Anti-CD47 antibodies and methods of use thereof

Abstract

Provided herein are antibodies, masked antibodies (e.g., activatable antibodies) and antigen-binding fragments that target human CD47, polynucleotides encoding them, therapeutic compositions thereof, and their use in treating CD47-positive diseases, such as cancer. The masked antibodies described herein can comprise an IgG1 Fc region.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to International Application No. PCT / CN2021 / 126597, filed October 27, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing Reference The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 695402001641seqlist.xml.txt, Recorded: October 14, 2022, Size: 288,462 bytes). [Technical field]

[0003] The present disclosure relates to antibodies, masked antibodies (eg, activatable antibodies) that target human CD47, and antigen-binding fragments thereof, polynucleotides encoding them, pharmaceutical compositions thereof, and uses thereof. [Background technology]

[0004] Binding of CD47, which is overexpressed on the surface of many types of cancer cells, to signal-regulatory protein alpha (SIRPα) on myeloid cells initiates an inhibitory signaling pathway that leads to a "don't' eat me" signal, resulting in malignant cells evading the host immune system. Therapies targeting the CD47 / SIRPα signaling axis have shown success in preclinical models and are currently in clinical trials for both solid and hematological malignancies. Anti-CD47 therapy has shown promising activity in early clinical trials, but challenges remain. For example, many different human cell types express CD47, including red blood cells (RBCs), making them a large antigen sink for anti-CD47 antibodies. Blockade of CD47 on RBCs, as with magrolimab (Hu5F9), causes transient anemia, necessitating step-up dosing in the clinic. It is worth noting that many anti-CD47 therapies, such as Hu5F9, TJC4, AK117, CC-90002, and IBI188, are IgG4 subclass type antibodies. IgG1 antibodies can guide immune cells with ADCC and ADCP capabilities to cells expressing the antigen targeted by IgG1 antibodies. Because CD47 is also widely expressed in normal tissues, it is generally believed that IgG1 subclass types should not be used for therapeutic anti-CD47 antibodies to prevent or limit on-target / off-tumor toxicity.

[0005] Therefore, there is a need for new anti-CD47 antibodies that can block the binding of CD47 to SIRPα without exhibiting the undesirable effect of binding to red blood cells and other non-cancerous cells. Summary of the Invention

[0006] To provide anti-CD47 antibodies with potent anti-tumor activity and reduced off-target effects, we developed a series of masked anti-CD47 antibodies (including activating anti-CD47 antibodies), each of which contains an N-terminal masking peptide and an IgG1 Fc region. These antibodies have dual anti-tumor activity: 1) blocking CD47-SIRPα interaction, allowing recognition and phagocytosis of tumor cells by macrophages, and 2) tumor killing through IgG1 Fc-mediated effector functions such as ADCC and ADCP. The masking peptide is designed to compete with the target binding site of the anti-CD47 antibody, thereby inhibiting on-target / off-tumor activity in healthy tissues and enabling anti-tumor activity in the tumor microenvironment (TME).

[0007] A masked antibody is designed to mask its antigen binding site with a masking site in a masking peptide, preventing the antibody from binding to its target in healthy tissues. The masking antibody may be activatable, also known as SAFEBODY™, where the masking moiety is designed to unmask in order to activate the antibody in a TME with specific activation conditions and allow the antigen binding site to bind to its target. For example, the masking peptide of an activatable antibody may contain a cleavable moiety that, when cleaved by a protease, removes the masking moiety from the antibody and activates the antibody to bind to CD47. In normal tissues and circulation, the masking moiety of an activatable anti-CD47 antibody functions to block binding to CD47 and reduce off-target effects associated with anti-CD47 antibodies, such as anemia. However, in the TME, where increased protease activity has been reported, the masking site is cleaved, allowing the activated anti-CD47 antibody to bind to CD47 on tumor cells. Thus, the activating anti-CD47 antibodies described herein exhibit enhanced anti-tumor activity and reduced toxicity to normal cells compared to existing anti-CD47 antibodies, both by inhibition of the CD47-SIRPα interaction and by inhibition of ADCC / ADCP effector function.

[0008] Thus, in some aspects, provided herein are antibodies (e.g., isolated antibodies), antigen-binding fragments, and masked antibodies (e.g., activatable antibodies) that bind to CD47 (e.g., human CD47). In some embodiments, the antibodies (e.g., isolated antibodies), antigen-binding fragments, and masked antibodies (e.g., activatable antibodies) have at least one (e.g., at least one, at least two, at least three, at least four, at least five, or all six) of the following functional properties: (a) bind to human CD47 with a KD of 500 nM or less; (b) are cross-reactive with monkey, rat, or dog CD47; (c) are capable of inhibiting tumor cell growth; (d) have a therapeutic effect against cancer; (e) block the binding between CD47 and SIRP proteins (e.g., SIRPα); and (f) induce antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP) against tumor cells expressing CD47.

[0009] Thus, in some embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from the N-terminus to the C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) that binds to human CD47; the masking peptide is linked to the N-terminus of the VH or VL; and the MM binds to the TBM in competition with human CD47. In some embodiments, 1) the VH comprises a first complementarity determining region (CDR-H1), a second complementarity determining region (CDR-H2) and a third complementarity determining region (CDR-H3), wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 182 or 183; CDR-H2 comprises the amino acid sequence of SEQ ID NO: 184 or 185; CDR-H3 comprises an amino acid sequence selected from SEQ ID NO: 186-189; and / or 2) the VL comprises a first complementarity determining region (CDR-L1), a second complementarity determining region (CDR-L2) and a third complementarity determining region (CDR-L3), wherein CDR-L1 comprises an amino acid sequence selected from SEQ ID NO: 190-193; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 194; and CDR-L3 comprises an amino acid sequence selected from SEQ ID NO: 195. or 196. In certain embodiments, the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:182, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:185, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:188; b) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:191, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:194, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:195. In some embodiments, the masked antibody comprises, from N-terminus to C-terminus, a first polypeptide comprising a masking peptide and a VL, and a second polypeptide comprising a VH. In another embodiment, the masked antibody comprises, from N-terminus to C-terminus, a first polypeptide comprising a masking peptide and a VH, and a second polypeptide comprising a VL.In further embodiments, the masked antibody comprises a first polypeptide comprising, from N-terminus to C-terminus, a masking peptide, a VL, and a VH. In yet further embodiments, the masked antibody comprises a first polypeptide comprising, from N-terminus to C-terminus, a masking peptide, a VH, and a VL. In some embodiments, the masking moiety (MM) comprises an amino acid sequence selected from SEQ ID NO:197-200. In one embodiment, the MM comprises the amino acid sequence of SEQ ID NO:199. In certain embodiments, the MM comprises the amino acid sequence of SEQ ID NO:137 and 167-181. In one embodiment, the MM comprises the amino acid sequence of SEQ ID NO:137. In some embodiments, the masking peptide, or a portion thereof (e.g., the MM), has a masking efficiency of at least about 50 as determined by Jurkat NFAT reporter assay. In some embodiments, the masking peptide, or a portion thereof (e.g., the MM), has a masking efficiency of at least about 100 as determined by Jurkat NFAT reporter assay. In some embodiments, the masked antibody comprises, from N-terminus to C-terminus, a first polypeptide comprising a masking peptide and a VL; and a second polypeptide comprising a VH. In certain embodiments, the masked antibody comprises a human IgG4 fragment crystallizable (Fc) region. In certain embodiments, the masked antibody comprises a human IgG1 Fc region. In some embodiments, the human IgG1 Fc region comprises two Fc domains, each of the two Fc domains comprising an S239D substitution and / or an I332E substitution (e.g., an S239D substitution and an I332E substitution). In some embodiments, the masked antibody binds to one or more amino acid residues selected from the group consisting of K39, W40, K41, F50, D51, G52, T99, E100, L101, and T102 of human CD47.In some embodiments, the masked antibody does not bind to one or more amino acid residues selected from the group consisting of L2, L3, F4, K6, N27, E29, A30, Q31, T34, E35, V36, Y37, D46, T49, A53, E97, R103, E104, G105, and E1062 of human CD47. In certain embodiments, the masked antibody binds to amino acid residues K39, W40, K41, F50, D51, G52, T99, E100, L101, and T102 of human CD47, and / or does not bind to amino acid residues L2, L3, F4, K6, N27, E29, A30, Q31, T34, E35, V36, Y37, D46, T49, A53, E97, R103, E104, G105, and E106 of human CD47.

[0010] In some embodiments, provided herein is a masked antibody comprising: (a) a masking peptide, comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM); (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) and binding to human CD47; and (c) an IgG1 Fc region (e.g., human IgG1 Fc region) or an IgG Fc region (e.g., human IgG Fc region) having enhanced antibody-dependent cellular cytotoxicity (ADCC) activity, wherein the masking peptide is linked to the N-terminus of the VH or VL, and the MM binds to the TBM in competition with human CD47. In some embodiments, the masked antibody comprises an IgG1 Fc region. In some embodiments, the masked antibody comprises an IgG Fc region having enhanced ADCC activity. In some embodiments, the human IgG1 Fc region comprises two Fc domains, each of which comprises an S239D substitution and / or an I332E substitution (eg, an S239D substitution and an I332E substitution).

[0011] In some embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from the N-terminus to the C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), said target binding moiety binding to human CD47; wherein the masking peptide is linked to the N-terminus of the VH or the VL; and the MM competes with human CD47 to bind to the TBM; wherein (a) the VH comprises a CDR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 35-37 and 39-50, a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 52-54 and 56-67, and a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 69-71 and 73-84; and (b) the VL comprises a CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 86-88 and 89-101, a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 87-89, and a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 89-110, and a CDR-L4 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 90-92, and a CDR-L5 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 93-94, and a CDR-L6 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 94-96, and a CDR-L7 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 95-97, and a CDR-L8 comprising an amino acid sequence selected A masked antibody comprising a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 103-105 and 106-118, and a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 120-122 and 124-135.

[0012] In certain embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from the N-terminus to the C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), and which binds to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; the MM binds to the TBM in competition with human CD47; the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, and 31; and / or the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, and 32.

[0013] In some embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from the N-terminus to the C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) that binds to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; the MM competes with human CD47 to bind to the TBM; wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:48, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:65, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:82; and 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:99, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:116, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:133. In certain embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) and binding to human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; and the VH comprises the amino acid sequence of SEQ ID NO:27 and / or the VL comprises the amino acid sequence of SEQ ID NO:28. In certain embodiments, the MM comprises the amino acid sequence of SEQ ID NO:137.

[0014] In some embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from the N-terminus to the C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) that binds to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; and the MM competes with human CD47 to bind to the TBM; wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:49, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:66, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:83; and 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:100, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:117, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:134. In certain embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) and binding to human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; and the VH comprises the amino acid sequence of SEQ ID NO:29 and / or the VL comprises the amino acid sequence of SEQ ID NO:30. In certain embodiments, the MM comprises the amino acid sequence of SEQ ID NO:137.

[0015] In some embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from the N-terminus to the C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) that binds to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; the MM competes with human CD47 to bind to the TBM; wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:50, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:67, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:84; and 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:101, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:118, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:135. In certain embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) and binding to human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; and the VH comprises the amino acid sequence of SEQ ID NO:31 and / or the VL comprises the amino acid sequence of SEQ ID NO:32. In certain embodiments, the MM comprises the amino acid sequence of SEQ ID NO:137.

[0016] In some embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from the N-terminus to the C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) that binds to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; the MM competes with human CD47 to bind to the TBM; wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:35, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:52, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:69; and 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:86, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:103, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:120. In certain embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) and binding to human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; and the VH comprises the amino acid sequence of SEQ ID NO:1 and / or the VL comprises the amino acid sequence of SEQ ID NO:2. In certain embodiments, the MM comprises the amino acid sequence of SEQ ID NO:137.

[0017] In some embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from the N-terminus to the C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) that binds to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; and the MM competes with human CD47 to bind to the TBM; wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:36, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:53, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:70; and 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:87, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:104, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:121. In certain embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) and binding to human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; and the VH comprises the amino acid sequence of SEQ ID NO:3 and / or the VL comprises the amino acid sequence of SEQ ID NO:4. In certain embodiments, the MM comprises the amino acid sequence of SEQ ID NO:137.

[0018] In some embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from the N-terminus to the C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) that binds to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; the MM competes with human CD47 to bind to the TBM; wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:37, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:54, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:71; and 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:88, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:105, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:122. In certain embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) and binding to human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; and the VH comprises the amino acid sequence of SEQ ID NO:5 and / or the VL comprises the amino acid sequence of SEQ ID NO:6. In certain embodiments, the MM comprises the amino acid sequence of SEQ ID NO:137.

[0019] In some embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from the N-terminus to the C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) that binds to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; the MM competes with human CD47 to bind to the TBM; wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:39, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:56, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:73; and 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:90, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:107, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:124. In certain embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) and binding to human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; and the VH comprises the amino acid sequence of SEQ ID NO:9 and / or the VL comprises the amino acid sequence of SEQ ID NO:10. In certain embodiments, the MM comprises the amino acid sequence of SEQ ID NO:137.

[0020] In some embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from the N-terminus to the C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), and which binds to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; and the MM competes with human CD47 to bind to the TBM; wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:40, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:57, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:74; and 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:91, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:108, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:125. In certain embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) and binding to human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; and the VH comprises the amino acid sequence of SEQ ID NO:11 and / or the VL comprises the amino acid sequence of SEQ ID NO:12. In certain embodiments, the MM comprises the amino acid sequence of SEQ ID NO:137.

[0021] In some embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from the N-terminus to the C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) that binds to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; the MM competes with human CD47 to bind to the TBM; wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:41, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:58, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:75; and 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:92, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:109, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:126. In certain embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) and binding to human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; and the VH comprises the amino acid sequence of SEQ ID NO:13 and / or the VL comprises the amino acid sequence of SEQ ID NO:14. In certain embodiments, the MM comprises the amino acid sequence of SEQ ID NO:137.

[0022] In some embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from the N-terminus to the C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) that binds to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; the MM competes with human CD47 to bind to the TBM; wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:42, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:59, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:76; and 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:93, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:110, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:127. In certain embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) and binding to human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; and the VH comprises the amino acid sequence of SEQ ID NO:15 and / or the VL comprises the amino acid sequence of SEQ ID NO:16. In certain embodiments, the MM comprises the amino acid sequence of SEQ ID NO:137.

[0023] In some embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from the N-terminus to the C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) that binds to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; the MM competes with human CD47 to bind to the TBM; wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:43, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:60, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:77; and 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:94, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:111, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:128. In certain embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) and binding to human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; and the VH comprises the amino acid sequence of SEQ ID NO:17 and / or the VL comprises the amino acid sequence of SEQ ID NO:18. In certain embodiments, the MM comprises the amino acid sequence of SEQ ID NO:137.

[0024] In some embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from the N-terminus to the C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) that binds to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; the MM competes with human CD47 to bind to the TBM; wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:44, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:61, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:78; and 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:95, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:112, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:129. In certain embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) and binding to human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; and the VH comprises the amino acid sequence of SEQ ID NO:19 and / or the VL comprises the amino acid sequence of SEQ ID NO:20. In certain embodiments, the MM comprises the amino acid sequence of SEQ ID NO:137.

[0025] In some embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from the N-terminus to the C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) that binds to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; the MM competes with human CD47 to bind to the TBM; wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:45, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:62, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:79; and 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:96, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:113, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:130. In certain embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) and binding to human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; and the VH comprises the amino acid sequence of SEQ ID NO:21 and / or the VL comprises the amino acid sequence of SEQ ID NO:22. In certain embodiments, the MM comprises the amino acid sequence of SEQ ID NO:137.

[0026] In some embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from the N-terminus to the C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) that binds to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; the MM competes with human CD47 to bind to the TBM; wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:46, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:63, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:80; and 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:97, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:114, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:131. In certain embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) and binding to human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; and the VH comprises the amino acid sequence of SEQ ID NO:23 and / or the VL comprises the amino acid sequence of SEQ ID NO:24. In certain embodiments, the MM comprises the amino acid sequence of SEQ ID NO:137.

[0027] In some embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from the N-terminus to the C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) and binds to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; and the MM competes with human CD47 to bind to the TBM; wherein 1) a) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:47, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:64, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:81; and 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:98, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:115, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:132. In certain embodiments, provided herein is a masked antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) and binding to human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; and the VH comprises the amino acid sequence of SEQ ID NO:25 and / or the VL comprises the amino acid sequence of SEQ ID NO:26. In certain embodiments, the MM comprises the amino acid sequence of SEQ ID NO:137.

[0028] In some embodiments of any one of the masked antibodies described above, the LM does not comprise a cleavable moiety (CM) comprising at least one cleavage site.

[0029] In some embodiments of any one of the masked antibodies described above, the masked antibody is an activatable antibody. Thus, in some aspects, provided herein is an activatable antibody comprising: (a) a masking peptide, comprising, from N-terminus to C-terminus, a masking portion (MM) and a linking portion (LM), wherein the LM comprises a cleavable portion (CM) comprising at least one cleavage site; and (b) a target binding portion (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), and binding to human CD47; the masking peptide is linked to the N-terminus of the VH or VL; and the MM competes with human CD47 to bind to the TBM. In a preferred embodiment, the activatable antibody has a higher binding affinity to human CD47 in vitro after CM cleavage than before CM cleavage. In some embodiments, 1) the VH comprises a first complementarity determining region (CDR-H1), a second complementarity determining region (CDR-H2) and a third complementarity determining region (CDR-H3), wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 182 or 183; CDR-H2 comprises the amino acid sequence of SEQ ID NO: 184 or 185; CDR-H3 comprises an amino acid sequence selected from SEQ ID NO: 186-189; and / or 2) the VL comprises a first complementarity determining region (CDR-L1), a second complementarity determining region (CDR-L2) and a third complementarity determining region (CDR-L3), wherein CDR-L1 comprises an amino acid sequence selected from SEQ ID NO: 190-193; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 194; and CDR-L3 comprises an amino acid sequence selected from SEQ ID NO: 195. or 196. In certain embodiments, the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 182, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 185, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 188; b) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 191, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 194, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 195.In some embodiments, an activatable antibody comprises, from N-terminus to C-terminus, a first polypeptide comprising a masking peptide and a VL, and a second polypeptide comprising a VH. In another embodiment, an activatable antibody comprises, from N-terminus to C-terminus, a first polypeptide comprising a masking peptide and a VH, and a second polypeptide comprising a VL. In a further embodiment, an activatable antibody comprises, from N-terminus to C-terminus, a first polypeptide comprising a masking peptide, a VL, and a VH. In yet a further embodiment, an activatable antibody comprises, from N-terminus to C-terminus, a first polypeptide comprising a masking peptide, a VH, and a VL. In some embodiments, the cleavable moiety (CM) cleavage site is a protease cleavage site of a protease selected from the group consisting of urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco etch virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE. In certain embodiments, the CM comprises an MMP-9 cleavage site that can be cleaved by MMP-9. In some embodiments, the CM comprises the amino acid sequence of SEQ ID NO:138. In some embodiments, the masking peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:139 and 152-166. In certain embodiments, the masking peptide comprises the amino acid sequence of SEQ ID NO:139. In some embodiments, the masking peptide, or a portion thereof (e.g., the MM), has a masking efficiency of at least about 50 as determined by a Jurkat NFAT reporter assay.In some embodiments, the masking peptide, or a portion thereof (e.g., MM), has a masking efficiency of at least about 100 as determined by Jurkat NFAT reporter assay. In some embodiments, the activatable antibody comprises, from N-terminus to C-terminus, a first polypeptide comprising the masking peptide and a VL; and a second polypeptide comprising a VH. In certain embodiments, the activatable antibody comprises a human IgG4 fragment crystallizable (Fc) region. In certain embodiments, the activatable antibody comprises a human IgG1 Fc region. In some embodiments, the human IgG1 Fc region comprises two Fc domains, each of the two Fc domains comprising an S239D substitution and / or an I332E substitution (e.g., an S239D substitution and an I332E substitution).

[0030] In some embodiments, provided herein is an activatable antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM), wherein the LM comprises a cleavable moiety (CM) comprising at least one cleavage site; and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) that binds to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; the MM competes with human CD47 to bind to the TBM; wherein (a) the VH comprises a CDR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 35-37 and 39-50, a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 52-54 and 56-67, and a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 69-71 and 73-84; and (b) the VL comprises a CDR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 69-71 and 73-84. an activatable antibody comprising a CDR-L1 having an amino acid sequence selected from the group consisting of SEQ ID NOs:86-88 and 89-101, a CDR-L2 having an amino acid sequence selected from the group consisting of SEQ ID NOs:103-105 and 106-118, and a CDR-L3 having an amino acid sequence selected from the group consisting of SEQ ID NOs:120-122 and 124-135.

[0031] In certain embodiments, provided herein is an activatable antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM), wherein the LM comprises a cleavable moiety (CM) comprising at least one cleavage site; and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), the target binding moiety binding to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; the MM binds to the TBM in competition with human CD47; the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, and 31; and / or the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, and 31; An activatable antibody comprising an amino acid sequence selected from the group consisting of NO:2, 4, 6, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 and 32.

[0032] In some embodiments, provided herein is an activatable antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM), wherein the LM comprises a cleavable moiety (CM) comprising at least one cleavage site; and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) that binds to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; and the MM competes with human CD47 to bind to the TBM; wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:48, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:65, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:82; 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:99, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:116, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:120. and (b) a target binding moiety (TBM) that binds human CD47, the target binding moiety comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), the target binding moiety binding to human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; the VH comprises the amino acid sequence of SEQ ID NO:27, and / or the VL comprises the amino acid sequence of SEQ ID NO:28. In certain embodiments, the masking peptide comprises the amino acid sequence of SEQ ID NO:139; the MM comprises the amino acid sequence of SEQ ID NO:137; and / or the CM comprises the amino acid sequence of SEQ ID NO:138. In some embodiments, an activatable antibody comprises, from N-terminus to C-terminus, a first polypeptide comprising a masking peptide and a VL; and a second polypeptide comprising a VH. In certain embodiments, the activatable antibody comprises a human IgG4 fragment crystallizable (Fc) region.In one embodiment, the first polypeptide comprises, from the N-terminus to the C-terminus, a light chain comprising a masking peptide and a VL, the first polypeptide comprising the amino acid sequence of SEQ ID NO:142; the second polypeptide comprises, from the N-terminus to the C-terminus, a heavy chain comprising a VH and a human IgG4 Fc domain, the second polypeptide comprising the amino acid sequence of SEQ ID NO:143. In certain embodiments, the activatable antibody comprises a human IgG1 Fc region. In one embodiment, the first polypeptide comprises, from the N-terminus to the C-terminus, a light chain comprising a masking peptide and a VL, the first polypeptide comprising the amino acid sequence of SEQ ID NO:148; the second polypeptide comprises, from the N-terminus to the C-terminus, a heavy chain comprising a VH and a human IgG1 Fc domain, the second polypeptide comprising the amino acid sequence of SEQ ID NO:149. In some embodiments, the human IgG1 Fc region comprises two Fc domains, each of the two Fc domains comprising an S239D substitution and / or an I332E substitution (e.g., an S239D substitution and an I332E substitution). In one embodiment, a first polypeptide comprises, from N-terminus to C-terminus, a light chain comprising a masking peptide and a VL, the first polypeptide comprising the amino acid sequence of SEQ ID NO:150; a second polypeptide comprises, from N-terminus to C-terminus, a heavy chain comprising a VH and a human IgG1 Fc domain, the second polypeptide comprising the amino acid sequence of SEQ ID NO:151.

[0033] In some embodiments, provided herein is an activatable antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM), wherein the LM comprises a cleavable moiety (CM) comprising at least one cleavage site; and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) and which binds human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; the MM competes with human CD47 to bind to the TBM; wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:49, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:66, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:83; 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:100, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:117, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:120. and (b) a target binding moiety (TBM) that binds human CD47, the target binding moiety comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), the target binding moiety binding to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; the MM binds to the TBM in competition with human CD47; the VH comprises the amino acid sequence of SEQ ID NO:29, and / or the VL comprises the amino acid sequence of SEQ ID NO:30. In certain embodiments, the masking peptide comprises the amino acid sequence of SEQ ID NO:139; the MM comprises the amino acid sequence of SEQ ID NO:137; and / or the CM comprises the amino acid sequence of SEQ ID NO:138.

[0034] In some embodiments, provided herein is an activatable antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM), wherein the LM comprises a cleavable moiety (CM) comprising at least one cleavage site; and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) that binds to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; the MM competes with human CD47 to bind to the TBM; wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:50, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:67, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:84; 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:101, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:118, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:120. and (b) a target binding moiety (TBM) that binds human CD47, the target binding moiety comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), the target binding moiety binding to human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; the VH comprises the amino acid sequence of SEQ ID NO:31, and / or the VL comprises the amino acid sequence of SEQ ID NO:32. In certain embodiments, the masking peptide comprises the amino acid sequence of SEQ ID NO:139; the MM comprises the amino acid sequence of SEQ ID NO:137; and / or the CM comprises the amino acid sequence of SEQ ID NO:138.

[0035] In some embodiments, provided herein is an activatable antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM), wherein the LM comprises a cleavable moiety (CM) comprising at least one cleavage site; and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) that binds to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; the MM competes with human CD47 to bind to the TBM; wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:35, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:52, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:69; 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:86, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:103, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:110. and (b) a target binding moiety (TBM) that binds human CD47, the target binding moiety comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), the target binding moiety binding to human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; the VH comprises the amino acid sequence of SEQ ID NO:1, and / or the VL comprises the amino acid sequence of SEQ ID NO:2. In certain embodiments, the masking peptide comprises the amino acid sequence of SEQ ID NO:139; the MM comprises the amino acid sequence of SEQ ID NO:137; and / or the CM comprises the amino acid sequence of SEQ ID NO:138.

[0036] In some embodiments, provided herein is an activatable antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM), wherein the LM comprises a cleavable moiety (CM) comprising at least one cleavage site; and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) that binds to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; the MM competes with human CD47 to bind to the TBM; wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:36, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:53, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:70; 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:87, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:104, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:106. and (b) a target binding moiety (TBM) that binds human CD47, the target binding moiety comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), the target binding moiety binding to human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; the VH comprises the amino acid sequence of SEQ ID NO:3, and / or the VL comprises the amino acid sequence of SEQ ID NO:4. In certain embodiments, the masking peptide comprises the amino acid sequence of SEQ ID NO:139; the MM comprises the amino acid sequence of SEQ ID NO:137; and / or the CM comprises the amino acid sequence of SEQ ID NO:138.

[0037] In some embodiments, provided herein is an activatable antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM), wherein the LM comprises a cleavable moiety (CM) comprising at least one cleavage site; and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) and which binds human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; the MM competes with human CD47 to bind to the TBM; wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:37, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:54, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:71; 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:88, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:105, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:110. and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:122. In certain embodiments, provided herein is an activatable antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM), wherein the LM comprises a cleavable moiety (CM) comprising at least one cleavage site; and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), and which binds human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; the VH comprises the amino acid sequence of SEQ ID NO:5, and / or the VL comprises the amino acid sequence of SEQ ID NO:6. In certain embodiments, the masking peptide comprises the amino acid sequence of SEQ ID NO:139; the MM comprises the amino acid sequence of SEQ ID NO:137; and / or the CM comprises the amino acid sequence of SEQ ID NO:138.

[0038] In some embodiments, provided herein is an activatable antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM), wherein the LM comprises a cleavable moiety (CM) comprising at least one cleavage site; and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) that binds to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; the MM competes with human CD47 to bind to the TBM; wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:39, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:56, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:73; 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:90, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:107, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:110. and (b) a target binding moiety (TBM) that binds human CD47, the target binding moiety comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), the target binding moiety binding to human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; the VH comprises the amino acid sequence of SEQ ID NO:9, and / or the VL comprises the amino acid sequence of SEQ ID NO:10. In certain embodiments, the masking peptide comprises the amino acid sequence of SEQ ID NO:139; the MM comprises the amino acid sequence of SEQ ID NO:137; and / or the CM comprises the amino acid sequence of SEQ ID NO:138.

[0039] In some embodiments, provided herein is an activatable antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM), wherein the LM comprises a cleavable moiety (CM) comprising at least one cleavage site; and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) that binds to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; the MM competes with human CD47 to bind to the TBM; wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:40, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:57, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:74; 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:91, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:108, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:110. and (b) a target binding moiety (TBM) that binds human CD47, the target binding moiety comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), the target binding moiety binding to human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; the VH comprises the amino acid sequence of SEQ ID NO:11, and / or the VL comprises the amino acid sequence of SEQ ID NO:12. In certain embodiments, provided herein is an activatable antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM), the LM comprising a cleavable moiety (CM) comprising at least one cleavage site; and (b) a target binding moiety (TBM) that binds to human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; the VH comprises the amino acid sequence of SEQ ID NO:11, and / or the VL comprises the amino acid sequence of SEQ ID NO:12. In certain embodiments, the masking peptide comprises the amino acid sequence of SEQ ID NO:139; the MM comprises the amino acid sequence of SEQ ID NO:137; and / or the CM comprises the amino acid sequence of SEQ ID NO:138.

[0040] In some embodiments, provided herein is an activatable antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM), wherein the LM comprises a cleavable moiety (CM) comprising at least one cleavage site; and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) and which binds human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; and the MM competes with human CD47 to bind to the TBM; wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:41, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:58, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:75; 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:92, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:109, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:110. and (b) a target binding moiety (TBM) that binds human CD47, the target binding moiety comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), the target binding moiety binding to human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; the VH comprises the amino acid sequence of SEQ ID NO:13, and / or the VL comprises the amino acid sequence of SEQ ID NO:14. In certain embodiments, the masking peptide comprises the amino acid sequence of SEQ ID NO:139; the MM comprises the amino acid sequence of SEQ ID NO:137; and / or the CM comprises the amino acid sequence of SEQ ID NO:138.

[0041] In some embodiments, provided herein is an activatable antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM), wherein the LM comprises a cleavable moiety (CM) comprising at least one cleavage site; and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) that binds to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; the MM competes with human CD47 to bind to the TBM; wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:42, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:59, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:76; 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:93, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:110, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:120. and (b) a target binding moiety (TBM) that binds human CD47, the target binding moiety comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL); the target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), the target binding moiety binding human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; the VH comprises the amino acid sequence of SEQ ID NO:15, and / or the VL comprises the amino acid sequence of SEQ ID NO:16. In certain embodiments, the masking peptide comprises the amino acid sequence of SEQ ID NO:139; the MM comprises the amino acid sequence of SEQ ID NO:137; and / or the CM comprises the amino acid sequence of SEQ ID NO:138.

[0042] In some embodiments, provided herein is an activatable antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM), wherein the LM comprises a cleavable moiety (CM) comprising at least one cleavage site; and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) that binds to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; the MM competes with human CD47 to bind to the TBM; wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:43, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:60, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:77; 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:94, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:111, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:112. and (b) a target binding moiety (TBM) that binds human CD47, the target binding moiety comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL); the target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), the target binding moiety binding human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; the VH comprises the amino acid sequence of SEQ ID NO:17, and / or the VL comprises the amino acid sequence of SEQ ID NO:18. In certain embodiments, the masking peptide comprises the amino acid sequence of SEQ ID NO:139; the MM comprises the amino acid sequence of SEQ ID NO:137; and / or the CM comprises the amino acid sequence of SEQ ID NO:138.

[0043] In some embodiments, provided herein is an activatable antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM), wherein the LM comprises a cleavable moiety (CM) comprising at least one cleavage site; and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) and which binds human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; the MM competes with human CD47 to bind to the TBM; wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:44, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:61, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:78; 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:95, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:112, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:120. and (b) a target binding moiety (TBM) that binds human CD47, the target binding moiety comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), the target binding moiety binding to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; the MM binds to the TBM in competition with human CD47; the VH comprises the amino acid sequence of SEQ ID NO:19, and / or the VL comprises the amino acid sequence of SEQ ID NO:20. In certain embodiments, the masking peptide comprises the amino acid sequence of SEQ ID NO:139; the MM comprises the amino acid sequence of SEQ ID NO:137; and / or the CM comprises the amino acid sequence of SEQ ID NO:138.

[0044] In some embodiments, provided herein is an activatable antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM), wherein the LM comprises a cleavable moiety (CM) comprising at least one cleavage site; and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) and which binds human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; the MM competes with human CD47 to bind to the TBM; wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:45, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:62, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:79; 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:96, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:113, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:120. and (b) a target binding moiety (TBM) that binds human CD47, the target binding moiety comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), the target binding moiety binding to human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; the VH comprises the amino acid sequence of SEQ ID NO:21, and / or the VL comprises the amino acid sequence of SEQ ID NO:22. In certain embodiments, the masking peptide comprises the amino acid sequence of SEQ ID NO:139; the MM comprises the amino acid sequence of SEQ ID NO:137; and / or the CM comprises the amino acid sequence of SEQ ID NO:138.

[0045] In some embodiments, provided herein is an activatable antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM), wherein the LM comprises a cleavable moiety (CM) comprising at least one cleavage site; and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) that binds to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; the MM competes with human CD47 to bind to the TBM; wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:46, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:63, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:80; 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:97, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:114, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:120. and (b) a target binding moiety (TBM) that binds human CD47, the target binding moiety comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), the target binding moiety binding to human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; the VH comprises the amino acid sequence of SEQ ID NO:23, and / or the VL comprises the amino acid sequence of SEQ ID NO:24. In certain embodiments, the masking peptide comprises the amino acid sequence of SEQ ID NO:139; the MM comprises the amino acid sequence of SEQ ID NO:137; and / or the CM comprises the amino acid sequence of SEQ ID NO:138.

[0046] In some embodiments, provided herein is an activatable antibody comprising: (a) a masking peptide comprising, from N-terminus to C-terminus, a masking moiety (MM) and a linking moiety (LM), wherein the LM comprises a cleavable moiety (CM) comprising at least one cleavage site; and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) that binds to human CD47; the masking peptide is linked to the N-terminus of the VH or the VL; and the MM competes with human CD47 to bind to the TBM; wherein 1) a) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:47, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:64, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:81; 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:98, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:115, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:120. and (b) a target binding moiety (TBM) that binds human CD47, the target binding moiety comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), the target binding moiety binding to human CD47; the masking peptide is linked to the N-terminus of the VH or VL; the MM binds to the TBM in competition with human CD47; the VH comprises the amino acid sequence of SEQ ID NO:25, and / or the VL comprises the amino acid sequence of SEQ ID NO:26. In certain embodiments, the masking peptide comprises the amino acid sequence of SEQ ID NO:139; the MM comprises the amino acid sequence of SEQ ID NO:137; and / or the CM comprises the amino acid sequence of SEQ ID NO:138.

[0047] In another aspect, provided herein is an isolated antibody, or antigen-binding fragment thereof, comprising a VH and a VL, and which binds to human CD47. In some embodiments, 1) the VH comprises a first complementarity determining region (CDR-H1), a second complementarity determining region (CDR-H2) and a third complementarity determining region (CDR-H3), wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 182 or 183; CDR-H2 comprises the amino acid sequence of SEQ ID NO: 184 or 185; CDR-H3 comprises an amino acid sequence selected from SEQ ID NO: 186-189; and / or 2) the VL comprises a first complementarity determining region (CDR-L1), a second complementarity determining region (CDR-L2) and a third complementarity determining region (CDR-L3), wherein CDR-L1 comprises an amino acid sequence selected from SEQ ID NO: 190-193; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 194; and CDR-L3 comprises an amino acid sequence selected from SEQ ID NO: 195. or 196. In certain embodiments, the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:182, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:185, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:188; b) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:191, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:194, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:195. In some embodiments, the isolated antibody comprises a human IgG4 Fc region. In another embodiment, the isolated antibody comprises a human IgG1 Fc region. In some embodiments, the human IgG1 Fc region comprises two Fc domains, each of the two Fc domains comprising an S239D substitution and / or an I332E substitution (e.g., an S239D substitution and an I332E substitution). In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fv, scFab and scFv. In some embodiments, the antibody or antigen-binding fragment binds to one or more amino acid residues selected from the group consisting of K39, W40, K41, F50, D51, G52, T99, E100, L101, and T102 of human CD47.In some embodiments, the antibody or antigen-binding fragment does not bind to one or more amino acid residues selected from the group consisting of L2, L3, F4, K6, N27, E29, A30, Q31, T34, E35, V36, Y37, D46, T49, A53, E97, R103, E104, G105, and E1062 of human CD47. In certain embodiments, the antibody or antigen-binding fragment binds to amino acid residues K39, W40, K41, F50, D51, G52, T99, E100, L101, and T102 of human CD47, and / or does not bind to amino acid residues L2, L3, F4, K6, N27, E29, A30, Q31, T34, E35, V36, Y37, D46, T49, A53, E97, R103, E104, G105, and E106 of human CD47.

[0048] In some embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein (a) the VH comprises a CDR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 35-37 and 39-50, a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 52-54 and 56-67, and a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 69-71 and 73-84; and (b) the VL comprises a CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 86-88 and 89-101, a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 103-105 and 106-118, and a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 120-122 and 124-135. In certain embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL, and which binds to human CD47, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, and 31; and / or the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, and 32.

[0049] In some embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:48, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:65, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:82; and 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:99, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:116, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:133. In certain embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein the VH comprises the amino acid sequence of SEQ ID NO:27, and / or the VL comprises the amino acid sequence of SEQ ID NO:28. In some embodiments, the isolated antibody comprises a human IgG4 Fc region. In one embodiment, the isolated antibody comprises a first polypeptide comprising a light chain comprising a VL and comprising the amino acid sequence of SEQ ID NO:140; and a second polypeptide comprising, from N-terminus to C-terminus, a heavy chain comprising a VH and a human IgG4 Fc domain and comprising the amino acid sequence of SEQ ID NO:141. In another embodiment, the isolated antibody comprises a human IgG1 Fc region. In one embodiment, the isolated antibody comprises a first polypeptide comprising a light chain comprising a VL and comprising the amino acid sequence of SEQ ID NO:144; and a second polypeptide comprising, from N-terminus to C-terminus, a heavy chain comprising a VH and a human IgG1 Fc domain and comprising the amino acid sequence of SEQ ID NO:145. In certain embodiments, the human IgG1 Fc region comprises two Fc domains, each of the two Fc domains comprising an S239D substitution and / or an I332E substitution (e.g., an S239D substitution and an I332E substitution).In one embodiment, the isolated antibody comprises a first polypeptide comprising a light chain comprising a VL, and comprising the amino acid sequence of SEQ ID NO:146; and a second polypeptide comprising, from N-terminus to C-terminus, a heavy chain comprising a VH and a human IgG1 Fc domain, and comprising the amino acid sequence of SEQ ID NO:147.

[0050] In some embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:49, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:66, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:83; and 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:100, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:117, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:134. In certain embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein the VH comprises the amino acid sequence of SEQ ID NO:29, and / or the VL comprises the amino acid sequence of SEQ ID NO:30. In some embodiments, the isolated antibody comprises a human IgG1 Fc region. In certain embodiments, the human IgG1 Fc region comprises two Fc domains, each of the two Fc domains comprising an S239D substitution and / or an I332E substitution (e.g., an S239D substitution and an I332E substitution).

[0051] In some embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:50, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:67, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:84; and 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:101, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:118, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:135. In certain embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein the VH comprises the amino acid sequence of SEQ ID NO:31, and / or the VL comprises the amino acid sequence of SEQ ID NO:32. In some embodiments, the isolated antibody comprises a human IgG1 Fc region. In certain embodiments, the human IgG1 Fc region comprises two Fc domains, each of the two Fc domains comprising an S239D substitution and / or an I332E substitution (e.g., an S239D substitution and an I332E substitution).

[0052] In some embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:35, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:52, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:69; and 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:86, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:103, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:120. In certain embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein the VH comprises the amino acid sequence of SEQ ID NO:1, and / or the VL comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, the isolated antibody comprises a human IgG1 Fc region. In certain embodiments, the human IgG1 Fc region comprises two Fc domains, each of the two Fc domains comprising an S239D substitution and / or an I332E substitution (e.g., an S239D substitution and an I332E substitution).

[0053] In some embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:36, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:53, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:70; and 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:87, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:104, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:121. In certain embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein the VH comprises the amino acid sequence of SEQ ID NO:3, and / or the VL comprises the amino acid sequence of SEQ ID NO:4. In some embodiments, the isolated antibody comprises a human IgG1 Fc region. In certain embodiments, the human IgG1 Fc region comprises two Fc domains, each of the two Fc domains comprising an S239D substitution and / or an I332E substitution (e.g., an S239D substitution and an I332E substitution).

[0054] In some embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:37, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:54, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:71; and 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:88, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:105, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:122. In certain embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein the VH comprises the amino acid sequence of SEQ ID NO:5, and / or the VL comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the isolated antibody comprises a human IgG1 Fc region. In certain embodiments, the human IgG1 Fc region comprises two Fc domains, each of the two Fc domains comprising an S239D substitution and / or an I332E substitution (e.g., an S239D substitution and an I332E substitution).

[0055] In some embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:39, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:56, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:73; and 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:90, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:107, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:124. In certain embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein the VH comprises the amino acid sequence of SEQ ID NO:9, and / or the VL comprises the amino acid sequence of SEQ ID NO:10. In some embodiments, the isolated antibody comprises a human IgG1 Fc region. In certain embodiments, the human IgG1 Fc region comprises two Fc domains, each of the two Fc domains comprising an S239D substitution and / or an I332E substitution (e.g., an S239D substitution and an I332E substitution).

[0056] In some embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:40, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:57, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:74; and 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:91, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:108, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:125. In certain embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein the VH comprises the amino acid sequence of SEQ ID NO:11, and / or the VL comprises the amino acid sequence of SEQ ID NO:12. In some embodiments, the isolated antibody comprises a human IgG1 Fc region. In certain embodiments, the human IgG1 Fc region comprises two Fc domains, each of the two Fc domains comprising an S239D substitution and / or an I332E substitution (e.g., an S239D substitution and an I332E substitution).

[0057] In some embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:41, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:58, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:75; and 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:92, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:109, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:126. In certain embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein the VH comprises the amino acid sequence of SEQ ID NO:13, and / or the VL comprises the amino acid sequence of SEQ ID NO:14. In some embodiments, the isolated antibody comprises a human IgG1 Fc region. In certain embodiments, the human IgG1 Fc region comprises two Fc domains, each of the two Fc domains comprising an S239D substitution and / or an I332E substitution (e.g., an S239D substitution and an I332E substitution).

[0058] In some embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:42, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:59, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:76; 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:93, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:110, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:127. In certain embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein the VH comprises the amino acid sequence of SEQ ID NO:15, and / or the VL comprises the amino acid sequence of SEQ ID NO:16. In some embodiments, the isolated antibody comprises a human IgG1 Fc region. In certain embodiments, the human IgG1 Fc region comprises two Fc domains, each of the two Fc domains comprising an S239D substitution and / or an I332E substitution (e.g., an S239D substitution and an I332E substitution).

[0059] In some embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:43, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:60, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:77; 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:94, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:111, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:128. In certain embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein the VH comprises the amino acid sequence of SEQ ID NO:17, and / or the VL comprises the amino acid sequence of SEQ ID NO:18. In some embodiments, the isolated antibody comprises a human IgG1 Fc region. In certain embodiments, the human IgG1 Fc region comprises two Fc domains, each of the two Fc domains comprising an S239D substitution and / or an I332E substitution (e.g., an S239D substitution and an I332E substitution).

[0060] In some embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:44, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:61, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:78; and 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:95, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:112, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:129. In certain embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein the VH comprises the amino acid sequence of SEQ ID NO:19, and / or the VL comprises the amino acid sequence of SEQ ID NO:20. In some embodiments, the isolated antibody comprises a human IgG1 Fc region. In certain embodiments, the human IgG1 Fc region comprises two Fc domains, each of the two Fc domains comprising an S239D substitution and / or an I332E substitution (e.g., an S239D substitution and an I332E substitution).

[0061] In some embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:45, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:62, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:79; and 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:96, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:113, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:130. In certain embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein the VH comprises the amino acid sequence of SEQ ID NO:21, and / or the VL comprises the amino acid sequence of SEQ ID NO:22. In some embodiments, the isolated antibody comprises a human IgG1 Fc region. In certain embodiments, the human IgG1 Fc region comprises two Fc domains, each of the two Fc domains comprising an S239D substitution and / or an I332E substitution (e.g., an S239D substitution and an I332E substitution).

[0062] In some embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:46, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:63, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:80; 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:97, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:114, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:131. In certain embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein the VH comprises the amino acid sequence of SEQ ID NO:23, and / or the VL comprises the amino acid sequence of SEQ ID NO:24. In some embodiments, the isolated antibody comprises a human IgG1 Fc region. In certain embodiments, the human IgG1 Fc region comprises two Fc domains, each of the two Fc domains comprising an S239D substitution and / or an I332E substitution (e.g., an S239D substitution and an I332E substitution).

[0063] In some embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein 1) a) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:47, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:64, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:81; and 2) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:98, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:115, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:132. In certain embodiments, provided herein is an isolated antibody or antigen-binding fragment thereof comprising a VH and a VL and binding to human CD47, wherein the VH comprises the amino acid sequence of SEQ ID NO:25, and / or the VL comprises the amino acid sequence of SEQ ID NO:26. In some embodiments, the isolated antibody comprises a human IgG1 Fc region. In certain embodiments, the human IgG1 Fc region comprises two Fc domains, each of the two Fc domains comprising an S239D substitution and / or an I332E substitution (e.g., an S239D substitution and an I332E substitution). In some embodiments, the isolated antibody or antigen-binding fragment binds to human CD47 with a KD of about 50 nM or less. In certain embodiments, the isolated antibody or antigen-binding fragment binds to human CD47 with a KD of about 10 nM or less. In some embodiments, the isolated antibody or antigen-binding fragment has a half maximal inhibitory concentration (IC50) for blocking human CD47 binding to human SIRPα in vitro of about 100 nM or less. In certain embodiments, the isolated antibody or antigen-binding fragment has a half maximal inhibitory concentration (IC50) for blocking human CD47 binding to human SIRPα in vitro of about 10 nM or less.In some embodiments, the isolated antibody or antigen-binding fragment completely blocks binding of human CD47 to human SIRPα in vitro when provided at a concentration of about 1 μM or greater. In some embodiments, the isolated antibody or antigen-binding fragment has a half-maximal effective concentration (EC50) of about 10 nM or less for binding to tumor cells in vitro, where the tumor cells comprise a B cell lymphoma cell line, a T cell lymphoma cell line, or a combination thereof. In some embodiments, the isolated antibody or antigen-binding fragment has a half-maximal effective concentration (EC50) of about 10 nM or less for increasing macrophage phagocytosis of tumor cells in vitro, where the tumor cells comprise a B cell lymphoma cell line, a T cell lymphoma cell line, or a combination thereof. In certain embodiments, the isolated antibody or antigen-binding fragment has a half-maximal effective concentration (EC50) of about 1 nM or less for increasing macrophage phagocytosis of tumor cells in vitro, where the tumor cells comprise a B cell lymphoma cell line, a T cell lymphoma cell line, or a combination thereof.

[0064] In some embodiments that can be combined with any of the preceding embodiments, the masked antibody, activatable antibody, isolated antibody, or antigen-binding fragment thereof is cross-reactive with CD47 polypeptides derived from at least one non-human species selected from the group consisting of cynomolgus monkey, rat, and dog. In certain embodiments that can be combined with any of the preceding embodiments, the masked antibody, activatable antibody, isolated antibody, or antigen-binding fragment thereof binds to cynomolgus monkey CD47.

[0065] In some aspects, provided herein is an isolated polynucleotide encoding one or more polypeptide chains of any of the masked antibodies, activatable antibodies, isolated antibodies, or antigen-binding fragments described herein.

[0066] In another aspect, provided herein is a vector comprising an isolated polynucleotide encoding one or more polypeptide chains of any of the masked antibodies, activatable antibodies, isolated antibodies, or antigen-binding fragments described herein.

[0067] In another aspect, provided herein is a host cell comprising a vector comprising an isolated polynucleotide encoding one or more polypeptide chains of any of the masked antibodies, activatable antibodies, isolated antibodies, or antigen-binding fragments described herein.

[0068] In yet another aspect, provided herein is a method of making a masked antibody, activatable antibody, isolated antibody, or antigen-binding fragment, comprising culturing a host cell comprising a vector comprising a polynucleotide encoding one or more polypeptide chains of any of the masked antibodies, activatable antibodies, isolated antibodies, or antigen-binding fragments described herein under conditions suitable for production of the masked antibody, activatable antibody, isolated antibody, or antigen-binding fragment.

[0069] In some aspects, provided herein is a pharmaceutical composition comprising a masked antibody, activatable antibody, isolated antibody, or antigen-binding fragment described herein and a pharma- ceutically acceptable carrier.

[0070] In another aspect, provided herein is a method of treating a CD47-positive disease or condition in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising an effective amount of any of the masked antibodies, activatable antibodies, isolated antibodies, or antigen-binding fragments described herein and a pharma- ceutically acceptable carrier. In some embodiments, the administration does not cause anemia in the subject. In some embodiments, the disease or condition is cancer. In certain embodiments, the cancer comprises B-cell lymphoma, T-cell lymphoma, or a combination thereof. In some embodiments, the cancer is selected from the group consisting of lymphoma, leukemia, head and neck cancer, gastric cancer, esophageal cancer, breast cancer, cervical cancer, bile duct cancer, colorectal cancer, ovarian cancer, thyroid cancer, uterine cancer, endometrial cancer, lung cancer, mesothelioma, and pancreatic cancer. In certain embodiments, the cancer is selected from the group consisting of triple negative breast cancer (TNBC), Her2+ gastroesophageal junction cancer (GEJ), small cell lung cancer (SCLC), diffuse large B cell lymphoma (DLBCL), acute myeloid leukemia (AML), head and neck squamous cell carcinoma (HNSC), gastric cancer (GC), invasive breast cancer (BRCA), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), ovarian serous cystadenocarcinoma (OV), thyroid cancer (THCA), uterine endometrial carcinoma (UCEC), HER2+ breast cancer, hormone receptor positive breast cancer, lymphoid neoplasm diffuse large B cell lymphoma (DLBC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), mesothelioma (MESO), and pancreatic adenocarcinoma (PAAD). In some embodiments, the masked antibody or antibody or antigen-binding fragment thereof is administered at a dose of at least about 0.6 mg / kg. In some embodiments, the pharmaceutical composition is administered at a frequency of at least once every three weeks or at least once every two weeks.

[0071] In some embodiments of any one of the methods of treating a CD47-positive disease or condition described above, the method further comprises administering to the subject an effective amount of one or more additional therapeutic agents, hi some embodiments, the one or more therapeutic agents comprise viral gene therapy, immune checkpoint inhibitors, targeted therapy, radiation therapy, chemotherapy, or any combination thereof. In certain embodiments, the one or more additional therapeutic agents comprise pomalyst, revlimid, lenalidomide, pomalidomide, thalidomide, DNA alkylating platinum-containing derivatives, cisplatin, 5-fluorouracil, cyclophosphamide, anti-CD47 antibody, anti-CTLA4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD20 antibody, anti-CD40 antibody, anti-DR5 antibody, anti-CD1d antibody, anti-TIM3 antibody, anti-SLAMF7 antibody, anti-KIR receptor antibody, anti-OX40 antibody, anti-HER2 antibody, anti-ErbB-2 antibody, anti-EGFR antibody, cetuximab, rituximab, trastuzumab, pembrolizumab, radiation therapy, single radiation, fractionated radiation, focal radiation, whole organ radiation, IL-12, IFNα, GM-CSF, chimeric antigen receptor, adoptively transferred T cells, anti-cancer vaccines, oncolytic viruses, or any combination thereof.

[0072] In some aspects, provided herein are methods of treating cancer comprising administering an anti-CD47 activatable antibody, wherein the anti-CD47 activatable antibody comprises human IgG1 Fc or IgG1 Fc with enhanced antibody-dependent cellular cytotoxicity (ADCC) activity, and wherein the anti-CD47 activatable antibody comprises a masking peptide comprising a cleavable moiety (CM) that comprises at least one cleavage site, wherein the CM when cleaved at the site of the cancer (e.g., the tumor microenvironment (TME)) has a higher binding affinity for human CD47 than when the CM is not cleaved.

[0073] The present application can be understood by reference to the following description taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0074] [Figure 1]The ability of the indicated antibodies to induce human RBC hemagglutination is shown. [Diagram 2] Figure 1 shows the ADCC activity of human NK cells from different donors against calcein-labeled CEM cells incubated with the indicated antibodies. The percentage of cell lysis is shown as the percentage of cytotoxicity. [Diagram 3] A and B show the in vivo antitumor efficacy of antibodies or isotype controls in the B-NDG / Raji-Luc mouse systemic model in two independent experiments. Data points represent group means and error bars represent standard deviations. A shows the first independent experiment. B shows the second independent experiment. [Figure 4] A and B show the in vivo antitumor efficacy of antibodies or isotype controls in the B-NDG / Raji mouse subcutaneous tumor model. Data points represent group means and error bars represent standard deviations. A shows the first independent experiment. B shows the second independent experiment. [Diagram 5] 1 shows the in vivo anti-tumor efficacy of antibodies or isotype controls in a SCID / Raji-Luc mouse systemic model. Data points represent group means and error bars represent standard deviation. [Figure 6] A and B show hematotoxicity in B-hSIRPα / hCD47 humanized mice of the indicated antibodies. A shows the change in RBC counts in peripheral blood before and after a single intraperitoneal injection of the antibodies. B shows the change in hemoglobin levels in peripheral blood before and after a single intraperitoneal injection of the antibodies. [Figure 7] AF shows the hematotoxicity and pharmacokinetics of the indicated antibodies in cynomolgus monkeys after a single intravenous injection. A shows the change in RBC ratio after dosing. B shows the change in hemoglobin percentage after dosing. C shows the change in reticulocyte percentage after dosing. D shows the change in hematocrit after dosing. E shows the change in platelet percentage after dosing. F shows the blood concentration of the indicated antibodies injected intravenously at the indicated doses in cynomolgus monkeys. [Figure 8] Plasma concentrations of the indicated antibodies injected intravenously at the indicated doses into CB17-SCID mice are shown. [Figure 9] A shows the clustering of CFSE-Raji and BMQC-Jurkat cells detected by flow cytometry. A shows the clustering of CFSE-Raji and BMQC-Jurkat cells when treated with a negative isotype control and a negative buffer control, illustrating the concept of trans vs. cis binding of antibodies to cells. B shows the clustering of CFSE-Raji and BMQC-Jurkat cells when treated with a positive control known to bind in trans to CFSE-Raji and BMQC-Jurkat cells. C shows the clustering of CFSE-Raji and BMQC-Jurkat cells when treated with the anti-CD47 antibody TY21446. D shows the clustering of CFSE-Raji and BMQC-Jurkat cells when treated with the anti-CD47 benchmark control antibody TAC2204. [Figure 10] (A-C) Clustering of fluorescent dye-labeled human red blood cells detected by flow cytometry. (A) Gating strategy to identify clustering between CFSE- and BMQC-red blood cells in flow cytometry. (B and C) Effect of test antibodies on clustering of human RBCs using RBCs from two different donors. [Figure 11] (A-C) shows human RBC clustering detected by flow cytometry. (A) shows identification of clustered red blood cells using a FSC-H / FSC-A gating strategy in flow cytometry. (B and C) show the effect of test antibodies on human RBC clustering using RBCs from two different donors. [Figure 12]A-E show the receptor occupancy (RO) of anti-CD47 antibodies binding in vivo to the extracellular domain (ECD) of human CD47 expressed in hSIRPα / hCD47 knock-in mice injected with Raji tumor cells. A and B show the RO percentage of the indicated antibodies binding to red blood cells (RBCs) and non-red blood cells (WBCs), respectively, after injection of the indicated antibodies at various doses and times. C shows the RO percentage of the indicated antibodies binding to Raji cells in subcutaneous tumor samples taken 72 hours after injection of the indicated antibodies. D shows the RO percentage of the indicated antibodies binding to Raji cells in the bone marrow of mice taken 72 hours after injection of the indicated antibodies. E shows the total number of blood cells per liter in mice 72 hours after injection of the indicated antibodies. Brackets with double asterisks (**) indicate that the two samples are significantly different at p<0.01, and brackets with "ns" indicate that the two samples are not significantly different. [Figure 13] AB shows the mapping of epitope residues on human CD47 bound by SIRPα, magrolimab, and TY21446. A shows the crystal structure of human CD47, where epitope residues on human CD47 bound by SIRPα, magrolimab, and TY21446 are shaded in dark grey. B shows the specific residues on human CD47 bound by SIRPα, magrolimab, and TY21446, which are shaded in grey. [Figure 14] A-D show the CD47 receptor occupancy (RO) of TAC2204 and TY26898 and their effects on blood cell counts and viability. A shows the CD47 RO of TAC2204 and TY26898 in tissues of mice treated with each antibody. B shows the relative CD47 RO of TY26898 vs. TAC2204 in tissues of mice treated with each antibody. C shows the total blood cell counts of blood taken from mice treated with TAC2204 and TY26898. D shows the blood cell viability of blood taken from mice treated with TAC2204 and TY26898. [Figure 15]A-D show the in vivo antitumor efficacy of TY26898, TY26896, TAC2204, or isotype control in different cancer models. Data points represent group means, and error bars represent standard deviation of the mean. A shows in vivo tumor inhibition by TY26898 and TAC2204 in OE19 HER2+GEJ cancer xenograft model in CB17 SCID mice, with Herceptin as positive control and vehicle negative control. B shows in vivo tumor inhibition by TY26898 and TAC2204 in MDA-MB-231 triple-negative breast cancer (TNBC) xenograft model in CB17 SCID mice, with PBS as negative control. C shows in vivo tumor inhibition in SHP-77 small cell lung cancer (SCLC) xenograft model in CB17 SCID mice by TY26898, TY26896, and TAC2204 with PBS as a negative control. D shows in vivo tumor inhibition in OV90 ovarian cancer xenograft model in CB17 SCID mice by TY26898 and TAC2204 with PBS as a negative control. E shows in vivo tumor inhibition in OE19 HER2+GEJ tumor model by TY26898 with PBS as a negative control. [Figure 16] AB shows CD47 receptor occupancy (RO) by TY26898 in different tumor models. A shows CD47 RO by TY26898 and TAC2204 in OE19 HER2+GEJ tumors, MDA-MB-231 TNBC tumors, and OV90 ovarian tumors. B shows CD47 RO in OE19 HER2+GEJ tumors from mice treated with 3 mg / kg or 10 mg / kg TY26898. [Figure 17]AB shows western blots and percentages of cleaved and intact TY26898 extracted from tumors of mice previously treated with TY26898. A shows cleaved and intact TY26898 in OE19 HER2+GEJ tumors from mice previously treated with TY26898. B shows cleaved and intact TY26898 in OV90 ovarian tumors from mice previously treated with TY26898. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0075] The following description sets forth example methods, parameters, etc. However, it should be appreciated that such description is not intended to limit the scope of the present disclosure, but is instead provided as a description of example embodiments.

[0076] A. Definition Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art.Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.In general, the nomenclature and techniques used in connection with antibody engineering, immunotherapy, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry described herein are those well known and commonly used in the art.

[0077] As used herein, each of the following terms has the meaning associated with it in this section.

[0078] The articles "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0079] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and those that are later modified, such as hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine. The term "amino acid analog" refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, but the C-terminal carboxy group, the N-terminal amino group, or a side chain functional group has been chemically modified to another functional group. The term "amino acid mimetic" refers to a compound that has a structure that is different from the general chemical structure of an amino acid, but functions similarly to a naturally occurring amino acid. As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)).

[0080] The term "amino acid substitution" or "amino acid residue substitution" refers to a change in one of the amino acid residues of an amino acid sequence relative to a reference sequence. An amino acid sequence can have any number of amino acid substitutions (e.g., 1, 2, 3, 4, 5, or more) relative to the reference sequence at any residue of the sequence. The term "variant" refers to a polypeptide or amino acid sequence having one or more amino acid substitutions relative to a reference amino acid sequence.

[0081] The term "antibody" is used herein in the broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, masked antibodies (e.g., activatable antibodies), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., single-chain variable fragments or scFvs), so long as they exhibit the desired biological activity.

[0082] The term "antibody" is an art-recognized term and may refer to an antigen-binding protein (i.e., an immunoglobulin) with a basic four-polypeptide chain structure consisting of two identical heavy (H) chains and two identical light (L) chains. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the isotype of the H chain. Each heavy chain has a variable region (abbreviated herein as VH) at the N-terminus followed by a constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain has a variable region (abbreviated herein as VL) at the N-terminus followed by a constant region at the other end. The light chain constant region consists of one domain, CL. The VL is juxtaposed to the VH, which is juxtaposed to the first constant domain (CH1) of the heavy chain. The pairing of the VH and VL together forms a single antigen-binding site. IgM antibodies consist of five basic heterotetrameric units with an additional polypeptide called the J chain and therefore contain 10 antigen-binding sites, whereas secreted IgA antibodies can form

[0083] The term "hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain that are hypervariable in sequence. HVRs may form structurally defined loops ("hypervariable loops"). Generally, naturally occurring four-chain antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). The HVRs are flanked by more conserved regions called framework regions (FWs). Each VH and VL is composed of three HVRs and four FWs, arranged from the amino terminus to the carboxy terminus in the following order: FW1, HVR1, FW2, HVR2, FW3, HVR3, FW4. HVRs generally contain amino acid residues from the hypervariable loops and / or "complementarity determining regions" (CDRs), which are those with the highest sequence variability and / or are involved in antigen recognition. Exemplary hypervariable loops are located at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) are located at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). With the exception of CDR1 of VH, the CDRs generally comprise the amino acid residues that form the hypervariable loops. The CDRs also comprise "specificity determining residues" or "SDRs", which are residues that contact the antigen. The SDRs are contained within regions of the CDRs called truncated CDRs or a-CDRs.Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) are located at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)).

[0084] Table I below provides exemplary CDR definitions according to various algorithms known in the art.

[0085] Table 1: CDR definition TIFF2024542978000001.tif40170 1 Residue numbering follows the nomenclature of Kabat et al., J. Biol. Chem. 252:6609-6616(1977); Kabat et al., US Dept. of Health and Human Services, "Sequences of proteins of immunological interest"(1991). 2 Residue numbering follows the nomenclature of Chothia et al., J. Mol. Biol. 196:901-917(1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948(1997). 3 Residue numbering follows the nomenclature of MacCallum et al., J. Mol. Biol. 262:732-745(1996); Abhinandan and Martin, Mol. Immunol.,45:3832-3839(2008). 4Residue numbering follows the nomenclature of Lefranc MP et al., Dev. Comp. Immunol., 27:55-77(2003); and Honegger and Plueckthun, J. Mol. Biol., 309:657-670(2001). 5 Residue numbering follows the nomenclature of Honegger and Plueckthun, J. Mol. Biol., 309:657-670 (2001).

[0086] The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 or more amino acids, usually as described in Fundamental Immunology Ch. 7 (Paul, W., ed., 2001). nd ed. Raven Press, NY) (1989).

[0087] L chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Antibodies can be assigned to different classes or isotypes depending on the amino acid sequence of the constant domain of their heavy chains (CH). The term "fragment crystallizable (Fc) region" refers to the heavy chain constant domain. There are five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, each with heavy chains designated α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu). The IgG class of antibodies can be further classified into four subclasses, IgG1, IgG2, IgG3, and IgG4, by the gamma heavy chains Y1-Y4, respectively.

[0088] The term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more portions of an antibody that retain the ability to bind to the antigen to which the antibody binds (e.g., CD47). Examples of "antigen-binding fragments" of antibodies include: (i) V L , V H , C L , and C H1 (ii) a Fab fragment, which is a monovalent fragment consisting of two Fab fragments linked by a disulfide bridge at the hinge region; and (iii) a F(ab') fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region. 2 Fragment; (iii) V H and C H1 (iv) an Fd fragment consisting of a V domain of a single arm of an antibody; L and V H (v) an Fv fragment consisting of a V domain; H (vi) a dAb fragment consisting of a domain (Ward et al., Nature 341:544-546 (1989)); (vi) containing isolated complementarity determining regions (CDRs).

[0089] The term "masked antibody" refers to an antibody or antigen-binding fragment thereof that contains a peptide that interferes with, prevents, reduces, blocks, inhibits, or competes with the binding of the target binding moiety (TBM) of the antibody or antigen-binding fragment thereof to a target. A masked antibody can be made by linking a masking peptide to the TBM of the antibody or antigen-binding fragment thereof.

[0090] The term "activatable antibody" refers to a masked antibody or antigen-binding fragment thereof that exhibits a first binding affinity to a target in an unactivated (e.g., inhibited, masked, and / or uncleaved) state and a second binding affinity to a target in an activated (e.g., uninhibited, unmasked, and / or cleaved) state, the second binding affinity being higher than the first binding affinity. Activatable antibodies can be generated by linking a masking peptide that includes an activatable moiety (e.g., a cleavable moiety (CM)) to a target-binding portion (TBM) of an antibody or antigen fragment thereof. Activatable antibodies are described, for example, in U.S. Patent Publication Nos. 2019 / 0241886 and 2021 / 0207126, the contents of both of which are incorporated herein by reference in their entireties.

[0091] "Target binding moiety (TBM)" refers to the structural portion of the antigen-binding portion of an antibody that binds to the antibody's target antigen. A TBM can include any combination of VH and VL, such as any VH or VL described herein.

[0092] "Masking peptide" refers to a structural portion of a masking antibody (e.g., an activatable antibody) that inhibits the binding of the TBM to a target antigen, typically comprising, from the N-terminus to the C-terminus, a masking portion (MM) and a linking portion (LM). The C-terminus of the masking peptide is typically linked to the N-terminus of the VH or VL of the masked antibody (e.g., an activatable antibody). In some embodiments, the masking peptide or a portion thereof prevents or inhibits the binding of the TBM to the target so efficiently that the binding of the TBM to the target is very low and / or below the limit of detection (e.g., the binding cannot be detected in an ELISA or flow cytometry assay). The masked antibodies (e.g., activatable antibodies) described herein may include one or more linkers, for example, located in the LM, between the MM and the LM, between the LM and the VH or VL, or between the TBM and the hinge region of the Fc.

[0093] The LM of the masking peptide can include a cleavable moiety (CM). The CM generally includes a cleavable amino acid sequence and serves as a substrate for an enzyme and / or a cysteine-cysteine ​​pair capable of forming, for example, a reducible disulfide bond. Thus, when terms such as "cleavage," "cleavable," and "cleaved" are used in connection with the CM, the terms encompass cleavage of the disulfide bond between the cysteine-cysteine ​​pair, for example, via enzymatic cleavage by a protease and reduction of the disulfide bond, which may occur upon exposure to a reducing agent. The amino acid sequence of the CM may overlap or be contained within the MM. An activatable antibody includes a CM configured to mediate activation of the antibody. For example, when the CM of the activatable antibody is intact (e.g., includes a cysteine-cysteine ​​disulfide bond that has not been cleaved and / or reduced by the corresponding enzyme), the masking peptide or a portion thereof may prevent or inhibit binding of the TBM to a target.

[0094] The term "masking efficiency" refers to the efficiency with which a masking peptide inhibits the binding of a TBM to a target antigen. Masking efficiency can be measured as the difference or ratio of a property (e.g., binding affinity to a target antigen) or activity (e.g., inhibition of binding of a target antigen to a ligand) of a masked antibody (e.g., an activatable antibody) having a TBM and a masking peptide to a corresponding unmasked antibody (a "parent antibody") having the same TBM but lacking the masking peptide. In an activatable antibody, masking efficiency can be measured as the difference or ratio of a property (e.g., binding affinity to a target antigen) or activity (e.g., inhibition of binding of a target antigen to a ligand) of an activatable antibody having a TBM and a masking peptide to an activatable antibody in a non-activated (e.g., inhibited, masked, and / or cleaved) state, to an activatable antibody in an activated (e.g., inhibited, unmasked, and / or cleaved) state, or to a parent antibody having the same TBM but lacking the masking peptide. For example, masking efficiency can be measured by dividing the EC50 of an activatable antibody that binds to a target antigen in an unactivated (e.g., inhibited, masked, and / or uncleaved) state by the EC50 or KD of an activatable antibody that binds to a target antigen in an activated (e.g., inhibited, unmasked, and / or cleaved) state, or by the EC50 or KD of a parent antibody that binds to the target antigen. EC50 values ​​can be measured, for example, by an ELISA assay, such as described in Example 5, or by a Jurkat NFAT reporter assay, such as described in U.S. Patent Application Publication No. 20210207126 A1. KD values ​​can be measured using surface plasmon resonance, for example, using any of the systems described herein.

[0095] The term "antibody derivative" or "derivative" of an antibody refers to a molecule that can bind to the same antigen (e.g., CD47) as the antibody and that contains the amino acid sequence of an antibody linked to an additional molecular entity. The amino acid sequence of an antibody that is included in the antibody derivative may be the full-length heavy chain, the full-length light chain, any portion(s) of the full-length heavy chain, any portion(s) of the full-length light chain, any other fragment(s) of an antibody, or a complete antibody. The additional molecular entity may be a chemical molecule or a biological molecule. Examples of the additional molecular entity include chemical groups, amino acids, peptides, proteins (enzymes, antibodies, etc.), and chemical compounds. The additional molecular entity may have any utility, such as use as a detection agent, label, marker, pharmaceutical, or therapeutic agent. The amino acid sequence of an antibody may be bound or linked to the additional molecular entity by chemical coupling, genetic fusion, non-covalent association, etc. The term "antibody derivatives" also includes chimeric antibodies, humanized antibodies, and molecules derived from amino acid sequence modifications of a CD47 antibody, such as conservative substitutions, additions, and insertions of amino acids.

[0096] The term "binding molecule" includes (1) antibodies, (2) antigen-binding fragments of antibodies, (3) masked antibodies (e.g., activatable antibodies), and (4) derivatives of antibodies, each as defined herein.

[0097] The terms "binding to CD47," "binding to CD47," "binding to CD47," or "binding to CD47" refer to a binding molecule (e.g., an antibody, antigen-binding fragment, or a masked antibody (e.g., an activatable antibody)) that has an affinity (K) of 100 nM or less in an in vitro assay, such as the Octet® RED96 assay as described in Example 2. D ) to human CD47.

[0098] The terms "CD47" and "CD47 receptor" are used interchangeably herein and include the human CD47 receptor, as well as variants, isoforms, and species homologs thereof. Thus, a binding molecule (e.g., an antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody)) may also bind to CD47 from species other than human. In other cases, a binding molecule (e.g., an antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody)) may be completely specific for human CD47 and may not exhibit species or other types of cross-reactivity. The term "CD47-ECD" refers to the extracellular domain of CD47.

[0099] The term "CD47" as used in this application includes human CD47 (e.g., UniProt Accession No. Q08722; NCBI Accession No. NP_001768), as well as variants, isoforms, and species homologs thereof (e.g., mouse CD47 (e.g., UniProt Accession No. Q61735; NCBI Accession No. NP_034711), rat CD47 (e.g., UniProt Accession No. P97829; NCBI Accession No. NP_062068), dog CD47 (e.g., UniProt Accession No. F1P6D7), cynomolgus monkey CD47 (e.g., UniProt Accession No. G7NZR3; NCBI Accession No. XP_005548289), rhesus monkey (e.g., NCBI Accession No. NP_001253446), etc.). Thus, a binding molecule (e.g., an antibody, antigen-binding fragment, or a masked antibody (e.g., an activatable antibody)) may also bind to CD47 from species other than human. In other cases, the binding molecule may be completely specific for human CD47 and may not exhibit species or other types of cross-reactivity.

[0100] The term "SIRPα" as used in this application refers to human SIRPα (e.g., UniProt Accession No. P78324), as well as variants, isoforms, and species homologs thereof (e.g., mouse SIRPα (e.g., UniProt Accession No. P97797), rat SIRPα (e.g., UniProt Accession No. P97710), canine SIRPα (e.g., UniProt Accession No. F1PK00), cynomolgus monkey SIRPα (e.g., NCBI Accession No. NP_001271679), etc.). Thus, the binding molecule (e.g., an antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody)) may also inhibit binding of CD47 from humans or other species to SIRPα from species other than human. In other cases, the binding molecule may be completely specific for the interaction of human CD47 and human SIRPα and may not exhibit species or other types of cross-reactivity.

[0101] The term "CD47 antibody" or "anti-CD47 antibody", as defined herein, refers to an antibody capable of binding to the human CD47 receptor.

[0102] The term "chimeric antibody" refers to an antibody that contains amino acid sequences derived from different animal species, such as those having a variable region derived from a human antibody and a murine immunoglobulin constant region.

[0103] The term "compete for binding" refers to the interaction of two antibodies in binding to their binding targets. A primary antibody competes with a secondary antibody for binding if the binding of the primary antibody to its cognate epitope is detectably reduced in the presence of the secondary antibody compared to the binding of the primary antibody in the absence of the secondary antibody. Alternatively, it may be, but is not necessarily, the case if the binding of the secondary antibody to its epitope is also detectably reduced in the presence of the primary antibody. That is, a primary antibody can inhibit the binding of a secondary antibody to its epitope without the secondary antibody inhibiting the binding of the primary antibody to its respective epitope. However, if each antibody detectably inhibits the binding of the other antibody to its cognate epitope to the same, greater or lesser extent, the antibodies are said to "cross-compete" with each other for binding of their respective epitope(s).

[0104] The term "epitope" refers to a portion of an antigen to which an antibody (or an antigen-binding fragment thereof) binds. Epitopes can be formed from both contiguous or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes can contain a variable number of amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance, deuterium hydrogen exchange combined with mass spectrometry, or site-directed mutagenesis, or any method used in combination with computational modeling of the complex structure of the antigen and its binding antibody and its variants. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed.(1996). Once the desired epitope of an antigen has been determined, antibodies against that epitope can be generated, for example, using the techniques described herein. Generation and characterization of antibodies can reveal information about the desired epitope. From this information, antibodies can be competitively screened for binding to the same epitope. An approach to accomplish this is to perform cross-competition studies to find antibodies that competitively bind to each other, i.e., that compete for binding to the antigen. A high-throughput process for "binning" antibodies based on cross-competition is described in PCT Publication No. WO03 / 48731.

[0105] The term "glycosylation site" refers to an amino acid residue that is recognized by a eukaryotic cell as a location for the addition of a sugar residue. The amino acids to which carbohydrates, such as oligosaccharides, are attached are typically asparagine (N-linked), serine (O-linked), and threonine (O-linked) residues. A particular attachment site is typically indicated by an amino acid sequence, referred to herein as a "glycosylation site sequence." The glycosylation site sequence for N-linked glycosylation is: -Asn-X-Ser- or -Asn-X-Thr-, where X can be any standard amino acid except proline. The terms "N-linked" and "O-linked" refer to the chemical groups that serve as the attachment site between a sugar molecule and an amino acid residue. N-linked sugars are added through an amino group and O-linked sugars are added through a hydroxyl group. The term "glycan occupancy" refers to the presence of a sugar moiety linked to a glycosylation site (i.e., the glycan site is occupied). When there are at least two potential glycosylation sites on a polypeptide, either none (0-glycan site occupied), one (1-glycan site occupied), or both (2-glycan site occupied) sites can be occupied by a sugar moiety.

[0106] The term "host cell" refers to a cell line that can be genetically engineered to produce a protein, protein fragment, or peptide of interest. Host cells include, but are not limited to, cultured cells such as rodent (e.g., rat, mouse, guinea pig, or hamster), e.g., CHO, BHK, NSO, SP2 / 0, YB2 / 0, or mammalian cultured cells derived from human tissue or hybridoma cells, yeast cells, insect cells, and cells contained within transgenic animals or cultured tissues. The term encompasses not only the particular subject cell, but also the progeny of such cells. Because certain modifications may occur in successive generations due to mutations or environmental influences, such progeny may not be identical to the parent cell, but are still within the scope of the term "host cell."

[0107] The term "human antibody" refers to an antibody whose entire light and heavy chain amino acid sequences are derived from human immunoglobulin genes. A human antibody may contain mouse glycosylation if produced in a mouse, a mouse cell, or a hybridoma derived from a mouse cell. Human antibodies can be prepared in a variety of ways known in the art.

[0108] The term "humanized antibody" refers to a chimeric antibody that contains amino acid residues derived from human antibody sequences. A humanized antibody can contain some or all of the CDRs or HVRs derived from a non-human animal or synthetic antibody, while the antibody framework and constant regions contain amino acid residues derived from human antibody sequences.

[0109] The term "illustrative antibody" or "exemplary antibody" refers to any one of the antibodies, masked antibodies (e.g., activatable antibodies), or antigen-binding fragments described in this disclosure and are defined as listed in Tables 3A-6. These antibodies may be of any class (e.g., IgA, IgD, IgE, IgG, and IgM). Thus, each of the antibodies identified above may be of any class, including, but not limited to, V, VL, VD, VL ... L and V H The present invention encompasses antibodies of all five classes that have identical amino acid sequences for the V domain. Furthermore, antibodies of the IgG class can be of any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). Thus, antibodies of each of the IgG subclasses identified above include antibodies of the V domain and V subclass. L and V H The present disclosure includes antibodies of all four subclasses that have identical amino acid sequences for the heavy chain constant regions. The amino acid sequences of the five classes and four IgG subclasses of human antibodies are known in the art. The amino acid sequences of the full-length heavy and light chains of each exemplary antibody of the IgG4 and IgG1 subclasses shown in Table 6 are provided in the present disclosure.

[0110] The term "isolated antibody" or "isolated binding molecule" refers to an antibody or binding molecule, as defined herein, that is: (1) not associated with naturally associated components that accompany it in its natural state; (2) free of other proteins from the same species; (3) expressed by a cell of a different species; or (4) not naturally occurring. Examples of isolated antibodies include CD47 antibodies that have been affinity purified using CD47, CD47 antibodies produced in vitro by a hybridoma or other cell line, and CD47 antibodies derived from transgenic animals.

[0111] The terms "isolated nucleic acid" and "isolated polynucleotide" refer to a nucleic acid molecule or polynucleotide of genomic, cDNA, or synthetic origin, or a combination thereof, that is separated from other nucleic acid molecules and polynucleotides that are present in the natural source of the nucleic acid. For example, with respect to genomic DNA, the term "isolated" includes nucleic acid molecules and polynucleotides that are separated from the chromosome with which the genomic DNA is naturally associated. For example, with respect to genomic DNA, the term "isolated" includes nucleic acid molecules or polynucleotides that are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an "isolated" nucleic acid or polynucleotide does not include sequences that naturally flank the nucleic acid or polynucleotide (i.e., sequences located at the 5' and 3' ends of the nucleic acid of interest).

[0112] "k a The term "k" refers to the association rate constant of a particular antibody-antigen interaction. d The term "dissociation rate constant" refers to the dissociation rate constant of a particular antibody-antigen interaction.

[0113] "K D The term "dissociation constant" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction. This is k d k a (i.e., k d / k a ) and is expressed as molar concentration (M). Dis used as a measure of the affinity of an antibody to bind to a binding partner (i.e., its "binding affinity"). D The smaller the K, the stronger the antibody binds (i.e., has a higher binding affinity) or the higher the binding affinity between the antibody and the antigen. For example, an antibody with a nanomolar (nM) dissociation constant will bind more tightly to a particular antigen than an antibody with a micromolar (μM) dissociation constant. D can be determined using methods well established in the art. D One method of determining is by using surface plasmon resonance, typically using a biosensor system such as a Biacore® system or an Octet® RED96 system. An assay procedure using the Octet® RED96 system is described in the Examples section of this disclosure.

[0114] The terms "subject," "patient," and "individual" are used interchangeably herein and may refer to a human or non-human animal. In some instances, "subject," "patient," or "individual" refers to a subject, patient, or individual in need of treatment for a disease or condition. "Non-human animal" may refer to any animal not classified as a human, such as farm animals, livestock, or zoo animals, sport animals, pet animals (e.g., dogs, horses, cats, cows, etc.), and animals used in research. Research animals may refer, but are not limited to, nematodes, arthropods, vertebrates, mammals, frogs, rodents (e.g., mice or rats), fish (e.g., zebrafish or pufferfish), birds (e.g., chickens), dogs, cats, and non-human primates (e.g., rhesus monkeys, cynomolgus monkeys, chimpanzees, etc.). In some embodiments, the subject, patient, or individual is a human.

[0115] The term "prevent" or "preventing" in relation to a particular disease state in a mammal refers to preventing or delaying the onset of the disease or preventing the manifestation of its clinical or subclinical symptoms.

[0116] As used herein, "sequence identity" between two polypeptide sequences refers to the percentage of amino acids that are identical between sequences.The amino acid sequence identity of polypeptides can be conventionally determined using known computer programs such as Bestfit, FASTA, or BLAST (see, for example, Pearson, Methods Enzymol. 183:63-98(1990); Pearson, Methods Mol. Biol. 132:185-219(2000); Altschul et al., J. Mol. Biol. 215:403-410(1990); Altschul et al., Nucelic Acids Res. 25:3389-3402(1997)). When using Bestfit or other sequence alignment programs to determine whether a particular sequence is, for example, 95% identical to a reference amino acid sequence, parameters are set such that the percentage of identity is calculated over the entire length of the reference amino acid sequence, allowing for gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence. This aforementioned method of determining the percentage of identity between polypeptides is applicable to all proteins, fragments, or variants disclosed herein.

[0117] The terms "treat", "treating" or "treatment" for a particular disease condition in a mammal refer to causing a desired or beneficial effect in a mammal having the disease condition. A desired or beneficial effect may include a reduction in the frequency or severity of one or more symptoms of the disease (e.g., tumor growth and / or metastasis, or other effects mediated by immune cell numbers and / or activity, etc.), or a halt or inhibition of further progression of the disease, condition, or disorder. In the context of cancer treatment in a mammal, a desired or beneficial effect may include inhibition of further growth or spread of cancer cells, killing of cancer cells, inhibition of recurrence of cancer, reduction of pain associated with cancer, or improved survival of the mammal. The effect may be either subjective or objective. For example, if the mammal is a human, the human may note improved energy or vitality, or reduced pain as subjective symptoms of an improvement or response to treatment. Alternatively, the clinician may note a reduction in tumor size or tumor burden based on physical exam, laboratory values, tumor markers, or x-ray findings. Some laboratory signs that a clinician may observe for response to treatment include normalization of tests such as white blood cell count, red blood cell count, platelet count, erythrocyte sedimentation rate, and various enzyme levels. In addition, a clinician may observe a decrease in detectable tumor markers. Alternatively, other tests, such as sonograms, nuclear magnetic resonance tests, and positron emission tests, may be used to assess objective improvement.

[0118] The term "CD47-positive disease" or "CD47-positive condition" refers to a disease or condition involving one or more cells having aberrant expression, amount, activity, or function of CD47, and / or which can be treated by modulating the binding of CD47 to one or more of its binding partners. For example, CD47-positive diseases include cancers characterized by tumors expressing high levels of CD47 compared to healthy tissue, which can be treated by inhibiting the binding of CD47 to one of its binding partners, such as SIRPα. Exemplary CD47-positive cancers are provided herein. CD47-positive cancers include lymphomas (e.g., diffuse large B-cell lymphoma (DLBCL) and lymphoid neoplasm diffuse large B-cell lymphoma (DLBC)), leukemias (e.g., acute myeloid leukemia (AML)), head and neck cancers (e.g., head and neck squamous cell carcinoma (HNSC)), gastric cancers (e.g., gastric cancer (GC) and Her2+ gastroesophageal junction (GEJ) cancer), breast cancers (e.g., breast invasive carcinoma (BRCA), HER2+ breast cancer, hormone receptor positive breast cancer and triple negative breast cancer (TNBC)), cervical cancers (e.g., These include, but are not limited to, cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), bile duct cancer (CHOL), colon cancer (e.g., colon adenocarcinoma (COAD)), ovarian cancer (e.g., ovarian serous cystadenocarcinoma (OV)), thyroid cancer (e.g., thyroid carcinoma (THCA)), uterine cancer (e.g., uterine endometrial carcinoma (UCEC)), endometrial cancer, lung cancer (e.g., lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), small cell lung cancer (SCLC)), mesothelioma (MESO), pancreatic cancer (e.g., pancreatic adenocarcinoma (PAAD)), and the like.

[0119] The term "vector" refers to a nucleic acid molecule capable of transporting a foreign nucleic acid molecule. The foreign nucleic acid molecule is linked to the vector nucleic acid molecule by recombinant techniques such as ligation or recombination. This allows the foreign nucleic acid molecule to be propagated, selected, further manipulated or expressed in a host cell or organism. The vector can be a plasmid, phage, transposon, cosmid, chromosome, virus, or virion. One type of vector integrates into the genome of the host cell upon introduction into the host cell, thereby replicating along with the host genome (e.g., non-episomal mammalian vectors). Another type of vector is capable of autonomous replication in the host cell into which it is introduced (e.g., bacterial vectors and episomal mammalian vectors having a bacterial origin of replication). Another specific type of vector capable of inducing the expression of an expressible foreign nucleic acid to which they are operably linked is generally called an "expression vector". Expression vectors generally have control sequences that drive the expression of an expressible foreign nucleic acid. Simpler vectors, known as "transcription vectors", are only capable of transcription and not translation: they can replicate in a target cell but not express. The term "vector" encompasses all types of vectors, regardless of their function. Vectors that are capable of directing the expression of expressible nucleic acids to which they are operatively linked are commonly referred to as "expression vectors".

[0120] The methods and techniques of the present disclosure are generally performed according to methods well known in the art and described in various general and more specific references cited and discussed throughout the specification, unless otherwise indicated. Such references include, for example, Sambrook and Russell, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY (2001), Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002), and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as the laboratory procedures and techniques, are well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, delivery, and treatment of patients.

[0121] B. Antibodies, antigen-binding fragments, and masked antibodies that bind human CD47 The present disclosure provides isolated binding molecules (e.g., antibodies, antigen-binding fragments, or masked antibodies (e.g., activatable antibodies)) that bind to human CD47, including CD47 antibodies, activatable CD47 antibodies, antigen-binding fragments of CD47 antibodies, and derivatives of CD47 antibodies. In some embodiments, the binding molecules are any of the antibodies, masked antibodies (e.g., activatable antibodies) and antigen-binding fragments described in relation to epitope binding, and the antibodies described in relation to CDRs, variable regions (VL, VH), and IgG (e.g., IgG4 and IgG4) light and heavy chains. In some embodiments, the present disclosure relates to antibodies, antigen-binding fragments, or masked antibodies (e.g., activatable antibodies) that bind human CD47 and have at least one (e.g., at least one, at least two, at least three, at least four, at least five, or all six) of the following functional properties: (a) bind to human CD47 with a KD of 500 nM or less; (b) are cross-reactive with monkey, rat, or dog CD47; (c) are capable of inhibiting tumor cell growth; (d) have a therapeutic effect against cancer; (e) block the binding between CD47 and a SIRP protein (e.g., SIRPα); and (f) induce antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP) against tumor cells expressing CD47. In some embodiments, the antibodies, antigen-binding fragments, or masked antibodies (e.g., activatable antibodies) disclosed herein can also block, e.g., completely block, the binding between CD47 and its ligand, SIRPα. Also provided herein are one or more anti-CD47 antibodies, antigen-binding fragments, or masked antibodies (e.g., activatable antibodies) that cross-compete for binding to human CD47 with one or more of the antibodies, antigen-binding fragments, or masked antibodies (e.g., activatable antibodies) described herein.

[0122] In some embodiments, when a cell expressing human CD47 (e.g., a human cell) is contacted with the antibody or antigen-binding fragment, the antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) alters (e.g., inhibits or enhances) one or more (e.g., one or more, two or more, three or more, etc.) activities of human CD47. Various CD47 activities are known in the art, including, but not limited to, inhibition of antibody-dependent cell-mediated phagocytosis (ADCP) by immune cells (e.g., macrophages); inhibition of ADCC by natural killer (NK) cells; and stimulation of cell-cell fusion, T cell activation, T cell proliferation, apoptosis, cell proliferation, cell survival, and cell adhesion (Sick et al. "CD47 update: a multifaceted actor in the tumor microenvironment of potential therapeutic interest." Br J Pharmacol. 2012;167(7):1415-1430). In some embodiments, the one or more CD47 activities is not CD47 binding to its ligand (e.g., SIRPα). Methods for measuring CD47 activities (e.g., inhibition of phagocytosis, inhibition of ADCC, etc.) are known in the art and include, for example, those methods described in Examples 9 and 10 below.

[0123] In some embodiments, the antibodies, antigen-binding fragments, and masked antibodies (e.g., activatable antibodies) described herein have enhanced antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP), function(s). Methods for measuring ADCC of antibodies and antigen-binding fragments are well known in the art. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be determined by assaying for cytotoxicity using a cytotoxic antibody such as that described by Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656(1998) can be evaluated in vivo, for example in animal models. The contribution of ADCC to tumor cell killing can be measured using a specific test using NK-92 cells transfected with high affinity FcR. The results are compared to wild-type NK-92 cells that do not express FcR. Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo, for example in animal models such as those disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656(1998).Methods for measuring the ADCC of antibodies and antigen-binding fragments include, for example, the methods described in Example 10 below. Methods for measuring the ADCC of antibodies and antigen-binding fragments are also well known in the art. To assess the ADCP activity of a molecule of interest, for example, an in vitro ADCP assay (see, for example, Bracher et al., 2007, J.Immunol.Methods 323:160-71) can be performed. Phagocytic cells useful for such assays include peripheral blood mononuclear cells (PBMCs), monocytes purified from PBMCs, or U937 cells differentiated into monocytic lineage. Alternatively or additionally, the ADCP activity of a molecule of interest can be assessed in vivo, for example, in an animal model (see, for example, Wallace et al., 2001, J.Immunol.Methods 248:167-82). Methods for measuring the ADCP of antibodies and antigen-binding fragments include, for example, the methods described in Example 9 below.

[0124] In some embodiments, the antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) does not cause hemagglutination (e.g., clustering) of red blood cells (RBCs) (e.g., human RBCs) in vitro. In certain embodiments, the antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) does not cause hemagglutination of human RBCs in vitro when provided at a concentration of up to about 50 nM, up to about 100 nM, up to about 500 nM, up to about 1000 nM, or up to about 5000 nM.

[0125] In some embodiments, the antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) has a therapeutic effect against cancer. In some embodiments, the antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) ameliorates one or more signs or symptoms of cancer. In some embodiments, a subject suffering from cancer goes into partial or complete remission upon administration of the antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody). In some embodiments, the cancer comprises B-cell lymphoma, T-cell lymphoma, or any combination thereof. In certain embodiments, the cancer is a B-cell lymphoma. In another embodiment, the cancer is a T-cell lymphoma. In some embodiments, the cancer is lymphoma (e.g., diffuse large B-cell lymphoma (DLBCL) and lymphoid neoplasm diffuse large B-cell lymphoma (DLBC)), leukemia (e.g., acute myeloid leukemia (AML)), head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSC)), gastric cancer (e.g., gastric cancer (GC) and Her2+ gastroesophageal junction (GEJ) cancer), breast cancer (e.g., breast cancer (BRCA), HER2+ breast cancer, hormone receptor positive breast cancer, and triple negative breast cancer (T NBC), cervical cancer (e.g., cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC)), bile duct cancer (CHOL), colon cancer (e.g., colon adenocarcinoma (COAD)), ovarian cancer (e.g., ovarian serous cystadenocarcinoma (OV)), thyroid cancer (e.g., thyroid carcinoma (THCA)), uterine cancer (e.g., uterine endometrial carcinoma (UCEC)), endometrial cancer, lung cancer (e.g., lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), small cell lung cancer (SCLC)), mesothelioma (MESO) or pancreatic cancer (e.g., pancreatic adenocarcinoma (PAAD)).

[0126] In some embodiments, the antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) binds to a different epitope of human CD47 than other antibodies. In some embodiments, the antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) binds to a different epitope of human CD47, such that the antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) binds to human CD47 in a cis configuration rather than a trans configuration. In some embodiments, the antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) binds to a different epitope of human CD47 in a cis configuration, such that the antibody causes less red blood cell agglutination than an antibody that binds to human CD47 in a trans configuration. In certain embodiments, the masked antibody binds to 1, 2, 3, 4, 5, 6, 7, 8, 9, or all 10 amino acid residues of human CD47 selected from the group consisting of K39, W40, K41, F50, D51, G52, T99, E100, L101, and T102. In certain embodiments that can be combined with the preceding embodiments, the masked antibody does not bind to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or all 20 amino acid residues of human CD47 selected from the group consisting of L2, L3, F4, K6, N27, E29, A30, Q31, T34, E35, V36, Y37, D46, T49, A53, E97, R103, E104, G105, and E106. In certain embodiments, the masked antibody binds to amino acid residues K39, W40, K41, F50, D51, G52, T99, E100, L101, and T102 of human CD47, and does not bind to any of amino acid residues L2, L3, F4, K6, N27, E29, A30, Q31, T34, E35, V36, Y37, D46, T49, A53, E97, R103, E104, G105, and E106 of human CD47.

[0127] In another aspect, the disclosure provides isolated antibodies that compete or cross-compete with any of the exemplary antibodies, antigen-binding fragments, or masked antibodies (e.g., activatable antibodies) of the disclosure, for binding to human CD47, such as TY25031, TY25034, TY25040, TY21446, TY1447, TY26294 (after CM cleavage), TY26896, TY26897, TY26898 (after CM cleavage), and TY26899 (after CM cleavage). In certain embodiments, the disclosure provides isolated antibodies that compete or cross-compete with any of the exemplary antibodies of the disclosure for binding to the same epitope on human CD47. The ability of an antibody to compete or cross-compete with another antibody for binding can be determined using standard binding assays known in the art, such as BIAcore analysis, ELISA assays, or flow cytometry. For example, an exemplary antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) of the present disclosure can be bound to human CD47 under saturating conditions, and then the ability of the test antibody to bind to CD47 can be measured. If the test antibody is able to bind to the CD47 simultaneously with the exemplary antibody, then the test antibody binds to a different epitope than the exemplary antibody. However, if the test antibody is not able to bind to the CD47 simultaneously, then the test antibody binds to the same epitope as, an epitope that overlaps with, or an epitope that is close to the epitope bound by the exemplary antibody. This experiment can be performed using a variety of methods, such as ELISA, RIA, FACS, or surface plasmon resonance.

[0128] In one aspect, the disclosure provides isolated antibodies, antigen-binding fragments, or masked antibodies (e.g., activatable antibodies) having particular complementarity determining regions (CDRs). The isolated antibodies, antigen-binding fragments, or masked antibodies (e.g., activatable antibodies) can include one or more (e.g., one, two, three, four, five, or six) of any of the CDRs described herein, in any combination.

[0129] In some embodiments, an isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) can contain an antibody heavy chain variable region (VH), an antibody light chain variable region (VL), or both. In some embodiments, the VH comprises a first complementarity determining region (CDR-H1), a second complementarity determining region (CDR-H2), and a third complementarity determining region (CDR-H3).

[0130] In some embodiments, CDR-H1 comprises an amino acid sequence according to formula (I): X1YX2IH (SEQ ID NO:182), where X1 is D, G, N, R, or S, and X2 is A, G, or W.

[0131] In some embodiments, CDR-H1 comprises an amino acid sequence according to formula (II): SGX1X2WX3 (SEQ ID NO:183), wherein X1 and X2 are each independently H or Y, and X3 is D, G, N, S, or T.

[0132] In some embodiments, CDR-H2 comprises an amino acid sequence according to formula (III): X1IX2X3X4GX5X6X7YX8PSLKS (SEQ ID NO:184), where X1 is A, E or R, X2 is S or Y, X3 is H, W or Y, X4 is D or S, X5 is D, N or S, X6 is K or T, X7 is R or Y and X8 is N or S.

[0133] In some embodiments, the CDR-H2 comprises an amino acid sequence according to formula (IV): X1IX2X3X4X5X6X7X8X9YAX10X11X12X13G (SEQ ID NO:185), where X1 is A, G, I, R or W, X2 is I, N, S or Y, X3 is G or P, X4 is A, N, S or V, X5 is F, G or S, X6 is G or S, X7 is G, S or T, X8 is A, P or T, X9 is K, N or Y, X10 is D or Q, X11 is K or S, X12 is F or V, and X13 is K or Q.

[0134] In some embodiments, the CDR-H3 comprises an amino acid sequence according to formula (V): X1X2X3X4X5X6FX7X8 (SEQ ID NO:186), where X1 is Q, R, S or Y, X2 is G, R, V or Y, X3 is G, H, I, P or Y, X4 is A, G, L or Y, X5 is A, G, P or Y, X6 is A, D, G, R, S or V, X7 is A or D, and X8 is V or Y.

[0135] In some embodiments, the CDR-H3 comprises an amino acid sequence according to formula (VI): X1X2X3GX4X5X6X7DX8 (SEQ ID NO:187), wherein X1 and X4 are each independently G or Y, X2 is A or G, X3 is R or Y, X5 and X6 are each independently A or Y, X7 is F or L, and X8 is V or Y.

[0136] In some embodiments, the CDR-H3 comprises an amino acid sequence according to formula (VII): X1X2X3X4X5X6GX7FDX8 (SEQ ID NO:188), where X1 is G or R, X2 is G or V, X3 is R or S, X4 is G or Y, X5 is G or S, X6 is F or Y, X7 is A or W, and X8 is V or Y.

[0137] In some embodiments, the CDR-H3 comprises an amino acid sequence according to formula (VIII): X1X2X3X4X5X6SX7X8YDX9FDX10 (SEQ ID NO:189), where X1 is D or H, X2 is R or Y, X3 is A or L, X4 is F or P, X5 and X9 are each independently A or G, X6 is G or S, X7 is G or T, X8 is S or Y, and X10 is I or Y.

[0138] In some embodiments, CDR-L1 comprises an amino acid sequence according to formula (IX): SASSX1VX2YX3Y (SEQ ID NO:190), where X1 is R or S, X2 is G, S, or T, and X3 is I or V.

[0139] In some embodiments, CDR-L1 comprises an amino acid sequence according to formula (X): RASQX1IX2X3X4LX5 (SEQ ID NO:191), where X1 is G or T, X2 is G or S, X3 is R or S, X4 is V or Y, and X5 is A or N.

[0140] In some embodiments, CDR-L1 comprises an amino acid sequence according to formula (XI): RASQX1VX2X3RX4LA (SEQ ID NO:192), where X1 is S or T, X2 is I or R, X3 is G or S, and X4 is L or Y.

[0141] In some embodiments, CDR-L1 comprises an amino acid sequence according to formula (XII): RASX1SVDFX2GX3SFLX4 (SEQ ID NO:193), wherein X1 is E or Q, X2 is H, V, or Y, X3 is F, I, or K, and X4 is A, D, or H.

[0142] In some embodiments, CDR-L2 comprises an amino acid sequence according to formula (XIII): X1ASX2X3X4X5 (SEQ ID NO:194), where X1 is A or D, X2 is N, S, or T, X3 is L or R, X4 is A, E, or Q, and X5 is S or T.

[0143] In some embodiments, the CDR-L3 comprises an amino acid sequence according to formula (XIV): X1QX2X3X4X5PX6T (SEQ ID NO:195), where X1 is A, H, Q or V, X2 is A, G, R, S or Y, X3 is G, I, L, S, T or Y, X4 is A, E, P, Q, R, S, T or Y, X5 is A, I, L, S, T or W, and X6 is F, H, L, W or Y.

[0144] In some embodiments, the CDR-L3 comprises an amino acid sequence according to formula (XV): QX1YX2SX3PX4X5X6T (SEQ ID NO:196), where X1 is H or Q, X2 is A, T, or V, X3 is S or W, X4 is P or R, X5 is G or V, and X6 is F or Y.

[0145] In some embodiments, an isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) can comprise one or more (e.g., one, two, three, four, five, or six) CDRs that comprise an amino acid sequence selected from the group consisting of SEQ ID NOs:182-196. In some embodiments, an isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) can comprise two or more (e.g., two, three, four, five, or six) CDRs, each of which comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:182-196.

[0146] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL. In some embodiments, the VH comprises CDR-H1, CDR-H2, and CDR-H3. In some embodiments, CDR-H1 comprises the amino acid sequence of SEQ ID NO: 182 or 183. In some embodiments, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 184 or 185. In some embodiments, CDR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 186-189. In certain embodiments, the VL comprises CDR-L1, CDR-L2, and CDR-L3. In some embodiments, CDR-L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 190-193. In some embodiments, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 194. In some embodiments, the CDR-L3 comprises the amino acid sequence of SEQ ID NO:195 or 196.

[0147] In some embodiments, an isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) can comprise a CDR-H1 having the amino acid sequence of SEQ ID NO:182 or 183; a CDR-H2 having the amino acid sequence of SEQ ID NO:184 or 185; a CDR-H3 having an amino acid sequence selected from the group consisting of SEQ ID NO:186-189; a CDR-L1 having an amino acid sequence selected from the group consisting of SEQ ID NO:190-193; a CDR-L2 having the amino acid sequence of SEQ ID NO:194; and / or a CDR-L3 having the amino acid sequence of SEQ ID NO:195 or 196.

[0148] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, wherein 1) the VH comprises CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO:182 or 183, CDR-H2 comprises the amino acid sequence of SEQ ID NO:184 or 185, and / or CDR-H3 comprises an amino acid sequence selected from SEQ ID NO:186-189; and / or 2) the VL comprises CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 comprises an amino acid sequence selected from SEQ ID NO:190-193; CDR-L2 comprises the amino acid sequence of SEQ ID NO:194; and / or CDR-L3 comprises the amino acid sequence of SEQ ID NO:195 or 196.

[0149] In certain embodiments, an isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:182; a CDR-H2 comprising the amino acid sequence of SEQ ID NO:185; and / or a CDR-H3 comprising the amino acid sequence of SEQ ID NO:188; and / or b) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:191; a CDR-L2 comprising the amino acid sequence of SEQ ID NO:194; and / or a CDR-L3 comprising the amino acid sequence of SEQ ID NO:195. In one embodiment, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, wherein 1) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:182, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:185, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:188; and b) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:191, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:194, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:195.

[0150] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, wherein the VH comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs:182-189 and / or the VL comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs:190-196. In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, wherein the VH comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs:182-189 and the VL comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs:190-196.

[0151] In certain embodiments, an isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, wherein 1) the VH comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO:182-183, one or more amino acid sequences selected from the group consisting of SEQ ID NO:184-185, and / or one or more amino acid sequences selected from the group consisting of SEQ ID NO:186-189; and / or 2) the VL comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO:190-193, the amino acid sequence of SEQ ID NO:194, and / or one or more amino acid sequences selected from the group consisting of SEQ ID NO:195-196. In certain embodiments, an isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, where 1) the VH comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO:182-183, one or more amino acid sequences selected from the group consisting of SEQ ID NO:184-185, and one or more amino acid sequences selected from the group consisting of SEQ ID NO:186-189; and 2) the VL comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO:190-193, the amino acid sequence of SEQ ID NO:194, and one or more amino acid sequences selected from the group consisting of SEQ ID NO:195-196.

[0152] In some embodiments, 1) the VH comprises the amino acid sequence of SEQ ID NO:182; the amino acid sequence of SEQ ID NO:185; and / or the amino acid sequence of SEQ ID NO:188; and / or 2) the VL comprises the amino acid sequence of SEQ ID NO:191; the amino acid sequence of SEQ ID NO:194; and / or the amino acid sequence of SEQ ID NO:195. In one embodiment, 1) the VH comprises the amino acid sequence of SEQ ID NO:182, SEQ ID NO:185, and SEQ ID NO:188; and 2) the VL comprises the amino acid sequence of SEQ ID NO:191, SEQ ID NO:194, and SEQ ID NO:195.

[0153] In some embodiments, an isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) can comprise one or more (e.g., one, two, three, four, five, or six) CDRs comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:35-136, or a variant thereof comprising up to five (e.g., one, two, three, four, or five) amino acid substitutions. An isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) can comprise two or more (e.g., two, three, four, five, or six) CDRs each comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:35-136, or a variant thereof comprising up to five (e.g., one, two, three, four, or five) amino acid substitutions, in any combination. In some embodiments, an isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) can comprise one or more (e.g., one, two, three, four, five, or six) CDRs of one or more exemplary antibodies shown in Tables 3A-3B, or a variant thereof containing up to five (e.g., one, two, three, four, or five) amino acid substitutions.

[0154] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL. In some embodiments, the VH comprises CDR-H1, CDR-H2, and CDR-H3. In some embodiments, the CDR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:35-51, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, the CDR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:52-68, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, the CDR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:69-85, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions. In certain embodiments, the VL comprises CDR-L1, CDR-L2, and CDR-L3. In some embodiments, CDR-L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:86-102, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions. In some embodiments, CDR-L2 comprises an amino acid sequence selected from SEQ ID NOs:103-119, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions. In some embodiments, CDR-L3 comprises an amino acid sequence selected from SEQ ID NOs:120-136, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions.

[0155] In some embodiments, the antibody comprises a CDR-H1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 35-51, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; a CDR-H2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 52-68, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; a CDR-H3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 69-85, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; a CDR-L1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 86-102, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or a CDR-L3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 120-136, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions.

[0156] In some embodiments, an isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, wherein 1) the VH comprises CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 35-51, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; CDR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 52-68, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or CDR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-56, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or 2) the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:69-85, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or 3) the VL comprises CDR-L1, CDR-L2 and CDR-L3, wherein CDR-L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:86-102, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; CDR-L2 comprises an amino acid sequence selected from SEQ ID NOs:103-119, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or CDR-L3 comprises an amino acid sequence selected from SEQ ID NOs:120-136, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions.

[0157] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, wherein the VH comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs:35-85, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or the VL comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs:86-136, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions. In certain embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, wherein 1) the VH comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 35-51, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 52-68, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 69-85, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or 2) the VL comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 86-102, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or one or more amino acid sequences selected from the group consisting of SEQ ID NOs:120-136, or variants thereof containing up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions.In certain embodiments, an isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, wherein 1) the VH comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO:35-51, one or more amino acid sequences selected from the group consisting of SEQ ID NO:52-68, and one or more amino acid sequences selected from the group consisting of SEQ ID NO:69-85; and 2) the VL comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO:86-102, one or more amino acid sequences selected from the group consisting of SEQ ID NO:103-119, and one or more amino acid sequences selected from the group consisting of SEQ ID NO:120-136. In some embodiments, an isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, where 1) the VH comprises one or more (e.g., one, two, three, four, five, or six) CDRs of the exemplary antibodies shown in Table 3A, or a variant thereof containing up to five (e.g., one, two, three, four, or five) amino acid substitutions; and / or 2) the VL comprises one or more (e.g., one, two, three, four, five, or six) CDRs of the exemplary antibodies shown in Table 3B, or a variant thereof containing up to five (e.g., one, two, three, four, or five) amino acid substitutions.

[0158] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions. In certain embodiments, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions. In some embodiments, an isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and / or VL of an exemplary antibody shown in Table 4A, or one or more amino acid sequences having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to a VH or VL of an exemplary antibody shown in Table 4A. An isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) can comprise any combination of any VH and any VL described herein.

[0159] In certain embodiments, an isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a light chain and / or a heavy chain (e.g., of an IgG, such as IgG1 or IgG4). In some embodiments, the heavy chain comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs:35-85, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or the light chain comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs:86-136, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a heavy chain and a light chain, wherein 1) the heavy chain comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 35-51, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 52-68, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 69-85, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or 2) the light chain comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 86-102, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or one or more amino acid sequences selected from the group consisting of SEQ ID NOs:120-136, or variants thereof containing up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions.

[0160] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH comprising a CDR-H1 comprising the sequence of NYAIH (SEQ ID NO:48), a CDR-H2 comprising the sequence of AISGSGSTYYADSVKG (SEQ ID NO:65), and a CDR-H3 comprising the sequence of RGSYGFGAFDY (SEQ ID NO:82), or a variant thereof comprising up to five (e.g., one, two, three, four, or five) amino acid substitutions relative to the CDR-H1, CDR-H2, and / or CDR-H3 sequences; and / or a VH comprising a CDR-L1 comprising the sequence of RASQTIGRYLN (SEQ ID NO:99), a CDR-L2 comprising the sequence of DASNRAT (SEQ ID NO:116), and a CDR-L3 comprising the sequence of QQRYPWPYT (SEQ ID NO:120). and a VL comprising a CDR-H1 sequence, a CDR-H2 sequence, and / or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions relative to the CDR-H1, CDR-H2, and / or CDR-H3 sequences.

[0161] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, wherein a) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:48, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; a CDR-H2 comprising the amino acid sequence of SEQ ID NO:65, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or a CDR-H3 comprising the amino acid sequence of SEQ ID NO:82, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or b) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:99, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or a CDR-L3 comprising the amino acid sequence of SEQ ID NO:133, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions. In certain embodiments, a) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:48, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:65, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:82; b) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:99, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:116, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:133.In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, wherein a) the VH comprises the amino acid sequence of SEQ ID NO:48, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:65, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or the amino acid sequence of SEQ ID NO:82, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or b) the VL comprises the amino acid sequence of SEQ ID NO:99, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:116, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; In certain embodiments, a) the VH comprises the amino acid sequence of SEQ ID NO:48, the amino acid sequence of SEQ ID NO:65, and the amino acid sequence of SEQ ID NO:82; b) the VL comprises the amino acid sequence of SEQ ID NO:99, the amino acid sequence of SEQ ID NO:116, and the amino acid sequence of SEQ ID NO:133. In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., activatable antibody) comprises one, two, three, four, five, or six CDRs of exemplary antibody TY21446 shown in Tables 3A-3B, or a variant thereof comprising up to five (e.g., one, two, three, four, or five) amino acid substitutions.

[0162] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH comprising CDR-H1, CDR-H2 and CDR-H3 of the VH comprising the amino acid sequence of SEQ ID NO:27, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:27; and / or a VL comprising CDR-L1, CDR-L2 and CDR-L3 of the VL comprising the amino acid sequence of SEQ ID NO:28, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:28. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO:27, or an amino acid sequence that has at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:27, and / or the VL comprises an amino acid sequence of SEQ ID NO:28, or an amino acid sequence that has at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:27. In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises the VH and / or VL of exemplary antibody TY21446 shown in Table 4A, or one or more amino acid sequences having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the VH or VL of TY21446 shown in Table 4A.

[0163] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, wherein a) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:49, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; a CDR-H2 comprising the amino acid sequence of SEQ ID NO:66, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or a CDR-H3 comprising the amino acid sequence of SEQ ID NO:83, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or b) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:100, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or CDR-L3 comprising the amino acid sequence of SEQ ID NO:134 or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions. In some embodiments, a) the VH comprises the amino acid sequence of SEQ ID NO:49, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:66, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or the amino acid sequence of SEQ ID NO:83, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or b) the VL comprises the amino acid sequence of SEQ ID NO:100, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:117, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; It includes the amino acid sequence of NO:134, or a variant thereof containing up to five (eg, 1, 2, 3, 4 or 5) amino acid substitutions.In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises one, two, three, four, five, or six CDRs of exemplary antibody TY21447 shown in Tables 3A-3B, or a variant thereof comprising up to five (e.g., one, two, three, four, or five) amino acid substitutions.

[0164] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH comprising CDR-H1, CDR-H2 and CDR-H3 of the VH comprising the amino acid sequence of SEQ ID NO:29, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:29; and / or a VL comprising CDR-L1, CDR-L2 and CDR-L3 of the VL comprising the amino acid sequence of SEQ ID NO:30, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:30. In certain embodiments, the VH comprises an amino acid sequence of SEQ ID NO:29, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:29; and / or the VL comprises an amino acid sequence of SEQ ID NO:30, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:30. In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises the VH and / or VL of exemplary antibody TY21447 shown in Table 4A, or one or more amino acid sequences having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the VH or VL of TY21447 shown in Table 4A.

[0165] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, wherein a) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:50, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; a CDR-H2 comprising the amino acid sequence of SEQ ID NO:67, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or a CDR-H3 comprising the amino acid sequence of SEQ ID NO:84, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or b) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:101, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or CDR-L3 comprising the amino acid sequence of SEQ ID NO:135, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions. In some embodiments, a) the VH comprises the amino acid sequence of SEQ ID NO:50, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:67, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or the amino acid sequence of SEQ ID NO:84, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or b) the VL comprises the amino acid sequence of SEQ ID NO:101, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:118, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; The amino acid sequence of NO:135, or a variant thereof containing up to five (eg, 1, 2, 3, 4 or 5) amino acid substitutions.In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises one, two, three, four, five, or six CDRs of exemplary antibody TY21449 shown in Tables 3A-3B, or a variant thereof comprising up to five (e.g., one, two, three, four, or five) amino acid substitutions.

[0166] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH comprising CDR-H1, CDR-H2 and CDR-H3 of the VH comprising the amino acid sequence of SEQ ID NO:31, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:31; and / or a VL comprising CDR-L1, CDR-L2 and CDR-L3 of the VL comprising the amino acid sequence of SEQ ID NO:32, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:32. In certain embodiments, the VH comprises an amino acid sequence of SEQ ID NO:31, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:31; and / or the VL comprises an amino acid sequence of SEQ ID NO:32, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:32. In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises the VH and / or VL of exemplary antibody TY21449 shown in Table 4A, or one or more amino acid sequences having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the VH or VL of TY21449 shown in Table 4A.

[0167] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, wherein a) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:35, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; a CDR-H2 comprising the amino acid sequence of SEQ ID NO:52, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or a CDR-H3 comprising the amino acid sequence of SEQ ID NO:69, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or b) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:86, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or CDR-L3 comprising the amino acid sequence of SEQ ID NO:120, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions. In some embodiments, a) the VH comprises the amino acid sequence of SEQ ID NO:35, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:52, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or the amino acid sequence of SEQ ID NO:69, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or b) the VL comprises the amino acid sequence of SEQ ID NO:86, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:103, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; The amino acid sequence of NO:120, or a variant thereof containing up to five (eg, 1, 2, 3, 4 or 5) amino acid substitutions.In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises one, two, three, four, five, or six CDRs of exemplary antibody TY25029 shown in Tables 3A-3B, or a variant thereof comprising up to five (e.g., one, two, three, four, or five) amino acid substitutions.

[0168] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH comprising CDR-H1, CDR-H2 and CDR-H3 of the VH comprising the amino acid sequence of SEQ ID NO:1, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:1; and / or a VL comprising CDR-L1, CDR-L2 and CDR-L3 of the VL comprising the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:2. In certain embodiments, the VH comprises an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:1; and / or the VL comprises an amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:2. In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises the VH and / or VL of exemplary antibody TY25029 shown in Table 4A, or one or more amino acid sequences having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the VH or VL of TY25029 shown in Table 4A.

[0169] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, wherein a) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:36, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; a CDR-H2 comprising the amino acid sequence of SEQ ID NO:53, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or a CDR-H3 comprising the amino acid sequence of SEQ ID NO:70, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or b) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:87, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; a CDR-L2 comprising the amino acid sequence of SEQ ID NO:104, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or a CDR-L3 comprising the amino acid sequence of SEQ ID NO:121, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions. In some embodiments, a) the VH comprises the amino acid sequence of SEQ ID NO:36, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:53, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or the amino acid sequence of SEQ ID NO:70, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or b) the VL comprises the amino acid sequence of SEQ ID NO:87, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:104, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; It includes the amino acid sequence of NO:121, or a variant thereof containing up to five (eg, 1, 2, 3, 4 or 5) amino acid substitutions.In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises one, two, three, four, five, or six CDRs of exemplary antibody TY25030 shown in Tables 3A-3B, or a variant thereof comprising up to five (e.g., one, two, three, four, or five) amino acid substitutions.

[0170] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH comprising CDR-H1, CDR-H2 and CDR-H3 of the VH comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:3; and / or a VL comprising CDR-L1, CDR-L2 and CDR-L3 of the VL comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:4. In certain embodiments, the VH comprises an amino acid sequence of SEQ ID NO:3, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:3; and / or the VL comprises an amino acid sequence of SEQ ID NO:4, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:4. In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises the VH and / or VL of exemplary antibody TY25030 shown in Table 4A, or one or more amino acid sequences having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the VH or VL of TY25030 shown in Table 4A.

[0171] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, wherein a) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:37, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; a CDR-H2 comprising the amino acid sequence of SEQ ID NO:54, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or a CDR-H3 comprising the amino acid sequence of SEQ ID NO:71, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or b) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:88, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or CDR-L3 comprising the amino acid sequence of SEQ ID NO:122, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions. In some embodiments, a) the VH comprises the amino acid sequence of SEQ ID NO:37, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:54, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or the amino acid sequence of SEQ ID NO:71, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or b) the VL comprises the amino acid sequence of SEQ ID NO:88, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:105, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; It includes the amino acid sequence of NO:122, or a variant thereof containing up to five (eg, 1, 2, 3, 4 or 5) amino acid substitutions.In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises one, two, three, four, five, or six CDRs of exemplary antibody TY25031 shown in Tables 3A-3B, or a variant thereof comprising up to five (e.g., one, two, three, four, or five) amino acid substitutions.

[0172] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH comprising CDR-H1, CDR-H2 and CDR-H3 of the VH comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:5; and / or a VL comprising CDR-L1, CDR-L2 and CDR-L3 of the VL comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:6. In certain embodiments, the VH comprises an amino acid sequence of SEQ ID NO:5, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:5; and / or the VL comprises an amino acid sequence of SEQ ID NO:6, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:6. In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises the VH and / or VL of exemplary antibody TY25031 shown in Table 4A, or one or more amino acid sequences having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the VH or VL of TY25031 shown in Table 4A.

[0173] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, wherein a) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:38, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; a CDR-H2 comprising the amino acid sequence of SEQ ID NO:55, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or a CDR-H3 comprising the amino acid sequence of SEQ ID NO:72, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or b) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:89, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or CDR-L3 comprising the amino acid sequence of SEQ ID NO:123, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions. In some embodiments, a) the VH comprises the amino acid sequence of SEQ ID NO:38, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:55, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or the amino acid sequence of SEQ ID NO:72, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or b) the VL comprises the amino acid sequence of SEQ ID NO:89, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:106, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or It includes the amino acid sequence of NO:123, or a variant thereof containing up to five (eg, 1, 2, 3, 4 or 5) amino acid substitutions.In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises one, two, three, four, five, or six CDRs of exemplary antibody TY25032 shown in Tables 3A-3B, or a variant thereof comprising up to five (e.g., one, two, three, four, or five) amino acid substitutions.

[0174] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH comprising CDR-H1, CDR-H2 and CDR-H3 of the VH comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:7; and / or a VL comprising CDR-L1, CDR-L2 and CDR-L3 of the VL comprising the amino acid sequence of SEQ ID NO:8, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:8. In certain embodiments, the VH comprises an amino acid sequence of SEQ ID NO:7, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:7; and / or the VL comprises an amino acid sequence of SEQ ID NO:8, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:8. In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises the VH and / or VL of exemplary antibody TY25032 shown in Table 4A, or one or more amino acid sequences having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the VH or VL of TY25032 shown in Table 4A.

[0175] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, wherein a) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:39, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; a CDR-H2 comprising the amino acid sequence of SEQ ID NO:56, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or a CDR-H3 comprising the amino acid sequence of SEQ ID NO:73, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or b) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:90, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or CDR-L3 comprising the amino acid sequence of SEQ ID NO:124 or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions. In some embodiments, a) the VH comprises the amino acid sequence of SEQ ID NO:39, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:56, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or the amino acid sequence of SEQ ID NO:73, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or b) the VL comprises the amino acid sequence of SEQ ID NO:90, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:107, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; It includes the amino acid sequence of NO:124, or a variant thereof containing up to five (eg, 1, 2, 3, 4 or 5) amino acid substitutions.In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises one, two, three, four, five, or six CDRs of exemplary antibody TY25033 shown in Tables 3A-3B, or a variant thereof comprising up to five (e.g., one, two, three, four, or five) amino acid substitutions.

[0176] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH comprising CDR-H1, CDR-H2 and CDR-H3 of the VH comprising the amino acid sequence of SEQ ID NO:9, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:9; and / or a VL comprising CDR-L1, CDR-L2 and CDR-L3 of the VL comprising the amino acid sequence of SEQ ID NO:10, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:10. In certain embodiments, the VH comprises an amino acid sequence of SEQ ID NO:9, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:9; and / or the VL comprises an amino acid sequence of SEQ ID NO:10, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:10. In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises the VH and / or VL of exemplary antibody TY25033 shown in Table 4A, or one or more amino acid sequences having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the VH or VL of TY25033 shown in Table 4A.

[0177] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, wherein a) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:40, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; a CDR-H2 comprising the amino acid sequence of SEQ ID NO:57, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or a CDR-H3 comprising the amino acid sequence of SEQ ID NO:74, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or b) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:91, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or CDR-L3 comprising the amino acid sequence of SEQ ID NO:125, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions. In some embodiments, a) the VH comprises the amino acid sequence of SEQ ID NO:40, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:57, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or the amino acid sequence of SEQ ID NO:74, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or b) the VL comprises the amino acid sequence of SEQ ID NO:91, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:108, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; The amino acid sequence of NO:125, or a variant thereof containing up to five (eg, 1, 2, 3, 4 or 5) amino acid substitutions.In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises one, two, three, four, five, or six CDRs of exemplary antibody TY25034 shown in Tables 3A-3B, or a variant thereof comprising up to five (e.g., one, two, three, four, or five) amino acid substitutions.

[0178] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH comprising CDR-H1, CDR-H2 and CDR-H3 of a VH comprising the amino acid sequence of SEQ ID NO:11, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:11; and / or a VL comprising CDR-L1, CDR-L2 and CDR-L3 of a VL comprising the amino acid sequence of SEQ ID NO:12, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:12. In certain embodiments, the VH comprises an amino acid sequence of SEQ ID NO:11, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:11; and / or the VL comprises an amino acid sequence of SEQ ID NO:12, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:12. In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises the VH and / or VL of exemplary antibody TY25034 shown in Table 4A, or one or more amino acid sequences having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the VH or VL of TY25034 shown in Table 4A.

[0179] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, wherein a) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:41, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; a CDR-H2 comprising the amino acid sequence of SEQ ID NO:58, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or a CDR-H3 comprising the amino acid sequence of SEQ ID NO:75, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or b) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:92, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or CDR-L3 comprising the amino acid sequence of SEQ ID NO:126, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions. In some embodiments, a) the VH comprises the amino acid sequence of SEQ ID NO:41, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:58, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or the amino acid sequence of SEQ ID NO:75, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or b) the VL comprises the amino acid sequence of SEQ ID NO:92, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:109, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; It includes the amino acid sequence of NO:126, or a variant thereof containing up to five (eg, 1, 2, 3, 4 or 5) amino acid substitutions.In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises one, two, three, four, five, or six CDRs of exemplary antibody TY25035 shown in Tables 3A-3B, or a variant thereof comprising up to five (e.g., one, two, three, four, or five) amino acid substitutions.

[0180] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH comprising CDR-H1, CDR-H2 and CDR-H3 of the VH comprising the amino acid sequence of SEQ ID NO:13, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:13; and / or a VL comprising CDR-L1, CDR-L2 and CDR-L3 of the VL comprising the amino acid sequence of SEQ ID NO:14, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:14. In certain embodiments, the VH comprises an amino acid sequence of SEQ ID NO:13, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:13; and / or the VL comprises an amino acid sequence of SEQ ID NO:14, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:14. In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises the VH and / or VL of exemplary antibody TY25035 shown in Table 4A, or one or more amino acid sequences having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the VH or VL of TY25035 shown in Table 4A.

[0181] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, wherein a) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:42, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; a CDR-H2 comprising the amino acid sequence of SEQ ID NO:59, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or a CDR-H3 comprising the amino acid sequence of SEQ ID NO:76, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or b) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:93, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or CDR-L3 comprising the amino acid sequence of SEQ ID NO:127, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions. In some embodiments, a) the VH comprises the amino acid sequence of SEQ ID NO:42, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:59, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or the amino acid sequence of SEQ ID NO:76, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or b) the VL comprises the amino acid sequence of SEQ ID NO:93, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:110, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; It includes the amino acid sequence of NO:127, or a variant thereof containing up to five (eg, 1, 2, 3, 4 or 5) amino acid substitutions.In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises one, two, three, four, five, or six CDRs of exemplary antibody TY25036 shown in Tables 3A-3B, or a variant thereof comprising up to five (e.g., one, two, three, four, or five) amino acid substitutions.

[0182] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH comprising CDR-H1, CDR-H2 and CDR-H3 of the VH comprising the amino acid sequence of SEQ ID NO:15, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:15; and / or a VL comprising CDR-L1, CDR-L2 and CDR-L3 of the VL comprising the amino acid sequence of SEQ ID NO:16, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:16. In certain embodiments, the VH comprises an amino acid sequence of SEQ ID NO:15, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:15; and / or the VL comprises an amino acid sequence of SEQ ID NO:16, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:16. In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises the VH and / or VL of exemplary antibody TY25036 shown in Table 4A, or one or more amino acid sequences having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the VH or VL of TY25036 shown in Table 4A.

[0183] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, wherein a) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:43, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; a CDR-H2 comprising the amino acid sequence of SEQ ID NO:60, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or a CDR-H3 comprising the amino acid sequence of SEQ ID NO:77, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or b) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:94, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; a CDR-L2 comprising the amino acid sequence of SEQ ID NO:111, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or a CDR-L3 comprising the amino acid sequence of SEQ ID NO:128, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions. In some embodiments, a) the VH comprises the amino acid sequence of SEQ ID NO:43, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:60, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or the amino acid sequence of SEQ ID NO:77, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or b) the VL comprises the amino acid sequence of SEQ ID NO:94, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:111, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; The amino acid sequence of NO:128 or a variant thereof containing up to five (eg, 1, 2, 3, 4 or 5) amino acid substitutions.In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises one, two, three, four, five, or six CDRs of exemplary antibody TY25037 shown in Tables 3A-3B, or a variant thereof comprising up to five (e.g., one, two, three, four, or five) amino acid substitutions.

[0184] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH comprising CDR-H1, CDR-H2 and CDR-H3 of the VH comprising the amino acid sequence of SEQ ID NO:17, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:17; and / or a VL comprising CDR-L1, CDR-L2 and CDR-L3 of the VL comprising the amino acid sequence of SEQ ID NO:18, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:18. In certain embodiments, the VH comprises an amino acid sequence of SEQ ID NO:17, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:17; and / or the VL comprises an amino acid sequence of SEQ ID NO:18, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:18. In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises the VH and / or VL of exemplary antibody TY25037 shown in Table 4A, or one or more amino acid sequences having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the VH or VL of TY25037 shown in Table 4A.

[0185] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, wherein a) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:44, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; a CDR-H2 comprising the amino acid sequence of SEQ ID NO:61, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or a CDR-H3 comprising the amino acid sequence of SEQ ID NO:78, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or b) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:95, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or CDR-L3 comprising the amino acid sequence of SEQ ID NO:129, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions. In some embodiments, a) the VH comprises the amino acid sequence of SEQ ID NO:44, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:61, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or the amino acid sequence of SEQ ID NO:78, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or b) the VL comprises the amino acid sequence of SEQ ID NO:95, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:112, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; It includes the amino acid sequence of NO:129, or a variant thereof containing up to five (eg, 1, 2, 3, 4 or 5) amino acid substitutions.In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises one, two, three, four, five, or six CDRs of exemplary antibody TY25038 shown in Tables 3A-3B, or a variant thereof comprising up to five (e.g., one, two, three, four, or five) amino acid substitutions.

[0186] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH comprising CDR-H1, CDR-H2 and CDR-H3 of the VH comprising the amino acid sequence of SEQ ID NO:19, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:19; and / or a VL comprising CDR-L1, CDR-L2 and CDR-L3 of the VL comprising the amino acid sequence of SEQ ID NO:20, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:20. In certain embodiments, the VH comprises an amino acid sequence of SEQ ID NO:19, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:19; and / or the VL comprises an amino acid sequence of SEQ ID NO:20, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:20. In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises the VH and / or VL of exemplary antibody TY25038 shown in Table 4A, or one or more amino acid sequences having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the VH or VL of TY25038 shown in Table 4A.

[0187] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, wherein a) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:45, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; a CDR-H2 comprising the amino acid sequence of SEQ ID NO:62, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or a CDR-H3 comprising the amino acid sequence of SEQ ID NO:79, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or b) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:96, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or CDR-L3 comprising the amino acid sequence of SEQ ID NO:130, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions. In some embodiments, a) the VH comprises the amino acid sequence of SEQ ID NO:45, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:62, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or the amino acid sequence of SEQ ID NO:79, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or b) the VL comprises the amino acid sequence of SEQ ID NO:96, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:113, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; The amino acid sequence of NO:130, or a variant thereof containing up to five (eg, 1, 2, 3, 4 or 5) amino acid substitutions.In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises one, two, three, four, five, or six CDRs of exemplary antibody TY25039 shown in Tables 3A-3B, or a variant thereof comprising up to five (e.g., one, two, three, four, or five) amino acid substitutions.

[0188] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH comprising CDR-H1, CDR-H2 and CDR-H3 of the VH comprising the amino acid sequence of SEQ ID NO:21, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:21; and / or a VL comprising CDR-L1, CDR-L2 and CDR-L3 of the VL comprising the amino acid sequence of SEQ ID NO:22, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:22. In certain embodiments, the VH comprises an amino acid sequence of SEQ ID NO:21, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:21; and / or the VL comprises an amino acid sequence of SEQ ID NO:22, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:22. In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises the VH and / or VL of exemplary antibody TY25039 shown in Table 4A, or one or more amino acid sequences having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the VH or VL of TY25039 shown in Table 4A.

[0189] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, wherein a) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:46, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; a CDR-H2 comprising the amino acid sequence of SEQ ID NO:63, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or a CDR-H3 comprising the amino acid sequence of SEQ ID NO:80, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or b) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:97, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and / or CDR-L3 comprising the amino acid sequence of SEQ ID NO:131, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:46, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:63, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or the amino acid sequence of SEQ ID NO:80, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or b) the VL comprises the amino acid sequence of SEQ ID NO:97, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:114, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; It includes the amino acid sequence of NO:131, or a variant thereof containing up to five (eg, 1, 2, 3, 4 or 5) amino acid substitutions.In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises one, two, three, four, five, or six CDRs of exemplary antibody TY25040 shown in Tables 3A-3B, or a variant thereof comprising up to five (e.g., one, two, three, four, or five) amino acid substitutions.

[0190] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH comprising CDR-H1, CDR-H2 and CDR-H3 of the VH comprising the amino acid sequence of SEQ ID NO:23, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:23; and / or a VL comprising CDR-L1, CDR-L2 and CDR-L3 of the VL comprising the amino acid sequence of SEQ ID NO:24, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:24. In certain embodiments, the VH comprises an amino acid sequence of SEQ ID NO:23, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:23; and / or the VL comprises an amino acid sequence of SEQ ID NO:24, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:24. In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises the VH and / or VL of exemplary antibody TY25040 shown in Table 4A, or one or more amino acid sequences having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the VH or VL of TY25040 shown in Table 4A.

[0191] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH and a VL, wherein a) the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:47, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; a CDR-H2 comprising the amino acid sequence of SEQ ID NO:64, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or a CDR-H3 comprising the amino acid sequence of SEQ ID NO:81, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or b) the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:98, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or CDR-L3 comprising the amino acid sequence of SEQ ID NO:132, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions. In some embodiments, a) the VH comprises the amino acid sequence of SEQ ID NO:47, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:64, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or the amino acid sequence of SEQ ID NO:81, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or b) the VL comprises the amino acid sequence of SEQ ID NO:98, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:115, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or It includes the amino acid sequence of NO:132, or a variant thereof containing up to five (eg, 1, 2, 3, 4 or 5) amino acid substitutions.In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises one, two, three, four, five, or six CDRs of exemplary antibody TY25041 shown in Tables 3A-3B, or a variant thereof comprising up to five (e.g., one, two, three, four, or five) amino acid substitutions.

[0192] In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises a VH comprising CDR-H1, CDR-H2 and CDR-H3 of the VH comprising the amino acid sequence of SEQ ID NO:25, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:25; and / or a VL comprising CDR-L1, CDR-L2 and CDR-L3 of the VL comprising the amino acid sequence of SEQ ID NO:26, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:26. In certain embodiments, the VH comprises an amino acid sequence of SEQ ID NO:25, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:25; and / or the VL comprises an amino acid sequence of SEQ ID NO:26, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:26. In some embodiments, the isolated antibody, antigen-binding fragment, or masked antibody (e.g., an activatable antibody) comprises the VH and / or VL of exemplary antibody TY25041 shown in Table 4A, or one or more amino acid sequences having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the VH or VL of TY25041 shown in Table 4A.

[0193] B-1. Isolated antibodies In some aspects, the disclosure includes an isolated antibody that binds to human CD47. In some embodiments, the isolated antibody binds to human CD47 in vitro with an EC50 of about 100 nM or less. In some embodiments, the isolated antibody has an in vitro IC50 for blocking human CD47 binding to human SIRPα of about 100 nM or less. In some embodiments, the antibody has a K of 50 nM or less as measured by surface plasmon resonance. D The antibody binds to human CD47 at a CDR, VH, VL, heavy chain and / or light chain sequence described herein. In certain embodiments, the antibody may be cross-reactive with at least one non-human species selected from the list consisting of cynomolgus monkey, rat and dog. The isolated antibodies described herein may be useful for treating CD47-positive diseases or conditions (e.g., cancer). The isolated antibodies of the present disclosure may comprise any of the CDR, VH, VL, heavy chain and / or light chain sequences described herein.

[0194] The CD47 antibodies described herein can be of any class, such as IgG, IgM, IgE, IgA, or IgD. Preferably, the CD47 antibodies are of the IgG class, such as IgG1, IgG2, IgG3, or IgG4 subclasses. CD47 antibodies can be converted from one class or subclass to another using methods known in the art. An exemplary method for producing antibodies of a desired class or subclass includes isolating a polynucleotide encoding a heavy chain of a CD47 antibody and a polynucleotide encoding a light chain of a CD47 antibody, isolating a sequence encoding a VH region, linking the VH sequence to a sequence encoding a heavy chain constant region of the desired class or subclass, expressing the light chain gene and the heavy chain construct in a cell, and harvesting the CD47 antibody.

[0195] The isolated antibodies described herein can comprise an Fc region of human IgG, IgM, IgE, IgA, or IgD. In a preferred embodiment, the isolated antibodies comprise an Fc region of human IgG of the IgG1, IgG2, IgG3, or IgG4 subclass. The Fc region can include one or more (e.g., 1, 2, 3, 4, 5, 10, or more) amino acid substitutions relative to the known sequence of an Fc region of a human antibody class or subclass.

[0196] In some embodiments, the isolated antibody comprises a first polypeptide comprising a light chain comprising a VL, and a second polypeptide comprising, from N-terminus to C-terminus, a heavy chain comprising a VH and an Fc region. In some embodiments, the Fc region is an IgG4 Fc region. In some embodiments, the Fc region is an IgG1 Fc region. In certain embodiments, the Fc region is an IgG1 Fc region comprising an S239D substitution and / or an I332E substitution, numbering according to Kabat. In certain embodiments, the Fc region is an IgG1 Fc region comprising an S239D substitution and an I332E substitution, numbering according to Kabat. IgG1 Fc regions comprising S239D and I332E substitutions are described in detail in Lazar et al. (Engineered antibody Fc variants with enhanced effector function. PNAS 103.11(2006):4005-4010). The first and second polypeptides can comprise any of the CDRs, VH, VL, heavy chains and / or light chains described herein. In some embodiments, a) the VL of the first polypeptide comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 190-196; and / or b) the VH of the second polypeptide comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 182-189. In some variations, a) the VL of the first polypeptide comprises the amino acid sequence of SEQ ID NOs: 191, 194, and / or 195; and / or b) the VH of the second polypeptide comprises the amino acid sequence of SEQ ID NOs: 182, 185, and / or 188. In certain embodiments, a) the VL of the first polypeptide comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs:31-85, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or b) the VH of the second polypeptide comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs:86-136, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions.In some variations, a) the VL of the first polypeptide comprises a CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 86-102, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 103-119, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 120-136, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or b) the VH of the second polypeptide comprises a CDR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 35-51, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and / or a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:69-85, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions. In certain embodiments, a) the VL of the first polypeptide comprises the amino acid sequence of SEQ ID NO:99, the amino acid sequence of SEQ ID NO:116, and / or the amino acid sequence of SEQ ID NO:133; and / or b) the VH of the second polypeptide comprises the amino acid sequence of SEQ ID NO:48, the amino acid sequence of SEQ ID NO:65, and / or the amino acid sequence of SEQ ID NO:82. In some embodiments, the VL of the first polypeptide comprises one, two or three CDRs of exemplary antibody TY21446 shown in Table 3B, or a variant thereof containing up to five (e.g., one, two, three, four or five) amino acid substitutions; and / or the VH of the second polypeptide comprises one, two or three CDRs of exemplary antibody TY21446 shown in Table 3A, or a variant thereof containing up to five (e.g., one, two, three, four or five) amino acid substitutions.In some embodiments, the VL of the first polypeptide comprises an amino acid sequence of SEQ ID NO:28, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the amino acid sequence of SEQ ID NO:28; and the VH of the second polypeptide comprises an amino acid sequence of SEQ ID NO:27, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:27. In certain embodiments, the first polypeptide comprises an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the VL of exemplary antibody TY21446 shown in Table 4A, or the VL of exemplary antibody TY21446 shown in Table 4A; and the second polypeptide comprises an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the VH of exemplary antibody TY21446 shown in Table 4A, or the VH of exemplary antibody TY21446 shown in Table 4A.

[0197] In some embodiments, provided herein is an isolated anti-CD47 antibody or antigen-binding fragment thereof comprising an IgG1 Fc region, wherein the anti-CD47 antibody comprises: a) a VH comprising the amino acid sequence of SEQ ID NO:48, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:65, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and the amino acid sequence of SEQ ID NO:82, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and b) an amino acid sequence of SEQ ID NO:99, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:116, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; An anti-CD47 antibody or antigen-binding fragment thereof comprising a VL comprising the amino acid sequence of NO:133, or a variant thereof containing up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions. In certain embodiments, provided herein is an isolated anti-CD47 antibody, or antigen-binding fragment thereof, comprising an IgG1 Fc region, wherein the anti-CD47 antibody comprises: a) a VH comprising the amino acid sequence of SEQ ID NO:48, the amino acid sequence of SEQ ID NO:65, and the amino acid sequence of SEQ ID NO:82, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions in each of SEQ ID NO:48, SEQ ID NO:65, and / or SEQ ID NO:82; and b) a VL comprising the amino acid sequence of SEQ ID NO:99, the amino acid sequence of SEQ ID NO:116, and the amino acid sequence of SEQ ID NO:133, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions in each of SEQ ID NO:99, SEQ ID NO:116, and / or SEQ ID NO:133.In some embodiments, provided herein is an isolated anti-CD47 antibody or antigen-binding fragment thereof comprising an IgG1 Fc region, wherein the anti-CD47 antibody comprises one, two, three, four, five, or six CDRs of exemplary antibody TY21446 shown in Tables 3A-3B, or a variant thereof comprising up to five (e.g., one, two, three, four, or five) amino acid substitutions. In certain embodiments, provided herein is an isolated anti-CD47 antibody, or antigen-binding fragment thereof, comprising an IgG1 Fc region, wherein the anti-CD47 antibody comprises a VH comprising an amino acid sequence of SEQ ID NO:27 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the amino acid sequence of SEQ ID NO:27; and a VL comprising an amino acid sequence of SEQ ID NO:28 or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:28. In some embodiments, provided herein is an isolated anti-CD47 antibody, or antigen-binding fragment thereof, comprising an IgG1 Fc region, wherein the anti-CD47 antibody comprises the VH and / or VL of exemplary antibody TY21446 shown in Table 4A, or one or more amino acid sequences having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the VH or VL of TY21446 shown in Table 4A, or an antigen-binding fragment thereof.In some embodiments, the anti-CD47 antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO:145, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the amino acid sequence of SEQ ID NO:145; and a light chain comprising an amino acid sequence of SEQ ID NO:144, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:144. In some embodiments, the anti-CD47 antibody comprises the heavy chain and / or light chain of exemplary antibody TY26896 shown in Table 6, or one or more amino acid sequences having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the heavy chain or light chain of exemplary antibody TY26896 shown in Table 6. In some embodiments, the IgG1 Fc region comprises an S239D substitution and / or an I332E substitution. In certain embodiments, the IgG1 Fc region comprises an S239D substitution and an I332E substitution. In certain embodiments, the human IgG1 Fc region comprises two Fc domains, each of which comprises an S239D substitution and / or an I332E substitution (e.g., an S239D substitution and an I332E substitution). In some embodiments, the anti-CD47 antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO:147, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the amino acid sequence of SEQ ID NO:147; and a light chain comprising an amino acid sequence of SEQ ID NO:146, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:146.In some embodiments, the anti-CD47 antibody comprises a heavy chain and / or a light chain of exemplary antibody TY26897 shown in Table 6, or one or more amino acid sequences having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to a heavy chain or a light chain of exemplary antibody TY26897 shown in Table 6. In some embodiments, the IgG1 Fc region has enhanced antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP) function.

[0198] In some embodiments, provided herein is an isolated anti-CD47 antibody or antigen-binding fragment thereof comprising an IgG1 Fc region, wherein the anti-CD47 antibody comprises: a) a VH comprising the amino acid sequence of SEQ ID NO:49, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:66, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and the amino acid sequence of SEQ ID NO:83, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and b) an amino acid sequence of SEQ ID NO:100, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:117, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; An isolated anti-CD47 antibody or antigen-binding fragment thereof, comprising a VL comprising the amino acid sequence of NO:134, or a variant thereof containing up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions. In certain embodiments, provided herein is an isolated anti-CD47 antibody, or antigen-binding fragment thereof, comprising an IgG1 Fc region, wherein the anti-CD47 antibody comprises a) a VH comprising the amino acid sequence of SEQ ID NO:49, the amino acid sequence of SEQ ID NO:66, and the amino acid sequence of SEQ ID NO:83, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions in each of SEQ ID NO:49, SEQ ID NO:66, and / or SEQ ID NO:83; and b) a VL comprising the amino acid sequence of SEQ ID NO:100, the amino acid sequence of SEQ ID NO:117, and the amino acid sequence of SEQ ID NO:134, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions in each of SEQ ID NO:100, SEQ ID NO:117, and / or SEQ ID NO:134.In some embodiments, provided herein is an isolated anti-CD47 antibody, or antigen-binding fragment thereof, comprising an IgG1 Fc region, wherein the anti-CD47 antibody comprises one, two, three, four, five, or six CDRs of exemplary antibody TY21447 shown in Tables 3A-3B, or a variant thereof comprising up to five (e.g., one, two, three, four, or five) amino acid substitutions. In certain embodiments, provided herein is an isolated anti-CD47 antibody, or antigen-binding fragment thereof, comprising an IgG1 Fc region, wherein the anti-CD47 antibody comprises a VH comprising an amino acid sequence of SEQ ID NO:29, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the amino acid sequence of SEQ ID NO:29; and a VL comprising an amino acid sequence of SEQ ID NO:30, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:30. In some embodiments, provided herein is an isolated anti-CD47 antibody, or antigen-binding fragment thereof, comprising an IgG1 Fc region, wherein the anti-CD47 antibody comprises the VH and / or VL of exemplary antibody TY21447 shown in Table 4A, or one or more amino acid sequences having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the VH or VL of TY21447 shown in Table 4A, or an antigen-binding fragment thereof. In some embodiments, the IgG1 Fc region comprises a S239D substitution and / or a I332E substitution. In certain embodiments, the IgG1 Fc region comprises a S239D substitution and a I332E substitution. In certain embodiments, the human IgG1 Fc region comprises two Fc domains, each of which comprises an S239D substitution and / or an I332E substitution (eg, an S239D substitution and an I332E substitution).In some embodiments, the IgG1 Fc region has enhanced antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP) function.

[0199] In some embodiments, provided herein is an isolated anti-CD47 antibody or antigen-binding fragment thereof comprising an IgG1 Fc region, wherein the anti-CD47 antibody comprises: a) a VH comprising the amino acid sequence of SEQ ID NO:50, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:67, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and the amino acid sequence of SEQ ID NO:84, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; and b) an amino acid sequence of SEQ ID NO:101, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; the amino acid sequence of SEQ ID NO:118, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions; An isolated anti-CD47 antibody or antigen-binding fragment thereof comprising a VL comprising the amino acid sequence of NO:135, or a variant thereof containing up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions. In certain embodiments, provided herein is an isolated anti-CD47 antibody, or antigen-binding fragment thereof, comprising an IgG1 Fc region, wherein the anti-CD47 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:50, the amino acid sequence of SEQ ID NO:67, and the amino acid sequence of SEQ ID NO:84, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions in each of SEQ ID NO:50, SEQ ID NO:67, and / or SEQ ID NO:84; and b) a VL comprising the amino acid sequence of SEQ ID NO:101, the amino acid sequence of SEQ ID NO:118, and the amino acid sequence of SEQ ID NO:135, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4 or 5) amino acid substitutions in each of SEQ ID NO:101, SEQ ID NO:118, and / or SEQ ID NO:135.In some embodiments, provided herein is an isolated anti-CD47 antibody, or antigen-binding fragment thereof, comprising an IgG1 Fc region, wherein the anti-CD47 antibody is an isolated anti-CD47 antibody, or antigen-binding fragment thereof, comprising one, two, three, four, five, or six CDRs of exemplary antibody TY21449 shown in Tables 3A-3B, or a variant thereof comprising up to five (e.g., one, two, three, four, or five) amino acid substitutions. In certain embodiments, provided herein is an isolated anti-CD47 antibody, or antigen-binding fragment thereof, comprising an IgG1 Fc region, wherein the anti-CD47 antibody comprises a VH comprising an amino acid sequence of SEQ ID NO:31, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the amino acid sequence of SEQ ID NO:31; and a VL comprising an amino acid sequence of SEQ ID NO:32, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to SEQ ID NO:32. In some embodiments, provided herein is an isolated anti-CD47 antibody, or antigen-binding fragment thereof, comprising an IgG1 Fc region, wherein the anti-CD47 antibody comprises the VH and / or VL of exemplary antibody TY21449 shown in Table 4A, or one or more amino acid sequences having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the VH or VL of TY21449 shown in Table 4A, or an antigen-binding fragment thereof. In some embodiments, the IgG1 Fc region comprises a S239D substitution and / or a I332E substitution. In certain embodiments, the IgG1 Fc region comprises a S239D substitution and a I332E substitution. In certain embodiments, the human IgG1 Fc region comprises two Fc domains, each of which comprises an S239D substitution and / or an I332E substitution (eg, an S239D substitution and an I332E substitution).In some embodiments, the IgG1 Fc region has enhanced antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP) function.

[0200] In some embodiments, the isolated antibody comprises a human IgG4 Fc region. The isolated antibody of the present disclosure can comprise an IgG4 Fc region and any combination of CDRs, VH, VL and / or light chains described herein. In some embodiments, the isolated antibody comprises a first polypeptide comprising a light chain comprising a VL, and a second polypeptide comprising, from N-terminus to C-terminus, a heavy chain comprising a VH and a human IgG4 Fc region. In certain embodiments, a) the VL of the first polypeptide comprises the amino acid sequence of SEQ ID NO:99, the amino acid sequence of SEQ ID NO:116, and / or the amino acid sequence of SEQ ID NO:133; and / or b) the VH of the second polypeptide comprises the amino acid sequence of SEQ ID NO:48, the amino acid sequence of SEQ ID NO:65, and / or the amino acid sequence of SEQ ID NO:82. In some embodiments, the VL of the first polypeptide comprises one, two or three CDRs of exemplary antibody TY21446 shown in Table 3B, or a variant thereof comprising up to five (e.g., one, two, three, four or five) amino acid substitutions; and / or the VH of the second polypeptide comprises one, two or three CDRs of exemplary antibody TY21446 shown in Table 3A, or a variant thereof comprising up to five (e.g., one, two, three, four or five) amino acid substitutions. In some embodiments, the VL of the first polypeptide comprises the amino acid sequence of SEQ ID NO:28, and / or the VH of the second polypeptide comprises the amino acid sequence of SEQ ID NO:27. In certain embodiments, the first polypeptide comprises an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the VL of exemplary antibody TY21446 shown in Table 4A, or the VL of exemplary antibody TY21446 shown in Table 4A; and the second polypeptide comprises an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the VH of exemplary antibody TY21446 shown in Table 4A, or the VH of exemplary antibody TY21446 shown in Table 4A.In certain embodiments, the first polypeptide comprises an amino acid sequence of SEQ ID NO:140, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the amino acid sequence of SEQ ID NO:140; and / or the second polypeptide comprises an amino acid sequence of SEQ ID NO:141, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the amino acid sequence of SEQ ID NO:141. In some embodiments, the first polypeptide comprises an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the light chain of antibody TY21446 shown in Table 6, or the light chain of an exemplary antibody TY21446 shown in Table 6; and / or the second polypeptide comprises an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the heavy chain of antibody TY21446 shown in Table 6, or the heavy chain of an exemplary antibody TY21446 shown in Table 6.

[0201] In some embodiments, the isolated antibody comprises a human IgG1 Fc region. The isolated antibody of the present disclosure can comprise an IgG1 Fc region and any combination of CDRs, VH, VL and / or light chains described herein. In some embodiments, the isolated antibody comprises a first polypeptide comprising a light chain comprising a VL, and a second polypeptide comprising, from N-terminus to C-terminus, a heavy chain comprising a VH and a human IgG1 Fc region. In certain embodiments, a) the VL of the first polypeptide comprises the amino acid sequence of SEQ ID NO:99, the amino acid sequence of SEQ ID NO:116, and / or the amino acid sequence of SEQ ID NO:133; and / or b) the VH of the second polypeptide comprises the amino acid sequence of SEQ ID NO:48, the amino acid sequence of SEQ ID NO:65, and / or the amino acid sequence of SEQ ID NO:82. In some embodiments, the VL of the first polypeptide comprises the amino acid sequence of SEQ ID NO:28, and / or the VH of the second polypeptide comprises the amino acid sequence of SEQ ID NO:27. In certain embodiments, the first polypeptide comprises an amino acid sequence of SEQ ID NO:144, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the amino acid sequence of SEQ ID NO:144; and / or the second polypeptide comprises an amino acid sequence of SEQ ID NO:145, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the amino acid sequence of SEQ ID NO:145.In some embodiments, the first polypeptide comprises an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the amino acid sequence of the light chain of antibody TY26896 shown in Table 6, or the light chain of an exemplary antibody TY26896 shown in Table 6; and / or the second polypeptide comprises an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the amino acid sequence of the heavy chain of TY26896 shown in Table 6, or the heavy chain of an exemplary antibody TY26896 shown in Table 6.

[0202] In some embodiments, the isolated antibody comprises a human IgG1 Fc region with one or more amino acid substitutions. The IgG1 Fc region can include any amino acid substitution known in the art to confer desired properties to an antibody having an IgG1 Fc region. In some embodiments, the IgG1 Fc region has enhanced antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP) function. In certain embodiments, the human IgG1 Fc region comprises an S239D substitution and / or an I332E substitution. In certain embodiments, the human IgG1 Fc region comprises two Fc domains, each of the two Fc domains comprising an S239D substitution and / or an I332E substitution (e.g., an S239D substitution and an I332E substitution). The isolated antibody of the present disclosure can comprise an IgG1 Fc region comprising an S239D substitution and / or an 332E substitution, and any combination of CDRs, VH, VL and / or light chains described herein. In some embodiments, the isolated antibody comprises a first polypeptide comprising a light chain comprising a VL, and a second polypeptide comprising, from N-terminus to C-terminus, a heavy chain comprising a VH and a human IgG1 Fc region comprising an S239D substitution and / or an I332E substitution. In certain embodiments, the isolated antibody has enhanced ADCC activity. In some embodiments, the isolated antibody comprises a first polypeptide comprising a light chain comprising a VL, and a second polypeptide comprising, from N-terminus to C-terminus, a heavy chain comprising a VH and a human IgG1 Fc region. In certain embodiments, a) the VL of the first polypeptide comprises the amino acid sequence of SEQ ID NO:99, the amino acid sequence of SEQ ID NO:116, and / or the amino acid sequence of SEQ ID NO:133; and / or b) the VH of the second polypeptide comprises the amino acid sequence of SEQ ID NO:48, the amino acid sequence of SEQ ID NO:65, and / or the amino acid sequence of SEQ ID NO:82. In some embodiments, the VL of the first polypeptide comprises the amino acid sequence of SEQ ID NO:28 and / or the VH of the second polypeptide comprises the amino acid sequence of SEQ ID NO:27.In certain embodiments, the first polypeptide comprises an amino acid sequence of SEQ ID NO:146, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the amino acid sequence of SEQ ID NO:146; and / or the second polypeptide comprises an amino acid sequence of SEQ ID NO:147, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the amino acid sequence of SEQ ID NO:147. In some embodiments, the first polypeptide comprises an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the amino acid sequence of the light chain of antibody TY26897 shown in Table 6, or the light chain of the exemplary antibody TY26897 shown in Table 6; and / or the second polypeptide comprises an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity to the amino acid sequence of the heavy chain of antibody TY26897 shown in Table 6, or the heavy chain of the exemplary antibody TY26897 shown in Table 6.

[0203] Furthermore, the antibodies provided in this disclosure can be monoclonal or polyclonal. In a preferred embodiment, the isolated antibodies are monoclonal.

[0204] Examples of specific isolated antibodies provided in this disclosure include those listed in Tables 3A-6. Also provided are the amino acid sequences of the heavy chain variable region, full-length heavy chains of the IgG1 and IgG4 subclasses, light chain variable regions, and full-length variable regions of these antibodies.

[0205] Also provided are anti-CD47 antibodies that competitively bind to the same epitope as any one of the anti-CD47 antibodies described herein, including the antibodies listed in Tables 3A-6.

[0206] In some embodiments, the isolated antibody, or antigen-binding fragment thereof, has a K of about 500 nM or less (e.g., about 500 nM or less, about 400 nM or less, about 300 nM or less, about 200 nM or less, about 150 nM or less, about 100 nM or less, about 90 nM or less, about 80 nM or less, about 75 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 25 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, about 0.1 nM or less, etc.). D In some embodiments, the isolated antibody, or antigen-binding fragment thereof, binds to human CD47 with a K of about 100 nM or less. D In some embodiments, the isolated antibody, or antigen-binding fragment thereof, binds to human CD47 with a K of about 50 nM or less. D In some embodiments, the isolated antibody, or antigen-binding fragment thereof, binds to human CD47 with a K of about 10 nM or less. D The isolated antibody or antigen-binding fragment thereof binds to human CD47 at D Methods for measuring KD may be performed using any method known in the art, including, for example, by surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assay, EMSA, etc. In some embodiments, KD is measured by surface plasmon resonance (see, for example, Example 2 below).

[0207] In some embodiments, the antibody, or antigen-binding fragment thereof, has a half maximal effective concentration (EC50) for binding to human CD47 in vitro of about 500 nM or less (e.g., about 500 nM or less, about 400 nM or less, about 300 nM or less, about 200 nM or less, about 150 nM or less, about 100 nM or less, about 90 nM or less, about 80 nM or less, about 75 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 25 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, about 0.1 nM or less, etc.). In some embodiments, the antibody, or antigen-binding fragment thereof, binds to human CD47 in vitro with an EC50 of about 100 nM or less. In some embodiments, the antibody, or antigen-binding fragment thereof, has an in vitro EC50 for binding to human CD47 of about 10 nM or less. In some embodiments, the antibody, or antigen-binding fragment thereof, has an in vitro EC50 for binding to human CD47 of about 1 nM or less. Methods for measuring the EC50 for binding of an antibody or antigen-binding fragment thereof to a target (e.g., CD47) may be performed using any method known in the art, including, for example, by ELISA, filter binding assay, EMSA, etc. In some embodiments, the EC50 is measured by ELISA (see, e.g., Example 2 below).

[0208] In some embodiments, the half maximal inhibitory concentration (IC50) of the antibody, or antigen-binding fragment thereof, for blocking human CD47 binding to human SIRPα in vitro is about 500 nM or less (e.g., about 500 nM or less, about 400 nM or less, about 300 nM or less, about 200 nM or less, about 150 nM or less, about 100 nM or less, about 90 nM or less, about 80 nM or less, about 75 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 25 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, about 0.1 nM or less, etc.). In some embodiments, the IC50 of the antibody, or antigen-binding fragment thereof, for blocking human CD47 binding to human SIRPα in vitro is about 100 nM or less. In some embodiments, the IC50 of the antibody or antigen-binding fragment thereof for blocking human CD47 binding to human SIRPα in vitro is about 25 nM or less. In some embodiments, the IC50 of the antibody or antigen-binding fragment thereof for blocking human CD47 binding to human SIRPα in vitro is about 10 nM or less. In some embodiments, the IC50 of the antibody or antigen-binding fragment thereof for blocking human CD47 binding to human SIRPα in vitro is about 5 nM or less. Methods for measuring the IC50 of the antibody or antigen-binding fragment thereof for binding to a target (e.g., CD47) may be performed using any method known in the art, including, for example, by ELISA, filter binding assay, EMSA, etc. In some embodiments, the IC50 is measured by ELISA (see, for example, Example 2 below).

[0209] In certain embodiments, when the antibody, or antigen-binding fragment thereof, is provided at a concentration of about 1 nM or greater (e.g., about 1 nM or greater, about 5 nM or greater, about 10 nM or greater, about 20 nM or greater, about 40 nM or greater, about 60 nM or greater, about 80 nM or greater, about 100 nM or greater, about 200 nM or greater, about 400 nM or greater, about 600 nM or greater, about 800 nM or greater, about 1 μM or greater, about 2 μM or greater, about 4 μM or greater, about 6 μM or greater, about 8 μM or greater, about 10 μM or greater, about 20 μM or greater, about 40 μM or greater, about 60 μM or greater, about 80 μM or greater, about 100 μM or greater, etc.), the antibody, or antigen-binding fragment thereof, completely blocks binding of human CD47 to human SIRPα in vitro. In some embodiments, the isolated antibody or antigen-binding fragment thereof completely blocks human CD47 binding to human SIRPα in vitro when the antibody or antigen-binding fragment thereof is provided at a concentration of about 1 μM or greater. In some embodiments, the antibody or antigen-binding fragment thereof completely blocks human CD47 binding to human SIRPα in vitro when the antibody or antigen-binding fragment thereof is provided at a concentration of about 100 nM or greater. In some embodiments, the antibody or antigen-binding fragment thereof completely blocks human CD47 binding to human SIRPα in vitro when the antibody or antigen-binding fragment thereof is provided at a concentration of about 100 nM or greater. In some embodiments, the antibody or antigen-binding fragment thereof completely blocks human CD47 binding to human SIRPα in vitro when the antibody or antigen-binding fragment thereof is provided at a concentration of about 10 nM or greater. As used herein, the term "completely blocking" or "completely blocking" refers to the ability of an antibody, antigen-binding fragment thereof to reduce binding of a first protein to a second protein by at least about 80% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, etc.).Methods for measuring the ability of an antibody or antigen-binding fragment thereof to block the binding of a first protein (e.g., CD47) to a second protein (e.g., SIRPα) are known in the art and include, but are not limited to, BIAcore analysis, ELISA assays, and by flow cytometry (see, e.g., Example 2 below).

[0210] In some embodiments, the antibody, or antigen-binding fragment thereof, has a half maximal effective concentration (EC50) of about 500 nM or less (e.g., about 500 nM or less, about 400 nM or less, about 300 nM or less, about 200 nM or less, about 150 nM or less, about 100 nM or less, about 90 nM or less, about 80 nM or less, about 75 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 25 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, about 0.1 nM or less, etc.) for binding to tumor cells in vitro. In some embodiments, the antibody, or antigen-binding fragment thereof, has an EC50 of about 50 nM or less for binding to tumor cells in vitro. In some embodiments, the antibody, or antigen-binding fragment thereof, has an EC50 of about 10 nM or less for binding to tumor cells in vitro. In some embodiments, the antibody, or antigen-binding fragment thereof, has an EC50 of about 5 nM or less for binding to tumor cells in vitro. In some embodiments, the antibody, or antigen-binding fragment thereof, has an EC50 of about 1 nM or less for binding to tumor cells in vitro. In certain embodiments, the tumor cells comprise a B cell lymphoma cell line (e.g., Raji cell line). In further embodiments, the tumor cells comprise a T cell lymphoma cell line (e.g., CEM cell line). Flow cytometry (see, e.g., Example 6 below). Methods for measuring the EC50 of an antibody or antigen-binding fragment thereof for binding to tumor cells may be performed using any method known in the art, including, for example, flow cytometry (see, e.g., Example 6 below).

[0211] In some embodiments, the antibody, or antigen-binding fragment thereof, has a half maximal effective concentration (EC50) of about 100 nM or less (e.g., about 100 nM or less, about 90 nM or less, about 80 nM or less, about 75 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 25 nM or less, about 20 nM or less, about 10 nM or less, about 5 nM or less, about 2 nM or less, about 1 nM or less, about 0.5 nM or less, about 0.4 nM or less, about 0.3 nM or less, about 0.2 nM or less, about 0.1 nM or less, etc.) for increasing macrophage phagocytosis of tumor cells in vitro. In some embodiments, the antibody, or antigen-binding fragment thereof, has an EC50 of 10 nM or less for increasing macrophage phagocytosis of tumor cells in vitro. In some embodiments, the antibody, or antigen-binding fragment thereof, has an EC50 of 5 nM or less for increasing macrophage phagocytosis of tumor cells in vitro. In some embodiments, the antibody, or antigen-binding fragment thereof, has an EC50 of 1 nM or less for increasing macrophage phagocytosis of tumor cells in vitro. In some embodiments, the antibody, or antigen-binding fragment thereof, when provided at a concentration of 1 μM or greater, results in maximal macrophage phagocytosis of tumor cells in vitro of about 20% or more (e.g., about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, etc.). In certain embodiments, when provided at a concentration of 1 μM or greater, the antibody, or antigen-binding fragment thereof, provides about a 50% or greater (e.g., about 50% or greater, about 2-fold or greater, about 3-fold or greater, about 4-fold or greater, about 5-fold or greater, about 10-fold or greater, etc.) increase in maximal macrophage phagocytosis of tumor cells in vitro compared to an antibody, or antigen-binding fragment thereof, that does not bind to CD47 at the same concentration. In certain embodiments, the tumor cells comprise a B cell lymphoma cell line (e.g., the Raji cell line). In further embodiments, the tumor cells comprise a T cell lymphoma cell line (e.g., the CEM cell line).Methods for measuring the EC50 for an antibody or antigen-binding fragment thereof for increasing macrophage phagocytosis of tumor cells in vitro are known in the art and include, for example, flow cytometry (see, e.g., Example 9 below).

[0212] In some embodiments, the antibody, or antigen-binding fragment thereof, has antibody-dependent cellular cytotoxicity (ADCC) activity. ADCC is mediated by immune cells (e.g., natural killer (NK) cells) and plays an important role in humoral immune responses (e.g., against infectious diseases, tumor cells, etc.). In some embodiments, the antibody, or antigen-binding fragment thereof, when provided at a concentration of 0.01 nM or greater, has ADCC activity that results in about 5% or greater (e.g., about 5% or greater, about 10% or greater, about 15% or greater, about 20% or greater, about 25% or greater, about 30% or greater, about 40% or greater, about 50% or greater, etc.) of tumor cell lysis. In some embodiments, the antibody, or antigen-binding fragment thereof, when provided at a concentration of 0.1 nM or greater, has ADCC activity that results in about 10% or greater (e.g., about 10% or greater, about 15% or greater, about 20% or greater, about 25% or greater, about 30% or greater, about 40% or greater, about 50% or greater, etc.) of tumor cell lysis. In some embodiments, the antibody, or antigen-binding fragment thereof, has ADCC activity that results in about 20% or more (e.g., about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, etc.) lysis of tumor cells when provided at a concentration of 1 nM or more. In certain embodiments, the tumor cells comprise a B cell lymphoma cell line (e.g., Raji cell line). In further embodiments, the tumor cells comprise a T cell lymphoma cell line (e.g., CEM cell line). Methods for measuring ADCC of an antibody or antigen-binding fragment thereof are known in the art and include, for example, the methods described in Example 10 below.

[0213] In some embodiments, the antibody, antigen-binding fragment, or activatable antibody is cross-reactive with monkey (e.g., cynomolgus monkey), rat, and / or dog CD47. In some embodiments, the antibody, antigen-binding fragment, or activatable antibody is cross-reactive with monkey CD47. In some embodiments, the antibody, antigen-binding fragment, or activatable antibody is cross-reactive with rat CD47. In some embodiments, the antibody, antigen-binding fragment, or activatable antibody is cross-reactive with dog CD47. In some embodiments, the antibody, antigen-binding fragment, or activatable antibody is cross-reactive with monkey and rat CD47; monkey and dog CD47; rat and dog CD47; or monkey, rat, and dog CD47. In some embodiments, the antibody, antigen-binding fragment, or activatable antibody is cross-reactive with monkey (e.g., cynomolgus), rat, and / or dog CD47 at about 100 nM or less (e.g., at about 1 nM, at about 10 nM, at about 25 nM, at about 50 nM, at about 75 nM, at about 100 nM). Methods for measuring the cross-reactivity of an antibody or antigen-binding fragment thereof are known in the art and include, but are not limited to, by surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assays, EMSA, and the like (see, e.g., Example 5 below).

[0214] In certain embodiments, the antibody, antigen-binding fragment, or activatable antibody is cross-reactive with cynomolgus CD47. In some embodiments, the antibody, or antigen-binding fragment thereof, has a half-maximal effective concentration (EC50) for binding to cynomolgus CD47 in vitro of about 100 nM or less (e.g., about 100 nM or less, about 90 nM or less, about 80 nM or less, about 75 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 25 nM or less, about 20 nM or less, about 10 nM or less, about 5 nM or less, about 2 nM or less, about 1 nM or less, about 0.5 nM or less, about 0.4 nM or less, about 0.3 nM or less, about 0.2 nM or less, about 0.1 nM or less, etc.). In some embodiments, the antibody, or antigen-binding fragment thereof, has an EC50 of about 10 nM or less for binding to cynomolgus CD47 in vitro. In some embodiments, the antibody, or antigen-binding fragment thereof, has an EC50 of about 5 nM or less for binding to cynomolgus CD47 in vitro. In some embodiments, the antibody, or antigen-binding fragment thereof, has an EC50 of about 1 nM or less for binding to cynomolgus CD47 in vitro.

[0215] The antibodies of the present disclosure can be produced by techniques known in the art, including conventional monoclonal antibody methodology, such as standard somatic cell hybridization techniques (see, e.g., Kohler and Milstein, Nature 256:495 (1975)), viral or oncogene transformation of B-lymphocytes, or recombinant antibody techniques, as described in detail herein below.

[0216] Hybridoma production is a very well-established procedure. A common animal system for preparing hybridomas is the mouse system. Techniques for immunization protocols and isolation of immune splenocytes for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known. One well-known method that can be used to generate human CD47 antibodies provided by the present disclosure includes the use of the Xeno Mouse™ animal system. Xeno Mouse™ mice are genetically engineered mouse strains that contain large fragments of human immunoglobulin heavy and light chain loci and are defective in the production of mouse antibodies. See, for example, Green et al., Nature Genetics 7:13-21 (1994) and WO2003 / 040170. Animals are immunized with CD47 antigen. The CD47 antigen is isolated and / or purified CD47, preferably CD47. It may be a fragment of CD47, such as the extracellular domain (ECD) of CD47, in particular the CD47 ECD comprising amino acid residues 34-108 or 34-93 of SEQ ID NO:1. Immunization of animals may be performed by any method known in the art. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990. Methods for immunizing non-human animals, such as mice, rats, sheep, goats, pigs, cattle, and horses, are well known in the art. See, for example, Harlow and Lane, supra, and U.S. Pat. No. 5,994,619. The CD47 antigen may be administered with an adjuvant to stimulate the immune response. Exemplary adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide), or ISCOM (immunostimulating complex). After immunization of an animal with CD47 antigen, an antibody-producing immortalized cell line is prepared from cells isolated from the immunized animal. Following immunization, the animals are sacrificed and lymph node and / or splenic B cells are immortalized.Methods for immortalizing cells include, but are not limited to, introducing oncogenes into them, infecting them with tumor viruses, culturing them under conditions that select for immortalized cells, subjecting them to oncogenic or mutating compounds, fusing with immortalized cells, e.g., myeloma cells, and inactivating tumor suppressor genes. See, e.g., Harlow and Lane, supra. When fusion with myeloma cells is used, it is preferred that the myeloma cells do not secrete immunoglobulin polypeptides (non-secretory cell lines). Immortalized cells are screened using CD47, a portion thereof, or cells expressing CD47. CD47 antibody-producing cells, e.g., hybridomas, are selected, cloned, and further screened for desirable properties, including robust growth, high antibody production, and desirable antibody properties, as discussed further below. Hybridomas can be grown in vivo in syngeneic animals, animals lacking an immune system, e.g., nude mice, or in cell culture in vitro. Methods for selecting, cloning, and growing hybridomas are well known to those skilled in the art.

[0217] The antibodies of the present disclosure can also be prepared using phage display or yeast display methods. Such display methods for isolating human antibodies are established in the art, such as Achim Knappik, et al., "Fully Synthetic Human Combinatorial Antibody Libraries (HuCAL) Based on Modular Consensus Frameworks and CDRs Randomized with Trinucleotides" J. Mol. Biol. (2000) 296, 57-86; and Michael J. Feldhaus, et al., "Flow-cytometric isolation of human antibodies from a non-immune Saccharomyces cerevisiae surface display library" Nat Biotechnol (2003) 21: 163-170.

[0218] B-2. Masked antibodies In some aspects, provided herein are masked antibodies that bind to CD47. In some embodiments, the masked antibodies comprise: (a) a masking peptide, comprising, from the N-terminus to the C-terminus, a masking moiety (MM) and a linking moiety (LM); and (b) a target binding moiety (TBM) that comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) and binds to human CD47. In certain embodiments, the masking peptide is linked to the N-terminus of the VH or VL. In preferred embodiments, the MM competes with human CD47 to bind to the TBM. The TBM can comprise one or more sequences of the anti-CD47 antibodies or antigen-binding fragments described herein, including antibodies or antigen-binding fragments described with respect to the specific amino acid sequences of the CDRs, variable regions (VL, VH), and / or light and heavy chains (e.g., IgG1, IgG2, IgG4). In some embodiments, the TBM comprises a full-length antibody light chain and / or a full-length antibody heavy chain of one or more anti-CD47 antibodies described herein.

[0219] In some embodiments, the present disclosure relates to masked antibodies that bind human CD47 and have at least one (e.g., at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or all thirteen) of the following functional properties: (a) a higher K binding to human CD47 compared to a parent antibody having the same TBM but lacking the masking peptide. D (b) a higher half maximal effective concentration (EC50) for binding to human CD47 compared to a parent antibody having the same TBM but lacking the masking peptide; (c) a higher half maximal effective concentration (IC50) for blocking human CD47 binding to human SIRPα in vitro compared to a parent antibody having the same TBM but lacking the masking peptide; (d) the masked antibody completely blocks human CD47 binding to human SIRPα in vitro when provided at a concentration of about 1 nM or greater; (e) a greater EC50 for binding to tumor cells in vitro compared to a parent antibody having the same TBM but lacking the masking peptide; (f) reduced binding to red blood cells (RBCs) in vitro compared to a parent antibody having the same TBM but lacking the masking peptide; (g) a higher EC50 for increasing macrophage phagocytosis of tumor cells in vitro compared to a parent antibody having the same TBM but lacking the masking peptide; (h) a greater than 500% EC50 for increasing macrophage phagocytosis of tumor cells compared to a parent antibody having the same TBM but lacking the masking peptide. (i) bind to human CD47 with a sub-nM KD; (j) are cross-reactive with monkey, rat, or dog CD47; (k) are capable of inhibiting tumor cell growth; (l) have one or more therapeutic effects against cancer; (l) inhibit the binding of CD47 to SIRP proteins (e.g., SIRPα); and (m) exhibit antibody-dependent cellular cytotoxicity (ADCC) against tumor cells expressing CD47.

[0220] In some embodiments, the masked antibody selectively binds to tissues or cells with high levels of CD47 expression. Without wishing to be bound by theory, because MM competes with human CD47 to bind to TBM, the masked antibody selectively binds to CD47 in cells or tissues where the expression level of CD47 is sufficiently high and therefore the local concentration of CD47 on the cell surface is sufficiently high. This phenomenon of selectively binding to a target (e.g., CD47) on tissues or cells with high expression levels of the target (e.g., CD47) may be referred to as selective target engagement. In certain embodiments, the selective target binding of the anti-CD47 masked antibodies described herein results in a reduction in on-target extratumoral effects (e.g., antigen sink effect, anemia) compared to an anti-CD47 antibody with the same TBM but lacking a masking peptide that includes a masking moiety.

[0221] In some embodiments, the masked antibody comprises a target binding moiety (TBM). In some embodiments, the TBM comprises an antibody light chain variable region and / or an antibody heavy chain variable region. In some embodiments, the TBM comprises an antibody light chain variable region. In some embodiments, the TBM comprises an antibody heavy chain variable region. In some embodiments, the TBM comprises an antibody light chain variable region and an antibody heavy chain variable region. In some embodiments, the antibody heavy chain variable region is C-terminal to the antibody light chain variable region. In some embodiments, the antibody light chain variable region is C-terminal to the antibody heavy chain variable region. In some embodiments, the TBM of the present disclosure comprises an antibody light chain variable region and / or an antibody heavy chain variable region having specificity for CD47. In some embodiments, the TBM comprises a full-length antibody light chain and / or a full-length antibody heavy chain. The antibody light chain can be a kappa or lambda light chain. The antibody heavy chain can be of any class (e.g., IgG, IgM, IgE, IgA, or IgD). In some embodiments, the antibody heavy chain is of the IgG class, such as IgG1, IgG2, IgG3 or IgG4 subclass. The antibody heavy chains described herein may be converted from one class or subclass to another class or subclass using methods known in the art. Any one or more of the TBMs described herein may incorporate any of the CDR sequences described herein (e.g., one, two or three of the CDR sequences of the heavy chain variable region and / or one, two or three of the CDR sequences of the light chain variable region), any of the heavy chain variable region sequences and / or any of the light chain variable region sequences of the anti-CD47 antibodies described herein.

[0222] In some embodiments, the masked antibody, ...

Claims

1. An antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), which binds to human CD47, a) the VH is CDR-H1 comprising the amino acid sequence of SEQ ID NO: 48; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 65, and comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 82; b) the VL is CDR-L1 comprising the amino acid sequence of SEQ ID NO: 99; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 116, and comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 133; An antibody or antigen-binding fragment thereof.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises the amino acid sequence of SEQ ID NO: 27 and / or the VL comprises the amino acid sequence of SEQ ID NO:

28.

3. The antibody or antigen-binding fragment thereof described in claim 1, wherein the antibody comprises a first polypeptide comprising a light chain including the VL, and the first polypeptide comprises the amino acid sequence of SEQ ID NO:

140.

4. The antibody described in claim 1, comprising a human IgG4 Fc region.

5. The antibody described in claim 1, wherein the antibody comprises, from the N-terminus to the C-terminus, a second polypeptide comprising a heavy chain including the VH and a human IgG4 Fc domain, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:

141.

6. 2. The antibody of claim 1, wherein the antibody comprises, from N-terminus to C-terminus, a first polypeptide comprising a light chain comprising the VL, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 140; and a second polypeptide comprising, from N-terminus to C-terminus, a heavy chain comprising the VH and a human IgG4 Fc domain, wherein the second polypeptide comprises the amino acid sequence of SEQ ID NO:

141.

7. The antibody of claim 1 , wherein the antibody comprises a human IgG1 Fc region.

8. The antibody described in claim 3, wherein the antibody comprises, from the N-terminus to the C-terminus, a second polypeptide comprising a heavy chain including the VH and a human IgG1 Fc domain, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:

145.

9. 2. The antibody of claim 1, wherein the antibody comprises, from N-terminus to C-terminus, a first polypeptide comprising a light chain comprising the VL, the first polypeptide comprising the amino acid sequence of SEQ ID NO: 140; and a second polypeptide comprising, from N-terminus to C-terminus, a heavy chain comprising the VH and a human IgG1 Fc domain, the second polypeptide comprising the amino acid sequence of SEQ ID NO:

145.

10. The antibody described in claim 1, wherein the antibody comprises, from the N-terminus to the C-terminus, a second polypeptide comprising a heavy chain including the VH and a human IgG1 Fc domain, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:

147.

11. 2. The antibody of claim 1, wherein the antibody comprises, from N-terminus to C-terminus, a first polypeptide comprising a light chain comprising the VL, the first polypeptide comprising the amino acid sequence of SEQ ID NO: 140; and a second polypeptide comprising, from N-terminus to C-terminus, a heavy chain comprising the VH and a human IgG1 Fc domain, the second polypeptide comprising the amino acid sequence of SEQ ID NO:

147.

12. (a) a masking peptide comprising, from the N-terminus to the C-terminus, a masking portion (MM) and a linking portion (LM); and (b) a target binding moiety (TBM) comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), wherein the TBM binds to human CD47.

1. A masked antibody comprising: a masking peptide linked to the N-terminus of the VH or the VL; The MM competes with human CD47 to bind to the TBM; and 1) the VH is CDR-H1 comprising the amino acid sequence of SEQ ID NO: 48; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 65, and comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 82; and 2) The VL is CDR-L1 comprising the amino acid sequence of SEQ ID NO: 99; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 116, and comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 133; Masked antibodies.

13. The masked antibody described in claim 12, wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 197-200.

14. The masked antibody described in claim 12, wherein the MM comprises the amino acid sequence of SEQ ID NO:

137.

15. A masked antibody described in claim 12, wherein the LM comprises a cleavable moiety (CM) comprising at least one cleavage site, and the activatable antibody has a higher binding affinity to human CD47 in vitro after CM cleavage than before CM cleavage.

16. The masked antibody of claim 15, wherein the cleavage site is a protease cleavage site of a protease selected from the group consisting of urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco etch virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE.

17. The masked antibody described in claim 15, wherein the CM comprises an MMP-9 cleavage site that can be cleaved by MMP-9.

18. The masked antibody described in claim 15, wherein the CM comprises the amino acid sequence of SEQ ID NO:

138.

19. The masked antibody described in claim 12, wherein the masking peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 139 and 152-166.

20. The masked antibody described in claim 12, wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:

139.

21. A masked antibody described in Claim 12, wherein the VH comprises the amino acid sequence of SEQ ID NO: 27 and / or the VL comprises the amino acid sequence of SEQ ID NO:

28.

22. The masked antibody described in claim 12, wherein the antibody comprises a first polypeptide comprising a light chain including the VL, and the first polypeptide comprises the amino acid sequence of SEQ ID NO:

142.

23. The masked antibody of claim 12, wherein the antibody comprises a human IgG4 Fc region.

24. The masked antibody of claim 12, wherein the antibody comprises a second polypeptide comprising, from the N-terminus to the C-terminus, a heavy chain comprising the VH and a human IgG4 Fc domain, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:

143.

25. The masked antibody of claim 12, wherein the antibody comprises: a first polypeptide comprising a light chain including the VL, the first polypeptide comprising the amino acid sequence of SEQ ID NO: 142; and a second polypeptide comprising, from the N-terminus to the C-terminus, a heavy chain comprising the VH and a human IgG4 Fc domain, the second polypeptide comprising the amino acid sequence of SEQ ID NO:

143.

26. The masked antibody of claim 12, wherein the antibody comprises a human IgG1 Fc region.

27. ​​The masked antibody of claim 12, wherein the antibody comprises a second polypeptide comprising, from the N-terminus to the C-terminus, a heavy chain comprising the VH and a human IgG1 Fc domain, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:

149.

28. The masked antibody of claim 12, wherein the antibody comprises: a first polypeptide comprising a light chain including the VL, the first polypeptide comprising the amino acid sequence of SEQ ID NO: 142; and a second polypeptide comprising, from the N-terminus to the C-terminus, a heavy chain including the VH and a human IgG1 Fc domain, the second polypeptide comprising the amino acid sequence of SEQ ID NO:

149.

29. The masked antibody of claim 12, wherein the antibody comprises a second polypeptide comprising, from the N-terminus to the C-terminus, a heavy chain comprising the VH and a human IgG1 Fc domain, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:

151.

30. The masked antibody of claim 12, wherein the antibody comprises: a first polypeptide comprising a light chain including the VL, the first polypeptide comprising the amino acid sequence of SEQ ID NO: 142; and a second polypeptide comprising, from the N-terminus to the C-terminus, a heavy chain comprising the VH and a human IgG1 Fc domain, the second polypeptide comprising the amino acid sequence of SEQ ID NO:

151.

31. An isolated polynucleotide encoding one or more polypeptide chains of an antibody or antigen-binding fragment thereof described in any one of claims 1-11, or one or more polypeptide chains of a masked antibody described in any one of claims 12-30.

32. A vector comprising the polynucleotide of claim 31.

33. A host cell comprising the vector of claim 32.

34. A method for producing an antibody or antigen-binding fragment thereof, or a masked antibody, comprising culturing a host cell described in claim 33 under conditions suitable for producing the masked antibody, or the antibody or antigen-binding fragment thereof.

35. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described in any one of claims 1-11, or a masked antibody described in any one of claims 12-30, and a pharmaceutically acceptable carrier.

36. A pharmaceutical composition as described in claim 35 for use in a method for treating a CD47-positive disease or condition in a subject in need of treatment, the method comprising administering an effective amount of the pharmaceutical composition to the subject, and the disease or condition is cancer.

37. The pharmaceutical composition of claim 36, wherein the method further comprises administering to the subject an effective amount of one or more additional therapeutic agents, wherein the one or more additional therapeutic agents comprise viral gene therapy, immune checkpoint inhibitors, targeted therapy, radiation therapy, chemotherapy, or any combination thereof.

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