NZ733261A

NZ733261A - Anti-cd47 antibodies and uses thereof

Info

Publication number: NZ733261A
Application number: NZ733261A
Authority: NZ
Inventors: Aaron Sato; Ryan Stafford; Junhao Yang
Original assignee: Celgene Corporation; Aaron Sato; Ryan Stafford; Junhao Yang
Priority date: 2014-12-30
Filing date: 2015-12-28

Abstract

Provided herein are compositions, methods and uses involving antibodies that specifically bind to human CD47. Also provided are uses and methods, such as therapeutic methods, diagnostic methods, and methods of making such antibodies.

Description

ANTI-CD47 ANTIBODIES AND USES THEREOF This application claims beneﬁt of US. Provisional Patent Application No. 62/098,291, ﬁled December 30, 2014, the disclosure of which is incorporated by reference herein in its entirety. nce to Sequence Listing Submitted Electronically This application incorporates by reference a Sequence Listing submitted with this application as text ﬁle entitled “Sequence_Listing_l2827228.txt” created on er 27, 2015 and having a size of 87,612 bytes. 1. FIELD Provided herein are dies (anti—CD47 antibodies) which speciﬁcally bind to CD47 and compositions comprising such antibodies, including pharmaceutical compositions, diagnostic compositions and kits. Also provided are methods of using anti-CD74 antibodies for therapeutic and diagnostic purposes, and methods for making such anti-CD47 antibodies, for example with cell-free (CF) s. 2. BACKGROUND CD47, also known as integrin—associated protein (IAP), ovarian cancer antigen 0A3, Rh-related antigen and MER6, is a multi-spanning transmembrane or belonging to the immunoglobulin amily. SIRPOL (signal-regulatory-protein or) expressed on macrophages interacts with CD47, and this interaction negatively ls effector function of innate immune cells such as host cell phagocytosis. CD47 expression and/or activity have been implicated in a number of diseases and disorders. Accordingly, there exists a need for therapies that target CD47, as well as better methods for making such therapies. 3. SUMMARY In one aspect, provided herein are antibodies (e.g., monoclonal antibodies), including antigen-binding nts thereof, which speciﬁcally bind to CD47 (e.g., human CD47), such as an extracelluar domain (ECD) of CD47. In speciﬁc aspects, such anti—CD47 antibody blocks CD47 binding to SIRPOL, promotes phagocytosis, has d or no Fc effector function (e.g., binding to FcyR, ADCC, or CDC) and/or has little or no ination (e.g., hemagglutination) activity.

WO 09415 In a speciﬁc aspect, provided herein is a monoclonal anti-CD47 antibody which speciﬁcally binds to human CD47, wherein the anti-CD47 antibody is a variant of a parental antibody, and wherein the anti-CD47 dy when produced using a cell-free (CF) expression system has a higher antibody expression titer or yield compared to the parental antibody when produced in the CF expression system. In a particular aspect, anti-CD47 antibodies provided herein which are expressed in a CF system, are sylated.

In one aspect, provided herein is a monoclonal anti-CD47 antibody which speciﬁcally binds to human CD47 (e.g., SEQ ID NO: 38 or 39), wherein the anti-CD47 antibody, when ed using a cell-free system, has a higher antibody expression titer or yield compared to a parental antibody produced using the ree system. In speciﬁc aspects, the the anti-CD47 antibody expression titer or yield is higher by at least 1 fold, at least 2 fold, or at least 3 fold compared to the parental dy. In c aspects, the the anti-CD47 antibody expression titer or yield is higher by at least 25%, 50%, 75%, or 100% compared to the parental dy.

In particular aspects, such anti-CD47 antibody is a zed antibody. In speciﬁc aspects, the cell-free system comprises using S30 cell—free t. In ular aspects, such cell-free system comprises prokaryotic disulﬁde bond isomerase Dst. In certain aspects, such anti- CD47 antibody is an IgG1 antibody. In certain aspects, such anti—CD47 antibody is an IgG4 antibody. In certain aspects, such anti-CD47 antibody is an IgG4 antibody comprising a S228P amino acid substitution according to the EU numbering index. In certain aspects, such anti- CD47 antibody is an IgG4 antibody comprising a S228P and L235E amino acid substitutions according to the EU numbering index.

In speciﬁc s, such parental antibody of an anti-CD47 antibody provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1.

In certain aspects, such parental dy comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12. In particular aspects, such parental antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, 3 or 4.

In particular s, an anti-CD47 antibody provided herein comprises (i) a heavy chain variable region comprising complementaiity determining region (CDR) l, 2, and 3 of antibody 2A1, and (ii) a light chain variable region comprising CDRl, CDR2, and CDR3 of antibody 2A1.

In speciﬁc aspects, an anti-CD47 antibody provided herein ses (i) a heavy chain variable region sing complementarity determining region (CDR) 1, 2, and 3 sing amino acid sequences GFNIKDYYLH (SEQ ID NO: 14), WIDPDQGDTE (SEQ ID NO: 15), and NAAYGSSSYPMDY (SEQ ID NO: 16), respectively, and (ii) a light chain variable region comprising CDR1, CDR2, and CDR3 comprising amino acid sequences KASQDIHRYLS (SEQ ID NO: 17), RANRLVS (SEQ ID NO: 18), and LQYDEFPYT (SEQ ID NO: 19), respectively.

In particular aspects, provided herein are D47 antibody comprising one or more amino acid modiﬁcations (e.g., amino acid substitutions) ve to a parental antibody. In c aspects, such one or more amino acid substitutions is in the framework region of the heavy chain variable region or light chain variable region. In speciﬁc aspects, such anti-CD47 antibody comprises 13 or 14 amino acid modiﬁcations (e.g., amino acid substitutions) in the framework region of the heavy chain variable region. In particular aspects, such anti-CD47 dy comprises 1 to 15 amino acid modiﬁcations (e.g., amino acid substitutions) in the framework region of the heavy chain variable region. In particular aspects, such amino acid modiﬁcations are conservative amino acid substitutions.

In c aspects, provided herein is a monoclonal anti-CD47I dy which speciﬁcally binds to CD47 (e. g., human CD47 such as SEQ ID NO: 38 or 39) and comprises a heavy chain variable region (VH) comprising the amino acid sequence: X1QXzQLVQSGAEVKKX3GX4SVKVSCKASGFNIKDYYLHWVRQAPGQX5LEWMGWIDP DQGDTEYAQKX6QXgRVTXgTXgDXmSX11STAYIVIELXIZSLRSX13DTAX14YYCNAAYGSS SYPMDYWGQGTTVTV (SEQ ID NO: 20), wherein the amino acid at position X1 is any amino acid or there is no amino acid at position X1, and wherein the amino acid at each of positions X2- X14 is any amino acid. In certain aspects, X1 is M or there is no amino acid at position X1, X2 is an amino acid with hydrophobic side chains such as M or V, X3 is T or P, X4 is S or A, X5 is an amino acid having aliphatic side chains such as A or G, X6 is F or L, X7 is D or G, X8 is an amino acid with hydrophobic side chains such as I or M, X9 is R or T, X10 is R or T, X11 is M or T, X12 is S or R, X13 is a negatively charged amino acid such as E or D, and X14 is an amino acid with hydrophobic side chains such as M or V. In ular aspects, the VH of an anti-CD47 antibody provided herein comprises the amino acid sequence of SEQ ID NO: 21. In certain aspects, the VH of an anti-CD47 antibody provided herein comprises the amino acid sequence of SEQ ID NO: 22. In speciﬁc aspects, an anti-CD47 antibody provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 5. In particular aspects, an anti—CD47 antibody provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 6. In certain aspects, an anti-CD47 antibody provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 7. In particular aspects, an anti-CD47 antibody provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 8. In speciﬁc aspects, an anti-CD47 antibody provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9. In certain aspects, an anti-CD47 antibody provided herein ses a heavy chain sing the amino acid sequence of SEQ ID NO: 10. In particular aspects, an anti-CD47 antibody provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 11.

In speciﬁc aspects, an anti-CD47 antibody provided herein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 13 without amino acid M at the N—terminus.

In c aspects, provided herein is a monoclonal anti-CD47 antibody, which cally binds to CD47 (e.g., human CD47 such as SEQ ID NO: 38 or 39), wherein the anti- CD47 antibody does not cause or promote ntial red blood cell depletion, , or both red blood cell depletion and anemia after administration. In certain s, such anti-CD47 antibody does not cause or promote substantial platelet depletion after administration. In particular aspects, such anti-CD47 antibody does not cause or promote ntial agglutination of cells after administration. In speciﬁc s, such anti-CD47 antibody does not cause or promote substantial lutination of red blood cells after administration. In n aspects, such D47 antibody inhibits (e.g., inhibits by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) CD47 from interacting with signal-regulatory-protein 0t ). In particular aspects, such anti-CD47 antibody promotes phagocytosis, such as macrophage-mediated phagocytosis of a CD47-expressing cell. In certain aspects, such anti- CD47 antibody provided herein does not cause or promote a signiﬁcant level of effector function.

In certain aspects, an anti—CD47 dy provided herein, when expressed using a cell-free system, exhibits lower (e. g., lower by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%) binding afﬁnity, or does not bind, to an FcyR ed to when expressed using CHO cells. In particular aspects, such lower binding afﬁnity is at least 1 log lower or at least 2 log lower. In certain aspects, the FcyR is FcyRI, FcyRIIA R131, FcyRIIA H131, FcyRIIB, or IA V158.

In particular aspects, an anti-CD47 dy provided herein is aglycosylated or has less (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% less) ylation when expressed using the cell free system compared to when expressed in CHO cells.

In speciﬁc aspects, provided herein is a monoclonal anti-CD47I antibody, which speciﬁcally binds to CD47 (e.g., human CD47 such as SEQ ID NO: 38 or 39), wherein the anti- CD47 antibody (i) promotes phagocytosis such as macrophage-mediated phagocytosis of a CD47-expressing cell, (ii) does not cause or promote a signiﬁcant level of hemagglutination of red blood cells after stration; (iii) does not cause or promote a signiﬁcant level of ADCC or CDC; and/or (iv) exhibits lower (e.g,, lower by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%) g afﬁnity, or does not bind, to an FcyR compared to when expressed using CHO cells or compared to a parental antibody.

In certain aspects, an anti-CD47 antibody provided herein is a bispeciﬁc antibody. In particular s, an anti—CD47 antibody provided herein is conjugated to an agent. In certain aspects, the agent is a label or a toxin.

In particular aspects, provided herein is a pharmaceutical composition comprising an effective amount of an anti-CD47 antibody provided herein or an antigen-binding fragment thereof. In speciﬁc aspects, the pharmaceutifcal composition provided herein further comprising a pharmaceutically acceptable carrier.

In speciﬁc aspects, provided herein is a polynucleotide comprising a nucleotide ce encoding a VH chain region, a VL chain region, or both a VL chain region and a VH chain region, of an anti—CD47 antibody described herein. In particular s, provided herein is a polynucleotide comprising a nucleotide sequence ng a heavy chain, a light chain, or both heavy chain and a light chain of an anti-CD47 dy described herein. In particular aspects, such polynucleotide comprises a tide sequence of any one of SEQ ID NOs: 26-32 encoding a heavy chain. In speciﬁc aspects, such polynucleotide comprises a nucleotide sequence of SEQ ID NO: 33 ng a light chain.

In particular aspects, provided herein is a population of polynucleotides comprising (i) a ﬁrst polynucleotide sing tide sequences encoding a VH or a heavy chain of an anti-CD47 antibody described herein, and (ii) a second polypeptide comprising nucleotide sequences encoding a VL or a light chain of an anti-CD47 antibody described herein. In certain aspects, such ﬁrst polynucleotide is operably linked to a ﬁrst promoter, and such second polynucleotide is operably linked to a second promoter.

In particular s, ed herein is a vector comprising one or more polynucleotides described herein.

In speciﬁc aspects, provided herein is a population of vectors comprising (i) a ﬁrst vector comprising nucleotide sequences encoding a VH or a heavy chain of an anti-CD47 dy bed herien, and (ii) a second vector comprising nucleotide sequences encoding a VL or a light chain of an anti-CD47 antibody described herein.

In ular aspects, provided herein is a composition for cell-free protein expression comprising a cell-free extract and one or more polynucleotides or vectors described herein. In speciﬁc aspects, the composition further comprising S30 cell-free extract. In ular s, the composition provided herein further comprises prokaryotic disulﬁde bond isomerase Dst.

In speciﬁc aspects, provided herein is a method of ng cancer, wherein the method comprises administering an D47 antibody described herein to a subject in need thereof in an amount sufﬁcient to treat the cancer in the subject.

In particular aspects, provided herein is a method of alleviating a symptom of a cancer, the method comprising stering an anti-CD47 antibody described herein to a subject in need thereof in an amount sufﬁcient to alleviate one or more ms of the cancer in the t.

In speciﬁc aspects, such method provided herein further comprises administering radiation or chemotherapy.

In speciﬁc aspects, such method provided herein further comprises administering r anti-cancer agent.

In speciﬁc aspects of the methods provided herein, the cancer is a hematological cancer. In a particular aspect, the cancer is a solid cancer. In a certain aspect, the cancer is multiple myeloma, non-Hodgkin’s lymphoma, acute myeloid leukemia (AML), breast cancer, bladder cancer, non-small cell lung cancer/carcinoma, hepatocellular carcinoma (HCC), sarcoma, or head and neck cancer.

In particular aspects, ed herein is an isolated cell comprising one or more polynucleotides or s described herein.

In c s, provided herein is an isolated cell comprising a population of polynucleotides or vectors described herein.

In speciﬁc aspects, provided herein is an isolated cell producing an D47 antibody or antigen-binding fragment described herein.

In particular aspects, provided herein is a population of host cells comprising (i) a ﬁrst host cell sing a polynucleotide comprising nucleotide sequences encoding a VH or a heavy chain of an anti-CD47 antibody described herein, and (ii) a second host cell comprising a polynucleotide comprising nucleotide sequences encoding a VL or a light chain of an anti-CD47 dy described herein.

In particular aspects, provided herein is a method of making an anti-CD47 antibody comprising expressing an anti-CD47 antibody described herein with a composition for cell-free protein expression described herein. In a certain aspect, such method further comprises purifying the anti-CD47 antibody.

In specific aspects, ed herein is a method of making an D47 antibody comprising expressing the anti-CD47 antibody with a cell bed herein. In a certain aspect, such method further comprises purifying the anti-CD47 antibody. 4. BRIEF DESCRIPTION OF THE FIGURES Figure 1A depicts the autoradiagram of anti-CD47 antibodies expressed with a cell- free (CF) system. Samples l-lO correspond to CF-expressed anti-CD47 IgGl (l), IgGl-Sm (2), IgGl-l3m (3), IgGl-l3mZ (4), IgG4P (5), IgG4P-5m (6), l3m (7), IgG4PE (8), IgG4PE- 5m (9), and IgG4PE-l3m (10) antibodies, respectively.

Figure 1B depicts a graph showing anti-CD47 dy titers (mg/L) obtained from aCF expression . Samples 1-10 correspond to CF—expressed anti-CD47 IgGl (1), IgGl- 5m (2), IgGl-l3m (3), IgGl-l3mZ (4), IgG4P (5), IgG4P-5m (6), IgG4P-l3m (7), IgG4PE (8), IgG4PE—5m (9), and IgG4PE-l3m (10) antibodies, respectively. s 2A-2F depict dual sensorgrams from Biacore analysis of anti-CD47 IgG1-5m (2A), IgG1-13m (2B), IgG1-13mZ (2C), IgG4P-5m (2D), IgG4PE-5m (2E), and l antibody (2F).

Figures 3A-3C depict graphs plotting speciﬁc heat capacity (kcal/mol/OC) versus temperature (°C) from thermostability analysis using Differential Scanning metry (DSC) for anti-CD47 IgGl-l3mZ (3A), IgGl-l3m (3B), and IgGl-Sm (3C) antibodies.

Figure 4 depicts a graph plotting anti—CD47 antibody plasma tration (pg/mL) versus time (hours) from pharmacokinetic s with anti-CD47 IgG4-PE antibody produced with CHO cells and anti-CD47 IgG1 and IgGl-Sm antibodies produced by the CF expression system.

Figure 5 depicts a graph plotting tumor volume (mm3) versus days after RPMI8226 tumor cell ation from in vivo mouse tumor xenograft studies using anti-CD47 IgGl-Sm antibodies produced by the CF expression system at doses of 1 mg/kg, 03 mg/kg, and 0.1 mg/kg (qwx3).

. DETAILED DESCRIPTION In one aspect, provided herein are antibodies (e.g., monoclonal antibodies), and antigen-binding fragments thereof, that cally bind to CD47 (e.g., human CD47). In c aspects, such anti-CD47 antibody blocks CD47 binding to , promotes phagocytosis, has reduced or no Fc effector on (e.g., binding to FcyR, ADCC, or CDC) and/or has little or no agglutination (e.g., hemagglutination) activity.

In a speciﬁc aspect, provided herein is a monoclonal anti-CD47 antibody which speciﬁcally binds to human CD47, wherein the anti-CD47 antibody is a t of a parental antibody, and wherein the anti-CD47 antibody when produced using a cell-free (CF) expression system has a higher dy expression titer or yield compared to the parental antibody when expressed in the CF system. In a particular , anti-CD47 antibodies provided herein which are expressed in a CF , are aglycosylated.

As used herein, the terms “CD47” or “integrin-associated protein” or “IAP” or “ovarian cancer antigen” or “0A3” or “Rh—related antigen” or “MER6” can be used interchangeably and refer to a multi-spanning transmembrane receptor belonging to the immunoglobulin superfamily. The amino acid sequence of an exemplary human CD47 is provided below (GenBank Accession No. Q08722.l (G1: 1 171879), incorporated herein by reference). The signal sequence (amino acids 1—18) is underlined. l MWPLVAALLL SAQL LFNKTKSVEF TFCNDTWIP CFVTNMLAQN T'i'.LVYVKWKF WO 09415 61 KGRDIYTTDG ALNKSTVPTD FSSAKIEVSQ LLKGDASLKM SHTG NYTCEVTELT 121 IELK YRVVSWFSPN ENIJIVIFPI FAILJFWGQF YRSG GMD?KTIA.L 181 VAGLVITVIV IVGAILFVPG EYSTKNATGT GTIVTSTGIL ILLHYYVFST AIGLTSFVIA 241 ILVIQVIAYI LAVVGLSLCI AACIPMHGP. LISG.SILAL AQ.LGLVYMK FVASNQKTIQ 301 PPRKAVEEPL NAFKESKGMM NDE (SEQ ID NO: 38) For clarity, the amino acid sequence of an exemplary human CD47 excluding the signal sequence is provided below. 1 QLJFNKTKSV EFTFCNDTVV IPCFVTNMEA.QNTTEVYVKW KFKGRDIY"F STVP 61 TDFSSAKIEV SQLLKGDASL KMDKSDAVSH TGNYTCEVTE LTREGETIIE LKYRVVSWFS 121 PNENILIVIF ?IFAIL4FWG QFGIKTLKYR SGGMD?KTIA L.VAGLVI"V IVIVGAIJFV 181 PGEYSLKNAT GLGLIVTSTG YYVF STAIGJTSFV IAILVIQVIA YILAVVGJSL 241 CIAACIPMHG ?LLISGJSIL ALAQLLGLVY MKFVASNQKT IQPPRKAVEE PLNAFKESKG 301 MMNDE (SEQ ID NO: 39) The terms red blood cell(s) and erythrocyte(s) are synonymous and used interchangeably herein.

The term agglutination refers to ar clumping, while the term hemagglutination refers to clumping of a speciﬁc subset of cells, i.e., red blood cells. Thus, hemagglutination is a type of agglutination. .1 Antibodies In a speciﬁc aspect, provided herein are antibodies which speciﬁcally bind to CD47 (e. g., human CD47). In particular aspects, ed herein are anti-CD47 antibodies comprising modiﬁcations in one or more amino acid residues (e.g., 5—13 amino acid substitutions in the framework region of the heavy chain variable region) that singly allow for better production in a cell-free (CF) expression sytem than the parental antibody without the modiﬁcations. In certain aspects, such D47 antibodies inhibit SIRPOL interaction with CD47, are sylated, promote phagocytosis, either in vivo or in vitro or both, have anti— tumor activity (e. g., without promoting agglutination, such as hemagglutination), and/or have low or no Fc effector function (e.g., binding to an FcyR, ADCC, or CDC).

In certain embodiments, antibodies or antigen-binding nts bed herein can comprise sequences that do not naturally exist within the antibody germline repertoire of an animal or mammal (e.g., human) in viva.

As used herein and unless otherwise speciﬁed, the terms “about” or “approximately” mean within plus or minus 10% of a given value or range. In instances where an integer is required, the terms mean within plus or minus 10% of a given value or range, rounded either up or down to the nearest integer, [Xsusedluxenrthetenns“anﬁbody”and‘ﬁnnnunogkﬂnﬂhf’and‘Tg”areteHnsofaﬁ and can be used hangeably herein and refer to a molecule with an antigen binding site that speciﬁcally binds an antigen.

Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, monospeciﬁc antibodies, multispeciﬁc antibodies (including bispeciﬁc antibodies), human antibodies, humanized antibodies, murine antibodies (e.g., mouse or rat antibodies), chimeric antibodies, synthetic antibodies, and tetrameric antibodies comprising two heavy chain and two light chain molecules. In speciﬁc embodiments, dies can include, but are not lirriiteacl tr) £111 £111til)()(1§/ liggllt (:llélill 111(311()111<:r, £111 l)()(13/ l1<3£1\/§I ll 111()11()111<3r, £111 l)()(l§l liggllt chain dimer, an antibody heavy chain dimer, an antibody light chain- antibody heavy chain pair, odies, heteroconjugate antibodies, single domain antibodies, and monovalent dies.

In a speciﬁc embodiment, antibodies can include antigen-binding nts or epitope binding fragments such as, but not limited to, single chain antibodies or single-chain Fvs (scFv) (e.g., including monospeciﬁc, bispeciﬁc, eta), camelized antibodies, affybodies, Fab fragments, F(ab’) fragments, 2 fragments, and disulﬁde-linked Fvs (dev). In certain embodiments, antibodies bed herein refer to polyclonal antibody populations.

Antibodies can be of any type (e.g, IgG, IgE, IgM, IgD, IgA or IgY), any class, (e.g., IgGl, Ing, IgGg, IgG4, IgA1 or IgAz), or any subclass (e.g., Inga or Igng) of immunoglobulin molecule. In n embodiments, antibodies bed herein are IgG antibodies, or a class (e.g., human IgG1, Ing, IgG3 or IgG4) or subclass thereof. In certain embodiments, antibodies @mﬂmﬂmmmmegGﬂmmwmuaghmmnkGQmawawmmmfhmmMn embodiments, IgG1 antibodies described herein comprise one or more amino acid substitutions and/or deletions in the constant region. In n embodiments, antibodies described herein are IgG4 antibodies (e.g., human IgG4) or a subclass thereof. In certain embodiments, IgG4 dies described herein comprise one or more amino acid substitutions and/or deletions in the constant region.

As used herein, an “antigen” is a moiety or molecule that contains an epitope to which an antibody can speciﬁcally bind. As such, an antigen is also speciﬁcally bound by an antibody.

As used herein, an “epitope” is a term in the art and refers to a zed region of an antigen to which an antibody can speciﬁcally bind. An epitope can be a linear epitope or a conformational, non-linear, or discontinuous, epitope. In the case of a polypeptide antigen, for example, an epitope can be contiguous amino acids of the polypeptide (a “linear” epitope) or an epitope can se amino acids from two or more ntiguous regions of the polypeptide (a “conformational,” “non-linear” or “discontinuous” epitope). It will be appreciated by one of skill in the art that, in general, a linear epitope may or may not be dependent on secondary, tertiary, or quaternary structure.

As used , the terms “immunospeciﬁcally binds,77 (4'1mmunospeciﬁcally recognizes,7) (( speciﬁcally binds,” and “speciﬁcally recognizes” are analogous terms in the context of antibodies and refer to molecules that bind to an n/epitope as such binding is understood by one d in the art. For example, a molecule (e.g., an antibody) that speciﬁcally binds to an antigen may bind to other peptides or polypeptides, generally with lower y as determined by, e.g., immunoassays, surface plasmon resonance assays, for example, BiacoreTM, KinExA platform (Sapidyne Instruments, Boise, ID), or other assays known in the art. In a speciﬁc embodiment, molecules that speciﬁcally bind to an antigen bind to the antigen with a Ka that is at least 2 logs, 2.5 logs, 3 logs, 4 logs or greater than the K21 when the molecules bind to another antigen. In another speciﬁc embodiment, molecules that speciﬁcally bind to an antigen do not cross react with other proteins. In another speciﬁc embodiment, molecules that speciﬁcally bind to an antigen do not cross react with other non-CD47 proteins.

As used herein, the term “monoclonal antibody” is a well known term of art that refers to an antibody obtained from a population of homogenous or substantially homogeneous dies. The term “monoclonal” is not limited to any particular method for making the antibody. Generally, a tion of monoclonal dies can be generated by cells, a population of cells, or a cell line. In speciﬁc embodiments, a “monoclonal antibody,” as used herein, is an antibody produced by a single cell or cell line wherein the antibody speciﬁcally binds to a CD47 epitope as determined, e.g., by ELISA or other n- binding or competitive binding assay known in the art or in the Examples provided herein. In particular embodiments, a monoclonal antibody can be a ic antibody or a zed antibody. In certain embodiments, a monoclonal antibody is a monovalent antibody or multivalent (e.g., bivalent) antibody. -1]- umsq Asundhmdmthﬁmnﬂhmvmmnmamnmaddﬁeﬁmtoanmnmoaddﬂmﬁsnma proteinogenic amino acid, or a ranslationally modiﬁed variant f. In particular, the term refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine or selenocysteine, or post-translationally modiﬁed variants f.

As used herein, the term “polyclonal antibodies” refers to an antibody population that includes a y of different antibodies that immunospeciflcally bind to the same and/or to different epitopes within an antigen or antigens.

As used , the terms “variable region” or “variable domain” refer to a portion of an antibody, generally, a portion of an antibody light or heavy chain, typically about the amino- tmhnnalllOto120annnoaddsinamnanneheavychahiandabmntheannno4ennnml90to100 ammomMsmanmmmhgndmm.meMemgmmcmmmﬁeammbmmWMWdammmmg mﬂmﬂRMMWMﬁmmemmEMIMMWﬁmmMmmmmﬁam mmFRsmmmﬁﬂmwﬂnmﬂlmmmmHoCJHmmddhaﬂmrFRLCDRLFRlCDRzFR} CDR3-FR4. t wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with antigen and for the specificity of the antibody for an epitope. In a speciﬁc embodiment, numbering of amino acid positions of antibodies described herein is according to the EU Index, as in Kabat el al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US. Department of Health and Human Services, NIH Publication No. 91-3242. In certain embodiments, the variable region is a human variable region. In certain embodiments, the le region ses murine (e.g, mouse or rat) CDRs and human framework regions GRS.hpMWMMmmmmmmmﬂwvmmmH%MnmammmewgdmmmonmmMmm primate) variable region. In certain ments, the variable region comprises murine (e.g., mouse or rat) CDRs and e (e.g., human or non-human primate) framework regions (FRs).

As a non-limiting example, a variable region described herein is obtained from ling two or more nts of human sequences into a composite human sequence.

In certain aspects, the CDRs of an antibody can be determined according to (i) the Kabat numbering system (Kabat el al. (1971) Ann. NYAcad. Sci. 190:382—391 and, Kabat el al. (1991) Seguences of Proteins of Immunological Interest, Fifth Edition, US. Department of Health and Human Services, NIH Publication No. 2); or (ii) the Chothia numbering scheme, which will be referred to herein as the “Chothia CDRs” (see, e.g., Chothia and Lesk, 1987, J. Mol. Biol., 1-917, Al-Lazikani e: 511., 1997, J. Mol. Biol., 273:927-948, Chothia el al., 1992, J, Mol. Biol, 227:799-817; Tramontano A ef al., 1990, J. Mol. Biol. 215(1):]75-82, and US. Patent No. 7,709,226); or (iii) the GeneTics (IMGT) numbering system, for example, as described in Lefranc, M.-P., 1999, The Immunologist, 7: 132-136 and Lefranc, M.-P. el‘ al., 1999, Nucleic Acids Res, 27:209—212 (“IMGT CDRs”), or (iv) MacCallum el al., 1996, J.

Mol. Biol, 262:732-745. See also, e.g., Martin, A., “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering, Kontermann and Dubel, eds, Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001).

With respect to the Kabat numbering system, CDRs within an antibody heavy chain mdmﬂememeMymﬂmHMammomMpmMmm31m3iwmwomMmMymnmdmkom or two additional amino acids, ing 35 red to in the Kabat numbering scheme as 35A and 35B) (CDRl), amino acid positions 50 to 65 (CDR2), and amino acid ons 95 to 102 @MB)UmyMKmemmmgwmmCMkwﬁmmmmmwhgmmmmmwMMe typically t at amino acid positions 24 to 34 (CDRl), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 . As is well known to those of skill in the art, using the Kabat numbering system, the actual linear amino acid sequence of the antibody variable domain can contain fewer or additional amino acids due to a shortening or lengthening of a FR and/or CDR and, as such, an amino acid’s Kabat number is not necessarily the same as its linear amino acid number.

Antibodies provided herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class, (e.g., IgGl, IgG2, IgG3, IgG4, IgA1 or IgAz), or any subclass (e.g., Inga or Ingb, or a mmmmmmaﬂoﬂmmmmgwmmnwbwk.mcmmmmmmmmMMJmMmmwdmmmw herein are IgG antibodies (e.g., human IgG), or a class (e.g., human IgG1, Ing, IgG3 or IgG4) or subclassthereof In speciﬁc aspects, provided herein is an antibody comprising an antibody light chain mmhwwmmmnaggwqmmwhgndmmamﬂwmydmm.“MhmwunwﬂwhgndmmJna c ment, the light chain of an antibody described herein is a kappa (K) light chain.

In another specific embodiment, the light chain of an antibody described herein is a lambda (9») light chain. In r embodiment, light chain is a mixed sequence, e.g., the variable portion of the light chain comprises kappa light chain sequences and the constant region of the light chain comprises lambda light chain sequences, or vice versa. In certain embodiments, the light chain of an antibody described herein is a human kappa light chain or a human lambda light chain.

Non-limiting examples of human constant region sequences have been described in the art, e.g., see US. Patent No. 5,693,780 and Kabat et a]. (1991) ces of Proteins of Immunological Interest, Fifth Edition, US. Department of Health and Human Services, NIH Publication No. 91- 3242.

In a speciﬁc aspect, provided herein is an antibody, e.g. a monoclonal antibody, which speciﬁcally binds to human CD47, wherein such an anti-CD47 antibody is a variant of a parental anti-CD47 antibody, wherein the anti-CD47 antibody, when produced using a cell-free (CF) expression system, has a higher antibody expression titer or yield ed to that of the al anti-CD47 antibody when expressed in the CF system, and n the anti-CD47 antibody ses one or more amino acid modiﬁcations, for example, 1-15 amino acid modiﬁcations, relative to the the parental anti-CD47 antibody. In a particular aspect, the one or more amino acid modiﬁcations, for example, 1-15 amino acid modiﬁcations, are within the heavy chain or VH (e.g., SEQ ID NO: 1). In a particular aspect, the one or more amino acid modiﬁcations, for e, l-15 amino acid modiﬁcations, are within the framework region of a VH (e.g., SEQ ID NO: 1). In a certain aspect, the anti-CD47 dy provided herein which is a t of a parental anti-CD47 antibody comprising the CDRs (e.g., Kabat CDRs) of the parental anti-CD47 antibody.

In a speciﬁc aspect, provided herein is an antibody, e.g. a monoclonal antibody, which speciﬁcally binds to human CD47, wherein such an anti-CD47 antibody is a variant of a parental anti-CD47 antibody, wherein the anti-CD47 dy, when ed using a cell-free (CF) sion system, has a higher antibody expression titer or yield compared to that of the al anti-CD47 antibody when expressed in the CF , and wherein the anti-CD47 antibody comprising one or more amino acid modiﬁcations, for example, 1-15 amino acid modiﬁcations, relative to the the parental anti-CD47 antibody. In a particular aspect, the one or more amino acid modiﬁcations, for example, 5 or 14 amino acid modiﬁcations, are within the heavy chain or VH (e.g., SEQ ID NO: 1). In a particular aspect, the one or more amino acid modiﬁcations, for example, 5, 10, 13 or 14 amino acid modiﬁcations, are within the framework region of a VH (e.g., SEQ ID NO: 1). In a particular aspect, the one or more amino acid modiﬁcations, for example, 5, 13 or 14 amino acid modiﬁcations are Within the framework region of a VH (e.g., SEQ ID NO: 1). In a certain aspect, the anti-CD47 antibody provided herein which is a variant of a parental anti-CD47 antibody comprising the CDRs (e.g., Kabat CDRs) of the parental anti-CD47I antibody. In certain aspects, such anti-CD47 antibody is an IgGl, IgG2, IgG3, or IgG4 isotype antibody. In certain aspects, such anti-CD47 antibody is an IgGl e antibody. In n aspects, such D47I antibody is an IgGl Z allotype isotype antibody. In certain aspects, such anti-CD47 dy is an IgG4, such as an IgG4P or IgG4PE, isotype antibody.

In a speciﬁc aspect, provided herein is an antibody, e. g. a monoclonal antibody, which speciﬁcally binds to human CD47, wherein such an D47 antibody is a variant of a parental anti-CD47 antibody, wherein the D47 antibody, when produced using a cell-free (CF) expression system, has a higher antibody sion titer or yield compared to that of the parental anti-CD47 antibody when expressed in the CF system. In speciﬁc embodiments, the parental anti-CD47 antibody is antibody AB6. 12 (see, e.g., US. Application Publication No. US 2014/0140989 A1, which is incorporated herein by reference in its entirety). The amino acid sequences of the heavy chain variable region (VH) and light chain le region (VL) of antibody AB6. 12 are provided below, wherein the Kabat CDRs are underlined. In a certain aspect, the anti-CD47 antibody provided herein is a variant of parental antibody AB6. 12, and comprises the CDRs (e.g, Kabat CDRs) of parental antibody AB6. 12, for e SEQ ID NOs: 14-19. In certain aspects, such anti-CD47 antibody is an IgGl, IgG2, IgG3, or IgG4 isotype antibody. In certain aspects, such anti-CD47 antibody is an IgGl isotype antibody. In certain aspects, such anti-CD47 antibody is an IgGl Z allotype isotype antibody. In certain s, such anti-CD47 antibody is an IgG4, such as an IgG4P or IgG4PE, isotype antibody.

Anti-CD47 antibody AB6. 12 heavy chain variable region (VH) (Kabat CDRs 1-3 are underlined, SEQ ID NOs: : QMQLVQSGAEVKKTGSSVKVSCKASGFNIKDYYLHWVRQABGQAL*ZWMGW TEYAQKF QDRVT I S TAYMELS SLRSEDTAMYYCNAAYGS S SYPMDYWGQGTTVTV (SEQ I D NO: Anti-CD47 dy AB6. 12 light chain variable region (VL) (Kabat CDRs 1-3 are underlined, SEQ ID NOs: 17-19): N: QMTQS PSAMSASVGDRVT I TCKAS Q) I HRYLSWFQQKPGKVPKI— L I YRANRLVS G VPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQYDEFPYTFGGGTKVEIK (SEQ ID NO: In a speciﬁc embodiment, an anti-CD47 described herein comprises one or more amino acid modiﬁcations (e.g,, 1—15 amino acid modiﬁcations), for e in the VH framework region, of a parental antibody, e.g., a parental dy selected from D47 dies described in US. Application Publication No. US 2014/0140989 A1, which is hereby incorporated by reference in its entirety, for example anti-CD47 dies described in Table 1 of US. Application Publication No. US 2014/0140989 A1 (e.g., anti-CD47 antibody 2A1, AB2.03, AB2.04, AB2.05, , AB2.07, AB2.08, , AB2.13, AB3.09, AB6.12, AB6.]3,AB6.14,AB6.17,AB10.13,AB10.14,AB11.05,AB12.05,AB15.05,AB16.05, , AB22.05, 5, AB24.05, and AB25.05).

In a speciﬁc aspect, provided herein is an antibody, eg. a monoclonal antibody, which speciﬁcally binds to human CD47, wherein such an anti-CD47 antibody is a variant of a parental anti-CD47 antibody, wherein the anti-CD47 antibody, when produced using a cell-free (CF) expression system, has a higher antibody expression titer or yield compared to that of the parental D47 antibody when expressed in the CF system, and wherein the anti-CD47 antibody comprises a VH comprising the following N—terminal to C-terminal sequence: XlQXgQLVQSGAEVKKXQGXQSVKVSCKASGFNIKDYYLHWVRQAPGQXéLEWMGWIDP DQGDTEYAQKxéQxZRVTxgrngxysxusrAYMELxESLRstDTAxﬂYYCNAAYGSs SYPMDYWGQGTTVTV (SEQ ID NO: 20), wherein the underlined amino acid residues for Xk Xﬂ are ordered from N-terminus to C-terminus, wherein X1 is M or there is no amino acid at position X1, X2 is an amino acid with hydrophobic side chains such as M or V, X3 is T or P, X4 is S or A, X5 is an acid having aliphatic side chains such as A or G, X; is F or L, X7 is D or G, X8 is an amino acid with hydrophobic side chains such as I or M, X9 is R or T, X10 is R or T, X11 is M or T, X12 is S or R, X13 is a negatively charged amino acid such as E or D, and X14 is an amino acid with hydrophobic side chains such as M or V.

In particular s, an anti-CD47 antibody described herein comprises a VH sing the sequence of SEQ ID NO: 20, wherein the amino acid at position X1 is any amino acid such as M, X2 is not M, X3 is not T, X4 is not S, X5 is not A, X6 is not F, X7 is not D, X8 is not I, X9 is not R, X10 is not R, X11 is not M, X12 is not S, X13 is not E, and/or X14 is not M. In particular aspects, any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of X1 to X14 are not these amino acids. In particular aspects, the VH amino acid sequence is not the VH amino acid sequence of antibody AB6. 12, for example, the VH amino acid sequence is not SEQ ID NO: 1.

In particular aspects, an D47 antibody described herein comprises a VH comprising the sequence of SEQ ID NO: 20, wherein the amino acid at position X7 is not G, X9 is not A and/or X11 is not S. In particular aspects, any 1, 2, or 3 of X7, X9 and X11 are not these amino acids. In particular aspects, when the amino acid at position X7 is G, then X8 is M and/or X10 is T, X9 is not A and/or X11 is not S.

In particular aspects, an anti-CD47 dy bed herein comprises a VH sing the sequence of SEQ ID NO: 20, n the amino acid at position X7 is not G, X8 is not M, X9 is not E, X10 is not T, and/or X11 is not T. In particular aspects, any 1, 2, 3, or 4 of X7 to X11 are not these amino acids. In particular aspects, when the amino acid at position X7 is G, then X; is M, Xlois T, X9 is not E, and X11 is T.

In a particular aspect, an anti—CD47 antibody described herein comprises a VH comprising the ce of SEQ ID NO: 20, n the VH does not comprise the amino acid ce of SEQ ID NO: 5, 6, 7, 8, 9, 10, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, of US. Application Publication No. US20l4/Ol40989 Al, which is incorporated herein by reference in its entirety. In a particular aspect, an anti-CD47 antibody described herein comprises a VH comprising the consensus sequence of SEQ ID NO: 20, wherein the VH does not se the ork regions of the amino acid sequence of SEQ ID NO: 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25,26, 27, 28, 29, or 30, of US. Application Publication No. US2014/0140989 A1, which is incorporated herein by reference in its entirety.

In particular aspects, X1 is M, X2 is V, X3 is P, X4 is A, X5 is G, X; is L, X7 is G, X8 is M, X9 is T, X10 is T, X11 is T, X12 is R, X13 is D, and/or X14 is V. In particular embodiments, any 1,2, 3,4, 5, 6, 7, 8, 9, 10, ll, l2, 13, or 14 ole to X14 are these amino acids.

In particular aspects, X1 is M, X2 is M, X3 is P, X4 is S, X5 is A, X6 is F, X7 is G, X8 is I, X9 is R, X10 is R, X11 is T, X12 is R, X13 is E, and/or X14 is V. In particular embodiments, any 1,2, 3,4, 5, 6, 7, 8, 9, 10, ll, 12, 13, or 14 ofX1 to X14 are these amino acids.

In a particular aspect, an anti—CD47 antibody provided herein is not antibody AB6. 12.

In a particular aspect, an anti-CD47 antibody provided herein does not comprise a VH (e.g., SEQ ID NO: 1) and/or a VL (e.g., SEQ ID NO: 12) ofantibody AB6.l2.

In a speciﬁc aspect, an anti-CD47 antibody provided herein, comprises one of the following VH amino acid sequences presented in Table 1.

Table 1: VH amino acid sequence 51;;ng VH amino acid sequence Consensus LVQSGA_LVKKX3GX4SVKVSCKASG\I—F---"DKYYLHWVRQAPGQX5 LEWMGWIDPDQGDTEYAQKXGQX-yRVTXgTX9DX_1_oSX_1_1STAYMELX_1_ZS QSX13DTAX14YYCNAAYGSSSYPMDYWGQGTTVTV LDMDYWGQGTTVTV MQMQLVQSGAEVKKPGSSVKVS C {AS GFN"_K WVRQA- _LWMG WI DPDQGDTEYAQKLQGRVTMTT3TSTSTAYMELRS LRSDDTAVYYCNAA YGS S SY9MDYWGQGTTVTV In a speciﬁc aspect, provided herein is an antibody, e. g. a monoclonal antibody, which speciﬁcally binds to human CD47, wherein such an anti-CD47 antibody is a variant of a al anti-CD47 antibody, wherein the anti-CD47 antibody, when produced using a cell-free (CF) expression system, has a higher antibody expression titer or yield compared to that of the al anti-CD47 antibody when expressed in the CF system, and wherein the anti-CD47 dy comprises a VH comprising SEQ ID NO: 21. In certain aspects, such anti—CD47 antibody is an IgGl, IgG2, IgG3, or IgG4 isotype dy. In certain s, such anti-CD47 antibody is an IgGl isotype antibody. In n aspects, such anti-CD47 antibody is an IgGl Z allotype isotype dy. In certain aspects, such anti-CD47 antibody is an IgG4, such as an IgG4P or IgG4PE, isotype antibody.

In a speciﬁc aspect, provided herein is an antibody, e. g. a monoclonal dy, which speciﬁcally binds to human CD47, wherein such an anti-CD47 antibody is a variant of a parental anti-CD47 antibody, wherein the D47 antibody, when produced using a cell—free (CF) expression system, has a higher antibody sion titer or yield compared to that of the parental anti-CD47 antibody when expressed in the CF system, and wherein the D47I antibody ses a VH comprising SEQ ID NO: 22. In certain aspects, such anti-CD47 antibody is an IgGl, IgG2, IgG3, or IgG4 isotype antibody. In certain aspects, such anti-CD47 antibody is an IgGl isotype dy. In certain aspects, such anti-CD47 antibody is an IgGl Z allotype isotype antibody. In certain aspects, such anti-CD47 antibody is an IgG4, such as an IgG4P or IgG4PE, isotype antibody.

In a particular aspect, an anti-CD47 antibody (IgGl—l3m) provided herein comprises an IgGl heavy chain comprising the amino acid sequence as set forth below: MQVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYLHWVRQAPGQGLEWMGWIDPDQGDTEYAQK LQGRVTMTTDTSTSTAYMELRSLQSDDTAVYYCNAAYGSSSYPMDYWGQGTTVTVSSASTKGPS SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTY:CNVNHKPSNTKVDKKVEPKSCDKTﬂTCPPCBAPELLGGPSVELEPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFWWYVDGVEVHNA{TKPQﬂﬂQYNSTYRVVSVLTVLIQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL3PSRJELTKNQVSLTCLVKGFYPSDIAVE BLNNYKTTPPVLDSDGSFFLYSKLTVDKSQWQQGWVFSCSVMHEALHNHYTQKSLSLS PGK (SEQ ID NO: 5) In a particular aspect, an anti-CD47 antibody (IgGl-l3mZ ) provided herein comprises an IgGl-Z allotype heavy chain comprising the amino acid sequence as set forth below: MQVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYLHWVRQAPGQGLEWMGWIDPDQGDTEYAQK LQGRVTMTTDTSTSTAYMELRSLQSDDTAVYYCNAAYGSSSYPMDYWGQGTTVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTY:CWVNHKPSNTKVDKKVEPKSCDKT{TCPPCBAPELLGGPSVELEPP{PKDTLM ISRTPEVTCVVVDVSHEDPEVKFWWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKA<GQPREPQVYTL3BSRELMTKNQVSLTCLVKGFYPSDIAVE WLSNGQBLNNYKTTPPVLDSDGSFFLYSKLTVDKSQWQQGWVFSCSVMHEALHNHYTQKSLSLS PGK (SEQ ID NO: 6) In a particular aspect, an D47 antibody (IgGl-Sm) provided herein comprises an IgGl heavy chain comprising the amino acid ce as set forth below: MQMQLVQSGAEVKKPGSSVKVSCKASGFN:KDYYLHWVRQAPGQALﬁWMGW DPDQGDTEYAQK FQGRVT TR)RSTSTAYMELRSLRSEDTAVYYCNAAYGSSSYPMDYWGQGTTVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV TQTYICWVNHKPSWTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFWWYVDGVEVHNAKTKP?ﬂﬁQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKAL?APIEKTISKAKGQPREPQVYTLPPSRDETTKNQVSLTCLVKGFYPSDIAVE WLSNGQPLNNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGWVFSCSVMHEALHNHYTQKSLSLS PGK (SEQ ID NO: 7) In a particular aspect, an anti—CD47 antibody (IgG4P-l3m) provided herein comprises an IgG4P antibody comprising the amino acid sequence as set forth below: WO 09415 MQVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYLHWVRQAPGQGLEWMGWIDPDQGDTEYAQK LQGRVTMTTDTSTSTAYMELRSLQSDDTAVYYCNAAYGSSSYPMDYWGQGTTVTVSSASTKGPS VFPLAPCSRSTS?STAALGCWVKDYTPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGBPCPBCPAPEFLGGPSVFLEEBKPKDTLMISR TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPQEEQFNSTYQVVSVLTVUHQDWLNGK?Y KCKVSNKGLBSSILKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSQLTVDKSRWQEGNVFSCSVM{EALHNHYTQKSLSLSLGK (SEQ ID NO: 8) In a particular aspect, an anti-CD47 antibody (IgG4P-5m) provided herein comprises an IgG4P heavy chain comprising the amino acid sequence as set forth below: MQMQLVQSGAEVKKPGSSVKVSCKASGFN:KDYYLHWVRQAPGQALﬁWMGW DPDQGDTEYAQK FQGRVTITRDRSTSTAYMELRSLRSZDTAVYYCNAAYGSSSYPMDYWGQGTTVTVSSASTKGPSJ.

VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTKTYTCWVDHKPSNTKVDKRVESKYGPPCP?CPAPEFLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSQL‘LJDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSS"?KTTSKAKGQPQEPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 9) In a particular aspect, an anti—CD47 dy (IgG4PE—l3m) provided herein comprises an IgG4PE heavy chain comprising the amino acid sequence as set forth below: MQVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYLHWVRQAPGQGLEWMGWIDPDQGDTEYAQK LQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCNAAYGSSSYPMDYWGQGTTVTVSSASTKGPS VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV TKTYTCNVDHKPSNTKVD{RVESKYGPPCPPCPAPjFEGGPSVELEBPKP{DTLMISR TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFVSTYRVVSVLTVLHQDWLNGKEY KCKVSN<GLPSS ﬁ<T SKAKGQPQEPQVYTLPPSQEEMTKWQVSLTCLVKGEYPSD AVHWﬁS NGQPENWYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 10) In a particular aspect, an anti—CD47 antibody (IgG4PE-5m) provided herein comprises an IgG4PE heavy chain comprising the amino acid ce as set forth below: MQMQLVQSGAEVKKPGSSVKVSCKASGFNIKDYYLHWVRQAPGQALﬂWMGW DPDQGDTEYAQK FQGRVTITRDRSTSTAYMELRSLRSEDTAVYYCNAAYGSSSYPMDYWGQGTTVTVSSASTKGPS VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTKTYTCWVDHKPSNTKVD{RVES{YGBPCPBCPAPEFEGGPSVFLFBBKP{DTLMISR TBjVTCVVVDVSQiDPEVQFNWYVDGVEV{NAKTKPREEQFNSTYQVVSVLTVWHQDWLNGK *J.Y KCKVSNKGLBSS"rXTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM{EALHNHYTQKSLSLSLGK (SEQ ID NO: L1) In a c aspect, provided herein is an antibody, e. g. a monoclonal dy, which speciﬁcally binds to human CD47, wherein such an anti-CD47 dy is a variant of a parental anti-CD47 antibody, n the anti-CD47 antibody, when produced using a cell-free (CF) expression system, has a higher antibody expression titer or yield compared to that of the parental anti-CD47 antibody when expressed in the CF system, and wherein the anti-CD47 antibody comprises a light chain comprising a kappa or lambda light chain constant region (e.g., human kappa or lambda light chain constant region), for e SEQ ID NO: 13.

In a speciﬁc aspect, provided herein is an antibody, e. g. a monoclonal dy, which speciﬁcally binds to human CD47, wherein such an anti-CD47 antibody is a variant of a parental anti-CD47 antibody, wherein the anti-CD47 antibody, when produced using a cell-free (CF) expression system, has a higher antibody expression titer or yield compared to that of the parental anti-CD47 dy when expressed in the CF system, and n the anti-CD47 antibody comprises (i) a VH bed herein (e.g., SEQ ID NO: 20, 21, or 22) or a heavy chain described herein (e.g., any one of SEQ ID NOsz5-l l), and (ii) a light chain comprising a kappa or lambda light chain constant region (e.g., human kappa or lambda light chain constant region), for example SEQ ID NO: 13, e.g., as set forth below (anti-CD47 antibody light chain (IgK)), or SEQ ID NO: 13 without the amino acid M at the N—terminus: QSPSAMSASVGDRVTITCKASQDIHRYLSWFQQKPGKVPKHLIYRANRLVSGVPSRFS ﬁbTLT SSLQPEDFATYYCLQYDEFPYTFGGGTKVﬁ KRTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSXDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGFC (SﬁQ 3 NO: L3) In a speciﬁc embodiment, an anti-CD47 described herein is not an anti-CD47 antibody described in US. Application Publication No. US 2014/0140989 A1, which is hereby incorporated by reference in its entirety, for example any one of anti-CD47 antibodies in Table 1 ofthe publication (e.g., anti-CD47 antibody 2A1, AB2.03, AB2,04, AB2.05, AB2.06, AB2.07, -2]- AB2.08, AB2.09, AB2.13, AB3.09, A3612, A3613, A3614, A3617, AB10.13, AB10.14, AB11.05, AB12.05, AB15.05, AB16.05, AB17.05, AB22.05, 5, 5, and AB25.05), or any antibody sing any of SEQ ID NOS: 5-30 of the publication.

In some embodiments, an anti-CD47 antibody provided herein or an antigen-binding fragment thereof is an IgG isotype. In some embodiments, the constant region of the antibody is of human IgG1 isotype, having an amino acid sequence: ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPE-GG PSVFLFPPKP SRTP EVTCVVVDVS HED?EVKFNW VHNA.KTKPREEQYE STYRVVSVLT VLHQDWLWGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE SLTC LVKGFYPSDI AVEWESNGQP ENNYKTT?PV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK (SEQ ID NO: 34) In some embodiments, the human IgG1 constant region is modiﬁed at amino acid Asn297 (Boxed, Kabat ing) to prevent to glycosylation of the antibody, for example Asn297Ala (N297A). In some embodiments, the constant region of the antibody is modiﬁed at amino acid Leu235 (Kabat Numbering) to alter Fc receptor interactions, for example Leu235Glu (L23 5B) or Leu235Ala ). In some embodiments, the constant region of the antibody is modiﬁed at amino acid Leu234 (Kabat Numbering) to alter Fc receptor interactions, e.g, Leu234Ala (L234A). In some embodiments, the constant region of the antibody is altered at both amino acid 234 and 235, for example Leu234Ala and Leu235Ala (L234A/L235A) (EU index ofKabat er a] 1991 Sequences ofProteins nological st).

In some embodiments, the constant region of an anti-CD47 antibody provided herein is of human IgG2 isotype, having an amino acid sequence: ASTKGPSVFP LAPCSRST87J. STAALGCLVK DYFPEPVTVS WNSGALTSGV LQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVER KCCVECPPCP APPVAGPSV? KDTW M"SRTPFVTC VVVDVSHEDP EVQFNWYVDG VEVHNAKTKB RELQ:ESTER VVSVLTVVHQ DWLNGKEYKC KVSNKGLPAP IEKTISKTKG QP?E?QVYTL MTKN QVSLTCLVKG EYPSDISVLW ESNGQBENNY KTTBBMLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA.LHNHYTQKSL SLSPGK (SEQ ID NO: 35) In some embodiments, the human IgG2 constant region is modiﬁed at amino acid Asn297 (Boxed, Kabat Numbering) to prevent to glycosylation of the antibody, e.g., Asn297A1a (N297A).

In some ments, the constant region of an D47 antibody provided herein is of human IgG3 isotype, having an amino acid sequence: ASTKGPSVFP LAPCSRSTSG GTAALGCLVK DYFPEPVTVS TSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YTCNVNHKPS NTKVDKRVEL KTPLGDTTHT CPRCPEBKSC JTBPBCBRCP TPBP CPRCPEBKSC DTPPPCPRCP GPSV HLEBBKPKDT PM SRTPﬁVT CVVVDVSHL‘LJ D PEVQFKWYVD GVEVHNAKTK PREEQYESTF RVVSVLTVLH QDWLNGKEYK CKVSNKALBA_P ﬁKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESSGQPENN YNTTPPMLDS DGSFFLYSKL TVDKSRWQQG NIFSCSVMHE ALHNEFTQKS K (SEQ ID NO: 36) In some embodiments, the human IgG3 constant region is modiﬁed at amino acid Asn297 (Boxed, Kabat Numbering) to prevent to glycosylation of the antibody, e.g, Asn297A1a (N297A). In some embodiments, the human IgG3 nt region is modiﬁed at amino acid 435 to extend the half—life, e.g., Arg43 5His (R43 5H) (EU index of Kabat et a! 1991 ces of Proteins ofImmunological Interest).

In some embodiments, the constant region of an D47 antibody provided herein is of human IgG4 isotype, having an amino acid sequence: ASTKGPSVFP LAPCSRSTSF STAALGCLVK DYFPRPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPECP APEFEGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PRﬁﬁQEESTY RVVSVLTVLH QDWLNGKEYK GLBS S ﬁKTTSKAK GQPQEPQVYT EMTK NQVSLTCLVK GEYBSDIAV: WESNGQPENN YKTTPPVLDS JGSEFLYSRL TVDKSRWQEG NVFSCSVMHE TQKS LSLSLGK (SEQ ID NO: 37) In some embodiments, the human IgG4 constant region is modiﬁed within the hinge region to prevent or reduce strand exchange, e.g., Ser228Pro ($228P). In other embodiments, the human IgG4 constant region is modiﬁed at amino acid 235 to alter Fc receptor interactions, e.g., Leu235G1u (L23 5E). In some embodiments, the human IgG4 constant region 2015/067642 is modiﬁed within the hinge and at amino acid 235, e.g., Ser228Pro and Leu235Glu (S228P/L23 5B). In some embodiments, the human IgG4 constant region is modiﬁed at amino acid Asn297 (Kabat Numbering) to prevent to glycosylation of the dy, e.g., Asn297Ala (N297A). In some embodiments of the invention, the human IgG4 constant region is modiﬁed at amino acid positions Ser228, Leu235, and Asn297 (e.g., S228P/L23 5E/N297A). (EU index of Kabat et a] 1991 Sequences ofProteins ofImmunological Interest). In other embodiments of the invention, the dy is of human IgG4 subclass and lacks glycosylation. In these embodiments the glycosylation can be eliminated by mutation at position 297 (Kabat numbering), for example N297A. In other embodiments, the glycosylation can be eliminated by production of the antibody in a host cell that lacks the ability for post-translational ylation, for example a bacterial or yeast derived system or a modiﬁed mammalian cell expression .

In some embodiments, the human IgG constant region is modiﬁed to alter antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC), e.g., the amino acid modiﬁcations described in Natsume et al., 2008 Cancer Res, 68(10): 3863-72; Idusogie et al., 2001 J Immunol, 166(4): 2571-5; Moore et al., 2010 mAbs, 2(2): 181- 189, Lazar et al., 2006 PNAS, 103(11): 4005—4010, Shields et al., 2001 JBC, 276(9): 6591— 6604; hagen et al., 2007 Cancer Res, 67(18): 8882—8890; Stavenhagen et al., 2008 Advan.

Enzyme Regul., 48: 152-164, Alegre et al, 1992 J Immunol, 148: 3461-3468, Reviewed in Kaneko and Niwa, 2011 gs, 25(1): 1-11.

In some embodiments, the human IgG constant region is modiﬁed to induce heterodimerization. For e, having an amino acid modiﬁcation within the CH3 domain at Thr3 66, which when ed with a more bulky amino acid, e.g., Try (T3 66W), is able to preferentially pair with a second CH3 domain having amino acid modiﬁcations to less bulky amino acids at positions Thr3 66, Leu3 68, and Tyr407, e.g., Ser, Ala and Val, respectively (T3 66S/L3 68A/Y407V). Heterodimerization via CH3 modiﬁcations can be further stabilized by the introduction of a disulﬁde bond, for example by ng Ser354 to Cys (S3 54C) and Y349 to Cys (Y3 49C) on opposite CH3 domains (Reviewed in , 2001 Journal of Immunological Methods, 248: 7—15).

In other aspects, the antibody lacks glycosylation, but is not modiﬁed at amino acid Asn297 (Kabat numbering). In these embodiments the glycosylation can, for example, be eliminated by production of the antibody in a host cell that lacks a post-translational glycosylation capacity, for e a bacterial or yeast derived system or a modiﬁed mammalian cell expression system. In certain aspects, such a system can be a CF expression system.

] In certain embodiments, an anti-CD47 antibody described herein or an antigen- binding fragment thereof comprises amino acid ces with certain percent identity relative to a al antibody.

The determination of percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a atical algorithm. A miting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA. 4 2268, modiﬁed as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA. 9025873 5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul ez‘ al., 1990, J.

Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program parameters set, e.g, to score 50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 2523389 3402. Alternatively, PSI BLAST can be used to perform an iterated search which detects distant relationships between les (Id) When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g, of XBLAST and NBLAST) can be used (see, e.g, National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov). Another preferred, non limiting example of a atical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11 17, Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.

When utilizing the ALIGN program for comparing amino acid ces, a PAM120 weight residue table, a gap length y of 12, and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined using techniques r to those described above, with or without allowing gaps. In ating percent identity, typically only exact s are counted.

In certain embodiments, an antibody described herein or an antigen-binding fragment thereof ses a VL domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 12, wherein the antibody cally binds to CD47. In certain embodiments, an antibody described herein or an antigen-binding fragment thereof comprises a VL domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 12, wherein the dy speciﬁcally binds to CD47, and wherein the antibody comprises CDRs (e.g., VL CDRs 1—3) that are identical to the CDRs (e.g., VL CDRs 1—3) of SEQ ID NO: 12 (e.g., SEQ ID NO: 17—19).

In certain embodiments, an antibody described herein or an antigen-binding nt thereof comprises a light chain having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 13, wherein the dy speciﬁcally binds to CD47. In certain embodiments, an antibody described herein or an antigen-binding fragment thereof ses a light domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 13, wherein the antibody speciﬁcally binds to CD47, and wherein the antibody ses CDRs (e.g., VL CDRs l—3) that are identical to the CDRs (e.g., VL CDRs 1—3) of SEQ ID NO: 13 (e.g., SEQ ID NO: 17—19).

In certain embodiments, an antibody described herein or an antigen-binding fragment thereof comprises a VH domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1, wherein the antibody speciﬁcally binds to CD47 and wherein the anti-CD47 antibody, when produced using a cell-free sion system, has a higher antibody expression titer or yield compared to the parental antibody when produced in the CF expression system. In certain embodiments, an antibody described herein or an antigen-binding fragment thereof ses a VH domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1, wherein the dy speciﬁcally binds to CD47, and wherein the antibody ses CDRs (e.g., VL CDRs 1-3) that are identical to the CDRs (e.g., VL CDRs 1-3) of SEQ ID NO: 1 (e.g., SEQ ID NO: 14-16).

In certain embodiments, an antibody described herein or an antigen-binding fragment thereof comprises a light chain having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 2, wherein the antibody speciﬁcally binds to CD47 and wherein the anti-CD47 antibody, when produced using a ree expression system, has a higher antibody sion titer or yield cmmmwKHMpmmmMmmwyWMnmmmwdmﬂwCFammﬁmwwwm.human embodiments, an antibody described herein or an antigen-binding nt thereof comprises a heavy domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 2, wherein the antibody speciﬁcally binds to CD47, and wherein the antibody ses CDRs (e.g., VL CDRs 1-3) that are cal to the CDRs (e.g., VL CDRs 1-3) of SEQ ID NO: 2 (e.g., SEQ ID NO: 17-19).

In certain embodiments, an antibody described herein or an antigen-binding fragment thereof comprises a light chain having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3, wherein the antibody speciﬁcally binds to CD47 and wherein the anti-CD47 antibody, when produced using a cell-free expression system, has a higher antibody expression titer or yield compared to the parental antibody when produced in the CF expression system. In certain embodiments, an antibody described herein or an antigen-binding fragment thereof comprises a heavy domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3, wherein the antibody speciﬁcally binds to CD47, and n the antibody comprises CDRs (e.g., VL CDRs 1-3) that are identical to the CDRs (e.g., VL CDRs 1-3) of SEQ ID NO: 3 (e.g., SEQ ID NO: 17-19).

In certain embodiments, an antibody bed herein or an antigen-binding fragment thereof comprises a light chain having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence ty to the amino acid sequence of SEQ ID NO: 4, wherein the antibody speciﬁcally binds to CD47 and wherein the anti-CD47 antibody, when ed using a cell-free sion system, has a higher antibody sion titer or yield compared to the parental antibody when produced in the CF expression system. In certain embodiments, an antibody bed herein or an antigen-binding fragment thereof comprises a heavy(knnaﬁihaxdngeuleast8096,atleast8596,atleast9096,atleast9596,atleast9896,orat least 99% sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein the antibody speciﬁcally binds to CD47, and wherein the antibody comprises CDRs (e.g., VL CDRs 1-3) that are identical to the CDRs (e.g., VL CDRs 1-3) of SEQ ID NO: 4 (e.g., SEQ ID NO: 17-19).

In certain aspects, anti-CD47 antibodies provided herein exhibit one or more desirable characteristics, such as, by way of non—limiting example, blocking of the interaction between CD47 and its ligand SIRPOL and/or promoting (e.g., inducing or increasing) ytosis, t promoting (e. g., inducing or increasing) hemagglutination of erythrocytes, as well as anti-tumor activity. For e, anti-CD47 antibodies provided herein block at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95%, or at least 99% of the interaction between CD47 and SIRPOL as compared to the level of interaction between CD47 and SIRPOL in the absence of the anti—CD47 antibody described herein.

In c s, anti-CD47 antibodies described herein promote (e..g, induce or increase) phagocytosis of cells, e. g., CD47-expressing cells (e.g., CCRF-CEM cells), for example, by macrophages. In one aspect, the level of phagocytosois in the presence of anti- CD47 antibodies described herein is increased by at least 5%, at least 10%, at least 20%, at least %, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, at least 150%, at least 200%, compared to the level of aphagocytosis in the presence of anti-CD47 antibodies described herein.

In speciﬁc aspects, anti—CD47 antibodies described herein do not promote (e..g, induce or increase), or cause a signiﬁcant level of, agglutination of cells, e.g., D47 antibodies described herein do not promote (e.. g, induce or increase), or cause a signiﬁcant level of, hemagglutination of red blood cells. In one aspect, the level of agglutination in the presence of anti-CD47 dies described herein is d by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% ed to the level of agglutination in the ce of anti-CD47 antibodies known to induce agglutination, such as MCA911 mouse anti-human CD47 antibody (BRIC126). In some aspects, anti-CD47 dies described herein do not promote (e.g., induce or increase), or cause a signiﬁcant level of, agglutination if the level of agglutination in the presence of anti- CD47 antibodies described herein is reduced by at least 5%, at least 10%, at least 20%, at least %, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% compared to the level of agglutination in the presence of existing anti-CD47 antibodies known to induce agglutination, such as MCA911 mouse anti-human CD47 antibody (BRIC126).

Anti-CD47 antibodies described herien also include monoclonal antibodies that speciﬁcally bind CD47, wherein the antibody does not promote (e.g., induce or increase), or cause a cant level of, ination, e. g., red blood cell hemagglutination (“RBC hemagglutination”).

In some aspects, the level of RBC depletion is determined by measuring the RBC count in a subject after administration of a ent, e. g., an anti-CD47 antibody described herein. In some embodiments, anti-CD47 antibodies described herein do not promote (e. g., induce or increase), or cause a signiﬁcant level of, RBC depletion if the RBC count in a subject after administration of an anti-CD47 antibody described herein is within the range of a normal, healthy subject. For example, the RBC count for a normal, healthy male human is about 4.7 to about 6.1 million cells per microliter of blood . For example, the RBC count for a , healthy female human is 4.2 to about 5.4 million cells per microliter of blood sample. In some aspects, anti-CD47 antibodies described herein do not promote (e.g., induce or increase), or cause a signiﬁcant level of, RBC depletion if the RBC count in a subject after administration (e.g., 5 min, 10 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 12 h, 24 h, 2 days, 4 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or more) of an D47 antibody described herein is at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 99.5% of the RBC count prior to administration. In speciﬁc aspects,anti-CD47 antibodies described herein do not promote (e. g., induce or increase), or cause a signiﬁcant level of, RBC ion if the RBC count in a subject after stration (5 min, 10 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 12 h, 24 h, 2 days, 4 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or more) of an anti47 antibody described herein is at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 99.5% of the RBC count in a subject after administration of a placebo treatment (e.g., vehicle). RBC counts are determined by rd methods in the art.

In speciﬁc aspects, anti-CD47 antibodies described herein do not promote (e.g., induce or increase), or cause a cant level of, platelet depletion. For example, stration of an D47 antibody described herein leads to a percentage of platelets remaining of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, Also, anti-CD47 antibodies described herien include but are not limited to antibodies that do not bind to, or have a low binding afﬁnity to, a Fcy receptor (FcyR). For example, the constant region of an anti-CD47 antibody, e. g., when produced using a CF expression system, has a lower binding afﬁnity to a FcyR than the constant region of an anti-CD47 dy, e.g., when produced using a host cell (e. g., CHO cells) expression system.

Those skilled in the art will recognize that it is possible to quantitate, without undue experimentation, the level of agglutination, e.g., the level of hemagglutination of RBCs. For example, those skilled in the art will recognize that the level of hemagglutination is ascertained by measuring the area of an RBC dot after performing a hemagglutination assay in the presence of anti-CD47 dies described, as described in the Examples below. In some cases, the area of the RBC dot in the presence of anti-CD47 antibody described heIien is compared to the area of the RBC dot in the e of an anti—CD47 antibody, e. g., in the presence of zero hemagglutination. In this manner, hemagglutination is quantiﬁed relative to a baseline l.

A larger RBC dot area corresponds to a higher level of hemagglutination. Alternatively, ometry of the RBC dot may also be utilized to quantitate lutination.

] Those skilled in the art will recognize that it is possible to quantitate, without undue mentation, the level of RBC depletion. For example, those skilled in the art will ize that the level ofRBC depletion is ascertained, e.g., by measuring the RBC count (i.e., the total number of RBCs in a sample of blood), e. g., by using a cell counter or a hemacytometer. Those of skill in the art will recognize that the RBCs in a sample of blood can optionally be isolated by fractionating whole blood using, e.g., centrifugation, prior to counting. In some cases, the RBC count in the presence of an anti-CD47 antibody described herein is compared to the RBC count in the absence of the CD47 antibody, e.g., in the presence of zero RBC ion. In this manner, the level of RBC depletion is normalized relative to a baseline l.

In speciﬁc aspects, anti-CD47 antibodies provided herein exhibit inhibitory activity, for example by inhibiting CD47 sion (e.g., inhibiting cell surface expression of CD47), activity, and/or signaling, or by interfering with the interaction between CD47 and SIRPoc. In certain aspects, anti-CD47 antibodies provided herein completely or partially reduce or otherwise modulate CD47 expression or activity upon binding to, or otherwise interacting with, CD47, e.g,, a human CD47. The reduction or modulation of a biological function of CD47 is te, signiﬁcant, or partial upon interaction between the antibodies and the human CD47 polypeptide and/or e. Anti-CD47 antibodies bed hereinare considered to completely inhibit CD47 expression or activity when the level of CD47 expression or activity in the presence of the antibody is decreased by at least 95%, e.g., by 96%, 97%, 98%, 99% or 100% as compared to the level of CD47 sion or activity in the e of interaction, e.g., g, with the antibody described herein. In a particular aspect, D47 antibodies are considered to signiﬁcantly inhibit CD47 expression or activity when the level of CD47 expression or activity in the presence of the CD47 dy is decreased by at least 50%, e. g., 55%, 60%, 75%, 80%, 85% or 90% as compared to the level of CD47 expression or activity in the absence of binding with a CD47 antibody described herein. In certain aspects, anti-CD47 antibodies are considered to partially inhibit CD47 expression or activity when the level of CD47 expression or activity in the presence of the antibody is decreased by less than 95%, e. g., 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85% or 90% as compared to the level of CD47 expression or activity in the absence of interaction, e.g., binding, with an antibody described herein.

In ular aspects, anti-CD47 antibodies provided herein comprise one or more non-natural amino acid residues at site—speciﬁc positions. See, e.g., US. Application Publication No. US 2014/0046030 A1, which is orated herein by reference in its entirety. In speciﬁc aspects, non-natural amino acid residues at site c positions has advantages for antibody tion yield, solubility, binding afﬁnity, and/or activity. Non—limiting examples of non- natural amino acids have been described, see, e.g., US. Application Publication No. US 2014/0066598 A1.

In a particular aspect, provided herein are anti-CD47 antibodies conjugated to a conjugation moiety or an agent such as a label or toxin. A conjugation moiety can be any conjugation moiety deemed useful to one of skill in the art. For instance, a conjugation moiety can be a polymer, such as polyethylene , that can improve the stability of the dy in vitro or in vivo. A conjugation moiety can have therapeutic activity, thereby yielding an antibody-drug conjugate. A conjugation moiety can be a lar payload that is harmful to target cells. A conjugation moiety can be a label useful for detection or diagnosis. In certain aspects, a conjugation moiety is linked to the antibody via a direct nt bond. In certain aspects, a ation moiety is linked to the antibody via a linker. In particular aspects, a conjugation moiety or a linker is attached via one of the non-natural amino acids of an anti-CD47 antibody. Exemplary conjugation moieties and linkers have been described, e.g., see US.

Application Publication No. US2014/0046030 Al, which is orated herein by reference in its entirety. .2 Antibody Production dies or an n-binding nts bed herein that immunospeciﬁcally bind to CD47 (e.g., ECD of human CD47) can be ed by any method known in the art, for example, by al synthesis or by recombinant expression techniques (e.g, CF expression systems).

Such methods can employ conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modiﬁcation, nucleic acid ization, and related ﬁelds within the skill of the art. These techniques are described, for example, in the references cited herein and are fully explained in the literature. See, e.g.,, Maniatis el al. (1982) Molecular Cloning: A Laboratog Manual, Cold Spring Harbor Laboratory Press, Sambrook et a]. , Molecular Cloning: A Laboratog Manual, Second Edition, Cold Spring Harbor Laboratory Press, Sambrook et a]. (2001) Molecular Cloning: A Laboratog; Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Ausubel el al., Current Protocols in Molecular My, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates) Gait (ed) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press, Birren et al. (eds) (1999) Genome Analysis: A Laboratogy Manual, Cold Spring Harbor Laboratory Press.‘ ] In speciﬁc aspects, anti-CD47I antibodies as provided herein can be produced using a CF expression system, for example, a CF expression system as known in the art, and, for example, as described in the Examples below. For example, CF sion systems can include cell-free extracts, such as S30 cell-free extracts, with Dst, and 20 amino acids (e.g., natural or non-natural), and ally, one or more of iodoacetamide, magnesium glutamate, ammonium glutamate, mM potassium glutamate, sodium pyruvate, AMP, GMP, UMP, and CMP, sodium oxalate, putrescine, spermidine, potassium phosphate, T7 RNAP, and oxidized (GSSG) glutathione. Heavy chain ds and light chain plasmids are added accordingly to the CF extract composition for polypeptide tion and puriﬁcation.

In some aspects, the CF expression system is an in vitro transcription and translation system as described in Yin et al., mAbs, 2012, 4:217-225, incorporated by reference in its -3 2- entirety. In some aspects, the cell-free system utilizes a ree extract from a eukaryoctic cell or from a prokaryotic cell. In some aspects, the prokaryotic cell is E. coli.

In particular embodiments, the CF expression system can utilize a system as described in US Application Publication No. US 2014/0315245, which is hereby incorporated by reference in its ty. For example, the CF sion system can comprise a bacterial extract having an ive phosphorylation system and components necessary for cell free protein synthesis and, in certain embodiments, can further comprise an exogenous protein chaperone, e.g., a protein disulﬁde isomerase (PDI), or a peptide-prolyl cis-trans isomerase. In speciﬁc embodiments, the PDI is a member of the Dsb (disulﬁde bond formation) family of E. coli, for example, DsbA or Dst. In certain embodiments, the CF expression system comprises a cell extract of E. coli strain SBDG028, 1, or SBDG044, as described in US Application Publication No. US 2014/0315245, which can, for example, be prepared according to Zawada et al., Biotechnology and Bioengineering (2011) vol. 108, No. 7.

In particular s, anti-CD47 antibodies provided herein comprise amino acid ations that allow for antibody production using CF expression systems better than the parental antibodies. In a particular aspect, anti-CD47 antibodies provided herein which are produced using CF expression systems are sylated.

Monoclonal dies can, for example, be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display logies, or a ation thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow el al., Antibodies: A Laboratog Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988), Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, NY, 1981), The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. For example, monoclonal antibodies can be produced recombinantly from host cells engineered to express an antibody bed herein (e.g., anti- CD47 antibody comprising the CDRs of any one of antibodies Ab235-Ab255) or a fragment thereof, for example, a light chain and/or heavy chain of such an antibody. r, the antibodies described herein or antigen-binding fragments thereof can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. Examples of phage display methods that can be used to make the antibodies described herein include those disclosed in an el al., 1995, J. l. Methods 182:41-50, Ames et 61]., 1995, J. Immunol. Methods 184:177-186, Kettleborough et al., 1994, Eur. J. l. 24:952-958; Persic et al., 1997, Gene 187:9-18, Burton ez‘ 61]., 1994, Advances in Immunology 57: 191-280, PCT Application No. PCT/GB91/Ol 134; International Publication Nos. WO 09, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/1 1236, WO 95/15982, WO 95/20401, and 3844; and US. Patent Nos. ,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 047, 5,571,698, ,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108.

Antibodies bed herein can, for example, include chimeric dies. A chimeric antibody is a molecule in which different ns of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody can contain a variable region of a mouse or rat monoclonal antibody fused to a constant region of a human antibody.

Methods for producing chimeric antibodies are known in the art. See, e.g, Morrison, 1985, Science 229: 1202; Oi e1al., 1986, BioTechniques 4:214; Gillies er al., 1989, J. Immunol.

Methods 125191—202, and US. Patent Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415.

Antibodies or antigen-binding nts ed using techniques such as those described herein can be isolated using standard, well known techniques. For example, antibodies or antigen-binding fragments can be suitably separated from, e.g., culture medium, ascites ﬂuid, serum, cell lysate, synthesis reaction material or the like by conventional immunoglobulin puriﬁcation procedures such as, for example, protein A-Sepharose,_hydroxylapatite chromatography, gel electrophoresis, dialysis, or afﬁnity chromatography. As used herein, an ted” or “puriﬁed” antibody is substantially free of cellular material or other proteins from the cell or tissue source from which the antibody is derived, or ntially free of chemical precursors or other chemicals when chemically synthesized, or from the components of the CF expression system used to produce the dies.

Antibodies bed herein include antibody fragments which ize speciﬁc CD47 antigens and can be generated by any technique known to those of skill in the art. For example, Fab and F(ab’)2 fragments described herein can be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab’)2 nts). A Fab fragment corresponds to one of the two identical arms of an antibody le and contains the complete light chain paired with the VH and CH1 domains of the heavy chain. A F(ab’)2 fragment contains the two antigen—binding arms of an antibody molecule linked by disulﬁde bonds in the hinge region. Alternatively, antibody fragments bed herein can routinely be ed via well known recombinant expression techniques. See, e.g., PCT publication No. WO 92/22324, MullinaX el al., 1992, BioTechniques 12(6):864-869; Sawai er al., 1995, AJRI34126-34; and Better et al, 1988, e 240: 1041- 1043.

Antibodies described herein can, for example, include humanized antibodies, e.g., deimmunized or composite human antibodies. A humanized antibody can comprise human constant region sequences. In certain embodiments, a humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgGl, Ing, IgG3 and IgG4. In certain embodiments, a humanized antibody can comprise kappa or lambda light chain constant sequences.

Humanized antibodies can be produced using a variety of ques known in the art, including but not limited to, CDR—grafting (European Patent No. EP 23 9,400, International publication No. wo 91/09967; and US. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneenng or resurfacing (European Patent Nos. EP 592,106 and EP 519,596, Padlan, 1991, Molecular Immunology 28(4/5):489-498, Studnicka el al., 1994, Protein Engineering 7(6):805- 814; and Roguska et al., 1994, PNAS 91:969-973), chain shufﬂing (US. Patent No. 332), and techniques disclosed in, e.g., US. Pat. No. 6,407,213, US. Pat. No. 5,766,886, WO 9317105 Tan et 61]., J. Immunol. 169: 1 1 19 25 (2002), Caldas et 61]., Protein Eng. 13(5):353-60 (2000), Morea er al., Methods 20(3):267 79 (2000), Baca el al., J. Biol. Chem. 272(16): 10678-84 (1997), Roguska el‘ al., Protein Eng. 9(10):895 904 , Couto el‘ al., Cancer Res. 55 (23 Supp):5973 s- 5977s (1995), Couto el al., Cancer Res. 55(8): 1717—22 , Sandhu JS, Gene 150(2):409-10 (1994), and Pedersen et 61]., J. Mol. Biol. 235(3):959-73 (1994). See also US.

Patent Pub. No. US 2005/0042664 A1 (Feb. 24, 2005), each of which is incorporated by reference herein in its entirety.

Antibodies described herein can, for example, be peciflc, e.g., iflc, antibodies. Methods for making peciflc (e. g, bispeciflc antibodies) have been described, see, for example, US. Patent Nos. 7951917, 6, 8227577, 5837242, 0, 5869620, 6132992, and 8586713.

Single domain antibodies, for example, dies lacking the light chains, can be ed by methods well-known in the art. See Riechmann et al., 1999, J. l. 23 1 125-3 8; Nuttall el al., 2000, Curr. Pharm. Biotechnol. l(3):253-263, Muylderman, 2001, J. Biotechnol. 74(4):277302; US. Patent No. 6,005,079; and International Publication Nos. WO 94/04678, WO 94/25591, and WO 01/44301.

Human antibodies can be produced using any method known in the art. For example, well known transgenic mice which are ble of expressing functional endogenous murine immunoglobulins, but which can express human globulin genes, can be used.

Alternatively, for example, phage display techniques, described above, can be ed.

Moreover, in some embodiments, human antibodies can, for example, be produced using mouse— human hybridomas. For example, human peripheral blood lymphocytes transformed with Epstein-Barr virus (EBV) can be fused with mouse myeloma cells to produce mouse—human hybridomas secreting human monoclonal dies, and these mouse—human hybridomas can be screened to determine ones which secrete human monoclonal antibodies that immunospeciflcally bind to a target antigen (e.g., ECD of human CD47). Such methods are known and are bed in the art, see, e.g., Shinmoto et al., Cytotechnology, 2004, 46: 19-23, Naganawa et al., Human dies, 2005, 1427—3 1.

] Antibody variable domains with the desired binding speciﬁcities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the ﬁrst heavy-chain constant region (CH1) containing the site ary for light-chain binding present in at least one of the fusions.

DNAs encoding the immunoglobulin chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispeciflc antibodies see, for example, Suresh et al., Methods in Enzymology, 121 :210 (1986).

According to another approach bed in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the ﬁrst dy molecule are replaced with larger side chains -3 6- (e.g. tyrosine or tryptophan). Compensatory “cavities” of cal or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This es a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

Bispeciflc antibodies can be prepared as full length antibodies or dy fragments (e.g. 2 bispeciflc antibodies). Techniques for generating bispecif1c antibodies from antibody fragments have been described in the literature. For example, iﬁc antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab’)2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and t intermolecular disulﬁde formation. The Fab’ fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab’-TNB tives is then reconverted to the Fab’-thiol by ion with mercaptoethylamine and is mixed with an equimolar amount of the other Fab’-TNB derivative to form the bispeciflc antibody. The ific antibodies produced can be used as agents for the selective immobilization of enzymes. .2.1 Polynucleotides, Cells and Vectors In certain aspects, provided herein are polynucleotides comprising a nucleotide ce encoding an antibody described herein or a fragment f (e.g., a variable light chain region and/or le heavy chain region) that immunospeciflcally binds to a CD47 antigen, and vectors, e.g., vectors comprising such polynucleotides for recombinant expression in host cells (e.g., E. coli and mammalian cells). In certain aspects, provided herein are cells (e.g., host cells). Also provided herein are methods of making the antibodies and antigen-binding fragments described herein.

In certain aspects, provided herein are polynucleotides compiising a nucleotide sequence encoding the light chain or heavy chain of an antibody described . In certain embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding the light chain and heavy chain of an antibody described herein. The cleotides can comprise nucleotide sequences encoding a light chain comprising the VL FRs and CDRs of dies described herein. The polynucleotides can comprise nucleotide sequences encoding a heavy chain comprising the VH FRs and CDRs of antibodies bed herein. In speciﬁc embodiments, a polynucleotide described herein encodes a VL chain region of SEQ ID NO: 13 or SEQ ID NO: 13 t amino acid M at the N—terminus. In speciﬁc embodiments, a polynucleotide described herein encodes a VH chain region of any one of SEQ ID NOs: 5-11 -22. In speciﬁc embodiments, a polynucleotide described herein encodes a light chain comprising the amino acid sequence of SEQ ID NO: 13. In speciﬁc embodiments, a polynucleotide bed herein encodes a heavy chain comprising the amino acid ce of any one of SEQ ID NOs: 5-11.

In speciﬁc embodiments, a polynucleotide described herein comprises a nucleotide sequence provided in Table 2 encoding a light chain or a heavy chain of an anti-CD47 antibody provided here.

Table 2: Anti-CD47 antibody VH and VL nucleotide sequences Nucleotide sequences D47 IgGl- 13m HC: ATGCAAGTCCAATTGGTCCAGAGCGGTGCGGAAGTCAAGAAACCGGGTGCAAG CGTCAAAGTTTCGTGCAAGGCGAGCGGTTTCAATATCAAAGACTATTATCTGCA TCGTCAGGCTCCGGGCCAAGGCCTGGAGTGGATGGGTTGGATCGATCC GGACCAGGGCGACACGGAGTACGCTCAGAAGCTGCAGGGTCGTGTTACCATGA CCACCGACACCAGCACGAGCACCGCGTACATGGAACTGCGCTCTCTGCGTTCGG ATGATACCGCGGTGTACTATTGCAACGCCGCGTACGGTAGCAGCAGCTATCCGA TGGATTATTGGGGTCAAGGCACTACGGTGACTGTCAGCAGCGCCAGCACCAAG GGCCCGTCCGTGTTTCCGCTGGCGCCAAGCTCCAAGAGCACCAGCGGTGGCACG GCCGCACTGGGTTGTCTGGTAAAAGATTACTTTCCTGAGCCGGTGACCGTGAGC TGGAATTCAGGTGCACTGACGTCCGGCGTTCACACGTTCCCGGCAGTTCTGCAG AGCTCCGGTTTGTACAGCCTGTCTAGCGTCGTGACGGTGCCGAGCAGCAGCCTG GGTACCCAAACCTACATTTGCAACGTTAACCATAAGCCGAGCAATACCAAAGTT GACAAGAAAGTCGAACCTAAGAGCTGTGATAAGACGCATACCTGTCCGCCGTG CCCGGCACCGGAACTGTTGGGCGGTCCGAGCGTGTTCCTGTTTCCGCCGAAGCC GAAAGATACCCTGATGATTAGCCGCACCCCTGAGGTGACGTGCGTGGTTGTGGA CGTTAGCCATGAGGATCCAGAGGTCAAATTCAATTGGTATGTCGATGGTGTTGA GGTTCACAATGCCAAGACCAAACCGCGTGAAGAACAGTACAATAGCACCTACC TGAGCGTGCTGACGGTCCTGCACCAGGACTGGCTGAACGGCAAAGAG TACAAGTGTAAGGTCAGCAACAAGGCGCTGCCAGCACCGATTGAAAAGACCAT TTCTAAAGCGAAAGGTCAGCCGCGTGAGCCGCAAGTCTATACCCTGCCGCCGTC GCGCGATGAGCTGACTAAAAACCAGGTTAGCCTGACGTGCCTGGTGAAAGGTTT CTACCCGAGCGACATCGCGGTGGAGTGGGAGAGCAACGGTCAACCGGAGAATA ACTACAAAACCACCCCACCGGTCTTGGACTCCGATGGCAGCTTCTTTCTGTACTC TAAACTGACCGTTGACAAAAGCCGTTGGCAACAGGGCAACGTCTTTAGCTGCAG CGTGATGCATGAGGCTCTGCACAACCACTACACCCAAAAATCCCTGAGCCTGAG CCCGGGTAAGTAA Anti-CD47 3mZ HC: ATGCAAGTCCAATTGGTCCAGAGCGGTGCGGAAGTCAAGAAACCGGGTGCAAG CGTCAAAGTTTCGTGCAAGGCGAGCGGTTTCAATATCAAAGACTATTATCTGCA CTGGGTTCGTCAGGCTCCGGGCCAAGGCCTGGAGTGGATGGGTTGGATCGATCC GGACCAGGGCGACACGGAGTACGCTCAGAAGCTGCAGGGTCGTGTTACCATGA CCACCGACACCAGCACGAGCACCGCGTACATGGAACTGCGCTCTCTGCGTTCGG ATGATACCGCGGTGTACTATTGCAACGCCGCGTACGGTAGCAGCAGCTATCCGA TGGATTATTGGGGTCAAGGCACTACGGTGACTGTCAGCAGCGCCAGCACCAAG TCCGTGTTTCCGCTGGCGCCAAGCTCCAAGAGCACCAGCGGTGGCACG GCCGCACTGGGTTGTCTGGTAAAAGATTACTTTCCTGAGCCGGTGACCGTGAGC TGGAATTCAGGTGCACTGACGTCCGGCGTTCACACGTTCCCGGCAGTTCTGCAG GGTTTGTACAGCCTGTCTAGCGTCGTGACGGTGCCGAGCAGCAGCCTG GGTACCCAAACCTACATTTGCAACGTTAACCATAAGCCGAGCAATACCAAGGTT GACAAAAAAGTTGAACCGAAATCTTGTGATAAAACTCATACCTGTCCGCCGTGC CCGGCGCCTGAGCTGTTGGGTGGTCCGTCGGTCTTTCTGTTCCCGCCGAAGCCG AAAGACACCCTGATGATTAGCCGCACCCCGGAAGTTACGTGCGTCGTCGTGGAT GTCAGCCACGAGGACCCGGAGGTTAAGTTCAATTGGTATGTCGATGGCGTGGA GGTTCACAACGCGAAAACCAAGCCGCGTGAGGAACAATACAATAGCACGTATC TGAGCGTGCTGACCGTGCTGCACCAAGATTGGCTGAATGGTAAAGAA TACAAGTGCAAAGTGAGCAACAAGGCATTGCCGGCACCGATCGAAAAGACGAT CAGCAAAGCGAAAGGCCAACCGCGTGAACCGCAGGTCTATACCCTGCCGCCGA GCCGTGAAGAAATGACGAAAAACCAAGTTAGCCTGACCTGTCTGGTGAAGGGC TWTTACCCGAGCGACATCGCCGTCGAGTGGGAGTCTAACGGCCAGCCGGAAAA CAATTACAAAACCACGCCGCCAGTCCTGGACAGCGACGGTAGCTTCTTTCTGTA TAGCAAGCTGACCGTCGATAAAAGCCGTTGGCAGCAGGGTAATGTGTTCAGCTG CAGCGTTATGCATGAGGCGCTGCACAATCACTATACCCAGAAATCCTTGTCCCT GTCCCCGGGTAAGTAA Anti-CD47 IgGl-Sm HC: ATGCAAATGCAATTGGTACAAAGCGGTGCGGAAGTAAAGAAACCGGGTTCGTC GGTAAAGGTTAGCTGTAAAGCTTCTGGCTTCAATATCAAGGATTACTATCTGCA CTGGGTGCGTCAGGCGCCAGGTCAGGCCTTGGAATGGATGGGCTGGATTGACCC GGATCAAGGTGACACCGAATATGCCCAAAAGTTTCAGGGTCGTGTGACCATCAC CCGTGACCGTAGCACCTCCACCGCATATATGGAGCTGCGTAGCCTGCGCAGCGA AGATACTGCGGTGTATTACTGCAATGCGGCCTATGGTAGCAGCTCCTATCCGAT GGATTACTGGGGCCAGGGTACCACGGTGACGGTTAGCAGCGCAAGCACCAAGG GCCCGAGCGTTTTCCCTCTGGCGCCGAGCAGCAAAAGCACTAGCGGCGGTACG GCAGCCCTGGGTTGTCTGGTTAAAGATTACTTTCCGGAACCGGTTACCGTGTCCT GGAACTCTGGCGCGCTGACCAGCGGTGTTCACACGTTTCCGGCGGTTCTGCAGA GCAGCGGTCTGTATTCTTTGAGCTCCGTCGTCACCGTCCCGTCTAGCTCGCTGGG CACGCAGACGTACATCTGCAATGTTAACCATAAGCCGAGCAATACCAAAGTTG ACAAGAAAGTCGAACCTAAGAGCTGTGATAAGACGCATACCTGTCCGCCGTGC CCGGAACTGTTGGGCGGTCCGAGCGTGTTCCTGTTTCCGCCGAAGCCG AAAGATACCCTGATGATTAGCCGCACCCCTGAGGTGACGTGCGTGGTTGTGGAC GTTAGCCATGAGGATCCAGAGGTCAAATTCAATTGGTATGTCGATGGTGTFEAG GTTCACAATGCCAAGACCAAACCGCGTGAAGAACAGTACAATAGCACCTACCG CGTGGTGAGCGTGCTGACGGTCCTGCACCAGGACTGGCTGAACGGCAAAGAGT ACAAGTGTAAGGTCAGCAACAAGGCGCTGCCAGCACCGATTGAAAAGACCATT TCTAAAGCGAAAGGTCAGCCGCGTGAGCCGCAAGTCTATACCCTGCCGCCGTCG CGCGATGAGCTGACTAAAAACCAGGTTAGCCTGACGTGCCTGGTGAAAGGTTTC TACCCGAGCGACATCGCGGTGGAGTGGGAGAGCAACGGTCAACCGGAGAATAA CTACAAAACCACCCCACCGGTCTTGGACTCCGATGGCAGCTTCTTTCTGTACTCT AAACTGACCGTTGACAAAAGCCGTTGGCAACAGGGCAACGTCTTTAGCTGCAG GCATGAGGCTCTGCACAACCACTACACCCAAAAATCCCTGAGCCTGAG CCCGGGTAAGTAA Anti-CD47 IgG4-13m HC: GTCCAATTGGTCCAGAGCGGTGCGGAAGTCAAGAAACCGGGTGCAAG CGTCAAAGTTTCGTGCAAGGCGAGCGGTTTCAATATCAAAGACTATTATCTGCA CTGGGTTCGTCAGGCTCCGGGCCAAGGCCTGGAGTGGATGGGTTGGATCGATCC GGACCAGGGCGACACGGAGTACGCTCAGAAGCTGCAGGGTCGTGTTACCATGA CCACCGACACCAGCACGAGCACCGCGTACATGGAACTGCGCTCTCTGCGTTCGG ATGATACCGCGGTGTACTATTGCAACGCCGCGTACGGTAGCAGCAGCTATCCGA TGGATTATTGGGGTCAAGGCACTACGGTGACTGTCAGCAGCGCCAGCACCAAG GGCCCGTCTGTGTTTCCGTTGGCACCGTGCAGCCGTAGCACTAGCGAATCCACT GCAGCGCTGGGTTGCCTGGTTAAGGACTATTTCCCGGAGCCGGTTACCGTGTCC TGGAACTCTGGCGCCCTGACCAGCGGTGTTCACACGTTTCCAGCCGTCCTGCAG AGCAGCGGTCTGTACAGCCTGAGCTCGGTGGTGACCGTTCCGAGCAGCTCTCTG GGTACCAAAACCTATACCTGTAATGTCGATCACAAACCGTCTAACACGAAGGTC GATAAACGTGTTGAAAGCAAGTACGGTCCGCCTTGTCCGCCGTGCCCGGCACCG GAGTTTCTGGGCGGTCCGTCCGTATTCCTGTTCCCGCCGAAACCGAAAGATACC TTGATGATTAGCCGTACGCCAGAGGTCACGTGCGTCGTGGTGGACGTTAGCCAA GAGGATCCGGAAGTCCAATTCAACTGGTACGTGGACGGTGTCGAGGTGCACAA TGCCAAAACCAAGCCGCGTGAAGAACAGTTTAACAGCACTTACCGCGTCGTTAG GACCGTGCTGCACCAAGATTGGCTGAATGGTAAAGAGTACAAGTGCA AGGTTAGCAATAAGGGTCTGCCGAGCAGCATCGAGAAAACCATTAGCAAGGCG AAAGGTCAACCGCGCGAGCCACAGGTCTACACGCTGCCGCCGAGCCAAGAAGA AATGACCAAAAATCAGGTTAGCCTGACTTGTCTGGTGAAAGGCTTCTACCCGAG CGATATTGCAGTTGAATGGGAGAGCAACGGCCAGCCTGAGAACAACTATAAGA CGACCCCGCCAGTGCTGGACAGCGATGGCAGCTTCTTTTTGTATTCTCGTCTGAC CGTGGACAAGTCCCGTTGGCAAGAGGGCAATGTGTTCAGCTGTTCTGTCATGCA CGAAGCGCTGCATAACCATTACACCCAGAAGTCCCTGAGCCTGTCGCTGGGCAA ATAA Anti-CD47 IgG4P-5m HC: ATGCAATTGGTACAAAGCGGTGCGGAAGTAAAGAAACCGGGTTCGTC GGTAAAGGTTAGCTGTAAAGCTTCTGGCTTCAATATCAAGGATTACTATCTGCA CTGGGTGCGTCAGGCGCCAGGTCAGGCCTTGGAATGGATGGGCTGGATTGACCC GGATCAAGGTGACACCGAATATGCCCAAAAGTTTCAGGGTCGTGTGACCATCAC CCGTGACCGTAGCACCTCCACCGCATATATGGAGCTGCGTAGCCTGCGCAGCGA AGATACTGCGGTGTATTACTGCAATGCGGCCTATGGTAGCAGCTCCTATCCGAT GGATTACTGGGGCCAGGGTACCACGGTGACGGTTAGCAGCGCAAGCACCAAGG CTGTGTTTCCGTTGGCACCGTGCAGCCGTAGCACTAGCGAATCCACTG CAGCGCTGGGTTGCCTGGTTAAGGACTATTTCCCGGAGCCGGTTACCGTGTCCT GGAACTCTGGCGCCCTGACCAGCGGTGTTCACACGTTTCCAGCCGTCCTGCAGA GCAGCGGTCTGTACAGCCTGAGCTCGGTGGTGACCGTTCCGAGCAGCTCTCTGG GTACCAAAACCTATACCTGTAATGTCGATCACAAACCGTCTAACACGAAGGTCG ATAAACGTGTTGAAAGCAAGTACGGTCCGCCTTGTCCGCCGTGCCCGGCACCGG AGTTTCTGGGCGGTCCGTCCGTATTCCTGTTCCCGCCGAAACCGAAAGATACCT TGATGATTAGCCGTACGCCAGAGGTCACGTGCGTCGTGGTGGACGTTAGCCAAG AGGATCCGGAAGTCCAATTCAACTGGTACGTGGACGGTGTCGAGGTGCACAAT GCCAAAACCAAGCCGCGTGAAGAACAGTTTAACAGCACTTACCGCGTCGTTAG CGTCCTGACCGTGCTGCACCAAGATTGGCTGAATGGTAAAGAGTACAAGTGCA AGGTTAGCAATAAGGGTCTGCCGAGCAGCATCGAGAAAACCATTAGCAAGGCG AAAGGTCAACCGCGCGAGCCACAGGTCTACACGCTGCCGCCGAGCCAAGAAGA WO 09415 AATGACCAAAAATCAGGTTAGCCTGACTTGTCTGGTGAAAGGCTTCTACCCGAG CGATATTGCAGTTGAATGGGAGAGCAACGGCCAGCCTGAGAACAACTATAAGA CGACCCCGCCAGTGCTGGACAGCGATGGCAGCTTCTTTTTGTATTCTCGTCTGAC CGTGGACAAGTCCCGTTGGCAAGAGGGCAATGTGTTCAGCTGTTCTGTCATGCA CGAAGCGCTGCATAACCATTACACCCAGAAGTCCCTGAGCCTGTCGCTGGGCAA ATAA Anti-CD47 -13m HC: ATGCAAGTCCAATTGGTCCAGAGCGGTGCGGAAGTCAAGAAACCGGGTGCAAG CGTCAAAGTTTCGTGCAAGGCGAGCGGTTTCAATATCAAAGACTATTATCTGCA CTGGGTTCGTCAGGCTCCGGGCCAAGGCCTGGAGTGGATGGGTTGGATCGATCC GGACCAGGGCGACACGGAGTACGCTCAGAAGCTGCAGGGTCGTGTTACCATGA CCACCGACACCAGCACGAGCACCGCGTACATGGAACTGCGCTCTCTGCGTTCGG CCGCGGTGTACTATTGCAACGCCGCGTACGGTAGCAGCAGCTATCCGA TGGATTATTGGGGTCAAGGCACTACGGTGACTGTCAGCAGCGCCAGCACCAAG GGCCCGTCTGTGTTTCCGTTGGCACCGTGCAGCCGTAGCACTAGCGAATCCACT GCAGCGCTGGGTTGCCTGGTTAAGGACTATTTCCCGGAGCCGGTTACCGTGTCC TGGAACTCTGGCGCCCTGACCAGCGGTGTTCACACGTTTCCAGCCGTCCTGCAG AGCAGCGGTCTGTACAGCCTGAGCTCGGTGGTGACCGTTCCGAGCAGCTCTCTG GGTACCAAAACCTATACCTGTAATGTCGATCACAAACCGTCTAACACGAAGGTC GATAAACGTGTTGAAAGCAAGTACGGTCCGCCTTGTCCGCCGTGCCCGGCACCG GAGTTTGAGGGCGGTCCGTCCGTATTCCTGTTCCCGCCGAAACCGAAAGATACC TTGATGATTAGCCGTACGCCAGAGGTCACGTGCGTCGTGGTGGACGTTAGCCAA GAGGATCCGGAAGTCCAATTCAACTGGTACGTGGACGGTGTCGAGGTGCACAA TGCCAAAACCAAGCCGCGTGAAGAACAGTTTAACAGCACTTACCGCGTCGTTAG CGTCCTGACCGTGCTGCACCAAGATTGGCTGAATGGTAAAGAGTACAAGTGCA AGGTTAGCAATAAGGGTCTGCCGAGCAGCATCGAGAAAACCATTAGCAAGGCG AAAGGTCAACCGCGCGAGCCACAGGTCTACACGCTGCCGCCGAGCCAAGAAGA AATGACCAAAAATCAGGTTAGCCTGACTTGTCTGGTGAAAGGCTTCTACCCGAG CGATATTGCAGTTGAATGGGAGAGCAACGGCCAGCCTGAGAACAACTATAAGA CGACCCCGCCAGTGCTGGACAGCGATGGCAGCTTCTTTTTGTATTCTCGTCTGAC CGTGGACAAGTCCCGTTGGCAAGAGGGCAATGTGTTCAGCTGTTCTGTCATGCA CGAAGCGCTGCATAACCATTACACCCAGAAGTCCCTGAGCCTGTCGCTGGGCAA ATAA Anti-CD47 IgG4PE-5m HC: ATGCAAATGCAATTGGTACAAAGCGGTGCGGAAGTAAAGAAACCGGGTTCGTC GGTAAAGGTTAGCTGTAAAGCTTCTGGCTTCAATATCAAGGATTACTATCTGCA CTGGGTGCGTCAGGCGCCAGGTCAGGCCTTGGAATGGATGGGCTGGATTGACCC AGGTGACACCGAATATGCCCAAAAGTTTCAGGGTCGTGTGACCATCAC CCGTGACCGTAGCACCTCCACCGCATATATGGAGCTGCGTAGCCTGCGCAGCGA AGATACTGCGGTGTATTACTGCAATGCGGCCTATGGTAGCAGCTCCTATCCGAT GGATTACTGGGGCCAGGGTACCACGGTGACGGTTAGCAGCGCAAGCACCAAGG GCCCGTCTGTGTTTCCGTTGGCACCGTGCAGCCGTAGCACTAGCGAATCCACTG CAGCGCTGGGTTGCCTGGTTAAGGACTATTTCCCGGAGCCGGTTACCGTGTCCT GGAACTCTGGCGCCCTGACCAGCGGTGTTCACACGTTTCCAGCCGTCCTGCAGA GCAGCGGTCTGTACAGCCTGAGCTCGGTGGTGACCGTTCCGAGCAGCTCTCTGG GTACCAAAACCTATACCTGTAATGTCGATCACAAACCGTCTAACACGAAGGTCG ATAAACGTGTTGAAAGCAAGTACGGTCCGCCTTGTCCGCCGTGCCCGGCACCGG AGTTTGAGGGCGGTCCGTCCGTATTCCTGTTCCCGCCGAAACCGAAAGATACCT TGATGATTAGCCGTACGCCAGAGGTCACGTGCGTCGTGGTGGACGTTAGCCAAG AGGATCCGGAAGTCCAATTCAACTGGTACGTGGACGGTGTCGAGGTGCACAAT GCCAAAACCAAGCCGCGTGAAGAACAGTTTAACAGCACTTACCGCGTCGTTAG GACCGTGCTGCACCAAGATTGGCTGAATGGTAAAGAGTACAAGTGCA AGGTTAGCAATAAGGGTCTGCCGAGCAGCATCGAGAAAACCATTAGCAAGGCG AAAGGTCAACCGCGCGAGCCACAGGTCTACACGCTGCCGCCGAGCCAAGAAGA AATGACCAAAAATCAGGTTAGCCTGACTTGTCTGGTGAAAGGCTTCTACCCGAG CGATATTGCAGTTGAATGGGAGAGCAACGGCCAGCCTGAGAACAACTATAAGA CGACCCCGCCAGTGCTGGACAGCGATGGCAGCTTCTTTTTGTATTCTCGTCTGAC CGTGGACAAGTCCCGTTGGCAAGAGGGCAATGTGTTCAGCTGTTCTGTCATGCA CGAAGCGCTGCATAACCATTACACCCAGAAGTCCCTGAGCCTGTCGCTGGGCAA ATAA Anti-CD47 IgK: ATGAACATCCAAATGACTCAATCCCCATCCGCAATGTCCGCATCCGTAGGTGAC CGCGTGACCATCACGTGCAAGGCGAGCCAGGATATTCATCGTTATCTGAGCTGG TTTCAACAGAAACCGGGCAAGGTTCCTAAGCATCTGATTTACCGCGCGAACCGC TTGGTTAGCGGTGTTCCGAGCCGTTTTAGCGGCAGCGGTTCTGGCACCGAGTTC ACCCTGACGATCTCCAGCCTGCAACCGGAAGATTTTGCGACGTACTACTGCCTG CAGTATGACGAGTTCCCGTATACCTTTGGTGGTGGTACGAAGGTGGAAATCAAA CGTACTGTGGCCGCTCCGAGCGTTTTCATTTTTCCGCCGTCGGATGAGCAATTGA AATCTGGTACCGCGAGCGTCGTTTGTCTGCTGAACAATTTCTACCCGCGTGAGG CTAAGGTGCAATGGAAGGTCGATAACGCGCTGCAGAGCGGTAATAGCCAGGAA AGCGTCACCGAACAGGATAGCAAAGACAGCACCTACTCTTTGAGCAGCACCCT GACCCTGAGCAAGGCCGACTATGAGAAACACAAAGTTTACGCATGTGAGGTCA CGCACCAGGGCCTGAGCAGCCCGGTGACCAAAAGCTTCAATCGTGGCGAATGC In certain embodiments, a polynucleotide provided herein is operably linked to a promoter for expression of such polynucleotide sequence in a host cell. In certain embodiments, the promoter is d form the genome of mammalian cells (e.g., othionein promoter) or from mammalian s (e.g., the adenovirus late er, the vaccinia virus 7.5K promoter).

In a speciﬁc embodiment, the expression of nucleotide sequences encoding antibodies described herein is regulated by a constitutive promoter, inducible promoter or tissue speciﬁc promoter.

In speciﬁc aspects, provided herein is a polynucleotide comprising a nucleotide sequence encoding an antibody comprising a light chain and a heavy chain, e.g., a separate light chain and heavy chain. With respect to the light chain, in a speciﬁc embodiment, a polynucleotide provided herein comprises a nucleotide sequence encoding a kappa light chain.

In r c embodiment, a polynucleotide provided herein comprises a nucleotide sequence encoding a lambda light chain. In yet r speciﬁc embodiment, a polynucleotide provided herein compiises a nucleotide sequence encoding an antibody described herein comprising a human kappa light chain or a human lambda light chain. For example, human constant region ces can be those bed in US. Patent No, 5,693,780.

] In a ular embodiment, a polynucleotide provided herein comprises a nucleotide sequence ng an antibody bed herein, which immunospecifically binds to a CD47 polypeptide, wherein the antibody comprises a heavy chain, wherein the constant region of the heavy chain comprises the amino acid sequence of a human gamma (y) heavy chain constant region, for example, human gamma (y) 1 heavy chain constant region, human gamma (7) 2 heavy chain constant region, human gamma (y) 3 heavy chain constant region, or human gamma (y) 4 heavy chain constant region. .2.2 Cells and s In certain aspects, provided herein are cells (e.g., host cells) sing (e.g., recombinantly) antibodies described herein (or an antigen-binding fragment f) which speciﬁcally bind to CD47 and related polynucleotides and expression vectors, for example, polynucleotides and expression vectors le for use in CF expression systems. Provided herein are vectors (e.g., expression vectors) comprising polynucleotides comprising nucleotide sequences encoding anti-CD47 antibodies or a fragment for recombinant expression in CF expression systems. In a particular aspect, provided herein are methods for producing an anti- CD47 antibody bed herein, comprising expressing such an antibody using a CF expression system, for example, under conditions resulting in improved antibody expression titer or yield.

In certain s, provided herein are s (e.g., expression vectors) comprising polynucleotides comprising tide sequences encoding anti-CD47 antibodies or a fragment for recombinant sion in host cells, e. g., in mammalian cells. Also provided herein are host cells comprising such vectors for recombinantly expressing anti-CD47 antibodies described herein (e.g., human or humanized antibody). In a particular , provided herein are methods for producing an antibody described herein, comprising expressing such an antibody using host cells, Recombinant expression of an antibody described herein (e.g., a full-length antibody, heavy and/or light chain of an antibody, or a single chain antibody described herein) that specifically binds to CD47 involves construction of an expression vector containing a polynucleotide that encodes the dy. Once a polynucleotide encoding an antibody molecule, heavy and/or light chain of an antibody, or a fragment thereof (e.g., heavy and/or light chain le domains) described herein has been obtained, the vector for the production of the antibody molecule can be produced by recombinant DNA technology using techniques well- WO 09415 known in the art. Thus, methods for preparing a protein by sing a polynucleotide containing an antibody or antibody nt (e.g., light chain or heavy chain) encoding tide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody or antibody fragment (e.g., light chain or heavy chain) coding ces and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in viva genetic recombination. Also provided are replicable vectors comprising a nucleotide sequence encoding an antibody le described herein, a heavy or light chain of an antibody, a heavy or light chain variable domain of an dy or a fragment f, or a heavy or light chain CDR, operably linked to a promoter. Such vectors can, for example, include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g, International Publication Nos. WO 86/05807 and WO 89/01036; and US. Patent No. ,122,464) and le domains of the antibody can be cloned into such a vector for expression of the entire heavy, the entire light chain, or both the entire heavy and light chains.

An expression vector can be transferred to a cell (e.g, host cell) by conventional techniques and the resulting cells can then be ed by conventional techniques to produce an antibody described herein or a fragment thereof. Thus, provided herein are host cells containing a polynucleotide encoding an antibody described herein or nts thereof, or a heavy or light chain thereof, or fragment thereof, or a single chain antibody described herein, operably linked to a promoter for expression of such sequences in the host cell. In certain embodiments, for the expression of double-chained antibodies, vectors encoding both the heavy and light chains, individually, can be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below. In certain embodiments, a host cell contains a vector comprising a polynucleotide encoding both the heavy chain and light chain of an antibody described , or a fragment thereof. In speciﬁc embodiments, a host cell ns two different vectors, a ﬁrst vector comprising a polynucleotide encoding a heavy chain or a heavy chain le region of an antibody described herein, or a fragment thereof, and a second vector comprising a polynucleotide encoding a light chain or a light chain variable region of an antibody described herein, or a nt thereof. In other embodiments, a ﬁrst host cell ses a ﬁrst vector compiising a polynucleotide encoding a heavy chain or a heavy chain variable region of an antibody described herein, or a fragment thereof, and a second host cell comprises a second 2015/067642 vector comprising a polynucleotide encoding a light chain or a light chain variable region of an antibody described herein. In speciﬁc embodiments, a heavy chain/heavy chain variable region expressed by a ﬁrst cell associated with a light chain/light chain variable region of a second cell to form an anti-CD47 antibody described herein or an antigen-binding fragment thereof. In certain embodiments, provided herein is a population of host cells comprising such ﬁrst host cell and such second host cell.

In a particular embodiment, provided herein is a population of vectors comprising a ﬁrst vector sing a polynucleotide encoding a light chain/light chain variable region of an anti-CD47 antibody described herein, and a second vector comprising a polynucleotide encoding a heavy chain/heavy chain variable region of an anti-CD47 antibody described herein.

A variety of host-expression vector systems can be ed to express antibody molecules described herein (see, e.g., US. Patent No. 5,807,715). Such host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently puriﬁed, but also represent cells which can, when transformed or ected with the appropriate nucleotide coding ces, express an antibody molecule described herein in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli and B. sublilz's) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences, yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression s containing antibody coding sequences; insect cell s infected with recombinant virus sion vectors (e.g., baculovirus) containing antibody coding ces; plant cell s (e.g., green algae such as domonas reinhardtii) infected with recombinant virus expression vectors (e.g., cauliﬂower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant d expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., cos, CHO, BHK, MDCK, HEK 293, N80, PER.C6, VERO, CRL7030, HsS78Bst, HeLa, and NIH 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of ian cells (e.g, othionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). In a speciﬁc ment, cells for expressing dies described herein or an antigen-binding fragment thereof are CHO cells, for example CHO cells from the CHO GS TM (Lonza). In a speciﬁc embodiment, a mammalian expression vector is pOptiVECTM or pcDNA3.3. In a particular embodiment, bacterial cells such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells), especially for the expression of whole recombinant antibody molecule, are used for the expression of a inant antibody molecule. For example, mammalian cells such as e hamster ovary (CHO) cells, in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an ive expression system for antibodies (Foecking et al., 1986, Gene 45: 101; and Cockett er al., 1990, Bio/Technology 8:2). In certain embodiments, antibodies bed herein are produced by CHO cells or NSO cells. In a speciﬁc embodiment, the expression of nucleotide sequences encoding antibodies bed herein which immunospeciflcally bind to CD47 is regulated by a constitutive promoter, inducible promoter or tissue speciﬁc promoter.

In bacterial systems, a number of expression vectors can be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such an antibody is to be produced, for the generation of pharmaceutical compositions of an dy molecule, vectors which direct the expression of high levels of fusion n products that are readily purifled can be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO 12: 1791), in which the antibody coding sequence can be ligated individually into the vector in frame with the lac Z coding region so that a fusion n is produced, pIN vectors e & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. For example, pGEX vectors can also be used to s foreign polypeptides as fusion proteins with hione 5-transferase (GST). In general, such fusion ns are soluble and can easily be puriﬁed from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. The pGEX s are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV), for example, can be used as a vector to express foreign genes. The virus grows in Spodoplera frugiperda cells. The antibody coding sequence can be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for e the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems can be utilized.

In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest can be ligated to an adenovirus ription/translation control x, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a sential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and e of expressing the dy molecule in infected hosts (e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 8 1:355-359). Speciﬁc initiation signals can also be required for efﬁcient ation of inserted antibody coding ces. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the d coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efﬁciency of expression can be enhanced by the inclusion of riate transcription er elements, transcription terminators, etc. (see, e.g., r el al., 1987, Methods in Enzymol. 153 :5 1-544).

In addition, a host cell strain can be chosen which modulates the expression of the inserted sequences, or modiﬁes and processes the gene product in the speciﬁc fashion desired.

Such modiﬁcations (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and speciﬁc mechanisms for the post-translational processing and modiﬁcation of proteins and gene products.

Appropriate cell lines or host systems can be chosen to ensure the correct ation and processing of the foreign protein expressed. To this end, eukaryotic host cells which s the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, Hela, cos, MDCK, HEK 293, NIH 3T3, W138, BT483, HsS78T, HTB2, BT20 and T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL703O and HsS78Bst cells. In certain embodiments, anti- CD47 antibodies described herein (e.g., an antibody comprising the CDRs of any one of antibodies Ab235-Ab255) are produced in mammalian cells, such as CHO cells.

For long-term, high-yield production of recombinant ns, stable expression cells can be generated. For example, cell lines which stably express an D47 antibody described herein or an antigen-binding fragment thereof can be engineered. In speciﬁc embodiments, a cell provided herein stably expresses a light chain/light chain le domain and a heavy chain/heavy chain variable domain which associate to form an antibody described herein or an antigen-binding fragment thereof.

In certain s, rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., er, enhancer, sequences, transcription ators, polyadenylation sites, etc), and a selectable marker. ing the introduction of the foreign DNA/polynucleotide, engineered cells can be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines which express an anti-CD47 antibody described herein or a fragment thereof. Such engineered cell lines can be particularly useful in screening and evaluation of compositions that interact ly or indirectly with the antibody molecule.

A number of selection systems can be used, including but not limited to, the herpes simplex virus thymidine kinase (Wigler el al., 1977, Cell 11:223), hypoxanthineguanine phosphoribosyltransferase lska & Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy ez‘ £11., 1980, Cell 22:8-17) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which s resistance to rexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77357, O’Hare el al., 1981, Proc. Natl. Acad. Sci. USA 78: 1527), gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc.

Natl. Acad. Sci. USA 782072); neo, which confers resistance to the aminoglycoside G—418 (Wu and Wu, 1991, Biotherapy 3:87-95, Tolstoshev, 1993, Ann. Rev. acol. l. 32:573- 596, Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. m. -217, May, 1993, TTB TECH 11(5):155—2 15), and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30: 147). Methods commonly known in the art of recombinant DNA technology can be routinely applied to select the d recombinant clone, and such methods are described, for e, in Ausubel et al. (eds), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993), Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994), Colberre-Garapin et al., 1981, J. Mol. Biol. 150: 1, which are incorporated by nce herein in their entireties.

The expression levels of an antibody molecule can be increased by vector ampliﬁcation (for a , see Bebbington and hel, The use of s based on gene ampliﬁcation for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is ampliﬁable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the ampliﬁed region is associated with the antibody gene, production of the antibody will also increase e et al., 1983, Mol. Cell. Biol. 3:257).

The host cell can be co-transfected with two or more expression vectors bed herein, the ﬁrst vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors can contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. The host cells can be co-transfected with different amounts of the two or more expression vectors, For example, host cells can be ected with any one of the following ratios of a ﬁrst expression vector and a second expression vector: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10,1:12, 1:15, 1:20, 1:25, 1:30,1:35,1:40,1:45, or 1:50.

Alternatively, a single vector can be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; and Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197-2199). The coding sequences for the heavy and light chains can comprise cDNA or genomic DNA. The expression vector can be monocistronic or multicistronic. A multicistronic nucleic acid uct can encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, or in the range of 2-5, 5-10 or 10-20 genes/nucleotide sequences. For example, a bicistronic nucleic acid construct can se in the following order a promoter, a ﬁrst gene (e.g., heavy chain of an antibody described ), and a second gene and (e.g., light chain of an antibody bed herein). In such an expression vector, the transcription of both genes can be driven by the promoter, whereas the translation of the mRNA from the ﬁrst gene can be by a cap—dependent scanning mechanism and the translation of the mRNA from the second gene can be by a cap-independent mechanism, e.g., by an IRES.

WO 09415 2015/067642 Once an antibody molecule described herein has been produced by inant expression, it can be puriﬁed by any method known in the art for puriﬁcation of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, afﬁnity, particularly by afﬁnity for the speciﬁc antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the ation of proteins. r, the antibodies described herein can be fused to logous polypeptide sequences described herein or otherwise known in the art to facilitate puriﬁcation.

In c embodiments, an antibody or antigen-binding fragment described herein is isolated or puriﬁed. Generally, an isolated antibody is one that is substantially free of other dies with different antigenic speciﬁcities than the isolated antibody. For example, in a particular embodiment, a preparation of an antibody described herein is substantially free of cellular material and/or chemical precursors. The language “substantially free of ar material” includes preparations of an dy in which the antibody is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, an antibody that is substantially free of cellular material es preparations of antibody having less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous protein (also referred to herein as a minating protein”) and/or variants of an antibody, for example, different post-translational modiﬁed forms of an antibody or other different versions of an dy (e.g., antibody fragments). When the antibody is recombinantly produced, it is also generally substantially free of culture medium, 1'.e., culture medium represents less than about %, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein preparation. When the antibody is produced by chemical synthesis, it is generally substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein, Accordingly, such preparations of the antibody have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In a c embodiment, antibodies bed herein are isolated or puriﬁed. .3 Pharmaceutical itions and Kits Provided herein are compositions, pharmaceutical compositions, and kits comprising one or more antibodies (e.g., anti-CD47 antibodies) described herein, or antigen-binding nts thereof, or conjugates thereof. In particular aspects, compositions (e.g., pharmaceutical compositions) described herein can be for in vitro, in vivo, or ex vivo uses. Non- limiting examples of uses include uses to modulate (e.g, inhibit or induce/enhance) CD47 activity and uses to manage or treat a disorder, for example, cancer. In speciﬁc embodiments, ed herein is a pharmaceutical composition sing an antibody (e.g., a humanized antibody) described herein (or an antigen-binding fragment f) and a pharmaceutically acceptable carrier or excipient.

As used herein, the term “pharmaceutically acceptable” means being approved by a regulatory agency of the Federal or a state government, or listed in the US. Pharmacopeia, European Pharmacopeia or other generally recognized Pharmacopeia for use in animals, and more particularly in .

Formulations containing one or more antibodies provided herein or an antigen- binding fragment thereof can be prepared for storage by mixing the antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co, Easton, PA; Remington: The Science and Practice of Pharmacy, 21st ed. (2006) Lippincott Williams & s, Baltimore, MD). Such formulations can, for example, be in the form of, e.g., lized formulations or aqueous solutions. Pharmaceutical carriers suitable for administration of the dies provided herein include any such rs known to those skilled in the art to be suitable for the particular mode of administration. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and trations employed, and include buffers such as phosphate, citrate, and other organic acids; and/or non-ionic surfactants such as TWEENTM, ICSTM or polyethylene glycol (PEG). ations to be used for in vivo stration can be sterile. This can be readily accomplished, for example, by ﬁltration through, e.g., sterile filtration membranes.

In specific aspects, the pharmaceutical compositions provided herein contain eutically effective amounts of one or more of the antibodies or antigen-binding fragments provided herein in a ceutically acceptable carrier. Such pharmaceutical compositions are useful in the tion, treatment, management or amelioration of a condition or disorder described herein or one or more symptoms thereof.

Compositions provided herein can contain one or more dies provided herein or an antigen-binding fragment f. In one embodiment, compositions are provided wherein WO 09415 antibodies or antigen-binding fragments described herein are formulated into suitable pharmaceutical preparations, such as solutions, suspensions, powders, sustained release formulations or s in sterile solutions or suspensions for parenteral administration, or as transdermal patch preparation and dry powder inhalers.

In one embodiment, compositions provided herein are formulated for single dosage administration. To formulate a composition, the weight fraction of compound is dissolved, suspended, dispersed or otherwise mixed in a selected carrier at an effective concentration such that the treated ion is relieved, prevented, or one or more symptoms are ameliorated.

In certain aspects, an antibody provided herein is included in the ceutically acceptable carrier in an effective amount sufﬁcient to exert a therapeutically useful effect in the e of, or with minimal or negligible, undesirable side effects on the patient treated.

Concentrations of anti-CD47 antibody in a pharmaceutical composition provided herein will depend on, e.g., the physicochemical characteristics of the antibody, the dosage schedule, and amount stered as well as other factors.

Pharmaceutical compositions described herein are provided for stration to humans or s (e.g., mammals) in unit dosage forms, such as sterile eral (e.g., intravenous) solutions or suspensions containing suitable quantities of the compounds or pharmaceutically acceptable derivatives thereof. Pharmaceutical compositions are also provided for administration to humans and animals in unit dosage form, such as tablets, capsules, pills, powders, granules, and oral or nasal solutions or suspensions, and oil-water ons containing suitable quantities of an anti-CD47 dy or pharmaceutically acceptable derivatives thereof.

The antibody is, in one embodiment, formulated and administered in unit—dosage forms or multiple-dosage forms. Unit-dose forms as used herein refers to ally discrete units suitable for human or animal (e.g., mammal) subjects and packaged dually. Each unit-dose contains a predetermined ty of an anti-CD47 dy sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical r, vehicle or diluent.

Examples of unit-dose forms include ampoules and syringes and individually packaged tablets or capsules. Unit-dose forms can be administered in fractions or multiples thereof. A multiple- dose form is a plurality of cal unit-dosage forms packaged in a single container to be administered in segregated unit-dose form. Examples of multiple—dose forms include vials, WO 09415 bottles of tablets or capsules or bottles. Hence, in speciﬁc s, le dose form is a multiple of unit—doses which are not segregated in packaging.

In n embodiments, one or more anti-CD47 dies described herein or an antigen-binding fragment thereof are in a liquid pharmaceutical formulation. Liquid pharmaceutically administrable compositions can, for example, be ed by dissolving, dispersing, or otherwise mixing an antibody and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, and the like, to thereby form a solution or suspension. In certain embodiments, a pharmaceutical composition provided herein to be administered can also contain minor amounts of nontoxic auxiliary substances such as wetting , emulsifying agents, solubilizing agents, and pH buffering agents and the like.

Methods of ing such dosage forms are known, or will be nt, to those skilled in this art, for example, see, e. g., Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA; Remington: The e and ce of Pharmacy, 21st ed, (2006) Lippincott Williams & Wilkins, Baltimore, MD.

Parenteral stration, in one embodiment, is characterized by injection, either subcutaneously, intramuscularly or intravenously is also contemplated herein. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or sion in liquid prior to injection, or as emulsions. The injectables, solutions and emulsions also contain one or more excipients. Suitable excipients are, for e, water, saline, dextrose, glycerol or ethanol. Other routes of administration may include, enteric administration, intracerebral administration, nasal administration, intraarterial administration, intracardiac administration, intraosseous infusion, intrathecal administration, and intraperitoneal administration.

] Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, e suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use and sterile ons.

The solutions can be either aqueous or nonaqueous.

If administered intravenously, suitable carriers include logical saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.

WO 09415 Pharmaceutically acceptable carriers used in parenteral ations include aqueous es, nonaqueous vehicles, crobial agents, isotonic agents, buffers, antioxidants, local anesthetics, ding and dispersing agents, emulsifying agents, tering or chelating agents and other pharmaceutically acceptable substances.

Pharmaceutical carriers also include ethyl l, polyethylene glycol and propylene glycol for water miscible vehicles; and sodium hydroxide, hydrochloric acid, citiic acid or lactic acid for pH adjustment.

In certain embodiments, intravenous or rten'al infusion of a sterile aqueous solution containing an anti-CD47 antibody or fragment described herein is an effective mode of ammmwwmrAmmmmmmMMmmaﬁwbmmwmmprwMMomMWMMn containing an anti-CD47 antibody bed herein injected as necessary to produce the desired pharmacological effect.

In specific embodiments, an anti-CD47 antibody described herein can be suspended mmMmmeMmTMEmdﬁmwMgmmewm®wmammm of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle.

] In other embodiments, the pharmaceutical formulations are lyophilized powders, which can be reconstituted for administration as solutions, emulsions and other mixtures. They can also be reconstituted and formulated as solids or gels.

Lyophilized powder can, for example, be prepared by dissolving an anti-CD47 antibody provided herein, in a suitable solvent. In some embodiments, the lyophilized powder is sterile. Suitable solvents can contain an excipient which improves the stability or other pharmacological ent of the powder or reconstituted solution, prepared from the powder.

Excipients that can be used include, but are not limited to, dextrose, al, fructose, corn syrup, l, glycerin, glucose, sucrose or other suitable agent. A suitable solvent can also contain a , such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, in one embodiment, about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides an example of a formulation. In one embodiment, the resulting solution will be ioned into vials for lyophilization. Lyophilized powder can be stored under appropriate conditions, such as at about 4 0C to room temperature.

Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier.

In certain aspects, anti—CD47 antibodies provided herein can be formulated for local administration or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, , and lotions and for application to the eye or for intracisternal or intraspinal application. Topical administration is contemplated for transdermal delivery and also for administration to the eyes or mucosa, or for inhalation therapies. Nasal solutions of the active compound alone or in combination with other pharmaceutically acceptable excipients can also be administered.

Anti—CD47 dies and other itions provided herein can also be formulated to be targeted to a particular tissue, organ, or other area of the body of the subject to be treated.

Many such targeting methods are well known to those of skill in the art. All such targeting methods are contemplated herein for use in the t compositions. For non-limiting examples oftargeting methods, see, e.g., US. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 751, 6,071,495, 082, 6,048,736, 6,039,975, 6,004,534, ,985,307, 366, 5,900,252, 5,840,674, 5,759,542 and 5,709,874. In some embodiments, anti-CD47 antibodies described herein are ed (or otherwise administered) to the visual organs, bone , gastrointestinal tract, lungs, brain, or joints. In speciﬁc embodiments, an anti-CD47 dy described herein is capable of ng the blood-brain barrier.

Provided herein is a pharmaceutical pack or kit comprising one or more containers ﬁlled with one or more of the ingredients of the pharmaceutical compositions described herein, such as one or more anti-CD47 antibodies provided herein. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the cture, use or sale of pharmaceuticals or biological products, which notice reﬂects approval by the agency of manufacture, use or sale for human administration.

Also provided herein are kits comprising one or more of the antibodies or antibody nts described herein. In one embodiment, a kit ses an antibody or antibody fragment bed herein, in one or more containers. In a specific embodiment, kits described herein contain a substantially purified CD47 antigen as a control. In another speciﬁc ment, the kits described herein further comprise a control antibody which does not react WO 09415 with a CD47 antigen. In another speciﬁc embodiment, kits described herein contain one or more ts for detecting the binding of a d antibody to a CD47 antigen (e.g, the antibody can be conjugated to a detectable substrate such as a ﬂuorescent compound, an enzymatic substrate, a ctive compound or a luminescent compound, or a second antibody which recognizes the ﬁrst antibody can be conjugated to a detectable substrate). In speciﬁc embodiments, a kit provided herein can include a recombinantly produced or chemically synthesized CD47 antigen. The CD47 antigen provided in the kit can also be attached to a solid support. In a more speciﬁc embodiment, the detecting means of the above described kit includes a solid support to which a CD47 antigen is attached. Such a kit can also include a non-attached reporter-labeled anti-human antibody or anti-mouse/rat antibody. In this embodiment, binding of the antibody to the CD47 antigen can be detected by binding of the said reporter-labeled antibody. .4 Uses and Methods In particular aspects, provided herein are methods of modulating CD47 activity with an anti-CD47 antibody or an antigen-binding fragment thereof described herein.

In speciﬁc embodiments, provided herein are s of inhibiting (e.g., partially inhibiting) a CD47 activity with an anti-CD47 antibody bed herein. In certain embodiments, provided herein are methods of ng or ng a condition or disorder, such as cancer, using an anti-CD47 antibody described herein. In certain embodiments, provided herein are methods of protecting t a condition or disorder, such as cancer, using an anti- CD47 antibody bed herein.

In particular embodiments, provided herein are methods for managing, ng, preventing or protecting against , comprising administering to a subject in need f a therapeutically ive amount of an antibody or an antigen-binding fragment described herein that binds speciﬁcally to CD47 (e. g., human CD47). In certain embodiments, provided herein is a method of alleviating, inhibiting or reducing the progression or severity of one or more symptoms associated with .

As used herein, “administer” or “administration” refers to the act of injecting or otherwise physically delivering a substance (e.g., a humanized anti-CD47 antibody provided herein or an antigen-binding fragment f) to a subject or a t (e.g., human), such as by l, topical, intradermal, parenteral, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical ry described herein or known in the art.

As used herein, the terms “effective amount” or “therapeutically effective amount” refer to an amount of a y (e.g., an antibody or ceutical composition provided herein) which is sufﬁcient to reduce and/or ameliorate the severity and/or duration of a given condition, disorder or disease (e.g, cancer, metastasis, or angiogenesis) and/or a symptom related thereto.

These terms also encompass an amount ary for the reduction, slowing, or amelioration of the ement or progression of a given disease, reduction, slowing, or amelioration of the recurrence, development or onset of a given disease, and/or to improve or enhance the prophylactic or therapeutic effect(s) of another therapy (e.g., a therapy other than an anti-CD47 antibody provided herein). In some embodiments, “effective amount” as used herein also refers to the amount of an antibody described herein to achieve a speciﬁed result.

] As used , the term “in combination” in the context of the administration of other therapies refers to the use of more than one therapy. The use of the term “in combination” does not restrict the order in which therapies are stered. The therapies may be administered, e.g., serially, sequentially, concurrently, or concomitantly.

As used herein, the terms “manage,77 (Cmanaging,” and “management” refer to the beneﬁcial effects that a subject derives from a therapy (e.g., a prophylactic or therapeutic agent), which does not result in a cure of a condition associated with CD47. In certain embodiments, a subject is administered one or more therapies (e.g, lactic or therapeutic agents, such as an antibody bed herein) to e” a condition or disorder described herein, one or more symptoms thereof, so as to prevent the progression or worsening of the condition or disorder.

As used herein, the terms “impede” or “impeding” in the context of a ion or disorder provided herein (e.g., autoimmune disorder, immunological disorder, cancer, or inﬂammation) refer to the total or l inhibition (e.g., less than 100%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5%) or blockage of the development, recurrence, onset or spread of a ion or disorder provided herein (e.g., cancer, metastasis, or angiogenesis) and/or symptom related thereto, resulting from the administration of a therapy or combination of therapies ed herein (e.g., a combination of prophylactic or therapeutic agents, such as an antibody described herein).

WO 09415 As used herein, the terms “subject” and “patient” are used interchangeably. As used herein, a subject is a mammal such as a non-primate (e.g, cows, pigs, horses, cats, dogs, goats, s, rats, mice, etc.) or a primate (e. g., monkey and human), for example a human. In one ment, the subject is a , e.g., a human, diagnosed with a condition or disorder ed herein (e.g., cancer, metastasis, or angiogenesis). In another embodiment, the t is a mammal, e. g., a human, at risk of developing a condition or disorder provided herein (e.g., cancer, asis, or angiogenesis). In another embodiment, the t is human.

In certain embodiments, CD47 is ampliﬁed in cells of a subject, e.g., the human subject. Identiﬁcation of cd47 ampliﬁcation in a sample from a subject can be performed by assays known to one of ordinary skill in the art, such as, e.g., quantitative reverse transcription PCR, immunoblot assays, DNA ﬁngerprinting, karyotyping (for example, by multicolor ﬂuorescence in situ hybridization (mFISH)), comparative genome hybridization, and gene sion proﬁling. As a non-limiting example, protein expression of tumor samples can be characterized using immunohistochemical assays to measure the amount of CD47 protein present in a sample.

Identiﬁcation of mutations or deltions in a sample from a subject can be performed by assays known to one of ordinary skill in the art, such as, e.g., DNA extraction, generation of complementary DNA, and cDNA sequencing. The cDNA sequence, for example, can be utilized to obtain the translation product by methods known to one of ordinary skill in the art. Genetic deletions and amino acid substitutions can be identiﬁed by, for example, comparing the sequence from the sample from the subject to a a wild type and/or consensus sequence.

In certain embodiments, CD47 is ampliﬁed in the subject treated in accordance with the methods provided . Identiﬁcation of CD47 ampliﬁcation in a sample from a subject is performed by assays known to one of ordinary skill in the art, such as, e.g., quantitative reverse transcription PCR or blot assays. Identiﬁcation of mutations or deletions in a sample from a subject are performed by assays known to one of ordinary skill in the art, such as, e.g., DNA extraction, tion of complementary DNA, and cDNA sequencing. The cDNA sequence, for example, is utilized to obtain the translation product by methods known to one of ordinary skill in the art. Genetic deletions and amino acid substitutions are identiﬁed by, for example, comparing the ce from the sample from the subject to a a wild type and/or COHSGHSUSSGQUCHCQ As used herein, the terms “therapies” and “therapy” can refer to any protocol(s), method(s), compositions, formulations, and/or agent(s) that can be used in the tion, treatment, management, or amelioration of a condition or disorder or symptom thereof (e.g., a condition or disorder provided herein (e.g, cancer) or one or more symptoms or condition associated ith). In certain embodiments, the terms “therapies” and “therapy” refer to drug therapy, adjuvant therapy, radiation, surgery, biological therapy, supportive therapy, and/or other ies useful in ent, management, tion, or amelioration of a condition or er or one or more symptoms thereof (e.g., cancer or one or more symptoms or condition associated ith). In certain embodiments, the term “therapy” refers to a therapy other than an anti- CD47 antibody described herein or pharmaceutical composition thereof. In speciﬁc embodiments, an “additional therapy” and ional ies” refer to a therapy other than a treatment using an D47 antibody described herein or pharmaceutical composition thereof.

In a speciﬁc embodiment, a therapy includes the use of an D47 antibody described herein as an adjuvant therapy. For example, using an D47 antibody described herein in conjunction with a drug therapy, biological therapy, surgery, and/or supportive therapy.

As used herein, “hematological cancer” refers to a cancer of the blood, and includes leukemia, lymphoma and myeloma among others. “Leukemia” refers to a cancer of the blood in which too many white blood cells that are ineffective in ﬁghting infection are made, thus crowding out the other parts that make up the blood, such as platelets and red blood cells. It is understood that cases of leukemia are classiﬁed as acute or chronic. Certain forms of leukemia include, by way of non-limiting example, acute lymphocytic ia (ALL); acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML); Myeloproliferative disorder/neoplasm (MPDS); and myelodysplasia syndrome. “Lymphoma” may refer to a n’s lymphoma, both indolent and aggressive non-Hodgkin’s lymphoma, Burkitt’s lymphoma, and follicular lymphoma (small cell and large cell), among others.

Myeloma may refer to multiple myeloma (MM), giant cell myeloma, chain myeloma, and light chain or Jones myeloma.

Non-limiting examples of a condition which can be d or managed with an anti- CD47 antibody described herein include hematological caner and/or solid tumors.

Diseases or disorders related to aberrant CD47 expression, activity and/or signaling include, by way of non-limiting example, hematological cancer and/or solid tumors.

Hematological cancers include, e.g., leukemia, lymphoma and myeloma. Certain forms of leukemia include, by way of non—limiting example, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), Myeloproliferative disorder/neoplasm (MPDS); and myelodysplasia syndrome. Certain forms of lymphoma include, by way of non-limiting example, Hodgkin’s lymphoma, both indolent and aggressive non-Hodgkin’s lymphoma, Burkitt’s lymphoma, and follicular lymphoma (small cell and large cell). Certain forms of myeloma include, by way of non-limiting example, multiple myeloma (MM), giant cell myeloma, heavy-chain myeloma, and light chain or Jones myeloma. Solid tumors include, e.g., breast tumors, ovarian tumors, lung tumors, pancreatic tumors, prostate tumors, melanoma tumors, ctal tumors, lung tumors, head and neck tumors, bladder tumors, esophageal tumors, liver tumors, and kidney tumors.

Symptoms associated with cancers and other neoplastic disorders include, for e, inﬂammation, fever, general malaise, fever, pain, often localized to the inﬂamed area, loss of appetite, weight loss, edema, headache, fatigue, rash, anemia, muscle weakness, muscle fatigue and abdominal symptoms such as, for example, nal pain, diarrhea or constipation.

In specific aspects, provided herein are anti-CD47 dies useful in treating, ng the progression of, impeding, preventing relapse of or alleviating a symptom of a cancer (e.g., MM, Nm, AML, breast , bladder cancer, non-small cell lung cancer/carcinoma, hepatocellular oma (HCC), a, and head and neck cancer). For example, the CD47 antibodies described herein are useful in treating logical malignancies and/or tumors, e.g., hematological ancies and/or . For example, the CD47 dies described herein are useful in treating CD47+ tumors. By way of miting example, the CD47 antibodies described herein are useful in treating non-Hodgkin’s lymphoma (NHL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic cytic leukemia (CLL), chronic enous leukemia (CML), multiple myeloma (MM), breast cancer, ovarian cancer, head and neck cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung , leiomyoma, leiomyosarcoma, glioma, glioblastoma, and so on. Solid tumors include, e.g., breast tumors, ovarian tumors, lung tumors (e.g., NSCLC), pancreatic tumors, prostate tumors, melanoma tumors, colorectal tumors, lung tumors, head and neck tumors, bladder tumors, geal tumors, liver tumors (e.g., hepatocellular carcinoma), sarcoma, and kidney tumors.

In a speciﬁc embodiment, provided herein is a method of treating cancer (e.g., a hematological disorder/cancer or solid cancer) in a subject comprising administering (e.g., administering concurrently or sequentially) to a subject in need thereof (i) an anti-CD47 antibody described herein or antigen-binding fragment thereof which cally binds to CD47 such as human CD47, and (ii) another anti-cancer agent. In certain embodiments, the anti-cancer agent is a chermotherapeutic agent (e.g., microtubule disassembly blocker, antimetabolite, topisomerase inhibitor, and DNA crosslinker or ng . In certain embodiments, the anti—cancer agent is a tyrosine kinase inhibitor (e.g., GLEEVEC® (imatinib mesylate) or SUTENT® (SU11248 or Sunitinib)). Other non-limiting examples of tyrosine kinse inhibitors include 706 and AMNIO7 (nilotinib). RADOOI, PKC412, ib ATM), erlotinib (TARCEVA®), sorafenib (NEXAVAR®), pazopanib (VOTRIENTTM), aXitinib, bosutinib, cediranib (RECENTIN®), SPRYCEL® (dasatinib), lapatinib (TYKERB®), lestaurtinib, neratinib, nilotinib (TASIGNA®), semaxanib, nib (PALLADIATM), anib (ZACTIMA TM), and vatalanib.

In a speciﬁc , provided herein is a method of treating cancer (e.g, a hematological disorder/cancer or solid ) in a subject comprising administering (e.g., administering concurrently or sequentially) to a subject in need thereof (i) an anti—CD47 antibody described herein or antigen-binding fragment thereof which cally binds to CD47 such as human CD47, and (ii) radiation therapy.

In a particular aspect, provided herein is a method of promoting (e.g., inducing or increasing) phagocytosis, e. g., macrophage mediated phagocytic killing of tumor cells, comprising contacting an effective amount of an anti-CD47 antibody described herein which speciﬁcally binds to human CD47 with tumor cells. Also provided herein is a method of promoting (e.g., inducing or increasing) phagocytosis, e.g., macrophage ed phagocytic killing of tumor cells, in a t in need thereof (e.g., a subject with tumor cells, such as tumor cells expressing CD47), comprising stering to the subject an effective amount of an anti- CD47 antibody described herein which cally binds to human CD47.

In a particular aspect, provided herein is a method of reducing tumor volumn, comprising contacting an effective amount of an anti-CD47 antibody described herein which speciﬁcally binds to human CD47 with the tumor. Also provided herein is a method of reducing tumor volumn in a t in need thereof (e.g., a subject with a tumor, such as a CD47 sing tumor), comprising administering to the subject an effective amount of an anti-CD47 antibody described herein which speciﬁcally binds to human CD47.

In a particular aspect, provided herein is a method of inhibiting cancer cell growth or proliferation, comprising contacting an ive amount of an anti—CD47 antibody described herein which speciﬁcally binds to human CD47 with cancer cells. Also provided herein is a method of inhibiting cancer cell growth or proliferation in a subject in need thereof (e. g., a subject with cancer cells, such as CD47 expressing cancer cells), comprising administering to the subject an effective amount of an D47 antibody described herein which speciﬁcally binds to human CD47. .4.1 Diagnostic Uses In one aspect, anti-CD47 antibodies bed herein and antigen-binding nts thereof, which speciﬁcally bind to an ECD of human CD47 can be used for stic purposes to detect, diagnose, or monitor a ion described herein (e.g., a ion involving CD47 and/or abnormal CD47 signaling and/or abnormal CD47 expression), such as cancer (e.g., colorectal cancer, gastric cancer, lung cancer, or melanoma). In speciﬁc embodiments, anti- CD47 antibodies described herein or an antigen-binding fragment thereof for use in diagnostic purposes are labeled. Methods provided herein for diagnostic purposes to detect, diagnose, or monitor a condition described herein can be in vitro methods, in situ methods, or ex vivo methods. Methods provided herein for diagnostic purposes to detect, diagnose, or monitor a condition described herein can be in viva methods.

] In certain embodiments, provided herein are methods for the detection of a condition described herein, such as cancer, comprising: (a) assaying the expression of CD47 in a sample of a subject using one or more dies described herein or an antigen-binding fragment thereof; and (b) comparing the level of CD47 expression with a control level, e.g., levels in normal tissue samples (e.g., from a t not having a condition described , or from the same patient before onset of the condition), whereby an increase or decrease in the assayed level of CD47 expression compared to the control level of CD47 expression is indicative of a condition described .

In certain embodiments, provided herein are s for the detection of cancer expressing CD47 (e.g., overexpressing CD47), comprising: (a) assaying the expression of CD47 in a sample of a subject using one or more antibodies described herein or an n-binding fragment thereof; and (b) comparing the level of CD47 expression with a control level, e.g., levels in normal samples (e.g., from a t not having cancer, a t having cancer that does not overexpress CD47, or from the same patient before onset of cancer). In speciﬁc s, an increase or decrease in the assayed level of CD47 sion compared to a control level of CD47 expression is indicative of cancer expressing CD47.

In a speciﬁc embodiment, provided herein is a method of diagnosing a CD47- expressing cancer in a patient, wherein the method comprises the steps of: (a) contacting a biological sample from the patient with one or more antibodies described herein or an antigen—binding nt thereof; (b) detecting binding of the dy or antigen-binding fragment to CD47 to determine a CD47 protein level in the biological sample from the patient; and (c) comparing the CD47 protein level with a standard CD47 protein level.

In a speciﬁc embodiment, provided herein is a method of ring CD47 protein level during ent of a CD47—expressing cancer in a patient, wherein the method ses the steps of: (a) contacting a biological sample from the patient with one or more antibodies described herein or an antigen-binding fragment thereof, (b) detecting binding of the antibody or antigen-binding fragment to CD47 to determine a CD47 protein level in the biological sample from the patient, and (c) comparing the CD47 protein level with a standard CD47 protein level.

Any sample (e.g., bodily ﬂuid or tissue sample) from a subject can be used in diagnostic methods provided herein. Non-limiting examples of samples which can be used in diagnostic methods ed herein include, serum sample, plasma sample, tissue , urine sample, tumor sample, and stool sample.

Antibodies described herein can be used to assay CD47 levels in a biological sample using classical immunohistological methods as described herein or as known to those of skill in the art (e.g., see Jalkanen el al., 1985, J. Cell. Biol. 101:976-985; and Jalkanen et al., 1987, J.

Cell . Biol. 105:3087-3096). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (1251, 1211), carbon (14C), sulfur (35$), tritium (3H), indium (1211n), and technetium (99Tc); luminescent labels, such as luminol; and ﬂuorescent labels, such as ﬂuorescein and rhodamine, and biotin.

In one embodiment, monitoring of a condition described herein (e.g., a condition ing CD47 and/or abnormal CD47 signaling and/or abnormal CD47 expression), such as , is d out by repeating the method for diagnosing for a period of time after initial sis.

Presence of the labeled le can be detected in the subject using methods known in the art for in vivo scanning. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the stic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as on emission aphy (PET), magnetic resonance imaging (MRI), and sonography. 6. EXAMPLES The examples in this section (1'. e., Section 6) are offered by way of illustration, and not by way of limitation. 6.1 Example 1: Cell-Free (CF) Antibody Production Uneff1cient assembly of the heavy and light chains of anti-CD47 antibody AB6. 12 has been observed when co-expressed in the cell—free (CF) system. One approach to improve this process e pre-addition of folded light chain into a heavy chain reaction. In another approach, heavy chain framework sequence ation surprisingly resulted in more efﬁcient assembly and co-expression of the heavy and light chains of anti-CD47 antibodies in the CF system. Characterization of these anti-CD47 dies with heavy chain framework sequence modiﬁcations are described in more detail below.

Small-Scale Production ree extracts with over-expression ofDst (2X Dst) were thawed to room ature and incubated with 50 uM iodoacetamide for 30 min. Cell-free reactions were run at °C for up to 10 hours containing 30% (v/v) iodoacetamide-treated extract with 8 mM magnesium glutamate, 10 mM ammonium glutamate, 130 mM potassium glutamate, 35 mM sodium pyruvate, 1.2 mM AMP, 0.86 mM each of GMP, UMP, and CMP, 2 mM amino acids for all 19 amino acids except ne which was added at 0.5 mM, 4 mM sodium e, 1 mM putrescine, 1.5 mM dine, 15 mM potassium phosphate,100 nM T7 RNAP, and 2 mM oxidized (GS SG) glutathione. The concentrations of heavy chain plasmid and light chain plasmid in the reactions were 7.5 ug/mL and 2.5 ug/mL tively. To label synthesized protein with 14C, 3.33% (v/v) l-[U-14C]-leucine (300 mCi/mmole, GE Life Sciences, Piscataway, NJ) was added to the reaction as well. In this experiment, the following heavy chains (HCs) paired with the same light chain (LC), SEQ ID NO: 13, were expressed.

SEQ 1]) sample # lgﬁ HC Description NO: Antibody name 1 IgGl parental 2 IgGl -parental 2 IgGl 5 mutations 7 IgGl-5m 3 IgGl 13 mutations 5 IgG1-13m IgGl Z allotype 13 mutations Z allotype 6 IgG1-13mZ 4 or 13 + 2 mutations IgG4P Parental 3 IgG4P—parental 6 IgG4P 5 ons 9 IgG4P-5m 7 IgG4P 13 mutations 8 IgG4P-13m 8 IgG4PE Parental 4 IgG4PE-parental 9 IgG4PE 5 mutations 11 IgG4PE-5m IgG4PE 13 mutations 10 IgG4PE—13m ] Exemplary nucleotide sequences encoding the above heavy chain sequences are provided at SEQ ID Nos. 23, 26, 27, 24, 28, 29, 25, 32 and 31, respectively.

For ducing SDS-PAGE, 4 uL of sample, 8 uL of deionized water (DI H20) and 4 uL of 4X LDS buffer (Invitrogen, ad, CA) were mixed before being loaded on gel. For reducing gel, 4 uL of sample, 1 uL of 1 M DTT, 7 uL of DI H20 and 4 uL of 4X LDS buffer (Invitrogen, Carlsbad, CA) were mixed and heated in hot blot at 70°C for 5 minutes. Samples were analyzed by 4~12% Bis-Tris SDS—PAGE gels (Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendations. Gels were dried and analyzed by autoradiography using a Storm 840 oImager after about 16 hours exposure. The autoradiography is shown in Figure 1A.

The data indicate that the CF expression titers of IgG1, IgG4P and IgG4PE were each dramatically improved by engineering the 5 mutations and the 13 mutations on their HC sequences. The titers are reported in Figure 1B.

Scale—Up Production — IgGl variants Scale—up CF production of IgG1 variant antibodies, such as IgG1-5m and IgG1-13mZ, and puriﬁcation were carried out. The up conditions are described in more details in the Tables (e.g., Tables 3-6) below. Both variants were made with essentially the same method; the most cant difference was in reaction temperature. IgGl-5m was expressed at 25°C and IgG1-13mZ was run at 30°C. IgG1-13mZ showed similar results at 25°C and 30°C, but IgGl- 5m showed higher titer at 25°C versus 30°C. The slight differences in reaction timing between the two variants are simply the result of scheduling convenience and not likely to have any cant impact on the process. Different extracts were used for the ts based on extract availability, not on speciﬁc extract requirements. Any t with over-expression ofDst would be expected to work similarly for these products.

IgG1-13mZ (IgGl, VHl-18 framework, l3+2 mutations) Scale-up Expression: S30 cell-free extract was d with 50 uM iodoacetamide (1AM) for 30 minutes at room temperature. After this treatment, the extract was combined with the reagents in Table 3 and erred to a bioreactor (Dci-BioLaﬁtte Evo Bioreactor, 10L maximum working volume).

The bioreactor controls were conﬁgured as listed in Table 4. After 6 hours of reaction, an additional 5 mM (ﬁnal concentration) ed glutathione was added to the reaction. The oxidized glutathione was prepared as a 250 mM stock solution with the pH adjusted to between 7 and 8 before on to the reactor. The on was run for a total of 15 hours before transferring to downstream sing.

Table 3 - Cell-free reaction components Final Concentration in CF Rea_ent reaction AMP 1.2 mM GM 0.86 mM UMP 0.86 mM CM 0.86 mM Sodium oohoshate 15 mM 19 amino acids excludin_ t rosine 2 mM each Oxalic Acid 4 mM Putrescine 1 mM Soermidine 1.5 mM Ammonium _lutamate 10 mM Potassium _lutamate 130 mM Ma_nesium _lutamate 8 mM 2015/067642 P ruvate 35 mM Oxidized lutathione 2 mM Bacterioo_hae T7 RNA 01 merase 0.02 _/L d ng li ht chain ' Plasmid encoding heaV chain .rotein 7.5 m_/L Pluronic—R 31R1 0.005% V/V IAM treated extract from E. coli strain 30% (V/V) SBDG028 Table 4 - Bioreactor control settings (5L reaction volume) Parameter Setpoint Temperature 30°C ”C3 no l Air ﬂow (sparger) 1.5 SLPM 100% air tion ion 200-400 RPM as needed for DO control (primary DO cascade Oxygen ﬂow 0-2 SLPM as needed for DO control (secondary DO s o ar_er cascade Anti-CD47 antibody IgG1-5m (IgGl, VH1-18 ork, 5 mutations) Scale-up Expression: S30 cell-free extract was treated with 50 uM iodoacetamide (1AM) for 30 minutes at room temperature. After this treatment, the extract was combined with the reagents in Table 5 and transferred to a bioreactor rius Biostat Q Bioreactor, 500 mL maximum working volume). The bioreactor controls were conﬁgured as listed in Table 6. After 5.5 hours of reaction, an additional 5 mM (ﬁnal concentration) oxidized glutathione was added to the reaction.

The oxidized glutathione was prepared as a 250 mM stock solution with the pH adjusted to between 7 and 8 before addition to the reactor. The reaction was run for a total of 15.7 hours before transferring to downstream processing.

Table 5 - Cell-free reaction components Final Concentration in CF Rea_ent reaction 0.86 mM 086 mM Sodium oohoshate 15 mM Bacterioo_hae T7 RNA .01 merase 0.02 _/L Plasmid encoding li_ht chain ' Plasmid encodin- heaV chain rotein 7.5 m_/L 1AM treated extract from E. coli strain 30% (V/V) SBDG03 1 Table 6 - Bioreactor control settings (0.5L reaction volume) |il||||||il||||||||Parameter Setpoint Temperature 25°C 'U no control Air ﬂow (sparger) 0.25 SLPM 80% air saturation Agitation 0 RPM as needed for DO control (primary DO cascade Oxygen ﬂow 0-1 SLPM as needed for DO control (secondary DO s o arer cascade 6.2 Example 2: e Analysis of CD47 Binding Material and Methods: Immobilization: Anti-human Fc (AHC) surfaces were ed by amine-coupling monoclonal mouse anti—human Fc IgG (included in the e Human Antibody Capture Kit, GE Life Sciences Cat # BR39) to a Biacore CM5 sensor chip e. The running buffer for the immobilization procedure for the immobilization procedure was HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% V/V P-2O as surfactant). The following was 2015/067642 performed in all four ﬂow cells to prepare surfaces amine-coupled anti-human Fc IgG. The CM5 chip surface was activated by injecting a 1:1 (v/v) mixture of 400 mM EDC and 100 mM NHS for 7 minutes at 10 uL/min. Following this treatment, anti-human IgG was diluted to 25 ug/mL in 10 mM sodium acetate buffer pH 5.0, and injected over all the flow cells at 10 uL/min for 7 minutes. Then, Ethanolamine was injected at 10 uL/min for 7 minutes to block all the surfaces.

This procedure resulted in immobilization levels of ~10,000 — 11,000 RU on the sensor chip.

] Kinetic Assays: For kinetic assays anti-CD47 antibodies were ed onto the anti- human Fc surfaces (surface preparation bed above). For e of the antibodies with human Fc, the antibodies were diluted to 10 ug/mL in HBS—EP+ running buffer. The antibody variants were immobilized by ﬂowing over ﬂow cell 2, 3 or 4 at a ﬂow rate of 10 uL/min for 12 seconds. The analyte (human CD47 n) was diluted into running buffer to prepare a serial dilution series created with a 2-fold dilution factor to give concentrations of3. 125, 6.25, 12.5, 25 and 50 nM. After anti-CD47 antibody capture, the CD47 analyte was injected over the ﬂow cells for 180 seconds (3 minutes) at 50 uL/min, and complex dissociation was monitored for 900 seconds (15 minutes). Buffer blanks were also run, and were used to reference the analyte binding data before fitting. Anti-human Fc surfaces were regenerated with two ond injections of 3M MgClz at 30 uL/min between each analyte binding cycle.

Kinetic data analysis: mental data for a given antibody — antigen ction were ﬁt using the 1:1 binding model, using global Rmax, global ka (association), global kd (dissociation) parameters, and constant RI (bulk refractive index) parameters. Fraction activity (% ligand activity) was determinined using the following a to calculate the relationship n the theoretical Rmax (Rmax Theo) and the experimental Rmax (Rmax exp). In the formula, stoichiometry represents the instances of interaction between dy and antigen, for instance, when the dy is the ligand, each of the antibody arms can interact with antigen, thus the stoichiometry is 2.

MW mméﬁes‘ £1anmI Eiganﬁ Eva-“1391 “i EX Sissﬁzioma‘tw \ hWW Egg; 4‘5"- $35!; é‘ﬁmam. em} Q‘s: mgmm $®d§lt§ =i—c . K. 3‘. mg}‘ ‘ ,ﬁmm was Results: Biacore analysis showed that all three anti-CD47 antibody variants with 5 mutations (regardless of isotype) have similar off—rate and afﬁnity, with fraction activity (% ligand activity) around 38%. Also, y for the antibody variants with 5 mutations agrees well with theparental antibody. The data is summarized in the table (Table 7) below.

Table 7: Biacore results summary CD47 IgG1— 636E+05 2.60E-03 4.09E-09 1234 1.83 37.4 PGRTV— CD47 IgG4P—5m 7.04E+05 2.54E-03 09 106.5 1.86 38.2 5Mutations CD47 IgG4PE—5m 5.94E+05 2.54E-03 4.27E-09 115.4 2.5 39.2 Control Anti-CD47 CF CD47 7.32E+05 2.31E-03 3.16E-09 99.68 1.36 39.4 Antibody Control The data for the D47 antibody variants 13m (with 13 mutations) and l3mZ (with 13 mutations and IgG1 Z allotype) displayed a very fast off-rate, and is of the kinetic parameters did not result in a reliable ﬁt based on the 1:1 g model.

Figures 2A-2E depict individual sensorgrams for dy variants m (Figure 2A), IgGl-l3m (Figure 2B), IgGl-l3mZ (Figure 2C), IgG4P-5m (Figure 2D), and IgG4PE-5m (Figure 2E), and Figure 2F depicts a sensorgram for anti-CD47 control antibody. 6.3 Example 3: Direct CD47 Cell Binding Assay To obtain binding curves, sample variants were titrated 1:2 in FACS buffer (PBS 2% FCS) starting from a high concentration of ~66 nM for each sample. 0.1 x 106 CCRF-EM cells were plated in a 96 well plate and incubated in 50 ul FACS buffer containing the indicated concentration of sample ts for 1 hour in ice. Cells were then washed with 150 pl FACS buffer and incubated in 50 ul FACS buffer containing the secondary antibody human-IgG— PE, Jackson ImmunoResearch) diluted 1:100, for 1hr in ice. Cells were then washed with FACS buffer and ﬁxed in 2% PFA for acquisition with LSR 11, DB ﬂow cytometer (BD Biosciences, San Jose, CA). Flow try analysis was med using FloJo software Version 9.6.4. To obtain titration curves and Kd, Mean Fluorescence Intensity (MFI) values were plotted against concentration (nM). Kd values were ated using Prism 6, Non linear regression curve ﬁt analysis, One Site Speciﬁc Binding with Hill Slope.

Results: All CH0 and CF anti-CD47 monoclonal antibodies showed equivalent binding Kd on surface of CCRF—CEM cells, which are human T lymphoblast cells (ATCC® 9TM). The calculated Kd (nM) values are presented in the table below (Table 8).

Table 8: 6.4 Example 4: Phagocytosis assay tion of human macrophages: Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats (white layer between red blood cells and plasma in a unit of whole blood after it has been spun down in a centrifuge) purchased from the Stanford Blood Center (Palo Alto, CA, USA). Buffy coats were diluted with PBS 2-fold and layered over ml NycoPrep 1.077 (Axis-Shield, , Scotland) in 50 ml Leucosep tubes (Greiner Bio One, Monroe, NC, USA) and centrifuged at 1,000 X g for 20 minutes. PBMCs were ted from the interface, washed with 35 ml PBS and centrifuged at 250 X g for 5 minutes. inating red blood cells were lysed with 10 ml ACK Lysing Buffer (Lonza, Allendale, NJ, USA) for 2 min and cells were diluted with 40 ml PBS and passed through a 40 um cell strainer (BD Biosciences, San Jose, CA, USA). Cells were centrifuged at 250 X g for 5 mins and resuspended in 30 ml RPMI media containing 10% FB S, 2 mM glutamine and penicillinstreptomycin.

PBMCs were counted and cultured at 5X106 cells/ml in RPMI media overnight.

The next day, CD14-positive monocytes were isolated with CD14 microbeads {Miltenyi Biotech, Auburn, CA, USA) using the cs Pro and cultured in RPMI media containing 5.0 ng/ml SF (Pepretech, Rocky Hill, NJ, USA) for 5“? days to obtain differentiated macrophages.

Cells were frozen down in Recovery Cell e ng Medium (Life Technologies, Grand , NY? USA).

Measurement of Phagocytosis Activity: Frozen er tresh human macrophages were cultured overnight in 96—well (20,000 cells in Oil nil RPMI media supplemented with 50 rig/ml M—CSF). The next day, rnedia was exchanged fer 50 ill RPMI nredia without M—CSP after one wash. CD47~pnsitive CCRF—CEM cells (ATCC, Manassas, VA, USA), passaged under l5xl06 cells/nil y, were laheled with 10 nM Cell’l‘race Oregon Green 488 {Life 'l'eehnolngies, Grand lsl and, NY, USA) for '30 minutes and washed 3x with RPMI niedia. Labeled CCRECEM cells (80,000 cells in 50 til) and anti—CD47 antibodies in 50 ill RPMI media were added to macrophages, AntinCDll’i antibodies were tested at a final enneentratinn (it‘d rig/ml tn l0 ltg/l‘lll.

Plates were briefly centriﬁiged and ted at 370C for 3' hours. Macrophages were washed 3;»; with PBS to ve CCRF—CEM cells and detached with 50 ill se (BD Biosciences, San Jose, CA, USA) at. 370C for 10 minutes. Maerenhages were collected, washed once with FACES wash buffer (PBS containing 0.20/25 FBS) and stained with antiuCl'JH—APC fer 15 . Cells were washed twice and ﬁxed in 4% paraforrnaldehyde for it) minutes. Cells were analyzed by flew eyternetry t0 determine the phagocytic index (9/6 Off CUM—positive cells that are Oregon Green labeled) Data was analyzed using GraphPad Prism to obtain dese~respense curves and half maximal effective concentration (Eng) values using the variable slope (4 parameters) non» linear regressinn analysis.

Results: Phagocytosis activity was Observed for all IgGl variants (CF and can), and lgG4P and ingPE from CHO enly. ECso values are presented in the table below (Table 9).

Table 9: -Anti—CD47 variants new (nit/r) lgGl lgG l ~parental~CllO 0‘3 lgG 1 ~paren tal~CF 0. 5 lgGl~5m~CF (R&D) 0.6 lgGl~5m~CF (PD) 0.3 lgG-“ll’ lngP—parental—CHQ 0. 5 lgG‘lP—parental —Cl'*‘ nd lgG‘lP—Sin—CF nd lgCrélPEparentalCHO TgG49Eparental—CP _ 6.5 Example 5: Hemagglutination Assay Published studies suggest that some anti-CD47 antibodies may cause hemagglutination of human red blood cells (RBCs). ore, hemagglutination assays were carried out to characterize anti-CD47 antibodies y to promote agglutination of RBCs.

Human RBCs were sourced from tive Research (Cat# IPLA-WB3). Human RBCs (2 mLs) were washed in 10 mLs of 1X dPBS (pH 7.4) and centrifuged for 10 minutes at 500g (1500rpm). The supernatant was aspirated, and human RBCs were washed twice, resuspended in 8 mL 1 x dPBS for a 20% solution of RBCs. Dilution for the Anti-Human RBC (Rabbit) antibody (Rockland Immunochemicals Inc., Catalog #109—4139, Lot 27233), positive control, was 1:64 with 1:3 serial dilutions (10X). The starting concentration for the samples was 1000 nM with 1:3 serial dilutions (10X). Each antibody ion were pipette (50 uL) to all wells of a U-bottom 96well plate. RBC solution (SOuL of 20% RBC solution) was added to all wells of the plate, and the plate was incubated at 37°C for at least 1.5 hours to 12 hours (Note: there is no visual difference in the s between 1.5 and overnight). Anti-Human RBC (Rabbit) dy and MCA911 (Mouse Anti-Human CD47 (clone BRIC 126, Abnova)) served as positive controls. Assays were visualized from the top of the plate. Negative (non— lutination) results appear as intact red dots, while positive (hemagglutination) s appear as a dispersed red mat.

Results: Only positive controls (rabbit anti-human RBC antibody and MCA911 (mouse anti-human CD47 antibody)) showed hemagglutination. No CH0 and cell free (CF)- expressed anti-CD47 monoclonal antibodies, ing IgGl-parental, IgG4P-parental, IgG4PE- al, IgG1—13mZ—CF, IgG1-13m-CF, IgGl-Sm-CF, IgG4P—5m-CF, IgG4PE-5m-CF, show evidence of hemagglutination. 6.6 Example 6: C1Q Binding ELISA : 96-well high protein binding ELISA plates (MaXSorp Nunc) were coated overnight at 4°C with sample anti-CD47 antibody variants diluted in 0.05 M Sodium Bicarbonate Buffer (pH 9). Samples were diluted at a high concentration of 133.4 nM (20ug/ml) and ed 1:2 in an 11-point dilution curve. Plates were washed three times in PBS, 0.05% Tween 20 and blocked for 1 hour at room temperature with ELISA Diluent (0.1 M NAPO4, 0.1 M NaCl, 0.1% gelatin, 0.05% Tween 20, 0.05% ProClin300). Plates were then washed again three times and incubated for two hours at room temperature with 2 ug/mL human Clq (AbD Serotec 2221-5504, 1mg/mL stock) diluted in ELISA Diluent. Plates were then washed three times and incubated with sheep anti-human Clq HRP (AbD c 2221-5004P) diluted 1:200 in ELISA Diluent for 1 hour at room temperature to detect bound Clq. Plates were then washed three times, then 100 uL of T1Vﬂ3 on was added. Reaction was quenched by adding 100 pl of 1M Phosphoric Acid and plate was read at 450 nM. Data are plotted using Prism6 as non-linear regression with log (inhibitor) vs. se —Variable slope (four parameters).

Results: IgGl-QNl-CHO shows activity in the ClQ ELISA assay,while IgG4P, IgG4PE, and scFv anti-CD47 monoclonal antibodies do not show activity (“NA”) in ClQ ELISA assay. EC50 values are ted in the table below (Table 10).

Table 10: Sample IgGl -parental-CHO IgGl tal—CF IgG1-13mZ-CF IgG1-13m-CF IgGl -5m-CF IgG4P-parental-CHO IgG4P-parental—CF IgG4P—5m-CF Anti-CD47 B6H12 scFv IgG4PE-parental-CHO IgG4PE-parental-CF IgG4PE-5m-CF 6.7 Example 7: Complement-Dependent Cytotoxicity (CDC) Assay Methods: CD47-expressing cell lines (Raji and/or CCRF) were harvested and re— suspended in CDC buffer (RPMI 1640, L-glutamine (100x stock), and 1% BSA) at 0.3 n cells per mL. Cells were then plated at 10,000 cells per well in a 96 well white tissue culture plate (Falcon) and incubated with sample anti-CD47 antibody variants at a ﬁnal concentration of ug/mL in CDC buffer at 37°C for 1 hour. Spin ﬁlters (Costar SpinX microcentrifuge tubes) were used to remove residual contaminants. Rabbit (7.5%) or human (3 0%) serum were then added at a ﬁnal tration of 2.5% and 10%, respectively and incubate for 2 hours at 37°C.

Sera were diluted in CDC assay . Cell death was then measured using the Cell Tox Glo kit (Promega G292) and following the manufacturer’s instructions. Plates were read on Envision luminescent plate reader (Luminescent 96 well full area program) and processed percent CDC activity as (Treated Cells-Spontaneous Cells)/(Total Lysis-Spontaneous Lysis)* 100. s: CDC activity was observed in Bn'c 126 antibodies, anti—CD20 IgG1 antibodies and anti-CD20 IgG4 antibodies, while CDC activity was not observed (“NA”) in any other antibodies tested. ECso values are presented in the table below (Table 11).

Table 11: Name CD20 IgG1 BRIC 126 (anti-CD47 dy) B6H12 (anti-CD47 antibody) IgG1-parental-CHO parental—CHO IgG4PE-parental-CHO IgG1-parental-CF IgG1-5m-CF IgG1-13m-CF IgG1-13mZ-CF IgG4P—parental—CF IgG4P-5m-CF Control IgG1 Isotype WO 09415 6.8 e 8: Antibody-Dependent Cytotoxicity (ADCC) Assay Methods: PBC were prepared from buffy coats. Buffy coats were diluted with PBS 2- fold and layered over 15 ml NycoPrep 1.077 (Axis-Shield, Dundee, nd) in 50 ml Leucosep tubes (Greiner Bio One, Monroe, NC, USA) and centrifuged at 1,000 x g for 20 minutes. PBMCs were collected from the interface and washed with 35 ml PBS and centrifuged at 250 x g for 5 minutes. Contaminating red blood cells were lysed with 10 ml ACK Lysing Buffer (Lonza, Allendale, NJ, USA) for 2 minutes and cells were diluted with 40 ml PBS and passed through a 40 um cell strainer (BD Biosciences, San Jose, CA, USA). Cells were centrifuged at 250 x g for minutes and resuspended in 30 ml RPMI media containing 10% PB S, 2 mM glutamine and penicillin-streptomycin. 10,000 CCRF-CEM or SKBR3 cells were co-cultured with 300,000 PBMCs per each well in a 96 well U bottom opylene plate. PBMC were prepared from human buffy coats ed from Stanford blood center Three-fold dilutions of the sample variants were added to each well in duplicates, starting from a highest tration of 222 nM and incubated in 37°C for 3 hours. Cells were then lysed in 50 uL of Glo reagent following manufacturer’s instructions. Plates were read on Envision luminescent plate reader (Luminescent 96 well full area program). Percent ADCC activity was calculated as (Treated Cells-Spontaneous Cells)/(Tota1 Lysis-Spontaneous Lysis)* 100.

] Results: ADCC activity was observed in Trastuzumab (CH0) and IgG1 parental- CHO, both expressed in CHO cells, while ADCC activity was not observed (“NA”) in any other anti-CD47 antibody (CHO cell expression or CF expression) tested. ECso values are presented in the table below (Table 12).

Table 12: Antibody Name IC50 (nM) Trastuzumab (CHO) 0.001 IgG4P-parental—CHO IgG4PE—parental—CHO Control IgG4 Isotype IgGl-parental-CF m-CF IgG 1 - 1 3m-CF IgGl- l 3mZ-CF IgG4P-parental-CF IgG4P-5m-CF IgG4PE—parental—CF IgG4PE-5m-CF 6.9 Example 9: Differential Scanning Calorimetry (DSC) (Thermostability) Analysis Method: Differential Scanning Calorimetry (DSC) was performed on a GE VP Capillary DSC ment. Analysis was performed from 20-100°C with 60°C/hr heating ramps.

Feedback mode was disabled, and a ﬁltering period of 105 was utilized. Pre-scan thermostating was set to 5 minutes. All samples were tested at a concentration of 1mg/mL in the following buffer: SOmM L-histidine, lSOmM NaCl, 2% trehalose, pH 6.0 s: Three different anti-CD47 IgGl antibody constructs were analyzed by DSC: 1) IgGl-l3mZ 2) IgGl-l3m 3) IgGl-Sm In particular, the melting transition of the Fab domain (TM2) was of interest, given that the three antibodies differed only in residues d in the Fab domain. As the data shows in Figure 3A, good ative unfolding is observed for the Fab transition for all three ucts, with IgGl-Sm exhibiting the highest TM2.

The table below (Table 13) summarizes the transition temperatures of the three different transitions for all three anti-CD47 IgGl constructs. As can be observed, only minor (<1.0°C) TM differences can be ed for the CH2 (TMl) and CH3 (TM3) transitions. The TM2 of IgGl-Sm, however, does show a 15°C stabilization over the other variants.

Table 13 In particular, the IgGl-Sm exhibits strikingly improved thermal stability when compared against CHO cell-culture derived IgG4PE (CHO ) reference standard e 3B). As can be observed, all thermal tions for the CH0 IgG4PE material lie below 75°C, while the IgGl-Sm is signiﬁcantly stabilized and denatures at higher temperatures, with the exception of the CH2 domain at 622°C.

To see whether the thermal stabilization observed in the IgGl context would also translate to the IgG4 scaffold, the following three anti-CD47 IgG4 variants were compared Via DSC: 1) IgG4PE-5m 2) IgG4P-5m 3) IgG4PE CHO ] As can be seen in the thermogram presented in Figure 3C, Fab region melting in the IgG4P and IgG4PE context is ed by 9°C, by introduction of the 5-mutations, when compared to IgG4PE CHO reference material.

Altogether, these results show that signiﬁcant thermal stabilization of the Fab domain can be achieved by the introduction of select mutations. As the Fab region remains unchanged between IgG1 and IgG4 scaffolds, the thermal stability gains seem to translate from one scaffold to r, indicating that this should also hold true for other IgGl isotypes. 6.10 Example 10: The cokinetics ties of anti-CD47 antibodies Methods: The anti-CD47 antibodies IgG4-PE CHO, IgGl-CF and IgGl-Sm-CF were administered by bolus intravenous injection to mice at dose levels of 3.0, 3.0 and 2.5 mg/kg, respectively, Plasma samples were collectd at selected times out to 28 days (672 hours) after , and the concentration of the respective protein determined by eimmunoassay. The pharmacokinetic parameters were then calculated using a non-compartmental approach with WinNonlin ‘v’ 5.3, Phoenix 64 (Certara, CA). The AUC was calculated using the linear trapezoidal rule for the ascending portion of the curve and the log trapezoidal rule for the descending portion. The al half-life was determined from a regression of the log of the plasma concentration versus time. The number of points used for the regression was determined by visual inspection of the data using a minimum of three terminal time points. The initial volume of distribution was calculated from the dose/plasma concentration extrapolated to zero time. All other parameters were calculated within WinNonlin using standard s.

] Results: The pharmacokinetics of IgG4—PE CHO, IgGl-CF and IgGl-Sm-CF were similar to each other. The clearances are low ing in vely long half-lives despite the volumes of distribution also being low, l for these types of compounds. The initial volume of distribution approximated blood volume whereas the volumes of distribution based on area and at steady-state were approximately half that of extracellular water. c0 Auc.ast AUC... Terminal Cl InitiaIV V. vs.

(Mg/mL) (ug*h/mL) (ug*h/mL) t1/z(h) (mL/h/kg) (mL/kg) (mL/kg) (mL/kg) 10063 11391 218.8 0.26 39.40627873 83.2 80 7009 7368 165 0.41 46.22496148 96.9 100.8 7021 7789 193.1 0.32 51.2295082 89.4 92.4 6.11 Example 11: In Vivo umor Activity The anti-tumor activity of anti-CD47 antibodies produced by the cell-free system were tested in vivo using a xenograft tumor model with the human myeloma cell line RPM18226.

Methods: NOD/SCID mice were injected aneously with RPMI 8226 cells.

Subsequently, mice were treated with vehicle control, hIgG4, or CF D47 antibodies, such as anti-CD47 IgGl-Sm, were administered (qwx3) at a dose of 1 mg/kg, 03 mg/kg, or 0.1 mg/kg.

Tumor volume were monitored.

Results: Figure 5 s a graph of tumor volume versus days after tumor cell inoculation. CF anti-CD47 IgGl—Sm antibody achieved tumor volume reduction (TVR) of 83% at a dose of 1 mg/kg and a TVR of 50% at a dose of 0.3 mg/kg. The tage of tumor free mice at termination is 25% (2/8) for the 1 mg/kg dose of CF anti—CD47 IgGl-Sm antibody.

WO 09415 All references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

] Other embodiments are within the following claims.

Claims

WHAT IS CLAIMED:

1. A monoclonal anti-CD47 antibody which speciﬁcally binds to human CD47, wherein the anti-CD47 antibody, when ed using a cell-free system, has a higher antibody expression titer or yield compared to a parental antibody ed using the cell-free system.

2. The anti-CD47 antibody of claim 1, wherein the antibody expression titer or yield is higher by at least 1 fold, at least 2 fold, or at least 3 fold compared to the parental antibody.

3. The anti-CD47 antibody of claim 1, which is a humanized antibody.

4. The anti-CD47 dy of claim 1, which is an IgGl antibody.

5. The anti-CD47 antibody of claim 1, which is an IgG4 antibody.

6. The D47 antibody of claim 1, which is an IgG4 antibody comprising a S228P amino acid substitution according to the EU numbering index.

7. The anti-CD47 antibody of claim 1, which is an IgG4 antibody comprising a $228P and L235E amino acid tutions according to the EU ing index.

8. The anti-CD47 antibody of any one of claims 1-7, wherein the parental antibody ses a heavy chain variable region comprising the amino acid sequence of SEQ IDNO: l.

9. The anti-CD47 antibody of claim 8, wherein the parental antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 124

10. The anti-CD47 antibody of claim 1, wherein the parental antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, 3 or 4.

11. The D47 antibody of claim 1, which comprises (i) a heavy chain variable region comprising complementarity determining region (CDR) 1, 2, and 3 of antibody 2A1; and (ii) a light chain variable region comprising CDRl, CDR2, and CDR3 of antibody 2A1.

12. The anti-CD47 antibody of claim 1, which comprises (i) a heavy chain variable region comprising complementarity determining region (CDR) 1, 2, and 3 comprising amino acid sequences GFNIKDYYLH (SEQ ID NO: 14), WIDPDQGDTE (SEQ ID NO: 15), and NAAYGSSSYPMDY (SEQ ID NO: 16), respectively; and (ii) a light chain variable region comprising CDRl, CDR2, and CDR3 comprising amino acid ces KASQDIHRYLS (SEQ ID NO: 17), RANRLVS (SEQ ID NO: 18), and LQYDEFPYT (SEQ ID NO: 19), respectively.

13. The anti-CD47 antibody of any one of claims 1—12, which comprises one or more amino acid modiﬁcations ve to the parental antibody.

14. The anti-CD47 antibody of claim 13, wherein the one or more amino acid substitutions is in the framework region of the heavy chain variable region or light chain le region.

15. The anti-CD47 dy of claim 13, which comprises 13 or 14 amino acid modiﬁcations in the framework region of the heavy chain variable region.

16. The anti-CD47 antibody of claim 13, which ses 1 to 15 amino acid modiﬁcations in the framework region of the heavy chain variable region.

17. The anti-CD47 antibody of any one of claims 13—16, wherein the amino acid modiﬁcations are vative amino acid substitutions

18. The anti-CD47 antibody of any one of claims 1-12, which comprises a heavy chain variable region (VH) comprising the amino acid sequence: