
107. The monoclonal antibody or antigen binding fragment thereof for use according to claim 106, wherein the activity of a downstream FAK target is increased or decreased.
108. The monoclonal antibody or antigen binding fragment thereof for use according to claim 107, wherein the downstream FAK target is selected from paxillin, ERK/MAPK, and Rho/Rac, and / or a combination thereof.
109. A monoclonal antibody or antigen binding fragment thereof for use in a method of decreasing and / or inhibiting FAK phosphorylation or auto-phosphorylation in a human subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 90.
110. The monoclonal antibody or antigen binding fragment thereof for use according to claim 109, wherein the ability of FAK to phosphorylate or auto-phosphorylate is decreased.
111. The monoclonal antibody or antigen binding fragment thereof for use according to claim 109, wherein the phosphorylation or auto-phosphorylation of FAK at one or more tyrosine amino acid residues is decreased.
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022148412A1 (en) * 2021-01-08 2022-07-14 北京韩美药品有限公司 Antibody specifically binding to cd47 and antigen-binding fragment thereof
US11692035B2 (en) 2016-10-21 2023-07-04 Arch Oncology, Inc. Therapeutic CD47 antibodies
US11702474B2 (en) 2019-12-17 2023-07-18 Pfizer Inc. Antibodies specific for CD47, PD-L1, and uses thereof
WO2023218378A1 (en) 2022-05-11 2023-11-16 Daiichi Sankyo Company, Limited Combination of an antibody specific for a tumor antigen and a cd47 inhibitor
WO2024074724A1 (en) 2022-10-07 2024-04-11 Caedo Oncology As Monovalent cd47 binding proteins
WO2024074730A2 (en) 2022-10-07 2024-04-11 Caedo Oncology As Divalent cd47 binding proteins

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018075960A1 (en) * 2016-10-21 2018-04-26 Tioma Therapeutics, Inc. Therapeutic cd47 antibodies

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11692035B2 (en) 2016-10-21 2023-07-04 Arch Oncology, Inc. Therapeutic CD47 antibodies
US11702474B2 (en) 2019-12-17 2023-07-18 Pfizer Inc. Antibodies specific for CD47, PD-L1, and uses thereof
WO2022148412A1 (en) * 2021-01-08 2022-07-14 北京韩美药品有限公司 Antibody specifically binding to cd47 and antigen-binding fragment thereof
WO2023218378A1 (en) 2022-05-11 2023-11-16 Daiichi Sankyo Company, Limited Combination of an antibody specific for a tumor antigen and a cd47 inhibitor
WO2024074724A1 (en) 2022-10-07 2024-04-11 Caedo Oncology As Monovalent cd47 binding proteins
WO2024074730A2 (en) 2022-10-07 2024-04-11 Caedo Oncology As Divalent cd47 binding proteins

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