US20180142019A1 - Therapeutic cd47 antibodies - Google Patents

Therapeutic cd47 antibodies Download PDF

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US20180142019A1
US20180142019A1 US15/871,802 US201815871802A US2018142019A1 US 20180142019 A1 US20180142019 A1 US 20180142019A1 US 201815871802 A US201815871802 A US 201815871802A US 2018142019 A1 US2018142019 A1 US 2018142019A1
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cancer
human
cells
tumor cells
cell
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Pamela T. Manning
Robyn PURO
Juan C. Almagro
Robert W. KARR
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Arch Oncology Inc
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Arch Oncology Inc
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Priority to US15/871,802 priority Critical patent/US20180142019A1/en
Assigned to TIOMA THERAPEUTICS, INC. reassignment TIOMA THERAPEUTICS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: Karr, Robert W., MANNING, PAMELA T., PURO, Robyn, ALMAGRO, JUAN C.
Assigned to Arch Oncology, Inc. reassignment Arch Oncology, Inc. CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: TIOMA THERAPEUTICS, INC.
Priority to CA3057139A priority patent/CA3057139A1/fr
Priority to CN201880028490.2A priority patent/CN111148535A/zh
Priority to PCT/US2018/023860 priority patent/WO2018175790A1/fr
Priority to SG11201908602U priority patent/SG11201908602UA/en
Priority to AU2018240377A priority patent/AU2018240377A1/en
Priority to JP2019551449A priority patent/JP7170331B2/ja
Priority to EP18771103.1A priority patent/EP3600393A4/fr
Assigned to Arch Oncology, Inc. reassignment Arch Oncology, Inc. CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: TIOMA THERAPEUTICS, INC.
Publication of US20180142019A1 publication Critical patent/US20180142019A1/en
Priority to US16/223,009 priority patent/US20190112373A1/en
Priority to US16/452,432 priority patent/US20190309066A1/en
Priority to US16/703,484 priority patent/US11692035B2/en
Priority to US16/942,531 priority patent/US20210079091A1/en
Assigned to Arch Oncology, Inc. reassignment Arch Oncology, Inc. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MANNING, PAMELA T., PURO, Robyn, ALMAGRO, JUAN C., Karr, Robert W.
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    • 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 inhibitory receptor protein ⁇ (SIRP ⁇ ) and thrombospondin-1 (TSP1).
  • SIRP ⁇ signal inhibitory receptor protein ⁇
  • TSP1 thrombospondin-1
  • anti-CD47 mAbs 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 SIRP ⁇ ; 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 expression on human tumor cells; 16
  • 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:1, 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 (V H ) 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 V H domain, examples of which are described herein.
  • the antibody or antigen binding fragment thereof may additionally comprise a light chain variable (V L ) domain, which is paired with the V H domain to form an antigen binding domain.
  • V L 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 V L domain, examples of which are described herein.
  • any given CD47 antibody or antigen binding fragment thereof comprising a V H domain paired with a V L 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:
  • anti-CD47 mAbs 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 (V H ) and a light chain variable domain (V L ), 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 V H 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 V L 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 V H and V L pairings or combinations in parts (i) through (xxxiv) may be preserved for anti-CD47 antibodies having V H and V L domain sequences with a particular percentage sequence identity to these reference sequences.
  • the V H and/or V L domains may retain identical CDR sequences to those present in the reference sequence such that the variation is present only within the framework regions.
  • CD47 antibodies may comprise a combination of a heavy chain (HC) and a light chain (LC), wherein the combination is selected from the group consisting of:
  • 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:
  • the anti-CD47 antibodies are characterized by:
  • the anti-CD47 antibodies are characterized by:
  • the anti-CD47 antibodies are characterized by:
  • the anti-CD47 antibodies are characterized by:
  • the anti-CD47 antibodies are characterized by:
  • 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:
  • 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 microvascular 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 microvascular 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 SIRP ⁇ ; 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 expression on human tumor cells;
  • the anti-CD47 mAbs disclosed can be full-length humanized antibodies with human frameworks and constant regions of the isotypes, IgA, IgD, IgE, IgG, and IgM, more particularly, IgG1, 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.
  • a F(ab′)2 fragment e.g., a F(ab′)2 fragment, a F(ab) fragment, a single chain Fv fragment (scFv), etc.
  • 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 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 mAbs 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 SIRP ⁇ 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 SIRP ⁇ 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 SIRP ⁇ 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 SIRP ⁇ 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 mAbs to Human OV10 Cells Expressing Human CD47. Binding of VLX4 humanized mAbs (VLX4hum_01 IgG1, VLX4hum_02 IgG1, VLX4hum_01 IgG4PE, and VLX4hum_02 IgG4PE) to human CD47 was determined using a OV10 cell line expressing human CD47 (OV10 hCD47) cell-based ELISA. OV10 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. 1B Binding of VLX4 Humanized mAbs to Human OV10 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 OV10 CD47 cell-based ELISA. OV10 hCD47 cells were plated into 96 well plates and were confluent at the time of assay. Various concentrations of VLX4 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.
  • VLX4hum_06 IgG4PE VLX4hum_07 IgG4PE
  • VLX4hum_12 IgG4PE VLX4hum_13 I
  • FIG. 2A Binding of VLX4 Humanized mAbs to Human RBCs (hRBCs). Binding of VLX4 humanized mAbs (VLX4hum_01 IgG, VLX4hum_02 IgG, 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 1 hr 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 1 hr with FITC-labelled donkey anti-human antibody. Cells were washed and antibody binding measured using flow cytometry.
  • VLX4hum_07 IgG4PE VLX4hum_12 IgG4PE
  • VLX4hum_13 IgG4PE VLX4hum_13 IgG4PE
  • FIG. 3A Binding of VLX8 Humanized mAbs to Human OV10 hCD47 Cells. Binding of VLX8 IgG4PE chimera (xi) or humanized mAbs (VLX8hum_01 IgG4PE, VLX8hum_04 IgG4PE, VLX8hum_07 IgG4PE, and VLX8hum_09 IgG4PE) to human CD47 was determined using an OV10 hCD47 cell-based ELISA. OV10 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. 3B Binding of VLX8 Humanized mAbs to Human OV10 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 OV10 hCD47 cell-based ELISA. OV10 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 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 1 hr with FITC-labelled donkey anti-human antibody. Cells were washed and antibody binding measured using flow cytometry.
  • 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 1 hr 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 OV10 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 OV10 human CD47 cell-based ELISA. OV10 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 OV10 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 OV10 hCD47 cell-based ELISA. OV10 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. 6A Specific Binding of VLX Humanized mAbs to CD47. Binding of VLX humanized mAb VLX4hum_07 IgG4PE to wildtype and CD47 knockout Jurkat cells was determined by flow cytometry. Various concentrations of mAbs were added to 1 ⁇ 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 mAbs to CD47. Binding of VLX humanized mAb VLX9hum_04 IgG2 to wildtype and CD47 knockout Jurkat cells was determined by flow cytometry. Various concentrations of mAbs were added to 1 ⁇ 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 mAbs to Human RBCs. Binding of VLX9 IgG2 xi or humanized VLX9 mAbs 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 1 hr with FITC-labelled donkey anti-human antibody. Cells were washed and antibody binding measured using flow cytometry.
  • FIG. 8A Binding of VLX Humanized mAbs to Human Aortic Endothelial Cells (HAEC). Binding of VLX humanized mAbs (VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_11 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 mAbs were added to 1 ⁇ 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 mAbs to Skeletal Human Muscle Cells (SkMC). Binding of VLX humanized mAbs (VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_11 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 mAbs were added to 1 ⁇ 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. 8C Binding of VLX Humanized mAbs to Human Lung Microvascular Endothelial Cells (HMVEC-L). Binding of VLX humanized mAbs (VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_11 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.
  • VLX4hum_07 IgG4PE VLX8hum_10 IgG4PE, VLX8hum_11 IgG4PE, VLX4hum_01 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2, VLX9hum_
  • HMVEC-L were removed from the flask using acutase.
  • Various concentrations of mAbs were added to 1 ⁇ 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 mAbs to Human Renal Tubular Epithelial Cells (RTEC). Binding of VLX humanized mAbs (VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_11 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 mAbs were added to 1 ⁇ 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 mAbs to Human Peripheral Blood CD3 + Cells. Binding of VLX humanized mAbs (VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_11 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 mAbs to Human Peripheral Blood Mononuclear Cells (PBMC). Binding of VLX humanized mAbs (VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_11 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 mAbs 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 mAb 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 mAbs were added to the wells for 1 hr 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 mAb 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 mAbs were added to the wells for 1 hr 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 mAb 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 mAbs were added to the wells for 1 hr 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 mAb 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 mAbs were added to the wells for 1 hr 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 mAbs Block SIRP ⁇ binding to CD47 on Human Jurkat Cells.
  • VLX4hum_01 IgG4PE VLX4hum_07 IgG4PE
  • VLX8hum_10 IgG4PE VLX4hum_11 IgG4PE
  • VLX9hum_06 IgG2 VLX9hum_08 IgG2
  • VLX9hum_08 IgG2 a control antibody or no antibody in RPMI containing 10% FBS for 30 min at 37° C.
  • FIG. 11 VLX4 CD47 Chimeric mAbs Increase Phagocytosis of Human Jurkat Cells by Human Macrophages.
  • Human macrophages were plated at a concentration of 1 ⁇ 10 4 cells per well in a 96 well plate and allowed to adhere for 24 hrs.
  • 5 ⁇ 10 4 CFSE-labelled human Jurkat cells and 1 ⁇ g/ml of the VLX4 chimeric mAbs (VLX4 IgG1 xi, VLX4 IgG1 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 CD14. Flow cytometry was used to determine the percentage of CD14 + /CFSE + cells in the total CD14 + population.
  • FIG. 12A VLX4 Humanized mAbs Increase Phagocytosis of Human Jurkat Cells by Human Macrophages.
  • Human macrophages were plated at a concentration of 1 ⁇ 10 4 cells per well in a 96 well plate and allowed to adhere for 24 hrs.
  • 5 ⁇ 10 4 CFSE-labelled human Jurkat cells and 1 ⁇ g/ml of antibody (VLX4hum_01 IgG1 and VLX4hum_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 VLX4 Humanized mAbs Increase Phagocytosis of Human Jurkat Cells by Human Macrophages.
  • Human macrophages were plated at a concentration of 1 ⁇ 10 4 cells per well in a 96 well plate and allowed to adhere for 24 hrs.
  • 5 ⁇ 10 4 CFSE-labelled human Jurkat cells and 1 ⁇ g/ml of antibody (VLX4 IgG4PE xi, VLX4hum_06 IgG4PE, VLX4hum_07 IgG4PE, VLX4hum_012 IgG4PE and VLX4hum_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 VLX8 CD47 Chimeric mAbs Increase Phagocytosis of Human Jurkat Cells by Human Macrophages.
  • Human macrophages were plated at a concentration of 1 ⁇ 10 4 cells per well in a 96 well plate and allowed to adhere for 24 hrs.
  • 5 ⁇ 10 4 CFSE-labelled human Jurkat cells and 1 ⁇ g/ml of the VLX8 chimeric mAbs (VLX8 IgG1 N297Q xi and VLX8 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 CD14. Flow cytometry was used to determine the percentage of CD14 + /CFSE + cells in the total CD14 + population.
  • FIG. 13B VLX8 Humanized mAbs Increase Phagocytosis of Human Jurkat Cells by Human Macrophages.
  • Human macrophages were plated at a concentration of 1 ⁇ 10 4 cells per well in a 96 well plate and allowed to adhere for 24 hrs. 5 ⁇ 10 4 CFSE-labelled human Jurkat cells and 1 ⁇ g/ml of antibody (VLX8 IgG4PE xi, VLX8hum_01 IgG4PE, VLX8hum_03 IgG4PE, VLX8hum_07 IgG4PE, VLX8hum_08 IgG4PE and VLX8hum_09 IgG4PE) were added to the macrophage cultures and incubated at 37° C.
  • VLX8 IgG4PE xi VLX8hum_01 IgG4PE, VLX8hum_03 IgG4PE, VLX8hum_07 IgG4PE, VLX8hum_08 IgG
  • 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. 14A VLX9 CD47 Chimeric mAbs Increase Phagocytosis of Human Jurkat Cells by Human Macrophages.
  • Human macrophages were plated at a concentration of 1 ⁇ 10 4 cells per well in a 96 well plate and allowed to adhere for 24 hours.
  • 5 ⁇ 10 4 CFSE-labelled human Jurkat cells and 1 ⁇ g/ml of the VLX9 chimeric mAbs (VLX9 IgG1 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 CD14. Flow cytometry was used to determine the percentage of CD14+/CFSE+ cells in the total CD14+ population.
  • FIG. 14B VLX9 Humanized mAbs Increase Phagocytosis of Human Jurkat Cells by Human Macrophages. Human macrophages were plated at a concentration of 1 ⁇ 10 4 cells per well in a 96 well plate and allowed to adhere for 24 hours.
  • VLX9 IgG2 xi 5 ⁇ 10 4 CFSE-labelled human Jurkat cells and 1 ⁇ g/ml of antibody (VLX9 IgG2 xi, VLX9hum_01 IgG2, VLX9hum_02 IgG2, VLX9hum_03 IgG2, VLX9hum_04 IgG2, VLX9hum_05 IgG2, VLX9hum_06 IgG2, VLX9hum_07 IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2 and VLX9hum_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 CD14. Flow cytometry was used to determine the percentage of CD14+/CFSE+ cells in the total CD14+ population.
  • FIG. 15A Induction of Cell Death in Human Jurkat Cells by Soluble VLX4 Humanized mAbs.
  • Jurkat cells (1 ⁇ 10 4 ) were incubated with 1 ⁇ g/ml VLX4 humanized mAbs (VLX4hum_01 IgG, VLX4hum_01 IgG4PE, VLX4hum_02 IgG1, VLX4hum_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 VLX4 Humanized mAbs.
  • Jurkat cells (1 ⁇ 10 4 ) were incubated with 1 ⁇ g/ml VLX4 humanized mAbs (VLX4hum_01 IgG, VLX4hum_01 IgG4PE, VLX4hum_02 IgG1, 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 the cells that are annexin V positive/7-AAD negative (annexin V + /7-AAD ⁇ ).
  • FIG. 15C Induction of Cell Death in Human Jurkat Cells by Soluble VLX4 Humanized mAbs.
  • Jurkat cells (1 ⁇ 10 4 ) were incubated with 1 ⁇ g/ml VLX4 humanized mAbs (VLX4hum_01 IgG, VLX4hum_01 IgG4PE, VLX4hum_02 IgG1, 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 mAbs.
  • Jurkat cells (1 ⁇ 10 4 ) were incubated with 1 ⁇ g/ml VLX4 humanized mAbs (VLX4hum_06 IgG4PE, VLX4hum_07 IgG4PE, VLX4hum_08 IgG4PE, VLX4hum_11 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 mAbs.
  • Jurkat cells (1 ⁇ 10 4 ) were incubated with 1 ⁇ g/ml VLX4 humanized mAbs (VLX4hum_06 IgG4PE, VLX4hum_07 IgG4PE, VLX4hum_08 IgG4PE, VLX4hum_11 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 V + /7-AAD ⁇ ).
  • FIG. 15F Induction of Cell Death in Human Jurkat Cells by Soluble VLX4 Humanized mAbs.
  • Jurkat cells (1 ⁇ 10 4 ) were incubated with 1 ⁇ g/ml VLX4 humanized mAbs (VLX4hum_06 IgG4PE, VLX4hum_07 IgG4PE, VLX4hum_08 IgG4PE, VLX4hum_11 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-AAD positive (annexin + /7-AAD + ).
  • FIG. 16A Induction of Cell Death in Human Jurkat Cells by Soluble VLX8 CD47 Chimeric mAbs.
  • Jurkat cells (1 ⁇ 10 4 ) were incubated with 1 ⁇ g/ml VLX8 chimeric mAbs (VLX8 IgG1 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 mAbs.
  • Jurkat cells (1 ⁇ 10 4 ) were incubated with 1 ⁇ g/ml VLX8 chimeric mAbs (VLX8 IgG1 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 negative (annexin V + /7-AAD ⁇ ).
  • FIG. 16C Induction of Cell Death in Human Jurkat Cells by Soluble VLX8 Chimeric mAbs.
  • Jurkat cells (1 ⁇ 10 4 ) were incubated with 1 ⁇ g/ml VLX8 chimeric mAbs (VLX8 IgG1 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 (1 ⁇ 10 4 ) were incubated with 1 ⁇ g/ml VLX8 humanized mAbs (VLX8hum_02 IgG4PE, VLX8hum_04 IgG4PE, VLX8hum_07 IgG4PE and VLX8hum_08 IgG4PE) and chimeric mAb 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 (1 ⁇ 10 4 ) were incubated with 1 ⁇ g/ml VLX8 humanized mAbs (VLX8hum_02 IgG4PE, VLX8hum_04 IgG4PE, VLX8hum_07 IgG4PE and VLX8hum_08 IgG4PE) and chimeric mAb 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-AAD negative (annexin V + /7-AAD ⁇ ).
  • FIG. 16F Induction of Cell Death in Human Jurkat Cells by Soluble VLX8 Humanized mAbs.
  • Jurkat cells (1 ⁇ 10 4 ) were incubated with 1 ⁇ g/ml VLX8 humanized mAbs (VLX8hum_02 IgG4PE, VLX8hum_04 IgG4PE, VLX8hum_07 IgG4PE and VLX8hum_08 IgG4PE) and chimeric mAb 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-AAD positive (annexin V + /7-AAD + ).
  • FIG. 17A Induction of Cell Death of Human Jurkat Cells by Soluble VLX9 Chimeric mAbs.
  • 1 ⁇ 10 4 Jurkat cells were incubated with 1 ⁇ g/ml of the VLX9 CD47 chimeric mAbs (VLX9 IgG1 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.
  • 1 ⁇ 10 4 Jurkat cells were incubated with 1 ⁇ g/ml of the VLX9 CD47 chimeric mAbs (VLX9 IgG1 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-AAD negative (annexin V + /7-AAD ⁇ ).
  • FIG. 17C Induction of Cell Death of Human Jurkat Cells by Soluble VLX9 Chimeric mAbs.
  • 1 ⁇ 10 4 Jurkat cells were incubated with 1 ⁇ g/ml of the VLX9 CD47 chimeric mAbs (VLX9 IgG1 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-AAD positive (annexin V + /7-AAD + ).
  • FIG. 17D Induction of Cell Death in Human Jurkat Cells by Soluble VLX9 Humanized mAbs.
  • Jurkat cells (1 ⁇ 10 4 ) were incubated with 1 ⁇ g/ml VLX9 humanized mAbs (VLX9hum_01 to 10 IgG2) and chimeric mAb 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 + ).
  • FIG. 17E Induction of Cell Death in Human Jurkat Cells by Soluble VLX9 Humanized mAbs.
  • Jurkat cells (1 ⁇ 10 4 ) were incubated with 1 ⁇ g/ml VLX9 humanized mAbs (VLX9hum_01 to 10 IgG2) and chimeric mAb 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-AAD negative (annexin V + /7-AAD ⁇ ).
  • FIG. 17F Induction of Cell Death in Human Jurkat Cells by Soluble VLX9 Humanized mAbs.
  • Jurkat cells (1 ⁇ 10 4 ) were incubated with 1 ⁇ g/ml VLX9 humanized mAbs (VLX9hum_01 to 10 IgG2) and chimeric mAb 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-AAD positive (annexin V + /7-AAD + ).
  • FIG. 18 Induction of Mitochondrial Depolarization in Human Raji Cells by Soluble VLX4, VLX8 and VLX9 Humanized mAbs.
  • 1 ⁇ 10 4 Raji cells were incubated with 10 ⁇ g/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_03 IgG2, VLX9hum_06 IgG2 and VLX9hum_08 IgG2), a negative IgG control antibody or 1 ⁇ M 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 mAbs cause an Increase in Cell Surface Calreticulin Expression on Human Raji Cells.
  • 1 ⁇ 10 4 Raji cells were incubated with 10 ⁇ g/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 ⁇ M 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 mAbs cause an Increase in Cell Surface Protein Disulfide-Isomerase A3 (PDIA3) Expression on Human Raji Cells.
  • VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l1 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2 a negative IgG control antibody or 1 ⁇ M of mitoxantrone as a positive control in RPMI media at 37° C. for 24 hours. Cells were washed and PDIA3
  • FIG. 21 Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase Cell Surface HSP70 Expression on Human Raji Cells. 1 ⁇ 10 4 Raji cells were incubated with 10 ⁇ g/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 ⁇ M 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. 1 ⁇ 10 4 Raji cells were incubated with 10 ⁇ g/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 ⁇ M 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. 23 Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase Release of Adenosine Triphosphate (ATP) by Human Raji Cells.
  • 1 ⁇ 10 4 Raji cells were incubated with ⁇ g/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 ⁇ M 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. 24 Soluble VLX4, VLX8 and VLX9 Humanized mAbs cause an Increase in Release of High Mobility Group Box 1 (HMGB1) by Human Raji Cells.
  • HMGB1 High Mobility Group Box 1
  • 1 ⁇ 10 4 Raji cells were incubated with 10 ⁇ g/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_03 IgG2, VLX9hum_06 IgG2 and VLX9hum_08 IgG2), a negative IgG control antibody or 1 ⁇ M of mitoxantrone as a positive control in RPMI media at 37° C. for 24 hours.
  • Cell-free supernatant was collected and analyzed using an HMGB1 immunoassay. The data are expressed as ng/ml of HMGB
  • FIG. 25 Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase CXCL10 Release by Human Raji Cells.
  • 1 ⁇ 10 4 Raji cells were incubated with 10 ⁇ g/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_03 IgG2, VLX9hum_06 IgG2 and VLX9hum_08 IgG2), a negative IgG control antibody or 1 ⁇ M 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 mAbs.
  • 1 ⁇ 10 4 Jurkat cells were incubated with 10 ⁇ g/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 ⁇ M 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. 1 ⁇ 10 4 Jurkat cells were incubated with 10 ⁇ g/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 ⁇ M 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.
  • 1 ⁇ 10 4 Jurkat cells were incubated with 10 ⁇ g/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 ⁇ M 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.
  • 1 ⁇ 10 4 Jurkat cells were incubated with 10 ⁇ g/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 ⁇ M 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.
  • 1 ⁇ 10 4 Jurkat cells were incubated with 10 ⁇ g/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 ⁇ M 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. 1 ⁇ 10 4 Jurkat cells were incubated with 10 ⁇ g/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1 ⁇ M 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 HMGB1 Release by Human Jurkat Cells.
  • 1 ⁇ 10 4 Jurkat cells were incubated with 10 ⁇ g/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_03 IgG2, VLX9hum_06 IgG2 and VLX9hum_08 IgG2), a negative IgG control antibody or 1 ⁇ M of mitoxantrone as a positive control in RPMI media at 37° C. for 24 hours. Cell-free supernatant was collected and analyzed using an HMGB1 immunoassay. The data are expressed as ng/ml of HMGB1 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 IgG1 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 ⁇ l) 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_11 IgG4PE) and the chimeric mAb VLX8 IgG4PE xi.
  • VLX8hum_01 IgG4PE VLX8hum_02 IgG4PE
  • VLX8hum_03 IgG4PE VLX8hum_08 IgG4PE
  • VLX8hum_09 IgG4PE VLX8hum_10 IgG4PE and
  • hRBCs were added to U-bottomed 96 well plates with equal volumes of the antibodies (75 ⁇ l) 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 ⁇ l) 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 ⁇ l) and incubated for 3 hrs at 37° C. and overnight at 4° C.
  • FIG. 35 VLX4 Humanized mAb 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 5 ⁇ 10 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 5 ⁇ /week for 4 weeks by intraperitoneal injection. Tumor volumes and body weights were recorded twice weekly.
  • FIG. 36 VLX8 Humanized mAb 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 5 ⁇ 10 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.
  • VLX8hum_10 IgG4PE or PBS (control) administration was initiated at this time. Mice were treated with 5 mg/kg of antibody 5 ⁇ /week for 4 weeks by intraperitoneal injection. Tumor volumes and body weights were recorded twice weekly.
  • FIG. 37 VLX9 Humanized mAb 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 5 ⁇ 10 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.
  • VLX9hum_08 IgG2 or PBS (control) administration was initiated at this time. Mice were treated with 5 mg/kg of antibody 5 ⁇ /week for 4 weeks by intraperitoneal injection. Tumor volumes and body weights were recorded twice weekly.
  • FIG. 38A Hemoglobin Levels in Blood Following Administration of a Humanized VLX9 mAb to Cynomolgus Monkeys by Intravenous Infusion.
  • VLX9hum_08 IgG2 or vehicle were administered as a one hour intravenous infusion a dose of 5 mg/kg on day 1 and a dose of 15 mg/kg on day 18. Hemoglobin levels were monitored throughout the study and normalized to control values.
  • FIG. 38B RBC Levels in Blood Following Administration of Humanized VLX9 mAbs to Cynomolgus Monkeys by Intravenous Infusion.
  • VLX9hum_08 IgG2 or vehicle was administered as a one hour intraveneous infusion a dose of 5 mg/kg on day 1 and a dose of 15 mg/kg on day 18.
  • RBC levels were monitored throughout the study and normalized to control values.
  • CD47 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.
  • 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.
  • 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.
  • Ig 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
  • mAbs of the present disclosure preferably exist in a homogeneous or substantially homogeneous population. Complete mAbs contain 2 heavy chains and 2 light chains.
  • 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 IgG1, 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 “HCDR1, 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 (H1, H2, H3) and three in VL (L1, L2, L3).
  • the CDRs used herein are as defined by Kabat except in H-CDR1, which is extended to include H1.
  • Ig heavy chains There are five types of mammalian immunoglobulin (Ig) heavy chains, denoted by the Greek letters ⁇ (alpha), ⁇ (delta), ⁇ (epsilon), ⁇ (gamma), and ⁇ (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, IgG1, 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 ⁇ , ⁇ , and ⁇ have a constant region composed of three tandem immunoglobulin (Ig) domains, and a hinge region for added flexibility.
  • Heavy chains t 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 ( ⁇ ) or lambda ( ⁇ ), 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, ⁇ or ⁇ , 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, EC 50 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, Calif., 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).
  • BLAST algorithm One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in (Altschul, S. et al., NCBI NLM 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
  • 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 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(1):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 (center-to-center) of any atom of a CDR amino acid in a three dimensional immunoglobulin model.
  • 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.
  • 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 mAbs can contain the set of CDRs from a single murine mAb 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 mAbs comprising a mixture of CDRs from the disclosed murine mAbs 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.
  • mAbs 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 mAbs 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 IgG1 isotype.
  • the human IgG1 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 IgG1 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).
  • 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
  • ADCC Antibody-Dependent Cellular Cytotoxicity
  • CDC Complement-Dependent Cytotoxicity
  • the human IgG1 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 IgG1 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), C1q binding, and altered binding to Fc receptors.
  • the human IgG1 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 IgG1 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 IgG1 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, H268Q/A330S/V309L/P331S, H268D/A330S/V309L/P331S, H268Q/A330R/V309L/P331S, H268D/A330R/V309L/P331S, H268D/A330R/V309L/P331S, E233D/A330R, E233D/A330S, E233D/P271G/A330R, E233D/P271G/A330S, G
  • 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 C1q 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 (Idusogie et al., 2001).
  • 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, V234A/G237A/P238S/H268A/V309L/A330S/P331S, H268Q/A330S/V309L/P331S, H268D/A330S/V309L/P331S, H268Q/A330R/V309L/P331S, H268D/A330R/V309L/P331
  • 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 (CH1 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 al., 1994); S228P, L235E and S228P/L235E (Reddy et al., 2000).
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer and “cancerous” refer 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.
  • susceptible cancer 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-nitrosothiols
  • 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. (2001 Nature 410 (6825): 212-5), CMF-1571, and A-350619 (reviewed in Evgenov et al. (2006) Nat. Rev.
  • 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.
  • treating 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.
  • 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.
  • anti-CD47 mAbs with distinct functional profiles 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 SIRP ⁇ ; 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
  • 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
  • SIRP ⁇ Signal Regulatory Protein alpha
  • TSP1 Thrombospondin-1
  • SIRP ⁇ on a phagocyte engages CD47 on a target cell
  • this interaction prevents phagocytosis of the target cell.
  • CD47 and SIRP ⁇ effectively sends a “don't eat me” signal to the phagocyte (Oldenborg et al. Science 288: 2051-2054, 2000).
  • Blocking the interaction of SIRP ⁇ and CD47 with an anti-CD47 mAb 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 SIRP ⁇ , 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 SIRP ⁇ , 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. 277: 10028-10036, 2002; Rebres et al. J.
  • the term “blocks SIRP ⁇ binding to human CD47” refers to a greater than 50% reduction of SIRP ⁇ -Fc binding to CD47 on cells by an anti-CD47 mAb 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 SIRP ⁇ 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 SIRP ⁇ binding to CD47 increase phagocytosis of cancer cells. SIRP ⁇ 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 mAb compared to either untreated cells or cells treated with a negative control antibody.
  • 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 (U.S. Pat. No. 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.
  • inducing cell death or “kills” and the like, are used interchangeably 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 treatment with an anti-CD47 mAb. These features may be quantitated in three cell populations: annexin V positive (annexin V + ), annexin V positive/7-AAD negative (annexin V + /7-AAD ⁇ ) and annexin V positive/7-AAD positive (annexin V + /7-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 mAb 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).
  • 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 mAb compared to the background obtained with a negative control, humanized isotype-matched antibody or no treatment.
  • Cell viability assays are described in NCI/NIH 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, Helene 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. 111: 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 B6H12 (Brown et al. J. Cell Biol., 1990, Oliveira et al.
  • 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 mAb 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 refers to no measurable binding to hRBCs at an anti-CD47 mAb 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 mAbs Because of this expression, it is expected that some anti-CD47 mAbs 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 mAbs 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 mAbs 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 mAb binding to these cells which is 8-fold or greater than the apparent Kd of the anti-CD47 mAb binding to a human tumor cell, wherein the tumor cell is OV10 hCD47.
  • minimal binding 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 mAb 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 CD3+ cells, and human peripheral
  • 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, U.S. Pat. No. 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 (U.S. Pat. No. 9,045,541, Petrova et al. Cancer Res 2015; 75(15 Suppl): Abstract no. 4271).
  • Hemagglutination has been shown to be reduced/eliminated by reducing the binding selectively to human RBCs, but not other cells, using a SIRP ⁇ -Fc fusion protein (Uger R. et al. Blood 2013; 122(21): 3935).
  • mouse anti-CD47 mAb 2A1 and humanized versions of 2A1 have been reported to block CD47/SIRP ⁇ but do not exhibit hemagglutination activity in a washed RBC assay (U.S. Pat. No. 9,045,541; Narla et al. Proc Am Assoc Cancer Res 58: 1200, 2017; abst 4694).
  • hemagglutination refers to cellular clumping
  • hemagglutination refers to clumping of a specific subset of cells, i.e., RBCs.
  • hemagglutination is a type of agglutination.
  • reduced hemagglutination refers to detectable agglutination activity of hRBCs at anti-CD47 mAb concentrations greater than or equal to 1.85 ⁇ g/ml, and no measurable activity at concentrations less than 1.85 ⁇ g/ml in a washed RBC assay, as visualized by discrete punctate dot compared to a diffuse pattern that represents hemagglutination.
  • no detectable hemagglutination refers to no visible or detectable agglutination activity of hRBCs at anti-CD47 mAb concentrations greater or equal to 0.3 pg/ml to a concentration less than or equal to 10 ⁇ g/ml in a washed RBC assay, as visualized by discrete punctate dot compared to a diffuse pattern that represents hemagglutination.
  • Some of the anti-CD47 antibodies described herein cause reduced or no detectable hemagglutination of human RBCs.
  • 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 mAbs 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 mAb compared to a negative control, humanized isotype-matched antibody or no treatment.
  • immediate reversal of NO pathway inhibition refers to 20-80% reversal of TSP1 inhibition of NO signaling by an anti-CD47 mAb 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 mAb compared to a negative control, humanized isotype-matched antibody or no treatment.
  • ICD immunogenic cell death
  • 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 (HMGB1), 4) annexin A1 release, 5) type I interferon release and 6) C-X-C motif chemokine ligand 10 (CXCL10) release.
  • DAMPs damage-associated molecular patterns
  • CDAMs 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 A1 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.
  • HMGB1 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 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.
  • 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 eIF2 ⁇ 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 Th17 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 HMGB1 and TLR4, as demonstrated by abrogation of the immune response by inhibition of HMGB1 and knock-out TLR4.
  • 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).
  • ATP-elicited production of IL-10 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.
  • HMGB1, a TLR4 agonist also contributes to the stimulation of the NLRP3 inflammasome in DCs and the secretion of IL-13.
  • 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, CD11c + CD11b + Ly6C hi cells, are particularly important for the induction of anticancer immune responses by anthracyclines.
  • Type I interferons bind to IFN- ⁇ and IFN- ⁇ 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. Science 350: 972-978, 2015 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 A1 (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 Fpr1 ⁇ / ⁇ 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).
  • obinutuzumab and tositumomab are forms of immunogenic cell death characterized by the release of HMGB1, HSP90 and ATP.
  • a type I anti-CD20 mAb did not cause release of HMGB1, HSP90 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 HMGB1 and ATP on dendritic cells. In contrast to the results reported by Cheadle et al., Zhao et al.
  • 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.
  • “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 mAb 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 A1, 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 mAb 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 mAb 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 mAb 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 PDIA3 expression on human tumor cells” refers to a statistically significant increase (p ⁇ 0.05) in PDIA3 expression by a soluble anti-CD47 mAb compared to the background obtained with a negative control, humanized isotype-matched antibody or no treatment.
  • “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 mAb 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 mAb 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.
  • “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).
  • 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:
  • the anti-CD47 antibodies are characterized by:
  • the anti-CD47 antibodies are characterized by:
  • the anti-CD47 antibodies are characterized by:
  • the anti-CD47 antibodies are characterized by:
  • the anti-CD47 antibodies are characterized by:
  • 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 SIRP ⁇ ; 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 expression on human
  • a monoclonal antibody, or an antigen binding fragment thereof which: binds to human CD47; blocks SIRP ⁇ 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 SIRP ⁇ 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.
  • 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 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 SIRP ⁇ , 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).
  • SIRP ⁇ an inhibitory receptor that prevents phagocytosis of CD47-bearing cells
  • Antibodies that block CD47 and prevent its binding to SIRP ⁇ 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; U.S. Pat. No. 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 mAb 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 mAb 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 mAb 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 mAb 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 mAb 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 mAb (Manna and Frazier (2003) J. Immunol. 170: 3544-53).
  • mAb 1F7 also blocks binding of SIRP ⁇ 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 mAb that can accomplish both functions may be superior to one that only blocks CD47/SIRP ⁇ binding.
  • anti-CD47 mAbs An additional mechanism by which anti-CD47 mAbs 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 mAb 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 A1, type I interferons and CXCL10 and tumor cell expression of calreticulin, PDIA3, HSP70 and HSP90.
  • IRI ischemia-reperfusion injury
  • 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.
  • 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 mAbs 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 mAb 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 TSP1-mediated CD47 signaling.
  • anti-CD47 mAb 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 mAb ameliorated atherosclerosis in multiple mouse models (Kojima et al. Nature., 2016).
  • 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-
  • 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 SIRP ⁇ , 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), CD154 (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
  • YERVOY® (ipilimumab; Bristol-Meyers Squibb) is an example of an approved anti-CTLA-4 antibody.
  • KEYTRUDA® pembrolizumab; Merck
  • OPDIVO® nivolumab; Bristol-Meyers Squibb Company
  • TECENTRIQ® (atezolizumab; Roche) is an example of an approved anti-PD-L1 antibody.
  • IRI Ischemia-Reperfusion Injury
  • a CD47 mAb 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 include: organ transplantation in which a mAb 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 mAb 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, rheumatoi
  • Anti-CD47 mAbs 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. Acad. Sci.
  • the agent that inhibits cyclic nucleotide phosphodiesterases can be selected from, tadalafil, vardenafil, udenafil, sildenafil and avanafil.
  • 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-aminosalicylic acid,
  • 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 mAb 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 SIRP ⁇ /CD47 binding such as antibodies to SIRP ⁇ , as well as soluble protein fragments of this ligand, or CD47 itself, inhibiting binding of, or interfering with binding of, SIRP ⁇ to CD47.
  • modulators of SIRP ⁇ /CD47 binding such as antibodies to SIRP ⁇ , as well as soluble protein fragments of this ligand, or CD47 itself, inhibiting binding of, or interfering with binding of, SIRP ⁇ 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, all 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 mAb 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 mAb, or an antigen binding fragment thereof, and assaying for binding of the anti-CD47 mAb to a patient's tumor sample, wherein binding of the anti-CD47 mAb to the patient sample is diagnostic of CD47 expression.
  • the patient's sample is a sample containing tumor cells.
  • binding of the anti-CD47 mAb 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 mAbs binding to cells in a patient's sample is associated with increased CD47 expression.
  • the anti-CD47 mAbs 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 mAbs 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 mAb to measure CD47 expression. Suitable methods may be selected with reference to the nature of any reporter moiety used to label the anti-CD47 mAbs 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.
  • HCDR1 HCDR2 HCDR3 Vx4-HCDR1 Vx4-HCDR2 Vx4-HCDR3 GYTFTNYVIH YIYPYNDGILYNEKFKG GGYYVPDY (SEQ ID NO: 1) (SEQ ID NO: 4) (SEQ ID NO: 7) Vx4-HCDR3 GGYYVYDY (SEQ ID NO: 8) Vx8-HCDR1 Vx8-HCDR2 Vx8-HCDR3 GYSFTNYYIH YIDPLNGDTTYNQKFKG GGKRAMDY (SEQ ID NO: 2) (SEQ ID NO: 5) (SEQ ID NO: 9) Vx9-HCDR1 Vx9-HCDR2 Vx9-HCDR3 GYTFTNYWIH YTDPRTDYTEYNQKFKD GGRVGLGY (SEQ ID NO: 3) (SEQ ID NO: 6) (SEQ ID NO: 10)
  • Vx4murH01 SEQ ID NO: 21
  • Vx4mur-H02 SEQ ID NO: 22
  • Vx4humH01 SEQ ID NO: 23
  • Vx4humH02 SEQ ID NO: 24
  • Vx9humH15 SEQ ID NO: 39
  • Vx9humH16 SEQ ID NO: 40
  • 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 IgG1, IgG1-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.
  • humanized antibodies 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.
  • CDRs complementarity determining regions
  • the humanized variable domains were then combined with a secretion signal and human kappa and human Fc IgG1, IgG1-N297Q, IgG2, IgG3, IgG4 S228P and IgG4 PE constant domains, cloned into a mammalian expression system, and transfected into CHO cells to generate humanized mAbs.
  • the humanized variants were expressed as full length IgG molecules, secreted into the medium and purified using protein A.
  • a non-glycosylated version (IgG1-N297Q) was created by site directed mutagenesis of heavy chain position 297 to change the asparagine to glutamine (Human Fc IgG1-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 hCD47 cells expressing cell surface human CD47.
  • OV10 hCD47 cells were grown in IMDM medium containing 10% heat inactivated fetal bovine serum (BioWest; S01520).
  • 3 ⁇ 10 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. Cells were washed, various concentrations of purified antibodies added in IMDM and incubated at 37° C.
  • 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.
  • VLX8 humanized mAbs 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_11 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 OV10 hCD47 tumor cells whereas VLX8hum_10 IgG4PE exhibited reduced to hRBCs ( FIG.
  • Table 1 shows the apparent binding affinities of VLX9 chimeric mAbs to human OV10 hCD47 cells and to human RBCs. All of the chimeric mAbs bound to OV10 hCD47 tumor cells with apparent binding constants in the picomolar range. Similarly, the humanized VLX9 mAbs bound to human OV10 hCD47 tumor cells in a concentration-dependent manner ( FIG. 5A and FIG. 5B ) with apparent affinities in the picomolar to nanomolar range (Table 2).
  • VLX9 chimeric mAbs bound to hRBCs with apparent Kd values in the picomolar range and these were similar to the apparent K d values obtained for OV10 hCD47 tumor cells by ELISA (Table 1).
  • the VLX9 humanized mAbs VLX9hum_01 IgG2, VLX9hum_02 IgG2 and VLX9hum_07 IgG2 exhibited reduced binding to human RBCs ( FIG. 7 , Table 2).
  • 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 mAbs to tumor cells and RBCs was unexpected as the VLX9 IgG2 chimeric mAbs all bound with similar apparent Kd values to both tumor and RBC CD47 (Table 1).
  • CD47 humanized mAbs were 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 1 ⁇ 10 4 cells were resuspended media and incubated with various antibody concentrations for one hour at 370 in 5% CO 2 . Cells were then washed twice with 1 ⁇ 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% CO 2 . Cells were then washed twice with 1 ⁇ PBS and resuspended in 1 ⁇ PBS. Median fluorescence intensity was determined by flow cytometry and the apparent binding affinities determined by non-linear fit (Prism GraphPad software).
  • 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 3 shows the apparent binding affinities of the humanized VLX4 and VLX8 mAbs 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 mAbs bind to mouse, rat, rabbit (data not shown) and cynomolgus monkey RBCs with apparent Kd values in the picomolar to nanomolar range.
  • Binding of soluble anti-CD47 mAbs 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 mAbs VLX4hum_07 IgG4PE, VLX8hum_11 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 1000 nM His-tagged human CD47 (Acro 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_11 IgG4PE, VLX9hum_08 IgG2 and VLX9hum_03 IgG2 are shown in Table 4.
  • 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.
  • 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 1 ⁇ 10 4 cells were incubated with various antibody concentrations for one hour at 37°, 5% CO 2 .
  • 1 ⁇ 10 4 cells were resuspended in the recommended media and incubated with various antibody concentrations for one hour at 37°, 5% CO 2 .
  • Cells were then washed twice with 1 ⁇ 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% CO 2 .
  • PBMC peripheral blood mononuclear cells
  • FcR blocking reagent Miltenyi Biotec
  • CD3 cells were detected using an allophycocyanin (APC)-labelled anti-CD3 antibody (BD BioSciences) which was added at the same time as the FITC-labelled goat anti-human IgG (H+L) antibody.
  • APC allophycocyanin
  • H+L goat anti-human IgG
  • 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.
  • 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).
  • Binding by soluble anti-CD47 mAbs to immobilized recombinant human CD47 and to human CD47 expressed on cells was measured in vitro.
  • His-CD47 AcroBiosystems
  • His-CD47 AcroBiosystems
  • 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).
  • an Anti-Human IgG (GE Lifesciences) was amine coupled to a CM5 chip on flow cells 1 and 2.
  • An Fc-tagged human CD47 (Acro 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 100 nM VLX8hum_11 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.
  • Jurkat cells were grown in RPMI medium containing 10% heat inactivated fetal bovine serum (BioWest; S01520). The cells were washed and 1 ⁇ 10 4 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 Alexa488, JacksonImmunoresearch) 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).
  • 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.
  • VLX4hum_07 IgG4PE FIG. 9C
  • VLX8hum_10 IgG4PE FIG. 9D
  • VLX9 antibody and VLX9 chimeric antibodies containing human Fc from isoytpes IgG1, IgG2 and IgG4PE did not display pH dependence (Table 6) whereas VLX9hum_04 as either an IgG, IgG2 or an IgG4PE demonstrated greater binding to His-CD47 at acidic pH (Table 7).
  • Table 8 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.
  • VLX8hum_11 Fab fragment and VLX9hum_08 Fab were tested for humanized mAbs VLX8hum_11 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_11 Fab did not.
  • the on-rate, off-rate and dissociation constants for VLX8hum_11 Fab and VLX9hum_08 Fab are shown in Table 9.
  • Table 10 illustrates the pH dependent binding exhibited by VLX9hum_04 IgG2 to CD47 expressed on Jurkat cells. No pH dependent binding was exhibited by VLX4hum_07 IgG4PE. This pH dependence of the VLX9 humanized mAbs imparts an additional desirable antibody property and therapeutic benefit in the treatment of cancer.
  • Murine VLX9 and mouse-human chimeric VLX9 Binding to CD47 is not pH Dependent KD (pM) KD (pM) pH 6 pH 8 VLX9 IgG (murine) 91 76 VLX9 IgG1-N297Q (xi) 99 135 VLX9 IgG2 (xi) 130 137 VLX9 IgG4PE (xi) 133 160
  • VLX9hum_04 Humanized mAbs Bind to CD47 in a pH Dependent Manner and Binding is not Isotype Specific KD (pM) KD (pM) pH 6 pH 8
  • VLX8hum_11 Fab and VLX9hum_08 Fab to Recombinant Human CD47 k a k d K D (nM)
  • SIRP ⁇ -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 ⁇ 10 6 Jurkat cells were incubated with humanized mAbs (5 ⁇ g/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 SIRP ⁇ -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 SIRP ⁇ -Fc bound to the Jurkat cells analyzed by flow cytometry.
  • the humanized VLX4, VLX8 and VLX9 mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_03 IgG2, VLX9hum_06 IgG2 and VLX9hum_08 IgG2) blocked the interaction of CD47 expressed on the Jurkat cells with soluble SIPR ⁇ , while the human control antibody (which does not bind to CD47) or media alone, did not block the CD47/SIRP ⁇ interaction.
  • Human derived macrophages were derived from leukapheresis of healthy human peripheral blood and incubated in AIM-V media (Life Technologies) for 7-10 days.
  • AIM-V media Life Technologies
  • macrophages were re-plated at a concentration of 1 ⁇ 10 4 cells per well in 100 ul of AIM-V media in a 96-well plate and allowed to adhere for 24 hrs.
  • the target human cancer cells (Jurkat) were labelled with 1 ⁇ M 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE; Sigma Aldrich) and added to the macrophage cultures at a concentration of 5 ⁇ 10 4 cells in 1 ml of AIM-V media (5:1 target to effector ratio).
  • VLX4, VLX8, and VLX9 CD47 mAbs (1 ⁇ g/ml) were added immediately upon mixture of target and effector cells and allowed to incubate at 37° C. for 2-3 hours.
  • VLX4 chimeric mAbs VLX4 IgG1 xi, VLX4 IgG1 N297Q xi, VLX4 IgG4PE xi, and VLX4 IgG4 S228P xi increased phagocytosis of Jurkat cells by human macrophages by blocking the CD47/SIRP ⁇ interaction. This enhanced phagocytosis is independent of Fc function.
  • humanized mAbs VLX4hum_01 IgG1, 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/SIRP ⁇ interaction. This enhanced phagocytosis is independent of Fc function.
  • VLX8 chimeric mAbs VLX8 IgG1 N297Q xi and VLX8 IgG4PE xi increase phagocytosis of Jurkat cells by human macrophages by blocking the CD47/SIRP ⁇ interaction. This enhanced phagocytosis is independent of Fc function.
  • VLX9 IgG1 N297Q xi, VLX9 IgG2 xi and VLX9 IgG4PE xi chimeric mAbs all increased phagocytosis of Jurkat cells by human macrophages by blocking the CD47/SIRP ⁇ interaction. This enhanced phagocytosis is independent of Fc effector function.
  • all of the humanized VLX9 IgG2 mAbs (VLX9hum_01 to _10 IgG2) increased phagocytosis of Jurkat cells.
  • 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 SIRP ⁇ binding to CD47.
  • Induction of cell death by soluble anti-CD47 mAbs is measured in vitro (Manna et al. (2003) J. Immunol. 107 (7): 3544-53).
  • 1 ⁇ 10 5 transformed human T cells Jurkat cells
  • soluble humanized VLX4, VLX8, and VLX9 CD47 mAbs (1 ⁇ g/ml) for 24 hrs at 37° C.
  • 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.
  • PS phosphatidylserines
  • PI propidium iodide
  • 7-AAD 7-aminoactinomycin D
  • annexin V and PI or 7-aminoactinomycin D (7-AAD) 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 V + /7-AAD ⁇ ) cells are observed in early stages of cell death and annexin V positive/7-AAD positive (annexin V + /7-AAD + ) cells are dead cells.
  • 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.
  • other cells e.g., NK cells, T cells, or macrophages
  • 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.
  • 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 V + /7-AAD ⁇ ( FIG. 15B and FIG. 15E ), or annexin V + /7-AAD + ( FIG. 15C and FIG. 15F ).
  • the humanized mAbs VLX4hum_01 IgG1, VLX4hum_01 IgG4PE, VLX4hum_02 IgG1, VLX4hum_02 IgG4PE, VLX4hum_06 IgG4 PE, VLX4hum_07 IgG4PE, VLX4hum_12 IgG4PE, and VLX4hum_13 IgG4PE caused increased PS exposure and cell death.
  • the humanized mAbs VLX4hum_08 IgG4PE and VLX4hum_11 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.
  • the soluble VLX8 chimeric and humanized mAbs induced increased PS exposure and cell death of Jurkat cells as measured by the % of the cells that are annexin V + ( FIGS. 16A , D), annexin V + /7-AAD ⁇ ( FIGS. 16B , E), or annexin V + /7-AAD + ( FIGS. 16C , F).
  • 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.
  • the soluble VLX9 chimeric and humanized 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 V + /7-AAD ⁇ ( FIG. 17B and FIG. 17E ), or annexin V + /7-AAD + ( FIG. 17C and FIG. 17F ).
  • the chimeric VLX9 IgG2xi mAb 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.
  • 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.
  • DAMP Damage-Associated Molecular Pattern
  • JC-1 dye Thermo; Catalogue #M34152.
  • Human Raji lymphoma cells ATCC, Manassas, Va.; Catalog # CCL-86
  • Cells were grown in RPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovine serum (BioWest; Catalogue # S01520), 100 units/mL penicillin, 100 lag mL streptomycin (Sigma; Catalogue # P4222) at densities less than 1 ⁇ 10 6 cells/mL.
  • Raji cells were plated in 96 well tissue culture plates at a density of 1 ⁇ 10 5 cells/ml RPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovine serum (BioWest; Catalog # S01520), 100 units/mL penicillin, 100 ⁇ g/mL streptomycin (Sigma; #P4222).
  • the humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 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 ⁇ g/ml. As a positive control for loss of mitochondrial membrane potential, cells were treated with 1 ⁇ M of chemotherapeutic anthracycline mitoxantrone. The cells were incubated at 37° C.
  • Some of the chimeric or humanized antibodies induce the loss of mitochondrial membrane potential in the tumor cell.
  • the percent of cells with mitochondrial membrane depolarization in all anti-CD47 mAb 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.
  • calreticulin was determined using a rabbit monoclonal antibody against calreticulin conjugated to Alexa Fluor 647 (Abcam; Catalogue #ab196159).
  • 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 lag mL streptomycin (Sigma; Catalogue # P4222) at densities less than 1 ⁇ 10 6 cells/mL.
  • cells were plated in 96 well tissue culture plates at a density of 1 ⁇ 10 5 cells/ml RPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovine serum (BioWest; Catalog # S01520), 100 units/mL penicillin, 100 ⁇ g/mL streptomycin (Sigma; #P4222).
  • the humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 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 ⁇ g/ml.
  • As a positive control for calreticulin exposure cells were treated with 1 ⁇ M of chemotherapeutic anthracycline mitoxantrone. The cells were incubated at 37° C.
  • the humanized antibodies induced the preapoptotic exposure of calreticulin on the tumor cell surface.
  • the percent of calreticulin positive cells in all anti-CD47 mAb 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.
  • PDIA3 Cell surface exposure of PDIA3 was determined using a mouse monoclonal antibody against PDIA3 conjugated to FITC (Abcam; Catalogue #ab183396).
  • 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 ⁇ g mL streptomycin (Sigma; Catalogue # P4222) at densities less than 1 ⁇ 10 6 cells/mL.
  • cells were plated in 96 well tissue culture plates at a density of 1 ⁇ 10 5 cells/ml RPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovine serum (BioWest; Catalog # S01520), 100 units/mL penicillin, 100 ⁇ g/mL streptomycin (Sigma; #P4222).
  • the humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 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 ⁇ g/ml.
  • As a positive control for PDIA3 exposure cells were treated with 1 ⁇ M of chemotherapeutic anthracycline mitoxantrone. The Raji cells were incubated at 37° C.
  • Some of the chimeric or humanized antibodies induce the preapoptotic exposure of PDIA3 on the tumor cell surface.
  • the percent of PDIA3 positive cells in all the soluble anti-CD47 mAb 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.
  • HSP70 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 ⁇ g mL streptomycin (Sigma; Catalogue # P4222) at densities less than 1 ⁇ 10 6 cells/mL.
  • RPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovine serum (BioWest; Catalogue # S01520), 100 units/mL penicillin, 100 ⁇ g mL streptomycin (Sigma; Catalogue # P4222) at densities less than 1 ⁇ 10 6 cells/mL.
  • cells were plated in 96 well tissue culture plates at a density of 1 ⁇ 10 5 cells/ml RPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovine serum (BioWest; Catalog # S01520), 100 units/mL penicillin, 100 ⁇ g/mL streptomycin (Sigma; #P4222).
  • the humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 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 ⁇ g/ml.
  • Raji cells were treated with 1 ⁇ M of chemotherapeutic anthracycline mitoxantrone. The cells were incubated at 37° C.
  • Some of the chimeric or humanized antibodies induce the preapoptotic exposure of HSP70 on the tumor cell surface.
  • the percent of HSP70 positive cells in all anti-CD47 mAb 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.
  • HSP90 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 ⁇ g mL streptomycin (Sigma; Catalogue # P4222) at densities less than 1 ⁇ 10 6 cells/mL.
  • RPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovine serum (BioWest; Catalogue # S01520), 100 units/mL penicillin, 100 ⁇ g mL streptomycin (Sigma; Catalogue # P4222) at densities less than 1 ⁇ 10 6 cells/mL.
  • cells were plated in 96 well tissue culture plates at a density of 1 ⁇ 10 5 cells/ml RPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovine serum (BioWest; Catalog # S01520), 100 units/mL penicillin, 100 ⁇ g/mL streptomycin (Sigma; #P4222).
  • the humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 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 ⁇ g/ml.
  • As a positive control for HSP90 exposure cells were treated with 1 ⁇ M of chemotherapeutic anthracycline mitoxantrone. The Raji cells were incubated at 37° C.
  • Some of the chimeric or humanized antibodies induce the preapoptotic exposure of HSP90 on the tumor cell surface.
  • the percent of HSP90 positive cells in soluble anti-CD47 mAb-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 humanized antibodies induce DAMPs from tumor cells and can lead to phagocytosis of tumor cells and processing of tumor antigen by innate immune cells.
  • ATP 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 ⁇ g mL streptomycin (Sigma; Catalogue # P4222) at densities less than 1 ⁇ 10 6 cells/mL.
  • cells were plated in 96 well tissue culture plates at a density of 1 ⁇ 10 5 cells/ml RPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovine serum (BioWest; Catalog # S01520), 100 units/mL penicillin, 100 ⁇ g/mL streptomycin (Sigma; #P4222).
  • the humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 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 ⁇ g/ml.
  • As a positive control for ATP release cells were treated with 1 ⁇ M of chemotherapeutic anthracycline mitoxantrone. The cells were incubated at 37° C.
  • 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 mAb 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.
  • HMGB1 protein Release of HMGB1 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 ⁇ g mL streptomycin (Sigma; Catalogue #P4222) at densities less than 1 ⁇ 10 6 cells/mL.
  • cells were plated in 96 well tissue culture plates at a density of 1 ⁇ 10 5 cells/ml RPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovine serum (BioWest; Catalog # S01520), 100 units/mL penicillin, 100 ⁇ g/mL streptomycin (Sigma; #P4222).
  • the humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 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 ⁇ g/ml.
  • HMGB1 release As a positive control for HMGB1 release, Raji cells were treated with 1 ⁇ M 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, 10 ⁇ l 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.
  • the humanized antibodies increased the release of HMGB1 protein from the tumor cells.
  • the amount of released HMGB1 protein in all anti-CD47 mAb 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 HMGB1 demonstrates that some of the chimeric or humanized antibodies induce release of DAMPs from tumor cells and can lead to dendritic cell activation.
  • CXCL10 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 ⁇ g mL streptomycin (Sigma; Catalogue # P4222) at densities less than 1 ⁇ 10 6 cells/mL.
  • cells were plated in 96 well tissue culture plates at a density of 1 ⁇ 10 5 cells/ml RPMI-1640 medium containing 5% (v/v) heat inactivated fetal bovine serum (BioWest; Catalog # S01520), 100 units/mL penicillin, 100 ⁇ g/mL streptomycin (Sigma; #P4222).
  • the humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 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 ⁇ g/ml.
  • Raji cells were treated with 1 ⁇ M of the chemotherapeutic anthracycline mitoxantrone. The cells were incubated at 37° C.
  • Some of the chimeric or humanized antibodies induce release of CXCL10 by human tumor cells. As shown in FIG. 25 , the amount of released CXCL10 in all anti-CD47 mAb treated cultures significantly increased (p ⁇ 0.05) compared to an isotype control. This increase in the release of CXCL10 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.
  • DAMP Damage-Associated Molecular Pattern
  • the humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 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.
  • the humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 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 mAb 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.
  • the percent of PDIA3 positive cells in soluble anti-CD47 mAb (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 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.
  • the percent of HSP70 positive cells in anti-CD47 mAb (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 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.
  • each of the anti-CD47 mAbs 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.
  • the percent of HSP90 positive cells in soluble anti-CD47 mAb (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 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.
  • the amount of released ATP in humanized anti-CD47 mAb (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2) treated cultures was significantly increased (p ⁇ 0.05) compared to an isotype control.
  • each of the anti-CD47 mAbs 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 humanized antibodies induce the release of ATP from tumor cells and can lead to dendritic cell migration through its cognate purinergic receptors.
  • the amount of released HMGB1 protein in anti-CD47 mAb (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 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 HMGB1 demonstrates that some of the chimeric or humanized antibodies induce DAMPs from tumor cells and can lead to dendritic cell activation.
  • 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 ⁇ l 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.
  • VLX4hum_01 IgG1 caused visible hemagglutination of hRBCs, whereas the humanized VLX4hum_01 IgG4PE mAb did not (mAb concentrations 50 ⁇ g/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.
  • 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_11 IgG4PE did not (mAb concentrations 50 ⁇ g/ml to 0.3 ng/ml).
  • VLX4hum_01 IgG4PE, VLX8hum_01 IgG4PE, VLX8hum_02 IgG4 PE, VLX8hum_03 IgG4 PE and VLX8hum_11 IgG4 PE imparts an additional desirable antibody property and a potential therapeutic benefit in the treatment of cancer.
  • 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 ⁇ g/ml). However, the amount of detectable hemagglutination caused by VLX9hum_07 was reduced compared to the VLX9 IgG2 chimeric mAb. Again, the reduced or lack of detectable hemagglutination by the VLX9 humanized mAbs imparts an additional desirable antibody property and a potential therapeutic benefit in the treatment of cancer.
  • 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.
  • 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, Calif.) within a 5% CO 2 atmosphere. Cultures were expanded in tissue culture flasks.
  • mice Female NSG (NOD-Cg-Prkdc scid I12rg tm1Wjl /SzJ) were obtained from Jackson Laboratory (Bar Harbor, Me.) at 5-6 weeks of age. Mice were acclimated prior to handling and housed in microisolator cages (Lab Products, Seaford, Del.) under specific pathogen-free conditions. Mice were fed Teklad Global Diet® 2920x irradiated laboratory animal diet (Envigo, Formerly Harlan; Indianapolis, Ind.) and provided autoclaved water ad libitum. All procedures were carried out under 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.
  • TSP1 binding to CD47 activates the heterotrimeric G protein Gi, which leads to suppression of intracellular cyclic AMP (cAMP) levels.
  • TSP1/CD47 pathway opposes the beneficial effects of the nitric oxide (NO) pathway in all vascular cells.
  • NO nitric oxide
  • 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).
  • stresses including, but not limited to, those resulting from wounding, inflammation, hypertension, metabolic syndrome, ischemia, and ischemia-reperfusion injury (IRI).
  • IRI ischemia-reperfusion injury
  • humanized anti-CD47 mAbs 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.
  • 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 ag mL streptomycin (Sigma; Catalogue # P4222) at densities less than 1 ⁇ 106 cells/mL.
  • cells will be plated in 96 well tissue culture plates at a density of 1 ⁇ 10 5 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 ⁇ g/mL streptomycin (Sigma; #P4222) for 24 hours and then transferred to serum free medium overnight.
  • humanized antibodies as disclosed herein purified from transient transfections in CHO 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 ⁇ g/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, Mich., Catalog #82100), will be added to half the wells at a final concentration of 1 ⁇ M. Five minutes later, the cells will be lysed with buffer supplied in the cGMP kit, and aliquots of each well assayed for cGMP content.
  • DEA diethylamine
  • TSP1 inhibition of cGMP 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 TSP on downstream effects resulting from the activation of NO signaling.
  • IRI ischemia-reperfusion injury

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JP2019551449A JP7170331B2 (ja) 2017-03-22 2018-03-22 固形及び血液癌の治療のための併用療法
EP18771103.1A EP3600393A4 (fr) 2017-03-22 2018-03-22 Polythérapie pour le traitement de cancers solides et hématologiques
SG11201908602U SG11201908602UA (en) 2017-03-22 2018-03-22 Combination therapy for the treatment of solid and hematological cancers
AU2018240377A AU2018240377A1 (en) 2017-03-22 2018-03-22 Combination therapy for the treatment of solid and hematological cancers
CN201880028490.2A CN111148535A (zh) 2017-03-22 2018-03-22 治疗实体癌和血液癌的组合疗法
PCT/US2018/023860 WO2018175790A1 (fr) 2017-03-22 2018-03-22 Polythérapie pour le traitement de cancers solides et hématologiques
CA3057139A CA3057139A1 (fr) 2017-03-22 2018-03-22 Polytherapie pour le traitement de cancers solides et hematologiques
US16/223,009 US20190112373A1 (en) 2016-10-21 2018-12-17 Therapeutic cd47 antibodies
US16/452,432 US20190309066A1 (en) 2017-03-22 2019-06-25 Combination therapy for the treatment of solid and hematological cancers
US16/703,484 US11692035B2 (en) 2016-10-21 2019-12-04 Therapeutic CD47 antibodies
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