AU2023266367B2 - Methods for manipulating phagocytosis mediated by CD74 - Google Patents

Abstract

#$%^&*AU2023266367B220250918.pdf##### ABSTRACT Methods are provided to manipulate phagocytosis of cells, including hematopoietic cells, e.g. circulating hematopoietic cells, bone marrow cells, etc.; and solid tumor cells. In some embodiments of the invention the circulating cells are hematopoietic stem cells, or hematopoietic progenitor cells, particularly in a transplantation context, where protection from phagocytosis is desirable. In other embodiments the circulating cells are leukemia cells, particularly acute myeloid leukemia (AML), where increased phagocytosis is desirable. ABSTRACT Methods are provided to manipulate phagocytosis of cells, including hematopoietic cells, e.g. circulating hematopoietic cells, bone marrow cells, etc.; and solid tumor cells. In some embodiments of the invention the circulating cells are hematopoietic stem cells, or hematopoietic progenitor cells, particularly in a transplantation context, where protection from phagocytosis is desirable. In other embodiments the circulating cells are leukemia cells, particularly acute myeloid leukemia (AML), where increased phagocytosis is desirable. 20 23 26 63 67 17 N ov 2 02 3 A B S T R A C T M e t h o d s a r e p r o v i d e d t o m a n i p u l a t e p h a g o c y t o s i s o f c e l l s , i n c l u d i n g 2 0 2 3 2 6 6 3 6 7 1 7 N o v 2 0 2 3 h e m a t o p o i e t i c c e l l s , e . g . c i r c u l a t i n g h e m a t o p o i e t i c c e l l s , b o n e m a r r o w c e l l s , e t c . ; a n d s o l i d t u m o r c e l l s . I n s o m e e m b o d i m e n t s o f t h e i n v e n t i o n t h e c i r c u l a t i n g c e l l s a r e h e m a t o p o i e t i c s t e m c e l l s , o r h e m a t o p o i e t i c p r o g e n i t o r c e l l s , p a r t i c u l a r l y i n a t r a n s p l a n t a t i o n c o n t e x t , w h e r e p r o t e c t i o n f r o m p h a g o c y t o s i s i s d e s i r a b l e . I n o t h e r e m b o d i m e n t s t h e c i r c u l a t i n g c e l l s a r e l e u k e m i a c e l l s , p a r t i c u l a r l y a c u t e m y e l o i d l e u k e m i a ( A M L ) , w h e r e i n c r e a s e d p h a g o c y t o s i s i s d e s i r a b l e .

Description

METHODSFOR METHODS FORMANIPULATING MANIPULATINGPHAGOCYTOSIS PHAGOCYTOSIS MEDIATED MEDIATED BYBY CD47 CD47

RELATEDAPPLICATION RELATED APPLICATION DATA DATA

This is This is aa divisional divisionalapplication applicationof of Australian Patent Australian Application Patent 2021240137, Application 2021240137, which which

is aa divisional is applicationofofAustralian divisional application Australian Patent Patent Application Application 2019253783, 2019253783, which is awhich is a divisional application divisional applicationofofAustralian Australian Patent Patent Application Application 2018201034, 2018201034, which is awhich is a divisional divisional 2023266367

application of application of Australian AustralianPatent Patent Application Application 2016203572, whichisisaadivisional 2016203572, which divisional application of application of Australian AustralianPatent Patent Application Application2014201010, whichisisaadivisional 2014201010, which divisional application application of of Australian AustralianPatent Patent Application Application2009205665, whichisisthe 2009205665, which the Australian Australian National National Phase ofPCT/US2009/000319 Phase of PCT/US2009/000319filedfiled 15 January 15 January 2009,2009, which which claimsclaims priority priority

from U.S. from U.S. Provisional Provisional Patent Patent Application Application Serial Serial Nos. US61/189,786 Nos. US 61/189,786 filed22 filed 22August August 2008, and 2008, andUSUS61/011,324 61/011,324 filed1515January filed January 2008. 2008. The The contents contents of each of each application application listed listed

in this in this paragraph paragraph areare fully fully incorporated incorporated by reference by reference herein.herein.

BACKGROUND BACKGROUND

[01]

[01] Thereticuloendothelial The reticuloendothelial system system(RES) (RES) is is a part a part of of thethe immune immune system. system. The RESThe RES consists ofof the consists thephagocytic phagocytic cellscells located located in reticular in reticular connective connective tissue, tissue, primarily primarily

monocytes and monocytes andmacrophages. macrophages. The consists The RES RES consists of 1) of 1) circulating circulating monocytes; monocytes; 2) 2) resident macrophages resident macrophages in in the the liver, spleen, liver, spleen, lymph lymphnodes, nodes, thymus, thymus, submucosal submucosal tissues tissues of of the respiratory the respiratory and andalimentary alimentarytracts, tracts,bone bone marrow, marrow, and connective and connective tissues; tissues; and 3) and 3) macrophage-likecells macrophage-like cellsincluding includingdendritic dendritic cells cells in inlymph lymph nodes, Langerhans nodes, Langerhans cellsininskin, cells skin, and microglial and microglial cells cells in in the the central central nervous nervoussystem. system. These These cells cells accumulate accumulate in lymph in lymph

nodesand nodes andthethe spleen. spleen. Thefunctions The RES RES functions topathogens, to clear clear pathogens, particulate particulate matter inmatter in circulation, and circulation, and aged aged or or damaged hematopoietic damaged hematopoietic cells. cells.

[02]

[02] To eliminate To eliminate foreign foreign cells cells or or particles particlesinin the innate the immune innate immune response, macrophage- response, macrophage-

mediatedphagocytosis mediated phagocytosisis isinduced induced when when the the phosphatidylserine phosphatidylserine receptor receptor (PSR) (PSR) reacts reacts to to phosphatidylserine(PS), phosphatidylserine (PS),which whichcan can bebe externalized externalized from from thethe membranes membranes of cells, of dead dead cells, such as such asapoptotic apoptotic and andnecrotic necroticcells. cells. In In turn, turn, the the interaction interactionbetween between PS andPSR PS and PSR plays plays

a crucial a crucial role role in in the the clearance clearance of of apoptotic apoptotic cells cells by by macrophages. Once macrophages. Once phagocytosis phagocytosis

has been has beenperformed performed by by macrophages, macrophages, the inflammatory the inflammatory response response is downregulated is downregulated by by an increase an increaseinin factors factors such suchasasIL-10, IL-10,TGF-B, TGF-β, and and prostaglandin prostaglandin E2 (PGE2). E2 (PGE2). The The strict strict balance between balance between thethe inflammatory inflammatory and and anti-inflammatory anti-inflammatory responses responses in innate in both both innate and and adaptiveimmunity adaptive immunity plays plays a critical a critical role role in maintaining in maintaining cellular cellular homeostasis homeostasis and and protecting protecting a host a hostfrom fromextrinsic extrinsic invasion. invasion.

[03]

[03] Thecausal The causalrelationship relationshipbetween between inflammation inflammation and and the neoplastic the neoplastic progression progression is a is a conceptwidely concept widelyaccepted. accepted.Data Data nownow support support the the concept concept of cancer of cancer immunosurveillance immunosurveillance - - that one that of the one of the physiologic functions of physiologic functions of the the immune system immune system is is to to recognize recognize andand destroy destroy

transformedcells. transformed cells. However, However, some some tumor tumor cellscells are capable are capable of evading of evading recognition recognition and and destruction by destruction the immune by the system. immune system. Once Once tumor tumor cells cells havehave escaped, escaped, the immune the immune system system mayparticipate may participate in in their their growth, growth, for for example example by promoting by promoting the vascularization the vascularization of tumors. of tumors.

[04]

[04] Both adaptive Both adaptiveandand innate innate immune immune cells participate cells participate in theinsurveillance the surveillance and theand the elimination of elimination of tumor cells, but tumor cells, but monocytes/macrophages monocytes/macrophages may may befirst be the the first lineline of of defense defense

in tumors, in tumors, asasthey theycolonize colonize rapidly rapidly and and secrete secrete cytokines cytokines that attract that attract and activate and activate

dendritic cells cells (DC) andNK NK cells, which in turn can initiate the adaptive immune 2023266367

dendritic (DC) and cells, which in turn can initiate the adaptive immune

responseagainst response againsttransformed transformed cells. cells.

[05]

[05] Tumors that Tumors that escape escape from from the the immune machinery can immune machinery be aa consequence can be consequence of of alterations occurring alterations occurring during during the the immunosurveillance phase.As As immunosurveillance phase. an an example, example, somesome tumortumor

cells develop cells deficienciesininantigen develop deficiencies antigen processing processing and presentation and presentation pathways, pathways, which which facilitate evasion facilitate evasionfrom from an an adaptive adaptive immune response, immune response, such such as the as the absence absence or abnormal or abnormal

functions of functions of components of the components of the IFN-y IFN-γreceptor receptor signaling signaling pathway. pathway. Other Other tumors tumors suppressthe suppress theinduction inductionof of

1A 1A danger proinflammatorydanger proinflammatory signals, signals, leading, forfor leading, example, example, to impaired to impaired DC maturation. DC maturation. Finally, Finally, 17 Nov 2023 the of the inhibition of the inhibition protective functions the protective of the functions of immune the immune system system mayfacilitate may also tumor tumor also facilitate escape, thethe suchas as escape,such overproduction overproduction of the the anti-inflammatory of anti-inflammatory cytokines cytokines IL-10 IL-10 and and TGF-B, TGF-fi, which can be which can many producedbybymany be produced tumor tumor cellsthemselves cells butbut themselves alsobyby also macrophages or Tor macrophages T cells. regulatorycells. regulatory

[06]

[06] A tumor A canbebe tumorcan viewed viewed as an organorgan an aberrant as aberrant initiated by a by initiated tumorigenic cancercancer a tumorigenic cell cell that that acquired the acquired indefinite proliferation for indefinite capacityfor the capacity proliferation throughaccumulated through accumulated mutations. view this view mutations.InInthis of aa tumor of tumor as abnormal asananabnormal organ, the the organ, principles of of principles normal normal cell cell stemstem biology can can biology be applied be applied 2023266367

better understand to better to how tumors understand how Many develop.Many tumorsdevelop. observations observations suggest that that suggest analogies analogies

between normal betweennormal stem stem cells and and cells tumorigenic cellscells tumorigenic are appropriate. are appropriate. Both normal Both normal stem stem cells cells tumorigenic andtumorigenic and havehave cells cells extensive extensive proliferative proliferative potential potential and the and the ability to ability give rise newrise give to to to new (normal or (normal tissues. Both abnormal) tissues, or abnormal) tumors and Both tumors normal tissues and normal are composed tissues are of composed of heterogeneous heterogeneous combinations combinations of cells, of cells, with with different phenotypic differentphenotypic characteristics andand characteristics different different

potentials. proliferative potentials. proliferative

[07]

[07] Stemcells Stem definedasas aredefined cells are cells thathave cellsthat thethe have ability perpetuate abilitytotoperpetuate themselves themselves through through

self-renewal generate andtotogenerate self-renewal and mature mature cells of of cells a particular a particular tissue tissue through through differentiation.In In differentiation.

mosttissues, most stemcells tissues, stem rare.As As arerare. cellsare a result, stem a result,stem cells cells must must be identified be identified prospectively prospectively

and purified carefully and purified in order carefully in to study order to their properties. studytheir Perhaps properties. Perhaps mostmost the the important important and and useful property usefulproperty of of cellscells stem stem is that is that of self-renewal. Through Through of self-renewal. this property, striking property,parallels this striking parallels can bebefound can between foundbetween cells cells stem stem and cancer tumors tumors cells: cells: and cancer may may often often originate originate from the from the transformation of normal transformation of stemcells, normalstem similar signaling cells,similar may pathwaysmay signaling pathways regulate regulate self-renewal self-renewal in in stem and cancer cells and stemcells cells, and cancercells, may cancersmay andcancers comprise rarerare comprise cells cells withwith indefinitepotential indefinite for potentialfor self-renewal self-renewal that drive tumorigenesis. that drive tumorigenesis.

[08]

[08] Study cell surface Studyofofcell markers surfacemarkers specific specific or or to to specifically upregulated specificallyupregulated in cancer in cancer cells cells is is in providing pivotal in pivotal targets for providing targets reducing growth for reducing depleting growthofofororforfordepleting cancer cancer cells. cells. Provided Provided

herein is herein markerfor is aa marker leukemia, myeloidleukemia, formyeloid especially especially a marker a marker for Acute for Acute Myeloid Myeloid Leukemia Leukemia

studies Ourstudies (AML). Our (AML). have have revealed revealed a role a role of this of this marker marker in helping in helping AML AML stem stem cells cells avoid avoid clearance by clearance phagocytosis. Methods by phagocytosis. are are Methods provided for for provided using thisthis using marker marker to increase to increase

phagocytosis of AMLstem of AML (AML cells(AML stemcells SCs), SCs), wellwell as as as to to improve as improve transplantation transplantation of of hematopoieticand hematopoietic progenitor andprogenitor stem stem cells. cells.

[09]

[09] Interestingly, certain Interestingly, markers certain areare markers showntotobe shown shared by be shared by leukemia stem cells leukemia stem and cells and hematopoietic cells (HSCs). hematopoietic stem cells During normal (HSCs). During HSCs development, HSCs normal development, migrate ectopic to toectopic migrate niches fetal and in fetal niches in and adult life via adult life blood stream. the blood via the Once stream. Once thethe in in blood blood stream, HSCsHSCs stream, must must navigate thevascular navigate the beds vascularbeds of the of the spleen spleen and liver and liver before before settling settling in a niche. in a niche. At At these these vascular macrophages beds,macrophages vascular beds, function function to remove to remove damaged cells andcells damaged foreign foreign particles and particles from from the blood stream. the blood Furthermore, during stream. Furthermore, states,macrophages inflammatory states, during inflammatory macrophages become more becomemore active. TheThe phagocytically active. phagocytically newly newly arriving cells cells stem stem arriving thus face face thusthe the possibility possibility of of being being

2 phagocytosed phagocytosed while while en en route, route, unless unless additional additional protection protection cancan be be generated. generated. Exploration Exploration of of 17 Nov 2023 mechanismsbybywhich mechanisms which thethe endogenous endogenous HSC avoid HSC avoid being being cleared cleared by phagocytosis by phagocytosis can can provide insight provide insight into intoways for improving ways for transplantation success improving transplantation success of of hematopoietic and hematopoietic and progenitor stem progenitor stemcells. cells. The presentinvention The present inventionsatisfies satisfies these, and other, these, and other, needs. needs.

SummaryOFofTHE SUMMARY Invention theINVENTION

[10]

[10] Methods are Methods areprovided providedtotomanipulate manipulatephagocytosis phagocytosisofofhematopoietic hematopoieticcells, cells, including including circulating hematopoietic circulating hematopoieticcells, cells,e.g.e.g. bone marrow bone marrowcells. cells. InInsome embodimentsofofthe some embodiments the 2023266367

invention the invention the circulating circulating cells cells are are hematopoietic hematopoietic stem stem cells, cells, or hematopoietic or hematopoietic progenitor progenitor

cells, particularly cells, particularly in a transplantation in a transplantationcontext, context,where where protection protection from phagocytosis from phagocytosis is is desirable. InIn other desirable. otherembodiments embodimentsthe the circulating circulating cells cells areare leukemia leukemia cells, cells, particularly particularly acute acute

myeloid leukemia myeloid leukemia(AML), (AML), where where increased increased phagocytosis phagocytosis is desirable. is desirable. In In certain certain embodiments of embodiments of the the invention, invention, methods are provided methods are provided to to manipulate manipulate macrophage macrophage phagocytosisofofcirculating phagocytosis circulating hematopoietic hematopoietic cells.InInyet cells. yetother otherembodiments embodiments of the of the invention, invention,

methodsare methods areprovided provided to to manipulate manipulate phagocytosis phagocytosis of solid of solid tumors. tumors.

[11]

[11] In some In embodiments some embodiments of of thethe invention,hematopoietic invention, hematopoieticstem stem or or progenitorcells progenitor cells are are protected from protected fromphagocytosis phagocytosisin in circulationbybyproviding circulation providinga host a host animal animal with with a CD47 a CD47 mimetic mimetic

molecule, which molecule, interacts with which interacts with SIRPa on phagocytic SIRPa on phagocytic cells, cells, such such as, as, macrophages, and macrophages, and

decreases phagocytosis. decreases phagocytosis. The TheCD47 CD47 mimetic mimetic maymay be soluble be soluble CD47; CD47; CD47CD47 coated coated on on the the surface of surface of the the cells cellstotobebeprotected, protected,a aCD47 CD47 mimetic mimetic that thatbinds bindstotoSIRPa SIRPa at at the the CD47 CD47

binding site, binding site, and the like. and the like. In In some someembodiments embodiments ofinvention, of the the invention, CD47 CD47 is is provided provided as a as a fusion protein, fusion protein, for for example solubleCD47 example soluble CD47 fused fused to Fc to an an fragment, Fc fragment, e.g.,e.g., IgG1 IgGI Fc, Fc, Fc, IgG2 lgG2 Fc, Ig A Ig Fcetc. A Fc etc.

[12]

[12] In other In other embodiments, embodiments, tumor tumor cells,cells, e.g. e.g. solid solid tumor tumor cells, cells, leukemia leukemia cells, cells, etc. areetc. are targeted for targeted for phagocytosis phagocytosisbybyblocking blocking CD47 CD47 on cell on the the cell surface. surface. It isItshown is shown that that leukemia leukemia

cells, particularly cells, particularlyAML AMLcells, cells,evade evademacrophage surveillance by macrophage surveillance by upregulation upregulation of of CD47 CD47

expression. Administration expression. Administration of of agents agents that that mask mask the the CD47CD47 protein, protein, e.g. e.g. antibodies antibodies that that bind bind to CD47 to CD47and and prevent prevent interaction interaction between between CD47 CD47 andare and SIRPa SIRPa are administered administered to a to a patient, patient, whichincreases which increasesthetheclearance clearance of AML of AML cellscells via phagocytosis. via phagocytosis. In other In other aspects, aspects, an an agent agent that masks that CD47isiscombined masks CD47 combinedwith withmonoclonal monoclonal antibodiesdirected antibodies directed against against one oneoror more more additional AMLSC additional markers, AMLSC markers, e.g.e.g. CD96, CD96, andlike, and the the like, which which compositions compositions can be can be synergistic synergistic

in enhancing in phagocytosis and enhancing phagocytosis and elimination elimination of of AMLSC AMLSC asascompared comparedto to thethe use use of of single single

agents. In agents. In other other embodiments, cells of embodiments, cells of solid solid tumors are targeted tumors are targeted for for phagocytosis phagocytosis by by blocking CD47 blocking CD47present present on on thethe cellsurface. cell surface. ;

[13]

[13] In another In another embodiment, embodiment, methods methods are provided are provided for targeting for targeting or depleting or depleting AML AML cancer cancer stemcells, stem cells, the themethod method comprising comprising contacting contacting a population a population of e.g. of cells, cells,blood e.g.from blood a from a leukemiapatient, leukemia patient, with with aareagent reagentthat thatspecifically specificallybinds bindsCD47 CD47 in order in order to target to target or deplete or deplete

3

AMLSC. AMLSC. In certain In certain aspects, thethe aspects, reagent reagent is an is an antibody antibody conjugated to a to conjugated a cytotoxic cytotoxic agent, e.g. e.g. agent, 17 Nov 2023

isotope, chemotherapeutic radioactive isotope, radioactive chemotherapeutic agent, toxin, etc. agent, toxin, In some etc. In embodiments, someembodiments, the the depletion is performed depletion is onananexexvivo performed on e.g. the purging cells, e.g. populationofofcells, v/Vopopulation of autologous purging of stem autologous stem

cell products cell products (mobilized peripheral blood (mobilized peripheral bone blood oror bone marrow) for for marrow) use in in autologous useautologous for patients transplantation for transplantation with acute patients with acute myeloid leukemia.In In myeloid leukemia. another another embodiment, embodiment, methods methods

are providedfor are provided targeting cancer for targeting cells of cancercells solid tumor of a solid in a human tumor in human subject byby subject administering administering

an antibody an CD47 againstCD47 antibodyagainst thethe to to subject. subject. 2023266367

Brief D BRIEF escription OF DESCRIPTION Drawings The DRAWINGS Of THE

[14]

[14] Figure 1. FACS Figure 1. of human analysis of FACS analysis HSCand human HSC progenitor CD47 andprogenitor from expression from CD47expression (MDS, syndrome(MDS, Myelodysplastic syndrome Myelodysplastic blue), Chronic blue),Chronic Myelogenous Myelogenous Leukemia, Leukemia, Accelerated Accelerated

Phase (CML Phase(CML AP,AP, green) and and green) normal normal marrowmarrow bone bone (red). (red).

[15]

[15] Figure 2. Figure ETvs. 2. ET FACS PV.FACS vs.PV. analysis analysis of CD47 of CD47 expression expression human myeloproliferative by humanbymyeloproliferative

disorders thrombocythemia essentialthrombocythemia suchasasessential disorders such (ET,(ET, blue) and and blue) polycythemia polycythemia vera (PV, (PV, green) veragreen) HSC, and lineage progenitor and HSC, progenitor cellscompared positive cells lineage positive with human compared with human normal bone normalbone marrow marrow

(red). (red).

[16]

[16] 3A. Progenitor Figure 3A. Normal ProfilesofofNormal ProgenitorProfiles Bone Bone Marrow Marrow (left), (left), post-polycythemic post-polycythemic

myelofibrosis withmyeloid myelofibrosis with metaplasia(PPMM) myeloidmetaplasia and CML (PPMM) and Crisis. Figure BlastCrisis. CMLBlast FACS 3B. FACS Figure3B. analysis ofhuman analysis of normal bone human normal bone marrow versus UMPD (red) versus marrow (red) (green) versus UMPD (green) (blue == ML) PV(blue versus PV ML)

versus atypical versus CML(orange), atypical CML HSC, (orange),HSC, progenitor and and progenitor lineage lineage positive cellcell positive CD47CD47 expression. expression.

[17]

[17] Figure 4. Figure 4. Increased CD47 Increased CD47 Expression by by Expression CMML CMML Progenitors Progenitors (blue) (blue) compared compared with with normalbone normal marrow bonemarrow (red) (red) with with disease disease progression. progression.

[18]

[18] Figures 5A-5B.(A) Figures 5A-5B. Normal ProfilesofofNormal ProgenitorProfiles (A)Progenitor bone bone marrow marrow (left) (left) versus AML AML versus (right). (right).

(B) FACS (B) ofof analysis FACSanalysis human human normal normal marrowmarrow bone bone (red) versus (red) versus AML (blue) (blue) AMLHSC, HSC, progenitor progenitor

and lineagepositive and lineage (blast)CD47 cell (blast) positive cell expression. CD47 expression.

[19]

[19] Figure 6. CD47 Figure 6. is More CD47 is Highly Expressed MoreHighly LSC LSC AML ExpressedononAML Compared Compared to Their NormalNormal to Their Counterparts. Counterparts. A. Relative CD47 A. Relative expression on CD47 expression bonemarrow normal bone on normal (Lin-(Lin- HSCHSC marrow CD34+CD38-CD90+)and CD34+CD38-CD90+) MPP andMPP (Lin-CD34+CD38-CD90-CD45RA-), (Lin-CD34+CD38-CD90-CD45RA-) as well as well as LSC as LSC (Lin- (Lin- CD34+CD38-CD90-) CD34+CD38-CD90-) andand bulk bulk leukemia leukemia cells from human cellsfrom AML humanAML samples waswas samples determined determined by by cytometry. Mean flow cytometry. flow Mean fluorescence intensity was fluorescence intensity was normalized cell size for cell normalized for against and against size and lineage positive cells lineage positive to account cells to account for analysis on for analysis days. The different days. on different The same sample samesample of normal of normal

marrow bonemarrow bone (red, or or n=3) (red,n=3) AML AML (blue, (blue, n=13) n=13) is indicated thethe by by is indicated same same symbol symbol the different in thein different

populations. The differences populations. The the mean between the differencesbetween mean expression HSCwith of HSC expression of (p=0.003), LSC(p=0.003), with LSC HSC with bulk HSCwith (p=0.001), MPP leukemia (p=0.001), bulk leukemia with LSC MPP with (p=0.004), and LSC (p=0.004), MPPwith and MPP leukemia bulk leukemia with bulk were (p=0.002)were (p=0.002) statistically significant using statisticallysignificant a 2-sided usinga 2-sided Student’s Student's t-test. t-test. The difference The difference

between the between meanexpression the mean LSCLSC AML expressionofofAML compared compared to bulk to bulk AML was was AMLnot not statistically statistically

4 significant with p=0.50 significant with p=0.50 using paired usinga apaired 2-sided 2-sided Student’s Student's t-test. t-test. B. Clinical and and B. Clinical molecular molecular 17 Nov 2023 characteristics of characteristics primary human of primary AML human AML samples samples manipulated manipulated in vitro in vitro and/or and/or in vivo. in vivo.

[20]

[20] Figure 7. Figure Antibody Anti-CD47Antibody 7. Anti-CD47 Stimulates Stimulates In Vitro In Vitro Macrophage Macrophage Phagocytosis Phagocytosis of Primary of Primary

Human AML Human AML AML LSC. AMLLSC. LSC were LSC were purified purified by FACS from from by FACS two primary two primary human AML AML human samples,labeled samples, withthe labeledwith fluorescentdyedye thefluorescent CFSE, CFSE, and incubated and incubated with mouse with mouse bone bone marrow- marrow- macrophages derived macrophages derived either either thethe in in presence presence of an an isotype of isotype control (A) (A) control or anti-CD47 or anti-CD47 antibody antibody

(B). These (B). These cells were assessed cells were assessed by microscopy immunofluorescencemicroscopy by immunofluorescence for presenceofof thepresence forthe fluorescently labeled LSC fluorescently labeled within the LSC within macrophages. the macrophages. The The (C) (C) phagocytic index index phagocytic was was 2023266367

determined for determined eachcondition for each calculating the condition bybycalculating number the number of of ingested ingested cells 100 100 perper cells macrophages. macrophages.

[21]

[21] Figure 8A-C. Monoclonal 8A-C. Monoclonal Antibodies Antibodies Directed Directed Against Against Human CD47 CD47 Human Preferentially Preferentially

Enable Human AML PhagocytosisofofHuman Enable Phagocytosis AML LSC by Human LSC by and Humanand Mouse Mouse Macrophages. A,B.A,B. Macrophages. LSC AMLLSC CFSE-labeled AML CFSE-labeled were were incubated human withhuman incubatedwith peripheral blood-derived macrophages peripheralblood-derived macrophages

(A) or mouse (A) mouse bone marrow-derived bonemarrow-derived macrophages macrophages (B) in in the (B) the presence presence of IgGI of IgG1 isotype isotype

control, anti-CD45 control, anti-CD45 IgG1, anti-CD47(B6H12.2) lgG1,ororanti-CD47 IgG1lgG1 (B6H12.2) antibody. antibody. These These were were cells cells assessed assessed

immunofluorescence by immunofluorescence by microscopy microscopy for the the presence for presence of fluorescently of fluorescently labeled labeled LSC within LSC within the the macrophages(indicated macrophages arrows). C. by arrows). (indicated by AML CFSE-labeledAML C. CFSE-labeled LSCLSC or normal bonebone or normal marrow marrow

CD34+cells CD34+ incubated wereincubated cellswere with with human human (left) or or (left) mouse mouse (right) (right) macrophages macrophages in in the the of the presence of presence indicated antibodies the indicated then assessed and then antibodies and phagocytosisby by assessedforforphagocytosis immunofluorescence immunofluorescence microscopy. microscopy. The phagocytic The phagocytic index index was was determined determined for each for each condition condition by the number calculating the by calculating of ingested number of per 100 cells per ingested cells macrophages.For 100 macrophages. AML ForAML LSC, LSC, the the differences between differences between isotype or anti-CD45 isotype or withblocking antibodywith anti-CD45antibody anti-CD47 blockinganti-CD47 antibody antibody treatment (B6H12.2 treatment(B6H12.2 andand BRIC126) were were BRIC126) statistically statistically significant significant with with p<0.001 p<0.001 for all for all pain/vise pairwise

with human comparisons with comparisons human and mouse andmouse macrophages. ForFor macrophages. human human macrophages, the the macrophages, differences between differences between AMLAML LSC LSC and normal normal and CD34+ CD34+ cells were statistically significantsignificant cells were statistically for for (p<0.001) and B6H12.2(p<0.001) B6H12.2 (p=0.002). BRIC126(p=0.002). and BRIC126

[22]

[22] Figure Anti-CD47 9. Anti-CD47 Figure 9. Antibody Antibody stimulates stimulates in vitro in vitro macrophage macrophage phagocytosis phagocytosis of primary of primary

human AML human LSC.AMLAML AMLLSC. LSC were LSC were purified purified by FACS fourfour fromfrom by FACS primary primary human AML AML human samples, labeled samples, the fluorescent with the labeled with dye CFSE, fluorescentdye CFSE, and incubatedwith andincubated human with human peripheral peripheral

macrophages blood macrophages blood either either in the the presence in presence of an isotype isotype isotype control,control, of an isotype matched matched anti- anti- CD45, ororanti-CD47 CD45, (A)These antibody. (A) anti-CD47antibody. assessed wereassessed cellswere Thesecells by immunofluorescence by immunofluorescence

microscopy for microscopy presence of the presence for the LSC within fluorescently-labeled LSC of fluorescently-labeled macrophages. The themacrophages. withinthe The phagocytic was indexwas phagocyticindex determined determined eacheach for for condition condition by calculating by calculating the number the number of ingested of ingested

cells per cells macrophages. 100 macrophages. per 100 TheThe (B)(B) macrophages macrophages were harvested, stainedstained were harvested, with a with a fluorescently labeled fluorescently anti-human labeled anti-human macrophage macrophage antibody, antibody, and analyzed and analyzed by flow cytometry. by flow cytometry.

hMac+CFSE+ hMac+CFSE+ double double positive identify macrophages eventsidentify positiveevents macrophages that have phagocytosed that have CFSE- phagocytosed CFSE-

Each LSC.Each labeled LSC. labeled sample sample is represented by aby is represented a different different color. color.

5

[23]

[23] Figure 10A-B: Figure 10A-B: AA Monoclonal Monoclonal Antibody Antibody Directed DirectedAgainst AgainstHuman CD47Inhibits Human CD47 Inhibits AML LSC AML LSC 17 Nov 2023

EngraftmentInInVivo. Engraftment Vivo.Three Three primary primary human human AML samples AML samples were incubated were incubated with IgG1with lgG1 isotype isotype control, anti-CD45 control, lgG1,ororanti-CD47 anti-CD45 IgG1, anti-CD47 lgG1 IgG1 antibody antibody (B6H12.2) (B6H12.2) prior prior to transplantation to transplantation into into

newborn NOG newborn NOG mice. mice. A portion A portion of of thethecells cellswas wasanalyzed analyzedforforcoating coatingbybystaining staining with with aa secondaryanti-mouse secondary anti-mouse IgG IgG antibody antibody and analyzed and analyzed by flow by flow cytometry cytometry (A). 13 (A). 13 weeks weeks later, later, mice were mice were sacrificed sacrificed and the bone and the bone marrow marrowwas was analyzed analyzed forfor thepercentage the percentage of of human human

CD45+CD33+ CD45+CD33+ myeloid myeloid leukemia leukemia cellscells by flow by flow cytometry cytometry (B). (B). The difference The difference in mean in mean

engraftment between engraftment between anti-CD47-coated anti-CD47-coatedcells cells and and both both isotype isotype (p<0.001) (p<0.001) and andanti-CD45 anti-CD45 2023266367

(p=0.003) (p=0.003) coated coated cells cells was statistically was statistically significant. significant.

[24]

[24] Figure 11. Figure 11. CD47 CD47 is upregulated is upregulated in murine in murine acuteacute myeloid myeloid leukemia. leukemia. Typical Typical stem and stem and progenitor plots progenitor plotsare areshown shown for for leukemic leukemic hMRPSbcrabl hMRP8bcrabl X xhMRP8bcl2 hMRP8bc/2 cells cells compared compared to to control non-leukemic control animals.Lin- non-leukemic animals. Lin- c-Kit+ c-Kit+ Sca-1+ Sca-1+ gated gated cells cells fromfrom control control bonebone marrow marrow (a) (a) and leukemic and leukemicspleen spleen (b)(b) andand Lin-Lin- c-Kit+ c-Kit+ Sca-1- Sca-1- gatedgated cells cells from from control control bone marrow bone marrow (c) (c) and leukemic and leukemicspleen spleen (d)(d) demonstrate demonstrate perturberances perturberances in normal in normal hematopoiesis hematopoiesis in leukemic in leukemic

mice. Frequency mice. Frequencyis isshown shown as as a percentage a percentage of entire of entire marrow marrow or spleen or spleen mononuclear mononuclear

fraction, (e) fraction. (e) Quantitative Quantitative RT-PCR RT-PCR shows shows that that CD47 CD47 is upregulated is upregulated in leukemic in leukemic BM cells.BM cells. Data are Data are shown shown from from 3 sets 3 sets of mice of mice transplanted transplanted with either with either leukemic leukemic or control or control

hRMPSbcrablX xhMRP8bc/2 hRMP8bcrabl hMRP8bc/2 BM cells BM cells and and thenthen sacrificed2-62-6weeks sacrificed weeks later.Results later. Resultswere were normalized to normalized to beta-actin beta-actin and 18SrRNA and 18S rRNA expression. expression. Fold Fold change change relative relative to control to control

transplanted whole transplanted Bcl-2+ BM whole Bcl-2+ BMcells cells was wasdetermined. determined.Error Errorbars bars represent represent 1 s.d.(f) (f) 1 s.d.

Histogramsshow Histograms show expression expression of CD47 of CD47 on gated on gated populations populations for leukemic for leukemic (gray) (gray) and and control control (black)mice. (black) mice.

[25]

[25] Figure 12. Figure 12. GMP GMP expansion expansion andand CD47CD47 upregulation upregulation in human in human myeloid myeloid leukemia, leukemia. a) a) Representative FACS Representative plots of FACS plots of myeloid myeloid progenitors progenitors (CD34+CD38+Lin-) including common (CD34+CD38+Lin-) including common myeloid progenitors myeloid progenitors(CMP), (CMP), megakaryocyte-erythroid megakaryocyte-erythroid progenitors progenitors (MEP) (MEP) and and granulocyte- granulocyte-

macrophageprogenitors macrophage progenitors (GMP) (GMP)inin normal normalbone bonemarrow marrow (BM) (BM) versus versus aCML, aCML, BC CML BC CML and and AML.b)b)Comparative AML. Comparative FACS FACS histograms histograms ofexpression of CD47 CD47 expression by(red; by normal normal (red; n=6) and n=6) acuteand acute myelogenousleukemic myelogenous leukemic(AML, (AML, blue;n=6) blue; n=6) hematopoietic hematopoietic stem stem cells(HSC; cells (HSC; CD34+CD38- CD34+CD38-

CD90+Lin-) and CD90+Lin-) andprogenitors progenitors (CD34+CD38+Lin-). (CD34+CD38+Lin-). c) Comparative c) Comparative FACS FACS histograms histograms of of CD47expression CD47 expressionby bynormal normal(red) (red) and and chronic chronic myelogenous myelogenousleukemia leukemiahematopoietic hematopoieticstem stem cells (HSC; cells (HSC;CD34+CD38-CD90+Lin) and CD34+CD38-CD90+Lin) and committed committed progenitors(CD34+CD38+Lin-). progenitors (CD34+CD38+Lin-). Upper Upper

panel: Normal panel: Normal(n=7) (n=7)versus versuschronic chronicphase phase CMLCML (n=4)(n=4) HSC, HSC, progenitors progenitors and lineage and lineage

positive cells. positive cells.Middle Middlepanel: panel:Normal Normal(n=7) (n=7)versus versus accelerated accelerated phase phase CML (n=7)HSC, CML (n=7) HSC, progenitors and progenitors andlineage lineagepositive positivecells. cells. Lower Lower panel: panel: Normal Normal (n=7)(n=7) versus versus blast blast crisis crisis CML CML (n=4) HSC, (n=4) HSC,progenitors progenitorsand and lineage lineage positive positive cells. cells.

[26]

[26] Figure 13. Figure 13. Over-expression Over-expression of of murine murine CD47 CD47 increases increases tumorigenicity tumorigenicity of MOLM-13 of MOLM-13 cells. cells, a) MOLM-13 a) MOLM-13 cells cells werewere transduced transduced with either with either controlcontrol virus virus or orexpressing virus virus expressing murine murine

6

CD47 formform cDNA CD47cDNA 2. The The resulting 2. resulting cell lines, cell lines, termed Tet or Tet termed or Tet-CD47, Tet-CD47, were transplanted were transplanted 17 Nov 2023

competitively into competitively RAG/common into gamma RAG/common gamma chain chain deficient mice with deficient mice MOLM-13 untransduced MOLM-13 with untransduced cells (5x105 cells (5x105 Tet (n=6) or Tet (n=6) Tet-47(n=8) or Tet-47 cells with (n=8)cells MOLM-13). 5x105MOLM-13). with 5x105 Mice Mice were analyzed were analyzed for for GFP CD45 humanCD45 and human GFP and chimerismwhen chimerism moribund. b) b) whenmoribund. MOLM-13 MOLM-13 chimerism in in chimerism tissues was hematopoietic tissues hematopoietic determined by was determined CD45 humanCD45 by human chimerism and and chimerism measurement measurement of of tumor lesion size. tumorlesion micecompetitively Survivalofofmice size, c)c)Survival transplantedwith competitivelytransplanted plus plus MOLM-13 withMOLM-13 Tet Tet or or Tet-CD47 Tet-CD47 MOLM-13 cells cells MOLM-13 was plotted. was plotted. Control Control miceofdied mice died large largeburden oftumor burden tumor at at the the site site of but had injection but of injection had no engraftment noengraftment in hematopoietic in hematopoietic tissues, tissues. d) Hematoxylin d) Hematoxylin and and eosin eosin 2023266367

sections of Tet-CD47 sections of MOLM-13 Tet-CD47MOLM-13 transplanted liver liver transplanted (200x). (200x). Periportal Periportal (arrow) (arrow) and sinusoidal and sinusoidal

tumor (arrowhead)tumor (arrowhead) infiltration is infiltration 1x106Tet evident, e)e) 1x106 is evident. Tet (n=5) or or (n=5) Tet-CD47 Tet-CD47 MOLM-13 MOLM-13 (n=4) (n=4) cells were cells were injected into the injected into rightfemur the right RAG2-/-, Gc-/- femurofofRAG2-/-, mice and Gc-/- mice and the were tissues were the tissues analyzed 50-75 days analyzed 50-75 later and days later of MOLM-13 chimerism of and chimerism MOLM-13 cells in bone cells in bone marrow was marrow was determined,f) f) determined. Survival Survival curve curve of mice of mice transplanted transplanted intrafemorally intrafemorally withor Tet with Tet or Tet-CD47 Tet-CD47

MOLM-13 MOLM-13 cells. Examples cells, g) g)Examples of liver tumor of livertumor formationandand formation hepatomegaly hepatomegaly in Tet-CD47 in Tet-CD47

MOLM-13 MOLM-13 transplanted transplanted mice mice versus versus control control transplanted mice.mice, transplanted GFP fluorescence GFP fluorescence

demonstrates demonstrates tumor tumor nodule nodule formation formation as well as well diffuse diffuse infiltration. infiltration.

[27]

[27] Figure 14. Figure CD47 14. CD47 over-expression over-expression prevents prevents phagocytosis phagocytosis of unopsonized of live MOLM-13MOLM-13 live unopsonized cells, a) Tet cells. or Tet-CD47 Tet or MOLM-13 Tet-CD47MOLM-13 cells cells werewere incubated incubated bonebone withwith marrow marrow derived derived

macrophages (BMDM) macrophages(BMDM) 4, 4,oror6 6hours 2, 2, forfor andphagocytic hoursand was indexwas phagocyticindex determined. Error determined.Error bars s.d. (n=6 represent11 s.d. bars represent for each (n=6 for point), b) timepoint). eachtime FACS b) FACS analysis of of analysis BMDMs BMDMs incubated incubated with with either Tet either Tet-CD47 or Tet-CD47 Tet or cells,c) c) cells. Photomicrographs Photomicrographs of BMDMs incubatedincubated of BMDMs with with Tet or Tet Tet- or Tet- MOLM-13 CD47MOLM-13 CD47 cells cells and2424hours atat2 2and (400X). d)d)Tet hours(400X). or Tet-CD47 Tetor MOLM-13 Tet-CD47MOLM-13 cells cells were were

transplanted intoRAG2-/-, transplanted into miceand Gc-/-mice RAG2-/-,Gc-/- spleen, and marrow, spleen, and marrow, macrophages were livermacrophages and liver were

analyzed 22 hours analyzed GFP+fraction later, GFP+ hours later. of macrophages fraction of macrophages are gated. Results are gated. are Results are representative of representative experiments. of 33 experiments.

[28]

[28] Figure Higher 15. Higher Figure 15. expression expression of CD47 of CD47 on MOLM-13 on MOLM-13 cells correlates cells correlates with tumorigenic with tumorigenic

potential and potential phagocytosis,a) a) evasionofofphagocytosis. and evasion Tet-CD47 Tet-CD47 MOLM-13 MOLM-13 cells were were divided cellsdivided into into high high and low expressing and low as described. clones as expressing clones Histograms show described. Histograms CD47expression show CD47 MOLM-13 in MOLM-13 expressionin high (black), high MOLM-13 lowlow (black), MOLM-13 (gray), andand (gray), mouse marrowmarrow bone bone mouse (shaded) (shaded) cells. Value Value obtained cells. obtained for MFI/FSC2 for MFI/FSC2 (x109) are shown, (x109) are shown. b) Micetransplanted b) Mice with CD47hi transplanted with MOLM-13 CD47hi MOLM-13 cells were cellswere given doxycycline given weeks.TheThe for 22 weeks. doxycyclinefor histograms histograms levellevel showshow of CD47 of CD47 expression expression in untreated in untreated

(shaded) (shaded) and treated (shaded) and treated with the mice, with (shaded) mice, values of the values MFI/FSC2 (x109) of MFI/FSC2 c) indicated, c) (xIO9) indicated. Survival of RAG2-/-,Gc-/- Survival of RAG2-/-,Gc-/- mice transplantedwith micetransplanted 106 CD47hl, 1 x CD47hi, withX 106 CD4710 CD47'0 MOLM-13 cells, orcells, MOLM-13 or CD47hi MOLM-13 CD47hiMOLM-13 with with cellscells doxycycline doxycycline administration after after administration 2 weeks 2 weeks post-transplant, post-transplant. d) d) Liver and spleen Liver and spleensize sizeofofmice miceatatnecropsy necropsy daysdays 75 75 or or after after transplant transplant 1 x 1106 withwith 106 CD47hi, x CD47hi, CD47¹ MOLM-13 CD4710MOLM-13 cells, CD47hl cells,ororCD47hi MOLM-13 MOLM-13 cells cells withwith doxycycline doxycycline administration after 22 administrationafter weeks post-transplant. weeks e) Bone post-transplant, e) marrowand Bone marrow spleenchimerism andspleen human chimerismofofhuman cells at mice at cellsinin mice

7 necropsy or 75 necropsy 75 days days after aftertransplant withwith transplant 1 x 1106106 CD47hl, CD47'° CD47hi, CD47 MOLM-13 cells, or MOLM-13 cells, or CD47101 CD47 17 Nov 2023

MOLM-13 MOLM-13 cells cells withwith doxycycline doxycycline administration administration after after 2 weeks 2 weeks post-transplant, post-transplant. f) Murinef) Murine CD47expression CD47 expressionononCD47 CD4710 MOLM-13 MOLM-13 cells cells engrafting engrafting in bone in bone marrow marrow (open) (open) compared compared

with original with original cell cellline line(shaded). (shaded). The values of The values of MFI/FSC2 MFI/FSC2 (x109) (x10) are are indicated, indicated. g) X2.5 g) 2.5 105x 105 CD47hl or CD47hi or CD47 CD47|0 MOLM-13 MOLM-13 cells cells were incubated were incubated with 5 with X 10 54 xBMDMs 104 for BMDMs for 2 hours. 2 hours. Phagocytic index Phagocytic index isisshown, shown. h) h)2.5 2.5x x105 CD47hl 105 RFP CD47hi RFPand andCD47l0 CD47'0MOLM-13 GFPcells MOLM-13 GFP cells were were incubatedwith incubated with5 5X x104 104BMDMs BMDMs for 2for 2 hours. hours. Phagocytic Phagocytic index index is shownis for shown threefor three separate separate

samples for samples for CD47hi CD47hi RFP (red) and RFP (red) and CD47l0 MOLM-13 CD47 MOLM-13 GFP GFP (green) (green) cells, cells. i) i)2.5 2.5Xx105 105 CD47hi CD47hi 2023266367

RFPand RFP andCD47¹ CD47|0 MOLM-13 MOLM-13 GFP cells GFP cells were were incubated incubated with with 5 x BMDMs 5 X 104 104 BMDMs for 24 for 24 hours. hours.

Photomicrographs Photomicrographs show show brightfield brightfield (top (top left), RFP left), RFP (topright), (top right), GFP GFP(bottom (bottom left), and left), andmerged merged (bottomright) (bottom right) images. images.

[29]

[29] 16. a) a)FACS Figure 16. Figure FACS analysis analysis of CD47 of CD47 expression expression of non-leukemic of non-leukemic Fas Fas Ipr/lpr Ipr/lpr hMRP8bcl-2 (blue) hMRP8bcl-2 (blue) and and leukemic leukemic Fas FasIpr/lpr Ipr/lpr hMRP8bcl-2 hMRP8bcl-2(green) (green)bone bone marrow marrow hematopoieticstem hematopoietic stem cells cells (c-kit+Sca+Lin-),myeloid (c-kit+Sca+Lin-), myeloid progenitors progenitors (c-kit+Sca-Lin-) (c-kit+Sca-Lin-) or or blasts blasts (c-(c-

kit lo kit lo Sca-Lin-). Sca-Lin-). b) Mouse b) Mouse bone bone marrow marrow was transduced was transduced with retrovirus with retrovirus containing containing p210 p210 bcr/abl as bcr/abl as previously previouslydescribed described24. Mice ²4. Mice were were sacrificed sacrificed whenwhen moribund moribund and theand the spleens spleens wereanalyzed. were analyzed.Expression Expression of CD47 of CD47 in c-Kit+ in c-Kit+ Mac-1 Mac-1+ cells+ in cells the in the spleens spleens of two of two leukemic leukemic

mice (unshaded mice (unshaded histograms) histograms) and and bone bone marrow marrowfrom fromaawild-type wild-type mouse (shadedhistogram) mouse (shaded histogram) are shown. are shown, c)c)Histograms Histogramsshow show expression expression of of CD47 CD47 on gated on gated populations populations forfor leukemic leukemic

hMRPSbcrablX xhMRP8bcl2 hMRP8bcrabl hMRP8bcl2 micemice (red), (red), hMRP8bcl2 hMRP8bcl2 non-leukemic non-leukemic (blue) (blue) and wild-type and wild-type

(green) mice. (green) mice. CD47 was stained CD47 was stained using using FITC FITC conjugated conjugated anti-mouse anti-mouse CD47 (Pharmingen). CD47 (Pharmingen).

[30]

[30] Figure 17. Figure 17. a) Expression of a) Expression of human humanCD47 CD47 (blackhistograms) (black histograms)on onhuman human leukemia leukemia cell cell

lines and lines cord blood and cord bloodHSCs HSCs is shown. is shown. Isotype Isotype control control staining staining is shown is shown in b) in gray. gray, CD47 b) CD47 MFI over MFI overbackground backgroundwaswas normalized normalized to cell to cell size size by dividing by dividing by FSC2. by FSC2. The obtained The value value obtained for each for cell type each cell type is is shown abovethe shown above thebar. bar.c) c)HL-60 HL-60 cells cells engraft engraft mouse mouse bone bone marrow. marrow. 5 X 5x 105 cells 105 cells were injected intravenously were injected into RAG2-/-, intravenously into Gc-/- animals RAG2-/-, Gc-/- animalsand and mice mice were were analyzed analyzed 4 4 weekslater. weeks later, d) d) Cells Cells were stained with were stained with CFSE andco-cultured CFSE and co-cultured with with BMDM. Phagocytic BMDM. Phagocytic

events were events werecounted counted after after 2h.ForFor 2h. irradiation,Jurkat irradiation, Jurkatcells cellswere weregiven givena adose dose of of 2 Gray 2 Gray and and

incubatedfor incubated for 16h 16hprior prior to to the the phagocytosis assay. phagocytosis assay.

[31]

[31] Figure 18. Figure 18. (a) (a) Analysis Analysisofofstem stemandand progenitor progenitor cells cells fromfrom bonebone marrow marrow of IAP+/+, of IAP+/+,

IAP+/-, and IAP+/-, IAP-/- mice. and IAP-/- mice. Stem Stem cells(left) cells (left) are gated on lineage- gated on lineage- c-Kit+ c-Kit+ Sca-1+ Sca-1+cells. cells. Myeloid Myeloid progenitors (right) progenitors (right) are gatedononlineage- are gated lineage- c-Kit+ c-Kit+ Sca-1+ Sca-1+ cells. cells. Frequency Frequency in bone in whole whole bone marrowisis shown marrow shownadjacent adjacenttotoeach each gated gated population,(b) (b) population. Colony Colony output output on day on day 7 of 7 of individually individually sorted LT-HSC. sorted LT-HSC. G-granulocyte, G-granulocyte,M-macrophage, M-macrophage, GM-granulocyte GM-granulocyte and and macrophage, GEMM-granulocyte, macrophage, GEMM-granulocyte, macrophage, macrophage, erythroid, erythroid, and megakaryocyte, Meg- and megakaryocyte, Meg- megakaryocyte. megakaryocyte. (c)(c) Survival Survival curve curve of recipient of recipient mice mice given given a radiation a radiation dose dose of 9.5 of 9.5 GrayGray and and transplanted with transplanted with the the cells cells shown. shown.Radiation Radiation control control micemice all died all died within within 12-15 12-15 days. days. n=5 n=5

8 for each for group, (d) each group. (d)Examples Examples of CD45.1/CD45.2 of CD45.1/CD45.2 chimerism chimerism plots plots at 4 weeks at 4 weeks post- post­ 17 Nov 2023 transplant. CD45.1 transplant. CD45.1 mice mice were transplanted with were transplanted with50 50LT-HSC LT-HSC (CD45.2) (CD45.2) and and X2 x 105CD45.1 105 CD45.1 helper marrow. helper marrow.Cells Cells are are gated gated on B220- on B220- CDS- side CD3- Mac-1+ Mac-1 + sidemid/hi scatter scatter mid/hi cells. cells. IAP-/- IAP-/- cells fail cells failtotoengraft, engraft.(e) Summary (e) of chimerism Summary of chimerismanalysis analysisofofmice mice transplanted transplanted with with either either 50 50 or 500 or 500 IAP+/+ IAP+/+ororIAP-/- IAP-/-cells. cells, (f) (f) IAP+/+ IAP+/+ororIAP-/- IAP-/-c-Kit c-Kit enriched enrichedcells cellswere wereincubated incubated with with wild-type BMDM. wild-type BMDM. Results Results indicate indicate mean mean phagocytic phagocytic index calculated index calculated from from three three separate separate samples.Error samples. Error bars bars represent represent 1 s.d. 1 s.d. (g) (g) Photomicrographs Photomicrographs of phagocytosis of phagocytosis assays assays taken taken after 22 hours. after Genotypeofofthe hours. Genotype the-Kit -Kit enriched enrichedcells cells is is shown. shown. 2023266367

132]

[32] Figure 19. Figure 19. (a) (a)Mice Micewere were mobilized mobilizedwith withCy/G Cy/Gand and bone bone marrow wasanalyzed marrow was analyzedonon day 2. day 2. Expression Expression levelofofCD47 level CD47 on c-Kit+ on c-Kit+ cells cells is is shown, shown. (b) (b) Myeloid Myeloid progenitor progenitor and and stem stem cell gates cell are shown gates are shownforforday day 2 mobilized 2 mobilized bonebone marrow. marrow. Histograms Histograms on left on left show show level of level of CD47expression CD47 expression in in marrow marrowLT-HSC LT-HSC and and GMPGMP for for steady-state(shaded steady-state (shaded histogram),day histogram), day2 2 mobilized (black mobilized (black line), line), and day5 5mobilized and day mobilized(gray (grayline). line), (c) (c)Relative RelativeMFI MFI of of CD47 CD47 for for GMP GMP on days on days0-5 0-5ofofCy/G Cy/Gmobilization. mobilization.Results Results were were normalized normalized so that so that steady steady state state GMP GMP were were equal to 100. equal to 100. (d) (d) Myeloid Myeloidprogenitor progenitorand and stem stem cell cell gates gates areare shown shown for day for day 2 bone 2 bone marrowmarrow

post-LPStreatment. post-LPS treatment.Histograms Histograms showshow levellevel of CD47 of CD47 expression expression onpost-LPS on day 2 day 2 post-LPS (black (black line), day line), day 55 post-LPS (dark gray post-LPS (dark gray shaded shadedhistogram), histogram),steady steadystate state(light (light gray gray shaded shaded histogram), and histogram), andIAP-/- IAP-/-(black (blackshaded shaded histogram) histogram) LT-HSC LT-HSC and(e) and GMP. GMP. (e) Evaluation Evaluation of KLS of KLS cells ininthe cells thehematopoietic organsofofIAP+/+ hematopoietic organs IAP+/+and and IAP-/- IAP-/- mice mice mobilized mobilized on days on days 2 through 2 through 5. 5. Twomice Two miceare areanalyzed analyzed perper genotype genotype per day. per day.

[33]

[33] Figure 20. Figure 20. (a) (a) CD47 CD47 expression expression level level of IAP+/+, of IAP+/+, IAP+/-, IAP+/-, and IAP-/- and IAP-/- LT-HSC. LT-HSC. The The numbersshown numbers shown are are the the MFI MFI for for eacheach group, group. (b) Donor (b) Donor chimerism chimerism analysis analysis for transplants for transplants of of IAP+/+(top) IAP+/+ (top) or or IAP+/- IAP+/- (bottom) (bottom) mice. mice.Mice Mice were were bled bled at at 2, 2, 8, 8,and and 40 40 weeks weeks postpost transplant. transplant.

2 Xx 106 2 106donor donor cells cells were were transplanted transplanted into sub-lethally into sub-lethally irradiated irradiated congenic congenic recipients. recipients.

[34]

[34] Figure 21A-D:Identification Figure 21A-D: Identificationand andSeparation Separation of Normal of Normal and Leukemic and Leukemic Progenitors Progenitors

From the From the Same SamePatient PatientBased BasedOnOn Differential CD47 Differential Expression. A.A.CD47 CD47 Expression. CD47 expression expression onon

the Lin-CD34+CD38- the LSC-enriched Lin-CD34+CD38- LSC-enriched fractionof fraction of specimen specimenSU008 SU008waswas determined determined by flow by flow

cytometry. CD47hi- cytometry. CD47hi- and and CD47lo-expressing CD47lo-expressing cells cells were identified were identified and purified and purified using using FACS. FACS. Theleft The left panels are gated panels are gatedononlineage lineagenegative negative cells,while cells, whilethe theright right panels panelsare aregated gatedonon Lin- Lin-

CD34+CD38- CD34+CD38- cells. B. B.Lin-CD34+CD38-CD47lo cells. Lin-CD34+CD38-CD47lo and Lin-CD34+CD38-CD47hi and Lin-CD34+CD38-CD47hi cells cells were were plated into plated into complete complete methylcellulose, methylcellulose, capable capable of supporting of supporting the of the growth growth of all all myeloid myeloid colonies. 14 colonies. days later, 14 days later, myeloid myeloid colony colonyformation formationwaswas determined determined by morphologic by morphologic

assessment. Representative assessment. Representative CFU-G/M CFU-G/M (left) and (left) andBFU-E BFU-E (right)are (right) arepresented. presented.C. C. Lin- Lin-

CD34+CD38-CD47lo CD34+CD38-CD47Io cells cells were were transplanted transplanted into 2into 2 newborn newborn NOG NOG mice. mice. later, 12 weeks 12 weeks the later, the mice were mice were sacrificed sacrificed and and the the bone bonemarrow marrow waswas analyzed analyzed for for the the presence presence of human of human

CD45+CD33+ CD45+CD33+ myeloid myeloid cellsand cells andhuman human CD45+CD19+ CD45+CD19+ lymphoid lymphoid cellscells by flow by flow cytometry. cytometry. D. D.

Normal bone Normal bone marrow marrowHSC, HSC, bulkSU008 bulk SU008 leukemia leukemia cells, Lin-CD34+CD38-CD47hi cells, Lin-CD34+CD38-CD47hi cells,Lin- cells, Lin-

9

CD34+CD38-CD47IO CD34+CD38-CD47lo cells,ororhuman cells, human CD45+ CD45+ cells cells purifiedfrom purified fromthe thebone bonemarrow marrow of of mice mice 17 Nov 2023

engrafted with engrafted with Lin-CD34+CD38-CD47lo Lin-CD34+CD38-CD47lo cells assessed cells were were assessed for the presence for the presence of the of the FLT3- FLT3- ITD mutation ITD mutationbybyPCR. PCR.TheThe wild wild type type FLT3 FLT3 and and the FLT3-ITD the FLT3-ITD products products are indicated. are indicated.

[35]

[35] Figure 22: Figure Increased CD47 22: Increased CD47Expression ExpressionininHuman Human AML AML is Associated is Associated with with Poor Poor

Clinical Outcomes. Clinical Event-free Outcomes. Event-free (A,C) (A,C) and overall and overall (B,D)(B,D) survival survival ofAML of 132 132patients AML patients with with normalcytogenetics normal cytogenetics (A,B) (A,B) andand the the subset subset of 74 of 74 patients patients without without the FLT3-ITD the FLT3-ITD mutation mutation (C,D). Patients (C,D). Patients were werestratified stratified into into low CD47andand low CD47 high high CD47 CD47 expression expression groups groups based onbased on an optimal an optimal threshold threshold (28% high, 72% (28% high, 72%low) low)determined determinedby by microarray microarray analysisfrom analysis from an an 2023266367

independent training independent training data set. The data set. significance measures The significance are based measures are basedonon log-likelihood log-likelihood

estimates of estimates of the p-value, when the p-value, treating the when treating the model with CD47 model with CD47expression expressionas as a binary a binary

classification. classification.

[36]

[36] Figure 23A-E: Figure 23A-E: AA Monoclonal MonoclonalAntibody AntibodyDirected Directed Against AgainstHuman Human CD47 CD47 Eliminates Eliminates

AMLInIn Vivo. AML Vivo. Newborn NOG Newborn NOG mice mice were were transplantedwith transplanted withAML AML LSC, LSC, andand 8-12 8-12 weeks weeks later, later,

peripheral blood peripheral blood (A,B) (A,B) and andbone bone marrow marrow (C-E) (C-E) werewere analyzed analyzed for baseline for baseline engraftment engraftment prior prior to treatment to with anti-CD47 treatment with anti-CD47(B6H12.2) (B6H12.2) or control or control IgG IgG antibody antibody (Day (Day 0). were 0). Mice Micetreated were treated with daily with daily 100 100 microgram microgram intraperitoneal intraperitoneal injections injections forfor 14 14 days, days, at the at the end end of which, of which, they they weresacrificed were sacrificed and andperipheral peripheralblood bloodand and bone bone marrow marrow were were analyzed analyzed for thefor the percentage percentage of of humanCD45+CD33+ human CD45+CD33+ leukemia. leukemia. A. Pre- A. Pre- and and post-treatment post-treatment human human leukemic leukemic chimerism chimerism in in the peripheral the peripheral blood bloodfrom fromrepresentative representative anti-CD47 anti-CD47 antibody antibody and control and control IgG-treated IgG-treated mice mice as determined as determined bybyflow flowcytometry. cytometry. B. B.Summary Summary of human of human leukemic leukemic chimerism chimerism in the in the peripheral blood peripheral bloodassessed assessedon on multiple multiple daysdays during during the course the course of treatment of treatment demonstrated demonstrated

elimination of elimination of leukemia in anti-CD47 leukemia in antibody treated anti-CD47 antibody treated mice mice compared comparedto to controlIgGIgG control

treatment (p=0.007). treatment (p=0.007). C. C. Pre- Pre- and and post-treatment post-treatmenthuman leukemic chimerism human leukemic chimerism in in the the bone bone marrowfrom marrow from representative representative anti-CD47 anti-CD47 antibody antibody or control or control IgG-treated IgG-treated mice mice as as determined determined

by flow by flow cytometry. cytometry.D.D.Summary of human Summary of leukemicchimerism human leukemic chimerisminin the the bone bone marrow marrowononday day 14 relative 14 relative to to day day 00demonstrated demonstrated a dramatic a dramatic reduction reduction in leukemic in leukemic burden burden in anti-CD47 in anti-CD47

antibody treated antibody treatedmice mice compared compared to control to control IgG treatment IgG treatment (p<0.001). (p<0.001). E. H&E of E. H&E sections sections of representative mouse representative mouse bone bone marrow marrow cavities cavities fromfrom mice mice engrafted engrafted with SU004 with SU004 post-treatment post-treatment

with either with either control control IgG IgG (panels 1,2) or (panels 1,2) or anti-CD47 antibody(panels anti-CD47 antibody (panels4,5). 4,5). IgG-treated IgG-treatedmarrows marrows were packed were packedwith with monomorphic monomorphic leukemic leukemic blasts,while blasts, while anti- anti- CD47-treated marrowswere CD47-treated marrows were hypocellular, demonstrating hypocellular, demonstrating elimination elimination of of the humanleukemia. the human leukemia.In Insome some anti-CD47 anti-CD47

antibody-treated mice antibody-treated that contained mice that contained residual residual leukemia, leukemia, macrophages macrophages were were detected detected

containing phagocytosed containing pyknotic cells, phagocytosed pyknotic cells, capturing capturing the elimination of the elimination of human leukemia human leukemia (panels 3,6 (panels 3,6 arrows). arrows).

[37]

[37] Figure 24. Figure 24. Increased IncreasedCD47 CD47 expression expression predicts predicts worse worse overall overall survival survival in DLBCL in DLBCL and and ovarian cancer. ovarian cancer.(A)(A)A A cohort cohort of of 230230 patients patients withwith diffuse diffuse large large B-cell B-cell lymphoma lymphoma (p=0.01). (p=0.01).

(B) A (B) cohort of A cohort of 133 patients with 133 patients with advanced stage advanced stage (lll/IV) ovarian (III/IV) carcinoma(p=0.04). ovarian carcinoma (p=0.04).

10

[38]

[38] Figure 25: Figure 25: Anti-CD47 Anti-CD47antibody antibody enables enables the the phagocytosis phagocytosis of solid of solid tumortumor stem stem cells cells in in 17 Nov 2023

vitro. The vitro. indicated cells The indicated cells were incubatedwith were incubated withhuman human macrophages macrophages in theinpresence the presence of IgG1of IgGI isotype, anti-HLA, isotype, anti-HLA, ororanti-CD47 anti-CD47 antibodies antibodies and and the phagocytic the phagocytic index index was was determined determined by by immunofluorescence immunofluorescence microscopy. microscopy. Statistics: Statistics: Bladder Bladder cancer cancer cellscells IgG1 lgG1 isotype isotype compared compared to to anti-HLA (p=0.93) anti-HLA (p=0.93) and andanti-CD47 anti-CD47 (p=0.01);normal (p=0.01); normal bladder bladder urothelium urothelium IgG1 lgG1 isotype isotype

compared compared to to anti-HLA anti-HLA (p=0.50) (p=0.50) and and anti-CD47 anti-CD47 (p=0.13); (p=0.13); ovarian ovarian cancer cancer cellsisotype cells IgG1 lgG1 isotype comparedtotoanti-HLA compared anti-HLA(p=0.11) (p=0.11)andand anti-CD47 anti-CD47 (p<0.001). (p<0.001). EachEach individual individual datadata point point

representsaa distinct represents distinct tumor or normal tumor or normaltissue tissue sample. sample. 2023266367

Detailed DESCRIPTION DETAILED Description OF Of THE TheEMBODIMENTS Embodiments

[39]

[39] Methods are Methods areprovided providedto to manipulate manipulate hematopoietic hematopoietic cells, cells, includingcirculating including circulating hematopoietic cells. hematopoietic In some cells. In someembodiments embodiments of the of the invention, invention, hematopoietic hematopoietic stemstem or or progenitor cells progenitor cells are are protected protectedfrom fromphagocytosis phagocytosis in circulation in circulation by by providing providing a host a host animal animal

with aa CD47 with CD47 mimetic mimetic molecule, molecule, whichwhich interacts interacts with with SIRPa SIRPa on phagocytic on phagocytic cells, cells, such as, such as, macrophages,and macrophages, anddecreases decreases phagocytosis.In other phagocytosis. In other embodiments embodiments leukemia leukemia cells cells are are targeted for targeted for phagocytosis byblocking phagocytosis by blockingCD47 CD47 on the on the cellcell surface. surface. In In other other embodiments, embodiments, cellscells

of solid of solid tumors aretargeted tumors are targetedforforphagocytosis phagocytosis by blocking by blocking CD47 CD47 on the on cellthe cell surface. surface. In In another embodiment, another embodiment, methods methodsareareprovided providedfor fortargeting targeting or or depleting depleting AML cancerstem AML cancer stem cells, the cells, the method comprisingcontacting method comprising contactingreagent reagentblood blood cellswith cells with an an antibody antibody thatthat

specifically binds specifically bindsCD47 CD47 in in order order to totarget targetorordeplete AMLSC. deplete In another AMLSC. In another embodiment, embodiment, methodsareare methods provided provided for for targeting targeting cancer cancer cellscells of a of a solid solid tumortumor in a human in a human subject subject by by administering an administering anantibody antibodyagainst againstCD47 CD47 to the to the subject. subject.

[40]

[40] Before the Before thepresent present invention invention is is further further described, described, it isto tobe be it is understood understood that that this this invention is invention is not not limited limited to to particular particularembodiments described,asassuch embodiments described, such may, may, of course, of course, vary. vary.

It isisalso It alsototobebeunderstood understood that that the the terminology usedherein terminology used hereinisisfor for the the purpose purposeofofdescribing describing particular embodiments particular only, embodiments only, andand is not is not intended intended to be to be limiting, limiting, since since the of the scope scope the of the present invention present invention will will be be limited limitedonly onlyby bythe theappended claims. appended claims.

[41]

[41] Wherea arange Where range of of values values is is provided, provided, it itisis understood understoodthat thateach each intervening intervening value, value, to to

the tenth the tenthofofthe theunit unitofof the the lower lower limit limit unless unless the context the context clearlyclearly dictatesdictates otherwise, otherwise, between between the upper the upperand andlower lower limitofofthat limit thatrange range andand any any otherother stated stated or intervening or intervening value value in thatin that stated range, stated range, isis encompassed encompassed within within the invention. the invention. The and The upper upper and lower loweroflimits limits these of these smaller ranges smaller ranges may mayindependently independently be be included included in the in the smaller smaller ranges ranges and also and are are also encompassed encompassed within within the the invention, invention, subject subject to any to any specifically specifically excluded excluded limit limit in stated in the the stated range. Where range. Wherethethe stated stated range range includes includes one one or both or both of the of the limits,ranges limits, ranges excluding excluding either either or or bothofofthose both thoseincluded included limits limits are are also also included included in the in the invention. invention.

11

[42]

[42] Methodsrecited Methods recitedherein hereinmay may be be carried carried outout in in any any order order of of the the recitedevents recited events which which is is 17 Nov 2023

logically possible, logically possible,asas well well as as the the recited recited orderorder of events. of events.

[43]

[43] Unlessdefined Unless definedotherwise, otherwise, allall technical technical andand scientific scientific terms terms usedused herein herein have have the the samemeaning same meaning as commonly as commonly understood understood by one ofby one ofskill ordinary ordinary skill in the artintothe art this which to which this invention belongs. invention Although any belongs. Although anymethods methodsandand materialssimilar materials similarororequivalent equivalent to to those those describedherein described hereincan can also also be be usedused in the in the practice practice or testing or testing of the of the present present invention, invention, the the preferred methods preferred methodsand and materials materials areare nownow described. described.

[44]

[44] All publications All mentionedherein publications mentioned herein areare incorporated incorporated herein herein by reference by reference to disclose to disclose 2023266367

and describe and describethe themethods methods and/or and/or materials materials in connection in connection with the with which which the publications publications are are cited. cited.

[45]

[45] It must It must be noted that be noted that as as used usedherein hereinand andin inthe theappended appended claims, claims, the the singular singular forms forms

“a”, “an”, "a", and"the" "an", and “the”include include plural plural referents referents unless unless the context the context clearly clearly dictatesdictates otherwise. otherwise. It is It is further noted further that the noted that the claims maybebedrafted claims may draftedtotoexclude excludeanyany optional optional element. element. As such, As such, this this

statementisis intended statement intendedtoto serve serveasasantecedent antecedent basis basis forfor use use of of such such exclusive exclusive terminology terminology as as “solely,” “only” "solely," "only"and and the the like like ininconnection connection with with the the recitation recitation of of claim claim elements, or use elements, or useofofaa “negative”limitation. "negative" limitation.

[46]

[46] Thepublications The publicationsdiscussed discussedherein herein are are provided provided solely solely forfor theirdisclosure their disclosureprior priorto to the the filing date filing dateofofthe thepresent presentapplication. application.Nothing Nothing herein herein is istotobe beconstrued construed as as an admissionthat an admission that the present the present invention invention is not is not entitled entitled to antedate to antedate such publication such publication by virtue by virtueinvention. of prior of prior invention. Further, the dates Further, of publication dates of publication provided maybebedifferent provided may differentfrom fromthe theactual actualpublication publicationdates dates whichmay which may need need to to be be independently independently confirmed. confirmed.

Definitions DEFINITIONS

[47]

[47] CD47polypeptides. CD47 polypeptides. TheThe three three transcript transcript variants variants of human of human CD 47 (variant CD 47 (variant 1, NM 1, NM 001777; variant 001777; variant 2, 2, NM 198793; and NM 198793; andvariant variant 3, 3, NM 001025079)encode NM 001025079) encode three three isoformsofof isoforms

CD47polypeptide. CD47 polypeptide. CD47 CD47 isoform isoform 1 (NP1 001768), (NR 001768), the longest the longest of theisoforms, of the three three isoforms, is 323 is 323 aminoacids amino acidslong. long.CD47 CD47 isoform isoform 2 (NP 2 (NP 942088) 942088) is 305isamino 305 amino acidCD47 acid long. long.isoform CD47 3isoform is 3 is 312 amino 312 aminoacids acids long. long. TheThe three three isoforms isoforms are identical are identical in sequence in sequence in theinfirst the first 303 303 aminoamino

acids. Amino acids. acids 1-8 Amino acids 1-8 comprise comprise the the signal signal sequence, amino acids sequence, amino acids 9-142 9-142comprise comprisethe the CD47immunoglobulin CD47 immunoglobulin likelike domain, domain, which which is the is the soluble soluble fragment, fragment, and amino and amino acids 143-300 acids 143-300

is the is the transmembrane domain. transmembrane domain.

[48]

[48] “CD47mimetics" "CD47 mimetics” include include molecules molecules that that function function similarly similarly to CD47 to CD47 by binding by binding and and activating SIRPa activating receptor. Molecules SIRPa receptor. Moleculesuseful usefulas as CD47 CD47 mimetics mimetics include include derivatives, derivatives,

variants, and variants, biologically active and biologically activefragments fragmentsof of naturally naturallyoccurring occurringCD47. CD47. AA"variant" "variant" polypeptide means polypeptide means a biologically a biologically active active polypeptide polypeptide as defined as defined below less below having having than less than 100%sequence 100% sequence identitywith identity witha anative nativesequence sequence polypeptide. polypeptide. Such Such variants variants include include

12 polypeptideswherein polypeptides or or whereinoneone more more amino amino acid residues acid residues are added added are at at or the N- N- or C-terminus theC-terminus 17 Nov 2023 of, within,the or within, of, or nativesequence; thenative sequence; from onetotoforty aboutone from about amino fortyamino acid acid residues areare residues deleted, deleted, optionally substituted and optionally and by one substituted by amino moreamino oneorormore acid acid residues; andand residues; derivatives derivatives of the of the above above polypeptides, wherein an polypeptides, wherein an amino residue has acid residue amino acid beencovalently has been modified so covalently modified the that the sothat resulting product resulting has aanon-naturally product has amino occurringamino non-naturallyoccurring acid. acid. Ordinarily, Ordinarily, a biologically active a biologicallyactive variant have an willhave variant will an amino sequence acidsequence aminoacid having having at least at least about about 90% amino 90% amino acid sequence acid sequence identity native sequence with aa native identity with sequence polypeptide, preferablyatatleast polypeptide,preferably 95%, about95%, leastabout more more preferably preferably at about 99%. least about at least Thevariant 99%. The polypeptides variantpolypeptides be be cancan naturally or or naturally non-naturally non-naturally glycosylated, glycosylated, 2023266367 i.e., the i.e., polypeptide has the polypeptide patternthat glycosylationpattern has aa glycosylation fromthe differs from thatdiffers pattern glycosylationpattern theglycosylation found corresponding the corresponding found inin the naturally naturally occurring occurring protein. TheThe protein. variant variant polypeptides polypeptides can can have have post-translational modifications post-translational not found modifications not found on natural CD47 the natural on the protein. CD47protein.

[49]

[49] Fragments thethe Fragmentsof of soluble soluble CD47, CD47, particularly particularly biologically biologically active active fragments fragments and/or and/or corresponding fragmentscorresponding fragments to functional to functional domains, domains, are of of interest. areinterest. Fragments Fragments of interest of interest will will typically be at typically be about 10 leastabout at least 10 aa to at aa to least about at least 15 aa about 15 length, usually aainin length, least about at least usually at 50 about 50 in length, aa in aa will usually but will length, but usually not exceedabout not exceed 142142 about aa in in length, aalength, where where the fragment the fragment will will havea astretch have amino stretchofofamino acids thatthat acids is identical is identical to CD47. to CD47. A fragment A fragment "at least aa in20 "at20least aa in length," for example, length," for example, is intendedtotoinclude is intended or or include2020 more more contiguous amino amino contiguous acids from, from, acidsfor for example,the example, polypeptideencoded thepolypeptide by aby encoded a cDNA cDNA for CD47. this context In thisIn context for CD47. "about" "about" includes includes the the particularly recited value particularly recited value or valuelarger or aa value smallerbybyseveral largerororsmaller (5,(5, several 3, 3, 4, 4, 1) 1) or or 2, 2, amino amino

acids. The acids. variantsdescribed proteinvariants Theprotein hereinare describedherein encoded areencoded by polynucleotides that that by polynucleotides are within are within

the scopeofofthe the scope invention. The theinvention. cancan code geneticcode Thegenetic be used be used to select the the to select appropriate appropriate codons codons

to construct to corresponding variants. the corresponding construct the polynucleotides may The polynucleotides variants.The may be produce usedtoto produce be used polypeptides, and polypeptides, and these maybebe polypeptides may these polypeptides used used to produce to produce antibodies antibodies by known by known

methods. methods.

[50]

[50] A "fusion" polypeptide A "fusion" is aa polypeptide polypeptide is polypeptide comprisinga apolypeptide polypeptide comprising or or portion one (e.g., one portion(e.g., or more or domains) moredomains) thereof thereof fused fused or bonded or bonded to heterologous to heterologous polypeptide. fusion A fusion Asoluble polypeptide. soluble CD47 protein,for CD47protein, example, forexample, will will share share at least one one at least biological biological property property in common in common with a with a native sequence native sequence soluble CD47 soluble CD47 polypeptide. Examples polypeptide.Examples of fusion of fusion polypeptides polypeptides include include

immunoadhesins,asasdescribed immunoadhesins, combine whichcombine above,which describedabove, a portion the CD47 a portionofofthe polypeptide CD47polypeptide with an with an immunoglobulin andepitope sequence,and immunoglobulin sequence, polypeptides,which taggedpolypeptides, epitopetagged comprise whichcomprise a a soluble CD47 soluble CD47 polypeptide portion thereof polypeptideororportion polypeptide". TheThe "tagpolypeptide". fusedtotoa a"tag thereof fused tag tag polypeptide enough hasenough polypeptide has residues residues to provide to provide an epitope an epitope against which which against an antibody an antibody can be can be made, yetisis short made,yet such enoughsuch short enough that that doesnotnotinterfere it itdoes withbiological interferewith activity of biological activity the CD47 of the CD47

polypeptide. Suitable polypeptide. polypeptides tagpolypeptides Suitabletag generally generally have have at least sixsix at least amino acidacid amino residues residues and and usually between usually about between about 6-60 6-60 amino acidacid amino residues. residues.

[51]

[51] A "functional A derivative" of "functional derivative" sequence native sequence of aa native polypeptide is is polypeptide a compound havinghaving a compound a a qualitative in common property in biological property qualitative biological common with sequence nativesequence witha anative polypeptide. polypeptide. "Functional "Functional

13 derivatives" include, derivatives" include, but but are not limited are not limited to, to, fragments of aa native fragments of native sequence sequence andand derivatives derivatives 17 Nov 2023 of aa native of native sequence sequence polypeptide polypeptide and and its fragments, its fragments, provided provided that have that they theya have a biological biological activity inincommon activity withaacorresponding common with corresponding native native sequence sequence polypeptide. polypeptide. The "derivative" The term term "derivative" encompasses encompasses bothboth amino amino acid acid sequence sequence variants variants of polypeptide of polypeptide and covalent and covalent modifications modifications thereof. Derivatives and thereof. fusion of and fusion of soluble CD47 CD47find finduse useasasCD47 CD47 mimetic mimetic molecules. molecules.

[52]

[52] Thefirst The first 142 aminoacids 142 amino acidsofofCD47 CD47 polypeptide polypeptide comprise comprise the extracellular the extracellular regionregion of of CD47(SEQ CD47 (SEQID ID NO:NO: 1). 1). The The three three isoforms isoforms havehave identical identical amino amino acidacid sequence sequence in in the the extracellular region, extracellular and thus region, and thusany any of of thethe isoforms isoforms are are canused can be be toused to generate generate soluble soluble 2023266367

CD47. "Soluble CD47. “SolubleCD47" CD47”isisaaCD47 CD47 proteinthat protein that lacks lacks the the transmembrane domain. Soluble transmembrane domain. Soluble CD47isissecreted CD47 secretedoutout of of thecell the cellexpressing expressingit itinstead insteadofofbeing beinglocalized localizedatatthe thecell cell surface. surface. Soluble CD47 Soluble CD47 may may be fused be fused to another to another polypeptide polypeptide to provide to provide for added for added functionality, functionality, e.g.e.g. to to increase the increase theinin vivo vivo stability. stability. Generally Generallysuch suchfusion fusionpartners partners areare a stable a stable plasma plasma protein protein

that is that is capable capableofofextending extending thethe in vivo in vivo plasma plasma half-life half-life of soluble of soluble CD47 protein CD47 protein when when presentas present asaa fusion, fusion, in in particular particularwherein wherein such such aa stable stable plasma plasmaprotein proteinisis an animmunoglobulin immunoglobulin constant domain. constant domain.In Inmost most cases cases wherewhere the stable the stable plasma plasma protein protein is is normally normally found in found a in a multimeric form, multimeric form,e.g., e.g.,immunoglobulins immunoglobulins or lipoproteins, or lipoproteins, in which in which theor same the same or different different polypeptidechains polypeptide chainsare arenormally normallydisulfide disulfideand/or and/ornoncovalently noncovalently bound bound to form to form an assembled an assembled

multichain polypeptide. multichain polypeptide. Soluble SolubleCD47 CD47 fused fused to human to human Ig G1Ighas G1been has described been described (Motegi (Motegi S. S. et al. et al.EMBO EMBO J.J.22(11): 22(11):2634-2644). 2634-2644).

[53]

[53] Stable plasma proteins Stable plasma proteins are areproteins proteins typically typically having having about about from from3030to to 2,000 2,000

residues, which residues, whichexhibit exhibitinin their their native native environment environmentan an extended extended half-life half-life in the in the circulation, circulation,

i.e. greater i.e. greater than than about 20 hours. about 20 Examples hours. Examples of suitable of suitable stable stable plasma plasma proteins proteins are are immunoglobulins,albumin, immunoglobulins, albumin, lipoproteins, lipoproteins, apolipoproteins apolipoproteins and and transferrin. transferrin. The extracellular The extracellular

region of region of CD47 CD47is is typicallyfused typically fused to to thethe plasma plasma protein protein at N-terminus at the the N-terminus of the of the plasma plasma protein or protein or fragment fragmentthereof thereofwhich which is is capable capable of conferring of conferring an extended an extended half-life half-life upon upon the the soluble CD47. soluble CD47.Increases Increases of greater of greater thanthan about about 100% 100% on theon the plasma plasma half-life half-life of theof the soluble soluble

CD47are CD47 aresatisfactory. satisfactory.

[54]

[54] Ordinarily, the Ordinarily, the soluble soluble CD47 CD47 isisfused fusedC-terminally C-terminallytotothe theN-terminus N-terminus of of thethe constant constant

region of region of immunoglobulins immunoglobulins in place in place of the of the variable variable region(s) region(s) thereof, thereof, however however N-terminal N-terminal

fusions may fusions mayalso alsofind finduse. use.Typically, Typically,such such fusions fusions retain retain at at least least functionallyactive functionally activehinge, hinge, CH2and CH2 andCH3 CH3 domains domains of the of the constant constant regionofofananimmunoglobulin region immunoglobulin heavy heavy chain, chain, which which

heavychains heavy chainsmay may include include lgG1, IgG1, lgG2a, IgG2a, lgG2b, IgG2b, IgG3, lgG3, IgG4, lgG4, IgA, IgE, IgA, IgM, IgM,and IgE,IgD, andusually IgD, usually one or one or aa combination combinationofofproteins proteinsininthe theIgG IgGclass. class.Fusions Fusions areare also also made made to the to the C-terminus C-terminus

of the Fc of Fc portion portion of of aa constant constantdomain, domain,ororimmediately immediately N-terminal N-terminal to the to the CH1 CHI ofheavy of the the heavy chain or chain or the thecorresponding corresponding region region of the of the light light chain. chain. This This ordinarily ordinarily is accomplished by is accomplished by constructing the constructing the appropriate appropriateDNA DNA sequence sequence and expressing and expressing it in recombinant it in recombinant cell culture. cell culture.

Alternatively, the Alternatively, the polypeptides polypeptides may besynthesized may be synthesized according according to to known known methods. methods.

14

[55]

[55] The precisesite Theprecise whichthe siteatatwhich made fusionis ismade thefusion is not is not critical; particularsites critical;particular maybe be sites may 17 Nov 2023

ordertoto optimize selected inin order selected biologicalactivity, thebiological optimizethe or binding secretion or activity, secretion characteristicsofof bindingcharacteristics CD47. The CD47.The optimal optimal site determinedbyby be determined will be sitewill routine experimentation. routineexperimentation.

[56]

[56] In some In some embodiments thehybrid embodimentsthe immunoglobulins are hybrid immunoglobulins assembledas are assembled monomers, asmonomers, oror

hetero- or hetero- or homo-multimers, and particularly as and particularly or tetramers. dimers or as dimers tetramers. Generally, these Generally, these assembled assembled immunoglobulins will will immunoglobulins knownknown havehave unit structures. basic A basicAfour unit structures. chain chain structural fourstructural unit isthe unit is which IgG, inwhich form in theform IgD, and IgG, IgD, and IgE fourchain exist. AAfour IgE exist. unit is chainunit repeatedinin the is repeated higher the higher molecularweight molecular IgM IgM immunoglobulins; weightimmunoglobulins; generally generally exists as a as exists a pentamer pentamer of basic basic four-chain of four-chain 2023266367

units held units together by held together bonds. IgAIgA disulfide bonds. by disulfide immunoglobulin, and and immunoglobulin, occasionally occasionally IgG IgG alsoalso immunoglobulin,maymay immunoglobulin, exist exist formform a multimeric in ainmultimeric in serum. In the In in serum. theofcase case of multimers, multimers,

eachfour each unit may chainunit four chain same thesame maybebethe or or different. different.

[57]

[57] Suitable CD47 Suitable CD47 mimetics and/orfusion mimeticsand/or maybebeidentified proteins may fusion proteins compound by compound identified by screeningbybydetecting screening theability detectingthe of an ability of an agent to mimic agentto thebiological mimicthe of CD47. activity of biologicalactivity One CD47. One

biological activity biological CD47 isis the of CD47 activity of SIRPa activationofofSIRPa theactivation receptor receptor on macrophages. In vitroIn on macrophages. vitro may assaysmay assays be be as aas conducted conducted a first first screen for for screen efficacy a a of of efficacy candidate candidate agent, andand agent, usually usually an an in vivo in assaywill vivo assay performed will bebeperformed to confirm to confirm the biological assay.assay. the biological Desirable Desirable agents agents are are effective inin temporarily effective blocking SIRPa temporarily blocking SIRP a receptor activation, Desirable receptor activation. agentsareare Desirableagents temporary nature, e.g. temporaryininnature, dueto e.g. due biological degradation. to biological degradation.

158]

[58] In assaysforfor vitro assays In vitro CD47 CD47 biological biological activity activity include, include, e.g. inhibition e.g. inhibition of phagocytosis of phagocytosis of of porcine cells by porcine cells macrophages, humanmacrophages, by human binding binding to SIRP to SIRP a receptor, a receptor, SIRP SIRP a a tyrosine tyrosine

etc. AnAn phosphorylation,etc. phosphorylation, assayassay exemplary exemplary for CD47 CD47 biological for biological activity activity contacts a humana contacts human compositioninin the macrophagecomposition macrophage candidate agent. presenceofof aa candidate the presence cells are Thecells agent. The incubated are incubated with candidateagent the candidate with the forabout agentfor andand minutes about3030minutes lysed. cell cell The The lysed. lysate lysate is mixed withwith is mixed anti- anti-

SIRP humanSIRP human a antibodies a antibodies to immunoprecipitate to immunoprecipitate SIRP SIRP a. a. Precipitated Precipitated proteins proteins are resolved are resolved

by SDS by SDS PAGE, then then PAGE, transferred transferred to nitrocellulose to nitrocellulose and probed and probed with antibodies specific specific with antibodies for for phosphotyrosine. agentuseful candidate agent phosphotyrosine. AA candidate CD47mimetic asCD47mimetic useful as increases increases SIRP SIRP a tyrosine a tyrosine

least10%, phosphorylationbybyatatleast phosphorylation toto20%, 10%,ororupup or or 20%, 50%, 50%, or 70% or 70% or or or 80% to up up or 80% to about about 90% 90% comparedtotothe compared phosphorylation observed of phosphorylation level of the level observed in the absence in the absence of agent. candidate agent. of candidate Another exemplaryassay Another exemplary CD47 CD47 for for assay biological biological activity activity measures measures phagocytosis phagocytosis of of cellsbybyhuman hematopoietic cells macrophages. AAcandidate human macrophages. as aa CD47 useful as agent useful candidate agent mimetic CD47 mimetic

results the down in the results in down regulation of phagocytosis regulation of 10%, about10%, least about phagocytosisbybyatatleast at at least about leastabout 20%, 20%, at at least about least about 50%, leastabout 50%,atatleast at at 70%, about70%, least least about about 80%, 80%, or up to about upabout or to 90% compared 90% compared to to of phagocytosis level of level phagocytosis observed absence observedininabsence of of candidate candidate agent. agent.

[59]

[59] Polynucleotide encoding Polynucleotideencoding soluble soluble CD47 CD47 or soluble or soluble CD47-Fc can be can CD47-Fc be introduced introduced into a into a suitable The The vector. expressionvector. suitable expression expression vector vector expression is introduced is introduced into a suitable suitable into a cell. cell. Expression generally vectorsgenerally Expressionvectors have have convenient convenient restriction sitessites restriction located near near located the promoter the promoter

sequence theinsertion provideforforthe sequencetotoprovide sequences. polynucleotidesequences. insertionofofpolynucleotide Transcription Transcription cassettes cassettes

15 maybebeprepared may preparedcomprising comprising a transcriptioninitiation a transcription initiation region, region,CD47 geneororfragment CD47 gene fragment 17 Nov 2023 thereof, and thereof, anda atranscriptional transcriptionaltermination termination region. region. The transcription The transcription cassettes cassettes may be may be introduced introduced into into a variety a variety of vectors, of vectors, e.g. plasmid; e.g. plasmid; retrovirus, retrovirus, e.g. lentivirus; e.g. lentivirus; adenovirus; adenovirus; and and the like, the like, where thevectors where the vectors areare able able to transiently to transiently or stably or stably be maintained be maintained in the in the cells, cells, usually for usually for aa period period of of at at least least about aboutone oneday, day,more more usually usually for for a period a period of least of at at least about about several days several daysto to several several weeks. weeks.

[60]

[60] Thevarious The variousmanipulations manipulations may may be carried be carried out out in in vitro vitro or be or may may be performed performed in an in an appropriate host, appropriate host,e.g. e.g.E.E.coli. coli.After Aftereach each manipulation, manipulation, the resulting the resulting construct construct may bemay be 2023266367

cloned, the cloned, the vector vector isolated, isolated, and the DNA and the DNA screened screened or sequenced or sequenced to ensure to ensure the correctness the correctness

of the of the construct. construct. The sequence The sequence maymay be screened be screened by restriction by restriction analysis, analysis, sequencing, sequencing, or or the the like. like.

[61]

[61] Soluble CD47 Soluble CD47cancan be be recovered recovered and purified and purified from from recombinant recombinant cell cultures cell cultures by by well- well- knownmethods known methods including including ammonium ammonium sulfatesulfate or ethanol or ethanol precipitation, precipitation, acid extraction, acid extraction, anion anion

or cation or cation exchange exchangechromatography, chromatography, phosphocellulose phosphocellulose chromatography, chromatography, hydrophobic hydrophobic

interaction chromatography, interaction affinity chromatography, chromatography, affinity chromatography, protein protein G affinity G affinity chromatography, chromatography, for for example,hydroxylapatite example, hydroxylapatitechromatography chromatography and lectin and lectin chromatography. chromatography. Most preferably, Most preferably, high high performanceliquid performance liquidchromatography chromatography ("HPLC") ("HPLC") is employed is employed for purification. for purification.

[62]

[62] Soluble CD47 Soluble CD47can canalso also be be recovered recovered from: from: products products of purified of purified cells,whether cells, whether directly isolated or directly or cultured; cultured; products productsof of chemical chemical synthetic synthetic procedures; procedures; and products and products

producedbyby produced recombinant recombinant techniques techniques from afrom a prokaryotic prokaryotic or eukaryotic or eukaryotic host, including, host, including, for for example,bacterial, example, bacterial, yeast yeast higher higherplant, plant, insect, insect, and and mammalian cells. mammalian cells.

[63]

[63] A plurality A plurality of of assays maybeberunrun assays may in inparallel parallelwith withdifferent different concentrations concentrationstotoobtain obtaina a differential response differential to the response to the various variousconcentrations. concentrations.As As known known in thein art, the determining art, determining the the effective concentration effective of an concentration of anagent agenttypically typically uses usesa arange range of of concentrations concentrations resulting resulting fromfrom

1:10, or 1:10, or other other log log scale, scale, dilutions. dilutions.The The concentrations maybebe concentrations may furtherrefined further refinedwith witha asecond second series of series of dilutions, dilutions,if if necessary. necessary.Typically, Typically,one oneofofthese theseconcentrations concentrations serves as aa negative serves as negative control, i.e. control, i.e. at at zero zeroconcentration concentration or below or below the oflevel the level of detection detection of the of the agent agent or at or at or below or below the concentration the concentration of of agent agentthat that does doesnot notgive giveaa detectable detectablechange changein in binding. binding.

[64]

[64] Compounds Compounds of interest of interest forfor screening screening include include biologically biologically active active agents agents of numerous of numerous

chemical classes, chemical classes, primarily primarily organic molecules, although organic molecules, although including including in in some someinstances instances inorganic molecules, inorganic molecules,organometallic organometallic molecules, molecules, immunoglobulins, immunoglobulins, chimeric chimeric CD47 proteins, CD47 proteins,

CD47related CD47 related proteins, proteins, genetic genetic sequences, sequences,etc. etc. AlsoAlso of interest of interest areare small small organic organic

molecules,which molecules, which comprise comprise functional functional groupsgroups necessary necessary for structural for structural interaction interaction with with proteins, particularly proteins, particularly hydrogen bonding,and hydrogen bonding, and typicallyinclude typically includeat atleast leastanan amine, amine, carbonyl, carbonyl,

hydroxyl or hydroxyl or carboxyl carboxylgroup, group,frequently frequentlyatatleast leasttwo twoofofthe thefunctional functionalchemical chemical groups. groups. The The candidateagents candidate agentsoften oftencomprise comprise cyclical cyclical carbon carbon or heterocyclic or heterocyclic structures structures and/or and/or aromatic aromatic

or polyaromatic or polyaromaticstructures structuressubstituted substitutedwith with oneone or more or more of theofabove the above functional functional groups. groups.

16 agents Candidateagents Candidate areare also also found found among among biomolecules, biomolecules, including including peptides, peptides, polynucleotides, polynucleotides, 17 Nov 2023 saccharides,fatty saccharides, steroids, purines, acids, steroids, fatty acids, derivatives,structural pyrimidines,derivatives, purines,pyrimidines, analogs structuralanalogs or or thereof. combinationsthereof. combinations

[65]

[65] Compounds Compounds areare obtained obtained from from a wide a wide variety variety of sources of sources including including librariesofof libraries

synthetic orornatural synthetic natural compounds. For compounds. example, numerous For example, meansare numerous means for random available for areavailable random

and directed and synthesisof of directedsynthesis a wide a wide variety variety of organic of organic compounds, compounds, including including biomolecules, biomolecules,

expressionof of including expression including randomized randomized oligonucleotides oligonucleotides and oligopeptides. and oligopeptides. Alternatively, Alternatively,

libraries naturalcompounds libraries ofofnatural formofofbacterial, theform compounds ininthe plant and fungal, plant bacterial, fungal, and animal are extractsare animalextracts 2023266367

available or available produced.Additionally, readily produced. or readily produced syntheticallyproduced naturalororsynthetically Additionally,natural libraries andand libraries compounds compounds are are readily readily modified modified through through conventional conventional chemical, chemical, physical physical and biochemical and biochemical

means, and means, may andmay usedused be be to produce to produce combinatorial combinatorial libraries. Known libraries.Known pharmacological pharmacological

may agentsmay agents be be subjected subjected to directed to directed or random or random chemical chemical modifications, modifications, such such as as acylation, acylation,

amidification, esterification,amidification, alkylation, esterification, alkylation, etc. to to etc. produce produce structural structural analogs. analogs.

[66]

[66] By phagocytosis” “manipulatingphagocytosis" By "manipulating is meant is meant an up-regulation an up-regulation or a down-regulation or a down-regulation in in phagocytosis about leastabout phagocytosisbybyatatleast 10%, 10%, or to or up to 20%, up 20%, or 50%, or 50%, 80% or or 70%oror70% up to or or 80% up to about about compared 90%compared 90% to level to level of phagocytosis of phagocytosis observed observed in absence in absence of intervention. Thus in Thus of intervention. the in the decreasing context ofofdecreasing context phagocytosis phagocytosis of circulating of circulating hematopoietic hematopoietic cells, particularly cells, particularly in a in a transplantation context, manipulating transplantation context, means phagocytosismeans manipulating phagocytosis a down-regulation a down-regulation in in phagocytosis about leastabout phagocytosisbybyatatleast 10%, 10%, or to or up to 20%, up 20%, or 50%, or 50%, 80% or or 70%oror70% up to or or 80% up to about about compared 90%compared 90% to level of of to level phagocytosis phagocytosis observed observed in absence in absence of intervention. of intervention.

[67]

[67] CD47 Agents inhibitors.Agents CD47inhibitors. of interest of interest as CD47 CD47 inhibitors as inhibitors specificspecific include include binding binding members members that that thethe prevent prevent binding of of binding CD47 SIRPSIRP withwith CD47 a receptor. a receptor. The term term “specific The"specific binding binding

member” member" or or member” “bindingmember" "binding as used as used herein herein to a to refersrefers a member member of a specific bindingbinding of a specific pair, pair, two molecules, i.e. two i.e. usuallytwo molecules, usually differentmolecules, twodifferent one one where molecules,where of molecules of the firstfirst (i.e.,(i.e., the molecules binding member) specific binding specific through member) through chemical chemical or physical or physical means means specifically specifically bindsbinds to the to the other other

(i.e., second molecule(i.e., molecule specific binding second specific member). binding member). CD47CD47 inhibitors inhibitors useful useful in the the methods in methods of of the invention the analogs, includeanalogs, invention include derivatives andand derivatives fragments fragments the original of theoforiginal specific specific binding binding

member. member.

[68]

[68] In embodiment, preferred embodiment, In aa preferred thethe specific specific binding binding member is an is member an antibody. antibody. The The term term or "antibody “antibody” or "antibody" is is moiety” “antibodymoiety" intended intended to include to include any polypeptide any polypeptide chain-containing chain-containing

molecular structurewith molecularstructure shapethat specific shape with aa specific to and fits to thatfits recognizesananepitope, and recognizes one one where epitope,where or non-covalent morenon-covalent or more binding binding interactions interactions stabilize the the stabilize complex betweenbetween complex the molecular the molecular

structure and the structure and Antibodies epitope.Antibodies theepitope. utilized utilized thethe in in present present invention may may invention be polyclonal be polyclonal

antibodies, monoclonal although monoclonal antibodies, although antibodies areare antibodies preferred preferred because they they because may may be be reproduced reproduced

cell culture bycell by recombinantly, cultureororrecombinantly, andbecan and can modified to reduceto be modified reduce their their antigenicity. antigenicity.

[69]

[69] Polyclonal antibodiescan Polyclonal antibodies a a raisedbyby canbeberaised standard standard protocol by by protocol injecting injecting a production a production

animal with an animal with antigenic composition. an antigenic See, composition. See, e.g., e.g., Harlow Harlow Lane,Lane, and and Antibodies: Antibodies: A A 17

LaboratoryManual, Laboratory Manual,Cold Cold Spring Spring Harbor Harbor Laboratory, Laboratory, 1988.1988. When utilizing When utilizing an entire an entire protein, protein, 17 Nov 2023

or aa larger or larger section section of of the the protein, protein, antibodies antibodies may maybe be raised raised by by immunizing immunizing the production the production

animal with animal with the theprotein proteinand anda suitable a suitable adjuvant adjuvant (e.g., (e.g., Freund’s, Freund's, Freund’s Freund's complete, complete, oil-in­ oil-in-

water emulsions, water emulsions,etc.) etc.)When When a smaller a smaller peptide peptide is utilized, is utilized, it isadvantageous it is advantageous to conjugate to conjugate

the peptide the peptide with withaalarger largermolecule moleculetoto make makean an immunostimulatory immunostimulatory conjugate. conjugate. Commonly Commonly

utilized conjugate utilized proteinsthat conjugate proteins thatare arecommercially commercially available available for such for such use include use include bovine bovine serumalbumin serum albumin (BSA) (BSA) andand keyhole keyhole limpet limpet hemocyanin hemocyanin (KLH). (KLH). In orderIntoorder raisetoantibodies raise antibodies to to particular epitopes, particular epitopes, peptides derived from peptides derived fromthe thefull full sequence sequence maymay be utilized. be utilized. Alternatively, Alternatively, 2023266367

in order in to generate order to generateantibodies antibodiestotorelatively relativelyshort shortpeptide peptideportions portionsofofthetheprotein protein target,a target, a superior immune superior immune response response may may be be elicited elicited if polypeptide if the the polypeptide is joined is joined to a to a carrier carrier protein, protein,

such as such asovalbumin, ovalbumin,BSABSA or KLH. or KLH. Alternatively, Alternatively, for for monoclonal monoclonal antibodies, antibodies, hybridomas hybridomas may may be formed be formedbybyisolating isolatingthe thestimulated stimulated immune immune cells, cells, suchsuch as those as those fromspleen from the the spleen of the of the inoculated animal. inoculated animal. These These cellsareare cells then then fused fused to to immortalized immortalized cells, cells, such such as as myeloma myeloma cells cells

or transformed or transformedcells, cells,which whichareare capable capable of replicating of replicating indefinitely indefinitely in cell in cell culture,thereby culture, thereby producingananimmortal, producing immortal, immunoglobulin-secreting immunoglobulin-secreting cell line. cell line. In addition, In addition, the antibodies the antibodies or or antigen binding antigen binding fragments fragmentsmaymay be be produced produced by genetic by genetic engineering. engineering. Humanized, Humanized, chimeric,chimeric,

or xenogeneic or xenogeneic human human antibodies,which antibodies, which produce produce lessless of immune of an an immune response response when when administeredtoto humans, administered humans,areare preferred preferred forfor use use in inthe thepresent present invention. invention.

[70]

[70] In addition In addition to to entire entire immunoglobulins immunoglobulins (or (or their their recombinant recombinant counterparts), counterparts),

immunoglobulin immunoglobulin fragments fragments comprising comprising the epitope the epitope binding binding site (e.g., site (e.g., Fab',Fab’, F(ab’)2, F(ab')2, or other or other

fragments) are fragments) are useful useful as as antibody antibody moieties moieties inin the the present presentinvention. invention. Such Such antibody antibody

fragmentsmay fragments maybe be generated generated fromfrom wholewhole immunoglobulins immunoglobulins by pepsin, by ricin, ricin, pepsin, papain,papain, or or other other protease cleavage. protease cleavage. "Fragment," “Fragment,” oror minimal minimal immunoglobulins immunoglobulinsmay may be be designed designed utilizing utilizing

recombinantimmunoglobulin recombinant immunoglobulin techniques. techniques. For instance For instance “Fv” immunoglobulins "Fv" immunoglobulins forthe for use in use in the present invention present inventionmay maybe be produced produced by linking by linking a variable a variable light light chain chain regionregion to a variable to a variable

heavychain heavy chainregion regionvia viaa apeptide peptide linker(e.g., linker (e.g., poly-glycine poly-glycine or or another anothersequence sequence which which doesdoes

not form not formanan alpha alpha helix helix or beta or beta sheetsheet motif). motif).

[71]

[71] Theefficacy The efficacy of of aa CD47 CD47 inhibitorisis assessed inhibitor assessedby by assaying assaying CD47CD47 activity. activity. The The above- above-

mentionedassays mentioned assays or or modified modified versions versions thereof thereof are are used. used. In anInexemplary an exemplary assay,assay, AML AML SCs SCs are incubated are incubatedwith withbone bone marrow marrow derived derived macrophages, macrophages, in the presence in the presence or of or absence absence the of the candidateagent. candidate agent.AnAn inhibitorofofthe inhibitor thecell cell surface surfaceCD47 CD47 will will up-regulate up-regulate phagocytosis phagocytosis by atby at least about least 10%,ororupuptoto20%, about 10%, 20%,oror50%, 50%, or or 70%70% or 80% or 80% or uportoupabout to about 90% compared 90% compared to the to the phagocytosisininabsence phagocytosis absence of the of the candidate candidate agent. agent. Similarly, Similarly, an inan in vitro vitro assayassay for levels for levels of of tyrosine phosphorylation tyrosine phosphorylationofofSIRPa SIRPa willshow will show a decrease a decrease in phosphorylation in phosphorylation byleast by at at least about about

10%, or 10%, or up up to to 20%, 20%, oror 50%, 50%,oror70% 70% or or 80%80% or to or up up about to about 90% 90% compared compared to to phosphorylationobserved phosphorylation observedin in absence absence of the of the candidate candidate agent. agent.

18

[72]

[72] In one In embodiment one embodiment of the the invention, of invention, the agent, the agent, or a pharmaceutical or a pharmaceutical composition composition 17 Nov 2023

comprising theagent, comprisingthe amount providedininananamount agent,isisprovided effective to to effective detectably detectably inhibit thebinding inhibitthe of of binding CD47 SIRPa CD47totoSIRPa receptor receptor present present on the the surface on surface of phagocytic of phagocytic cells. The The cells. effective effective amount amount is is determined via empirical determined via testing routine empirical testing in the routine in art. The the art. effective amount The effective may amount may vary vary thethe dependingonon depending number number of cells beingbeing of cells transplanted, transplanted, site site of of transplantation transplantation and factors and factors

specific toto the specific transplant thetransplant recipient. recipient.

[73]

[73] Theterms The “phagocytic terms"phagocytic cells" and cells”and “phagocytes” "phagocytes" usedused are are interchangeably hereinherein interchangeably to to refer refer to aa cell to is capable that is cell that capable of There phagocytosis.There of phagocytosis. areare three three main main categories categories of phagocytes: of phagocytes: 2023266367

macrophages,mononuclear macrophages, cellscells mononuclear (histiocytes (histiocytes and monocytes); and monocytes); polymorphonuclear polymorphonuclear

leukocytes leukocytes (neutrophils) and and (neutrophils) dendritic cells.cells. dendritic

[74]

[74] The term"biological Theterm sample" "biological sample" encompasses encompasses a variety a variety of sample of sample types obtained from an from types obtained an and can organism and organism diagnostic or usedinina adiagnostic canbebeused assay. The monitoring assay. or monitoring encompasses term encompasses The term blood andother blood and samples liquid samples otherliquid of of biological origin,solid biologicalorigin, tissue samples, solid tissue as as such samples,such a biopsy a biopsy

specimenor ortissue specimen culturesor or tissuecultures cells derived cellsderived therefrom and and therefrom the progeny the progeny thereof. thereof. The The term term encompasses samples encompassessamples that have thathave been been manipulated manipulated in any way way in any after after their their procurement, procurement, such treatment such asasbybytreatment with with reagents, reagents, solubilization, or or solubilization, enrichment for for enrichment certain certain components. components.

The encompasses termencompasses Theterm a clinical a clinical and alsoand sample,sample, includes includes also cells cells in cell culture, culture, in cellcell cell supernatants, lysates, serum, cell lysates, supernatants,cell serum, plasma, fluids, and biological fluids, plasma,biological tissue samples. and tissue samples.

[75]

[75] Hematopoietic stem Hematopoieticstem cells cells (HSC), (HSC), as used as used herein, herein, refers to a to refers a population population of cells of cells having having

the ability self-renew,and ability toto self-renew, give rise to give and to hematopoietic lineages. allhematopoietic to all rise to lineages. Such cell Suchcell populations have populations been havebeen described described in detail in inthe in detail Hematopoietic art. Hematopoietic theart. progenitor progenitor cells cells include include

the myeloid progenitors (CMP), committed progenitors myeloid committed committed lymphoidcommitted thelymphoid (CMP),the progenitors progenitors (CLP), (CLP),

megakaryocyte progenitors, and megakaryocyteprogenitors, progenitors. The multipotent progenitors. and multipotent earliest known The earliest lymphoid- known lymphoid- restricted cell in restricted cell adult mouse inadult bone mouse bone marrow marrow is the is the common common lymphocyte lymphocyte progenitor progenitor (CLP), (CLP), and the and known earliest known theearliest myeloid-restricted cellcell myeloid-restricted thethe is is common common myeloid myeloid progenitor progenitor (CMP). (CMP). Importantly, these cell Importantly, these possess populations possess cell populations an an extremely highhigh extremely level level of lineage of lineage fidelity fidelity in in in in

vitro and vitro and in vivo developmental in vivo assays. developmental assays. A complete A complete description description of these of these cell subsets cell subsets may may be found be Akashi foundininAkashi al.al. et et (2000) (2000) Nature Nature 404(6774):U.S. 404(6774):193, U.S. Patent 193, Patent no. 6,465,247; no. 6,465,247; and and published application published USSN application USSN 09/956,279 09/956,279 (common myeloid myeloid (common progenitor); Kondo etKondo progenitor); et al. al. (1997) (1997) Cell 91(5):661-7, Cell and International 91(5):661-7, and WO99/10478 application WO99/10478 International application (common (common lymphoid lymphoid progenitor); progenitor);

and isis reviewed and Kondo reviewedbybyKondo et al. et al. (2003) AnnuAnnu (2003) Rev Immunol. Rev Immunol. 21:759-806, 21:759-806, each of which of each is which is herein incorporatedbybyreference. specifically incorporated herein specifically The The reference. composition may be may composition beatfrozen frozen liquid at liquid and temperaturesand nitrogen temperatures nitrogen forfor stored stored long of of periods longperiods time, being time,being capable useuse of of capable on thawing. on thawing.

For such For the cells composition, the such aa composition, cells usually willwill be stored usually be stored 10% in a in DMSO, a 10% 50% DMSO, FCS, 40% 50%FCS, 40%

RPMI1640 RPMI medium. 1640medium.

[76]

[76] Populationsofofinterest Populations for use interest for use in the methods in the methods of theinvention ofthe includesubstantially invention include pure substantially pure compositions,e.g. compositions, least about e.g. atat least HSC,HSC, 50% about50% at least aboutabout at least 75% HSC, leastat 75%atHSC, about about least85% 85% 19

HSC, at HSC, at least least about about 95% HSCorormore; 95% HSC more;or or may maybebecombinations combinationsofof one oneor or more morestem stemand and 17 Nov 2023

progenitor cells progenitor cells populations, populations, e.g. e.g. white white cells cells obtained obtained from fromapheresis, apheresis,etc. etc.Where Where purified purified

cell populations cell populations are are desired, desired, the the target target population population may bepurified may be purified in in accordance withknown accordance with known techniques. ForFor techniques. example, example, a population a population containing containing white white blood blood cells, cells, particularly particularly including including

blood or blood or bone bonemarrow marrow samples, samples, are stained are stained with reagents with reagents specific specific for markers for markers present present of of hematopoieticstem hematopoietic stem andand progenitor progenitor cells, cells, whichwhich markers markers are sufficient are sufficient to distinguish to distinguish the the major stem major stem and andprogenitor progenitor groups. groups. TheThe reagents, reagents, e.g.antibodies, e.g. antibodies,may may be be detectably detectably

labeled,orormay labeled, may be indirectly be indirectly labeled labeled in theinstaining the staining procedure. procedure. 2023266367

[77]

[77] Any combination Any combinationofofmarkers markers may may be that be used used are thatsufficient are sufficient to select to select for for the the stem/progenitorcells stem/progenitor cells of of interest. interest. AAmarker marker combination combination of interest of interest maymay include include CD34 CD34 and and CD38,which CD38, which distinguishes distinguishes hematopoietic hematopoietic stemstem cells, cells, (CD34+, (CD34+, CD38') CD38`) from progenitor from progenitor cells, cells, whichare which areCD34+, CD34+,CD38*). CD38+). HSC HSC are lineage are lineage markermarker negative, negative, and positive and positive for expression for expression of of CD90. CD90.

[78]

[78] In the In the myeloid myeloid lineage lineage are are three three cell cellpopulations, populations,termed CMPs, termed CMPs, GMPs, and MEPs. GMPs, and MEPs. These cells These cells are are CD34+ CD34+CD38+, CD38+, they they areare negative negative forfor multiplemature multiple maturelineage lineagemarkers markers including early including earlylymphoid lymphoid markers markers such as CD7, such as CD7, CD10, CD10,and and IL-7R,andand IL-7R, they they areare further further

distinguished by distinguished bythe the markers markers CD45RA, CD45RA, an isoform an isoform of that of CD45 CD45 that can can negatively negatively regulateregulate at at least some least someclasses classes of of cytokine cytokine receptor receptor signaling, signaling, and and IL-3R. IL-3R. These characteristics These characteristics are are CD45RA"IL-3Ra10 CD45RA IL-3Ral0 (CMPs), (CMPs), CD45RA*IL-3Ra CD45RA+IL-3Ral0 (GMPs), (GMPs), andand CD45RA' CD45RA IL-3RcT IL-3Ra (MEPs). (MEPs).

CD45RA' CD45RA IL-3Ral0 IL-3Ra10 cells cells givegive riserise to to GMPs GMPs and and and MEPs MEPs and at at least oneleast thirdone third generate generate both both GMand GM and MegE MegE colonies colonies on a on a single-cell single-cell level. level. All three All three of the of the myeloid myeloid lineage lineage progenitors progenitors

stain negatively stain negatively for for the the markers Thy-1(CD90), markers Thy-1 (CD90),IL-7Ra IL-7Ra (CD127); (CD127); and with and with a panel a panel of lineage of lineage

markers, which lineage markers, lineage markers markers may include CD2; may include CD2; CD3; CDS;CD4; CD4;CD7; CD7; CDS; CD8; CD10; CD10; CD11b; CD11b;

CD14; CD19; CD14; CD19;CD20; CD20;CD56; CD56; and and glycophorinA A(GPA) glycophorin (GPA)ininhumans humansand andCD2; CD2; CD3; CD3; CD4; CD4; CDS; CD8;

CD19;IgM; CD19; IgM;Ter110; Ter110; Gr-1 Gr-1 in mice. in mice. WithWith the exception the exception ofmouse of the the mouse MEP all MEP subset, subset, all of the of the progenitor cells progenitor cells are are CD34 CD34 positive. positive. In the In the mousemouse all of all theofprogenitor the progenitor subsetssubsets may be may be further characterized further as Sca-1 characterized as Sca-1negative, negative,(Ly-6E (Ly-6E andand Ly-6A), Ly-6A), and and c-kitc-kit high.high. In human, In the the human, all three all three of ofthe thesubsets subsets are are CD38+. CD38+

[79]

[79] Common Common lymphoid lymphoid progenitors, progenitors, CLP, express CLP, express low of low levels levels c-kitof(CD117) c-kit (GD117) on their on celltheir cell surface. Antibodies surface. Antibodiesthat thatspecifically specifically bind bind c-kit c-kit in in humans, mice,rats, humans, mice, rats, etc. etc. are are known knownin inthe the art. Alternatively, art. Alternatively,thethe c-kit c-kit ligand, ligand, steel steel factor factor (Slf) (SIf) may may betoused be used to identify identify cells expressing cells expressing

c-kit. The c-kit. CLPcells The CLP cells express expresshigh highlevels levels of of the the IL-7 IL-7 receptor receptor alpha alpha chain chain (CDw127). (CDw127). Antibodies that Antibodies that bind bind to to human human oror totomouse mouse CDw127 CDw127 are known are known in the in the art. art. Alternatively, Alternatively, the the cells are cells are identified identifiedby bybinding binding of ofthe the ligand ligandtotothe thereceptor, receptor,IL-7. IL-7.Human CLPs Human CLPs express express low low levels of levels of CD34. Antibodies CD34. Antibodies specific specific forfor human human CD34 CD34 are commercially are commercially available available and well and well known in known in the the art. art. See, See, for forexample. example,Chen Chen et et al. al.(1997) Immunol (1997) ImmunolRev Rev 157:41-51. 157:41-51. Human Human

CLPcells CLP cells are also also characterized characterizedas asCD38 positive and CD38 positive and CD10 positive. The CD10 positive. CLPsubset The CLP subset 20 also has also hasthe thephenotype phenotype of lacking of lacking expression expression of lineage of lineage specific specific markers, markers, exemplified exemplified by by 17 Nov 2023

B220, CD4, B220, CD4,CD8, CDS,CD3, CDS, Gr-1 Gr-1 and and Mac-1. Mac-1. Thecells The CLP CLP are cellscharacterized are characterized as lacking as lacking

expressionofofThy-1, expression Thy-1, a marker a marker that that is characteristic is characteristic of hematopoietic of hematopoietic stem The stem cells. cells. The phenotype of phenotype of the the CLP maybebefurther CLP may further characterized characterizedas asMel-14', Mel-14,CD4310, CD43 , HSA10, CD45+and HSA , CD45+ and common common cytokine cytokine receptor receptor y chain Y chain positive. positive.

[80]

[80] Megakaryocyteprogenitor Megakaryocyte progenitor cells cells (MKP) (MKP) cells cells are are positive positive for for CD34 expression, and CD34 expression, and tetraspanin CD9 tetraspanin antigen. The CD9 antigen, CD9antigen The CD9 antigen is is aa 227-amino 227-amino acid acid molecule molecule with with 44 hydrophobicdomains hydrophobic domains and and 1 N-glycosylation 1 N-glycosylation site. site. The antigen The antigen is widely is widely expressed, expressed, but is but is 2023266367

not present not present on on certain certain progenitor progenitor cells cellsininthe thehematopoietic hematopoieticlineages. lineages. The The MKP cells MKP cells

expressCD41, express CD41, also also referred referred to the to as as glycoprotein the glycoprotein llb/llla llb/Illa integrin, integrin, which which is platelet is the the platelet receptor for receptor for fibrinogen fibrinogen and several other and several other extracellular extracellular matrix matrix molecules., molecules., for for which which antibodies are antibodies are commercially commercially available,for available, forexample example from from BD Biosciences, BD Biosciences, Pharmingen, Pharmingen, San San Diego, CA., Diego, CA., catalog catalognumber number 340929, 340929, 555466. 555466. The The MKP MKP cells arecells are positive positive for expression for expression of of CD117,which CD117, which recognizes recognizes the the receptor receptor tyrosine tyrosine kinase kinase c-Kit.c-Kit. Antibodies Antibodies are commercially are commercially

available, for available, forexample fromBDBDBiosciences, example from Biosciences, Pharmingen, Pharmingen, San Diego, San Diego, CA,No. CA, Cat. Cat. No. 340529. 340529.

MKPcells MKP cellsare arealso alsolineage lineagenegative, negative,and andnegative negative forexpression for expression of of Thy-1 Thy-1 (CD90). (CD90).

[81]

[81] The phrase The phrase"solid “solid tumor” as used tumor" as used herein herein refers refers to to an an abnormal massofoftissue abnormal mass tissue that that usually does usually doesnot notcontain containcysts cysts or or liquidareas. liquid areas.Solid Solidtumors tumors may may be benign be benign or malignant. or malignant.

Different types Different types of solid solid tumors are named tumors are namedforfor thetype the typeofofcells cellsthat thatform formthem. them.Examples Examples of of solid tumors solid are sarcomas, tumors are sarcomas,carcinomas, carcinomas, lymphomas lymphomas etc. etc.

[82]

[82] Anti-CD47antibodies. Anti-CD47 antibodies.Certain Certain antibodies antibodies that that bind bind CD47 prevent CD47 prevent its interaction its interaction with with SIRPareceptor. SIRPa receptor.Antibodies Antibodies include include free free antibodies antibodies and and antigen antigen binding binding fragments fragments derivedderived

therefrom, and therefrom, and conjugates, conjugates, e.g. e.g.pegylated pegylatedantibodies, antibodies,drug, drug,radioisotope, radioisotope,or or toxin toxin

conjugates, conjugates, andand the the like.like.

[83]

[83] Monoclonalantibodies Monoclonal antibodies directed directed against against a specific a specific epitope, epitope, or combination or combination of epitopes, of epitopes,

will allow will allow for for the the targeting targeting and/or depletion of and/or depletion of cellular cellular populations expressingthethe populations expressing marker. marker.

Various techniques Various techniquescancan be utilized be utilized usingusing monoclonal monoclonal antibodies antibodies to screentoforscreen for cellular cellular populations expressing populations expressingthethe marker(s), marker(s), and include and include magnetic magnetic separation separation using antibody- using antibody-

coated magnetic coated magnetic beads, beads, "panning" "panning" withwith antibody antibody attached attached to a to a solid solid matrix matrix (i.e., (i.e., plate), plate), andand

flow cytometry flow cytometry(See, (See,e.g., e.g.,U.S. U.S. Pat. Pat. No. No. 5,985,660; 5,985,660; and Morrison and Morrison et al. 96:737-49 et al. Cell, Cell, 96:737-49 (1999)). These (1999)). Thesetechniques techniques allow allow for screening for the the screening of particular of particular populations populations of in of cells; cells; in immunohistochemistry of immunohistochemistry of biopsy biopsy samples; samples; in in detecting detecting the the presence presence of of markers markers shed by shed by

cancercells cancer cellsinto intothethe blood blood and and otherother biologic biologic fluids,fluids, and and the the like. like.

[84]

[84] Humanized Humanized versions versions of such of such antibodies antibodies arewithin are also also the within theofscope scope of this invention. this invention.

Humanized Humanized antibodies antibodies are are especially especially useful useful for for in vivo in vivo applications applications in humans in humans due due to to their their

lowantigenicity. low antigenicity.

21

[85]

[85] Thephrase The phrase “bispecific "bispecific antibody” antibody" refers refers to atosynthetic a synthetic or recombinant or recombinant antibodyantibody that that 17 Nov 2023

recognizesmore recognizes more than than oneone protein. protein. Examples Examples include include bispecific bispecific antibodies antibodies 2B1,2B1, 520C9xH22, 520C9xH22,

mDX-H210, mDX-H210, andand MDX447. MDX447. Bispecific Bispecific antibodies antibodies directed directed against against a combination a combination of epitopes, of epitopes,

will allow will allow for for the targeting and/or depletion the targeting depletion of of cellular cellular populations expressing the populations expressing the combination of combination of epitopes. epitopes. Exemplary Exemplarybi-specific bi-specific antibodies antibodies include include those thosetargeting targeting a a combination of combination of CD47 anda acancer CD47 and cancer cellmarker, cell marker,such suchas, as,CD96, CD96, CD97, CD97, CD99, CD99, PTHR2, PTHR2,

HAVCR2 HAVCR2 etc. etc. Generation Generation of bi-specific of bi-specific antibody antibody is is described described in the in the literature, for literature, for example, example,inin USPN USPN 5989830, 5989830, USPNUSPN 5798229, 5798229, which which are are incorporated incorporated herein byherein by reference. reference. 2023266367

[86]

[86] The terms"treatment", Theterms "treatment","treating", "treating", "treat" "treat" and the like and the like are usedherein are used hereintotogenerally generallyrefer refer to obtaining to obtaining aa desired desired pharmacologic pharmacologicand/or and/orphysiologic physiologiceffect. effect. The Theeffect effectmay may be be prophylactic in prophylactic in terms termsofofcompletely completelyor or partiallypreventing partially preventing a disease a disease or symptom or symptom thereofthereof

and/or may and/or maybe be therapeutic therapeutic in terms in terms of a partial of a partial or complete or complete stabilization stabilization or cureor cure for a for a diseaseand/or disease and/oradverse adverse effect effect attributable attributable to the to the disease. disease. "Treatment" "Treatment" as used as used herein herein covers any covers anytreatment treatment of of a disease a disease in ainmammal, a mammal, particularly particularly a human, a human, and includes: and includes: (a) (a) preventing the preventing the disease diseaseororsymptom symptom fromfrom occurring occurring in a in a subject subject whichwhich may bemay be predisposed predisposed

to the disease to diseaseororsymptom symptombut but has has not been not yet yet been diagnosed diagnosed as it; as having having it; (b) inhibiting (b) inhibiting the the diseasesymptom, disease symptom, i.e.,arresting i.e., arrestingits its development; development;oror(c) (c)relieving relieving the the disease diseasesymptom, symptom, i.e., i.e.,

causing regression causing regressionofofthe thedisease diseaseororsymptom. symptom.

[87]

[87] Theterms The terms"recipient", “recipient”, "individual", “individual”, “subject”, "subject",“host”, "host",and and“patient”, "patient",used usedinterchangeably interchangeably

herein and herein andrefer refertotoany anymammalian mammalian subject subject for whom for whom diagnosis, diagnosis, treatment, treatment, or is or therapy therapy is desired, particularly desired, particularly humans. humans.

[88]

[88] A "host A "host cell", cell", as as used herein, refers used herein, refers to to aa microorganism microorganism or or a eukaryotic a eukaryotic cell cell or or cellline cell line cultured as cultured asa aunicellular unicellularentity entitywhich whichcancan be, be, or been, or has has been, used asused as a recipient a recipient for a for a recombinantvector recombinant vectorororother othertransfer transferpolynucleotides, polynucleotides,and andinclude includethe theprogeny progeny of of thethe original original

cell which cell has been which has beentransfected. transfected.ItItisis understood understoodthat thatthe theprogeny progeny of aofsingle a single cellcell maymay not not necessarily be necessarily becompletely completely identicalininmorphology identical morphology or genomic or in in genomic or total or total DNA DNA complement complement

as the as theoriginal originalparent, parent,duedue to natural, to natural, accidental, accidental, or deliberate or deliberate mutation. mutation.

[89]

[89] The terms"cancer", Theterms "cancer", "neoplasm", "neoplasm", "tumor", "tumor", and "carcinoma", and "carcinoma", areinterchangeably are used used interchangeably herein to herein to refer refer to to cells cells which exhibit relatively which exhibit relativelyautonomous growth, autonomous growth, so so that that they they exhibit exhibit an an

aberrantgrowth aberrant growth phenotype phenotype characterized characterized by a significant by a significant lossofofcell loss of control control of cell proliferation. proliferation.

In general, In general, cells cells of of interest interest for for detection detection or or treatment treatmentininthe thepresent present application application include include

precancerous(e.g., precancerous (e.g.,benign), benign), malignant, malignant, pre-metastatic, pre-metastatic, metastatic, metastatic, and non-metastatic and non-metastatic

cells. Detection cells. Detection ofof cancerous cancerous cellscells is of is of particular particular interest. interest. The The term term as "normal" "normal" used in as the used in the context of context of "normal "normalcell," cell,"isis meant meantto to refer refer to to a cell a cell of untransformed of an an untransformed phenotype phenotype or or exhibiting a a morphology exhibiting of aa non-transformed morphology of non-transformed cell cell of of the the tissue tissue type type being being examined. examined.

"Cancerousphenotype" "Cancerous phenotype" generally generally refers refers to to anyany of of a variety a variety ofof biologicalphenomena biological phenomenathatthat are are characteristic of characteristic of aa cancerous cell, which cancerous cell, phenomena which phenomena can can vary vary with with the type the type of cancer. of cancer. The The

22 cancerousphenotype cancerous phenotype is generally is generally identified identified by by abnormalities abnormalities in,in, forforexample, example, cell cell growth growth or or 17 Nov 2023 proliferation (e.g., proliferation (e.g., uncontrolled growth ororproliferation), uncontrolled growth proliferation), regulation regulation ofofthe thecell cellcycle, cycle,cell cell mobility, cell-cell mobility, cell-cell interaction, interaction,orormetastasis, metastasis,etc.etc.

[90]

[90] "Therapeutictarget" "Therapeutic target" refers refers to to aa gene or gene gene or geneproduct productthat, that, upon uponmodulation modulation of of itsitsactivity activity (e.g., by (e.g., by modulation modulationof of expression, expression, biological biological activity, activity, andlike), and the the like), can provide can provide for for modulationofofthe modulation thecancerous cancerous phenotype. phenotype. As used As used throughout, throughout, "modulation" "modulation" is meant is meant to to refer refer to an to increaseororaa decrease an increase decreasein in theindicated the indicatedphenomenon phenomenon (e.g.,(e.g., modulation modulation of a biological of a biological

activity refers activity refers to to an anincrease increasein ainbiological a biological activity activity or aor a decrease decrease in a biological in a biological activity). activity). 2023266367

MethodsFOR METHODS Transplantation forTRANSPLANTATION

[91]

[91] Methodsare Methods areprovided provided to to manipulate manipulate phagocytosis phagocytosis of circulating of circulating hematopoietic hematopoietic cells.cells. In In someembodiments some embodiments of invention of the the invention the circulating the circulating cells cells are hematopoietic are hematopoietic stem or stem cells, cells, or hematopoieticprogenitor hematopoietic progenitor cells,particularly cells, particularlyinin a atransplantation transplantationcontext, context,where where protection protection

from phagocytosis from phagocytosisis is desirable. desirable. In other In other embodiments embodiments the circulating the circulating cells cells are are leukemia leukemia

cells, particularly cells, particularly acute acutemyeloid myeloid leukemia (AML), where leukemia (AML), whereincreased increased phagocytosis phagocytosis is is desirable. desirable.

[92]

[92] In some In embodiments some embodiments of of thethe invention,hematopoietic invention, hematopoieticstem stemor or progenitorcells progenitor cells are are protected from protected fromphagocytosis phagocytosisin in circulationbybyproviding circulation providinga host a host animal animal with with a CD47 a CD47 mimetic mimetic

molecule, which molecule, which interacts interacts with with SIRPa SIRPaon on macrophages macrophages and decreases and decreases macrophage macrophage

phagocytosis.The phagocytosis. The CD47 CD47 mimetic mimetic may may be be soluble soluble CD47; CD47; CD47 CD47 coated oncoated on the the surface ofsurface the of the cells to cells to be be protected, protected, aa CD47 mimeticthat CD47 mimetic thatbinds bindstotoSIRPa SIRPaat at thethe CD47 CD47 binding binding site,site, and and the the like. In like. In some some embodiments embodiments of theofinvention, the invention, CD47 CD47 is is provided provided as protein, as a fusion a fusionfor protein, for examplesoluble example solubleCD47 CD47 fused fused to Fc to an an fragment, Fc fragment, e.g., e.g., IgG1IgGI Fc, lgG2 Fc, IgG2 Fc,A Ig Fc, Ig Fc Aetc. Fc etc.

[93]

[93] Methodsforforgenerating Methods generating proteins proteins lacking lacking the the transmembrane transmembrane region region are wellare wellinknown known in the art. the art. For For example, a soluble example, a soluble CD47 CD47can canbebe generated generated by by introducing introducing a stop a stop codon codon

immediately before immediately before the the polynucleotide polynucleotide sequence sequenceencoding encodingthethetransmembrane transmembrane region. region.

Alternatively, thethepolynucleotide Alternatively, sequence polynucleotide sequenceencoding encoding the the transmembrane region can transmembrane region canbebe replaced by replaced by aa polynucleotide polynucleotide sequence sequenceencoding encoding a fusion a fusion protein protein such such as lgG1 as IgG1 Fc. Fc. Sequence Sequence forfor Fc Fc fragments fragments from different from different sources sources are available are available via publicly via publicly accessible accessible

database including database including Entrez, Entrez, Embl, Embl, etc. etc. For For example, example, mRNA mRNA encoding encoding human human lgG1 IgG1 Fc Fc fragmentisis provided fragment providedbybyaccession accession number number X70421. X70421.

[94]

[94] The subject The subject invention invention provide provide for for methods for transplanting methods for transplanting hematopoietic hematopoietic stem or stem or

progenitor cells progenitor cells into into aa mammalian recipient.A need mammalian recipient. A need for for transplantation transplantation may may be caused be caused by by genetic or genetic or environmental environmental conditions, conditions, e.g. e.g. chemotherapy, chemotherapy, exposure exposure to radiation, to radiation, etc. etc. The The cells for cells fortransplantation transplantation may be mixtures may be mixturesofofcells, cells, e.g. e.g. buffy buffy coat coat lymphocytes froma adonor, lymphocytes from donor, or may or maybebepartially partially or or substantially substantially pure. pure. The Thecells cellsmay may be be autologous autologous cells, cells, particularly particularly if if

23 removedprior removed priortotocytoreductive cytoreductive or or other other therapy, therapy, or or allogeneic allogeneic cells, cells, andand may may be for be used used for 17 Nov 2023 hematopoieticstem hematopoietic stemororprogenitor progenitor cellisolation cell isolation and and subsequent subsequent transplantation. transplantation.

[95]

[95] Thecells The cells may maybebecombined combined withwith the the soluble soluble CD47 CD47 mimetic mimetic prior prior to to administration. administration. For For example,the example, thecells cellsmay maybe be combined combined with with the mimetic the mimetic at a concentration at a concentration of fromofabout from 10about 10 pg/ml, about ug/ml, about100 100 pg/ml, ug/ml, about about 1 mg/ml, 1 mg/ml, about about 10 mg/ml, 10 mg/ml, etc., atetc., at a temperature a temperature of from of from about 4°, about 4°, about about10°, 10°,about about25°25° about about 37°,37°, for for a period a period of time of time sufficient sufficient to coat to coat the the cells, cells,

where in where in some someembodiments embodimentsthethe cellsare cells aremaintained maintainedononice. ice. InIn other other embodiments embodimentsthe the cells are cells are contacted with the contacted with the CD47 CD47mimetic mimeticimmediately immediately priortotointroduction prior introduction into into the the 2023266367

recipient, where recipient, the concentrations where the concentrationsofofmimetic mimeticare areasasdescribed described above. above.

[96]

[96] Thecomposition The compositioncomprising comprising hematopoietic hematopoietic stem stem or progenitor or progenitor cellscells and aand a CD47 CD47 mimeticmimetic

is administered is administered in in any anyphysiologically physiologically acceptable acceptable medium, medium, normally normally intravascularly, intravascularly,

althoughthey although theymay may also also be be introduced introduced into into bonebone or other or other convenient convenient site, where site, where the the cells cells mayfind may findananappropriate appropriate site site forfor regeneration regeneration and and differentiation. differentiation. Usually, Usually, at least at least 1x1051x105

cells will cells willbe beadministered, administered, preferably preferably 1x106 1x106 or or more. Thecomposition more. The composition maymay be introduced be introduced by by injection, catheter, injection, catheter,ororthe thelike. like.

MyeloproliferativeDISORDERS, MYELOPROLIFERATIVE DisordersLEUKEMIAS, , LeukemiasAND , andMYELODYSPLASTIC Myelodysplastic Syndrome SYNDROME

[97]

[97] Acute leukemias Acute leukemias are arerapidly rapidly progressing progressing leukemia leukemia characterized characterized by by replacement replacement of of normalbone normal bone marrow marrow by blast by blast cellscells of a of a clone clone arising arising from malignant from malignant transformation transformation of a of a hematopoieticcell. hematopoietic cell. The Theacute acute leukemias leukemias include include acuteacute lymphoblastic lymphoblastic leukemia leukemia (ALL) (ALL) and and acute myelogenous acute myelogenous leukemia leukemia (AML). (AML). ALL ALLoften ofteninvolves involves the the CNS, CNS, whereas whereasacute acute monoblasticleukemia monoblastic leukemia involves involves thethe gums, gums, and and AML involves AML involves localized localized collections collections in anyinsite any site (granulocytic sarcomas (granulocytic sarcomas ororchloromas). chloromas).

[98]

[98] Thepresenting The presentingsymptoms symptoms are usually are usually nonspecific nonspecific (e.g.,(e.g., fatigue, fatigue, fever, fever, malaise, malaise, weight weight

loss) and loss) and reflect reflect the the failure failure of of normal hematopoiesis. normal hematopoiesis. Anemia Anemia and thrombocytopenia and thrombocytopenia are are very common very (75to common (75 to 90%). 90%). The TheWBC WBC count count may may be be decreased, decreased, normal,ororincreased. normal, increased. Blast Blast cells are cells usually found are usually found inin the the blood bloodsmear smear unless unless the the WBC WBC count count is markedly is markedly decreased. decreased.

The blasts The blasts of of ALL ALL can canbebedistinguished distinguishedfrom fromthose thoseofofAML AML by histochemical by histochemical studies, studies,

cytogenetics, immunophenotyping, cytogenetics, immunophenotyping, and molecular and molecular biologybiology studies. studies. In addition In addition to smearsto smears with the with the usual usual stains, stains, terminal terminal transferase, transferase,myeloperoxidase, myeloperoxidase, Sudan Sudan black black B, andB,specific and specific and nonspecific and nonspecificesterase. esterase.

[99]

[99] ALLisis the ALL the most mostcommon common malignancy malignancy in children, in children, with with a peak a peak incidence incidence from 3ages from ages to 5 3 to 5 yr. It yr. It also also occurs occurs in in adolescents andhas adolescents and hasa asecond, second, lower lower peak peak in adults. in adults. Typical Typical treatment treatment

emphasizesearly emphasizes earlyintroduction introduction of of an an intensive intensive multidrug multidrug regimen, regimen, which whichmay may include include

prednisone,vincristine, prednisone, vincristine, anthracycline or asparaginase. anthracycline or asparaginase.Other Other drugs drugs and combinations and combinations are are cytarabine and cytarabine etoposide, and and etoposide, cyclophosphamide. Relapse and cyclophosphamide. Relapse usually usually occurs occurs in in thebone the bone marrowbut marrow butmaymay alsoalso occur occur in the in the CNS CNS or or testes, testes, alone alone or concurrent or concurrent with with bone bone marrow. marrow. 24

Althoughsecond Although second remissions remissions can can be induced be induced in many in many children, children, subsequent subsequent remissions remissions tend tend 17 Nov 2023

to be to bebrief. brief.

[100]

[100] The incidence The incidence of of AML increaseswith AML increases with age; age; it it isisthe themore more common acuteleukemia common acute leukemiainin adults. AML adults. AMLmaymay be associated be associated with with chemotherapy chemotherapy or irradiation or irradiation (secondary (secondary AML).AML).

Remissioninduction Remission induction rates rates areare lower lower than than with with ALL, ALL, and long-term and long-term disease-free disease-free survival survival reportedly occurs reportedly occursininonly only2020toto40%40% of patients. of patients. Treatment Treatment differs differs most most fromin ALL from ALL that in that AMLresponds AML responds to to fewer fewer drugs. drugs. The basic The basic induction induction regimen regimen includes includes cytarabine; cytarabine; along along with with daunorubicinororidarubicin. daunorubicin idarubicin.Some Some regimens regimens include include 6-thioguanine, 6-thioguanine, etoposide, etoposide, vincristine, vincristine, 2023266367

and prednisone. and prednisone.

[101]

[101] Polycythemia vera Polycythemia vera(PV) (PV) is anis idiopathic an idiopathic chronicchronic myeloproliferative myeloproliferative disorder disorder

characterized by characterized by an increase in an increase in Hb concentration and Hb concentration RBCmass and RBC mass (erythrocytosis). PVPV (erythrocytosis).

occurs in occurs in about about2.3/100,000 2.3/100,000 people people perper year; year; more more often often in males in males (about (about 1.4:1). 1.4:1). The The mean mean age at age at diagnosis diagnosisisis 60 60 yr yr (range, (range, 15 15 to to 90 90 yr; yr; rarely rarely in inchildhood); childhood); 5% of patients 5% of patients are are < < 40 yr 40 yr

at onset. at onset. The The bone bonemarrow marrow sometimes sometimes appears appears normalnormal but usually but usually is hypercellular; is hypercellular;

hyperplasia involves hyperplasia involves all allmarrow marrow elements elements and replaces marrow and replaces marrowfat. fat. There There is is increased increased production and production andturnover turnover of of RBCs, RBCs, neutrophils, neutrophils, and platelets. and platelets. Increased Increased megakaryocytes megakaryocytes

maybebepresent may presentininclumps. clumps.Marrow Marrow ironiron is is absent absent in >in 90% > 90% of patients, of patients, eveneven when when

phlebotomy phlebotomy has has notnot been been performed. performed.

[102]

[102] Studies of Studies of women women with with PV PV who who are heterozygous are heterozygous at the at the X-chromosome-linked X-chromosome-linked locus for locus for G6PDhave G6PD haveshown shown thatRBCs, that RBCs, neutrophils, and neutrophils, and platelets platelets have have the thesame same G6PD isoenzyme, G6PD isoenzyme,

supporting supporting a clonal a clonal origin origin of this of this disorder disorder at a at a pluripotent pluripotent stem stem cell cell level. level.

[103]

[103] Eventually, about Eventually, about25% 25% of patients of patients havehave reduced reduced RBC survival RBC survival and failand fail to adequately to adequately

increase erythropoiesis; increase erythropoiesis;anemia anemiaand and myelofibrosis myelofibrosis develop. develop. Extramedullary Extramedullary hemopoiesis hemopoiesis

occursininthe occurs thespleen, spleen, liver, liver, andand other other sitessites with with the potential the potential for cell for blood blood cell formation. formation.

[104]

[104] Withouttreatment, Without treatment,50% 50% of symptomatic of symptomatic patients patients die within die within 18 diagnosis. 18 mo of mo of diagnosis. With With treatment, median treatment, mediansurvival survivalisis7 7toto1515yr. yr.Thrombosis Thrombosis is the is the most most common common cause cause of of death, death, followed by followed by complications complications ofof myeloid myeloidmetaplasia, metaplasia,hemorrhage, hemorrhage, and and development development of of leukemia. leukemia.

[105]

[105] Theincidence The incidenceofoftransformation transformationinto intoananacute acuteleukemia leukemiais is greater greater ininpatients patientstreated treatedwith with radioactive phosphate radioactive phosphate (32P) (32P) or or alkylating alkylating agents agents than than in those in those treated treated with phlebotomy with phlebotomy

alone. PV alone. PVthat thattransforms transformsinto intoacute acuteleukemia leukemia is more is more resistant resistant to induction to induction chemotherapy chemotherapy

than de than de novo novoleukemia. leukemia.

[106]

[106] BecausePVPV Because is is thethe only only form form of of erythrocytosis erythrocytosis forfor which which myelosuppressive myelosuppressive therapy therapy may may be indicated, be indicated, accurate diagnosisisis critical. accurate diagnosis critical. Therapy Therapy must be individualized must be individualized according accordingto to age, age, sex, medical sex, medicalstatus, status, clinical clinical manifestations, manifestations, and and hematologic findings. hematologic findings.

[107]

[107] Myelodysplastic syndrome Myelodysplastic (MDS)is isa agroup syndrome (MDS) group of of syndromes syndromes (preleukemia, (preleukemia, refractory refractory

anemias, Ph-negative anemias, Ph-negative chronic chronic myelocytic myelocytic leukemia, leukemia, chronic chronicmyelomonocytic myelomonocytic leukemia, leukemia,

25 myeloidmetaplasia) myeloid metaplasia)commonly commonly seen seen in older in older patients. patients. Exposure Exposure to carcinogens to carcinogens may may by be by be 17 Nov 2023 implicated. MDS implicated. MDS is characterized is characterized by clonal by clonal proliferation proliferation of hematopoietic of hematopoietic cells,cells, including including erythroid, myeloid, erythroid, myeloid, and megakaryocytic and megakaryocytic forms. forms. The The bonebone marrow marrow is normal is normal or hypercellular, or hypercellular, and ineffective and ineffective hematopoiesis hematopoiesiscauses causes variable variable cytopenias, cytopenias, the the mostmost frequent frequent beingbeing anemia. anemia.

Thedisordered The disorderedcell cellproduction productionisisalso alsoassociated associatedwith withmorphologic morphologic cellular cellular abnormalities abnormalities in in marrowand marrow and blood. blood. Extramedullary Extramedullary hematopoiesis hematopoiesis may occur, may occur, leadingleading to hepatomegaly to hepatomegaly and and splenomegaly.Myelofibrosis splenomegaly. Myelofibrosis is is occasionally occasionally present present at at diagnosis diagnosis or or maymay develop develop during during the the courseof course of MDS. MDS.TheThe MDS MDS cloneclone is unstable is unstable and tends and tends to progress to progress to AML.to AML. 2023266367

[108]

[108] Anemiaisisthe Anemia themost most common common clinical clinical feature, feature, associated associated usually usually with with macrocytosis macrocytosis and and anisocytosis. Some anisocytosis. Some degree degree of thrombocytopenia of thrombocytopenia is on is usual; usual; bloodon bloodthesmear, smear, the platelets platelets vary in vary in size, size, and and some appear some appear hypogranular. hypogranular. The The WBC WBC count count may be may be increased, normal, normal, increased, or or decreased.Neutrophil decreased. Neutrophil cytoplasmic cytoplasmic granularity granularity is abnormal, is abnormal, with with anisocytosis anisocytosis and variable and variable

numbersof ofgranules. numbers granules. Eosinophils Eosinophils alsoalso may abnormal may have have abnormal granularity. granularity. A monocytosis A monocytosis is is characteristic ofofthethechronic characteristic myelomonocytic chronic myelomonocyticleukemia leukemiasubgroup, subgroup, and and immature myeloid immature myeloid

cells may cells occurinin the may occur theless lesswell welldifferentiated differentiated subgroups. The subgroups. The prognosis prognosis is highly is highly

dependenton dependent onclassification classification and and on on any any associated associated disease. ResponseofofMDS disease. Response MDSto to AMLAML

chemotherapy chemotherapy is is similartotothat similar that of of AML, AML,after after age ageand andkaryotype karyotype areare considered. considered.

Treatment of TREATMENT OF CCANCER ancer

[109]

[109] Theinvention The inventionprovides providesmethods methods for for reducing reducing growth growth of cancer of cancer cells cells by increasing by increasing their their clearancebybyphagocytosis, clearance phagocytosis, through through thethe introduction introduction of of a CD47 a CD47 blocking blocking agent, agent, e.g. e.g. an anti- an anti-

CD47antibody. CD47 antibody.In certain In certain embodiments embodiments the cancer the cancer cellsbemay cells may AML be stemAML stem cells. In cells. other In other embodiments, the embodiments, thecancer cancercells cells may maybebe those those of of a solidtumor, a solid tumor,such such as,as, glioblastoma, glioblastoma,

melanoma melanoma etc. etc. By By blocking blocking the the activity activity ofof CD47, CD47, thethe downregulation downregulation of phagocytosis of phagocytosis that that is is foundwith found withcertain certain tumor tumor cells, cells, e.g. e.g. AML cells, AML cells, is prevented. is prevented.

[110]

[110] In addition In additiontotoCD47, CD47, we have discovered we have discovered aa number numberofofmarkers markersspecific specific to to AML AMLSC. SC. These include These include CD96, CD96, CD97, CD99, PTHR2, CD97, CD99, PTHR2, HAVCR2 HAVCR2 etc.These etc. These markershave markers havebeen been disclosed inin U.S. disclosed U.S.Patent PatentApplication Application No.No. 61/011,324, 61/011,324, filedfiled on January on January 15,and 15, 2008 2008 are and are herebyincorporated hereby incorporatedbybyreference. reference.

[111]

[111] "Reducing "Reducing growth growth of cancer of cancer cells" cells" includes, includes, butlimited but is not is not to, limited to, reducing reducing proliferation proliferation of of cancercells, cancer cells, and andreducing reducingthethe incidence incidence of aofnon-cancerous a non-cancerous cell becoming cell becoming a cancerous a cancerous

cell. Whether cell. Whether aa reduction reduction in in cancer cancercell cell growth growthhas hasbeen been achieved achieved can can be readily be readily

determined using determined usinganyany known known assay, assay, including, including, butlimited but not not limited to, [3H]-thymidine to, [3H]-thymidine

incorporation; counting incorporation; countingcell cell number number over over a period a period of time; of time; detecting detecting and/or and/or measuring measuring a a markerassociated marker associatedwith withAML, AML, etc. etc.

[112]

[112] Whethera substance, Whether a substance, or aor a specific specific amount amount of the of the substance, substance, is effective is effective in treating in treating

cancer can cancer can be be assessed assessedusing usingany anyofofa avariety variety of of known knowndiagnostic diagnostic assays assaysfor for cancer, cancer,

26 including, but including, but not not limited limited to to biopsy, biopsy, contrast contrast radiographic studies, CAT radiographic studies, CATscan, scan,andand detection detection 17 Nov 2023 of aa tumor of markerassociated tumor marker associated with with cancer cancer in in thethe blood blood of of thethe individual.TheThe individual. substance substance can can beadministered be administered systemically systemically or locally, or locally, usually usually systemically. systemically.

[113]

[113] As an As an alternative alternative embodiment, an agent, embodiment, an agent, e.g. e.g. aa chemotherapeutic drug that chemotherapeutic drug that reduces cancercell cancer cell growth, growth,can can be be targeted targeted to atocancer a cancer cellconjugation cell by by conjugation to aspecific to a CD47 CD47 specific antibody. Thus, antibody. Thus,ininsome some embodiments, embodiments, the invention the invention provides provides a method a method of delivering of delivering a druga drug to aa cancer to cell, comprising cancer cell, administering aa drug-antibody comprising administering drug-antibodycomplex complex to to a subject, a subject, wherein wherein thethe

antibodyisis specific antibody specific for for aa cancer-associated polypeptide,andand cancer-associated polypeptide, thethe drug drug is one is one thatthat reduces reduces 2023266367

cancercell cancer cell growth, growth, aa variety variety of of which are known which are knownininthe theart. art. Targeting Targetingcancan be be accomplished accomplished

by coupling by coupling(e.g., (e.g.,linking, linking,directly directlyororviaviaa linker a linker molecule, molecule, either either covalently covalently or or non- non- covalently, so covalently, so as astotoform forma drug-antibody a drug-antibody complex) complex) a druga to drug to an antibody an antibody specific specific for a for a cancer-associatedpolypeptide. cancer-associated polypeptide.Methods Methods of coupling of coupling a drug a drug to antibody to an an antibody are well are well known known in in the art the art and neednot and need notbe beelaborated elaboratedupon upon herein. herein.

[114]

[114] In certain In certain embodiments, a bi-specificantibody embodiments, a bi-specific antibody maymay be used. be used. For example For example a bi-specific a bi-specific

antibody in antibody in which one antigen which one antigen binding binding domain domain isis directed directed against against CD47 andthe CD47 and theother other antigen binding antigen binding domain is directed domain is directed against againsta acancer cancer cell cellmarker, marker,such suchas, as,CD96 CD96 CD97, CD97,

CD99, PTHR2, CD99, PTHR2,HAVCR2 HAVCR2 etc.etc. maymay be be used. used.

[115]

[115] Depletion of Depletion of AMLSC AMLSC is useful is useful in the in the treatment treatment of AML. of AML. Depletion Depletion can be can be achieved achieved by by several methods. several methods.Depletion Depletion is is defined defined as as a reduction a reduction in in thethe targetpopulation target population by by up up to to about about

30%,ororupuptotoabout 30%, about 40%, 40%, or to or up upabout to about 50%, 50%, or up or to up to about about 75% or 75% more.or Anmore. An effective effective depletion is depletion is usually usually determined determinedbyby thethe sensitivityofofthe sensitivity theparticular particulardisease diseasecondition conditiontotothe the levels of levels of the the target target population. population. Thus Thusininthe thetreatment treatment of of certain certain conditions conditions a depletion a depletion of of evenabout even about20% 20% could could be be beneficial. beneficial.

[116]

[116] A CD47 A CD47 specificagent specific agent that that specificallydepletes specifically depletesthe thetargeted targetedAMLSC AMLSC is used is used to contact to contact

the patient the patientblood blood in in vitro vitro or or in vivo, in vivo, wherein wherein after after the contacting the contacting step, step, there is there is a reduction a reduction in in the number the numberofofviable viableAMLSC AMLSC in the in the targeted targeted population. population. An effective An effective dose dose of antibodies of antibodies for for such aapurpose such purposeis is sufficienttotodecrease sufficient decrease the the targeted targeted population population to desired to the the desired level,level, for for exampleasas example described described above. above. Antibodies Antibodies for purposes for such such purposes may havemay have low antigenicity low antigenicity in in humansoror may humans maybebehumanized humanizedantibodies. antibodies.

[117]

[117] In one In embodiment one embodiment of of thethe invention,antibodies invention, antibodiesforfordepleting depletingtarget targetpopulation populationare areadded added to patient to patient blood blood in in vivo. vivo. InInanother anotherembodiment, embodiment, the antibodies the antibodies are added are added to the to the patient patient

blood ex blood exvivo. vivo. Beads Beads coated coated with with thethe antibody antibody of interest of interest cancan be be added added to the to the blood, blood, target target

cells bound cells to these bound to thesebeads beadscancan then then be be removed removed from from the blood the blood using using procedures procedures common common in the in the art. art.InIn one oneembodiment embodiment the the beads beads are are magnetic magnetic and and are are removed using a removed using a magnet. magnet.

Alternatively,when Alternatively, whenthe the antibody antibody is biotinylated, is biotinylated, it is possible it is also also possible to indirectly to indirectly immobilize immobilize the the antibody onto antibody ontoa asolid solidphase phase which which has has adsorbed adsorbed avidin, avidin, streptavidin, streptavidin, or like. or the the like. The The solidsolid

phase, usually phase, usually agarose agaroseororsepharose sepharose beads beads are are separated separated from from the blood the blood by by brief brief

27 centrifugation. Multiple centrifugation. Multiple methods methods fortagging for taggingantibodies antibodies andand removing removing such such antibodies antibodies and and 17 Nov 2023 any cells any cells bound to the bound to the antibodies antibodies are are routine routine in in the the art. art.Once the desired Once the desired degree degree of of depletion has depletion hasbeen beenachieved, achieved, thethe blood blood is returned is returned to the to the patient. patient. Depletion Depletion of target of target cells cells ex vivo ex vivo decreases decreasesthe theside sideeffects effectssuch such as as infusion infusion reactions reactions associated associated withwith the the intravenousadministration. intravenous administration.An additional An additional advantage advantage is thatisthe that the repertoire repertoire of available of available antibodies isis expanded antibodies expanded significantly significantly as this as this procedure procedure does does not notto have have to beto limited be limited to antibodies with antibodies with low low antigenicity antigenicity in in humans orhumanized humans or humanized antibodies. antibodies. 2023266367

Example11 EXAMPLE CD47IS CD47 is a AM arker OF MARKER MyeloidLEUKEMIAS of MYELOID Leukemias Materials and Materials and Methods Methods

[118]

[118] Immunohistochemistry. Cytospins Immunohistochemistry. Cytospins ofof double doublesorted sortedmyeloid myeloidprogenitor progenitor populations populations

(CMP,GMP), (CMP, GMP), IL-3Ra IL-3Ra highhigh CD45CD45 RA+and RA+ cells cells and CD14+c-kit+lin- CD14+c-kit+lin- cells cells were were performed performed using using a Shandon a Shandon cytospin cytospin apparatus. apparatus. Cytospins Cytospins were stained were stained with Giemsa with Giemsa diluted diluted 1/5 with 1/5 H20 with H20 for 10 for min followed 10 min followed by by staining staining with with May-Grunwald May-Grunwaldforfor2020minutes. minutes.Cytospins Cytospins werewere

analyzedwith analyzed withthe theaid aid of of aa Zeiss microscope. Zeiss microscope.

[119]

[119] HumanBone Human Bone Marrow Marrow and and Peripheral Peripheral Blood Blood Samples. Samples. Normal Normal bone bone marrowmarrow samplessamples

wereobtained were obtainedwith withinformed informed consent consent from from 20 -20 25-year 25 year old paid old paid donors donors whohepatitis who were were hepatitis A, B, A, B, CC and andHIV HIVnegative negativebyby serology(All serology (All Cells). Cells). CMML CMML bone bone marrow marrow samples samples were were obtained with obtained with informed informedconsent, consent, from from previously previously untreated untreated patients, patients, at Stanford at Stanford University University

Medical Center. Medical Center.

[120]

[120] HumanBone Human Bone Marrow Marrow HSCHSC and Myeloid and Myeloid Progenitor Progenitor Flow-Cytometric Flow-Cytometric Analysis Analysis and and CellCell

Sorting. Mononuclear Sorting. Mononuclear fractions fractions were were extracted extracted following following Ficoll centrifugation Ficoll density density centrifugation according to according to standard methodsand standard methods andanalyzed analyzedfresh freshororsubsequent subsequent to to rapid rapid thawing thawing of of samples previously samples previously frozen frozeninin90% 90% PCS FCS and 10%DMSO and 10% DMSOin in liquid nitrogen. liquid nitrogen. In In some cases, some cases,

CD34+cells CD34+ cells were wereenriched enriched from from mononuclear mononuclearfractions fractions with with the the aid aid of of immunomagnetic immunomagnetic

beads (CD34+ beads (CD34+Progenitor ProgenitorIsolation Isolation Kit, Kit, Miltenyi Miltenyi Biotec, Biotec, Bergisch-Gladbach, Bergisch-Gladbach, Germany). Germany).

Prior to Prior to FACS analysisandand FACS analysis sorting, sorting, myeloid myeloid progenitors progenitors werewere stained stained with lineage with lineage markermarker

specific phycoerythrin specific phycoerythrin(PE)-Cy5-conjugated (PE)-Cy5-conjugatedantibodies antibodiesincluding CD2 including CD2RPA-2.10; RPA-2.10; CD11b, CD11b,

ICRF44; CD20, ICRF44; CD20,2H7; 2H7;CD56, CD56, B159; B159; GPA,GPA, GA-R2GA-R2 (Becton (Becton Dickinson Dickinson - PharMingen, - PharMingen, San San Diego), CD3,S4.1;CD4, Diego), S3.5;CD7, CD3,S4.1;CD4, S3.5; CD7,CD7-6B7; CD7-6B7; CD8,CDS, 3B5; 3B5; CD10,CD10, 5-1B4,5-1B4, CD14, CD14, TUK4; TUK4; CD19, SJ25-C1 CD19, SJ25-C1(Caltag, (Caltag, South San Francisco, South San Francisco, CA) CA) and and APC-conjugated anti-CD34, HPCA- APC-conjugated anti-CD34, HPCA- 2 (Becton 2 (BectonDickinson-PharMingen), Dickinson-PharMingen), biotinylated biotinylated anti-CD38, anti-CD38, HIT2 HIT2 (Caltag) (Caltag) in addition in addition to PE-to PE- conjugated anti-IL-3Ra, conjugated anti-IL-3Ra, 9F5 (Becton Dickinson- 9F5 (Becton Dickinson- ParMingen) ParMingen) and andFITC-conjugated FITC-conjugated anti- anti-

CD45RA, CD45RA, MEM56 MEM56 (Caltag) (Caltag) followed followed by staining by staining with Streptavidin with Streptavidin -Texas -Texas Red Red to to visualize visualize CD38-BIOstained CD38-BIO stainedcells cells and andresuspension resuspensionin inpropidium propidium iodideto toexclude iodide exclude dead dead cells. cells.

Unstainedsamples Unstained samplesandand isotype isotype controls controls were were included included to assess to assess background background fluorescence. fluorescence.

28

[121]

[121] Following staining, Following staining,cells cellswere analyzed were analyzedand andsorted sortedusing usinga amodified modifiedFACS Vantage FACS Vantage 17 Nov 2023

(Becton Dickinson (Becton Dickinson Immunocytometry Systems, Mountain Immunocytometry Systems, Mountain View, View, CA) CA) equipped equipped with with a 599 599 nm nm

dye laser dye laser and anda a488 488nm nm argon argon laser. laser. Double Double sortedsorted progenitor progenitor cells (HSC) cells (HSC) were identified were identified

as CD34+ as CD34+CD38+ CD38+andand lineagenegative. lineage negative. Common Common myeloid myeloid progenitors(CMP) progenitors (CMP)were were identified based identified based on CD34+CD38+ on CD34+ CD38+ IL-3Ra+ IL-3Ra+ CD45RA- CD45RA- lin- staining lin- staining and their and their progeny progeny

including granulocyte/macrophage including granulocyte/macrophage progenitors progenitors(GMP) were CD34+CD38+IL-3Ra+ (GMP) were CD34+CD38+IL-3Ra+ CD45RA+ CD45RA+ while while megakaryocyte/erythrocyte megakaryocyte/erythrocyte progenitors(MEP) progenitors (MEP) were were identifiedbased identified based on on

CD34+CD38+ CD34+ CD38+ IL-3Ra IL-3Ra - CD45RA- - CD45RA- lin-staining lin- staining (Manz, (Manz, PNAS 11872). PNAS 11872). 2023266367

CD47Expression CD47 Expression by by Normal Normal versus versus Myeloproliferative Myeloproliferative andProgenitors and AML AML Progenitors

[122]

[122] Peripheral blood Peripheral bloodand and bone bone marrow samples were marrow samples wereobtained obtained with with informed informed consent consent from from

patients with patients with myeloproliferative myeloproliferativedisorders disordersandand acute acute myelogenous myelogenous leukemialeukemia at at Stanford Stanford University Medical University MedicalCenter Center according according to Stanford to Stanford IRBHIPAA IRB and and regulations. HIPAA regulations. PeripheralPeripheral

blood or blood or bone bonemarrow marrow mononuclear mononuclear cellscells (1 - (1 - 5x106 5x106 cells) cells) werewere stained stained with with lineage lineage cocktail cocktail

as above as but excluding above but excluding CD7, CD7, CD11b andCD14. CD11b and CD14.Subsequently, Subsequently, samples samples were were stained stained with with

CD14PEPE(1/25), CD14 (1/25), CD47 CD47FITC FITC(1/25), (1/25), CD38 CD38Bio Bio(Bio) (Bio) and and c-kit c-kit APC APC (1/25) (1/25) or orCD34 CD34 APC or APC or

FITC(1/50) FITC (1/50)for for 45 45 min minfollowed followedbybywashing washingandand staining staining with with Streptavidin Streptavidin Texas Texas Red (1/25) Red (1/25)

for 45 for 45 min and finally min and finally resuspension in propidium resuspension in propidiumiodide. iodide. Discussion Discussion

[123]

[123] Here we Here weshow show thatCD47 that CD47 overexpression overexpression is characteristicofofprogression is characteristic progression of of human human myeloproliferative disorders myeloproliferative disorders to to AML AML(see (see Figures Figures 1 - 15B). - 5B). CD47 CD47 controls controls integrin integrin function function

but also but also the the ability ability of of macrophages macrophages toto phagocytose phagocytose cells, cells, depending depending onlevel on the the level of of CD47 CD47 expression. Thus, expression. Thus, aberrant aberrant CD47 CD47expression expressionmay may allow allow LSCLSC to evade to evade bothboth innate innate and and

adaptive host adaptive host immunity. immunity.

[124]

[124] HumanCD47 Human CD47 expression expression analysis analysis waswas performed performed via via FACSFACS on human on human normal,normal, pre- pre­ leukemicmyeloproliferative leukemic myeloproliferativedisorder disorder(MPD) (MPD) or AML or AML HSC, HSC, progenitors progenitors and lineage and lineage positive positive

cells derived cells derived from marrowororperipheral from marrow peripheralblood. blood.MPDMPD samples samples (n=63)(n=63) included included polycythemia polycythemia

vera (PV; vera (PV; n=15), n=15), post-polycythemic post-polycythemic myeloid myeloid metaplasia/myelofibrosis metaplasia/myelofibrosis (PPMM/MF; n=5), (PPMM/MF; n=5),

essential thrombocythemia essential (ET; thrombocythemia (ET; n=8), n=8), atypical atypical chronic chronic myelogenous myelogenous leukemia leukemia (aCML; (aCML; n=2), n=2), CML(n=7), CML (n=7), chronic chronic eosinophilic eosinophilic leukemia leukemia (CEL; (CEL; n=1), n=1), chronic chronic myelomonocytic leukemia myelomonocytic leukemia

(CMML;n=n=13) (CMML; 13)and andacute acute myelogenous myelogenousleukemia leukemia(AML; (AML;n=12). n=12).AsAs we we have have observed observed with with

the transgenic the transgenic leukemic leukemicmouse mouse models, models, progression progression of human of human myeloproliferative myeloproliferative disorders disorders

to AML to (n=12) was AML (n=12) wasassociated associatedwith with an an expansion expansionofof the the GMP GMP pool(70.6%+/- pool (70.6%+/-S.D. S.D.2.15) 2.15) comparedwith compared with normal normalbone bonemarrow marrow (14.7%+/- (14.7%+/- S.D. S.D. 2.3). 2.3). Furthermore, Furthermore, FACSFACS analysis analysis

revealed that revealed that CD47 CD47 expression expression firstincreased first increased 1.7 1.7 fold fold in in AMLAML compared compared with normal with normal HSC HSC and then and thenincreased increasedtoto2.2 2.2fold foldgreater greaterthan thannormal normal with with commitment commitment of AMLofprogenitors AML progenitors to to the myeloid the myeloid lineage. lineage. CD47 CD47was was over-expressed over-expressed by by AMLAML primitive primitive progenitorsandand progenitors their their

progenybut progeny butnot notbybythe themajority majorityofofMPD MPD (MFI(MFI 2.3+/-S.D. 2.3+/-S.D. 0.43)0.43) compared compared with normal with normal bone bone 29 marrow(MFI marrow (MFI 1.91.9 +/-S.D. +/-S.D. 0.07). 0.07). Thus, Thus, increased increased CD47 expression CD47 expression is diagnostic is a useful a useful diagnostic 17 Nov 2023 markerfor marker for progression progressiontotoAML AML and and in in addition addition represents represents a novel a novel therapeutic therapeutic target. target.

Example22 Example Humanand Human andmouse mouse leukemias leukemias upregulateCD47 upregulate CD47 to to evade evade macrophage macrophage killing killing

[125]

[125] CD47 CD47 FacilitatesEngraftment, Facilitates Engraftment, InhibitsPhagocytosis, Inhibits Phagocytosis, andand is More is More Highly Highly Expressed Expressed on on AMLLSC. AML LSC. WeWe determinedexpression determined expression of ofCD47 CD47 on on human human AML LSC and AML LSC and normal normal HSC by HSC by flow cytometry. flow cytometry. HSC (Lin-CD34+CD38-CD90+) from HSC (Lin-CD34+CD38-CD90+) from three three samples samples of of normal normal human human 2023266367

mobilized peripheral mobilized peripheralblood bloodand andAML AML LSC (Lin-CD34+CD38-CD90-) LSC (Lin-CD34+CD38-CD90-) from from seven seven samples samples of of human AML human AMLwere were analyzedfor analyzed forsurface surface expression expression of of CD47 (Figure 6). CD47 (Figure 6). CD47 was CD47 was expressed at expressed at low low levels levels on on the the surface surface of of normal normalHSC; HSC; however, however, on average, on average, it was it was

approximately5-fold approximately 5-foldmore morehighly highlyexpressed expressedon on AML AML LSC, LSC, as as as well well as leukemic bulk bulk leukemic blasts.blasts.

[126]

[126] Anti-Human CD47 Anti-Human CD47Monoclonal Monoclonal Antibody Antibody StimulatesPhagocytosis Stimulates Phagocytosisandand Inhibits Inhibits Engraftment of Engraftment of AML LSC.In Inorder AML LSC. ordertototest test the the model model that that CD47 CD47overexpression overexpressionononAML AML LSCprevents LSC prevents phagocytosis phagocytosis of these of these cells cells through through its interaction its interaction with SIRPa with SIRPa on effector on effector

cells, we cells, haveutilized we have utilized aa monoclonal monoclonal antibody antibody directed directed against against CD47CD47 known known to disrupt to disrupt the the CD47-SIRPa CD47-SIRPa interaction. interaction. The hybridoma The hybridoma producing producinga mouse-anti-human a mouse-anti-human CD47CD47 monoclonal antibody, monoclonal antibody, termed termed B6H12, B6H12,waswas obtained obtained fromfrom ATCCATCC andtoused and used to produce produce

purified antibody. purified First, we antibody. First, conducted we conducted in in vitrophagocytosis vitro phagocytosis assays. assays. Primary Primary human human AML AML LSCwere LSC werepurified purified by by FACS FACSfrom fromtwo twosamples samples of of human human AML, AML, and and then then loaded loaded with with the the fluorescent dye fluorescent CFSE.These dye CFSE. These cellswere cells were incubated incubated with with mouse mouse bone bone marrow-derived marrow-derived

macrophagesand macrophages and monitored monitored using using immunofluorescence immunofluorescence microscopy microscopy (Figure (Figure 7) and 7) and flowflow

cytometry(Figure cytometry (Figure9)9)totoidentify identifyphagocytosed phagocytosed cells. cells. In both In both cases, cases, no phagocytosis no phagocytosis was was observedininthe observed thepresence presenceof of an an isotype isotype control control antibody; antibody; however, however, significant significant phagocytosis phagocytosis

wasdetected was detected with with thethe addition addition of the of the anti-CD47 anti-CD47 antibody antibody (Figure (Figure 9). blockage 9). Thus, Thus, blockage of of humanCD47 human CD47 with with a monoclonal a monoclonal antibody antibody is capable is capable of stimulatingthe of stimulating thephagocytosis phagocytosisofof these cells these cellsbybymouse mouse macrophages. macrophages.

[127]

[127] Wenext We nextinvestigated investigatedthe theability ability of of the the anti-CD47 anti-CD47antibody antibodyto to inhibitAML inhibit AMLLSC LSC

engraftmentininvivo. engraftment vivo. Two Twoprimary primary human human AML samples AML samples were untreated were either either untreated or with or coated coated with the anti-CD47 the anti-CD47antibody antibodyprior priortoto transplantation transplantation into into NOG NOG newborn newborn mice. mice. 13 weeks 13 weeks later, later, the the mice were mice were sacrificed sacrificed and and analyzed analyzed for forhuman human leukemia leukemia bone marrowengraftment bone marrow engraftmentby byflow flow cytometry (Figure cytometry (Figure 10). 10). The The control control mice mice demonstrated leukemic engraftment demonstrated leukemic engraftment while while mice mice transplanted with transplanted with the theanti-CD47-coated anti-CD47-coated cells cells showed showed little little to to no no engraftment. engraftment. These These data data indicate that indicate that blockade blockadeofofhuman humanCD47CD47 with awith a monoclonal monoclonal antibodyantibody is able is able to to AML inhibit inhibit AML LSCengraftment. LSC engraftment.

[128]

[128] CD96isis aa Human CD96 HumanAcute Acute Myeloid Myeloid Leukemia Leukemia Stem Stem Cell-SpecificCell Cell-Specific CellSurface SurfaceMolecule. Molecule. CD96, CD96, originally originally termed termed Tactile, Tactile, was identified was first first identified as a Tas aT cell cell surface surface molecule molecule that that is highly is highly

30 upregulatedupon upregulated upon T cellactivation. T cell activation.CD96 CD96 is expressed is expressed at lowatlevels low levels on resting on resting T and T NKand NK 17 Nov 2023 cells and cells is strongly and is strongly upregulated uponstimulation upregulated upon stimulationininboth bothcell cell types. types. ItIt is is not not expressed expressed onon other hematopoietic other hematopoieticcells, cells, and andexamination examination of itsexpression of its expression pattern pattern showed showed that that it is itonly is only otherwisepresent otherwise presentononsome some intestinalepithelia. intestinal epithelia. The The cytoplasmic cytoplasmic domain domain of CD96 of CD96 contains contains a a putativeITIM putative ITIMmotif, motif, butbut it isnotnot it is know know if this if this functions functions in signal in signal transduction. transduction. CD96 CD96 promotes promotes adhesionofofNKNKcells adhesion cellstototarget target cells cells expressing CD155, expressing CD155, resultingininstimulation resulting stimulationofofcytotoxicity cytotoxicity of activated of activatedNKNK cells. cells.

[129]

[129] Preferential Cell Preferential CellSurface SurfaceExpression Expression of of Molecules Molecules Identified Identifiedfrom fromGene Gene Expression Expression 2023266367

Analysis. Beyond Analysis. Beyond CD47 CD47 and CD96, and CD96, severalseveral molecules molecules described described in U.S.Application in U.S. Patent Patent Application No. 61/011,324 No. 61/011,324 are are known known to to be be expressed expressed on on AML AMLLSC, LSC, including: CD123, including: CD123,CD44, CD44, CD99 CD99

and CD33. and CD33.

[130]

[130] Tumorprogression Tumor progressionisischaracterized characterized bybyseveral severalhallmarks, hallmarks,including including growth growthsignal signal independence, independence, inhibitionofofapoptosis, inhibition apoptosis,andand evasion evasion of the of the immune immune system, system, among among others. others. Weshow We show here here thatexpression that expressionofofCD47, CD47, a ligand a ligand forfor themacrophage the macrophage inhibitorysignal inhibitory signal regulatory protein regulatory proteinalpha alpha (SIRPa) (SIRPa) receptor, receptor, isisincreased increased ininhuman and mouse human and mouse myeloid myeloid

leukaemiaand leukaemia and allows allows cellstotoevade cells evade phagocytosis phagocytosis and increase and increase their their tumorigenic tumorigenic potential. potential.

CD47, also CD47, alsoknown known as integrin as integrin associated associated protein protein (IAP), (IAP), is immunoglobulin-like is an an immunoglobulin-like transmembranepentaspanin transmembrane pentaspaninthat that is is broadly broadly expressed expressed in inmammalian tissues. We mammalian tissues. provide We provide

evidence that evidence that CD47 CD47 isis upregulated upregulated in in mouse mouseand and human human myeloid myeloid leukaemia leukaemia stem stem and and progenitor cells, progenitor cells, as well as as well as leukemic leukemicblasts. blasts.Consistent Consistent with with a biological a biological role role forfor CD47 CD47 in in myeloid leukaemia myeloid leukaemia development developmentandand maintenance, maintenance, we demonstrate we demonstrate that ectopic that ectopic over-over­

expressionofofCD47 expression CD47 allows allows a myeloid a myeloid leukaemia leukaemia cell line cell line to grow to grow in mice in mice that that areB,T,and are T, B, and NK-cell deficient, NK-cell deficient, whereas whereas it itisisotherwise otherwise cleared cleared rapidly rapidly when when transplanted transplanted into into these these recipients. The recipients. Theleukemogenic leukemogenic potential potential of CD47 of CD47 is also is also shownshown to be dose-dependent, to be dose-dependent, as as higher expressing higher expressing clones clones have have greater greater tumor tumorforming formingpotential potential than than lower lowerexpressing expressing clones. We clones. Wealso alsoshow show that that CD47 CD47 functions functions in in promoting promoting leukemogenesis leukemogenesis by inhibiting by inhibiting

phagocytosisofofthe phagocytosis theleukemic leukemiccells cellsbybymacrophages. macrophages.

[131]

[131] CD47isis significantly CD47 significantly upregulated upregulatedininleukemic leukemicFaslpr/Ipr Fas! XxhMRP8bcl2 hMRP8bcl2 transgenic transgenic bone bone

marrow, and marrow, and inin leukemic leukemichMRP8bcr/abl hMRP8bcr/abl x hMRP8bcl2 X hMRP8bcl2 mice. mice. Transcripts Transcripts for CD47 for CD47 are are increased in increased in leukemic leukemic hMRP8bcr/abl hMRP8bcr/abl Xx hMRP8bcl2 hMRP8bcl2 bone bone marrow marrow 3-4 3-4 foldfold by by quantitative quantitative

RT-PCRand RT-PCR and 6-7fold 6-7 foldinin c-Kit c-Kit enriched enriched leukemic leukemic marrow relative totohealthy marrow relative healthyhMRP8bcl2+ hMRP8bcl2+

bone marrow bone marrow(Figure (Figure11e). lie).Leukemic Leukemic spleen spleen had anhad an expansion expansion of the granulocyte of the granulocyte

macrophage macrophage progenitor progenitor (GMP) (GMP) population population as as as well wellc-Kit+ as c-Kit+ Sca-1+ Sca-1+ Lin-stem Lin-stem and progenitor and progenitor

subsetsrelative subsets relative to to control control mice, mice, which whichwere were of of thethe same same genotype genotype as leukemic as leukemic mice butmice but failed to failed todevelop develop disease (Figure11a-d). disease (Figure 11a-d).Expression Expression levels levels forfor CD47 CD47 protein protein werewere foundfound to to begin increasing begin increasing in in leukemic leukemicmice micerelative relativetoto control control mice miceatat the the stage stageofofthe the Flk2- Flk2- CD34- CD34-C-c- Kit+ Sca-1+ Kit+ Lin-long-term Sca-1+ Lin- long-termhematopoietic hematopoietic stem stem cellcell (LT-HSC) (LT-HSC) (Figure (Figure 11f). 11f). This increased This increased

31 level of level of expression expressionwas maintained inin GMP was maintained and Mac-1+ GMP and Mac-1 +blasts, blasts, but but not not 17 Nov 2023 megakaryocyte/erythroid megakaryocyte/erythroid restrictedprogenitors restricted progenitors (MEP) (MEP) (Figure (Figure 11f).11f). The increase The increase in CD47in CD47 between leukemic between leukemicand andnormal normalcells cells was wasbetween between 3 to2020 3 to fold. All fold. Allmice micethat thatdeveloped developed leukaemia that we leukaemia that haveexamined we have examined from from hMRP8bcr/abl hMRP8bcr/abl x hMRP8bcl2 X hMRP8bcl2 primary primary (n=3) (n=3) and and Ipr/lpr X hMRP8bcl2 primary (n=14) and secondary secondary secondarytransplanted mice transplanted (n=3), mice Fas Fas (n=3), lpr/lpr x hMRP8bcl2 primary (n=14) and secondary (n=19) mice, (n=19) mice, and and hMRP8bcl2 hMRP8bcl2 X xhMRP8bcl2 hMRP8bcl2 primary primary (n=3)and (n=3) andsecondary secondary (n=12) (n=12) mice mice had had increased CD47 increased expression. We CD47 expression. Wehave have alsofound also foundincreased increasedCD47 CD47 expressionininmice expression micethat that received p210bcr/abl received p210bcr/abl retrovirally-transduced retrovirally-transduced mouse mouse bone marrowcells bone marrow cells that that developed developed 2023266367 leukemia. leukemia.

[132]

[132] FACS-mediated FACS-mediated analysis analysis of human of human hematopoietic hematopoietic progenitor progenitor populations populations was performed was performed

on blood on blood and andmarrow marrow derived derived from from normal normal cord cord bloodblood and mobilized and mobilized peripheral peripheral blood (n=16) blood (n=16)

and myeloproliferative and myeloproliferative disorders disorders (MPDs) (MPDs)including includingpolycythemia polycythemia veravera (PV; (PV; n=16),n=16),

myelofibrosis (MF; myelofibrosis (MF; n=5), n=5), essential essentialthrombocythemia thrombocythemia (ET; (ET; n=7), n=7), chronic chronic myelomonocytic myelomonocytic

leukaemia (CMML; leukaemia (CMML;n=11) n=11)and and atypical chronic atypical chronic myeloid myeloid leukaemia leukaemia (aCML; (aCML;n=1) n=1)asaswell well as as blast crisis blast crisis phase chronic myeloid phase chronic myeloidleukaemia leukaemia (CML; (CML; n=19), n=19), chronic chronic phase phase CML CML (n=7) and(n=7) and acute myelogenous acute myelogenous leukaemia leukaemia (AML;(AML; n=13).n=13). This analysis This analysis demonstrated demonstrated that granulocyte- that granulocyte-

macrophageprogenitors macrophage progenitors(GMP) (GMP) expanded expanded in MPDs in MPDs with with myeloid myeloid skewed skewed differentiation differentiation

potential including atypical potential atypical CML, proliferative phase CML, proliferative CMML phase CMML and and acuteacute leukaemia leukaemia including including

blast crisis blast crisisCML CML and and AML (Figure 12a). AML (Figure 12a). AML AMLHSCHSC and and progenitors progenitors uniformly uniformly exhibited exhibited

higher levels higher levels of of CD47 expressioncompared CD47 expression compared with with normal normal controls controls (Figure (Figure 12b);every 12b); every sample from sample from BC-CML BC-CML and and AML AML had had elevated elevated levelsofofCD47. levels CD47. Moreover, Moreover, progression progression from from

chronic phase chronic CMLtotoblast phase CML blastcrisis crisis was was associated associated with with aa significant significant increase increase in in CD47 CD47

expression(Figure expression (Figure12c). 12c).Using Using the the methods methods described described in thisinstudy, this study, we havewe have found found that that humanCD47 human CD47 proteinexpression protein expression in in CML-BC increased 2.2 CML-BC increased 2.2 fold fold ininCD90+ CD90+ CD34+ CD38-Lin- CD34+ CD38- Lin- cells relative cells relativetotonormal normal (p=6.3 x 105), (p=6.3 x 10'5), 2.3 2.3 fold fold in in CD90- CD34+ CD90- CD34+ CD38- CD38- Lin- Lin- cellscells relative relative to to normal(p=4.3 normal (p=4.3X x10-5), 10'5), and and2.4 2.4fold fold in in CD CD34+ 34+CD38+ CD38+ Lin- Lin- cells cells (p=7.6 (p=7.6 1066x (Figures ICT6) (Figures 12b- 12b- 12c); however, 12c); however,using usinga anewer newer optimized optimized staining staining protocol protocol we observed we have have observed that that CD47 is CD47 is increased approximately increased approximately 10 10 fold fold ininAML AML and BC-CMLcompared and BC-CML compared to to normal normal human human HSCs HSCs

and progenitors. and progenitors.

[133]

[133] It was It then asked was then askedwhether whether forced forced expression expression of mouse of mouse CD47 CD47 on onleukemic human human cells leukemic cells wouldconfer would conferaacompetitive competitiveadvantage advantage in forming in forming tumors tumors in mice. in mice. MOLM-13 MOLM-13 cells, are cells, which which are derived from derived fromaapatient patient with with AML AML5a,5a,were were transduced transduced withwith Tet-MCS-IRES-GFP Tet-MCS-IRES-GFP (Tet) or (Tet) Tet- or Tet- CD47-MCS-IRES-GFP CD47-MCS-IRES-GFP (Tet-CD47) (Tet-CD47) (Figure (Figure 13a), 13a), andand stable stable integrants were integrants werepropagated propagatedonon the basis the basis of of GFP GFPexpression. expression.The The cellscells werewere then then transplanted transplanted intravenously intravenously in a in a competitive setting competitive setting with untransduced MOLM-13 with untransduced MOLM-13 cells cells intointo T, and T, B, B, NK anddeficient NK deficient recombination activating recombination activating gene gene 2, 2, common gamma common gamma chain chain deficient(RAG2-/-, deficient (RAG2-/-,Gc-/-) Gc-/-) mice. mice. Only cells Only cells transduced with Tet-CD47 transduced with wereable Tet-CD47 were abletotogive giverise rise toto tumors tumorsininthese thesemice, mice,

32 efficiently engrafting efficiently bone engrafting bonemarrow, marrow,spleen spleenand and peripheral peripheral blood blood (Figures (Figures 13a-b). The 13a-b). The 17 Nov 2023 tumorswere tumors werealso alsocharacterized characterized by by large large tumor tumor burden burden in the in the liver liver (Figures (Figures 13b, 13b, 13g), 13g), which which is particularly is particularly significant significant because the because the liveris isthought liver thought to have to have the highest the highest number number of of macrophages macrophages of of anyany organ, organ, withwith estimates estimates that that Kupffer Kupffer cells cells may may comprise comprise 80% of80% of the the total total tissue macrophage tissue population. These macrophage population. Thesecells cells also also make makeupup 30%30% of the of the sinusoidal sinusoidal lining, lining, thereby strategically thereby strategically placing placingthem them at sites at sites of entry of entry into liver. into the the liver. Hence, significant Hence, significant engraftmentthere engraftment therewould would have have to disable to disable a macrophage a macrophage cytotoxic cytotoxic response. response. In addition In addition to to developing tumor developing tumor nodules, nodules, the the Tet-CD47 Tet-CD47MOLM-13 MOLM-13 cellscells exhibited exhibited patterns patterns of of hepatic hepatic 2023266367 involvementtypically involvement typically seen seenwith withhuman human AML,AML, with with leukemic leukemic cells infiltrating cells infiltrating the the liver liver with with a a sinusoidal and sinusoidal and perivenous perivenous pattern.(Figure pattern. (Figure13d). 13d).Overall, Overall, Tet-CD47 Tet-CD47 MOLM-13 MOLM-13 transplanted transplanted mice died mice died more morequickly quicklythan thanTet TetMOLM-13 MOLM-13 transplanted transplanted mice, mice, which which had virtually had virtually no no engraftmentofofleukemic engraftment leukemic cells cells in in hematopoietic hematopoietic tissues tissues (Figure (Figure 13c).13c). Tet-MOLM-13 Tet-MOLM-13 mice mice still had still had significant mortality,most significant mortality, most likely likely duedue to localized to localized growth growth at the at theofsite site of injection injection (retro- (retro- orbital sinus) orbital sinus)with withextension extensionintointo the the brain. brain.

[134]

[134] Since CD47 Since CD47hashas beenbeen shownshown to be important to be important for the for the migration migration of hematopoietic of hematopoietic cells, cells, andisisknown and known to modulate to modulate bindingbinding to extracellular to extracellular matrix either matrix proteins, proteins, eitherinteraction by direct by direct interaction or via or via its its effect effecton on integrins, integrins,one one possibility possibilityforfor thethelack of of lack growth growthofofTet TetMOLM-13 cellsinin MOLM-13 cells

mice was mice wastheir theirinability inability to to migrate migrate to to niches. niches. To Totest testthis this possibility, possibility, Tet Tet MOLM-13 MOLM-13 or or Tet- Tet-

CD47MOLM-13 CD47 MOLM-13 cellscells were were directly directly injected injected intointo the the femoral femoral cavity cavity of immunodeficient of immunodeficient mice.mice.

Tet-CD47MOLM-13 Tet-CD47 MOLM-13 cellscells were were able able to to engraft engraft all bones all bones and other and other hematopoietic hematopoietic tissues tissues of of recipient mice, recipient mice, whereas TetMOLM-13 whereas Tet MOLM-13 cellscells had had minimal, minimal, if any, if any, engraftment engraftment only only at site at the the site of injection of injection (Figure (Figure 13e). Mice Micetransplanted transplanted in in thismanner this mannerwithwith Tet-CD47 Tet-CD47 MOLM-13 MOLM-13 cells cells died at died at approximately approximately 50-60 50-60 daysdays post-transplant post-transplant (n=4), (n=4), whereas whereas mice mice that that received received Tet Tet MOLM-13 MOLM-13 (n=5) (n=5) cells cells remained remained alivealive forleast for at at least 75 days 75 days without without signssigns of disease of disease at which at which

point they point wereeuthanized they were euthanizedforfor analysis.These analysis. These results results suggest suggest a function a function otherother than than or in or in addition to addition to migration migration or or homing for CD47 homing for CD47ininMOLM-13 MOLM-13 engraftment. engraftment.

[135]

[135] Completelack Complete lackofofCD47 CD47hashas beenbeen shown shown to result to result in phagocytosis in phagocytosis of transplanted of transplanted murinemurine

erythrocytes and erythrocytes andleukocytes, leukocytes,via vialack lackofofinteraction interaction with with SIRPa SIRPa onon macrophages. macrophages. Thus, Thus, we we tested whether tested whetherover-expression over-expression of CD47 of CD47 could could prevent prevent phagocytosis phagocytosis of live,of live, unopsonized unopsonized

MOLM-13 MOLM-13 cells. WeWe cells. incubatedTet incubated TetororTet-CD47 Tet-CD47MOLM-13 MOLM-13 cells cells withbone with bone marrow marrow derived derived

macrophages(BMDM) macrophages (BMDM)forfor 2-24 2-24 hoursand hours and assessed assessed phagocytosis phagocytosis by by countingthe counting thenumber number of ingested of ingested GFP+ GFP+cells cells under undera amicroscope microscope or or by by evaluating evaluating thethe frequency frequency of GFP+ of GFP+

macrophages using macrophages using aa flow flow cytometer. cytometer. Expression ExpressionofofCD47 CD47 dramaticallylowered dramatically lowered macrophage macrophage clearance clearance of these of these cells cells at all at all time time points points tested,whereas tested, whereas Tet-MOLM-13 Tet-MOLM-13 were were quickly phagocytosed quickly in aa manner phagocytosed in mannerthat thatincreased increased over overtime time(Figures (Figures 14a-c). 14a-c). WeWe also also

injected MOLM-13 injected cells into MOLM-13 cells into mice mice and and analyzed analyzedhematopoietic hematopoieticorgans organs2 2hours hourslater laterfor for evidenceofofmacrophage evidence macrophage phagocytosis. phagocytosis. Macrophages Macrophages in bone spleen, in bone marrow, marrow,and spleen, and liver all liver all 33 had higher had higher GFP+ GFP+fraction fraction when wheninjected injected with with Tet Tet MOLM-13 cellsasascompared MOLM-13 cells comparedto to CD47 CD47 17 Nov 2023 expressing cells. expressing cells. This This indicates indicates that that CD47 over-expression can CD47 over-expression cancompensate compensate for for pro- pro- phagocyticsignals phagocytic signalsalready alreadypresent present on on leukemic leukemic cells, cells, allowing allowing themthem to survive to survive when when they they wouldotherwise would otherwisebebecleared cleared by by macrophages. macrophages.

[136]

[136] Recentreport Recent reportindicates indicates that that lack lack of CD47 of CD47 reactivity reactivity acrossacross speciesspecies might might mediate mediate xenorejectionsofoftransplanted xenorejections transplantedcells. cells.Furthermore, Furthermore, a recent a recent studystudy has demonstrated has demonstrated that that humanCD47 human CD47 is unable is unable to interact to interact withwith SIRPa SIRPa from C57BI/6 from C57BI/6 mice, mice, but but istoable is able reacttowith react with receptor from receptor fromnon-obese non-obese diabetic diabetic (NOD) (NOD) mice,mice, which which arepermissive are more more permissive for humanfor human cell cell 2023266367

engraftmentthan engraftment thanC57BI/6 C57BI/6 mice. mice. Furthermore, Furthermore, wealso we have have also observed observed that HL-60that HL-60 cells, a cells, a humanpromyelocytic human promyelocytic cellline cell linewith withhigher higherlevels levels of of human human CD47 CD47 expression expression than than MOLM-13, MOLM-13,

are able are able to to engraft engraft mice andcause mice and causeleukaemia. leukaemia. Jurkat Jurkat cells, cells, a human a human T-lymphocyte T-lymphocyte cell line, cell line,

are very are very high high for for human human CD47 CD47 and and are phagocytosed are phagocytosed by macrophages by murine murine macrophages in vitro at in a vitro at a muchlower much lower ratethan rate than MOLM-13. MOLM-13. Thus, Thus, ourindicate our data data indicate that that the the ability ability of cells of cells to engraft to engraft

mice in mice in vivo vivo or or evade phagocytosis evade phagocytosis in in vitrobybymouse vitro mouse macrophages macrophages correlates correlates withlevel with the the level of human of CD47expression. human CD47 expression.

[137]

[137] To model To modelthe thetumorigenic tumorigenic effect effect of of having having high high versus versus low low CD47CD47 expression, expression, we we sorted sorted clones of clones of murine murine CD47 expressingMOLM-13 CD47 expressing MOLM-13 cells cells intohigh into highand andlow lowexpressers. expressers.When When adjusted for adjusted for cell cell size, size,CD47 densityon CD47 density onthe theCD47 CD4710 MOLM-13 MOLM-13 cells cells was was approximately approximately equal equal to mouse to bonemarrow mouse bone marrow cells,whereas cells, whereasCD47hi CD47hi MOLM-13 MOLM-13 cellscells had had approximately approximately 9 fold 9 fold

higher expression, higher expression, an anincrease increasecommensurate commensurate with with the change the change seen seen in CD47inexpression CD47 expression on on primary leukemic primary leukemiccells cellscompared compared to their to their normal normal counterparts counterparts (Figure (Figure 15a).15a). When When high or high or low expressing low expressingcells cellswere were transplanted transplanted into into recipients, recipients, onlyonly mice mice transplanted transplanted with with high high expressing cells expressing cells succumbed to disease succumbed to disease byby7575days daysofofage age (Figure15c). (Figure 15c).Furthermore, Furthermore, organomegalywas organomegaly was more more pronounced pronounced in mice in mice transplanted transplanted with with high high expressing expressing cells cells (Figure 15d). (Figure 15d). Mice Micereceiving receivingCD47 CD4710 MOLM-13 MOLM-13 cells had cells still still notable had notable liver liver masses. masses.

However, the However, the masses masseswere were invariably1-2 invariably 1-2 large large nodes nodesthat that were werewell-encapsulated well-encapsulated and and physically segregated physically fromthe segregated from theliver liverparenchyma, parenchyma,in in marked marked contrast contrast to tumor to tumor masses masses from from CD47hiMOLM-13 CD47hi MOLM-13 cellscells whichwhich consisted consisted of hundreds of hundreds of masses of small small masses scatteredscattered throughout throughout

the parenchyma. the parenchyma. Thus, Thus, these these large large tumor tumor masses masses consist consist of cells of cells which which havehave foundfound

macrophage macrophage free-niches free-niches to grow to grow in separate in separate from from the main the main organ organ body. body. As As expected, expected, the the infiltration of of infiltration MOLM-13 cells in MOLM-13 cells in bone marrowandand bone marrow spleen spleen of recipient of recipient mice mice waswas much much higherhigher

for mice for transplantedwith mice transplanted withCD47hi CD47hlMOLM-13 MOLM-13 cellscells as well as well (Figure (Figure 15e).15e). Weexamined We also also examined level ofofCD47 the level in two CD47 expression in two mice that received mice that received CD47l0 MOLM-13 CD47 MOLM-13 cells cells but but had had

significant marrow significant engraftment.In In marrow engraftment. both both cases, cases, thethe persisting persisting cellsafter cells after7575days days hadhad muchmuch

higher levels higher levels of of CD47 CD47than than thethe originalline original line(Figure (Figure15f), 15f),indicating indicatingthat thata astrong strongselection selection pressureexists pressure exists in in vivo vivo for for high high levels levels of ofCD47 expressionononleukemic CD47 expression leukemic cells.In In cells. total,these total, these data indicate data indicate that that CD47 expression CD47 expression level level is isa asignificant significantfactor factor in in tumorigenic potential, and tumorigenic potential, and 34 that in that in aa heterogeneous heterogeneous population population of leukemic of leukemic cells, cells, strongstrong selection selection exists exists for for those those 17 Nov 2023 clones with clones with high high CD47 CD47 expression. expression.

[138]

[138] Wethen We thenasked asked if if higher higher CD47 CD47 expression expression levellevel would would provide provide added added protection protection againstagainst

macrophagephagocytosis. macrophage phagocytosis. WeWe performed performed an an in in vitro phagocytosis vitro phagocytosis assay assay with with CD47hl and CD47hi and

CD47'0 CD47 MOLM-13 MOLM-13 red fluorescent red fluorescent protein protein (RFP) (RFP) expressing expressing cells. cells. After After incubation incubation with with

macrophages,far macrophages, far greater greater numbers numbersofofCD47 CD47|0 cells cells were were phagocytosed phagocytosed as compared as compared to to CD47hicells CD47hi cells (Figure (Figure 15g). 15g).If Ifphagocytic phagocytic indices indices areare compared compared for control for control MOLM-13 MOLM-13 cells, cells, bulk (un-sorted) bulk (un-sorted)CD47 CD47 MOLM-13 cells, CD47, MOLM-13 cells, CD47l0, andand CD47hi CD47hi MOLM-13 MOLM-13 cells, cells, thenthen a direct a direct 2023266367

correlation between correlation CD47 between CD47 expression expression levellevel and and ability ability to evade to evade phagocytosis phagocytosis can becan seenbe seen (Figure 14a, (Figure 14a, Figure Figure15f). 15f).Furthermore, Furthermore,when whenCD47l0 RFPMOLM-13 CD47 RFP MOLM-13 cells cells andand CD47hl CD47hi GFPGFP

MOLM-13 MOLM-13 cells cells were were co-incubated co-incubated with with macrophages macrophages in thewells, in the same samethe wells, low the low expressing expressing

cells were cells far more were far likely to more likely tobe be phagocytosed (Figure15h, phagocytosed (Figure 15h,15i). 15i).Thus, Thus,inina amixed mixed population population

of cells of cells with with varying varying levels levels of of CD47 expression,the CD47 expression, thelow low expressing expressing cells cells areare more more likely likely to to be cleared be cleared by byphagocytic phagocyticclearance clearance over over time. time.

[139]

[139] Wealso We also titrated titrated CD47 CD47 expression expression using using another anothermethod. Since CD47 method. Since is expressed CD47 is expressed in in MOLM-13 MOLM-13 cells cells using using a Tet-OFF a Tet-OFF system, system, we utilized we utilized the Tet-inducible the Tet-inducible promoter promoter elementelement to to control expression control ofCD47 expression of CD47in in MOLM-13 MOLM-13 cells.cells. Beginning Beginning twoafter two weeks weeks after transplantation transplantation

with CD47hi with CD47hiMOLM-13 MOLM-13 cells, cells, a cohort a cohort of mice of mice was was givengiven doxycycline doxycycline and followed and followed for for up to up to 75 days 75 days post-transplant. post-transplant. During During this this time time course, course, none of the none of the mice mice given given doxycycline doxycycline succumbed succumbed to to disease disease or had or had largelarge infiltration infiltration of of MOLM-13 MOLM-13 cells cells in hematopoietic in hematopoietic organs organs (Figures 15b-d). (Figures 15b-d). AtAtthe thedoses dosesof of doxycycline doxycycline used used in this in this experiment, experiment, muCD47 muCD47 expression expression

in MOLM-13 in cells was MOLM-13 cells wasreduced reducedtotolevels levels below belowthat that of of normal normal mouse mousebone bone marrow, marrow, butbut

notably not notably not completely completely absent absent (Figure (Figure 15b). 15b). Thus, Thus, a sustained a sustained highhigh level level of CD47 of CD47

expressionisis required expression required for for robust MOLM-13 robust MOLM-13 survival survival in in hematopoietic hematopoietic organs. organs.

[140]

[140] Manyexamples Many examples of tumor of tumor clearance clearance by T, by B, T, andB,NKand NKhave cells cells have been been described described in the in the literature, indicating literature, that indicating a healthy that a healthyimmune systemisis essential immune system essential for for regulating regulating nascent nascenttumor tumor growth. However, growth. However,to to date, date, fewfew examples examples have have been produced been produced indicating indicating that macrophage- that macrophage-

mediatedphagocytosis mediated phagocytosis cancan check check tumortumor development. development. Collectively, Collectively, our studies our studies reveal reveal that that ectopic expression ectopic expressionofofCD47 CD47cancan enable enable otherwise otherwise immunogenic immunogenic tumor tumor cells to cells grow to grow rapidly rapidly in aa T, in T, B, B, and and NK-cell deficient deficient host. host. Furthermore, this is Furthermore, this is likely likelytotoreflect a mechanism reflect a mechanism used used

by human by human myeloid myeloid leukemias leukemias to evade to evade the immune the host host immune system system since since CD47 CD47 is consistently is consistently

upregulatedininmurine upregulated murineandand human human myeloid myeloid leukemias, leukemias, including including allofforms all forms of and chronic chronic and acute myeloid acute myeloidleukaemia leukaemia tested tested thus thus far.Thus, far. Thus, it itappears appears likelythat likely thattumor tumorcells cellsare arecapable capable of being of being recognized recognizedasas a target a target by activated by activated macrophages macrophages and cleared and cleared through through phagocytosis. ByByupregulating phagocytosis. upregulating CD47, CD47,cancers cancers areare able able to to escape escape this this form form of of innate innate

immune immune tumor tumor surveillance. surveillance.

35

[141]

[141] This form This form of of immune immune evasion evasion is particularlyimportant is particularly important since since these these cancers cancers often often occupy occupy 17 Nov 2023

sites of sites of high high macrophage macrophage infiltration.CD47CD47 infiltration. was cloned was first first cloned as an tumor as an ovarian ovarian celltumor cell marker, indicating marker, indicating that that it it may play aa role may play role in in preventing preventing phagocytosis phagocytosis ofofother othertissue tissuecancers cancers as well. as well. Furthermore, Furthermore,solid solidtumors tumors often often metastasize metastasize to macrophage to macrophage rich tissues rich tissues such assuch as liver, lung, liver, lung, bone marrow,and bone marrow, and lymph lymph nodes, nodes, indicating indicating that that they they must must be ablebe to able to escape escape macrophage-mediatedkilling macrophage-mediated killing in in those those tissues. tissues. Finding Finding methods to disrupt methods to disrupt CD47-SIRPa CD47-SIRPa

interaction may interaction thus prove may thus prove broadly broadlyuseful useful inin developing developingnovel noveltherapies therapiesfor forcancer. cancer. PreventingCD47-SIRPa Preventing CD47-SIRPa interaction interaction is doubly is doubly effective effective sincesince antigens antigens from phagocytosed from phagocytosed 2023266367

tumor cells tumor cells may may be be presented presented by by macrophages to activate macrophages to activate an an adaptive adaptive immune response, immune response,

leadingtotofurther leading furthertumor tumor destruction. destruction.

Methods Methods

[142]

[142] Mice. hMRP8bcrabl, Mice. hMRPSbcrabl, hMRP8bcl2, hMRP8bcl2,and andFas Faslpr/Ipr transgenic transgenic mice mice were were created created as as previously described previously describedand and crossed crossed totoobtain obtaindouble transgenics. double hMRP8bcl2 transgenics. hMRP8bcl2 homozygotes homozygotes

were obtained by were obtained by crossing crossing heterozygote heterozygote mice mice to to each other. C57BI/6 each other. C57BI/6 Ka Kamice micefrom fromour our colony were colony wereused used as as a source a source of wild-type of wild-type cells. cells. For transplant For transplant experiments, experiments, cells cells were were transplanted into transplanted intoC57BI/6 C57BI/6RAG2'7' common RAG2 common gamma gamma chain chain (Gc)(Gc)''' mice mice given given a radiation a radiation dose dose

of 44 Gy of Gyusing usinggamma gamma rays rays from from a cesium a cesium irradiator irradiator (Phillips). (Phillips). Primary Primary mouse leukemias mouse leukemias

were transplanted were transplanted into into CD45.2 C57BI6/Ka mice CD45.2 C57BI6/Ka micegiven givena aradiation radiation dose dose of of 9.5 9.5 Gy. Gy. Mice Mice were euthanized were euthanized when moribund. when moribund.

[143]

[143] Mousetissues. Mouse tissues. Long Longbones bones were were flushedwith flushed withPBS PBS supplemented supplemented withwith 2% fetal 2% fetal calfcalf serumstaining serum stainingmedia media (SM) (SM) Spleens Spleens and livers and livers were dissociated were dissociated using frosted using frosted glass glass slides slides in SM, in thenpassed SM, then passed through through a nylon a nylon mesh. mesh. All samples All samples were treated were treated with ACKwith ACK lysis lysis buffer buffer to lyse to lyse erythrocytes erythrocytes prior prior to to further further analysis. analysis.

[144]

[144] Quantitative Quantitative RT-PCR Analysis. RT-PCR Analysis. Bone Bonemarrow marrowwas was obtainedfrom obtained fromleukemic leukemic hMRP8bcr/ablXxhMRP8bcl2 hMRP8bcr/abl hMRP8bcl2 mice mice or or hMRP8bcl2 hMRP8bcl2 control control mice. mice. Cells Cells werewere c-Kit c-Kit enriched enriched

using c-Kit using c-Kit microbeads microbeads and an autoMACS and an autoMACS column column (Miltenyi). RNARNA (Miltenyi). was was extracted extracted using using

Trizol reagent Trizol (Invitrogen) and reagent (Invitrogen) and reverse reversetranscription transcriptionperformed performed using using SuperScriptll SuperScriptII reverse reverse

polymerase (Invitrogen). polymerase (Invitrogen). cDNA corresponding to cDNA corresponding to approximately approximately 1000 1000 cells cells was used per was used per PCRreaction. PCR reaction. Quantitative Quantitative PCR wasperformed PCR was performedwith witha aSYBR SYBR green green kitkitonon anan ABI ABI Prism Prism

7000 PCR 7000 PCR(Applied (AppliedBiosystems) Biosystems)machine machineatat50°C 50°C for2 2minutes, for minutes, followed followed by by 95°C 95°Cfor for 10 10 minutesand minutes andthen then 40 40 cycles cycles of of 95°C 95°C for for 15 minutes 15 minutes followed followed by for by 60°C 60°C for 1 minute. 1 minute. Beta- Beta- actin and actin 18SRNA and 18S RNA were were used used as controls as controls for cDNA for cDNA quantity quantity and results and results of of CD47 CD47 expression were expression normalized. Sequences were normalized. for 18S Sequences for 18SRNA RNA forwardand forward and reverse reverse primers primers were were

TTGACGGAAGGGCACCACCAG TTGACGGAAGGGCACCACCAG and GCACCACCACCCACGGAATCG, and GCACCACCACCCACGGAATCG, respectively, respectively, for for beta-actin were beta-actin ITCCTTCTTGGGTATGGAAT were TTCCTTCTTGGGTATGGAAT andand GAGCAATGATCTTGATCCTC, GAGCAATGATCTTGATCCTC, and forand for CD47 were CD47 wereAGGCCAAGTCCAGAAGCATTC AGGCCAAGTCCAGAAGCATTO andand AATCATTCTGCTGCTCGTTGC. AATCATTCTGCTGCTCGTTGC.

36

[145]

[145] HumanBone Human Bone Marrow Marrow and and Peripheral Peripheral Blood Blood Samples. Samples. Normal Normal bone marrow bone marrow samplessamples 17 Nov 2023

wereobtained were obtainedwith withinformed informed consent consent fromfrom 20 -20 25-year 25 year old paid old paid donors donors whohepatitis who were were hepatitis A, B, A, B, CC and andHIV HIVnegative negative by by serology serology (All (All Cells).Blood Cells). Blood andand marrow marrow cells cells were were donated donated by by patients with patients with chronic chronic myelomonocytic myelomonocytic leukemia leukemia (CMML), (CMML), chronicchronic myeloidmyeloid leukemialeukemia (CML), (CML), and acute and acute myelogenous myelogenousleukemia leukemia(AML) (AML) andand were were obtained obtained with with informed informed consent, consent, from from

previously untreated previously untreated patients. patients.

[146]

[146] Cell lines. Cell lines. MOLM-13 cells were MOLM-13 cells were obtained obtained from from DSMZ. DSMZ.HL-60 HL-60 and and Jurkat Jurkat cells cells were were

obtained from obtained fromATCC. ATCC. Cells Cells werewere maintained maintained in Iscove’s in Iscove's modified modified Dulbecco’s Dulbecco's media media (IMDM) (IMDM) 2023266367

plus 10% plus 10%fetal fetal bovine bovineserum serum (FBS) (FBS) (Hyclone). (Hyclone). To fractionate To fractionate MOLM-13 MOLM-13 cellsthose cells into into those with with high and high low CD47 and low CD47expression, expression,Tet-CD47 Tet-CD47 MOLM-13 MOLM-13 cellscells werewere stained stained withwith anti-mouse anti-mouse

CD47 Alexa-680 CD47 Alexa-680 antibody antibody (mlAP301). The highest (mIAP301). The highest and and lowest lowest 5% of mouse 5% of mouseCD47 CD47 expressing cells expressing cells was sorted on was sorted a BD on a BDFACSAria FACSAriaandand re-grown re-grown in IMDM+10%FCS in IMDM+10%FCS for 2 for 2 weeks. The weeks. The cellswere cells were sorted sorted forfor threemore three more rounds rounds of selectionfollowing of selection followingthe thesame same protocol to protocol to obtain obtain the thehigh highandand low low expressing expressing cellscells used used in study. in this this study. To red To obtain obtain red fluorescent protein fluorescent protein (RFP) (RFP)constructs, constructs,thethemCherry mCherry RFP RFP DNA DNA was wasinto cloned cloned into Lentilox Lentilox 3.7 3.7 (pLL3.7) empty (pLL3.7) emptyvector. vector.Lentivirus Lentivirus obtained obtained from from thisthis construct construct waswas thenthen used used to infect to infect cell cell

lines. lines.

[147]

[147] Cell staining Cell staining and andflow flowcytometry. cytometry.Staining Staining for for mouse mouse stem stem and and progenitor progenitor cells wascells was performedusing performed usingthe thefollowing followingmonoclonal monoclonal antibodies: antibodies: Mac-1, Mac-1, Gr-1,Gr-1, CD3, CD3, CD4,B220, CD4, CD8, CDS, B220, and Ter1 and Ter119 conjugatedtotoCy5-PE 19 conjugated Cy5-PE (eBioscience) (eBioscience) werewere used used in lineage in the the lineage cocktail, cocktail, c-Kitc-Kit PE- PE-

Cy7 (eBioscience), Cy7 (eBioscience), Sca-1 Sca-1 Alexa680 AlexaSSO (e13-161-7, (e13-161-7, produced producedin inourour lab), lab), CD34 CD34 FITC(eBioscience), CD16/32(FcGRII/lll) APCAPC FITC(eBioscience), CD16/32(FcGRII/III) (Pharmingen), (Pharmingen), and CD135(Flk-2) and CD135(Flk-2) PE PE (eBioscience) were (eBioscience) used asaspreviously were used previously described described to to stain stain mouse mousestem stem andand progenitor progenitor

subsets. Mouse subsets. Mouse CD47 CD47 antibody antibody (clone (clone mlAP301) mIAP301) was assessed was assessed using biotinylated using biotinylated antibody antibody producedininour produced ourlab. lab. Cells Cellswere werethen thenstained stainedwith withstreptavidin streptavidinconjugated conjugated Quantum Quantum Dot Dot 605 605 (Chemicon).Samples (Chemicon). Samples werewere analyzed analyzed using using a FACSAria a FACSAria (Beckton(Beckton Dickinson). Dickinson).

[148]

[148] For human For humansamples, samples,mononuclear mononuclear fractions fractions were were extracted extracted followingFicoll following Ficolldensity density centrifugation according centrifugation tostandard according to standardmethods methods and analyzed and analyzed fresh fresh or or subsequent subsequent to rapid to rapid thawing of thawing of samples previously frozen samples previously frozen inin90% 90% FCS and 10% FCS and 10%DMSO DMSO in liquidnitrogen. in liquid nitrogen. InIn somecases, some cases,CD34+ CD34+ cells cells were were enriched enriched fromfrom mononuclear mononuclear fractions fractions with with the of the aid aid of immunomagneticbeads immunomagnetic beads (CD34+ (CD34+ Progenitor Progenitor Isolation Isolation Kit, Miltenyi Kit, Miltenyi Biotec, Biotec, Bergisch- Bergisch-

Gladbach,Germany). Gladbach, Germany). Prior Prior to FACS to FACS analysis analysis and sorting, and sorting, myeloid myeloid progenitors progenitors were stained were stained

with lineage with lineage marker markerspecific specificphycoerythrin phycoerythrin (PE)-Cy5-conjugated (PE)-Cy5-conjugated antibodies antibodies including including CD2 CD2 RPA-2.10; CD11b, RPA-2.10; GDI 1b,ICRF44; ICRF44;CD20, CD20, 2H7; 2H7; CD56, CD56, B159; B159; GPA, GPA, GA-R2GA-R2 (Becton (Becton Dickinson Dickinson - - PharMingen, San PharMingen, SanDiego), Diego), CD3,S4.1;CD4, CD3,S4.1;CD4,S3.5; S3.5;CD7, CD7,CD7-6B7; CD7-6B7; CDS, CD8, 3B5;3B5; CD10, CD10, 5-1B4, 5-1B4,

GDI4, TUK4; CD14, TUK4;CD19, CD19,SJ25-C1 SJ25-C1 (Caltag,South (Caltag, SouthSan SanFrancisco, Francisco, CA) CA) and and APC-conjugated APC-conjugatedanti- anti- CD34, HPCA-2 CD34, HPCA-2 (Becton (Becton Dickinson-PharMingen), Dickinson-PharMingen), biotinylated anti-CD38, biotinylated anti-CD38, HIT2 HIT2(Caltag) (Caltag) in in

37 addition to addition to PE-conjugated anti-IL-3Ra, 9F5 PE-conjugated anti-IL-3Ra, (Becton Dickinson- 9F5 (Becton Dickinson- ParMingen) ParMingen)and andFITC- FITC- 17 Nov 2023 conjugated anti-CD45RA, conjugated anti-CD45RA, MEM56 MEM56 (Caltag) (Caltag) followedbyby followed stainingwith staining with Streptavidin Streptavidin -Texas -Texas

Redtoto visualize Red visualize CD38-BIO CD38-BIO stained stained cells. cells.

[149]

[149] Following staining, Following staining, cells cellswere were analyzed using aa modified analyzed using modified FACS FACS Vantage Vantage (Becton (Becton

Dickinson Immunocytometry Dickinson ImmunocytometrySystems, Systems,Mountain Mountain View, View, CA)CA) equipped equipped withwith a 599 a 599 nm nm dye dye laser and laser and a 488 nm a 488 nmargon argonlaser laseroror aa FACSAria. FACSAria.Hematopoietic Hematopoietic stem stem cells cells (HSC) (HSC) were were

identified asasCD34+ identified CD34+ CD38+ CD90+ CD38+ CD90+ andand lineage lineage negative.Anti-human negative. Anti-human CD47 CD47 FITCFITC (clone (clone

B6H12, Pharmingen) B6H12, Pharmingen)was was used used to to assess assess CD47 CD47 expression expression in all in all human human samples. samples. Fold Fold 2023266367

change for change for CD47 CD47expression expressionwas wasdetermined determinedbybydividing dividing the the average average mean meanfluorescence fluorescence intensity ofofCD47 intensity CD47 for forallall thethe samples of of samples CML-BC, CML-BC,CML-CP, CML-CP, or or AML by the AML by the average average mean mean fluorescence intensity fluorescence intensity ofof normal normal cells cells for fora a given given cell cellpopulation. population. Common myeloid Common myeloid

progenitors (CMP) progenitors (CMP) were identified based were identified basedononCD34+ CD34+ CD38+ IL-3Ra+CD45RA- CD38+ IL-3Ra+ CD45RA- lin-staining lin- staining and their and their progeny progenyincluding including granulocyte/macrophage granulocyte/macrophage progenitors progenitors (GMP) (GMP)were were CD34+CD38+IL-3Ra+ CD34+CD38+IL-3Ra+ CD45RA+ CD45RA+ Lin- while Lin- while megakaryocyte/erythrocyte megakaryocyte/erythrocyte progenitors progenitors (MEP) (MEP)

wereidentified were identified based onCD34+ based on CD34+ CD38+ CD38+ IL-3RaIL-3Ra - CD45RA- - CD45RA- Lin- staining. Lin- staining.

[150]

[150] To determine To determinethethedensity density of of mouse mouse or human or human CD47, CD47, cellsstained cells were were stained with saturating with saturating

amountsofof anti-CD47 amounts anti-CD47antibody antibody and andanalyzed analyzedonona aFACSAria. FACSAria. Since Since forward forward scatter scatter is is directly proportional directly proportional to to cell cell diameter, and density diameter, and densityisis equal equaltotoexpression expression level level perper unitunit of of surface area surface areaweweused used FloJo FloJo software software to calculate to calculate geometric geometric mean fluorescent mean fluorescent intensity intensity of of the CD47 the CD47channel channel andand divided divided by the by the geometric geometric mean mean of the of the forward forward scatterscatter value squared value squared

(FSC2)to (FSC2) to obtain obtain an an approximation approximationfor fordensity densityofofCD47 CD47 expression expression on the on the membrane. membrane.

[151]

[151] Engraftment of Engraftment of MOLM-13 MOLM-13 cellswaswas cells assessed assessed by using by using anti-human anti-human CD45 CD45 PE-Cy7 PE-Cy7 (Pharmingen), anti-mouse (Pharmingen), anti-mouse CD45.2 APC(clone CD45.2 APC (cloneAL1-4A2), AL1-4A2),and andanti-mouse anti-mouseCD47 CD47 Alexa-680 Alexa-680

(mlAP301). All (mIAP301). All samples sampleswere wereresuspended resuspended in in propidium propidium iodidecontaining iodide containingbuffer buffer before before analysis to analysis to exclude deadcells. exclude dead cells. FACS FACS data data waswas analyzed analyzed usingusing FloJo FloJo software software (Treestar). (Treestar).

[152]

[152] Lentivira! preparation Lentiviral andtransduction. preparation and transduction.pRRL.sin-18.PPT.Tet07.IRES.GFP.pre, pRRL.sin-18.PPT.Tet07.IRES.GFP.pre, CMV, CMV, VSV,and VSV, andtet tettrans-activator trans-activator (tTA) (tTA) plasmids plasmidswere wereobtained obtained from from Luigi Luigi Naldini.TheThe Naldini. fulllength full length murine cDNA murine for CD47 cDNA for form 22 was CD47 form was provided providedbybyEric EricBrown (UCSF). Brown (UCSF).The TheCD47 CD47 cDNA cDNA construct was construct was ligated ligatedinto thethe into BamHI/Nhel BamHI/Nhelsite of Tet-MCS-IRES-GFP. site of Tet-MCS-IRES-GFP. Plasmid Plasmid DMA was DNA was

transfected into transfected into 293T 293Tcells cells using usingstandard standard protocols.TheThe protocols. supernatant supernatant was harvested was harvested and and concentratedusing concentrated usinga aBeckman Beckman LM-8LM-8 centrifuge centrifuge (Beckman). (Beckman). Cellstransduced Cells were were transduced with Tet with Tet or Tet-CD47-MCS-IRES-GFP or Tet-CD47-MCS-IRES-GFP and and tTA tTA lentivirusfor lentivirus for48 48hours. hours. GFP+ GFP+ cellswere cells were sortedtoto sorted purity and purity and grown for several grown for several generations generationstotoensure ensurestability stability of of the the transgenes. transgenes.

[153]

[153] Injections. Cells Injections. Cells were wereinjected injectedintravenously intravenously into into thethe retro-orbitalsinuses retro-orbital sinusesof of recipient recipient

mice ororvia mice via the the tail tail vein vein as noted. For as noted. Forintra-femoral intra-femoralinjections, injections, cells cells were wereinjected injectedinto into the the femoral cavity femoral cavity ofofanesthetized anesthetizedmice miceinina avolume volumeofof20 20piplusing usinga a27-gauge 27-gauge needle. needle. An An

isofluorane gas isofluorane gas chamber chamberwaswas usedused to anesthetize to anesthetize micemice when when necessary. necessary.

38

[154]

[154] MOLM-13cell MOLM-13 cell engraftment. engraftment. Animals Animals were were euthanized euthanized when moribund and when moribund and bone bone 17 Nov 2023

marrow,spleen, marrow, spleen,andand liver liver harvested. harvested. Peripheral Peripheral bloodblood was obtained was obtained by tail by tail ofbleed bleed the of the animals1 1 hour animals hourprior priortotoeuthanization. euthanization.Engraftment Engraftment of MOLM-13 of MOLM-13 cells incells in marrow, marrow, spleen, spleen, and peripheral and peripheralblood bloodwas was determined determined as described as described above.above. Tumorinburden Tumor burden in the the liver was liver was determinedbybycalculating determined calculatingthe thearea areaofofeach eachvisible visibletumor tumornodule nodule using using thethe formula formula ((length ((length in in

mm+ +width mm widthininmm)/2) mm)/2)*T *n. AreaArea of each of each nodule nodule was added was then then added together together per per liver. liver.

[155]

[155] Doxycyclineadministration. Doxycycline administration.Doxycycline Doxycycline hydrochloride hydrochloride (Sigma) (Sigma) was to was added added to drinking drinking

water at water at aa final final concentration of 11 mg/mL. concentration of mg/mL. Drinking Drinking water water was was replaced replaced every every 4 days 4and days and 2023266367

protectedfrom protected from light. light. In addition, In addition, mice mice received received a 10 ug abolus 10 pg bolus of doxycycline of doxycycline by i.p. by i.p. injection injection once aa week. once week.

[156]

[156] Bone marrow Bone marrowderived derivedmacrophages macrophages (BMDM). (BMDM). Femurs Femurs and tibias and tibias werewere harvested harvested fromfrom

C57BI/6KaKamice C57BI/6 mice andand thethe marrow marrow was flushed was flushed and placed and placed into a into a sterile sterile suspension suspension of PBS.of PBS. The bone The bone marrow marrow suspension suspension was was grown grown in in IMDM IMDMplus plus 10% 10%FBSFBS with10 10 with ng/mL ng/mL of of recombinant murine macrophage recombinant murine macrophage colony colony stimulatingfactor stimulating factor(MCSF, (MCSF, Peprotech) Peprotech) forfor 7-10 7-10

days. days.

[157]

[157] In vitro In vitrophagocytosis phagocytosisassays. assays. BMDM were BMDM were harvested harvested by by incubation incubation in intrypsin/EDTA trypsin/EDTA (Gibco) for (Gibco) for 55 minutes minutesand and gentle gentle scraping. scraping. Macrophages Macrophages were at were plated plated at 5cells 5 X 104 x 104percells per well in well in aa 24-well 24-well tissue tissue culture culture plate plate(Falcon). (Falcon).After After 24 24 hours, hours, media media was replaced was replaced with with serum-freeIMDM. serum-free IMDM. After After an additional an additional 2 hours, 2 hours, 2.5 X2.5 105x Tet 105or Tet or Tet-CD47 Tet-CD47 MOLM-13 MOLM-13 cells cells wereadded were addedto to thethe macrophage macrophage containing containing wells wells and incubated and incubated at 37 C°at 37the for C° indicated for the indicated times. After times. After co-incubation, co-incubation, wells wells were werewashed washed thoroughly thoroughly withwith IMDMIMDM 3 times 3 times and examined and examined

under an under an Eclipse Eclipse T5100 T5100(Nikon) (Nikon) using using an anenhanced enhancedgreen green fluorescentprotein fluorescent protein (GFP) (GFP)oror TexasRed Texas Red filter set filter set (Nikon). (Nikon). The Thenumber number of GFP+ of GFP+ or cells or RFP+ RFP+ within cells within macrophages macrophages was was countedand counted andphagocytic phagocytic index index was was calculated calculated usingusing the formula: the formula: phagocytic phagocytic index=number index=number

of ingested of ingested cells/(number cells/(numberofof macrophages/100). macrophages/100). At At least least200 200macrophages macrophages were counted were counted

per well. per well. For Forflow flowcytometry cytometry analysis analysis of of phagocytosis phagocytosis macrophages macrophages were harvested were harvested after after incubation with incubation with MOLM-13 MOLM-13 cells cells using using trypsin/EDTA trypsin/EDTA and gentle and gentle scraping. scraping. Cells Cells were stained were stained

with anti-Mac-1 with anti-Mac-1 PEPEantibody antibody andand analyzed analyzed on a on BD aFACSAria. BD FACSAria. Fluorescent Fluorescent and brightfield and brightfield

images were images weretaken takenseparately separatelyusing usingananEclipse EclipseT5100 T5100 (Nikon),a asuper (Nikon), super high high pressure pressure

mercurylamp mercury lamp(Nikon), (Nikon),anan endow endow green green fluorescent fluorescent protein protein (eGFP) (eGFP) bandpass bandpass filter (Nikon) filter (Nikon) a a Texas Red Texas Redbandpass bandpass filter (Nikon), filter (Nikon),and andaa RT RT Slider Slider(Spot (SpotDiagnostics) Diagnostics)camera. camera. Images Images

weremerged were merged with with Photoshop Photoshop software software (Adobe). (Adobe).

[158]

[158] For in For in vivo vivo assays, assays,marrow marrow fromfrom leg long leg long bones, bones, spleen, spleen, andwere and liver liverharvested were harvested 2 2 hours after hours after injecting injecting target targetcells cellsinto RAG2 into RAG2 /',Gc'/' mice. Gc+ mice. TheyThey were were prepared prepared into single into single cell cell suspensions in suspensions in PBS plus 2% PBS plus FCS.Cells 2% FCS. Cellswere werelabeled labeled with with anti-human CD45Cy7-PE anti-human CD45 Cy7-PE and and

anti-mouseF4/80 anti-mouse F4/80 biotin(eBiosciences). biotin (eBiosciences). Secondary Secondary stainstain was performed was performed with Streptavidin- with Streptavidin-

39

ARC(eBiosciences). APC (eBiosciences). Cells Cellsthat thatwere were human human CD45-, CD45-, F4/80+F4/80+ were considered were considered to be to be 17 Nov 2023

macrophages, macrophages, andand GFP+ GFP+ cellscells in this in this fraction fraction was was assessed. assessed.

EXAMPLE33 EXAMPLE Hematopoieticstem Hematopoietic stem andand progenitor progenitor cells cells upregulate upregulate CD47 CD47 to facilitate to facilitate mobilization mobilization

and homing and homingtotohematopoietic hematopoietic tissues tissues

[159]

[159] Weshow We showhere herethat thathematopoietic hematopoietic stem stem cells cells (HSCs) from CD47 (HSCs) from CD47deficient deficient (IAP+) (lAP'7') mice mice

fail toto engraft fail engraft wild-type wild-type recipients. Asexpected, recipients. As expected, these these cells cells areare rapidly rapidly cleared cleared by by host host 2023266367

macrophages, macrophages, whereas whereas IAP+/+ IAP+/+ HSCsHSCs are When are not. not. stem Whenandstem and progenitor progenitor cells are cells forcedaretoforced to divide and divide entercirculation and enter circulation using using cyclophosphamide/G-CSF cyclophosphamide/G-CSF or lipopolysaccharide, or lipopolysaccharide, CD47 CD47 is is rapidly up-regulated rapidly onthese up-regulated on thesecells. cells.We We propose propose that that higher higher levels levels of CD47 of CD47 in stemincells stem cells during stress during stress hematopoiesis hematopoiesisandand mobilization mobilization provides provides addedadded protection protection againstagainst

phagocytosisbybyactivated phagocytosis activatedmacrophages macrophages of the of the reticuloendothelial reticuloendothelial system. system. In support In support of this of this

hypothesis, we hypothesis, weshow show that that IAP+/'cells IAP+/- cellstransplanted transplantedinto intowild-type wild-typerecipients recipients lose lose engraftment engraftment over time, over time, whereas wild-type donor whereas wild-type cells do donor cells do not. Weconclude not. We concludethat thatphagocytosis phagocytosisisisa a significant physiological significant physiological mechanism that mechanism that clears clears hematopoietic hematopoietic progenitors progenitors over and over time, time, and that CD47 that over-expression CD47 over-expression is is required required toto prevent prevent phagocytic phagocytic clearance. clearance.

[160]

[160] HSCshave HSCs have thethe ability ability toto migrate migrate to to ectopic ectopic niches niches in fetal in fetal andand adult adult lifelife viavia thethe blood blood

stream. Furthermore, stream. Furthermore, HSCs HSCscancan be be prodded prodded intointo thethe circulation using circulation using aa combination combination of of cytotoxic agents cytotoxic agentsand andcytokines cytokines that that first expand first expandHSCHSC numbers numbers in situ. in situ. Once Once in in the the blood blood stream, HSCs stream, mustnavigate HSCs must navigatethe the vascular vascular beds beds of of the the spleen spleen and and liver. liver. Macrophages at Macrophages at

these sites these sites function function to to remove removedamaged damaged cellscells and and foreign foreign particles particles fromfrom the blood the blood stream. stream.

Furthermore,during Furthermore, duringinflammatory inflammatory states,macrophages states, macrophages become become more phagocytically more phagocytically active. active. Hence,additional Hence, additionalprotection protectionagainst againstphagocytosis phagocytosis might might be be required required for for newly newly arriving arriving stem stem

cells at cells at these thesesites. sites.

[161]

[161] Wedetermined We determinedifif CD47 CD47expression expressionononbone bonemarrow marrow stem stem andand progenitor progenitor cellshad cells hada a role in role in regulation regulationofofnormal normal hematopoiesis. CD47expression hematopoiesis. CD47 expressionhas has been been shown shown to beto be essential for essential for preventing preventing phagocytosis phagocytosisof of redred blood blood cells, cells, T-cells,andand T-cells, whole whole bone bone marrowmarrow

cells in cells in aa transplant setting. Thus, transplant setting. Thus,wewe asked asked if lack if lack of CD47 of CD47 would would preventprevent HSCs HSCs from from engrafting after engrafting afterbeing being delivered delivered intravenously. intravenously. To To test test this, this,we we employed the CD47 employed the CD47 knockout mouse knockout mouse(IAP) (IAP'7'). TheseThese mice develop mice develop normally normally and do and not do not display display any any gross gross abnormalities. They abnormalities. Theydo,do, however, however, die die veryvery quickly quickly after after intraperitoneal intraperitoneal bacterial bacterial challenge challenge

because because neutrophils neutrophils fail fail to migrate to migrate to theto the gut gut quickly. quickly. In addition, In addition, cells fromcells thesefrom these mice fail mice fail to transplant to transplantinto intowild-type wild-type recipients, recipients, but but they they will will engraft engraft in IAP-/- in IAP-/- recipients. recipients.

[162]

[162] Wefirst We first examined stem and examined stem andprogenitor progenitor frequencies frequencies in in IAP+/' IAP+/-and and IAP'7' mice. When IAP mice. When examining for examining for cells cells in in the the stem stem and andmyeloid myeloidprogenitor progenitorcompartment, compartment, there there was was no no difference between difference betweenthese these mice mice andand wild-type wild-type micemice (Figure (Figure 18a).18a). Wetested We then then tested stem stem cells cells

40 from these from thesemice mice forfor theirability their abilitytoto form formcolonies coloniesin inan an in in vitroassay. vitro assay. We sorted We sorted highly highly 17 Nov 2023 purified Flk2- purified Flk2- CD34- KLS CD34- KLS stem stem cells cells from from these these micemice and plated and plated them them onto methylcellulose onto methylcellulose in the in presenceofofa astandard the presence standard cytokine cytokine cocktail. cocktail. We examined We examined colony colony formation formation at day 7 at day 7 and found and foundthat thatthere therewas wasnono major major difference difference between between wild-type wild-type and and IAP-/- IAP-/- stem stem cells cells in in the the numberand number and type type of of colonies colonies formed formed (Figure (Figure 18b). 18b).

[163]

[163] Wethen We thenasked asked if bone if bone marrow marrow cellscells from from lAP^'could IAP+ mice mice rescue could recipient rescue recipient mice mice from from the effects the effects of of lethal lethal irradiation. irradiation.Typically, Typically,a adose dose of of 22 xX 105 105 bone marrow bone marrow cellswill cells willrescue rescue 100%ofofwild-type 100% wild-typerecipient recipientmice miceininthis this assay. assay. WeWe found found thatthat IAP'7' IAP+ bonebone marrow marrow could could not not 2023266367

rescuethese rescue theserecipients recipients(Figure (Figure18c). 18c). However, However, administration administration of these of these cellsprolong cells did did prolong lifespan; normally, lifespan; normally, mice die between mice die betweendayday 12 12 andand 15 after 15 after irradiation,butbut irradiation, mice mice that that received received

IAP_/'bone IAP+ bonemarrow marrow lived lived about about 7 to 710todays 10 days longerlonger (Figure (Figure 18c). 18c). Weyet We do not do know not yet the know the reason for reason for the the prolongation prolongation of of lifespan lifespan ininthis thiscase, case,although althoughwe we have observed that have observed that multipotent progenitors multipotent progenitorsand and megakaryocyte megakaryocyte erythrocyte erythrocyte progenitors progenitors can survival can prolong prolong survival after lethal after lethal irradiation, irradiation,and andthat thatcontribution contributionfrom from these cells following these cells following transplant of whole transplant of whole

bonemarrow bone marrowmaymay havehave contributed contributed to the to the elongation elongation of survival of survival time. time.

[164]

[164] Next, we Next, we sorted sorted Flk-2 Flk-2'CD34 CD34' KLS KLS stem stem cells cells from from wild-type wild-type andcells and IAP lAP"7' and cells and transplanted them transplanted theminto intowild-type wild-typerecipients recipientsalong alongwith with2 2X x105 105competitor competitor cells.None cells. None of the of the

mice which mice which received received IAP+ IAP'7'HSCs, HSCs,atateither either aa dose doseofof 50 50or or 500 500had hadany anyengraftment engraftmentofof donorcells, donor cells, indicating indicating that thatCD47 CD47was was indeed indeed required required for thefor the ability ability ofcells of these theseto cells to transplant (Figure transplant (Figure 18d-e). 18d-e).We We speculated speculated that that this this wastodue was due to phagocytosis phagocytosis of the of the cells cells which lacked which lacked CD47, CD47,asashas hasbeen been shown shown for for erythrocytes erythrocytes andand T-cells.To To T-cells. test test this, we this, we enriched c-Kit+ enriched c-Kit cells cellsfrom fromthe thebone bonemarrow marrow of of wild-type wild-typeand andlAP'7' mice and IAP mice and co-incubated co-incubated themwith them withbone bonemarrow marrow derived derived macrophages. macrophages. lAP"7' IAP+ stem andstem and progenitor progenitor cells werecells were readily readily phagocytosed inin this phagocytosed this assay, whereas wild-type assay, whereas wild-type cells cells were were only only minimally minimally phagocytosed phagocytosed

(Figure 18f-g). (Figure Interestingly, when 18f-g). Interestingly, incubatedwith when incubated withIAP+ IAP''' macrophages, macrophages, there there was was significantly less significantly lessphagocytosis of IAP phagocytosis of lAP'7' cells,confirming cells, confirmingthat thatmacrophages macrophagesfrom from thesethese mice mice are indeed abnormal are indeed abnormalin intheir theirphagocytic phagocyticcapacity. capacity.

[165]

[165] Since mobilizationofofstem Since mobilization stem andand progenitor progenitor cellscells involves involves several several steps steps in which in which they they comeinto come into contact contact with with macrophages (egressfrom macrophages (egress fromthe themarrow marrow sinusoids,entry sinusoids, entryinto into the the marrowand marrow and liversinusoids, liver sinusoids, andand in the in the splenic splenic marginal marginal zone), zone), we if we asked asked CD47 if isCD47 up- is up- regulated in regulated in the themarrow marrow of of mice mice which have been which have beeninduced inducedtotoundergo undergomobilization. mobilization. The The

most commonly most commonly used used protocol protocol involvesadministering involves administeringthe thedrug drugcyclophosphamide cyclophosphamide (Cy), (Cy),

whichkills which kills dividing dividing (mainly (mainly myeloid progenitor) cells, myeloid progenitor) cells, followed followed by by treatment with granulocyte treatment with granulocyte colony stimulating colony stimulating factor factor (G-CSF). (G-CSF).ThisThis involves involves administering administering cyclophosphamide cyclophosphamide on the on the first day, first day, and then giving and then giving G-CSF G-CSF every every day day thereafter. thereafter. By convention, By convention, the day the first firstafter day after cyclophosphamide cyclophosphamide administration administration is called is called day day 0. peak 0. The The numbers peak numbers of stem of stem cells cells in the in the bonemarrow bone marrowis is achieved achieved on day on day 2; from 2; from days days 3-4 egress 3-4 they they egress from from the bonethe boneinto marrow marrow into

41 the periphery, and their periphery, and numbersininthe their numbers andand spleen thespleen liver liver reach reach a peak a peak at 5;day at day 5; 17 Nov 2023 progenitors are myeloerythroid progenitors myeloerythroid are also mobilized. There also mobilized. rise in characteristic rise Thereisisa acharacteristic the in the frequency cells and stemcells frequencyofofstem progenitors myeloidprogenitors andmyeloid during thethe during mobilization mobilization response. response.

[166]

[166] Thus, administered weadministered Thus, we this this mobilization mobilization protocol to to protocol wild-type wild-type mice and and mice sacrificed mice mice sacrificed on through days2 2through on days We We 5. 5. found was awas therethere that that found a notable notable increase of CD47ofonCD47 increase c-Kit+ c-Kit+ onbone bone cellsonondayday marrowcells marrow 2 (Figure 2 (Figure 19a). 19a). We that We found there thatwas foundthere approximately a four-folda was approximately four-fold increase in increase level of the level in the CD47 ononstem of CD47 andand stem progenitor progenitor cells on on cells of mobilization 2 of2 mobilization day day (Figure (Figure

increase Theincrease 19b). The 19b). was was seen seen at all at all levels of of levels the the myeloid myeloid progenitor progenitor hierarchy, hierarchy, as LT-HSCs as LT-HSCs 2023266367

well as as well as GMPs as GMPs displayed displayed this this increase in in increase CD47 CD47 expression expression (Figure (Figure when5, 5, day By dayBy 19b). 19b). when egress from the egress from marrowhas the marrow largelyhalted, haslargely the levels halted, the of CD47 levels of CD47 had nearly returnedtoto nearly hadreturned levels. In normallevels. normal Figure 19c, In Figure mean the mean 19c, the fluorescence fluorescence intensity of of intensity CD47 CD47 expression expression on on GMPs GMPs is shown onon is shown days of mobilization. 0 to0 5toof5mobilization. days CD47 levels levels CD47 are are actually actually subnormal subnormal following following day myeloablationononday myeloablation butbut 0, 0, they they quickly quickly rise rise a a to to peak peak on day on day 2. expression 2. The quicklyquickly The expression lowers thereafter lowers and the thereafter and by day levels by the levels equivalentto are equivalent day55 are state. steadystate. to steady

[167]

[167] Endotoxins are also Endotoxins are thought to also thought to bone contribute to to contribute marrowmobilization. bone marrow may Thismay mobilization. This represent response physiologicalresponse represent aaphysiological to infection, to infection, where where normal marrowmarrow normal output output of of immune immune cells needs cells to be needs to increasedtotoclear beincreased theoffending clearthe pathogens. offendingpathogens. Lipopolysaccharide (LPS) (LPS) Lipopolysaccharide is is a cell a wall component cell wall gram-negative component ofofgram-negative bacteria. bacteria. It binds It binds to the to the lipid binding lipidbinding protein protein (LBP) (LBP)

in the serum, in the serum, which then canthen whichcan form form a complex with with a complex GDI CD1411 411 and and toll-like toll-like receptor receptor 4 (TLR-4) 4 (TLR-4)

12 onmonocytes, 12 on macrophages, monocytes,macrophages, and dendritic and dendritic cells. cells. This results This results in activation in activation of of macrophagesand macrophages pro-inflammatory response. resultsinin aa pro-inflammatory andresults LPS response. LPS administrationhas administration also hasalso shown beenshown been to to increase thethe increase phagocytic phagocytic capacity capacity of macrophages. This mayThis of macrophages. be due thedue maytobe to the fact that LBP-LPS fact that complexes LBP-LPS complexes as as act act opsonins. opsonins.

[168]

[168] We tested if Wetested LPS administration if LPS mice would in mice administration in affect CD47 would affect CD47 expression in stem expression in and stem and progenitor cells. Mirroring progenitor cells. pattern Mirroringthethepattern seen seen in Cy/G in Cy/G induced induced mobilization, mobilization, LPS LPS caused caused expansion stem expansionofofstem andand progenitor progenitor cells cells by 2 post post 2 days bydays treatment, treatment, followed followed by migration by migration to to the spleen and the spleen liver (Figure andliver dayday 19d).OnOn (Figure19d). 2 after LPSLPS 2 after administration, stemstem administration, and progenitor and progenitor

cells in cells the marrow in the marrow had up-regulated hadup-regulated CD47 CD47 a similar to atosimilar degree degree as in in Cy/G asCy/G mobilization. mobilization. By By day 5, day whenthe 5, when inflammatory theinflammatory response has has response resolved, resolved, the levels the levels of the of the protein had had protein dropped dropped

steady-state to steady-state to levels levels (Figure 19d).19d). (Figure

[169]

[169] Since CD47 Since CD47 waswas consistently consistently up-regulated up-regulated in the in the mobilization mobilization response, response, we decided we decided to to test the ability test the stem abilityofof and progenitor stemand cellsto progenitor cells followingCy/G. mobilizefollowing tomobilize Cy/G. The CD47 TheCD47 knockout knockout

mousehashas mouse defects in in defects of of migration migration neutrophils sitesof neutrophilstotosites inflammationsand of inflammation8 of of and dendritic cells dendriticcells to secondary to organs. TheThe lymphoid organs. secondary lymphoid exact exact role of of role CD47 CD47 in migration of of in migration these these cellsisis cells unknown, may it itmay unknown,butbut relate to to relate poor poor integrin integrin association association in the in the circulation circulation (CD47 binds binds (CD47 to to several integrins) several or lack integrins) or ofinteraction lack of with interaction SIRPa with on endothelial SIRPa on cells. Hence endothelial cells. Hence we reasoned wereasoned that if CD47 that if was CD47 was involved involved in the the migration in migration capacity capacity of these theseincells of cells in the mobilization the mobilization

42 response,then response, thenIAP-/- IAP-/-mice micewould would display display reduced reduced numbers numbers of cells of cells in the in the peripheral peripheral organs organs 17 Nov 2023 after Cy/G. after Cy/G.

[170]

[170] To test To test this this hypothesis weadministered hypothesis we administered Cy/G Cy/G to both to both wild-type wild-type and and knockout knockout mice mice and and sacrificed mice sacrificed mice on on days 2-5. For days 2-5. Foreach eachmouse, mouse, we we analyzed analyzed the the number number of stem of stem and and progenitor cells progenitor cells in in marrow, spleen,and marrow, spleen, andliver. liver. WeWe decided decided to use to use the the crude crude KLS population KLS population

as aa surrogate as surrogate for for HSCs HSCsbecause because numbers numbers of CD34- of CD34- cells drops cells drops considerably considerably in proliferative in proliferative

states, making states, accuratecalculation making accurate calculationofofLT-HSC LT-HSC numbers numbers difficult. difficult. Since Since GMP GMP are theare the most most expanded expanded of of allallthe thepopulations populations in mobilization, in mobilization, we we decided decided to analyze to analyze their their numbers numbers as as 2023266367

well. ToTocalculate well. calculate absolute absolute progenitor progenitor count, count, the the total total cellularity cellularity of marrow, of marrow, spleen, andspleen, liver and liver was estimated was estimated by by counting counting the the mononuclear mononuclear cell cell number in the number in the whole whole organ organ byby hemocytometer. For hemocytometer. Forbone bonemarrow, marrow,leg leglong long bones boneswere wereassumed assumedto to represent15% represent 15% of of the the

total marrow. total Thisnumber marrow. This number was was then multiplied then multiplied by thebyfrequency the frequency of the of thepopulation cell cell population to to determineananabsolute determine absolute count. count.

[171]

[171] Wefound We foundthat thatthere there waswas littledifference little differenceininmobilization mobilizationofofKLS KLSor or GMPGMP between between wild- wild- type and type andIAP IAPV' mice mice (Figure (Figure 19e). 19e). ThereThere was awas a modest modest decreasedecrease in the of in the ability ability IAP of lAP '" mice mice to move to moveprogenitors progenitorstotothe thespleen spleen by by dayday 3, but 3, but by by days days 4 and 4 and 5 they 5 they had restored had restored normal normal

numbersof of numbers cellstotothe cells theperiphery. periphery.TheThe marrow marrow and compartments and liver liver compartments lookedtosimilar looked similar to wild-type mice. wild-type mice. Hence, Hence,IAPIAP'7' micemice do have do not not have a significant a significant mobilization mobilization defect. defect.

[172]

[172] HeterozygoteIAP+/ Heterozygote IAP+/"erythrocytes erythrocytes have have roughly roughly the the halfhalf thethe amount amount of CD47 of CD47 as wild-type as wild-type

erythrocytes and erythrocytes and platelets. platelets. There There is is also alsoaa dose dose dependent increase in dependent increase in the the amount of amount of

phagocytosisthat phagocytosis thatoccurs occurs in immunoglobulin in immunoglobulin opsonized opsonized IAP+/' erythrocytes IAP+/- erythrocytes and platelets and platelets

relative to relative to wild-type. Ourobservation wild-type. Our observation that that CD47 CD47 levels levels increase increase in states in states of stress of stress and and mobilization led mobilization led us ustotohypothesize hypothesize that that cells cells that that were were genetically genetically hemizygous hemizygous for for CD47 CD47 might be might bemore moreprone prone to to phagocytosis phagocytosis and and clearance clearance by macrophages by macrophages overHence, over time. time. weHence, we askedifif IAP+ asked IAP+/'stem stem cells cells would would be disadvantaged be disadvantaged relative relative to wild-type to wild-type stem in stem cells cells in long- long­ term contribution to term contribution to hematopoiesis. hematopoiesis.

[173]

[173] Wefirst We first analyzed analyzedthe thelevels levelsofofCD47 CD47 expressed expressed on IAP+/+, on IAP+/+, IAP+/‘, IAP+/-, and and IAP'7'cells. IAP stem stem cells. FACS analysis FACS analysis of of CD34 CD34'Flk-2 Flk-2'KLS KLSstem stemcells cells revealed revealed that that the the MFI of CD47 MFI of on CD47 on heterozygoteHSCs heterozygote HSCswaswas indeed indeed at roughly at roughly half half the the level level of wild-type of wild-type stem stem cells cells (Figure (Figure 20a). 20a).

Wethen We thentransplanted transplantedthese thesecells cellsand andexamined examined their their abilitytotoengraft ability engraft and andproduce produce hematopoieticcells hematopoietic cellsinin aarecipient. recipient. WeWe gave gave congenic congenic wild-type wild-type recipient recipient mice mice 475 Gy,475 a Gy, a sublethal dose sublethal doseofofirradiation. irradiation. WeWe then then transplanted transplanted one one cohort cohort of recipients of recipients with with 2 2x10 6 X 106

wild-type whole wild-type wholebone bonemarrow marrow cells, cells, andand another another withwith the the samesame dose dose of IAP+ofbone IAP+/' bone marrow marrow cells. Such cells. Such aadose dosewould would be be expected expected to contain to contain roughly roughly 50-100 50-100 HSCs. HSCs. Since granulocyte Since granulocyte

chimerismininthetheperipheral chimerism peripheral blood blood is aisgood a good surrogate surrogate marker marker of stem of stem cell cell we fitness, fitness, we analyzedcells analyzed cellsfrom fromthethe blood blood of these of these recipients recipients at periodic at periodic intervals. intervals. When wild-type When wild-type

marrowwas marrow was transplanted transplanted intointo wild-type wild-type recipients, recipients, granulocyte granulocyte chimerism chimerism was maintained was maintained

43 for up for up to to 40 40 weeks. However,when weeks. However, when IAP+/‘ IAP+ cellscells were were transplanted,3 3out transplanted, outofof5 5mice micelost lost 17 Nov 2023 donorchimerism donor chimerism over over time,despite time, despite having having a successful a successful engraftment engraftment initially initially (Figure (Figure 20b). 20b).

[174]

[174] Wehave We have observed observed thatthat CD47CD47 is up-regulated is up-regulated on the on the surface surface of hematopoietic of hematopoietic cells in cells in the progression the progressionofofleukemia. leukemia. We also We have havefound also an found an analogous analogous increase increase in in of the level the level of CD47expression CD47 expression when when mice mice were stimulated were stimulated to mobilize to mobilize stem andstem and progenitor progenitor cells cells to the to the periphery using periphery usingCy/G, Cy/G,ororwhen when they theywere were challenged challengedwith LPS. But LPS. with But why why is is CD47 CD47 upregulatedininthese upregulated thesestates? states?Various Various studies studies havehave described described a dose-dependent a dose-dependent effect effect for for CD47ininthe CD47 theprevention prevention of of phagocytosis. phagocytosis. IAP+/' IAP+/- erythrocytes erythrocytes and and platelets, platelets, which which have have half half 2023266367

the level the level of of CD47 CD47as as wild-type wild-type cells, cells, are are phagocytosed phagocytosed more than more readily readily than their their normal normal counterparts. Evidence counterparts. Evidence also also indicates indicates that that thethe levelofofCD47 level CD47 expression expression on cells on cells correlates correlates

well with well with the the ability ability of of the the cell cell to to engage theSIRPa engage the SIRPa inhibitory inhibitory receptor receptor on on macrophages. macrophages.

RecentlyDanska Recently Danskaet et al alreported reported thatthe that theability ability of of NOD-SCID mice NOD-SCID mice to support to support transplantation transplantation

of human of hematopoietic human hematopoietic cells cells correlated correlated with with a mutation a mutation in the in the SIRPalpha SIRPalpha receptor receptor in these in these

mice. Here mice. Herewewe show show thatthat stemstem and progenitor and progenitor cells cells that that express express higherhigher levelslevels of CD47 of CD47 are are less likely less likely to to be becleared clearedby by phagocytosis. phagocytosis.

[175]

[175] Thesestudies These studiespoint pointtotoa arole rolefor forCD47 CD47 up-regulation up-regulation in protecting in protecting hematopoietic hematopoietic stem stem cells during cells during states states when theyare when they aremore moreprone prone to to being being phagocytosed phagocytosed by macrophages, by macrophages, such such as post-myeloablation as post-myeloablationand and during during mobilization.Macrophages mobilization. Macrophages have have the the function function of removing of removing

agedorordamaged aged damaged cells cells that that they they encounter; encounter; it seems it seems thatthat they they can can eliminate eliminate damaged damaged stem stem cells as cells as well. Thus, healthy well. Thus, healthy recovering recovering stem stemcells cells might might up-regulate up-regulate CD47 CD47 duringa during a mobilization response mobilization responsetotoprevent prevent clearance, clearance, whereas whereas damaged damaged stemfail stem cells cellstofail do to so do andso and are cleared. are cleared. We Wespeculate speculate that that this this isisa amechanism by which mechanism by which the the hematopoietic hematopoietic system system

self-regulates itself self-regulates itself totoensure ensure that that only only healthy, healthy, undamaged cells undamaged cells areare permitted permitted to survive to survive

and proliferate and proliferate and utilize resources and utilize during high resources during high stress stress states. states. The mobilizationof The mobilization of HSC HSCandand

progenitors into progenitors into the the bloodstream bloodstreamandand thence thence to hematopoietic to hematopoietic sitessites following following LPS induced LPS induced

inflammationisisvery inflammation veryinteresting; interesting; HSC HSC migrate migrate fromfrom bloodblood to marrow to marrow using integrin using integrin a4B1 a4(31 (Wagers and (Wagers andWeissman, Weissman, Stem Stem Cells Cells 24(4): 1087-94, 24(4):1087-94, 2006) 2006) and and the the chemokine chemokine receptor receptor

CXCR4 CXCR4 (WrightDEDEetetal., (Wright al., JJ Exp Exp Med Med195(9): 195(9): 1145-54, 1145-54, 2002). 2002). We Wehave haveshown shown previously previously

that integrin that integrina4(31 a4ß1binds bindstoto VCAM1 on hematopoietic VCAM1 on hematopoietic stroma (Mikaye KK et stroma (Mikaye et al. al. JJ Exp Exp Med Med

173(3):599-607,1991); 173(3):599-607, 1991);VCAM1 VCAM1 is also is also the the vascular vascular addressin addressin on vessels on vessels that inflammatory that inflammatory

T cells T cells use to recognize use to recognizeand andenter enter localsites local sitesofofcell cell death deathand andinflammation. inflammation. In In addition addition to to

expressingthe expressing theintegrin integrin associated associatedprotein proteinCD47, CD47, itinerant itinerant HSCHSC express express functional functional integrin integrin

a4pi, leading a4B1, leadingto to the the speculation speculationthat that migrating migrating hematopoietic hematopoieticstem stem andand progenitors progenitors in states in states

of inflammation of inflammationmay maynotnot only only re-seed re-seed marrow marrow hematopoiesis, hematopoiesis, but alsobut also participate participate in localin local inflammationasaswell. inflammation well.

44

Materials and Materials and Methods Methods 17 Nov 2023

[176]

[176] Mice. C57BI/6 Mice. CD45.1and C57BI/6 CD45.1 andC57BI/6 C57BI/6CD45.2 CD45.2 (wild-type)mice (wild-type) micewere were maintained maintained in in ourour

colony. IAP-/- colony. IAP-/- mice micewere were obtained obtained fromfrom Eric Eric BrownBrown (University (University of California, of California, San San Francisco). These Francisco). Thesewere werebred bredonon C57BI6/J C57BI6/J background background and and crossed crossed with with our wild-type our wild-type

colony. colony.

[177]

[177] Screening.IAP+/- Screening. IAP+/-were were crossed crossed to each to each otherother to generate to generate IAP-/-IAP-/- and IAP+/- and IAP+/- offspring. offspring.

Mice were Mice were screened screenedbybyPCR PCR of tailDNA. of tail DNA. The The following following primers primers werewere used:used: 3’ 3' Neo- Neo- GCATCGCATTGTCTGAGTAGGTGTCATTCTATTC; GCATCGCATTGTCTGAGTAGGTGTCATTCTATTC 5’ 5' IAP- IAP- 2023266367

TCACCTTGTTGTTCCTGTACTAC TCACCTTGTTGTTCCTGTACTAC AAGCA; AAGCA; 3' 3’ IAP-TGTCACTTCGCAAGTGTAGTTCC. IAP-TGTCACTTCGCAAGTGTAGTTCC.

[178]

[178] Cell staining and Cell sorting. Staining and sorting. Staining for for mouse mousestem stem andand progenitor progenitor cells cells was was performed performed

using the using the following following monoclonal antibodies: Mac-1, monoclonal antibodies: Mac-1, Gr-1, Gr-1, CD3, CD3,CD4, CD4, CDS, CD8, B220, B220, and and Ter119conjugated Ter119 conjugatedto to Cy5-PE Cy5-PE (eBioscience) (eBioscience) were were used used in theinlineage the lineage cocktail, cocktail, c-Kitc-Kit PE-Cy7 PE-Cy7

(eBioscience), Sca-1 (eBioscience), Sca-1Alexa680 AlexaSSO (el3-161-7, (e13-161-7, produced produced in ourin lab), our lab), CD34 CD34 FITC(eBioscience), FITC(eBioscience),

CD16/32(FcGRII/lll) APC CD16/32(FcGRII/III) (Pharmingen), and APC (Pharmingen), and CD135(FIk-2) CD135(Flk-2) PE PE(eBioscience) (eBioscience) were were used used as as previously described previously described to tostain stainmouse mouse stem stem and progenitor subsets and progenitor subsets 21 22. Mouse 21 22. Mouse CD47 CD47

antibody (clone antibody (clonemIAP301) mlAP301)was was assessed assessed using biotinylated using biotinylated antibody antibody producedproduced in in our lab. our lab. Cells were Cells then stained were then stained with with streptavidin streptavidin conjugated conjugated Quantum QuantumDotDot 605 605 (Chemicon). (Chemicon).

Sampleswere Samples were analyzed analyzed using using a FACSAria a FACSAria (Beckton (Beckton Dickinson). Dickinson).

[179]

[179] CD34-Flk2- CD34- Flk2-KLS KLS stem stem cells cells were were double-sorted double-sorted usingusing a BD aFACSAria. BD FACSAria. Peripheral Peripheral blood blood cells were cells obtainedfrom were obtained from tailvein tail veinbleed bleed andand red red cellscells werewere eliminated eliminated by Dextran by Dextran T500 T500 (Sigma)precipitation (Sigma) precipitation and and ACK ACK lysis.Cells lysis. Cellswere were stained stained with with anti-CD45.1 anti-CD45.1 APC,APC, anti-CD45.2 anti-CD45.2

FITC, anti-Ter119 FITC, anti-Ter119 PE PE (Pharmingen), (Pharmingen), anti-B220 anti-B220 Cy5-PE (eBiosciences), anti-CD3 Cy5-PE (eBiosciences), anti-CD3 Cascade Cascade

Blue, and Blue, anti-Mac-1 Cy7-PE and anti-Mac-1 Cy7-PE(eBiosciences). (eBiosciences). Granulocytes Granulocyteswere were Ter119- Ter119- B220- B220- CD3-CD3-

Mac-1+ SSC Mac-1+ SSChi. hi. Cells Cells were analyzed using were analyzed using aa BD BD FACSAria. FACSAria.

[180]

[180] All samples All wereresuspended samples were resuspended in propidium in propidium iodide iodide containing containing bufferbuffer beforebefore analysis analysis to to excludedead exclude deadcells. cells. FACS FACS data data was was analyzed analyzed usingusing FloJo FloJo software software (Treestar). (Treestar).

[181]

[181] In vitro In vitro colony forming assay. colony forming assay.LT-HSC LT-HSC werewere directly directly cloneclone sorted sorted into ainto a 96-well 96-well plate plate containing methycellulose containing methycellulose media media (Methocult (Methocult 3100) 3100) that thatwas was prepared prepared as as described. described. The The

mediawas media was also also supplemented supplemented with with recombinant recombinant mouse mouse stem cellstem cell(SCF), factor factorinterleukin (SCF), interleukin (IL)-3, IL-11, (IL)-3, IL-11, granulocyte-macrophage colony granulocyte-macrophage colony stimulating stimulating factor factor (GM-CSF), (GM-CSF), thrombopoietin thrombopoietin

(Tpo) and (Tpo) and erythropoietin erythropoietin (Epo). (Epo).Colonies Colonieswere scored were forfor scored CFU-G, CFU-M, CFU-G, CFU-M,CFU-GM, CFU- CFU-GM, CFU-

GEMM,and GEMM, and Meg. Meg.

[182]

[182] Cell transfers. Cell transfers. For wholebone For whole bone marrow marrow transfers, transfers, IAP+/+, IAP+/+, IAP+/-, IAP+/-, or IAP-/- or IAP-/- cells cells were were freshly isolated freshly isolatedfrom fromleg leglong longbones. bones. Cells Cells were were counted using aa hemacytometer counted using hemacytometer and and

resuspended in resuspended in PBS+2% FCSatat100uL. PBS+2% FCS IOOuL.ForFor some some experiments,CD45.1 experiments, CD45.1 cellsfrom cells from C57BI/6KaKaCD45.1 C57BI/6 CD45.1 mice mice werewere used used as donors as donors into CD45.2 into CD45.2 wild-type wild-type mice. mice.

45

[183]

[183] For sorted For sorted cells, cells, cells cells were sortedinto were sorted intoPBS PBS buffer buffer at at thethe correct correct dose dose (i.e. (i.e. 50 50 or 500 or 500 17 Nov 2023

cells per cells per tube) tube) and resuspended and resuspended in in100uL 100uL of of PBS+2% PBS+2% FCS. FCS. For For competition competition experiments, experiments, 2 2 x 105 X 105 freshly freshlyisolated whole isolated wholebone bonemarrow marrow cells cellsfrom fromC57BI/6 C57BI/6 CD45.1 CD45.1 were addedto were added to the the IOOuLstem 100uL stemcell cellsuspension. suspension.

[184]

[184] C57BI/6KaKaCD45.1 C57BI/6 CD45.1 or C57BI/6 or C57BI/6 J CD45.2 J CD45.2 recipient recipient miceirradiated mice were were irradiated using ausing a cesium cesium sourceatat the source the doses dosesindicated. indicated.Sub-lethal Sub-lethal dose dose was was 4.75 4.75 Graylethal Gray and and lethal dose dose was was a split a split doseofof9.5 dose 9.5Gray. Gray.Cells Cells were were transferred transferred usingusing a 27-gauge a 27-gauge syringe syringe into theinto the retro-orbital retro-orbital

sinuses of sinuses of mice miceanesthetized anesthetizedwith withisofluorane. isofluorane. 2023266367

[185]

[185] Mobilization assay. Mobilization assay. Mice Micewere were mobilized mobilized withwith cyclophosphamide cyclophosphamide (Sigma) (Sigma) (200 (200 mg/kg) mg/kg) and G-CSF and G-CSF(Neupogen) (Neupogen) (250 (250 pg/kg) ug/kg) asas previouslydescribed. previously described. Bacterial BacterialLPS LPSfrom fromE.E.coli coli 055:B5(Sigma) 055:B5 (Sigma) was was administered administered at aatdose a dose ofmg/kg of 40 40 mg/kg into into the peritoneal the peritoneal cavity. cavity.

[186]

[186] For analysis For analysis of of mobilized mobilizedorgans, organs,whole whole spleen, spleen, whole whole liver, liver, and and leg long leg long bonesbones were were preparedinin aasingle prepared singlecell cell suspension. suspension.Cell Celldensity density was was determined determined usingusing a hemacytometer a hemacytometer

to determine to determine overall overall cellularity cellularity of hematopoietic of hematopoietic cells cells in in organs. these these organs.

[187]

[187] Enrichment of Enrichment of c-Kit+ c-Kit+cells. cells.Whole mouse Whole mousemarrow marrow was stained with was stained with CD117 microbeads CD117 microbeads

(Miltenyi). c-Kit+ (Miltenyi). c-Kit cells cellswere were selected selectedon on an an AutoMACS Midicolumn AutoMACS Midi column (Miltenyi)using (Miltenyi) usinga a magneticseparator. magnetic separator.

[188]

[188] In vitro phagocytosis In vitro assay.BMDM phagocytosis assay. BMDM were were prepared prepared as previously as previously described. described. c-Kit c-Kit enriched bone enriched bonemarrow marrow cells cells werewere stained stained with with CFSE CFSE (Invitrogen) (Invitrogen) prior prior to thetoassay. the assay. 2.5 2.5 x 105 c-Kit 105 c-Kitenriched enrichedcells cellswere wereplated with plated 5 x5 104 with macrophages. X 104 macrophages. Macrophages andc-Kit Macrophages and c-Kit cells were cells obtainedfrom were obtained fromeither eitherIAP+/+ IAP+/+ororIAP IAP'7' mice. mice. Cells Cells werewere incubated incubated for 2 for 2 hours hours and and phagocyticindex phagocytic indexwas wasdetermined. determined. Photographs Photographs were taken were taken as described as described previously. previously.

Example44 Example

CD47isisananIndependent CD47 Independent Prognostic Prognostic Factor Factor and and Therapeutic Therapeutic Antibody Antibody TargetTarget on on HumanAcute Human AcuteMyeloid MyeloidLeukemia LeukemiaCells Cells

[189]

[189] Acutemyelogenous Acute myelogenous leukemia leukemia (AML)(AML) is organized is organized as a cellular as a cellular hierarchy hierarchy initiated initiated and and maintainedbybya asubset maintained subset of of self-renewing self-renewing leukemia leukemia stemstem cellscells (LSC). (LSC). We hypothesized We hypothesized that that increased CD47 increased CD47expression expressionononAML AML LSCLSC contributes contributes to to pathogenesis pathogenesis by inhibitingtheir by inhibiting their phagocytosisthrough phagocytosis through thethe interaction interaction of of CD47 CD47 with with an inhibitory an inhibitory receptor receptor on phagocytes. on phagocytes.

We found We found that that CD47 CD47was wasmore more highlyexpressed highly expressedononAMLAML LSC LSC than than theirtheir normal normal counterparts, and counterparts, andthat thatincreased increased CD47 CD47 expression expression predicted predicted worse survival worse overall overall survival in 3 in 3 independentcohorts independent cohorts of of adult adult AMLAML patients. patients. Furthermore, Furthermore, blocking blocking monoclonal monoclonal antibodies antibodies

against CD47 against preferentially enabled CD47 preferentially enabledphagocytosis phagocytosisofofAML AML LSC by macrophages LSC by macrophagesin in vitro, vitro,

and inhibited and inhibited their their engraftment engraftmentininvivo. vivo. Finally, Finally,treatment treatmentof of human human AML-engrafted AML-engrafted mice mice with anti-CD47 with anti-CD47antibody antibody eliminated eliminated AMLAML in vivo. in vivo. In summary, In summary, increased increased CD47 expression CD47 expression

46 is an is independentpoor an independent poor prognostic prognostic factor factor thatthat can can be targeted be targeted on human on human AML stemAML cellsstem cells 17 Nov 2023 with monoclonal with monoclonalantibodies antibodiescapable capable of of stimulating stimulating phagocytosis phagocytosis of LSC. of LSC.

RESULTS RESULTS

[190]

[190] CD47isisMore CD47 More Highly Highly Expressed Expressed on AML on AML LSCTheir LSC than thanNormal Their Normal Counterparts Counterparts and is and is Associatedwith Associated withthe theFLT3-ITD FLT3-ITD Mutation. Mutation. In ourIninvestigation our investigation of several of several mouse mouse models models of of myeloid leukemia, myeloid leukemia, we identified increased we identified increasedexpression expressionofofCD47 CD47 on mouseleukemia on mouse leukemiacells cells comparedtotonormal compared normalbone bone marrow. marrow. This This prompted prompted investigationofofCD47 investigation CD47 expression expression on on humanAML human AMLLSCLSC and and their their normal normal counterparts. counterparts. Using Using flow flow cytometry,CD47 cytometry, CD47 was was moremore 2023266367

highly expressed highly expressed on on multiple multiplespecimens specimens of ofAML LSC than AML LSC than normal normal bone bonemarrow marrow HSC HSC and and

MRP(Figure MPP (Figure6). 6). This This increased increased expression expression extended extendedtotothe thebulk bulkleukemia leukemiacells, cells, which which expressedCD47 expressed CD47 similarly similarly to to theLSC-enriched the LSC-enriched fraction. fraction.

[191]

[191] Examinationofofa asubset Examination subset of of these these samples samples indicated indicated that that CD47 CD47 surfacesurface expression expression

correlated with correlated withCD47 CD47 mRNA expression. ToTo mRNA expression. investigate CD47 investigate CD47expression expressionacross across morphologic, cytogenetic,and morphologic,cytogenetic, and molecular molecular subgroups subgroups of gene of AML, AML,expression gene expression data fromdata a from a previously described previously describedcohort cohortofof285 285adult adultpatients patientswere were analyzed analyzed (Valk (Valk et al.,2004 et al., 2004 N Engl N Engli J J Med 350, Med 350, 1617-1628). 1617-1628). NoNosignificant significant difference differenceinin CD47 CD47expression expressionamong FAB (French- among FAB (French- American-British) subtypes American-British) was found. subtypes was In most found,In most cytogenetic cytogenetic subgroups, subgroups, CD47 CD47 was was expressedatatsimilar expressed similarlevels, levels,except exceptforfor cases cases harboring harboring t(8;21)(q22;q22), t(8;21)(q22;q22), a favorable a favorable risk risk group which group whichhad had a statistically significant a statistically significant lower CD47expression. lower CD47 In molecularly expression,In molecularly characterized AML characterized subgroups,no no AML subgroups, significantassociation significant associationwas was found found between between CD47 CD47 expression and expression and mutations mutationsininthe thetyrosine tyrosine kinase kinasedomain domain of of FLT3 FLT3 (FLT3-TKD), (FLT3-TKD), over-over­

expression of expression of EVI1, EVI1, or or mutations mutations in in CEBPA, CEBRA,NRAS, NRAS, or KRAS. or KRAS. However, However, higherhigher CD47 CD47 expression was expression was strongly strongly correlated correlated with with the the presence of FLT3-ITD presence of FLT3-ITD (p<0.001), (p<0.001), which which isis observedininnearly observed nearlyone one thirdofofAML third AML withwith normal normal karyotypes karyotypes and isand is associated associated with with worse worse overall survival. overall survival. This This finding finding was separatelyconfirmed was separately confirmedin in two two independent independent datasets datasets of of 214 214 and 137 and 137AML AML patients patients (Table (Table 1).1).

47

Table Clinicaland Table 1:1: Clinical Molecular andMolecular Characteristics Characteristics of AML of AML Samples Samples from from the the Validation Validation 17 Nov 2023

Cohort and Comparison Cohort Comparison Between LowCD47 Between Low CD47 HighCD47 andHigh CD47and Expression Groups ExpressionGroups

Clink nl Featur*’ Clinical Feature* Overall Overall Low CD47 LowCD47 High CD47 High CD47 Ft Pt n=U7 p=137 n=95 n=95 n=37 o=37 Aee. vrs. Age, VFS. 026 0.26 Median Median Range Range 46 16-dO 16-60 47 24-dO 24-60 iJ^O46 16-60 ilO’/L WBC,x10'/L WBC, <0.01 <0.01 Median Median 24 24 17 17 35 35 Range____ ______________ Range 1-238 1-238 1-178 1-178 1-238 1-238 reviewedFAB Centrally reviewed Centrally FAB 029 0.29 Classification, no.no. Clasiification, (96)(lb) 1 a , MO MO 11(8) 11(8) 9(9) 2(5) 2 (5)

16(17) 22(32) Ml 28(20) 28 (20) 16(17) (32) 2023266367

MI 2tQ M2 3606) 36 (26) 22(23) 22 (23) 11(30) 11 (30) Ml 33 C4) 33 (24) 25 06) 25:(20) 80» 8 (22) MA 19(14) 1607) 33(8) (8) M5 M5 19(14) 16(17) M6 M6 2 (1) 2 0) 2 0) 2(2) 0 (0) 0(0) Vnclaisified___ _____ ;___ Unclassified ____6(4)6 (f) 4(4) _____ 4.(4) 0(0) 0.00 ____ /ITi-ITD,no.no.(°6) FLT3-ITD, («*) <0.05 <0.05 Negative Negative 84(61) 84 (61) 63(66) 63 (66) 17(46) 17 (46)

Positive____________ Positive

FLT3-TKD, no.no. FLTi-TKS>, (%a) (96) smx..-........... _____ 53 (39) 32 (34) 20 (54)

0J4 0.24 Negative Negative 109(87) 109 (87) 78(89) 78 (89) 27(79) 27 (79) Poativ»__ Positive :......1703)___ 17 (13) 10(11)______ (11) 01}_____ _ 77(21) NPMI, no. (95) no.(96) 010 0.10

WUd-Tvpe Wild-Type (45) 55(45) 55 41(49) 41 (49) 10 (30) 10 (30)

MatatgO Mutated 66 (55) 66(55) 43.(51)______23.(70) 43 (51) 23 (70)

CEBPA^ CEBPA. no. (Oil) no.(96) 1 Wild-Type Wild-Type 100(86) 100 (86) 70(86) 70 (86) 27(87) 27 (37)

Mutated ■________ Mutated 16 04) . 16 (14) 11(14)_____ 11(14) 03)........... 44(13) MLL-PTD. MLL-PTD, no.no. (95) (96) 1 1 Negative Negative 121 121 (93) (93) 83(92) 83 (92) 34(94) 34 (94) Positive______ Positive .JL©._ 9 (7) H8) 7 (8) 2(6) 2 (6)

Event-free survival Event-free survival MWIjV 0.004 Median,mos. Median, mos. 14 14 17.1 17.1 6JS 6.8

Disease-free Disease-free at 3at 3 yrs, yrs, 9% (9598 96 (95% CI) Cl) ..36 (27-44) .. 41(31-52) 36 (27-44) 41 (31-52) 22(8^36) 22 (8-36)

survival Overall survival Overall 0.002 0.002 Median,mos. Median, inos. 18.5 18.5 22.1 22.1 9.1 9.1

Alive 96 44 vrs, Aliveatat3 3yrs, (9556 (95% CT) Cl) 39(31-48) 39 (31-48) 44(33-5?) 44 (33-55) 26(12-41) 26 (12-41) remissionrate. Completeremission > Complete rate.no. (44) no. (96) CRafter CR Induction, no. (94) 1st Induction, after 1st (%) 60(4696) 60 (46%) 46 (4896) 46 (48%) 14(3894) 14 (3896) 0J3 0.33 1 CRafter CR Induction,no. 2ndInduction, after 2nd (94) no. (%) 84(7494) 34 (74%) 64(7544) 64 (75%) 20(6996) 20 (6996) 0.63 0.63 Randomization Randomization toto 2ndary 2ndary consolidative consolidative tlierapy therapy Allogeneic-HSCT, Allogeneic-HSCT, no. (94) no.(%) 29(2294) 29 (22%) 25(2644) 25 (2690) 41 (1196) (11%) 0.09 0.09 Autolngous-HSCT, Autologous-HSCT, no.no.(40)(94) 23 (1794) 23 (17%) 17 (1896) 17 (13%) 66 (1696) (16%) 0J>8 0.98

* Tabulated clinical and molecular cbaractenstks at WBC . Tabulated clinical and molecular characteristics at diagnosis. indicatesWBC diagnosis. indicates whire blood whileFAB, cell count; blood cell covm.t, FAB, French-American-Biitish, FLT3-ITD, French-American-British; FLT3-TTD,internal internaltandem duplicationof tandemduplication ofthe FITSgene the FLT3 gene(for (&r1010cases missing withmissing caseswith FIT3-ITDstatus, FLT3-ITD status,the FLT3-ITD predicted FLT3-ITD thepredicted status based statusbased on ongenegate expression expression waswas substituted substituted using using method of method of Bullinger el Bullinger or al, 2003);FLT3-TKD, al,200S); FLT3-TKD, tyrosine kinase domain tyrosine hinase mutationof domain mutation the FZT3 of the gene;NPMI, FLT3 gene; XPM1, mutation mutation of of thethe NPUlgene; NPMI .)£Li-PTD, gene;MLL-PTD partialpartial tandem tandem duplication duplication of tire of the MILLXCLL gene; gene; andand CE3PA, CEBPA, mutation mutation of tire of the CEBPA CEBPA gene.gene. CR, remission.CRCR ccanpleteremission. CR complete was was assessed assessed both both after fust and afterfirst and second regimens, which induction regimens, second induction which comprised ICE comprised ICE (idamhicin, etoposide, (idarubicin, cytarabice) or etoposide, cytarabine) A-HAM or A-HAM (all-tnuir (all-grans retípoic acid acid retinoic and high-dose and high-dose cynarabinecytarebine plus phis mitoxantrone). Autologous-HSCT: aurologous autologous mitoxantrone). Aurologous-HSCI: transplantation; transplantation; Allogeneic-HSCT, ADogeneic-HSCT, allogeneic Transplantation. altogeneic transplantation.

and clinical characteristics at diagnosis with low patients with low patients between f P value compares differences in molecular + P value compares differences in molecular and clinical characteristics at diagnosis between

CD47mRNA high CD47 and high and mRKA expression expression values. values. CD47 CD47 expression expression was was dichotomized dichommized on an on based based an optimal optimal cut point for for cut point overall survival stratification that overall survival stratification that we identified on anon vie identified independent microarraymictoanay an independent dataset dataset published published (Valk (Valk el al 2004) at al, 2004)

described as described as in in supplemental supplemental methods methods.

[192]

[192] Identification and Identification and Separation Normal SeparationofofNormal andand Leukemic Leukemic Progenitors Progenitors From the Samethe From Same Patient Based Patient Differential CD47 OnDifferential Based On CD47 Expression. the LSC-enriched Expression. InIn the Lin-CD34+CD38- LSC-enrichedLin-CD34+CD38- of specimen fraction of fraction SU008, specimen SU008, a rare a rare population population of CD47lo-expressing cells cells of CD47lo-expressing was detected, was detected, in in addition to addition themajority to the CD47h'-expressing majorityCD47hi cells -expressing cells (Figure (Figure 21A). A). These 21These populations populations were were isolated cell sorting fluorescence-activated cell isolated by fluorescence-activated >98% (FACS) toto>98% sorting (FACS) purity purity and either and either

transplanted into transplanted newborn into newborn mice mice NOGNOG or plated or plated into complete into complete methylcellulose. methylcellulose. The The CD47 CD47hi cells failed cells engraft ininvivo toengraft failed to form any orform vivoor any colonies in vitro, colonies in as can vitro, as can be with observedwith be observed some some

specimens. AMLspecimens. AML

[193]

[193] However, CD47l0 the CD47 However, the cells with normal engraftedwith cellsengrafted hematopoiesisinin myelo-lymphoid hematopoiesis normal myelo-lymphoid vivo and vivo and formed numerous formednumerous morphologically morphologically normal normal myeloid myeloid colonies colonies in vitro in vitro (Figure (Figure 21B,C). 21B,C).

48

This specimen This specimen harbored harbored the the FLT3-ITD FLT3-ITD mutation, mutation, which which was was detected detected in the in the bulk bulk leukemia leukemia 17 Nov 2023

cells (Figure cells (Figure 21D). 21D). The purified CD47hi The purified CD47hi cells cells contained contained the the FLT3-ITD FLT3-ITDmutation, mutation,andand therefore, were therefore, werepart partofofthe theleukemic leukemic clone, clone, while while the CD47l0 the CD47 cells cells did not.did not.cells Human Human cells isolated from isolated miceengrafted from mice engraftedwith withthe theCD47 CD47l0 cellscells contained contained only only wild wild type type FLT3,FLT3, indicating indicating

that the that the CD47|0 cells contained CD47' cells containednormal normal hematopoietic hematopoietic progenitors. progenitors.

[194]

[194] Increased CD47 Increased CD47Expression Expression in Human in Human AML AML is is Associated Associated withClinical with Poor Poor Clinical Outcomes. WeWe Outcomes. hypothesized hypothesized thatincreased that increasedCD47 CD47 expression expression on on human human AML AML contributes contributes

to pathogenesis. to pathogenesis.From From thisthis hypothesis, hypothesis, we predicted we predicted thatwith that AML AML with expression higher higher expression of of 2023266367

CD47would CD47 would be associated be associated with with worseworse clinical clinical outcomes. outcomes. Consistent Consistent with with this this hypothesis, hypothesis,

analysis of analysis of aa previously previously described describedgroup groupofof285 285 adultAML adult AML patients patients withwith diverse diverse cytogenetic cytogenetic

and molecular and molecularabnormalities abnormalities (Valk (Valk et et al.,2004) al., 2004)revealed revealed that that a dichotomous a dichotomous stratification stratification of of

patients into patients intolow lowCD47 CD47 and high CD47 and high CD47 expression expression groups groups was was associated associated with with aa significantly increased significantly increased risk risk of ofdeath death in inthe thehigh highexpressing group(p=0.03). expressing group (p=0.03).The The association association

of overall of overall survival survivalwith withthis thisdichotomous stratification ofof dichotomous stratification CD47 CD47 expression wasvalidated expression was validatedininaa second test cohort second test cohort ofof 242 242adult adultpatients patients(Metzeler (Metzeleretetal., al., 2008 2008Blood) Blood)with withnormal normal karyotypes(NK-AML) karyotypes (NK-AML) (p=0.01). (p=0.01).

[195]

[195] Applyingthis Applying this stratification stratification to to aa distinct distinctvalidation validationcohort cohort of of 137 adult patients 137 adult patients with with normal karyotypes normal karyotypes (Bullinger (Bullinger et et al., al., 2008 Blood 111, 2008 Blood 111,4490-4495), 4490-4495),we we confirmed confirmed the the prognostic value prognostic valueofofCD47 CD47 expression expression for both for both overall overall and event-free and event-free survival survival (Figure (Figure 22). 22). Analysis of Analysis of clinical clinical characteristics characteristicsofof the low the and low andhigh highCD47 expressiongroups CD47 expression groupsin inthis thiscross- cross- validationcohort validation cohort also also identified identified statistically statistically significant significant differences differences in blood in white whitecell blood cell (WBC) (WBC) count and count andFLT3-ITD FLT3-ITD status, status, andand no differences no differences in rates in rates of complete of complete remission remission andoftype and type of consolidative therapy consolidative therapyincluding includingallogeneic allogeneictransplantation transplantation(Table (Table1).1).Kaplan-Meier Kaplan-Meier analysis analysis

demonstrated demonstrated that that high high CD47CD47 expression expression at diagnosis at diagnosis was significantly was significantly associated associated with with worseevent-free worse event-freeand andoverall overallsurvival survival(Figure (Figure2222A,B). A,B).Patients Patients in in the the low low CD47 CD47 expression expression

grouphad group hada amedian median event-free event-free survival survival of of 17.1 17.1 months months compared compared to 6.8 to 6.8 months months in the in the high high CD47expression CD47 expression group, group, corresponding corresponding to a hazard to a hazard ratio ratio of of (95% 1.94 1.94confidence (95% confidence interval interval 1.30 to 1.30 to 3.77, 3.77, p=0.004). Foroverall p=0.004). For overallsurvival, survival, patients patients in in the the low low CD47 expression CD47 expression group group had had

a median a median ofof 22.1 22.1 months monthscompared compared to to 9.19.1 months months in the in the high high CD47 CD47 expression expression group, group,

correspondingtotoa ahazard corresponding hazard ratio ratio of of 2.02 2.02 (95%(95% confidence confidence interval interval 1.37 1.37 to to p=0.002). 4.03, 4.03, p=0.002). WhenCD47 When CD47 expression expression waswas considered considered as aascontinuous a continuous variable, variable, increased increased expression expression

wasalso was alsoassociated associatedwith witha aworse worse event-free event-free (p=0.02) (p=0.02) andand overall overall survival survival (p=0.02). (p=0.02).

[196]

[196] Despite the Despite the association association with with FLT3-ITD (Table 1), FLT3-ITD (Table 1), increased increased CD47 expression atat CD47 expression

diagnosis was diagnosis was significantlyassociated significantly associated with with worse worse event-free event-free and overall and overall survival survival in the in the subgroupofof7474patients subgroup patientswithout withoutFLT3-ITD, FLT3-ITD, when when considered considered either either as a as a binary binary classification classification

(Figure 22C,D) (Figure 22C,D)ororasasa acontinuous continuous variable variable (p=0.02 (p=0.02 forfor both both event-free event-free andand overall overall survival). survival).

In multivariable In multivariableanalysis analysisconsidering age, considering FLT3-ITD age, FLT3-ITDstatus, status,and andCD47 CD47 expression as aa expression as

49 continuous variable, continuous variable, increased increasedCD47 expression remained CD47 expression remained associated associated with with worse worse event- event- 17 Nov 2023 free survival free survival with with aa hazard ratio of 1.33 (95% hazard ratio confidence (95% confidence interval1.03 interval 1.03toto1.73, 1.73,p=0.03) p=0.03) andand overall survival overall survival with with a a hazard ratio of hazard ratio of 1.31 1.31 (95% (95%confidence confidence interval interval 1.00 1.00 to 1.71, to 1.71, p=0.05) p=0.05)

(Table2). (Table 2). Table 22 Table

Outcome Measure/variables considered Outcome Measure/Variables Considered HR HR 95%CIa 95% P P Event-freesurvival Event-free survival CD47expression, CD47 expression,continuous, continuous, perper 2-fold 2-fold increase increase 1.33 1.33 1.03-1.73 1.03-1.73 0.03 0.03 flO-lTD, positive FLT3-ITD, positive vs. VS. negative negative 2.21 2.21 1.39-3.53 1.39-3.53 <0.001 <0.001 Age, per per year year 1.03 1.00-1.05 0.03 2023266367

Age, 1.03 1.00-1.06 0.03

Overall survival Overall survival CD47expression, CD47 expression,continuous, continuous,perper 2-fold 2-fold increase increase 1.31 1.31 1.00-1.71 1.00-1.71 0.05 0.05 7173-no, positive FLT3-ITD, positive vs. negative negative 2.29 2.29 1.42-3.68 1.42-3.68 <0.001 <0.001 Age, per Age, per year year_____________ 1.03 1.03 1.01-1J0S 1.01-1.06 0.01 0.01

[197]

[197] Monoclonal Antibodies Monoclonal AntibodiesDirected DirectedAgainst Against Human Human CD47 Preferentially CD47 Preferentially Enable Enable

Phagocytosis of Phagocytosis of AML LSCby AML LSC byHuman Human Macrophages. Macrophages. We We hypothesized hypothesized thatthat increased increased CD47 CD47

expression on expression on human humanAMLAML contributes contributes to pathogenesis to pathogenesis by inhibiting by inhibiting phagocytosis phagocytosis of of leukemiacells, leukemia cells, leading leadingusustotopredict predictthat thatdisruption disruptionofofthe theCD47-SIRPa CD47-SIRPa interaction interaction with with a a monoclonalantibody monoclonal antibody directed directed against against CD47CD47 will preferentially will preferentially enable enable the phagocytosis the phagocytosis of of AMLLSC. AML LSC.Several Severalanti-human anti-humanCD47 CD47 monoclonal monoclonal antibodieshave antibodies havebeen beengenerated generatedincluding including somecapable some capableof of blocking blocking the the CD47-SIRPa interaction (B6H12.2 CD47-SIRPa interaction (B6H12.2 and and BRIC126) and others BRIC126) and others unable totododososo unable (2D3) (2D3) (Subramanian (Subramanian et2006 et al., al., Blood 2006 107, Blood 107, 2548-2556). 2548-2556). The The ability of ability of these antibodies these antibodies to to enable phagocytosis of enable phagocytosis of AML LSC,orornormal AML LSC, normal human human bonebone marrow marrow

CD34+cells, CD34+ cells, by by human humanmacrophages macrophages in vitrowas in vitro was tested.Incubation tested. IncubationofofAML AML LSCLSC withwith

humanmacrophages human macrophages in presence in the the presence of isotype of IgG1 IgGI isotype control control antibody antibody or mouse or mouse anti-human anti-human

CD45IgG1 CD45 IgGI monoclonal monoclonal antibody antibody did result did not not result in significant in significant phagocytosis phagocytosis as determined as determined by by either immunofluorescence either microscopy immunofluorescence microscopy (Figure (Figure 8A)flow 8A) or or flow cytometry. cytometry. However, However, addition addition of of the blocking the blocking anti-CD47 anti-CD47 antibodies antibodies B6H12.2 B6H12.2 and BRIC126,but and BRIC126, butnot not the the non-blocking non-blocking 2D3, 2D3, enabled phagocytosis of enabled of AML LSC(Figure AML LSC (Figure 8A,C). 8A,C). No Nophagocytosis phagocytosis of of normal normal CD34+ CD34+cells cells wasobserved was observed with with any any of of thethe antibodies antibodies (Figure (Figure 8C). 8C).

[198]

[198] Monoclonal Antibodies Monoclonal Antibodies Directed DirectedAgainst AgainstHuman CD47Enable Human CD47 EnablePhagocytosis PhagocytosisofofAML AML LSCbybyMouse LSC Mouse Macrophages. Macrophages. The CD47-SIRPa The CD47-SIRPa interaction interaction has been has been implicated implicated as a as a critical critical regulator of regulator of xenotransplantation xenotransplantationrejection rejectionininseveral several cross cross species species transplants; transplants; however, however,

there are there are conflicting conflicting reports of of the ability ability ofofCD47 fromone CD47 from onespecies species to to bind bind andand stimulate stimulate

SIRPaof of SIRPa a different a different species. species. In order In order to directly to directly assess assess theofeffect the effect of inhibiting inhibiting the the interaction of interaction of human CD47 human CD47 with with mouse mouse SIRPa, SIRPa, the in the in vitro vitro phagocytosis phagocytosis assays assays described described

above were above wereconducted conductedwith withmouse mouse macrophages. macrophages. Incubation Incubation of LSC of AML AMLwith LSCmouse with mouse macrophages macrophages in in thethe presence presence of IgGI of IgG1 isotype isotype control control antibody antibody or mouse or mouse anti-human anti-human CD45 CD45 IgGI monoclonal IgG1 monoclonal antibody antibody diddid notnot result result in in significantphagocytosis significant phagocytosisas as determined determined by either by either 50 immunofluorescence immunofluorescence microscopy microscopy (Figure 8B) or8B) (Figure However,However, flow cytometry. floworcytometry. addition addition of the of the 17 Nov 2023 blocking anti-CD47 antibodies blocking andBRIC126, B6H12.2and antibodies B6H12.2 the the not not but but BRIC126, non-blocking 2D3, 2D3, non-blocking phagocytosis enabledphagocytosis enabled of of AML LSC LSC AML (Figure (Figure 8B,C). 8B,C).

[199]

[199] A Antibody Directed Monoclonal Antibody A Monoclonal Against Human Directed Against CD47 HumanCD47 Inhibits LSC LSC AML InhibitsAML Engraftment and Engraftmentand Eliminates AMLAML Eliminates in Vivo. in Vivo. The ability of the of The ability the blocking blocking anti-CD47 antibody antibody anti-CD47 B6H12.2 AML targetAML B6H12.2tototarget LSCLSC in vivo was was in vivo tested. tested. First, First, a pre-coating a pre-coating strategy strategy was utilized was utilized in in which AML which LSC AMLLSC were were purified by by purified FACS FACS and incubated and incubated lgG1 lgG1 with with isotype isotype control, control, anti­ anti-

human CD45, humanCD45, or or anti-human anti-human CD47 CD47 antibody. antibody. An aliquot An aliquot of the of the cells waswas cells analyzed analyzed for for 2023266367

coating by staining coating by secondary with aa secondary staining with antibody antibody demonstrating both both that that demonstrating anti-CD45 anti-CD45 and and anti- anti- antibody CD47antibody CD47 bound bound the cells the cells (Figure 10A).10A). (Figure The remaining The remaining cells cells were were transplanted transplanted into into NOG newborn NOG newborn mice mice that that were were analyzed forfor analyzed leukemicengraftment leukemic 13 13 engraftment weeks weeks later (Figure later(Figure 10B). In all 10B). In but one all but one mouse, the isotype mouse, the control and isotype control anti-CD45antibody and anti-CD45 cells coatedcells antibodycoated exhibited long-term exhibited engraftment. However, leukemic engraftment. long-term leukemic However, most transplantedwith micetransplanted mostmice cells withcells with anti-CD47 coated with coated antibody anti-CD47antibody had no no had detectable detectable leukemia leukemia engraftment. engraftment.

[200]

[200] Next, treatmentstrategy Next, aa treatment waswas strategy utilized utilized in which in which mice were first engrafted wereengrafted micefirst with with LSC LSC humanAMLAML human and then then administered and administered daily intraperitoneal daily intraperitoneal injections of 100 of injections 100 micrograms micrograms

either mouse of either of anti-CD47 IgGororanti-CD47 mouse IgG antibody antibody 14 14 forfor days, withwith days, leukemic leukemic engraftment engraftment

determined pre- determined post-treatment. Analysis andpost-treatment. pre- and thethe Analysisof of peripheral blood peripheralblood showed near near showed completeelimination complete leukemiaininmice circulating leukemia eliminationofofcirculating anti-CD47 withanti-CD47 treatedwith micetreated antibody, antibody, often often

after dose, with no single dose, after aa single responseinincontrol no response mice(Figure controlmice 23A,B). (Figure23A,B). Similarly, was therewas Similarly,there a a significant reduction significant in leukemic reduction in leukemic engraftment bone thebone engraftmentininthe marrow marrow of mice of mice treated treated anti-anti- with with

CD47 while antibody,while CD47antibody, leukemic leukemic involvement involvement increased increased in control in control IgG-treated IgG-treated mice mice (Figure (Figure 23 C,D). Histologic 23 C,D). analysisofofthe Histologicanalysis marrow bonemarrow thebone identified identified monomorphic monomorphic leukemic blasts blasts leukemic in in control IgG-treated mice control IgG-treated 23E, (Figure23E, mice(Figure panels and and 1,2)1,2) panels cleared cleared hypocellular areas areas hypocellular in in anti- anti- CD47 antibody-treated CD47antibody-treated mice mice 23E,23E, (Figure (Figure panels 4,5). 4,5). panels themarrow In the Inbone marrow bone of of some some anti- anti- antibody-treated CD47antibody-treated CD47 mice mice that that contained contained residual residual leukemia, leukemia, macrophages macrophages were detected were detected

containing phagocytosed containing pyknotic cells, phagocytosed pyknotic capturing the cells, capturing the elimination of human elimination of leukemia human leukemia

23E, panels (Figure 23E, (Figure 3,6). panels3,6).

[201]

[201] We report here Wereport theidentification herethe of higher identification of expression of higher expression AML CD47 ononAML of CD47 LSC LSC compared compared to to their normalcounterparts theirnormal andand counterparts hypothesize that that hypothesize increased increased expression of CD47of expression CD47 humanAMLAML on human on contributes contributes to pathogenesis to pathogenesis by inhibiting by inhibiting phagocytosis of of phagocytosis these these cells cells

through the interaction through the of CD47 interaction of with CD47with SIRPa. SIRPa. Consistent Consistent this this with with hypothesis, hypothesis, we we demonstrate increased expression that increased demonstrate that CD47 expressionofofCD47 in human AML isAML in human is associated associated with with decreased overall decreased survival. We overall survival. also demonstrate We also disruption of thatdisruption demonstratethat CD47-SIRPa the CD47-SIRPa of the with monoclonal interaction with interaction antibodies directed monoclonal antibodies CD47preferentially against CD47 directed against enables preferentially enables AML phagocytosisofofAML phagocytosis LSC LSC by macrophages by macrophages in vitro, vitro, inhibits in inhibits the engraftment the engraftment of AML of AML LSC, LSC,

51 and eliminates and eliminatesAML AMLin in vivo.Together, vivo. Together, these these results results establish establish the the rationale rationale for for considering considering 17 Nov 2023 the use the use of of an anti-CD47monoclonal an anti-CD47 monoclonal antibody antibody as aasnovel a novel therapy therapy for human for human AML. AML.

[202]

[202] Thepathogenic The pathogenic influence influence of CD47 of CD47 appears appears mechanistically mechanistically distinct distinct from thefrom two the two main complementing main complementingclasses classesofofmutations mutationsinina amodel modelproposed proposed forfor AMLAML pathogenesis. pathogenesis.

According to According to this this model, model, class class I I mutations, mutations, which whichprimarily primarily impact impactproliferation proliferation and and apoptosis(for apoptosis (for example, example,FLT3 FLT3 and and NRAS), NRAS), andIIclass and class II mutations, mutations, which primarily which primarily impair impair hematopoietic cell hematopoietic cell differentiation differentiation(for(for example, CEBPA, example, CEBPA,MLL, MLL,and and NPM1), cooperate in NPM1), cooperate in leukemogenesis. leukemogenesis. As demonstrated As demonstrated here,contributes here, CD47 CD47 contributes to pathogenesis to pathogenesis via via a distinct a distinct 2023266367

mechanism, mechanism, conferring conferring a survival a survival advantage advantage to LSC to LSC and progeny and progeny blasts blasts throughthrough evasion evasion of of phagocytosis by phagocytosis by the the innate innate immune system. While immune system. Whilestrategies strategies for for the the evasion evasion of of immune immune

responses have responses have been beendescribed described for for many manyhuman humantumors, tumors,wewe believethat believe that increased increased CD47 CD47 expression represents expression represents the the first first such such immune evasionmechanism immune evasion mechanism withwith prognostic prognostic and and

therapeutic implications therapeutic implications for for human AML. human AML.

[203]

[203] Higher CD47 Higher CD47Expression Expressionisis aaMarker MarkerofofLeukemia LeukemiaStem Stem Cells Cells andand Prognostic Prognostic forfor

Overall Survival Overall Survival in in AML. AML AML. AML LSC LSC are enriched are enriched in theinLin-CD34+CD38- the Lin-CD34+CD38- fraction, fraction, which in which in normalbone normal bonemarrow marrow contains contains HSC HSC and andTheMPP. MPP. The identification identification of cell surface of cell surface moleculesmolecules

that can that distinguish between can distinguish leukemic between leukemic andand normal normal stemstem cellscells is essential is essential forfor flow flow cytometry- cytometry-

based assessment based assessmentof of minimal minimal residual residual disease disease (MRD) (MRD) andthefordevelopment and for the development of of prospective separation prospective separationstrategies strategiesfor for use useinin cellular cellular therapies. Several candidate therapies. Several candidatemolecules molecules have recently have recently been been identified, identified,including CD123, including CD96, CD123, CLL-1, CD96, andand CLL-1, nownow CD47. CD47.CD123 CD123 was was

the first the firstmolecule moleculedemonstrated demonstratedtotobe bemore more highly highlyexpressed expressed on on AML LSCcompared AML LSC compared to to normalHSC-enriched normal HSC-enriched populations. populations. We previously We previously identified identified AML LSC-specific AML LSC-specific expression expression

of CD96 of comparedtotonormal CD96 compared normalHSC, HSC, andand demonstrated demonstrated thatthat only only CD96+, CD96+, and and not CD96-, not CD96-,

leukemia leukemia cells cells were were able able to engraft to engraft in in vivo. vivo.

[204]

[204] CLL-1 was CLL-1 was identifiedasasanan identified AML AML LSC-specific LSC-specific surface surface molecule molecule expressed expressed on most on most AMLsamples AML samplesand and notnormal not normalHSC; HSC; importantly,the importantly, the presence presenceofofLin-CD34+CD38-CLL-1+ Lin-CD34+CD38-CLL-1 + cells in cells in the the marrow ofseveral marrow of severalpatients patientsinin hematologic hematologic remission remission was was predictive predictive of relapse. of relapse.

Here we Here we demonstrate demonstratethat that not not only onlyisis CD47 CD47more more highly highlyexpressed expressedononAML AML LSC compared LSC compared

to normal to HSCand normal HSC andMPP, MPP, but but that that thisdifferential this differential expression expression can can be used to be used to separate separate normalHSC/MPP normal HSC/MPPfrom from LSC. LSC. This This is the isfirst the first demonstration demonstration of theofprospective the prospective separation separation of of normalfrom normal fromleukemic leukemic stem stem cells cells in the in the samesame patient patient sample, sample, and the and offers offers the possibility possibility of of LSC-depletedautologous LSC-depleted autologous HSC HSC transplantation transplantation therapies. therapies.

[205]

[205] We initially identified Weinitially higher identified expression higher of of expression CD47 CD47on on AML LSC,but AML LSC, butnoted notedthat that expressioninin bulk expression bulkblasts blastswas was the the same. same. Because Because of we of this, this, we decided decided to utilize to utilize published published

geneexpression gene expressiondata data on on bulk bulk AMLAML to investigate to investigate thethe relationship relationship between between CD47 CD47 expression expression

and clinical and clinical outcomes. Consistent with outcomes. Consistent with our our hypothesis, hypothesis, we found that we found that increased increased CD47 CD47 expressionwas expression wasindependently independently predictive predictive of of a worse a worse clinical clinical outcome outcome in AML in AML patients patients with with a a

52 normalkaryotype, normal karyotype,including includingthe thesubset subset without without thethe FLT3-ITD FLT3-ITD mutation, mutation, whichwhich is theislargest the largest 17 Nov 2023 subgroupofofAML subgroup AML patients. patients. As this As this analysis analysis was dependent was dependent on the relative on the relative expression expression of of CD47mRNA, CD47 mRNA, a quantitativePCR a quantitative PCR assay assay forfor AML AML prognosis prognosis maymay be based be based on level on the the level of of CD47expression. CD47 expression. SuchSuch an assay an assay could could be be utilized utilized in risk in risk adapted adapted therapeutic therapeutic decision decision making,particularly making, particularly in in the the large large subgroup ofAML subgroup of AML patients patients withnormal with normal karyotypes karyotypes who who lack lack the FLT3-ITD the FLT3-ITDmutation. mutation.

[206]

[206] Targeting of Targeting of CD47 CD47onon AMLAML LSC LSC with Therapeutic with Therapeutic Monoclonal Monoclonal Antibodies Antibodies Cell Cell surface molecules surface molecules preferentially preferentially expressed on AML expressed on AMLLSC LSC compared compared to normal to their their normal 2023266367

counterpartsare counterparts arecandidates candidates fortargeting for targetingwith withtherapeutic therapeuticmonoclonal monoclonal antibodies. antibodies. ThusThus far, far, several molecules several have been molecules have been targeted targeted on on AML AMLincluding including CD33, CD33,CD44, CD44, CD123, CD123, and and now now CD47. CD33 CD47. CD33 is is thetarget the target of of the the monoclonal monoclonal antibody antibody conjugate conjugate gemtuzumab ozogamicin gemtuzumab ozogamicin

(Mylotarg), which (Mylotarg), is approved which is forthe approved for the treatment treatmentofofrelapsed relapsedAML AMLin in older older patients.Targeting patients. Targeting of CD44 of with aa monoclonal CD44 with monoclonal antibody antibody was wasshown showntotomarkedly markedlyreduce reduce AML AML engraftment engraftment in in mice, with mice, with evidence evidencethat thatitit acts acts specifically specifically on on LSC toinduce LSC to inducedifferentiation. differentiation. AAmonoclonal monoclonal antibodydirected antibody directedagainst againstCD123 GDIwas 23 was recently recently reported reported to efficacy to have have efficacy in reducing in reducing AML AML LSCfunction LSC functionininvivo. vivo. Here Here we we report report that that a monoclonal a monoclonal antibody antibody directed directed against against CD47 CD47 is is able to able to stimulate phagocytosis ofAML phagocytosis of AMLLSCLSC in vitroand in vitro and inhibitengraftment inhibit engraftmentin in vivo. vivo.

[207]

[207] Several lines Several lines of of evidence suggestthat evidence suggest thattargeting targetingofof CD47 CD47 with with a a monoclonal monoclonal antibody antibody

likely acts likely by disrupting acts by disruptingthe theCD47-SIRPa CD47-SIRPa interaction, interaction, thereby thereby preventing preventing a phagocytic a phagocytic

inhibitory signal. inhibitory signal. First, First,two twoblocking blocking anti-CD47 antibodiesenabled anti-CD47 antibodies enabledAMLAML LSC LSC phagocytosis, phagocytosis,

while one while onenon-blocking non-blocking antibody antibody did not, did not, even even thoughthough all bind all three threethebind thesimilarly. cells cells similarly. Second,ininthe Second, thecase caseofofthe theB6H12.2 B6H12.2 antibody antibody usedused for for mostmost of our of our experiments, experiments, the isotype- the isotype-

matchedanti-CD45 matched anti-CD45 antibody, antibody, which which also also bindsbinds LSC, LSC, failedfailed to produce to produce the effects. the same same effects. In In fact, the fact, B6H12.2 the B6H12.2 antibody antibody is mouse is mouse isotype isotype lgG1, iswhich IgG1, which is less effective less effective at at engaging engaging mouseFcFc mouse receptors receptors than than antibodies antibodies of of isotype isotype lgG2a IgG2a or lgG2b. or IgG2b.

[208]

[208] For human For human clinical clinical therapies, therapies,blocking CD47 blocking CD47ononAML LSC with AML LSC with humanized humanized monoclonal antibodies monoclonal antibodies promotes promotesLSC LSC phagocytosis phagocytosis through through a similar a similar mechanism, mechanism, as as indicated by indicated by the the human human macrophage-mediated macrophage-mediated in phagocytosis in vitro vitro phagocytosis (Figure(Figure 8A,C). 8A,C). Higher Higher CD47expression CD47 expression is is detected detected on on AMLAML LSC; LSC; however, however, CD47 isCD47 is expressed expressed on normalon normal tissues, tissues, including bone including bonemarrow marrow HSC. HSC. We identified We identified a preferential a preferential effecteffect of anti-CD47 of anti-CD47 antibodies antibodies in in enabling the enabling the phagocytosis phagocytosis of ofAML AML LSC comparedtotonormal LSC compared normalbone bonemarrow marrow CD34+ CD34+ cells cells by by humanmacrophages human macrophagesin in vitro. InInfact, vitro. fact, no no increased increased phagocytosis phagocytosis of of normal CD34+cells normal CD34+ cells comparedtotoisotype compared isotypecontrol controlwas was detected, detected, demonstrating demonstrating thatthat blocking blocking CD47 CD47 with with monoclonalantibodies monoclonal antibodies is isa aviable viabletherapeutic therapeuticstrategy strategyfor for human human AML. AML.

[209]

[209] The experimental The experimental evidence evidencepresented presentedhere hereprovides providesthe therationale rationale for for anti-CD47 anti-CD47 monoclonal antibodies monoclonal antibodies as as monotherapy monotherapyforfor AML. AML. However, However, such antibodies such antibodies may be may be equally, if equally, if not notmore more effective effective as as part part of ofaacombination strategy. The combination strategy. Thecombination combinationof of an an anti- anti-

53

CD47antibody, CD47 antibody, able able to block to block a strong a strong inhibitory inhibitory signal signal for phagocytosis, for phagocytosis, with awith a second second 17 Nov 2023

antibody able antibody able to to bind bind aa LSC-specific LSC-specificmolecule molecule (forexample (for example CD96) CD96) and engage and engage Fc receptors Fc receptors

on phagocytes, on phagocytes,thereby thereby delivering delivering a strong a strong positivesignal positive signalfor forphagocytosis, phagocytosis,maymay result result in ain a synergistic stimulus synergistic stimulus for for phagocytosis phagocytosisandand specific specific elimination elimination of of AMLAML LSC. LSC. Furthermore, Furthermore,

combinations of combinations of monoclonal antibodies to monoclonal antibodies to AML LSCthat AML LSC that include include blocking blocking anti-CD47 and anti-CD47 and

humanIgG1 human IgGI antibodies antibodies directed directed against against twotwo other other cellcell surface surface antigens antigens will will be be more more likely likely toto

eliminate leukemia eliminate leukemiacells cellswith withpre-existing pre-existingepitope epitopevariants variantsororantigen antigenloss lossthat thatare arelikely likelytoto recurinin patients recur patientstreated treated with with a single a single antibody. antibody. 2023266367

EXPERIMENTAL PROCEDURES EXPERIMENTAL PROCEDURES

[210]

[210] HumanSamples. Human Samples.Normal Normal human human bone bone marrow marrow mononuclear mononuclear cells cells were were purchased purchased

from AllCells from AllCells Inc. Inc. (Emeryville, (Emeryville, CA). CA).Human Human acute acute myeloid myeloid leukemia leukemia samples samples (Figure (Figure 1A) 1A) were obtained were obtained from frompatients patients atat the the Stanford Stanford University University Medical Medical Center Centerwith withinformed informed consent, according consent, according to toan anIRB-approved IRB-approved protocol protocol(Stanford IRB# (Stanford IRB#76935 76935and and 6453). 6453).Human Human

CD34-positive CD34- positivecells cells were wereenriched enrichedwith withmagnetic magnetic beads beads (Miltenyi (Miltenyi Biotech). Biotech).

[211]

[211] Flow Cytometry Flow CytometryAnalysis Analysis and andCell Cell Sorting. Sorting. AApanel panelofofantibodies antibodies was wasused used forfor

analysis and analysis and sorting sortingofofAML AML LSC (Lin-CD34+CD38-CD90-, LSC (Lin-CD34+CD38-CD90-, where where lineage lineage included included CDS, CD3,

CD19, and CD19, and CD20), CD20), HSC HSC(Lin-CD34+CD38-CD90+), (Lin-CD34+CD38-CD90+),and and MPPMPP (Lin-CD34+CD38-CD90- (Lin-CD34+CD38-CD90- CD45RA-) CD45RA-) as as previously previously described described (Majeti (Majeti et al., et al., 2007). 2007). Analysis Analysis of CD47 of CD47 expression expression was was performed with performed with an an anti-human anti-human CD47 PE antibody CD47 PE antibody (clone (clone B6H12, BD Biosciences, B6H12, BD Biosciences, San Jose San Jose

CA). CA).

[212]

[212] GenomicDNA Genomic DNA Preparationand Preparation and AnalysisofofFLT3-ITD Analysis FLT3-ITDbybyPCR. PCR. Genomic Genomic DNA DNA was was isolated from isolated from cell cell pellets pellets using using the theGentra CentraPuregene Puregene Kit according Kit according to thetomanufacturer's the manufacturer’s protocol (Gentra protocol (Centra Systems, Systems,Minneapolis, Minneapolis, MN). MN). FLT3-ITD FLT3-ITD statusstatus was screened was screened by PCR by PCR using using primers that primers that generated generateda wild-type a wild-type product product of 329 of 329 bp ITD bp and andproducts ITD products of variable of variable larger larger sizes. sizes.

[213]

[213] Anti-HumanCD47 Anti-Human CD47 Antibodies.Monoclonal Antibodies. Monoclonal mouse mouse anti-human anti-human CD47 antibodies CD47 antibodies

included: BRIC126, included: BRIC126, lgG2b IgG2b (Abeam, Cambridge, MA), (Abcam, Cambridge, MA), 2D3, 2D3, IgG1 lgG1 (Ebiosciences. (Ebiosciences. San San Diego, Diego, CA), and CA), B6H12.2, IgG1. and B6H12.2, IgGI. The TheB6H12.2 B6H12.2hybridoma hybridoma waswas obtained obtained from from thethe American American Type Type

Culture Collection Culture Collection (Rockville, (Rockville, MD). Antibodywas MD). Antibody waseither eitherpurified purified from fromhybridoma hybridoma supernatant supernatant

using protein using protein GG affinity affinity chromatography according chromatography according to to standard standard procedures procedures or obtained or obtained from from BioXCell (Lebanon, BioXCell (Lebanon,NH). NH).

[214]

[214] Methylcellulose Colony Methylcellulose Colony Assay. Methylcellulose colony Assay. Methylcellulose colony formation formation was was assayed by assayed by

plating sorted plating sortedcells cellsinto intoa a6-well 6-well plate, plate, each each well well containing containing 1 ml of1 complete ml of complete methylcellulose methylcellulose

(Methocult GF+ (Methocult H4435,Stem GF+ H4435, Stem CellTechnologies). Cell Technologies).Plates Plateswere wereincubated incubatedfor for1414days daysatat 37°C, then 37°C, thenscored scoredbased basedon on morphology. morphology.

[215]

[215] In Vitro In VitroPhagocytosis PhagocytosisAssays. HumanAML Assays. Human AML LSCLSC or normal or normal bonebone marrow marrow CD34+ CD34+

cells were cells were CFSE-labeled and incubated CFSE-labeled and incubated with with either eithermouse mouse or or human macrophages human macrophages in inthe the

54 presenceofof7 7ug/ml presence pg/mlIgG1 lgG1 isotype isotype control, control, anti-CD45 anti-CD45 IgG1,IgGI, or anti-CD47 or anti-CD47 (clones (clones B6H12.2, B6H12.2, 17 Nov 2023

BRIC126, oror2D3) BRIC126, 2D3) antibody antibody forfor 2 hours. 2 hours. Cells Cells were were thenthen analyzed analyzed by fluorescence by fluorescence

microscopy toto determine microscopy determinethethe phagocytic phagocytic index index (number (number of cells of cells ingested ingested per per 100 100 macrophages). macrophages). In In some some cases, cases, cellscells werewere then then harvested harvested and stained and stained with aeither with either mouse a mouse or human or humanmacrophage macrophage marker marker and phagocytosed and phagocytosed cells cells were were identified identified by flow by flow cytometry cytometry as as macrophage+CFSE+. macrophage+CFSE+ Statistical Statistical analysis analysis using using Student’s Student's t-test t-test was was performed performed with with GraphRadPrism GraphPad Prism(San (SanDiego, Diego, CA). CA).

[216]

[216] In Vivo In Vivo Pre-Coating Pre-Coating Engraftment Engraftment Assay. LSCisolated Assay. LSC isolated from from AML AMLspecimens specimens were were 2023266367

incubated with incubated with 28 28 ug/mL ug/mLofofIgG1 IgGIisotype isotypecontrol, control, anti-CD45 anti-CD45IgG1, lgG1,ororanti-CD47 anti-CD47IgG1 lgG1 (B6H12.2)antibody (B6H12.2) antibody at at 4°C4°C for for 30 minutes. 30 minutes. A small A small aliquot aliquot of cells of cells was stained was then then stained with with donkeyanti-mouse donkey anti-mousePE PE secondary secondary antibody antibody (Ebioscience) (Ebioscience) and analyzed and analyzed by flow by flow cytometry cytometry to to assesscoating. assess coating.Approximately Approximately105105 coated coated LSC LSC weretransplanted were then then transplanted intoirradiated into each each irradiated newborn NOD.Cg-Prkdcscidll2rgtm1Wjl/SzJ newborn NOD.Cg-Prkdcscidll2rgtm1Wjl/Sz (NOG) (NOG) mouse. mouse. Mice Mice were were sacrificed sacrificed 13 13 weeks weeks

post-transplantation and post-transplantation and bone marrowwas bone marrow wasanalyzed analyzed forfor human human leukemia leukemia engraftment engraftment

(hCD45+hCD33+) (hCD45+hCD33+) by cytometry by flow flow cytometry (Majeti(Majeti et al.,et2007 al., Cell 2007Stem CellCell Stem Cell 1, 635-645). 1, 635-645). The The presence of presence of human humanleukemia leukemiawas was confirmed confirmed by by Wright-Giemsa Wright-Giemsa staining staining of of hCD45+ hCD45+ cells cells

and FLT3-ITD and FLT3-ITDPCR. PCR. Statisticalanalysis Statistical analysis using using Student’s Student's t-testwaswas t-test performed performed with with GraphPadPrism GraphPad Prism(San (SanDiego, Diego, CA). CA).

[217]

[217] In Vivo In Vivo Antibody Antibody Treatment Treatment of of AML Engrafted Mice. AML Engrafted Mice. 1-25x105 1-25x105FACS-purified FACS-purified LSC LSC were transplanted were transplanted into into NOG pups. Eight NOG pups. Eight to to twelve twelve weeks later, human weeks later, AMLengraftment human AML engraftment (hCD45+CD33+ (hCD45+CD33+ cells) cells) was was assessed assessed in the in the peripheral peripheral blood blood and andmarrow bone bonebymarrow by tail tail bleed bleed and aspiration and aspirationofofthethefemur, femur, respectively. respectively. Engrafted Engrafted mice then mice were were then with treated treated with daily daily intraperitoneal injections intraperitoneal injections of 100 micrograms of 100 micrograms of anti-CD47 of anti-CD47 antibody antibody or IgGorcontrol IgG control for 14 for 14 days. On days. On day day1515mice micewere were sacrificed and sacrificed andthe theperipheral peripheral blood blood and and bone bonemarrow marrow were were

analyzedfor analyzed for AML. AML.

[218]

[218] AMLPatients, AML Patients, Microarray Microarray Gene GeneExpression ExpressionData, Data,and andStatistical Statistical Analysis. Analysis. Gene Gene

expressionand expression andclinical clinical data datawere were analyzed analyzed for for three three previously previously described described cohorts cohorts of adult of adult

AMLpatients: AML patients:(1) (1)a atraining training dataset datasetofof285 285patients patientswith withdiverse diversecytogenetic cytogenetic andand molecular molecular

abnormalities described abnormalities describedby by ValkValk et al., et al., (2) (2) a test a test dataset dataset of 242ofpatients 242 patients with with normal normal karyotypesdescribed karyotypes describedby by Metzeler Metzeler et al., et al., andand (3) (3) a validation a validation dataset dataset of patients of 137 137 patients with with normalkaryotypes normal karyotypes described described by Bullinger by Bullinger et al. et al. The The clinical clinical end end points points analyzed analyzed included included

overall and overall event-free survival, and event-free survival, with with events defined as events defined as the the interval interval between studyenrollment between study enrollment and removal and removalfrom from thethe study study owing owing to a to a lack lack of complete of complete remission, remission, relapse, relapse, or death or death from from any cause, any cause,with withdata datacensored censored for for patients patients whowho did did not not havehave an event an event at theatlast the follow-up last follow-up visit. visit.

[219]

[219] FLT3-ITDPCR. FLT3-ITD PCR. All All reactions reactions werewere performed performed in a volume in a volume of 50 of 50 ul pi containing containing 5 5 ul of pi of 10x PCR 10x PCRbuffer buffer (50mM KCL/10nM (50mM KCL/10nM Tris/2mM Tris/2mM MgCI2/0.01% MgC12/0.01% gelatin),1 1ulpiof gelatin), of 10mM 10mMdNTPs, dNTPs, 2 2 55 units of units Taq polymerase of Taq polymerase (Invitrogen), (Invitrogen), 1 1 ul ul of of 10uM 10pMforward forwardprimer primer11F11F (5'-(5- 17 Nov 2023

GCAATTTAGGTATGAAAGCCAGC-3’) GCAATTTAGGTATGAAAGCCAGC-3') and reverse and primer reverse primer 12R 12R (5- (5'-

CTTTCAGCAl I I l GACGGCAACC-3’), CTTTCAGCATTTTGACGGCAACC-3'), and 10-50 and 10-50 ng genomic ng of of genomic DNA. DNA. PCRPCR conditionsfor conditions for amplification of amplification of the the FLT3 gene FLT3 gene were were 40 cycles 40 cycles of denaturation of denaturation (30 at (30 sec sec at 95°C) 95°C) annealing annealing

(30 sec (30 sec at at 62°C), andextension 62°C), and extension(30 (30sec secatat72°C). 72°C).

[220]

[220] Preparation ofofMouse Preparation Mouseand andHuman Human Macrophages. BALB/cmouse Macrophages. BALB/c mousebone bonemarrow marrow mononuclearcells mononuclear cells were were harvested harvested and and grown in IMDM grown in containing 10% IMDM containing 10% FBS FBSsupplemented supplemented with 10 with 10 ng/ml ng/mLrecombinant recombinant murine murine macrophage macrophage colony colony stimulating stimulating factor factor (M-CSF,(M-CSF, 2023266367

Peprotech,Rocky Peprotech, Rocky Hill,NJ) Hill, NJ) forfor 7-10 7-10 days days to allow to allow terminal terminal differentiation differentiation of of monocytes monocytes to to macrophages. Human macrophages. Human peripheralblood peripheral bloodmononuclear mononuclear cellswere cells were prepared prepared from from discarded discarded

normal blood normal blood from from the the Stanford Stanford University University Medical Medical Center. Center. Monocytes wereisolated Monocytes were isolated by by adheringmononuclear adhering mononuclear cells cells to to cultureplates culture platesfor forone onehour hour atat 370C, 37°C, afterwhich after which non-adherent non-adherent

cells were cells removedbyby were removed washing. washing. TheThe remaining remaining cellscells werewere >95% >95% GDI CD14 and 4 and CD11b GDI 1b positive. positive. Adherent cells Adherent cells were were then then incubated incubated in inIMDM plus 10% IMDM plus humanserum 10% human serum (ValleyBiomedical, (Valley Biomedical, Winchester, VA)for Winchester, VA) for7-10 7-10 days days to allow to allow terminal terminal differentiation differentiation of monocytes of monocytes to to macrophages. macrophages.

[221]

[221] In vitro In vitro phagocytosis assay. BMDM phagocytosis assay. BMDM or peripheral or peripheral bloodblood macrophages macrophages were were harvestedbybyincubation harvested incubationinintrypsin/EDTA trypsin/EDTA (Gibco/lnvitrogen) (Gibco/Invitrogen) forfor 5 minutes 5 minutes followed followed by gentle by gentle

scraping. 55 xx 104 scraping. 104 macrophages macrophageswerewere plated plated in each in each well well of a of a 24-well 24-well tissue tissue culture culture plateplate in in 10%IMDM 10% IMDM containing10%10% containing FBS.FBS. AfterAfter 24 hours, 24 hours, mediamedia was replaced was replaced with serum-free with serum-free

IMDMandand IMDM cells cells were were cultured cultured an additional an additional 2 hours. 2 hours. LSCfluorescently LSC were were fluorescently labeled labeled with with CFSEaccording CFSE accordingtotothe themanufacturer's manufacturer’s protocol protocol (Invitrogen). (Invitrogen). 2 2x X104 104CFSE-labeled CFSE-labeled LSC LSC

were added were addedtotothe themacrophage-containing macrophage-containing wells wells along along with with 7 pg/mL 7 ug/mL of IgGI of IgG1 isotype isotype

(Ebiosciences), anti-CD45 (Ebiosciences), (clone HI30, anti-CD45 (clone HI30,Ebiosciences), Ebiosciences),or or anti-CD47 anti-CD47 antibody, antibody, and and incubatedfor incubated for 22 hours. hours. Wells Wellswere were then then washed washed 3 times 3 times with with IMDM IMDM and examined and examined under an under an Eclipse T5100 Eclipse T5100immunofluorescent immunofluorescent microscope microscope (Nikon) (Nikon) using using an enhanced an enhanced green fluorescent green fluorescent

protein filter protein filter able to to able detected detectedCFSE fluorescence. The CFSE fluorescence. The number number of CFSE of CFSE positive positive cellscells within within

macrophageswas macrophages was counted counted andand thethe phagocytic phagocytic index index waswas determined determined as the as the number number of of ingested cells ingested cellsper per100 100macrophages. At least macrophages. At least 200 macrophageswere 200 macrophages were counted counted perper well. well.

Flourescentand Flourescent andbrightfield brightfield images imageswere were taken taken separately separately and and merged merged with Image with Image Pro Pro Plus Plus (MediaCybernetics, (Media Cybernetics,Bethesda, Bethesda, MD). MD). In Figure In Figure 22A,B, 22A,B, the three the three left images left images are presented are presented

at 200x at 200xmagnification, magnification,with with thethe anti-CD47 anti-CD47 right right imageimage at 400xatmagnification. 400x magnification. For flow For flow cytometryanalysis cytometry analysisofofphagocytosis, phagocytosis, the the cells cells were were then then harvested harvested fromwell from each each well using using trypsin/EDTA. Cell trypsin/EDTA. Cell suspensions suspensions were werethen thenstained stained with with aa mouse mousemacrophage macrophage antibody antibody

anti-mouse F4/80-PECy7 anti-mouse F4/80-PECy7(Ebiosciences) (Ebiosciences)oror anti-human anti-humanCD14-PECy7 CD14-PECy7 (Ebiosciences) (Ebiosciences) andand

analyzed on analyzed on aa FACSAria. FACSAria.Phagocytosed Phagocytosed LSC LSC were were defined defined as either as either CFSE+F4/80+ CFSE+F4/80+ or or CFSE+CD14+ CFSE+CD14+ cellswhen cells when incubatedwith incubated withmurine murineor or human humanmacrophages, macrophages, respectively. respectively.

56

[222]

[222] Microarray Gene Microarray Expression Data. Gene Expression Data. Panel PanelA AofofSupplemental SupplementalFigure Figure2222describes describes 17 Nov 2023

the main the mainmicroarray microarray datasets datasets analyzed analyzed herein, herein, including including the training, the training, test,test, and validation and validation

cohorts. Training cohorts. Training Set: Set: Gene Geneexpression expression data, data, cytogenetics cytogenetics data, data, and and molecular molecular datathe data for for the 285 and 285 and 465 465 patients patients with with AML profiled with AML profiled withAffymetrix HG-U133A Affymetrix HG-U133A and and HG-U133 Plus 2.0 HG-U133 Plus 2.0 microarraysbybyValk microarrays Valketetal. al. and andJongen-Lavrencic Jongen-Lavrencic et al. et al. respectively,were respectively, were obtained obtained fromfrom the the GeneExpression Gene ExpressionOmnibus Omnibus using using thethe corresponding corresponding accession accession numbers numbers (GSE1159 (GSE1159 and and Outcome GSE6891). Outcome GSE6891). datadata werewere only only available available forfor thethe former former dataset,and dataset, andthethe correspondingclinical corresponding clinicalinformation informationwere were kindly kindly provided provided byauthors. by the the authors. Thisiscohort This cohort is 2023266367

presentedasasthethe presented “training” "training" dataset. dataset. The The latter latter dataset dataset was toused was used to univariate confirm confirm univariate associations with associations withkaryotype karyotypeandand molecular molecular mutations mutations described described in the in the former. former. However, However,

these two these twodatasets datasets overlapped overlapped in that in that 247 247 of the of 285the 285 patients patients in the in the first firstwere study study were included in included in the the second, second,and and were were accordingly accordingly excluded excluded in validation in validation of theofassociation the association of of FLT3-ITD with FLT3-ITD with CD47 CD47expression expressionininthe the2nd 2nddataset. dataset. Using UsingNetAffx4, NetAffx4,RefSeq5, RefSeq5,andand thethe

UCSCGenome UCSC Genome Browser6, Browser6, we identified211075_s_at we identified 211075_s_at and and 213857_s_at 213857_s_at as as Affymetrixprobe Affymetrix probe sets on sets onthe theU133 U133 PlusPlus 2.0 microarray 2.0 microarray mapping mapping exclusively exclusively to constitutively to constitutively transcribed transcribed

exonsofofCD47. exons CD47.TheThe geometric geometric mean mean of theof the base-2 base-2 logarithms logarithms of two of these these twosets probe probe wassets was employedininestimating employed estimatingthethemRNA mRNA expression expression level level for CD47, for CD47, and corresponding and corresponding statistical statistical

measures measures forfor associations associations withwith FAB classification, FAB classification, karyotype, karyotype, and molecular and molecular mutations. mutations.

Becausethe Because thedata dataprovided provided by Valk by Valk et as et al. al. GSE1159 as GSE1159 were Affymetrix were Affymetrix intensityintensity

measurements, measurements, we we converted converted thesethese intensities intensities to normalized to normalized base-2base-2 logarithms logarithms of ratios of ratios to to allow comparison allow to the comparison to the corresponding corresponding measurements measurements from from cDNA cDNA microarrays microarrays usingusing a a conventionalscheme. conventional scheme. Specifically, Specifically, we we first(1) first (1)normalized normalizedrawraw data data using using CEL CEL filesfiles fromfrom all all 291 microarrays 291 microarrayswithin withinthis this dataset datasetusing usinggcRMA8, gcRMA8,thenthen (2) (2) generated generated ratios ratios by dividing by dividing the the intensity measurement intensity measurement forfor each each gene gene on aon a given given arrayarray by average by the the average intensity intensity of theofgene the gene across all across all arrays, arrays, (3) (3) log-transformed (base2) log-transformed (base 2) the the resulting resulting ratios, ratios, and and (4) (4) median centered median centered

the expression the expressiondata dataacross acrossarrays arrays then then across across genes. genes. For For the the assessment assessment of theofprognostic the prognostic value of value of CD47, we employed CD47, we employedthe theprobe probeset set213857_s_at 213857_s_at from from thethe AffymetrixHG-U133A Affymetrix HG-U133A and HG-U133 and HG-U133PlusPlus 2.0 2.0 microarrays, microarrays, givengiven its similar its similar expression expression distribution distribution (Supplemental (Supplemental

Figure 3B), Figure 3B), and andconsidering consideringits its position position within within the mRNA transcriptasascompared mRNA transcript compared withwith cDNAcDNA

clones on clones on the the Stanford StanfordcDNA cDNA microarrays microarrays as annotated as annotated within within the NetAffx the NetAffx resource. resource.

[223]

[223] Test Set: Test Set: Gene Geneexpression expression and and clinical clinical data data forfor thethe 242242 adult adult patients patients withwith NKAML NKAML

profiled with profiled with Affymetrix HG-U133A Affymetrix HG-U133A and and HG-U133 HG-U133 Plus 2.0Plus 2.0 microarrays microarrays by et by Metzeler Metzeler al. et al. were obtained were obtained from from the the Gene Gene Expression Expression Omnibus Omnibus using using the corresponding the corresponding accession accession

numbers(GSE12417). numbers (GSE12417). SinceSince rawwere raw data datanot were not available available for thisfor this dataset, dataset, for purposes for purposes of of assessingthe assessing theprognostic prognosticvalue valueofofCD47, CD47, we we employed employed the normalized the normalized datasets datasets provided provided by by the authors the authors (base 2 logarithms) (base 2 logarithms) and and assessed expression of assessed expression of CD47 CD47using usingthe theprobe probeset set 213857_s_at 213857_s_at on on thethe corresponding corresponding microarrays. microarrays.

57

[224]

[224] Validation Set: Validation Set: Gene Gene expression expression datadata for 137 for the the patients 137 patients with normal with normal karyotype karyotype 17 Nov 2023

AMLprofiled AML profiledwith withcDNA cDNA microarrays microarrays by Bollinger by Bullinger et al.etwere al. obtained were obtained from thefrom the Stanford Stanford

MicroarrayDatabase10. Microarray DatabaselO.TheThe corresponding corresponding clinical clinical information information including including outcome outcome data data and and FLT3mutation FLT3 mutationstatus statuswere were kindly kindly provided provided by by the the authors. authors. Using Using the the original original annotations annotations of of microarray features microarray featuresas aswell wellasasSOURCE11, RefSeqS,and SOURCE11, RefSeq5, andthe theUCSC UCSC Genome Genome Browsers, Browser6,

we identified we identified IMAGE:811819 as aa sequence IMAGE:811819 as sequence verified verified cDNA clone mapping cDNA clone mappingtotothe the constitutively transcribed constitutively transcribed 3’ 3'terminal terminalexon exon of ofCD47 onthe CD47 on the corresponding corresponding cDNA cDNA microarrays. microarrays.

[225]

[225] Details of Details of Treatment: Treatment:AMLAML patients patients described described byetValk by Valk et al. (training al. (training set), set), were were 2023266367

treated according treated according to to several several protocols protocols ofofthe theDutch-Belgian Dutch-Belgian Hematology-Oncology Hematology-Oncology

Cooperativegroup. Cooperative group.The The majority majority (90%) (90%) of the of the NK-AML NK-AML patients patients described described by Metzeler by Metzeler et al. et al. (test set) (test set)were weretreated perper treated protocol AMLCG-1999 protocol AMLCG-1999ofofthe German the German AML Cooperative Group, AML Cooperative Group, with all with all patients patients receiving receiving intensive intensive double-induction andconsolidation double-induction and consolidation chemotherapy. chemotherapy. All All 137 NK-AML 137 NK-AML patients patients described described by Bullinger by Bullinger et al. et al. (validationset) (validation set)received receivedstandard-of-care standard-of-care intensified treatment intensified treatment regimens (protocol AML regimens (protocol HD98A),which AML HD98A), which included included 2 courses 2 courses of of induction therapy induction therapywith withidarubicin, idarubicin,cytarabine, cytarabine,andand etoposide, etoposide, one consolidation one consolidation cycle cycle of of high-dose cytarabine high-dose cytarabine and mitoxantrone (HAM), followed and mitoxantrone followed by random assignmenttotoa alate random assignment late consolidation cycle consolidation cycle of of HAM HAM versus versus autologous autologous hematopoietic hematopoietic cell transplantation cell transplantation in case in case no no HLAidentical HLA identical family family donor donorwas wasavailable availablefor forallogeneic allogeneichematopoietic hematopoietic celltransplantation. cell transplantation.

[226]

[226] Statistical Analysis. Statistical We Analysis. We used used two two tailed tailed t-tests t-tests and and analysis analysis of variance of variance for thefor the estimation of estimation of significant significant differences differences inin CD47 CD47 expression expression levellevel across across subgroups subgroups of AML of AML basedononmorphologic, based morphologic, cytogenetic, cytogenetic, and and molecular molecular categorizations. categorizations. Associations Associations between between

the high the high and andlow lowCD47 CD47 groups groups and baseline and baseline clinical, clinical, demographic, demographic, and molecular and molecular featuresfeatures

wereanalyzed were analyzedusing using Fisher’s Fisher's exact exact and and Mann-Whitney Mann-Whitney rank rank sum sum tests fortests for categorical categorical and and continuousvariables, continuous variables, respectively. respectively. Two-sided Two-sidedp-values p-values of of lessthan less than 0.05 0.05 were were considered considered to to indicatestatistical indicate statistical significance. significance.

[227]

[227] The prognosticvalue Theprognostic valueofofCD47 CD47 expression expression was measured was measured through through comparison comparison of the of the event-free and event-free andoverall overall survival survival of of patients patients with with estimation estimation of of survival survival curves curves by bythe theKaplan- Kaplan- Meier product Meier productlimit limit method methodandand the the log-rank log-rank test. test. Within Within this this analysis, analysis, we first we first derived derived a a binary classification binary classification of ofAML patients into AML patients into High HighCD47 CD47andand Low Low CD47 CD47 expression expression groups groups by by comparing the comparing the expression expression of ofCD47 CD47 (as (as measured by 213857_s_at measured by 213857 withinwithin GSE1159) GSE1159) relative relative

to an to optimal threshold. an optimal threshold. This Thisthreshold thresholdwas was determined determined using using X-Tile16, X-Tile16, a method a method which which we we employedto tomaximize employed maximize the chi-square the chi-square statistic statistic between between the the two two for groups groups for the expected the expected

versus observed versus observed number number of deaths. of deaths. This stratification This stratification segregates segregates the 261the AML 261 AML patients patients with available with available outcome outcome data data intointo two two unequally unequally sizedsized groups, groups, with with 72% of 72% of patients patients with with lowest expression lowest expression considered considered CD47 CD47low, low,and and28%28% withwith highest highest expression expression considered considered

CD47high. CD47 high.These These two two groups groups have have different different overall overall survival survival withwith a hazard a hazard ratioratio of 1.42 of 1.42 for for

58 the CD47 the CD47 high high group, group, and and a corresponding a corresponding uncorrected uncorrected p-value p-value of 0.033,ofwhich 0.033, which requires requires 17 Nov 2023 cross-validation cross-validation to to avoid avoid the the risk risk of overfitting. of overfitting.

[228]

[228] Accordingly, weweassessed Accordingly, assessed the validity the validity and robustness and robustness of risk of risk stratification stratification using using CD47expression CD47 expression by applying by applying this this optimal optimal threshold threshold to antoindependent an independent test of test cohort cohort 242 of 242 NK-AMLpatients NK-AML patientsdescribed describedbybyMetzeler Metzeleretetal. al. Notably, Notably,despite despitethe thepresence presenceof ofother other variables potentially variables potentially confounding associationswith confounding associations withsurvival survival(including (includingmore more advanced advanced age, age, and differing and differing therapies), therapies), derivation derivation of of an an optimal optimalcutpoint cutpointusing usingthethe 242242 NK-AML NK-AML patients patients

within the within the test test dataset datasetyielded yieldeda similar a similar stratification, with stratification, with74% 74% of patients of patients with with lowest lowest 2023266367

expressionconsidered expression considered CD47 CD47 low,low, and and 26% highest 26% with with highest expression expression considered considered CD47 CD47 high. high.

[229]

[229] Next,wewe Next, assessed assessed the validity the validity ofstratification of this this stratification in a cross-validation in a cross-validation cohort ofcohort 137 of 137 uniformly treated uniformly treatedNK-AML NK-AML patients patients described described by Bullinger by Bullinger et al. this et al. Within Within this validation validation

dataset, we dataset, wecould couldsimilarly similarly define define two twogroups groupsofofsimilar similarsize size(i.e., (i.e., 72% and28% 72% and 28% with with lowest lowest

and highest and highestCD47 CD47 levels, levels, respectively), respectively), and and thesethese two groups two groups had significantly had significantly different different

outcomeswhen outcomes when assessed assessed for overall for overall survival survival (Figure (Figure 22B,22B, p=0.002, p=0.002, hazardhazard ratio 2.02, ratio 2.02, 95% 95% Cl 1.37 CI 1.37 to to 4.03), 4.03), and and event-free event-freesurvival survival(Figure (Figure223A, 223A,p=0.004, p=0.004, hazard hazard ratio ratio 1.94, 1.94, 95% 95% CI Cl 1.30 to 1.30 to 3.77). 3.77). Of Of the the 137 137patients, patients,5 5did didnot nothave have reliable reliable measurements measurements for when for CD47 CD47 when using the using the data data selection selection and andnormalization normalizationcriteria criteria described bythe described by theauthors. authors.

[230]

[230] To determine To determinethethe robustness robustness of this of this association, association, we also we also examined examined the predictive the predictive

value of value of CD47 CD47 expression expression whenwhen the validation the validation cohort cohort was divided was divided intoandlow into low andCD47 high high CD47 expressiongroups expression groups based based on expression on expression relative relative to the to the median, median, or asora as a continuous continuous variable. variable.

As above, As above, higher higher CD47 CD47 expression expression waswas associated associated withwith worse worse event-free event-free and and overall overall

survival. Of survival. Of the the 137 137patients patientsstudied, studied,a asubset subsetofof 123 123 patients patients hadhad available available survival survival data, data,

CD47expression CD47 expression data, data, andand FLT3-ITD FLT3-ITD statusstatus reported. reported. WithinWithin this cohort, this cohort, we assessed we assessed the the relationship ofof CD47 relationship expression level CD47 expression level as asa acontinuous continuousvariable variablewith withoutcome outcome using using

univariate Cox univariate Coxproportional-hazards proportional-hazards analysis,with analysis, withevent-free event-free survival survival or or overallsurvival overall survivalasas the dependent the dependentvariable. variable. WeWe used used multivariateCox-proportional multivariate Cox-proportionalhazards hazardsanalysis analysiswith with event-free survival event-free survival or or overall overall survival survival as as the the dependent variableand dependent variable and FLT3-ITD FLT3-ITD status, status, age,age,

and continuous and continuousexpression expression level level ofof CD47 CD47 as directly as directly assessed assessed independent independent variables. variables.

[231]

[231] Associations of Associations of CD47 with other CD47 with other covariates covariates (eg, (eg, NPM1, CEBPA) NPM1, CEBPA) were were limitedbyby limited

sample size sample size and andmissing missingdata datafor forcovariates. covariates. The TheWald Waldtest testwas was used used to assess to assess the the significance of significance of each covariate inin multivariate each covariate multivariate analyses. analyses. Univariate Univariate and andmultivariate multivariate proportional-hazardsanalyses proportional-hazards analyses werewere done done using using the thefunction coxph coxph in function in the R the R statistical statistical package. package.

59

Example55 Example 17 Nov 2023

CD47isisaaPrognostic CD47 PrognosticFactor Factor and and Therapeutic Therapeutic Antibody Antibody Target Target on Solid on Solid TumorTumor CancerCancer Stem Stem Cells Cells

[232]

[232] Wehave We have found found thatthat increased increased CD47 CD47 expression expression is associated is associated with with worse worse clinical clinical outcomes inin diffuse outcomes diffuse large large B-cell B-celllymphoma lymphoma (DLBCL) andovarian (DLBCL) and ovarian carcinoma carcinoma(Figure (Figure 24). 24). Additionally, wewehave Additionally, have now found that now found that anti-CD47 anti-CD47 antibodies antibodies enable enable the the phagocytosis phagocytosis of of cancerstem cancer stemcells cellsfrom from bladder bladder cancer, cancer, ovarian ovarian carcinoma, carcinoma, and medulloblastoma and medulloblastoma in vitro in vitro with human with humanmacrophages macrophages (Figure (Figure 25). 25). 2023266367

60

2023266367 16 Jan 2024

Whatisis claimed What claimedis: is:

1. 1. A method A methodofoftreating treating acute acute myeloid myeloidleukemia leukemia(AML) (AML) by by increasing increasing phagocytosis phagocytosis of of the AML the cell in AML cell in aa human subject,the human subject, themethod method comprising comprising administering administering a pharmaceutical a pharmaceutical

composition comprising: composition comprising:

(i) (i)aa first antibody first antibodythat prevents that preventsthe thebinding binding of ofCD47 onananAML CD47 on AML cellcell to to SIRPα SIRPa 2023266367

receptor receptor on on aa phagocytic phagocyticcell, cell, thereby thereby targeting targeting the theAML cell for AML cell forphagocytosis; phagocytosis; and and

(ii) (ii)aasecond antibody second antibody that that specifically specifically binds binds to CD123. to CD123.

2. 2. The methodofofclaim The method claim1,1, wherein whereinthe theantibody antibodythat that disrupts disrupts the the binding binding of ofCD47 with CD47 with

SIRPα specifically binds SIRPa specifically to CD47. binds to CD47.

3. 3. Themethod The methodof of claim claim 1 or 1 or claim claim 2, 2, wherein wherein the the firstantibody first antibody is is a humanized a humanized or or chimeric monoclonalantibody. chimeric monoclonal antibody.

4. 4. Themethod The methodofofclaim claim1 1ororclaim claim2,2, wherein whereinthe thesecond second antibody antibody is is a ahumanized humanizedor or chimeric monoclonalantibody. chimeric monoclonal antibody.

5. 5. The method The method of of claim claim 1, 1, wherein wherein the the firstantibody first antibody andand thethe second second antibody antibody are are

provided as aa bispecific provided as bispecific antibody antibody binding binding to to CD47 andtotoCD123. CD47 and CD123.

6. 6. Use of: Use of:

(i) (i)aafirst antibody first antibody that that prevents thebinding prevents the bindingof of CD47 CD47 on anon an acute acute myeloidmyeloid leukemia leukemia

(AML) cell to (AML) cell to SIRPa SIRPαreceptor receptor on on a phagocytic a phagocytic cell,cell, thereby thereby targeting targeting the the AML AML cell for cell for

phagocytosis; and phagocytosis; and

(ii) (ii)aasecond antibody second antibody that that specifically specifically binds binds to CD123, to CD123,

in in the the manufacture of aa medicament manufacture of medicament forfor treatingAML treating AMLby by increasing increasing phagocytosis phagocytosis

of of the the AML cell in AML cell in aa human subject. human subject.

7. 7. The useofofclaim The use claim6,6,wherein whereinthe theantibody antibody thatdisrupts that disruptsthe thebinding bindingofofCD47 CD47 with with

SIRPα specifically binds SIRPa specifically to CD47. binds to CD47.

8. 8. The use The use ofof claim claim 6 or 6 or claim claim 7, wherein 7, wherein the first the first antibody antibody is a humanized is a humanized or chimeric or chimeric

monoclonal antibody. monoclonal antibody.

61

2023266367 16 Jan 2024

9. 9. Theuse The useofofclaim claim6 6ororclaim claim7,7,wherein wherein thethe second second antibody antibody is a is a humanized humanized or or chimeric monoclonalantibody. chimeric monoclonal antibody.

10. 10. The useofofclaim The use claim 6, 6, wherein wherein the the first first antibody antibody andsecond and the the second antibody antibody are provided are provided

as a bispecific as a bispecificantibody antibody binding binding to toCD47 andto CD47 and to CD123. CD123. 2023266367

62

Progenitors Progenitors

HSC HSC

100

100 100- 100-

80

80 80- 80- x X ffi

60 i 60-

60 2 60- FIG. 1

O O FIG. 1

40

40 55 40- S? 40-

20

20 20- 20-

■ > | nii| \V TTJ ' * I

0

0 0 nr™ tt rrm| 0 ......... "I

102 103

101

10°

10-1

103

10° 102

101

10-1 10'1 10° 101 102 103 10-1 10° 101 102 103

Fluor: CD47 FITC

Fluor: CD47 FITC Fluor: CD47 FITC Fluor: CD47 FITC Lineage +

Lineage +

HSC Progenitors HSC Progenitors 100

100 100- 100-

100 100-

80 80

80- 80-

80 80- x 60

60 «o 60- ro 60-

60 |60- 2 2 FIG. 2

40 40

o 40- o 40- FIG. 2

40 ® 40- 5? 5? 55 20

20 20- 20-

20 20- superscript(3) 10 102 101 100 10-1 0

0 ''■“I

0 0 ’“T 0 4 0 102 103

100 101

10-1

10-1 10° 101 102 103 10-1 10° 101 102 103 10-1 10° 101 102 103 10'1 10° 101 102 103 Fluor: CD47 FITC Fluor: CD47 FITC

Fluor: CD47 FITC Fluor: CD47 FITC Fluor: CD47 FITC Fluor: CD47 FITC

PPMM CML Blast Crisis

Normal BM Normal BM PPMM CML Blast Crisis

103

103

10³ 103- . LU o Ui 111 CL CL CL

102

102 102 co102i 2 102- «102i a: tc CO CO co

0.99 89.5

10 ¹ ■

101 SS.S.W'.::

62.8 V

37.9 0.99

10

2.48 —101 ji2Sj — io1- . 2.48 -10'i FIG. 3A

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10° W

10°

10° ^10°- >,10° n sz -C D, CL IP' Q.

0.66

28

27.6 27.6 ( 0.66

10-1

10- 10-1 10'1 i ■ "i *■"1 10'1 28 - 10'1

102 10³

102 10³

102 10° 101

10°

103 101 10-1

10° 10-1

10-1 10 1 10-1 10° 101 102 103 10-1 10° 101 102 103 10-1 10° 101 102 103 Fluor: CD45RA FITC

Fluor: CD45RA FITC

Fluor: CD45RA FITC Fluor: CD45RA FITC Fluor: CD45RA FITC Fluor: CD45RA FITC ho

100 100-

100 100-

100 100- 80 80-

80

80 80- 80- x 60

60 $60-

60 I 60- S ^6°- s 40

O 40-

40

40 0 40- FIG. 3B

5 40' 5? 5? FIG. 3B 20

20 20-

20 20- 20- 0

0 fVW]

0 *'■"1 ■PWt 0 0 \3 o 102 10³

10° 101

10-1

102 103

10° 101

10³

102

10° 101 10-1

10-1 10-1 10° 101 102 103 10-1 iq0 io1 102 103 10-1 10° 101 102 103 Fluor: CD47 FITC

Fluor: CD47 FITC

Fluor: CD47 FITC Fluor: CD47 FITC Fluor: CD47 FITC Fluor: CD47 FITC

3/41 3/41 2023266367

1001 100

80- 80 Normal Normal CMML CMML

a 606°- S o 40- as 40 %

20- 20

0-» 0 I ""I ......... "I T-rrrmf 0.1 0.1 1 1 10 10 100 100 1000 1000

Fluor: CD47 Fluor: FITC CD47 FITC

FIG. 44 FIG.

Normal AML Normal AML

10³ 103 103 1 103 1 ui 111 Q. 102.

102

210 « w 1°2 ■ ra Wm K n «?

21.5 68.1

16.2

40.8 10 ¹

10 1 =J 1011 40.8- d 101 i ■. I.'-' yffsawEiv*

FIG. 5A £ £ & FIG. 5A •V5

10°

10° >10° 1 > 10° £ JC o. 0. ^Slif

0.77

31.2 0.77

10-1

10-1 10‘1 10‘1 H*, *""l

103

102

101

102 10°

103

10° 10-1

101

10-1 lO"1 10° 101 102 103 10’1 10° 101 102 103 FITC CD45RA Fluor: FITC CD45RA Fluor: Fluor: CD45RA FITC Fluor: CD45RA FITC -t* Lineage + Cells

Progenitor Lineage + Cells

Progenitor

HSC HSC Normal

Normal

Normal 100 Normal

Normal Normal 100-

100 AML

100- AML

100 100-

AML AML

AML AML Vi 80 80-

80 Vi 80-

80 80- J x 60

u ® 60- | 60

60 s 60-

60 s S FIG. 5B

FIG. 5B 40

°n 40

40 o 40- ° 40-

40 5? s?

20

20-

20 20-

20 20- 0

TTTTTr

0 7 • ■ I 1,1 '| ■ * •i 0

0 0 %-! 10° 101 102 103

-1 101 102 103

10°

10-1

102 10³

10°

101 10 ¹

10-1 10° 101 102 103

103

102

10°

10-1 10 ¹ 10-1 10° 101 102 103 FITC CD47 Fluor: Fluor: CD47 FITC Fluor: CD47 FITC

Fluor: CD47 FITC

Fluor: CD47 FITC Fluor: CD47 FITC

+ NBM1 4- A

100 SU031 100: o NBM1 o SU031

X c

SU032 o 4* ^ a NBM2 a SU032

SU001 •«

NBM2 NBM3 o NBM3 a SU001 v

SU004 SU009 (U □v • SU004 o SU009 b a OA

SU016

SU008 a *** A o a SU008 v SU016 X

SU027

SU014 Ill *0 ■ SU014 x SU027 * r-

+ SU030

SU018 FIG. 6A 'Sf ♦ SU018 + SU030 FIG. 6A

* SU035

SU028 a ▼ SU028 * SU035 o

10 10 ! o o V Sa v<o V V & ■O' V* G

Primary/ Fit-3 ITD Primary/ De Novo/

Cytogenetics % CD34

Gender FAB

WHO Classification

Sample Fit-3 ITD % CD34 WHO Classification FAB

Age Cytogenetics

De Novo/ Relapsed

Secondary Sample Age Gender Secondary Relapsed specified not-otherwise AML cn

Normal

Female Primary

SU001 Relapsed Negative SU001 59 Female Primary Relapsed Normal Negative 99 AML not-otherwise specified specified not-otherwise AML M2 4^

Female Normal

Primary 99 38

SU004 Relapsed Positive SU004 47 Female Primary Relapsed Normal Positive 38 AML not-otherwise specified specified not-otherwise AML M5

Normal

Male De Novo

Primary M2 M5 M1

SU008 3

59 47 64 Positive SU008 64 Male Primary De Novo Normal Positive 3 AML not-otherwise specified dysplasia multilineage with AML M1 AML with multilineage dysplasia MDS antecedent without Normal

Male De Novo

Primary M2

73

SU009 43 Negative SU009 43 Male Primary De Novo Normal Negative 73 M2 without antecedent MDS specified not-otherwise AML Normal

Male NS

Primary De Novo 18

59

SU014 Positive SU014 59 Male Primary De Novo Normal Positive 18 AML not-otherwise specified dysplasia multilineage with AML NS AML with multilineage dysplasia MDS antecedent without Normal

Male De Novo

Primary NS

59

SU016 10

Positive SU016 59 Male Primary De Novo Normal Positive 10 NS without antecedent MDS specified not-otherwise AML Normal

Male Primary

SU018 Relapsed Positive SU018 21 Male Primary Relapsed Normal Positive 55 AML not-otherwise specified specified not-otherwise AML M5

Male Failed to grow Unknown

Primary Relapsed

SU027 SU027 61 Male Primary Relapsed Failed to grow Unknown 10 AML not-otherwise specified specified not-otherwise AML M5

Female Complex

Primary M5 M5 M5

21 61 53 55 10 50

SU028 Relapsed Positive SU028 53 Female Primary Relapsed Complex Positive 50 AML not-otherwise specified dysplasia multilineage with AML M5 AML with multilineage dysplasia MDS antecedent without Normal

Female De Novo

Primary M4

SU030 53 40

Positive

SU030 53 Female Primary De Novo Normal Positive 40 M4 without antecedent MDS t(16;16)(p13;q22) or inv(16)(p13;q22) with AML Female M4eo

De Novo

Primary

SU031 inv (16)(p13;q22) Negative

SU031 31 Female Primary De Novo inv (16)fp13;g22) Negative 97 AML with inv(16)(p13;g22) ort(16;16){p13;g22) specified not-otherwise AML M4eo

Normal

Male De Novo

Primary

SU032 Negative

SU032 47 Male Primary De Novo Normal Negative 68 AML not-otherwise specified specified not-otherwise AML M5

Male Failed to grow

De Novo

Primary M5 M5

SU035 31 47 46 97 68 98

Negative

SU035 46 Male Primary De Novo Failed to grow Negative 98 AML not-otherwise specified M5 FIG. 6B

FIG. 6B

6/41 6/41 2023266367

%

i p|p©x a i 20x 20x

FIG. 7A FIG. 7A FIG. 7B FIG. 7B

Effect ofAnti-CD-47 Effect of Anti-CD-47ononHuman Human LSCs LSCs

0.8 0.8

0.7 0.7

0.6 £ 0.6 -o £ 0.5 0.5 £ 0.4 S' 0.4 o 0.3 S* 0.3 £ 0.2 Q- 0.2 0.1 0.1

0 0 A A iQ & ab no oo" N 5b ft SUO20 ftN #' 5^ ‘ft <5 ‘ft ft

Cell type Cell type

FIG. 7C FIG. 7C

7/41 7/41

Isotype lgG1 Isotype IgG1 Anti-CD45 Anti-CD45 Anti-CD47 (B6H12.2) Anti-CD47 (B6H12.2) •■-.T4 2023266367

FIG. 8A FIG. 8A

Isotype lgG1 Isotype IgG1 Anti-CD45 Anti-CD45 Anti-CD47 (B6H12.2) Anti-CD47 (B6H12.2) ■M r*.\ f? \ •. • .* * >4

FIG. 8B FIG. 8B

Macrophages HumanMacrophages Human Mouse Macrophages Mouse Macrophages 70- 70 70 70 a NBM1 NBM1 60 x 60 NBM2 o NBM2 60 x 60 <D NBM3 v NBM3 o 50 | 50 50 1 50 NBM4 o NBM4 »« «DM SU001 A SU001 40 ~ 40 40 ~ 40 >1 >> SU008 0 SU008 30 g 30 30 o 30 SU009 ▼ SU009 cn o> SU014 * SU014 20 5 20 20 1 20 SU016 ♦ SU016 Q. Q. dk. 10 SU018 o SU018 10 10 10 *s»s SU028 x SU028 I* 0 ♦* 0 0 0 SU032 a SU032 SU035 • SU035 & s C?' ^ oO"

-y GO a? J* ST // "o

FIG. 8C FIG. 8C

Percent hMac+CFSE+ Phagocytic Index NJ 03 4^ o o o o o

0.6

10 O.O. 0.4 0.8

0.2 (X>

20 CT>

30 ^

O. o o o o o o k> A vs1. $ % *> <s>- o o 9 O €>- CD ^ % 4^ 8/41

o c o co.

FIG. FIG. %- □ c □

IgG1 lsotype Anti-CD45 o s CD CD > \

9A

9B ro oo O. o. > > t> t ► >

9/41 9/41

10CH 100 A 80-j 80 SU028 SU028 v\ m IgG1 Isotype IgGI Isotype 60^ 60 Anti-CD45 Anti-CD45 2023266367

40-; 40 E3 Anti-CD47 Anti-CD47 20-; 20 0 0 TT 10 superscript(3) * I * * ' *1

0 0 103 104 104 105 105

10(H 100 80r SU018 80 SU018 >9 4 60 60 t 40: 40 20: 20 0J 0 * ' I ■ I »»• * ■ i rry I i '-lm-rf-i I I j H*

0 0 103 10³ 104 104 105 105

100: I 100 Ira ^ 60: SU004 80 SU004 60 40i i 40 20^ § 20 FIG. 10A FIG. 10A l- d> 0J 0 TT a. 0 0 103 104 105

Fluorescence Fluorescence

10°1 S 100 ®0o © ® ■■□■ SU028 •■aSU028 CD c SU018 ooaSU018 □ + 10: o SU004 OQA SU004 co 10 o A CO □ O O & 1l 1 ■’t A a u 0.1- ■= 0.1 □ AAAXmA AMM 5? <d & <0 FIG. 10B FIG. 10B s# O P O N .Ok' o \^> Vs

3. 10

103 10 ³

103 io3^ 103^ 103^ 103! -,5‘M&k. .a

GMP 2. GMP GWiP

0.92%

102 210

102

210 GMP 0.1% 10 io2^ _ 102; 0.92% _ 102l m 0.1% H CN cj* 5

MPP WIPP MPP

1 MEP

MEP MPP S 101^

10

110

10 10 £ 101- jc 101 5 101^

0.02% 0.044%

LT-HSC 0.82% 0.044%

LT-HSC 0.02%

1.1% u_ LT-HSCW Ll_ LT-HSC ■BSSx ; £T &

0.009% 0.004%^

0.004% q.009% u. U. CMP

CMP yerfipf

ST-HCC ST-HCC CMP CMP

ST-HCC ST-HCC -.im. 0.41%

0.20% iY

0.14% 0

0 0

0.006% 0.41%

0 0: 0.006% 0- 0.14% 0: 0,20% 0: €■: ■ m I m 'TTt^ 104 superscript(3) 10 102 101 10 1 102 103 10 4 10 ¹ 103

10 ¹ 104

102

0 0

103 0

102 104

0 0 101 102 103 104 0 101 102 103 104 0 1 01 102 1 03 1 04 0 101 102 103 104 CD34

CD34

CD34 CD34 CD34 CD34

CD34 CD34 FIG. 11d

FIG. 11c

FIG. 11b

FIG. 11a FIG. 11a FIG. 11b FIG. 11c FIG. 11d S ffl

10 + ID­ o 100

100 CM 100

LT-HSC GMP

LT-HSC GMP 4^ 80

80 'S 9- x 80 x 80i

beta actin m □ beta actin 60

60 | 60 1 6o; *«-

18S RNA +-> 018S RNA 40

40 c 7. o 40 o 40^ «u ' 20

20 sz ^ 20 O'] ^ 20: . ,1UH .... '/Winn o 6- 0

0 10° 102

10 ¹

10³ 104

'a c 102 310

10 104 10°

(U o 10° 101 102 103 IQ4 10° 101 102 103 104 l_ CD47

CD47

re4 CD47 CD47 a 4- 100

100 E 100 100 Mac-1+ MEP

o Mac-1+ MEP 80

o 3- ° 80 x 80 80 O o s D) 2- 60

| 60 60

c I E 60

FIG. 11e TO 1 FIG. 11e FIG. 11f

40 40

JC I o 40 o 40 FIG. 11f O 20

20

5s 20 55 20

9 8 7 6 5 4 3 2 1 0 2 0 m 1

Healthy Leukemic

Leukemic o Healthy Leukemic Leukemic 0 0

Ql ....

Bcl-2+ C- u.

BM c-Kit 10 superscript(3) 10 ¹

10 1

10° 103

102

102 104

104 10°

Bcl-2+ c- BM c-Kit 10° 101 102 103 104 10° 101 102 103 104

enriched BM

Kit enriched BM CD47

enriched BM CD47

Kit enriched BM CD47 CD47

Normal BM BC CML

aCML AML Normal BM aCML BCCML AML superscript(3) 10 10³ 103

10³ M

23 ;>a

102 102

102

102 ffiioH 'M •I gjio2- R1 £io2- S102^ re re re re ■MB* a: •a. q; q; i on cad

110 110

10 110 100 ?101- ‘■101- ri l:\ 2101- -J Kl * 31°1i 1 V '"’s&W I^IO0! I U r~

010

10° 010'

010 pi#? ui iu - m10° r*. «' x: ..'J £ ,ii£ >» sz mr >> W „ ‘T’* Q. a. CL

-1 1 rjp rm

10-1 10-1

10- 'I »«H I|

10-1 Iv 10’1 .•: gn Tr*nr TmT I 10'1 10-1

10-1 103

102 10³

102

101

103

102

10° 10° 103

10°

101 10° 102

101

101

10-1 10-1 10-1 10-1 10° 101 102 103 10-1 10° 101 102 103 10-1 10° 101 102 103 10-1 100 101 102 103 FITC CD45RA Fluor: FITC CD45RA Fluor: FITC CD45RA Fluor: FITC CD45RA Fluor: Fluor: CD45RA FITC Fluor: CD45RA FITC Fluor: CD45RA FITC Fluor: CD45RA FITC ■>

CD45RA CD45RA JL

FIG. 12a FIG. 12a

100 100 100- 100- Progenitors

^0,s

HSCs HSCs

80

80 80- 80- « X

60

60 5 60- 2 60- E E

40 40 ° 40- ° 40- 35 5?

20 20 20- 0 20-

i t i I iitij * i i 11iiij I |IIM| 11 ui] •

0 0 1 I I | lill] 0 * |i»'| I I | llll]

102 101 102

10° 10°

101 10 superscript(3)

10-1 10-1 10 superscript(3)

10~1 10° 101 102 103 10-1 10o IQ1 102 103 FIG. 12b

Fluor: CD47 FITC Fluor: CD47 FITC

Fluor: CD47 FITC Fluor: CD47 FITC FIG. 12b

CD47 CD47 >

% of max 0% of max 0 % of max ro ct) co o N> -C» 05 00 O 0 20 40 60 80 40 60 80 N) 20 -(s* 40 05 60 00 80 O 100 100 % 20 o o o o o o 100 -1.0 O O O O O o o o o o o O• 10-1 O o- TJ -g —. j 2 gj r* o'i O no o H o C o H ; n oJ *n : X CD47 ^ go ro h w o oPLT FITC

HSC o O PLT FITC

a & oOJ j o} Flour: CD47 -'1 oM j M oJ 104 superscript(3) 10 102 0101 A o^ 103 102 101 10° 10-1 10° 101 102 10³

oCO j co % of max 0 % of max % of max ro 05 co o 0 20 40 60 80 100 ro 05 00 o 0 K5 20 40 05 60 CD 80 O 100 o o o o o o % 20 40 60 80 100 O O O O O O o o o o o o q. q 10-1 —k o ■ 3 i ■o i T3 ■0 o'i o O' r o' 10° o C H o O H n -j n (O ■n o-1^ o M 101 o o h 3 PLT FITC a i o i PLT FITC O i 4^ O' O' 102 O O' -4 co Flour: CD47 w “J to Progenitors 5 0) o' 0101 102 103 104 oj 4^ 10 superscript(3)

oco i ³ 10 102 101 10° 10-1 CO % of max 0 % of max 0 % of max ro 4^ 05 60 00 80 o to 45k 05 03 O 0 20 40 40 60 80 lo 20 4^ 40 05 60 CO 80 O 100 100 20 100 _iO O O O O O o o o o o o O O O O O O q 10-1 q- 10-1 o- -0 -o O' 10° 2 o n oi 10° o H o C H c rs TI i r 10 ¹ T| O ' 101 oM o H O' 3 : Lin+ PLT FITC H O oJ o + PLT FITC oJ 102 co 102 Flour: CD47

° oJ M -vl ho Oj i 0101 102 103 104 4^ O O' 10 ³ 103 CO co o o o S s CD > CML AP

o CML CP CML BC TJ -o O m Q 2023266367

ro FIG. 12c o wzi 17 Nov 2023

13/41 13/41

Tet-vector Tet-vector Tet-CD47 Tet-CD47 2023266367

103 103 103

h- 102 102 101 10 1 102 102 10 1 101 m 0 0 0 0 Q O 0 10 1 102 10 superscript(3)

0 101 102103 0 10 1 102 10 superscript(3)

0 101 102103 3 E GFP GFP

103; 10 ³ 103- 103

io2 : 102

io1 101 m 0 lO2^ 102

io1 i 101 WT i 14.9 14.9 0 YT^S.- ID 0- 0 0] 0 D Ttwy o ■n-T

3 0 101 102 0 101 104 103 104 102 103 0 0 101 101 102 102 TIO3 104 103 104 £ > huCD45 huCD45 103- 103

102 102 : io1 : 10 ¹ L A A rr 0■ 98.9 98.9 Q T'T O 3 0 10101¹ 102 0 104 103104 102 10³ £ > GFP GFP

FIG. 13a FIG. 13a

14/41 14/41

Marrow Chimerism Marrow Chimerism Spleen Chimerism Spleen Chimerism 151 = 25 25- c 15 cu 120- 20 E 2023266367

510- 10 J: 15 15- sn |io- 10 o 55- •• 0 H<D S 55- *• a. 00 •-A 9 CL 0 0 ■f* Tet-CD47 Tet-CD47 Tet Tet Tet-CD47 Tet-CD47 let Tet

Blood Chimerism Blood Chimerism Liver Engraftment Liver Engraftment = 75 75- % 250 250- TO E |£200- 200 = 50 50- I 2 150 150- j= | ! 100 100- §25- 25 e o 50- o 50 •••• a 0 ■t -f®- 3 0 0 <0 0 Tet-CD47 Tet-CD47 Tet Tet Tet-CD47 Tet-CD47 Tet Tet

FIG. 13b FIG. 13b

«100- 100 Tet-CD47-MOLM Tet-CD47-MOLM £ 75 75- J. — Tet-MOLM Tet-MOLM » 50 50- c « 25 25- s ~r FIG. 13c FIG. 13c <D a o 0 0 0 25 50 25 50 7575100 100125 125 Days Days

FIG. 13d FIG. 13d

15/41 15/41

Injected femur Chlmerism Injected femur Chimerism femur Chimerisnn Contratatenal femur Chimerism

100- i 100 2023266367

1001 ra 100 •• E E = 7575- I 75'75 •»

| 50- 50 50- 1 50 m O S * 0. 25- 25 l 25- 25

0 0 0 0 Tet MOLM Tet MOLM Tet-CD47 Tet-CD47 Tet MOLM Tet MOLM Tet-CD47 Tet-CD47 MOLM MOLM MOLM MOLM

FIG. 13e FIG. 13e

100 100 4 1 75- | 75 Tet MOLM Tet MOLM 50- 1 50 Tet-CD47 MOLM —t—Tet-CD47 MOLM c <u 25- 2 25 o> Q. 0 0 t I-----1-----1----1-----1-----1 0 10 20 30 40 50 0 50 60 60 7070 80 80 Days Days

13f FIG. 13f FIG.

\umm & m FIG. 13g FIG. 13g

phagocytosis of Efficiency 12 Efficiency of phagocytosis 12

Macs only Macs only

MOLM only MOIM only

200 200::

9

120 120 9-

150 ^ iso:;

TET MOLM □TET MOLM ffl CD o 6-

100

100 £ 100::

TETCD47 MOLM 100- 0TETCD47 MOLM X 0 o.: o

50 (U I % 50:: 97.5

244

0

100

80 •o 80- 0 £0 :: 244 i7.5 105 104 superscript(3) 10 102 0

6 3# 0 £ T 0 -4™, 0+

102 103 104 105 102 103 104 105 102103104105

60 -£60- GFP

GFP o GFP GFP

FIG. 14a o FIG. 14a

15 15

TET47

40 TET47

15 ro 40- TET47 15: TET47 -C

MOLM MOLM

Q- MOLM MOLM w

10 “210

20 2h 4h

20- 10 2h 4h

T I 0 1 10: I o o

5

0 1 5 0 ■ o 5 o 5-

#

2h 6h

4h 2h 4h 6h CD 6.26

4.27

95.7 93.7

95.7 4.27 93.7 6.26

Time(h) Time(h) 105 104 superscript(3) 10 102 4^ 0

0 0 rZ m»Vn 0“ 1fiii! >mrr^,TnTirT 102 103 104 105

102 1 03 1 04 1 05 102103104105

MOLM

TET CD47 MOLM 2h GFP

GFP

TE-T GD47 MGLM;2hi WWB m GFP GFP N

10 TET

TET

: TET TET 8 MOLM MOLM

/T* 8: MOLM MOLM 2h 4h

;> A' 2h £ 6 4h 0 •.* I 6\ / 0 <+. 4 *+ 'S # o #

% 2: % 2 40.9

28.5 59.5

71.5

71.5 + 28.5, 59.5 + 40.9, 105 104 superscript(3) 10 102 105 104 superscript(3) 10 102 8 6 4 2 0

8 6 4 2 0

24h

M

TETCD47 MOLM 124h 1 1 0 4 0 102103164105 102103104105 GFP GFP

GFP GFP FIG. 14b

FIG. 14b FIG. 14c

FIG. 14c RJ

can ten

ht

Tet- Tet- it/ Tet-

105 105 105

105

105 105 Tet- 105- \ 105 let- 105 \ Tet-

Mock CD47 Mock

Mock CD47 CD47

Mock CD47 Mock CD47 Mock CD47

octs

104 104 104 OCII

104 104 104 104 104 104 104 104

MOLM- MOLM-

MOLM- MOLM- 1 MOLM- MOLM-

104 3) 10³

103 103

10³ 10³

10 103 103 103; 103 103 103-

13 13

V._,

2102 102

102 102 102

102 io2 io2 a* 102: •r^^P 102:^ 102- 't -w :

0 0

0 0

0 0 04^£^ ni n qWv»„.*... . oogp i • nn n-r-l 0 10110210³

101 102 103

10 ¹ 102 103 0

0 0

0 10110210³

0 10110210³

10110210³ 0 lOHO2^3 0 lOMO2^3 0 101102103 0 101102103 0 101102103 0 101102103

4^

105 105 105 \ ■ \ \ Tet

Tet

Tet Tet Tet 4 Tet

104 10 o

104 104 104-

MOLM- MOLM- MOLM- MOLM- . MOLM- . MOLM-

310

510 410° 310 Bone marrow 310

13 103 103 13

103

13 Liver Spleen

.'•ii* Bone marrow Liver iil Spleen

4102 402

402 41 o2 3# no2; S'." CL 0:

0

0 LL Ows-fc,.'..... S: oi

101102 10³

0

0 0 10110210³

10110210³ (3 "o'" io1io2io3 > o 0 > 101102103 Jtim (3 0 101102103

huCD45 huCD45

huCD45 huCD45 huCD45 huCD45

FIG. 14d FIG. 14d

18/41 18/41

muCD47l0 muCD47¹0 MOLM-13 MOLM-13 884 884 100- 100 mouse BM mouse BM A muCD47hi 2023266367

muCD47hi 1041 1041 MOLM-13 MOLM-13 80- 80 8056 8056 x 1 60 60- o 100- 100 S? 40 40- muCD47hl muCD47hi mouse BM BM % MOLM-13 MOLM-13 mouse 1085 1085 80- 80 +DOX 20- 20 +DOX x 174 174 | 60 60- 0 0 11 r1 • pi i 1' i' m,i IT#H ' mill ' 'iu"\ o o 0 io3 103 104 104 105 105 as 40 40- muCD47 muCD47 FIG. 15a FIG. 15a 20- 20

0 0 11 MM ‘ * inH ‘ iu “i 0 0 103 104 104 105 muCD47 muCD47 FIG. 15b FIG. 15b 110-1 110 100 100 90- 90 80- 80 ro > 70 70- I 60 60- -i i CD47 lo CD47 lo MOLM-13 MOLM-13 U) - 50 50- 1 CD47 hi hi MOLM-13 CD47 MOLM-13 ++ DOX DOX o 40 40- I 30 30- • CD47 CD47 hi hi MOLM-13 MOLM-13

20- 20 10- 10 0 0 I 0 0 25 25 50 50 75 75 100 100

Days post-transplant Days post-transplant

FIG. 15c FIG. 15c

Bone Marrow

Liver Liver Bone Marrow

10.0 30 10.0-+ 30-1 E 9 .52

7.5 7.5- <u

20 E 20- .la. O) 0 9 © S 9

6.0 x 6.0- O ra 9 9 9

10 s c 10-

2.5 2.5- <u 9 O *• 9 9 01 CL

0.0 ■1^9------------^9^99—

0 0.0 t t 0 t CD47 hi +

CD47 lo

CD47 hi

CD47 lo CD47 hi

CD47 hi CD47 hi CD47 !o CD47 hi CD47 hi CD47 lo CD47 hi + DOX

DOX DOX DOX co 4^ Spleen

Spleen Spleen Spleen l.l-i 9 45t

1.1 1.0 1.0- 9 0.9- 9

0.9 0.8 ~ 0.8- 0.7- 9 I £: | 30- § 0.6- 99 Z 25- % 0.5- O 20- 99 S 0.4- ■£ 15— 0.3- 9 45 40 35 30 25 20 15 10

0.2- 9 8 io- 0.1- 9999 CD 5- Q. 9 m 9?-.a

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 50

0.0 t t 0 CD47 hi CD47 lo CD47 hi +

CD47 hi

CD47 lo

CD47 hi CD47 hi CD47 lo CD47 hi CD47 hi CD47 lo CD47 hi + DOX

DOX DOX DOX FIG. 15e

FIG. 15d FIG. 15d FIG. 15e

% of max 05 CO 0 ho 20 4^ % 40 60 80 o 100 o o o o o o . . I I I ■ I I I I I___1 3 0 o= 3 00 o 00 884 T| ji. 05 3 —v = CD O 10600 o o C o- 10³ o 19100 O W o D •P» muCD47 -0 CJl FIG. 15f Tjum o4^. _i. = Time o-5 104 105 cn 0 5Phagocyte Index 10 ho 15 20 ho 25 o cn p o oi CJl n O —i 2h 3 S Time CD cn □ FIG. 15g S3 ^3 CQ oooo r- o r- O ^ JSk 0^ JSk 2* ■•■T I I -vl 3* —1 q“ mCD47hi mCD47lo MOLM-13 00 — CO U MOLM-13- 2023266367

twz 17 Nov 2023

21/41 21/41

Competetivephagocytosis Competetive phagocytosis

30 30

^muCD47hi MOLM-13 muCD47hi MOLM-13 2023266367

25- 25 muCD47lo MOLM-13 muCD47lo MOLM-13

S 20 T3 20- I £ - 15 15- o o O) £ 10 10- Q.

5- 5

0 0 Sample1 1 Sample Sample 22 Sample Sample 33 Sample

FIG. 15h FIG. 15h

o-'' O ‘&Zj- 1 m' 'VJ -f — 1^-' 'w"

■i, O- 4- -s -.r. Q 3 Jo) O ^ ' if c2 O f) 2/- O " 4 C iS '<£? P': ■' '1>‘

V7 ' v’5 /i ■si « 2S S.'S I"?*

M 51 J irf i « mm -S* l\v* *22 fr-. W‘ (! kSfts-^v* u

FIG. 15I FIG. 151

Cells Stem Hematopoietic Blasts

Myeloid Progenitors Hematopoietic Stem Cells Myeloid Progenitors Blasts

100

100

100 100 100- 100i

80

80

80 80- 80- 80-

60

60

60 S I 60- 60- 160; s«

40

40

40 o 40- o 40- 40- 5S s? s?

20

20

20 20- 20- 20- rTTTTOt i iTjiiuj

0 I I 1 1IIUJ HIW1J J'IU|

0 I 11 i •ft * 'niiiuf-rpriiwi

0 0 rtB| ' I'illliJ 0 0 1000

100

0.1

1000

100

0.1

1000

100 1 10

0.1 1 10

1 10 0.1 1 10 100 1000 0.1 1 10 100 1000 0.1 1 10 100 1000 FITc CD47 Fluor: Fluor: CD47 FITc

Fluor: CD47 FITc Fluor: CD47 Flic Fluor: CD47 Flic Fluor: CD47 Flic rv) N3

FIG. 16a FIG. 16a

100 100-

80 Lin- Mac-1+

80- GMP

GMP Lin- Mac-1+ X 100

100

60 1 60-

80 80

4" 80: 80:

40 60 60

2 40- « 60- $ 60- S? 40 £ 40- 40

I 40-

20 20- o o 20 20

% ^20: ss 20: 0 0

U|tU|| t^MU| ' T*i r piiipf 104 10³ 102 101 10° 0 04 ^O0 101 102 103 104 0 m 104

10³

102

10° 10 ¹

105

104

103

0102 0102 103 104 105 10° 101 102 103 104 CD47 CD47

CD47 CD47 CD47 CD47 FIG. 16c

FIG. 16b FIG. 16b FIG. 16c

23/41 23/41

100-F 100 100 i HL60 HL60 Kasumi-1 Kasumi-1 80-■ 80 8£> 80 X x 60- 2 60 60- SS 60 E E 2023266367

40.. o 40 40- ° 40 s? S? % 20-• 20- 20 20 0” 0 * fUl'l 11 UOT"1" ! ^ ll"T 1 *" 1 0 10 superscript(3)

0 102 10³ 0 104 103 104 105 0 0 102 102 104 103 104 105 CD47 CD47 CD47 CD47

100-- 100 100- 100 K562 K562 Jurkat Jurkat 80- 80 80- 80 x X 60-- 5 60 2 60- 60 E E 40-. o 40 o 40- 40 S? 5? 20-' 20 20- 20

0 FUT t- Uli 1 0 0 0 0 102 102 103 103 104 104 105 105 0 0 102 102 103 103 104 104 105 105 CD47 CD47 CD47 CD47 100-f 100 100- 100 IM0LM-13 MOLM-13 U937 U937 SO-- 80 SO- 80 x x 60-- 2 60 2 60- 60 E E 4Q.. o 40 o 40- 40 5? S? 20-: 20 20- 20

0 i|i j-r. i... . . .fri-j ‘iFIT u i miu i i'v , . 0 0 I||ww»4i|(mii®|"| »vi n u| 0 in 0 0 102 102 103 10³ 104 104 105 105 0 0 102 102 103 10³ 104 104 105 105 CD47 CD47 CD47 CD47

100-F 100 A HSC CB HSC CB 80-- 80 x 60- 2 60 E FIG. 17a FIG. 17a 40 ° 40 55 % 20 20-

0+IIM'i'M 1 T rr**B| * 0 i..ri|iuii| :

00 1021 02 1031 03 1 04 104 1 05 105 CD47 CD47

24/41 24/41 900 900

813 813 800- 800 2023266367

700- 700

600- 600 cn < ? 500- 500 x CM < ° 400- 400 LL =5 LL s 300- 300 245.6 245.6

1J 198 183 200 200 183

96 96 100 100 81.6 81.6

0 0 HS60 Kasumi-1 HS60 Kasumi-1 K562 K562 41.6 41.6

Jurkat Jurkat 1 MOLM- U937 MOLM- U937 CB CB HSC HSC I FIG. 17b FIG. 17b

13 13

105 105 1 -r • 60 60 ■ MOL MM MOL 104"j 104 &.'■. 50- 50 s Live Jurkat Live Jurkat

M ■■ x □ IrradiatedJurkat Irradiated Jurkat ■ 0) ^76.8» 76.8 "g 40 40- T 103 10³ .2 ) 16.1 16.11V . > 30 30- m 0 o

I o 0 i1-.; ■«fr a h i 20 20- o3 ’m- £. Ja •.nr'. CL E * )»y 1" i k 'I'urnj 10- 10 0 0 103 103 104 104 105 105

> O' 0 huCD47 huCD47

FIG. 17c FIG. 17c FIG. 17d FIG. 17d

25/41 25/41

105^ 105 105- 105

\±fl± - GMP 104- 10 ²4

MPP MPP 104‘ 104 •jS m GMP 0.96% 0.96% 2023266367

I m CM LT-HSC- 0.037% 0.037% '&• IAP+/+ IAP+/+ ^3. LT-HSCj ^ 103 Q£ •* * r ar 2 u_ 0.004% 0.004% ^lO3- 103 MEP ST-HSC ag? I ST-HSC ■I ■vm 0.90%

Or 0 pt)0.018% 0.018% 0* 0 ai1 mts& • . CMP CMP 0.27% 0.27% TT r^T 1 "T 10 superscript(3) ■^T *mr rTT ‘T 10 superscript(3) t^T

0 0 102 102 103 104 104 105 105 0 0 102 102 103 104 104 105 105

CD34 CD34 CD34 CD34

105- 105 105- 105

JZ* .v* -?

104- 1044 104' 5 GMP MPP jjjjjjjj S 104 ?'ix GMP :0.91% MPP 0.91% CM 0.055% 0.055% D£ IAP-/- IAP-/- i 103 LT-HSC 103, LT-HSC 6- 10 superscript(3) ********¥7* LL. 0.006% - / 0.006% ST-HSC

[I ST-HSC |)0.026% u. 103- 0 0 v|K MEP 0.91% “! 0.026% < 'i. Or 0 CMP CMP •» ?Sv’- ^sr- 0.43% 0.43% nr * ---j . -'1 nTT * "’i i

0 102 103 104 10s 10 superscript(3)

o 102 103 104 104 105 0 102 103 104 105 0 CD34 CD34 CD34 CD34

105’ 105 105- 105

104^ 104 ■M GMP 1041 - 104 GMP

IAP+/- IAP+/- CM LT-HSC 1Q3T LT-HSC ^ 10³ MPP MPP 0.052% 0.052% S & m • rimm 0.91% 0.91%

0.007%"/ io3-j mep :S MH? z 0.007% 74 3 f/ ST-HSC ST-HSC 0.013% 0.013% £ 10³ 0 0 . sMlI * ■A MEP 0.81%

u 1 .1 Oi 0 : CMP CMP V R! 0.46% 0.46% r—j 1 I nTT t-~i • "i * ’”i 10 superscript(3)

0 0 102 102 103 104 104 105 105 0 0 102 102 103 10 ³ 104 104 10s 105

CD34 CD34 CD34 CD34

18a FIG. 18a FIG.

26/41 26/41

Day 77 methycellulose Day methycellulose colony colony formation formation 2023266367

100% 100% 90% 90% V//7/7//7/, 80% 80% 70% 70% 60% Empty Empty 60% V7?m Meg Meg 50% 50% ESS3 GEMM 40% 40% GEMM 1///J GM 30% GM fyXXXl M 30% M 20% 20% E Z3 G 10% 10% 0% 0% IAP+/+ IAP+/+ IAP-/- IAP-/-

FIG. 18b FIG. 18b

Rescue Rescue of of lethal lethal irradiation irradiation 110i 110 100 100 15 90- 90 I I > 80- 80 - U - •- Irradiation Irradiation only only i t 70- 70 i I: 2x10A5IAP-/- 2x10^5 IAP-/- BM BM + irradiation + irradiation i 60- 60 1 --J-- 2x10^6 2x10A6IAP-/- IAP-/- BM BM + irradiation + irradiation ~ 50- 50 i I o 40- 40 i I —^ 2x10^5 2x10A5IAP+/+ IAP+/+BMBM + irradiation + irradiation g 30- I 30 i I- 2x10A6IAP+/+ 2x10^6 IAP+/+BMBM + irradiation + irradiation o! 20- 20 i I i 2x10A6BMBM(IAP-/- 2x10^6 (IAP-/-recipient) recipient) 10- 10 i I 0 0 0 0 10 10 20 20 30 30 40 40 50 50 60 60 70 70 Days Days

FIG. 18c FIG. 18c

10s-

105 105- CM +/+ cj1 -I- m • •, !$104- Q104- Q o o O A < A ro 6103- < 103 H o 4^ u_ mSf v E iI ‘ i 4 ^

oi •O; v 05 96.9

w B:E. 11 1 f*l “T ’T 105

105 0 103 104 105 0 103 104 10s <APC-A>: CD45-1 <APC-A>: CD45-1

FIG. 18d FIG.

28/41 28/41

Peripheral blood Peripheral blood Peripheral blood Peripheral blood granulocyte chimerism granulocyte chimerism granulocyte chimerism granulocyte chimerism 65i 65 lOOi 100 60- 60 ■w m U. 55- 55 2023266367

o 50- 50 o 75 75- § 45- 45 c •§ 40- o 40 TJ - 35- 35 ■£ 50 50- g 30- 30 9 O S 25- 25 o fe 20- 20 9 25- S. 15- 15 Q. 25 10- 10 5- 5 0 0 ♦ 0 0 ■9-®- > In % “b % <b x\x X x\x X x\x X x\x X s s s § s s s \^ $ \V- 50 Flk2- 50 Flk2- CD34- KLS CD34- KLS 500 Flk2- 500 Flk2- CD34- KLS CD34- KLS

FIG. 18e FIG. 18e w L"' '«< \-v e

' A? C< • r *' Efficiency of Efficiency of Phagocytosis Phagocytosis 70 70 e

.•> •''••S' 60 60 .? 4f' c 8 50 50 ■a c -f fx r o 40 40 ♦3

8 30 30 r,,r*' X O) r-^4 ■, X ra V ; i ■

& 20 Q. 20 Q . fr' .-v ’ %-i •U, f

10 10 c* ■ —• >.v • , . o 0 'n IAP+/+c-Kit+ IAP+/+ c-Kit+ IAP-/- c-Kit+ IAP-/- c-Kit+ • r- G sV • 4; r.-

FIG. 18f FIG. 18f MiF^©4aKs=

FIG. 18g FIG. 18g

CD47-FITC MFI % of Max (normalized) 0 M 4^ 40 CXi 60 CO 80 O 20 100 ho Ki GO CO o o o o o o oi 50 o 100 01 150 o 200 cn 250 o 300 cn 350 OOOOOOOO oJ o ■n oJ M 1 Q o ■■■': . V ■T’c 4^> CD47 co. 2 -0 ho CO o- o FIG. 19a Q) 5 c-Kit+

3 7 GO oJi 0 102 103 104 105 cn 4

U1 c-Kit 0 o o 103 o104 o 105 CO Ol Tl A Q 0 CO FIG. 19c o- 10³ o cn co o u Jl^

WaF^'fm Sca-1 104 o-fc. 1 o- 105 cn Flk-2 y y FcyR,I/HI 0 510 0 101 102 o 103 o 104 o o o 103 o 105 oCn o 4^ cn 4^ oro y> o; o: -?r*4 ■ 5 MEP X "n oJ LT-HSC oJ N3 o N3 p Q o a oj CO

Sv 2 -i ■ O oJ GO CO.

m GO CD34 CD34 4^ ^Hjg oj o: CO -tv ■Fv co 5 o FIG. 19b ■H MP o CT -a s CMP s GMP X T) T3 o- CO oJ 105 104 superscript(3) 10 102 0 ST-HSC Ol 0 102 10³ 104 105 Ol o % of Max 0 % of Max NO 4Sv CT) 00 O ro -Pv ct) oo o 0 60 80 20 40 60 80 20 40 100 100 o o o o o o o o o o o o X o= O’ o o- ho. M 2023266367

o *<•.* .■ • ■. .“*,i=^=aaaate»_1__1 o D o- O o- CO A CD47 CO CD47 ~4 -4 q oj o- Q 4^-' 0) 4^ LT-HSC s o GMP ■o oJ o- 0 102 10³ 104 105 0102 10³ 104 105 cn- cn imz 17 Nov 2023

-10s

100 GMP GMP

GMP GMP

80 fe- •• 80-

104 ■104 I Wl- x

60 q; | 60- >-

MEP CMP o MEP ICMP-: ■

103 -103 1

40 u. o 40-

105 105^ *=c. Wl-'i

0 m- ■0 s? #„

20 20- •* . ‘rW ;iO-

0 1 * I'"'' TrV 0 .iMi ■flT' *'a it'1*-

104 0 104i il'4 0 103 104 105 103 104 105

m,:-. 105 104 103 102 0 CD47

10s 104 103 102 0 CD47 CD34

CD34 *: s>: GO fv o

103 ° 103. JSs

10s ji 8 -10s 100

LT-HSC LT-HSC

104 ^.-y- 80

mm -104 80- ?T“ x ■ :■

0 0 SB?-- & 60

CM | 60- { o A MPP -103 MPP 103

LT-HSl ? 40-

LT-HSC 40

TrTVTJ T u. %

2? V’

0 103 104 105 0 103 104 10s 20

ST-HSC 0

felsc ■0 20-

Sca-1 Sca-1 “ j—■»• - - 0

0 m 0

105 104 103 102 0 0 103 104 105 103 104 105

105 104 103 102 0

CD47 CD47

CD34 CD34

FIG. 19D FIG. 19D

Liver

Spleen

Bone Marrow Bone Marrow Spleen Liver

1.00E+07 1.00E+06 1.00E+07 1.00E+06

1.00E+06 1.00E+06

1.00E+06 1.00E+06 7

1.00E+05 ..... *

1.00E+05 N 1.00E+05 T77' W 1.00E+05 .... OT / «

1.00E+05 □ 1.00E+05 _1 {// / r 00 1.00E+04 o

1.00E+04

1.00E+04 w 1.00E+04 ® 1 .OOE+04 ©1.00E+04 (!) X2 13 sx E i E t

1.00E+03 IAP+/+ 1

3

IAP+/+ 1 E r—

IAP+/+ 1 - IAP+/+ 1 2 1.00E+03 —•—IAP+/+ 1 1.00E+03

if

1.00E+03 —^IAP+/+ 1 IAP+/+ 2

z

IAP+/+ 2 z 1.00E+03

IAP+/+ 2 z 1.00E+03 — — •«— - IAP+/+ 2 —-«—IAP+/+ 2 —IAP+/+ 2 IAP-/- 1

IAP-/- 1 1.00E+02 IAP-/- 1 ....IAP-/- 1 1.00E+02 IAP-/- 1 ....*-— IAP-/- 1

IAP-/- 2 IAP-/-2

—— IAP-/-2 IAP-/-2 —— IAP-/- 2 —IAP-/-2 1.00E+02

1.00E+02 5 Day 4 Day 3 Day 2 Day 5 Day 4 Day 3 Day 2 Day 1.00E+01 1.00E+02 1.00E+01 5 Day 4 Day 3 Day 2 Day 1.00E+02 Day 2 Day 3 Day 4 Day 5 Day 2 Day 3 Day 4 Day 5 Day 2 Day 3 Day 4 Day 5

FIG. 19E

FIG. 19E

32/41 32/41

1001 100

80- 80 IAP+/+ 489 IAP+/+ 489 X I 60 60- 1 IAP+/- 271 IAP+/- 271 lAP-/- 32 IAP-/- 32 2023266367

o 40 40- & vii 20- 20 Vr'

o 0 4^ 0 0 103 103 104 105 104 105 CD47 CD47

FIG. 20a FIG. 20a

70 70 60 60 IAP+/+ IAP+/+ 50 50 o 40 c 40 o T3 30 ■L ♦ —Mouse Mouse 11 30 \ s? —Mouse Mouse 22 20 20 * -x ---*■..... Mouse Mouse 33 10 10 ■x- Mouse44 Mouse ----- x----- Mouse Mouse 55 0 0 0 0 10 10 20 20 30 30 40 40 50 50

Weekspost Weeks posttransplant transplant

70 70 60 60 \ IAP+/- IAP+/- \ 50 50 c 40 40 7- O T3 —Mouse 30 30 ♦ Mouse 11 s? —Mouse Mouse 22 20 20 A. ..... Mouse Mouse 33 10 10 -x Mouse44 Mouse ----- x----- Mouse Mouse 55 0 0 A-1 * 0 0 10 10 20 20 30 30 40 40 50 50

Weekspost Weeks posttransplant transplant

FIG. 20b FIG. 20b

33/41 33/41

Lineage Negative Lineage Negative Lin-CD34+CD38- Lin-CD34+CD38- 2023266367

A105 105 1 A105 1 105

104 104 1 ' 'f *. < 104 104 I Pre-Sort Pre-Sort si:-. 103, 10³

M 103, 103 • 1 1 99 99 102 102 & 00 Y- O 00^ CO i •ft

a n- cn Q 0 U3 pm Ttrrm 7 w ^“iMr'Vi

0 0 103 103 104 104 105 105 0 0 10³ W 105 ib3' 104 1b5' CD34 CD34 CD47- CD47

4-105 105 1 A105- 4105 .

io 104 4-, i 104i 104

Lin-CD34+CD38- Lin-CD34+CD38- 10 superscript(3)

CD47hiSort CD47hi Sort 103, 103- 103 (T- %i k. 0 0 100 100 102 102 t 00 CO i o 0°= CO Q O U= 0 n- 99 99 co l*n *i 10 superscript(3) 1^*'I'i

0 0 103 104 104 105 105 0 0 ■ "1b3 10³ "1b4 104 "1b5' 105 CD34 CD34 > CD47 CD47 >

4105 105 ' AlO5- A105

104 104 ■ 104^ 104

Lin-CD34+CD38- Lin-CD34+CD38- 10 superscript(3)

CD47loSort CD471o Sort 103 103- 'sa 103 1 © 100 100 0 0 102 102 00 CO © © i o 0 Q n- CO O 0 u] . )H1 ^ * *‘“i 98 98 — ' T * l*n,J .* r * i co 0 0 103 103 104 104 105 105 0 0 ’1b3" 10³ “To4'"To5' 104 105 CD34 CD34 ■> CD47- CD47 >

FIG. 21A FIG. 21A

34/41 34/41

c? . A * * V m % 2023266367

^v. yxb % > * r> % c' •* !? k; %* ,• *>V,T$!r^ -’•a* % *

! ;-

FIG. 21B FIG. 21B

A c A 105 105 ‘ 105 105 ]

■Mt Jm 104 104 104 104 90 1 Lin-CD34+CD38- Lin-CD34+CD38-

mlr% CD471o: Mouse1 CD47lo: Mouse 1 103- 10³ fX r\ 10³ t’'* W i’Bi LD ;-.V. v.; On ** 0 Vi v On O 0 Q 3 ""4.5 i ^

o 6 6 Q E r-m o 7.5 oio2...io3 "To4 "To5’ *•1

o 0 ‘WWW 10³ 104 105 0102 10 superscript(3) 104 105

hCD45- hCD45 CD33 CD33 >

>k c ^^ c 105 105 105 1 1 __ *r-^. / It^5 104 104 104 104 89^-45^, - 89 % jl -» Lin-CD34+CD38- Lin-CD34+CD38- CD471o: Mouse2 CD47lo: Mouse 2 103 103- .5 10³ * ■: '• *•-’

m f it S 05 Q 0 O) 005 nr- o fr’4 4 Q tX.

9.5 E PTTfT o 9.5 0 0 10³ 'To4"’To5' 'To3 104 105 r"6T62 0102 10³ To5’ To4 105 To3 104 hCD45 hCD45 > CD33 CD33 >•

FIG. 21C FIG. 21C

35/41 2023266367

SU008 ■JE h- CM 'f % =H= Q Q 03 03 oi o■ <o 3 co u QQ oo co CO CO o o c E E ro .2 Q Q E E O O 2 2 3 03 + + m CO X rf 3 CO CO + + 15 03 Q Q m in '3‘ E i_ Ot O Q ■'3- Q o 3 .£ .E O o Z HI JZ JZ

400 <— FLT3-ITD WT FLT3 300

1 2 3 4 5 6

FIG. 21D

NK-AML Patients

NK-AML Patients NK-AML Patients

100 NK-AML Patients

100 nooi -=■100- S5 ro

80

80 80-

60 ¥| 8°- 60-

60 CD47 Low Z 60: CD47 Low

CD47 Low 40

40 2 40- CD47 Low _ CD47 Highly

CD47 High 1 4°~

20

20 CD47 High i 20- CD47 High g 20- c

P=0.004 P=0.002 P=0.004 «

0

0 | 0 > 0- P=0.002 O 8

4 6

2

0

8

6

4

2

0 ui 0 2 4 6 8 0 2 4 6 8

Years

Years Years Years

No. at Risk: No. at Risk:

16

37

CD47 High: 95 14

30 CD47 High: 95 30 14 3 1 95 37 16 4 1

3 40

CD47 Low: 37 8

95 37 10

10

3 30

8 CD47 Low: 37 8 3 0 0 37 8 3 0 0 FIG. 22B

FIG. 22A FIG. 22A FIG. 22B CO Oi -fe.

Patients FLT3-ITD-Negative Patients FLT3-ITD-Negative g FLT3-ITD-Negative Patients FLT3-ITD-Negative Patients 100

100 13100 ^100- > g. 80

80 80- so: E | 60- 60

60 « 60- CD47 Low

CD47 Low 4> CD47 Low 1CD47 Low 40

40 U 40- 1 w 40- 1 CD47 High

CD47 High

CD47 High ll CD47 High 20

20 4. 20- « 20- P=0.03

c

P=0.06 v P=0.03 0

> 0 T o 4 8

4 6

2

8 0

6

2

00 4 6 8 0 2 4 6 8 iLU o 0 P=0.062

Years Years

Years Years

No. at Risk: No. at Risk:

CD47 High: 58 13

30

12

24 CD47 High: 58 24 12 2 1 58 30 13 2 1

CD47 Low: 16 10

20

5 3

58 16

10

20

5 3 CD47 Low: 16 5 3 0 0 16 5 3 0 0 FIG. 22D

FIG. 22C

FIG. 22C FIG. 22D

37/41 37/41

Day Pre Day00 -- Pre Day Post 14-- Post Day14

105- 105 105^ 105 HP'^^S 71 6835 68 104i 2023266367

104- 104 104 - IgG IgG 104 “ 103 103^ 103

0 •; 0^ o tmm 0 0 ^23 (22 23 ‘ ' ‘ ''“"i TTTnair cv^rsy 1 ’rrTTTT > >i'“<j * * • ■ p i 11“ i 0 0 102 102 103 103 104 104 10s 105 0 102 0 102 103 10 ³ 104 104 10s 105

105^ 105 105- 105

77 b^s 77^£ 98 104. 104 J w4-. m 104 V Anti- Anti- A 3 ”' 10 Superscript(3)

103 o 103- 10³ CD47 CD47

m mm ■\02-- 2 10 Q 0 0 = o 0= E 1 i jiJi |j I l|IHtf ^24 24 0 'i'".i..I1" 0 0 »»i • ■* | v •«*• *u| ‘ 1 MfuiJ r'‘““Ir • 10 superscript(3)

105 103 104 105 0 102 0 102 103 103

> 104 104 105 I0 102 0 102 104 105

hCD45 hCD45 FIG. 23A FIG. 23A lOCh 100 SU004-1 SU004-1 QQ SU004-2 SU004-2 a. 80- 80 anti- anti- SU004-3 SU004-3 CD47 CD47 + fO SU028-1 SU028-1 co 60- 60 SU028-2 Q SU028-2 O SU004-4 SU004-4 + 40- m 40 SU004-5 -o- SU004-5 Q * ^ SU028-3 SU028-3 IgG IgG O JZ 20- 20 SU028-4 SU028-4 & SU028-5 SU028-5 S 04>---r 0 0 0 5 5 10 10 15 15 of Antibody Days of Days Treatment Antibody Treatment

FIG. 23B FIG. 23B

38/41 38/41 2023266367

Day00-- Pre Day Pre Day14 Day 14-- Post Post

105l 105

104- 104 = 12 rf?§' 12 " 105t 105

104- 104 7 7 m (ft igG IgG 103, 103 u 103- 103 I] : 0= 0 ll t o= 0 ? ^92 P! I* ' '1 "“I lW87 n r 87 PTTffH| I < • | M.Uj » iij | a ii jHt iJ1 ' r«l «nijf ■v 92 f I*-i| Ttwy

o 0 io2 102 103 103 104 104 io5 105 o 0 102 102 io3 103 io4 104 io5 105

io5-: 105 39 105^ 105 99

A 104i 104 j V m 104- 104

Anti- Anti- 103- 103 103i 103 CD47 CD47

in Q u E 0 0

1 fMaJeo60 M " " *j ' i i |»u« j i 102- 102

Oi 0 1 111*>ijj 'i "ii 1 T 0 0 TTWf

0 102 103 0 102 10 superscript(3) 104 105 IO4 IO5 0 0 102 102 103 103 104 104 105 105

> HCD45 hCD45 FIG. 23C FIG. 23C

39/41 39/41

3401 340

320- 320 □ □ SU004 SU004 .2 □ SU018 SU018 E 300-1- 300 ■ SU028 SU028 2023266367

® 180t 180 □ 160- 1 I 160 -i E S? 140140- 120- z'Z 120 J- tn 100- 2 £ 100 3 80- ■Si 80 □ ■ DC .E □ 60- 60 □ o s? 40- 40 ■ 20- 20 □ 0 0 'P' IgG IgG Anti-CD47 Anti-CD47

Treatment

FIG. 23D FIG. 23D

%.;v ■^1 V < %" f.V ; ■ 9' 2 ■ iV? 33 %/ * “ M?' -/• * •. s., rS s •»-'. - : r

.oCT ; •• V ■' S %. &

V. ■ ■

100X\ 100X t. 200X 200X .... Vbhi'-- 200X

i-: T.-;i 4 4 5 5 6 VrV:V/:'l ' ■ ■ • {V"' f :•-r vc- > FX,4-'-:u \ 4:;., mms/i ••iv';. '

fcxm . - Vs *?%::* A > 100X 100X v w 200X 200X /v>- - ‘ AV. -1 200X

FIG. 23E FIG. 23E

100

1.0 1.0

CD47 high -*-CD47 high

80

CD47 low -*-CD47 low 5? 80-

0.8 1 0.8- 'E To

60 > 60- TTTTTTTm

0.6 « 0.6- CD47 High

CD47 High — 3 CD47 Low

O CD47 Low —

40 (0 40- ■«*

*

0.4 £ 0.4- <*--**• 15 4^ 2 KXK*—HrM- ■If TT O

20 ^ 20- 4^

0.2 ■§ 0.2- O

0 CL 0 T T T

p=0.01 p=0.01 150 200

50

0 100

0 50 100 150 200

0 0 Months

15

10

5

0 0 5 10 15 Months

Number at risk Number at risk

Follow-up (years) Group:CD47 High

Follow-up (years) Group:CD47 High 0

5

37 8 0

37 8 5 0 0 Group:CD47 Low

Group:CD47 Low

FIG. 24A 1

2

12

46

96

FIG. 24A 96 46 12 2 1 FIG. 24B

FIG. 24B

80 n

Normal Bladder S Normal Bladder

0

70 9 Bladder Cancer 70- A ■ Bladder Cancer O

60 Ovarian Cancer

x 60- □ Ovarian Cancer

V a> -0- Medulloblastoma

+ "D + T H Medulloblastoma

50 c 50- o o

40 40- A -¥■ o o

30 A x o o am so- -0-

X

+

20 CL 2°- + x ~Q- *■ 0

10 10- X JL ■> +

+ ©

AV AT T A

0 0 T T T T T T T T T

Anti-HLA Anti-CD47

lgG1 isotype lgG1 isotype Anti-HLA Anti-CD47

Antibody FIG. 25 Antibody

FIG. 25