^■' A TIGEKS.

CROSS REFERENCE TO RELATED APPLICATIONS.

This application claims the ^' saelit.oi U.S., Provisional Application Natn&er

62/242,685, filed October 16, 2015, under 0,S,C, §11 , the entire content of which iiicof porated by reference .

BACKGROUND OF DISCLOSURE

Despite decades of attanrpts, Cttrative imnroao logical therapy against cancer has beers ver difficult to achieve, with, the todamenial ads beiftg a dgen-reee on capacity; either by antibodies or through T cells, (via the T cell receptor) (Cousin-Prankel, Scie e (2013) 342; 1432). Anribo y )ased i M»80therapfes have been ed extensively against cmc in .instances where the target antigen is up-regulated in iaisor cells as compared to normal cells; (e.g., Her-2 in amplified breast cancer), or in eases where the tanw ceil express an. antigen that can be..recopk©d by the antibod or an antibody-toxin conjugat (<?.*., ^■ Rilti imab against. GD20) (Baselga et al., Anmk f¾ieo¾¾i? (2O01.) 12:S 5).. While clinical trials iis ng antibody-hased iirsmnnotherapies haw shown improved. patie survival i a limited number of e&ncer types (usually when combined with standard, chemotherapy}, these effects are often accompanied by significant safety and efficacy concerns (Cousin* Frankei Cancer,. Science (2013) 342:1432).

Effective I cell therapie against cancers have ^' been, even more difficult 10 achieve clirheaiiy (Sehfnltt: et al. Mm. G&w n r, (2009) 26(11); l2M Aa effective T cell therapy against cancer relies on a f cell with a hig affinity bindin directed against an antigen an a cancer cell. Chimeric antigen receptor T ceils (C AR, T ceils) are widel used to recognize antigens o ceils with both, high aCJnity and specificity and without the requirement for accessory recognition rnoleeoles, sach as HLA antigens to "present ^' ' peptides. The I cell receptor of a C AR. I cells is ^: ^swa ed" with an aniigen ) Wing heavy and light chains, iiwrsby obviating the need for HLA accessory molecules. The recomhiaant C R T recepto is fnsed to signaling domains leading to acdvaiion of the T ceil upon bindin of the CA T receptor to the target antigen.

The clinical use of CAR. T ceils has been limited to targeting a .narrow range of eel! su face antigens, farther snpporling the need for improved and novel; approaches in, the freairnent of cancer . In particnlar, new approaches are needed for diseases such as scale

vim with the CAR T cells.

SUMMARY OF DISCLOSURE

The present disclosure is based, at least in part, oa the discovery that agents om isin aw antigen-binding Iragj tent thai binds a lineage-specific eell-s &ce .antigen. (e.g., immune cells expression a chimeric receptor that..targets CD.33) selectively cause cell death of cells e p e iag. e Bm¾ge»spe $ifie eeli-sur&ce antigen, whereas cells that ate deficient for the antigen (e.g., genetically en ineered .hematopoietic cells) evade eel! death caused thereby. Based on such imdings, it would have been expected that immunotherapies involving the combination of an agent targeting a lineage-specific cell-smrfk¾ antigen, for example _* CAR-T cells targeting CD33, and hematopoietic cells that are deficient in the lineage-specific cell-sitrtace antigens (e.g., CDS 3) would provide an efficacious method of treatment for hematopoietic malignancies.

One aspect of the present disclosure provides methods for treating a hematopoietic malignancy, the meihod comprising administering to a subject in need thereof (i) an effect ve amount of an agent targeting a iineage-specific cell-surface antigen, wherein the agent comprises an antigen-binding fragment that hinds the lineage-specific cell-surface antigen; and (B) a populatio of hematopoietic cells that are deficient in the lineage-specific eel!.- surface antigen _; . In som embodiments, the agent can be an ^' iftimyae cel.! (e.g., a T cell) expressing a chimeric receptor thai comprises the antigen-bindi g fragme t that hinds tie lineage-specific cell-surface antigen. In. some embodiments, the imnmne cells, the hematopoietic cells, or boil are allogeneic or anto gons. fit some embodiments, the hematopoietic cells ar henmtopdiedc stem cells: (e.g. , CD34 CD3 ^' 3 ^* HSCs}.. I some embodiments, the hematopoietic stem cells can be obtained from bone marrow ceils or peripheral blood mononuclear ceils (PBMCs). In some einnodinis¾ts, the hei«aiopoietie cell is &k &pmmc

In some embodiments, the hematopoietic stem cells $m be obiawt8d ¾m hone marrow cells or peripheral blood mononuclear ceils (FBMCs).

Also provided hereto are methods of producin a cell thai is deficient in a lineage- specific ceU-snriace antigen as described herein. The methods comprises providing a cell, and introducing into the cell (i) a nucleic a id that comprises a nucleotide sequence of a C iSP -Cas system guide EN A (gRNA), which hybridizes to a portion of the nucleotide sequence thai encodes the lineage-specific cell-suriace antigen {e.g. , CD33, CD 1 % and (ii) a Cas endonuclease (e.g. , Cas9 or Cpf 1% The nucleic acid tha comprises a nucleotides sequence of a CRISPR as system gRNA and a Cas nuclease may, for example, he encoded on the same nucleic acid or on different nucleic acids, or introduce into the cell as a preformed rtboB«clet protein complex.. In some embodim nts^ the portion of the nucleotide sequence to whic the gRNA hybridizes consists of I - 22 nucleotides. In some examples, (lie gRN eomprises the nucleotide ^; sequence of SEQ ID NO: 1 1 or 28-31,

la some mbodim nts; the cell is a hem to oietic cell, such as a hematopoietic stem cell (£¾ CD3 ^' ) ,

Also within the scope of the present disclosure are kits comprising an immune eel! of claim B l 3 an agent that target a lineage-specific, eelkmriaee antigen, which comprises an antigen bindin fragment thai binds the lineage-specific cell-surface antigen; and iii) a population of hematopoietic cells (eg,., hematopoietic stem cells) that are deficient in the lineage-specific cell-surface antigen. In some embodiments* the agent that targets the lineage-specific, cell-surface antigen is an immune ceil, expressing a chimeric receptor, which comprises the antigen-binding fragment the lineage-specific celi-si &ce antigen.

Further, the present disclosure provides pharmaceutical compositions comprising any of the immune cells targeting a lineage-specific ce!l-sqrface antigen and/or any of the hematopoietic ceils that are deficient io the lineage-specific ceil-sitr&ce antigen for us in treating a hematopoietic malignancy; as well as uses of the immune cell s and hematopoietic cells for maiiit&etiuln : a medi ca ent for use to treating a hematopoietic m lignaney .

The. details .of one of more embo iments of the disclosure are set forth in the description below. Other features or advantages of the present, disclosure will be apparent from the detailed description of several embodiments and also from the appended claims. BRIEF DESCRIPTION OF XB£. DRAWINGS

f&s l¾ltewi¾ drawl ngs form part .of the present apecificaiiosi and are included to ^■ further demonstrate certa n aspects of the preset disclosure, which/can be better understood: by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein,

FIGURE 1 presents exemplary illustration, of type 0, type type % and type 3 lineage-specific antigens

.FIGURE 2 is a schematic showing an immune ceil expyessiag a chimeric receptor thai targets the type 0 iineage-specifk eell-sarface antigen, CD3 )7. Multipk rayeloma (MM) cells expressing CD307 as- well other cells expressing CP307, such as plasma cells,, are targeted by the immune ceils expressing the at.ti-'CD307cliii«eric receptor:

FIGURE 3 is a schematic showing an immune ceil express ig a chimeric receptor that targets the type 2 lineage-specific eel-sariace antigen, CD33. Acute myeloid leukemia (AMU) cells expressing CD33. Boman - ^' hematopoietic ^' . stem cells (HSC) are genetically engineered to be deficient in CD33 and- therefore- .not .recognized by the immune cells expressing the anti-CBSS c imeric receptor. 1¾e HSC a e able to give rise to myeloid ceils,

FIGURE 4 is a schematic showing genome editing using a CRISPR Cas system, A sgRNA hybridizes to a portion of an exoh of a lineage-specific celi-smlace antigen, and the Cas9 endonuclease cleaves upstream of the Pfotospacer Adjacent Motif PAM) Sequence (5 « NGG-3 ⁵ }, The sequences, from, top to bottom, correspond to S.EQ 1 ^' HQs; 45 and 46.

FIGURE 5 is a schematic showing a genome editing strategy using the CRJSPR Cas9 system -to disrupt CD33. A PX458 vector encoding a Cas9 protein and a guide RNA targeting CD33 was mjcleol cte into .-S62 cells, hitman leufceoiic ceil line, Flo cytometry was -performed on the cell population asmg.an anti-Ci>33 antibody prior to (top plot) and after (bottom plot) delivery of Cas9 and guide RNA to the ceils. The genome editing resulted in the deteti on of coding regio of the gene and a significant redaction, in CD33 ftom the cell surface.

FIGURE 6 is .a schematic showing a genome editing strategy using the G I FR GasS system to disrupt CD45RA. A PX458 vector encoding a Cas^ protein thd t guide RNA targeting C 45HA was .ft icleofeeieil into TIB-67.reticulum ceil sarcoma mouse macrophage- like cells. Flow cytometry was performed on the ceil population asitig an:anti:-CP45 A antibody prior to (to plot) and after (bottom plot) delivery of Cas9 and guide UNA to the cells,. The genome editing resulted in the deletion of a coding region, of the gene and a significant reduction in C.D45RA from the cell surface, FiiJlJME 7 slows schematics; example Mnw ie receptors comprisiig ¾>¾e ~ n¾i fi&gmentS: -iiat target- CD33 ·: A: a ge eric chimeric receptor targeting CD3 _: .

comprising an &nii-CD33 scfv, hinge domain, Iransi erabrane domain, co~stimalatery domain, and signaling domain. B; chimeric receptor targeting CD33 comprising an aoti- CD33 scFv, hinge domain, from CDS, transmembrane domain from CD _? and intracellular domains from CD28 and CD3 C; a chimeric receptor targeting€033 comprising an -anti- CD33 scFv, ^' hinge domain torn CDS, transmembrane domain ftora CDS, and intracellular domains from ICG ^' S (or CD27, - IBB, or QX-40) and CD3 . ft a chimeric receptor targeting C033 comprising an anti-CD33 scFv* h nge domain from C¾ transmembrane domain from. CDS, and intracellular domains from OX40, CD28, and C 3

FiGURE 8 is a schematic of an imtminotoxia.

FIGURE ^' 9 shows expression of anti ^« CD33 chimeric receptors expressed m K562 cells transduced with art empty vector or vector encoding an aati~CD33 chimeric receptor. A: Wester blot using a. primary antibody: that recognizes CD3 The table provides the estimated ^' molecular weight of each of the chimeric receptors tested. B Flow cytometric analysis showing an. increase in the population of cells that stain positive for the anti~CE)33 cMrnoric receptor,

IGURE 10 shows the astf CD33 chimeri receptors bind to GD33. A: Ponceaa stained protein gel. laaes ^' .1 ,3,5 :€033 molecule. Lanes 2,4,6; CD33 moi + APC

Conjiigate. B: Western h»iftg pr tty.-8Btibody that reco nises€Ρ3ζ. Lanes 1, , and 5 contain the chimeric receptors co- ncubated withCD33 molecules, and lanes 2, 4, a il 6 contain the chimeric receptors co ncubated with a CD33-APC conjugate. C: Flow cytometric- analysis showing an increase in the population of cells that express anii-CD33 chimeric receptors and bind 033.

FiGURE 11 shows cytotoxicity of K562 cells by NK92. cells expressing the indicated chimeric receptors. A; CART! and CART2 compared to e t^ El Vzs vector. B:,CART3: compared t empty HTVzsG vector,.

FIGURE 12 shows cytotoxicity (expressed as percent cytotoxicity on- the.y- xis) of K562 ceils ^: -deficient in€033 by NK¾ celis expressing the ^' indicated chimeric receptors. A: imsorfed population of 562 cells p-retreated with CD33~targeiirig C ISPR/Cas reagents, B: single clones of KS S2 cells deficient in C033, The columns, from left to right, correspond to empty HIV¾sG vector, CART 1 , CARD, and CA ^' RT3.

DETAILED DESCRIPTION OF DISCLOSURE

Oaaoer iffimunotherapies targeting autigens present on the cell, ssriaee of a cancer cell is particularly challenging when, the target antigen is also present on the cell surface- of normal, non-cancer cells that are .required or critically involved in the development .and/or survival of the subject. Targeting these antigens may lead to deleterious effects in the subject dm to cytot xic: effects of the inimiinotherapy toward s ch cells in addition, to the cancer eeils ₊

The ntetliods,.»ucieic ^: acids, and cells desc ibe herein allow or targeting of antigens (#,.g., type I. or type 2 antigens) thai are present not only on cancer cells but also cells critical for the development and/or survival of the subject The method involves; (I } reducing the nu ber of cells eariying the target lineage-specific cell-siiriace _: antigen, usin an agent ihat targets such an antigen; aad (2) replacement of the normal cells (e.g., non-cancer ceils) that present the antigen at d thus can be killed doe to administration of the agen with, hematopoietic ceils that are deficient for the ikeage-speeifc eell-surface antigen. The

wthods ^ gWT»e- ^iting ^' -metltods) for making such.

vectors, soate .DM . o any sequence, soate ^' RN o any sequence, aucec

and primers. One or more nudeoiides within a polynucleotide can further be modified.: The ssqaen.ee of imeleotides may e intem ted by non-uueleoi ide components, A !ynucieot ide may also be modified after polymerisation, such as by conjugation with a labeling agent.

The term ^w hyb.rid-¾atioa ^> ' refers to a reaction in which one or raor olynucleotides react to form a complex that is stabilized, vi hydrogen bonding ^' between the bases of the

present scosure, ncu ng, Jg , Ι , Ig , I , an g , uta e g sa types ncu e IgGl, IgG2„ IgG3 and Ig04. An aniihody light or heavy dmifi variable region, consists of a framework region interrupted by three liypervwiable regi.otvs., referred O: as complementarity determining regions (CDRs). The C ^' D s of me present antibodies or antigen-bindi ng portions can be from, a non-human or a human source. The .framework of the present antibodies or antigen-binding portions can be human., humanized, non-human (eg,, a murine

CAR can also comprise a chimeric fosion protei comprising an extraceUular antigen recognition domain, a transmembrane domain and an mtrac-eHular signaling domain comprising at least two functional signaling domains derived from one or more co- stimulatory moiecule(s) and a functional signaling domain derived from a stimulatory Molecule. The: antigen rseognM nucleic aciti sequenc

: ca« ton tain arty I ease; eci iC _j aRiigen-hindirt aMihody fragment. : The _: amibodv fragmeftl can comprise o e o more CPRs, the variable region (or portions thereof), the constant region (or portions thereof), or combinations of any of the ^' fore-going.

The term '"signaling domain" refers to the functional ^' portion of a protein which acts by transmitting information within the cell to regulate cellular acti vity via defined signaling pathways by generating second messengers or ranctmning as effectors b responding to such messengers.

The term ¾la ^5:> or alternatively ¾eta ehaiif , "CI -zeta" or ^' " B-zet-T is defined as the protein provided as GenBank accession numbers P _m 932t? } _> ΚΡ_000725, or

XP 01 1508447: or the equivalent residues from a uon. mman, species, e.g., moose, rodent, .monkey* ape arid die like, and a.* ^* a stimulator domain" or alternativel a ^w CD3««eta stimulatory do ai " or a "TCR-zeia stimulatory domain" is defined as the amino acid residues from, tie cytoplasmic domain of «¾ zeta chain that are sufficient to fiaicttonally transmit an initial signal necessary for T ceil activation

The term "geneticall .en ineered" or "genetically modified" refers to cells bein manipulated by genetic engineering, for exam le by genome editing. Thai is, the cells contain a heterologous sequence which, does sot naturally occur in -said cells. Typically, the heterologous sequence is introduced via a vector system or other means for introducing nucleic acid -molecules .into cells including liposomes. The heterologous nucleic acid molecule may be integrated into the genome of the cells or may be present extra- clmimosomally, e,$., in the form -of piasmids. The term also includes embodiments- of introducing genetically engineered, isolated CAR. polypeptides into the cell.

The term ' ologous" refers to any material derived from the same individual to whom it is later to ^' be re-introduced into the same individual

The tetni "allogeneic" refers to any inaterial .deri ed: from a _: different animal of the $ame ^: species a the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when, the genes at one or more loci are not identi cal.

The term "cell lineage" refers to cells with a common ancestry and developing fern the -same type of ideniif able cell into specific identifiahte/ftractiosing ceils. The cell lineages used herein include, but are not limited to, respiratory, prostatic, pancfeatie, mammary, renal, intestinal, neural, skeletal, vascular, hepatic, hematopoietic, muscle or cardiac cell lineages.

The term ¾ bition" when, used in reference to gene expression or function of a lineage specific an tigen refers to decrease in the level of gene expression or function of the lineage specific aitige !iere t¾e inlAiilon is a result of ialerfergoce w;iifi gene expression w ;¾rtctioii. he idiibitlft«-i - - ^' edmp.e6-; ₅ in which case there is no deteciaMe express on or ¾nctk% or st rnay be partial Partial inhibition can range from Pea complete inhibition to a near absence of tMiibi ion. By eliminating ¾niienlar target cells, CAR T ceils may effectively in ibit the overall expression of particular cell lineage.

Cells such as hematopoietic cells ..that are "deficient in. a lineage^spexific antigen" refers to cells having a substantially reduced expression level of the lineage-specific anti gen as compared with their tmtiifaliy-occarfiiig counterpart, e.g., endogenous henmtopoietie ceils of the same ty e, or cells that do not. express the Hneap-speeific antigen; ie,, not detectable by a routine assay such as FACS, In some instances* tie express level of a lineage-specific antigen of cells thai are "deficient in the antigen- ' cm be lower than about 40% (e.g., 30%, 20%, 15%, 10%, 5% or lower) of the expression level of the same I½eage ^» specific antigen of the »at«mlly-oecurring 'Counterpart. As used hete¾ _; the term "about" refers to a particular value.*/' 5%. For example, an expression level of about 40% may include any amount of expression bd eeR 3S%~45%,

Ageff tar ^

Aspects of the disclostrii ^rovide agents targetin 3 lineage-specific eeii-swia.ee antigen, for example on a target cancer cell, Siteh an agent may comprise an antigen-binding fr gniem th binds and targets th lineage-specific cell-surface antigen, in some instances, the antigen-binding fragment can be a single chain antibody (scFv) specifically binding to the lineage-specific antigen.

A , Limege-Specffic€(dl ~Swfa.ee Antigens

As used herein, the terms 'lineage-specific cell-surface antigen'" and "ceil-surface lineage-specific antigen ^*5 may be used interchangeably and refer to any antigen that is stv&ientiy present on the surface of a cell and is ^' associated with one or more populations of ceil lineagei $X : For example, the antigen may be present: on one or more populations of ceil iioeaga(s) and absent (or at reduced levels) on the celf surface of other cell populations, in general, lineage-specific cell-surface antigens can be classified based o a number of factors such as whether the antigen and/or the populations of cells that present the antigen are required for survival and/or development of the host orga.uis.ra. A stusmary of exemplary types of lineage-specific antigens is pro vide in Table 1 below . See also FIGURE i . ^■ aMei€&^^ Spsit e Anligeiis

and Borkitts Lyrapto na)

As shown in Table 1 and FIGURE 1, type $ lineage-specirle eei stirfaee antigens are necessar for the Tissue homeostasis, and survival, and cell types carrying type 0 hrieage- speciite ce|i-siu1¾ee rotli a hi be also ecessa y: for snryival of the siirjeet Tbti , given the /im ortance of ty e Oltneage-speeiie ceil-surince a i.t ge s 0 cells carrying type Ii0eage«speei c eeiVsnrface riiigens, m homeostasis and survival, targeting this category of antigens may be challenging using conventional CAR T ceil innnuwotberapies, as the inhibition or .removal of $ueh antigens and ceil: carryi»g snob antigens may be detrimental to fee survival of the subject. Consequently, lineage-Specific eei-sarfaee antigens (such as type 0 lineage-specific antigens) aiid or the cell types that cany such aniigeos may be required for the survival, for ^" exam l because it pedonm a vital m)«~reduudant &nt¾on in the subject,

or genera ted using standard techniques, "including' immunisation, of an animal- wi th the antigen of interest followed by conventional .monoclonal antibod methodologies e.g., the standard somatic cell hybridisation technique of Kobler and Milsteia, Netwe- (1975) 256: 495, as discussed above. The antibodie or nucleic acids encoding for he antibodies may be •sequenced using any standard DMA. or protein sequencing techniques.

In some em odinmts,, the cell-surface lineage-specific antigen that is targeted using fee methods and cells described ^' herein is a. cell-surface lineage-specific antigen of leakocyies & subjjo is soti of teakcic te. lii sot¾ etitodtaMis, ihe cdl-stirfsce lae ge-specific a¾Ige« is as Mtigeii that Is associated w fc Biyeloid; el!s. n softie embo ments, i¾e cell- surllce Htieage-specifsc aBiigen. is a cluster of difeeiiiiai en anlige»s (C s), Examples of ^■ CD .antigens include withoutliraitati ft, CD la, CD lb, C ic, CDld, CDle, CD2, CD3, CD34 Cm CD3g, CD4, CDS, CD6, CD7, CD8a,€D8b, CD¾ CDIO, CDll a, CD J lb, CD! !c _s CDI 1 d _; CDw 12, CDI 3, CDI 4, CD .15, CDI6, CD16b, CD 17, CDI 8, CDi. CD20 _> CD21 , CD22, CD23, CD24,€025,€026, 0027, CD28, CD29, CD30, CD31, CD32a, CD32b, CD32c, CD33, C034, CD3S, CD36, CD37, CD3S, W CB40, CD41, CD42a _? CD42b, CD 2c, C042<L CB43 CD44, CD45 _: CD45RA, CB451.B, CD45RC, CD45M), CD46, CD47, CWK C049a C»49b, CD49c, CD4¾€D49s, CD49f, CD50, CD$J _? CD$2, CD53, CD54, CD55, CDS 6, CD57, CD58, CD59, CD6¾ CD61, CD62E, CD62L, CD62P, CD63, CD6¾ CD65, CD65s, CD66a, CD66b _; CD66C, CD66F,€068, C069, CD70, CD 1, CD72, CD73, C.D?4 _: CD75, CD75S, CD77, CD79a, CD79b, CD VCD8 ^' ., CD82, CD83-, €084, CD85A, CD85C, CD85D CD85B CD85F, CD85G, CD8.5H,€0831 CD85J, CD85K, CD86, GD87, CD88, CD89, CD90, GD9 L GD92, CD93 , CD94, CD95, CD96, €097, CD98, CD99, CD99R, CDIOO, CDIOI, CD!.02, CD!i , CDi04,€Bi05, CDI06, CDl07a, CD1 7b, CDlOS. CDI09, CDI 10, CD 1 11, CDI 12, CD113, CDI 14, CDI 13, CDI 16, CDI 17, CDi Ϊ 8, CDI 19, CD120a, CD120b, CD12Ia, CDl21b, CD121a,€D12Ih CD 122, CDI23, CD 124, CDI25, CD 1 6, CD127, CD129, CD 1.30, CD131 , CD132, CDI 33, CD J 34 _» CDi ¾ CD ΪΜ, CDi37,:CDL38, CD139, CDL4¾, CDI 40k CD! 41, CD! 42, C J43, CDI4 CDwl45, CDI46, CD! 47, CD 148 _* CDS 50* CDI52, CD152, CDI53, €0)54, CD155, CDI 56a, CD 156b, CDI 56c, CD.I57, CDI58M, CBIS8¾2, CDJ58d, CD]58e]/e2, CD! 58i€015%, CDlSSh, CBI 581, CD1 S8j ₄ CD158k CDi:59a, CDi59c; _! CD 160, CDI 61, CDI 63, CDI 4, CDi 5, CD166 CDI67a, CDI68, CD169, CD170, .CDI 7I , CD372a, CDi72b, CD172g, CD173, CD1 4, CD175, CD 175s, CDI 76. CD.177, CD.178, CDI 79a ₅ ,CD179b ₅ GD]:80, CDi 81 ₅ CDI 82, CDI83, CDI 84, _: CD185, CDi 86, CD;] 91, CD192, CDI93, CD194, CDI95, CDi9 CD 197. CDwl98 _s GDwl99, CD20i, CD201, DSOS , CD203c, _: CD204, CD205, CD206, CD207, CD208, CD209, CD210a, CPwSlOb, CD212, CD 3ai , CD2i 3s2,€0215, CD21.7, CD218a, CDS b, GD22 , GD221, CD222, CD223, CD224, C0225, CD226, CD227, CD228, CD229, CD230, CD231, CD232 _:! CD233, CD234, CD235¾ CD23Sb, CD236, CD236 , CD238, CD239, CD24¾€ϋ241 ,€0242, CD243, CD244, CD245, CD246, CD247, CD248, GD249, CD252, C 253, CD254, CD256, CD257, CD258, CD261, CD262, CD263, CD264, CD265, CD266, CD267, CD268, CD269, CD270 _; CD271 _; CD272, CD273 _$ CD274, CD27S, CD276, CD277, CD278, CD279, CD280, €0281, CD282, CD2f S, CD284, CD2¾ C0 , CD289, CD29¾ CK¾ GDw293 _s CD294, CD295, C02 , CD297, CD29&, CD299, CDSO il, CD3Q0c _¥ C Mte, GB301, CD302, CD3 3, CD304, CD3 5, 306,€03078, CD307h _; CD307c, CD3 7e, CD309,

CD3 I2, CD3 CD3I5, CDS 16, CD317, CD31S, C0319, CD320, CD32L CD322, C0324, CD325, CD326, CB327, CD328, CD329, CD331, CD332, CD333, C0334, CD335, C033S, CD331 CD»S _; Om% CD340, CB344, C03 9, C035O, CD35 I, CD352, CD S3, CD354, C03SS, C0357, C0358, C0359, C036O, C0361, CD362 and C0363. See

w w.bdbioscienee^

In some embodiments, the eel-sisr&ce ii age-speciiie antigen is CD 19,€020, CD ^] l, CDI23, CD56, CD34, CD 1 C033, CD6i¾, C0 1, C061, C062, CD23$a, CD 146, CD326, LMP2, CD22, C052, C0HK CD3/TCR, CD79/BC.R, and CD26, !a some embodiments,, die cell»sur.ike. tineage^specific antigen is CD33 of GDI ,

Alte¾ tvety or .in addit on^ the cell-surface lineage-specific anti n may be a cancer antigen, fox example a cell-snriace ^' iiueage-specific antigen that is differentially present on cance celts. In some eiribodirne s, the easeer antigen is an antigen that is specific to a tissue or cell lineage . - Examples of eeU~$urface eage-specific antigen that are associated with a specific type of cancer include, Uhotrt limitation,€020,€022 (Han-Hodgkin's lym homa, B-cell lymphoma, ebronie lymphocytic leukemia (CLL», CD52 (B-cell CLL), C033 (Acute myelogenous leukemia (AML)), CD 10 (g l fKl) (Common (pre-B) acute lymphocytic leukemia and malignant melanoma), CD3/T~cell. tsceptor (ICR) (T-eell lymphoma and leukemia), C079/B-ceil receptor (BCR) (B-cell: lymphoma and leakeniia), CD26 (epithelial and lymphoid malignancies), human leukocyte antigen- (BLA)-DR, KL-A- DP and HLA-DQ (iynip oid malignancies), R.CAS1 (gynecological carcinonias, biliary adenocarcinomas and ductal adenocarcinomas of the pancreas) as well a s prostate specific membrane antigen, in some embodiments, the cell-surface antigen C-0 ^' 33 and is associated with AML cells.

& lnti$&i~8miing Fmgmmi

Any antibody or .an antigen-hln ing ftagment thereof c n be used for cpnsi acting tbs agent that, targets a !ineage-speeiiic cell-surface aBiigen as described herein. Such an antibody or antigen-binding fragment can be prepared by a conventional method, for ^• example, the hybridoma technology or recombinant- technology.

For exam le, antibodies speciiic to a lineage-specific antigen of interest can be made by the conventional hybridoma technology. The lineage-specific antigen, which may be coupled ίο a carder protein suc , ss KTH, can fee used to imMui e a lost animal for generating antibodi s binding to that complex. Tire route and schedule of Iramiirii^ai &K « the host animal are generall in keeping with established and eonyentkwl techniques for antibody stimulation and productioa, as further described herein, General techniques for production of mouse,, human feed, and Imam antibodies are known in the art- and are

described herein. It is contemplated that any .mammalian subject inchrding humans or antibody producing cells therefrom can be sti ulated to serve as the basis fo production of ^* maBitaalian, including human hybiidoma eel! Maes. Typically, the host animal is inoculated mtraperit neatly, rat muscuiariy, orally, subeirtanaousl , miraplantar^ and/or ii tod rraall with an amount of im unogeii, tnclediug as described .herein,

Kybridornas can be prepared from the lymphocytes and immortalized myeloma ceils using tie general somatic ceil hybridization t hn que of Kohler, , and Milsiein, C. (.1 75) Nature 256:495-497 or as modified by Buck, D, „ et aL M Vitro, 1.8:377-381 (1982), Available myeloma lines, including but not limited to X63-Ag8„653 and those from the Salk Institute, Gel! Distribution Center, San Biego,, Cali£, USA, may be used in the hybridization. Generally, die technique involves fksing myeloma cells and lymphoid cells using a fnSogen such as polyethylene glycol, or by electrical means well known, to those skilled in the art. Ate the fusion, the cells are separated from the fusion medium and grown in a selective growth medium, such as h>'poxa»thme- .inopterin-thyi»idi«e (MAT) medium, to eliminate tmhybrkfeed parent cells. Any of the media described herein, supplemented with or without serum, can be used, for culturing bybrkfoaias that secrete monoclonal antibodies. As another alternative to the cell fusion tedmiqus, EBV inimortafeed B cells may be used to produce the TCE-like monoclonal antibodies described herein, The hybridoraas are expanded and subclonsci if desired, and supernatasfs are assayed, for antHmmuaogea activity by conventional immunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, or lluorescence i tnauoassa },

1 lyhndoroas that may be used as source of antibodie enc m ass all derivatives , progeny ceils o f the parent bybridonias tliat produce monoclonal an tibodies capable of bindin to a lineage-specific antigen, Hybridomas that produce such antibodies may be grown m vitro or i n vi vo using known procedures. The monoclonal antibodies may be isolated from tie culture media or body fluids, by conventional immuaoglobulm purification procedures such as ammonium sulfate precipitation, gel elecuOphoresis, dialysis, chromaiography, and itrafilimiion, If desired, IJndesired. activity if present, cm be removed, for example, by ranning the preparation over adsorbents made of the i ramunogen attached to a solid phase and. ehtiing at off th i«iffiu¾oge¾.

Immunization of a host an maS ift : a target antigen or a fegnlent cont inin tbie target amino acid sequence conjugated to a protein that- is iromunogeaie in s¾e species to be »»mam¾ed _s e.g., keyhole iiiBpet he.taoeyaoi;n, serum albumin, bovine thyrog!obulin, or soybean trypsin .mhibitor using a bifactional or derivatkrag agent, for example maieintidobenxoyi suifosiiccinimide ester (conjugation hrough cysteine residues), N-hydroxysucrintmide (through lysine residues), gtutaraklehyde, succinic anhydride, SOCl or i -C^ , where R and Ri are different aikyl groups, can yield a population of antibodies (e.g... monoclonal antibodies}.

If desired, m antibody of interest (e.g. _t roduced, by a hybridoms) may be sequenced. and die polynucleotide sequence may then be cloned into a vector for expression or propagation. The sequence encoding the an tibody of interest ma be -maintained i vector in a host cell and. fee host cell can then be expanded and frozen for future use. In an alternative, the polya ucieotide sequence ma be used for .genetic manipulation to ¾man»ze" the antibody or to impro ve the affinity {affinity f aiurai.ioa} _} or other characteristics of the _: antibody. For example, the constant region may engineered to more resemble hu an constan regions to avoid immune response if the antibody is used in clinical trials an treatments in hurnans. it may be desirable to genetically manipulate the antibody sequence to obtain greater -affinity to the lineage-specific, antigen, ft will be apparent to one of skill in. the art that one o more polynucleotide changes can be made to the antibody and still maintain its binding specificity to the target antigen.

^' In other embodiments, fully human antibodies cau.be obtained by using, commercially available mice that have been engi eered to express specific human immunoglobnliu proteins. Transgenic auiroals thai .are designed to produce a more desirable (e.g> _t felly bo.roa antibodies) or more robust immune response may also be used for generation of humanized or human antibodies. Examples of seek iechuology are eBOmouseRXM fi¾n¾ Amgeu, inc. (Fremont Calif and f- feM.Ah-Mo«sel¾-TM and TC MouselM from. Medarex, lac,

(Princeton, f In another alternative, antibodies may -be made reijombmant!y by phage display or yeast technology. See;, ibr example, U.S. Fat os.. 5,365,332; 5,580,717;

S J33. _S ?43; and 6,265,1.50; and Winter et al. _:! (1994) Anna. lev. Immunol, 12;4B-45S, and . Alternatively, the phage display teelmology ( cCafferty elaf , ( 1990) Nature 34 ;552-553} can be used to produce human antibodies and antibody fragments in vitro, from

imaiunoglobnittii variable (V) domain gene repertoires &¾« aniuuHunized donors.. Ajftigeii' iidiag ftaga¾nts of m l»ti¾cf $ttibo4y aiiibody) can be ?epa*ed Y routine n hods. For example, P{a ')2 fragmeniS άίίί be roduced: by pepsin digestion of so antibody uMieeule, and Fab fragments thai cm be generated by reducing the disulfide bridges of F(ah ^f ) fragments.

Genetically engineered antibodies, such as human zed antibodies, chimeric antibodies, .single-chain antibodies, _, and bi-specific antibodies _* c be produced vis, e.g.., cweatiosal recontniiant technology. In one example, DNA -encoding a.monoclonal antibodies specific to a target aatigen can. be readily isolated and sequenced using conventional procedures ( .g. _^ by using oligonucleotide probes that are capable of binding speci fically to genes encoding the heav and. light chains of the monoclonal antibodies). The hybridoma ceils serve as a preferred source of such DNA, Qnee isolated, the DNA may be placed into one or .owe expression vectors, which are then transfeeled mo host cells such as.E, coll celts, simian COS cells, Chinese hamster ovar (CHO) cells, _: or myeloma ceils that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in die WjntbihStit hosfe ^' elts. See, &g., PCT Publicatio No,.■ WO; 87/04462. The DNA can. then be Modified, for example, by substituting the coding sequence for h man heavy and light chain consta t d mai s in place of the homologous murine sequences, Morrison et al., (1984) Pr , Nat. mi Set. $ J MS 1 , or by covaleotly joining to the inuuunogiobuiin coding sequence all or part of the coding sequence for a uon- inimunogiobuire polypeptide. In that manner, genetically engineered antibodies, such as "chimeric' ^* or "hybrid" antibodies; can be prepared that: have the binding specificity of a target antigen,

Techniques developed for the production of "chimeric antibodies' ^* are well known is the art See, e.g., Morrison et al (1984) Pmc, Hail Am4- * tSA -81, 6851; Neuberger et al, (1984) Nature 312, 604; and Takeda et-al. (1984) Nature 314:452,

Methods for constructing humanized antibodies are also well known in th art. See, e.g., Queen et al,, Proc, NatlAcml ScL USA, &6;10 29-1ί ί>33 (1989), la one ex&Mple, variable regions _: of i VH- and YL of a. parent non-human, antibody are subjected to three- dimessional fflo!eewiar modeling anal sis followin methods known in the art Next, framework amino acid residues predicted to be important for the for maiion. of the correct COR snueiures are identified using the same molecular modeling analysis. & parallel, human VH and VL chains having amino acid sequences that are homologous to those of the parent non-human antibody are identi fied from any antibody gene database using the parent V f md VL s queiices as search -peries. Hurnan VH and ¥L acceptor ^: genes ate i¾e» selected.

^■ Fhe CDR regions withiB ¾e _: sslected ¾«r»an aecej&or genes cm be,!^laced w¾ i¾e _: CDR regwas from the parent non-human antibody or functional vari nts thereof. When necessary, residues wiihia the framework regions of the parent chai» that are predicted to be important in interacting with the CDR. regions (see above description} can be used to substitute for the correspomdlag residues in the human- acceptor genes.

A single-chain antibody caw be prepared via recombinant technology by linking a nucleo ide sequence coding fo a heavy chain variable region and a nucleotide sequence coding for a light chain variable region. Preferably, a flexible linker is incorporated between, fee two variable regions. Alternatively, techniques described for the production of single chain antibodies (U.S. Patent Nos. 4,946,778 and 4,704,692) can be adapted to produce a phage or yeast scFv I ibrary and scf v clones specific to a lineage-specific antigen, can be identified from tSie library following routine procedures. Positive closes can -be. subjected to further screening to MeRtlfy those that hiu Ineage-specific antigen,

la some ¾sta«ees _:! laeage-specitle- antigen of i tetest: is- CD33 an d the ^■ antigen- binding fragment specifically binds D33, for example, huma CD33. Amino acid and nucleic- acid sequences of an exemplary heavy chain variable region and light chain variable region of an anii -h ma CD33 antibody are provided below. The CD sequences are s own in boldface and underlined in the amino acid sequences.

Amino acid sequence of anti-CD33 Light Chain Variable Region (SEQ ID NO 13)

EIVL QSBGSL¾ySPG£R^

SVp F S SG p-n¾TISS Q EDXAi :« ⁽ ^ ttcli e acid seimenee o¾^ ID MO: I)

GAS¾KGTGC?GAGC AG^

G& GX;GCA G&GCAGGC^

GSG.G¾GCCGG¾eA S¾

GCGAGGGG&GG GCTGGAGA CAAGAGG

The anii-CD33 antibody binding fragment for use in constructing the agent that targets CD33 as described hereto may comprise the same heavy chain and/or light chain CDR regions as those in SEQ ID NO:12 and SEQ ID NO: 13, Suc antibodies may comprise amino acid residue variations m one or more of the farae ork regions. In some instances, fee a»ti-CD33 antibody fragment may comprise a heavy chain-variable region that shares at least ?ø% sequence identity (e.g., 75%, 80%, 85%, 90%, 95%, or higher) with SEQ ID NO; 12 aiid/or may comprise a light chain variable region that shares at least 70% sequence identity (e,.g., 75%, 80%, 85%, 9 %, 95%, _. or higher) wife SBQ ID NO; ! 3,

The "percent identity'' of two mi o acid seque ces is determined using fee algorithm Of arlin and Altscboj Proe/Hati, Acad. Sci. USA 87;226 ~68, 1990, modified: as hi Karta and. Akscbn! Proc. Natl Acad. Sci, USA 90;58?3-77, 1993. Such an algorithm ¼ incorporated into fee NBLAS ^' i and XBLAST programs (version 2,0) of Altsc ^' hui, et al. J. Mai. Biol. 215;403-1 , 1 90. BLAST protein searches can be performed with the XBLAST program _* se fe^SO, wordiengifrf to obtain amino acid sequences homologous to the protein molecules of the present disclosure. Where gaps exist between two sequences. Gapped BLAST can be utilized as described in Altschii! et al., Nud Acids Res. 25(17):3389-3402, .1997. When tilis n BLAST and. Gapped BLAST progmms, fee default parameters of fee respective ograms (e.g,, XBLAST and BLAST) can be used. ϋ, Immune C k ΕψΜ^^ Chimeric eceptors

to som mbodiments, the agen that: targets a liaea -specifi e ce!l-snrince antigen as described herein is ^; an immune ceil th t expresses a chimeric receptor, which comprises an aniige?}-bffid¾g fisgmeni ( g, a si |ie^¼t».a»tiboil ):capay^-0lbmdJi¾ to fee iraea - speeife antigen (e.g.., CD33 or CD19), ^· Recognit o of a target cell (e.g , a cancer ceil) having fee lineage-specific antigen on its cell surface by the antigen-binding fragment of the chimeric receptor transduces an activation signal to the signaling doraain(s) (e.g., to- siitftuiatory .signaling domain and/or the cytoplasmic signali ng domain) of fee chimeric receptor, whic . may ^* activate an effector iioctlos In fe mtmsm cell pressing he hhx receptor,.

As use herein, a chimeric receptor e^ that can be expressed on the surface of a host ceil an comprises an anageii-bWirig Fragment th t binds to a eeli-surface !ineage-spedfse asltgea. in general, chimeric receptors comprise at least two domains that are derived from different Molecules, in addition to the antigen- bmding fragment described herein, th c raeric receptor may iitrther comprise one or more of a binge domain, a transmembrane domain, at least one co-stimulatory domain, and cytoplasmic signaling domain, in some embodiments, th chimeric receptor comprises from N tOTiim!S to C terraitms, an. an:tige»-¾iading fragment that binds to a. cell~sm1¾ee lineage- speciilc antigen, a hinge domain, a transmembrane domain, and a. cytoplasmic signaling domain. In some-embodiments, the chimeri receptor farthe comprises at least one eo- Stimulatory domain,

In some embodiments, the chimeric recep tors described herein com ise a bin ge domain, wh ch may fee located between the antigeb-Bindhig fagmeiit and a _:

transmembrane domain, A binge domain is an amino acid, segment that is generally found- etvveen two- domains of a protein and may allow for flexibility ^' of the protein and movement of one or ^' both of the domains relative to one another. Any amino acid sequence that provides such flexibility and movement of the antigen-bmding fragment ieiai¾ t ^' 8» thef domain, of the chimeric receptor can he used,.

The hinge domain ma contain abont 10-200 amino acids,, e-g., 15-1,50 amino acids, 20-100 amino acids, or 30-69 amino acids. In some embodiments, the hinge domain may be of about 1 ti _, 11, 12, 1.3, 14 _S 15, 16, 17, 18, 1 % 20, 21, 22, 23, 2 25, 26, 27, 28, 2% 30, 35, 40, 5, 50, 5.5, 60, 65, 0, 75; 80, 85, 0, 5, 100, 110, 120, 130, _, 140, 150, 60, 170, 110, 190, or 200 ammo acids in length.

¾--S nae-e^l ii^^,-t iSi n e domain is ί¾·h βg|-.ciQi]ft¾ή ofs·ti u^¾tiy· occurring protein, Hinge domains of any protein known in the art to com pris e a b inge domain, are compatible i¾r use in, he chimeric receptors described herein. In some embodiment, the hinge domain is at least a portion, of a hinge domain of a -naturally oecurring Olein and confers flexibility to the chimeric receptor. In some embodiments, fee hinge domain is of CO or CP2S a. I some enibodimeffis, the hinge -do ain is a portion of the hinge domain of CD8o;, e.g., a iragment containing at least 15 (eg,, 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8a or CD28 o. Hinge domains of antibodies _y s :k as m ¾G _? IgA, IgM, igl¾ IgD atnisody. ate also compatible for m in the chimeric reeepters described he.t¾¾. in seme wdimeois, the inge domain is the hinge domain thai joins the constant, domains. CU I an CH2 of an antibody, in some enrbodinients, the Mage doatam is of an antibody and comprises th hinge domain of the antibody and one or more constant -regions of the antibody, in some embodiments, the hinge domain comprises the hinge . domain of an antibody and the CH3 constant region of the antibody, la some embodiments, fe hinge domain comprises the hinge domain of an antibody and the C.H2 and CH3 constant regions of the antibody, in some embodiments, the antibody is an IgG, IgA, IgM, tgfi or IgD antibody. In some embod ments, the antibod is as IgG antibody. In. some embodiments* he antibody is m Ig L iaG2, ¾G3, or IgG4 antibody., in some embodiments, the hinge re ion comprises the hinge region and the CH2 and C.H3 constant regions of an IgCJl aniibody . la some embodiments, the hinge region comprises the hinge region, and. the CH3 constant region of a lgCJl antibody.

Also wiihtii the scope of the present disclosure are ehinwie receptors eomprssieg a hinge domain that is a n.on.-naiaraily ^' QCCKfrmg.p¾piide. In sor»embodiffi£ s,, the hinge ^' domain between the C-temimiis of the extracellular iigartd-biridirsg domain o f i c receptor and the N e.anio.us of the {ransmembrane domain is a peptide linker, such as a (G!:y¾Ser}n linker, wherein x and a, independently can be an integer- between 3 and 12. inebiding 3, 4, 5, 6, ?., 8, % 0, 1.1, 12, or more.

Additional peptide linkers that may be used in a hinge domain of the chimeric receptors described herein are known in the art. See, eg,, Wriggers et al. Current Trends in Peptide Science (2005) 80(6}: 736-746 and PCX Publication WO 2012/088461.

in- some embodiments, the chimeric receptors described herein may comprise a .transmembrane domain. The iransmembraae domain for use in the chimeric receptors can he in any form o n hi the art. As n eci herein, a a sme m !e domain" refers to any protein straciyxe that is thermodynaffiicaliy stable n a ceil membrane, preferably a eaksryotte ceil membrane. Transmern rarie domairis compatible for wse in ihe chimeric receptors used herein m y be pbiaiaed from, a taaiial!y occarrin protein. Alternatively,, me iransaiembrane domain, may be a y mhetie. noa¾tarally occurring protein segment .g . ₅ a hydrophobic protein segment that is thermodynamically stable in a cell membrane.

Transmembrane domains, are classified based on the transmembrane domain topology, incl uding the numbe of passes thai the transmembrane domain makes across the membrane and the orientati on of the protein.: For example, single-pass membrane proteins cress: the cell iMtoi onse, and malti-pass ros a i e preteif!S cross the eel membrane at least twice 2, % 4, _S <¾ 7 or more itnes). In some embodiments, tie

transmembrane domak-is a single^pass .tessmem iW doraaifiu. In. som embedments ^' , the transmembrane domain is a single-pass transmembrane domain that orients the N terminus o f t e chimeric receptor to the extracellular side of the cell and the C terminus of the chimeric receptor to the intracellular side of the cell. In some embodiments, the transmembrane domain is obtained from- a single pass transmembrane protein, in some embodiments, tfee transmembrane domain is ofCD8u. In some embodiments, lite

transmembrane domain, is of C028. m some embo i ents, the transmembrane domain is ofiCOS.

In some embedments, the chime ic receptors described herein comprise one or more costimit!atory signaling domains. The term "eo^stimulatoty signaling domain.," as tjsed herein, refers to at least portion of a protein that mediates signal transduction wi thin a cell t induce an immune esponse;, such as aa effector function. The co-stimulatory signaling domain of the chimeric receptor described herein can be cytoplasmic signaling domain from & eo-stimatatory protein.,, hich trans uces a signal and modidates responses mediated by immune cells, such as T cells, NK cells, macrophages, neutrophils, or eosinophils.

In some embodiments, the chimeric receptor comprises more than one (at least 2 _> 5. 4. or more) co-siimnlatory signaling domains. I some embodiments, the chimeric receptor comprises more than one eo-sttmiilatory signaling domains obtained fro

different costimulatory proteins, i some embodiments, the chimeric receptor does not. comprise a co-stimntatory signaling domain.

In. general, many immune cells require, co-stimulation, in ..addition to stiimwlation of an aMigen-specafic signal to promote cell proliferation, differentiation and suraval, and to: acti vate effector func tions of the cell. Activation of ¾ C0-:S imalato0 stgriahng domain in a host eetl (e.g., an immune cell) .-may induce the ceil to increase or decrease the Reduction and secretion of: cytokines, phagocytic properties, proliferation, differentiation, survival: and/or cytotoxicity. The eo-stimt atory signaling domain of any co-stimnlatofy protein, may be compatible for use- in. th chimeric receptors described herein,. The iype s) of eo~ stimulatory signaling domainis selected based on factors such the type of the immune cells in winch the chimeric receptors would be expressed (e.g., primary T cells, T cell Sines, NK ceil lines) and the desired immune effector Ikociion (e.g., cytotoxicity),.

Examples of co-siimulator signaling domains for use in the chimeric receptors can be the c to lasmic si aling domain of co-soffiiikior ^• pto^i^ ^' tetidiiig _: * without limitation; CD27 _; CD2L ^' .4~l BB _; m, CD3¾ Cd40, MH _. ¾ lyniphocyte fi&¾¾n~assQC5 ed. antigen-l (JLFA % CD2, CD?, LIGHT, HKG2C, B7-H3. In some embodiments, the co~ stimulatory domain is derived from 4-1BB, CD28, or ICOS. In some embodiments, the costimulatory domain is deri ved from. CB28- and chimeric receptor comprises a second co* ^' stimulatory domain from.4-1 BB or ICOS.

in some embod ments, the eosiiroulaiory domain Is a its-ion domain eoorprising more than one cosiiraulalory domain or portions of more than one eostifimktory domains, in some embotliments, the costirouiatorj' domain is a fusion of cosiinmlaiory domains tem CD28 and ICOS.

in some embodiments, ihe chimeric receptors described herein comprise a

c to lasmic sigriaiing domain. Any cytoplasmic signaling domain can be used i the chimeric receptors described herein in general a cytoplasmic signaling domain relays a signal, such as interaction of an extracellular ligand-bindiiig domain with its iigand, to stimulate a cel ular response, snch as Indticmg an _: effector funeiion of die Cell (e;g. ,

cytotoxicity},

As wil be evident to one o f ordinary ski ll in the % a factor invol ed cell, activation is the hosphor lat on of imn¾usoreceptor tyrosine-based activation motif (ITAM) of a cytoplasmic signaling domain. Any ITAM-containing domain known m the art may be used to construct ihe chimeric receptors described herein. In general, an ITAM motif ma comprise two repeats of the amino acid, sequence Υχ, ^χ Ι, ϊ separated by 6-8 amino acids, wherein each x is independently any amino acid, producing the conserved motif YxxlJx(6~8)YxxL I, In some embodiments, the ^■ cytoplasmic signaling domain is om >3 .

Exemplary chimeric receptors are provided, in Tables 2 and 5 below.

Tahle 2; Exemplary components of a chimeric receptor

.Chimeric receptor ex on nt 1 Amino acid ser enee

Aiitigea-binding ^' fragment 1 Ligh chain- GSTSSGSG PGSGEGSTKG

1 (SEQ-JD NO: 14VHeavy chain

CD28 eostitm atory domain- 1 lEVMYPPPYLDNEKSNGTiieVKGKHLCPSP

1 LFPG.PSKP.FWVLVVVGGVXACYSLLVTVA

1 FI1FW VR SKRS&LL SD YMNMIT PG TR

The nucleic acid sequence of exemplary compoaenls for construction of a c feimseric receptor are provided Mow.

CD28.iarace¾te si nalng donmin>D A-H¾im» (SEQ ID NO: 3)

ATTGAAGTTA.TGTATCCTCCTCCTTACCTAGAGAATG¾GAAGAGCAATGGAACCATTA TGCA GTGftAAGGGAAACACC^TTG CGAAG CCCCTAT TCCCGGACC TCTAAGCCCTTTTGGG GCfGGTGG GGT GG GGAGTGC GGC TG

A T TCTGGGTGAGGAGTAAGAGGAGGAGGCfCCTGCACAGTGACTACA GAACATGACTCC CCGCCGCGCGGGGCCCAGCCGCAAGCA ^' ACGAGCCC ATGCGGeACCACGGGACT CGCAG

AAG G C'iA'

ACGTGGCGGGGACCCTGAGAIGGGGGG AGAA?GAAC?GCAGAAAGAT. GA?GGCGG¾^

CGCCGGAGGGG ^' eAAGGGGCA

C ACGACGGCCTTCACATGCAGGCCCTGCCGCC CGC

ICOS iBtraeeiyaF signaling domaffi-D AAaian (SEQ IB NO: 4)

GTATCAATTT TGATCCiCCTC

GAATCACAAGTTTGTTGCGAGCTGAAGTTCTGGTTACCCATAGGATGTGGAGCCTT GT G

tAGTCTG ¾'TTTGGGATGCA¾

GTGCACGAGCC AAGGGTGAA ACA G CATGAGAGCAG G CAGAGCC AGA^TC AG ACTCACAGATGTGACCCTAAGAGTGAAG TCAGCAGGAGCGCAGACGCGCGCGGGTACCAGC AGGGCTGI&CAA¾GAACiGCAG AG¾

AAGGCGAGGGGCGGAGGGGCA¾GGGGCACGA GGCC :T&CCAGGG C C¾G¾ACAGCC¾CC GGAClCCTAGG :GCCCTfCAC¾ ^: GC¾GGCCCTGGCCCCTCGC

CD2MC S COSTIMUtATORY SIGNALING REGiON-ΟΝΑΉιΐΒΐ (SEQ IP NO: - 5) :T ¾¾GiiAT M¾CT

T T ^ G G ACAG TTT^

XGGTGGXGGTGGTTGGTGGAGTCCTGGGTTGC AXAGCX GCTAGGAACAGTGGCCTXTATT ATiiTCTGGGTGAGGAGTAAGA GAGGAGGCTCCXGCACAGTG

AG GAACACAGCCAAAAAATGTAGACTGACAGA GXGACGGTAAGAGTGAAGTTCAGGAGG GCGCAGACGCCCCCGCGXACCAGCAGGGCCAGAACCAGCTCTATMCGAGC ^' XCAAXCXAGGA

GCCGAGAAGGAAGAACGCTCAGGA

AGGCG ACAGTGAGATTGGGAXGAAAGGCGAGCGGCGGAGGGGC GGGGGAGGAXGGCCTT ACCAGGG C CAGTACAGCGACGAAGGACACCTACGAGGCCCTTCACATGCAGGCCCTGCG CCCTGGC

!a some en*h dim &is fe an antigen bkdmg imgiient ibai teds to C 33 and. comp ises a heavy ehaia variable region which has ike: same CDRs as the CJDRs in SEQ I ^' D NO; 12 and a light chain variable region, which has the same CDRs as the CDRs- in SEQ ID NO: 13. la some embodiment the antigea- biudmg fragment comprises a heavy chain variable ^■ region as provided by SEQ ID NO: 12 and a lig t chain variable region, as provided by SEQ ID NO: 13, in some embodiow ts, the chimeric receptor farther comprises at least a transmembrane domain and a

cytoplasmic signaling domain. in some embodiments, the chimeric receptor farther comprises a hinge domain and/or co~stirodatory sipaling domain.

Table 3 provides .exemplary dilraeric receptors described herei». The exemplary constructs have from N-iernimiis to C-teoninns, the s«rigen«-bm4iiig fragment, the transinembraae domain, and a cy toplasmic signaling domain, in some examples, the chimeric receptor .further comprises a hinge domain, located between the antigen-binding fragment and the transmembrane domain. In some example, the chimeric receptor iurt er comprises one o more co-stinmteiof domaias., which may be located between the inni membrane do ain nd the c oplasmic signalin domain. Table ^Emi iar ciiiffiferic receptors

Amino acid seqtiences of t e example chimeric rece tor^ listed, is. Table 3 above are provided below;

C ART ! mim acid se uenc (SEQ ID NO; 20) SSSGKP S ESS ¾

' GHDDI::SYi¾I §^

CART2 wim ae sequeace (SEQ ID NO:2I)

MiiLQS LI J2LS CS J.SE I LTQSFGSL.AVS FGE RVf S CKS S QSVF S S _: SQKiY¾¾ YQQ I FGQS

PRFI J Y»¾ S RESGVFDRFXGS OS TP fm SSVQ PB:DMIYYCHQYLSBR GQGT¾I, IKBSS

^SGSGKPGS EGSiK Q QLQyP^

∑YPGMDDISYMQKFQ Km<mD^^

SAL3:MSIMVFSHF¥FVFLPAKFTTTPAPRPPTP^

CARTS amioo acid sequence (SEQ ID NO: 22)

M¾QSLLLEG C

?RLLJ ASTRE-S VI X¾F¾

fSGS<mPGS £GS RGQ G:¾^^

l G DDIS MQKFQ K LTADKSSf^

SSALSMSIMYFSHFVPVFAA^

FVLvVvGGVLCYSLL^TVAFII ?ryRSKRS]RLLaSDmOTPBBe FTi^YQJ?yAPPR T Y ^' S RGFC LLYIFKQPF¾RP QT QESD CSCRF?EEEI!GGCELRV F3RMDAPAYQQGQf?¾XjYii

ELNLGRPEEYDVLDKERGRDPE GGKPREEHPQ^

1,YQGAS¾.T ^: DTY0ALEMQ¾LPPH

CARTS m > acid sequence (SEQ ID NO.23}

1¾;SLRX _; LGTV&^

FELEI TRB^

SGSGKPGS EGS KGQ^QI<Q¾FGAB^PGASVKHSCK¾3GY^FTSY I9¾RQ PGQGLEW^G I PGKDDXSYMQKFQG ATLTA- SS'TTAYHQLSSIATSEDSAtTfYGARFVRLRYP ^' GQGTT T ^' v SimLSHSIMYFSHHVPVFLPAKP^

LPIGC FV XLGGILICILT KKYSSSVHD KGEYMFMRAVMf KKSRLTDVTLTKRGREKLL

YIFKO F yO OEEDGCS^

PVEF¾ R;GRDPE G(¾R

DTYS&LH OALPRR

CARTSdual amlwo acid se asttce (SEQ 3D NO: 25)

MK:LQSLLLLG ACSISE ^' XJLT&S GS VSPSSR MSCKS 3¾ΚΜ¾Ϊ Υ$0;Ipi¾S ^:

PREt'l 5¾¾3J'RESG PEPE SGSGTREtLTISSvQPE:DXiΑΪYYC ^' E YLSS fEQQGfί¾¾ ^' ΕI&RG8 _: SGSGRP SSE Sl'KGQV&LQQFG^

IYPG DiSYSiQKFG;GKATETAB: SSTT¾YMQ;LSSLTSEBSAYYYGARVRLRYFD GQG rV V

SS¾L;SNS,IMYFSHFVPVFL:PAKP TPAPR

F E VGyLACYSLLVfmFIIF V SK

RS

CA T6 affliiio add sequence (SEQ ID NO: 26)

HKLQSL «LGTVAC3ISIQ TQT SSLSASI^^^ISCSASQDIS yi^KQQKPI ⁾ GT KX»LISa

TSRIHSGVPSRFSGSGSSTDYSLT S^^

SGBGSfKGI4ESGFGLVAFSGS^

ALKGRLTIIKDNSKSQ^^

CART? amino acid sequence (SEQ ID NO: 27} LL I Y¾AS TKESG^PDEf 'ί&¾ GSfiaf FTL X SS VQI¾ DLAIYY CBQ Y AS SBTf G¾Gf Κί,Ε ί REGS :S 5¾KPG« SG3 K 0 ¾Lq¾

I Y pGMDDX S Y QKFQGKA IiTAyKS S mYWQ ^' LS SLf SETJS AVYYCARS VKLRY? ^' D?S ¾GTTV?V

SSIS M pppYLDME ^

VRS RSRL HSDY HM^

!1<MLGRREEYDVLDKRBGRDPEMGGKPREKEP

QGLSTA ^' DTYDALHMQALPP

Hacleic: acid seq«ettce$ of the example chimeric receptors Ikied Table 3 above are provided below:

GGT¾f GTGAGGSGGGG ^' G G¾¾C GGCGACC

:GGCC ^: &&G TC¾ ^

GC GTGAC TGA C^GC AGA CA^

GGCCTGGIATCAGCAGATCGGCGGCCAGAGCCCCAG&CTGCTGATGiACTGGGCCA GCACCAGAGA AAGCGGCGTGCCGGATAGASTCAGGGGCAGCGGCrCTGGCAGGGAC CACCCTGACAATCAGGAG

GGGCACGAAGC GGAAATCAAGGGSGGCAGGACA&GCGGGAGCGGAAAGCCTGG¾ C^GGGG¾GGG CTCTACC¾AGGQCCAQG GCAGC GC^

GAAG&TGTCCXGCAAGGCCAGCGGCTAGACC TCAGGAGCTACTACATGCAGTGGATGAAGCAGAe CGGTGGAGAGGGCGTGGAATGGG GGGAG GATGTACGCCGGCAACGACGACA CAGCl'AGAaCC GA¾G TGCAGGGCAAGGCGACCCfSAGGGCGGaC¾AG CTAGCAGCACGGCCTAGATGCAGGTG G

CAGGCTGACCAiGGAGGACAGCGCCG GTACTAC!G

XGtGTGGGGCCAGGGAACCACCG GACCG GTC AGCGCCCTGAGCAACAGCA CA^GTAC TCAG CGACTfCGTSCCCGiGTXfCTGCCCGCGAA

AGCCGCTACAA CGCGAGCCAGCCXC ¾ CTCTG¾G¾GGCGAGGC G

AGCCGTGGACACGAGAGGCCTGGATATCTACATCrGGGGGCCAGTGGGCGGGACCTGTGG GGTGCT GC:TGCi TCTC CGTGATCACCA&GAGAGGC

CATGCGGCCCG GCAGACCAGCCAGGAAGAGGACGGCTGTAGCTGCCGGTTGCCCGAGGAAGAAG AGGGGGGTGCGAG TGAGAGTGAAGT GAGGAGAAGCGCGGACGGCGGTGGCTATCAGCAGGGCCA

AGGCftGGGACCG^GAGA GGGCGGCAAGCCGAGACGGAAGA CCTCAGGAAGGCCTGTA AACiGA ¾C? C¾ AAA^

C&&GGGC C CG&TGGAC T T C C AGGGC CTG AGC¾CCGC CACC AGGAC ACCfATG&C GCCCTGCft

CAR.T2 t im aci . ^■ sequenc (SEQ _. ID NO: 39)

GG G¾CGTG¾GGGSCCGefGSAC

GGtCIGT CAiC&GCG^

GCGCGTGACCATGAGCTGCAAGAGCAGGCAGAGCG GTTCTTCAGCAGCTCCCAGAAGAACTACCT GGCCTGGTATCAGC&GATCCCCGGCCAGAGCCCCAGAC GCTGATC ACTGGGCCAGCACCAGAGA AftGCGGCGXGCCCGATAGATTCACCGGCAGCGGCrC GGCACGGAC TCAGCG GACAA eAGCAG GAAGATGTCC?GGAAGGCCAGCSGG?ACACCfTCACC^^

CCCTGGACAGGGGC?GGAASGGGSGGGAGTGA ^' TCI¾C€CC

GA^GTTCCAGSGCAAGGCC^CCC GACCGCCGAG.AAGTCT^GCACCACCGCCTACATGCAGCTGTC GaGGGTG C¾GGG¾GGAC¾GCGCCGTGTAG ¾GrGCGCGAGAGA¾GTGGGSC GGGS AGT CG¾

Cf?0SiGCCGGTGf?I¾^^

CG GCAG CC S C^

TmCAGCCtGCTCGTGACtGTGGCCTTCA CATCrT TGGGTGCGCAGCAAGCGG C AGAGTGCT GCACAGCGACTACATGAACATGACCCCCAGAAGGCCAGGCCCCACGCGGAAGCAC ATCAGCCT a CGGGCCSGCCAGAGACT CGCCGGGmCGGGTGCAGAGTGA^GTTCAGCAGAACiGGCC ACfSCGCC GCCT¾ ^: CAGC¾GGGCCA£5AACC GC ^' TGTACAACGAGCTGAACCTGGGCAGACeGGAAGAGmGQA CG GGTGGAGAAGAGAAGAGGCCGGGA^^

GGAASGCCTGTATAACGAACIGCAGAAAGACAAGATGGGC AGGCCTACTCGGAGATCGGCAtGAA

GGGCG CGGCGGAG GGG SGGACACGA GGAGTGTAGCAGGGCC GAGCACCGCCACCAASGA

GAGCSATGAGGGCGTGCAGATGGASGGGCiGGCCC^

AG

CART4dual Riicleic acid sequence (SEQ ID ^' NO: 41)

GG-FGTGG GAGCGGCCGGTGAACTGGCCAGCATG GGCTGC¾STCTGTGCTGC GGTGGGCACCG GGCCTGCAGCA CAGCATCCAGATGACCCAGACCACCAGCAGCGTSAGCGCCAGCCTGGGGGATAG AGTC&CCMCASCT^ ¾CCG'GC&£CGS

CX GfTCf iC C ^

CGGCAGC&CAAGGGGCTCTGGCAAGCCXGGAfG GGCGAGGGCTC ^{^} ^

GGCCC GGACSGG GGCG'CCTAGCGAGAGGC GTC GTGACGTGTACCGTGTCGGGCGTGSCCCT GCG GACTATGGCG GTGC GGA CAGACAGCGCGCCAGAAAGGGCCTGGAAT GCTGGGAGTGA GTGGGGCAGCGAGACAAGCTACTAGA¾CAGGGCGGTGAAGTCCCGSGTGSGCA CATGAAGGAGA¾ C^CCAAGAGCCAGGTGTTCCTG GAiGAAGAGGGTGGAGACCGACGAGACCGGGAiG3¾C?A£¾£ GGGCAAGCAGTAC AG AGGGCGGCAGGi'AGGCGAiGGAGmefG

GCGmCGACAAGCCGTGCCGC AGACGGCmG

tCGGAGGCCCGAGGCTTCTAGACCAGCTGC GGCGGAGCCGXGCACACCAGAGGACTGGACAAGGC CTGC GGGtGCTGGTGGXCGTGGGCGGAG GCTGGCGTGTTAAGCCTGCTCG^GACAGGGGCC T CA GATCTTTAGGGTGCGCGTGAAGTTG&GCCGCAGGGGGGATGCCGCTGGCTATCAGG AGGGAC&

GAAGCAGCTG ACAAGGAGCTGAACGTGGGCAGACGGGAAGKGTACGACGTGC GGACAAGAGAAG AGGCCGGGAGCGTGAGATGGGCGGGAAGCC€AGA¾GA¾A

ACFGGAGAAAGAC GA GGCGGAGGCCTACAGCGAGA CGGGATGAAGGGCGAAGGGCGGAGAGG G GGGCGAGGATGGAC G A GASGGGC GAGCAGCGCGACCAAGGAGAGCTAIGACSCCGTGGA GA GC&GGG C GGGGCCTCGGTGAAATTCATCGAGGT AACFA'TTC AG

CARI6 nucleic& sequence SBQ ID NO: 43}

GG GTGGIGAGCGGCCGCTGAAC GGGCAC

GGCGfGCAGGAfCAGCAXCC&GATGACGCAGACCAGC&GCAGG

ATG&CC CASC^GC&GA^

CGACGGCACCGTGAAGC GGTGATCTAGCACACCAGGAGAG GCACAGGGGCGTGCCGTCTAGATT TGCGGCAGCGGGTCCGGCACCGACTACAGv TGACCA CTCCAACG GGAA

TAGC ACT ^' CTGTCAGCAAGGCAACACCG GCCCrACACCTTGGGCGGAGGCACCAAGCTGGAAAT CGGCAGGACAAGCGGCTCTGGCAAGCCTGGAfCTGGCGAGGG

GGCCCSGGAGTGG GGCCCGGAGGCAGAGCGTGrCTGTGAGGI'GiACCGTGTGCGGGGGG GCCT GGCTGACTATGGGG G CCTGGATCAGACAGCCG CCAGAAAGGGCCTGGAATGGCTGGGAG GAT C GGGGCAGCGAGACAACCTAGTACAACAGCGCCCTGAAG CCCGGCTGACCA CA CAAGGACAA CTCC&kG&GCCAG iGi^

A« of the- chimeric reeepicrs described herek cm b e prepared by roaliae methods, such as recomi>i«ai\i technology. etods lor rs rmg fte c¾iffieric receptors ■teeitiittvolve geReratiori of a nacieic acid that encodes a poly eptid co«iprisi»$: each of the domains of the chimeric recep tors, ineluding fee tvi ^biodkg Mg»«t mi optaoal!y, the hinge omain, tire transmembrane d main, at least one co-stimulatory signaling domain, and the cy toplasmic signaling domain. In some embodimeats, a nocleic acid encoding each of the components of chimeric r eepior are joined together us i ng recombinant, teenaoS gy, Sequences of each of the, c m one ts: of the chimeric receptors may be obtained •via routine teehuoiogy, . _; , PCI..a plillcaiios from any ens: ofa variety of Sour ees known in the art. I» somt embodiments, sequence of ne or more of the cojnpofteais-of the chimeric receptors me obtained ίτοη» a human cell. Alternatively, the sequences of one or more components of the chimeric receptors can be synthesized. Sequences of each, of the components (e.g. , domains) can. be joined directly or indirectly (e.g., using a nucleic acid sequence encoding a peptide linker} to tbn» nucleic acid sentience encoding the chimeric receptor, using methods such as ΚΆ araplifkaiion o ligation. Alternatively, the nucleic acid encoding the chimeric receptor may be synthesized. In. sortie embodiments, the nucleic acid is DNA, In other embodiments,, the nucleic acid, is RNA.

Mutation of one or more residues wite one or more of the components of the chimeric receptor (e.g., the antigeft¾»diag ftagment, etc)., prior to or after joining the sequences of each of the components, in some embodiments, one or more mutations in a component of the chimeric receptor maybe made to modulate (increase or decrease): the affinity oi the component for a target (e.g., the aniigen-hindirig fragment for the target antigen) and/or nipdnfate the acti vity of the component,

Any of the chimeric receptors described herein can be. introduced into a s itable immune cell for expression via conventional technology . In some embodiments, the immune ceils are T cells, such as primary T cells or T cell l ines. Alteniative!y, the immune ceils cm be UK cells, such as established NK cell lines (e.g., K-92 cells). In some embodiments, the immune cells are T cells that express CDS (CDS") or CDS and.€04 {0 ⁾ 8 05 ^* )■ in some embodiments, the T ceils are T ceils of an established I ceil Sine, for example, 2.93 ceils or Jurfcat. celts.

Primary T cells ma be obtained from any source, such as peripheral blood, mononuclear cells (PBMCs), bone marrow, tissues such as spleen, lymph node, thymus,, or tumor tissue. A source suitable for obtaining the type of immune ceils desired would be evident to one of skill m the art. In. some embodiments, the opulation o immune ceils is derived from a Immau patient haying a hematopoietic malignancy, such as from the bone marrow or from PBMCs obtaine from th patient, m some .embodiments, the population of immmie cells is derived from a health donor, In some embodiments, the imm ne cell are obtained from the subject to whom the immune cells expressing the chimeric recep tors will he subsequently administered. Immune cells that are administered to the same subject From Which the cells were obtained, are referred to as autologous ceils, whereas immune cells thai are obtained from a s^ not the subject to v iorn the ceils will b sdffiMisiejeti ate referred to as allogeneic eels.

The type of host cells desired way be ex anded, witiun & popu fieft of cells obtained by c -incttbatkg the cells with stimoktory raofecules, for example, ti-iSDS an mii~CB2B antibodies may be used for expansion of T cells.

To construct the immune cells thai express any of the chimeric receptor constructs described herein, expression vectois tor stable or transieal expression of the chimeric receptor eons ct ma be constructed via conventional .methods as described herein tad introduced into immune host cells. For xample, nucleic acids encoding the chimeric receptors ma be cloned into a suitable expression vector, suc as a viral vector in operable linkage to a. suitable pro-motet The nucleic acids and the vector may be contacted, under suitable conditions, ^' it a .restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined with, a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to tire ierrnini of the nucleic acid encoding he chimeric receptors . The synthetic: linkers may contain nucleic acid sequences iluii correspond to particular restriction site ½. the vector, Th sel ction of expression, vectors^ksmids/v irai vectors-would depend on the type of host cells for expression of the chimeric receptors, but should be suitable i r integration and replication in eukaryotfc cells,

A variety of promoters can be used for expression of the chimeric receptors described herein, including _* without limitation, cytomegalo irus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-i LTR, Maloney murine leukemia vims (MMLV) LTR, myeoloprollferative sarcoma virus (MPSV) LTR, spleen, fecus-forming vims (SFFV) LTR, the simian virus 40 (SV40) early promoter, herpes simplex tk virus rompter, elongation factor l-alpha (BFl-a) promoter with or without the EF I -a in iron. Additional promoters tor expression of the chimeric recep tors include any ednstiiutively active promoter in as immune- ceil Alternatively, any regulatable promoie m y be used, such that its expression can fee modulated ^' within an immune cell.

Additionally, the vector may contain, for example, some, or all f the -.following: a selectable marker gene, sneh. asrthe-aeomycia-gene-fpr sdectiMof stable or transient transfeciafits in host cells: enhaocer/promaier sequences -.from- the Immediate early gene of human CMV -for high levels of transcription; transcription termination and R A processing signals from SV40 for nvRNA stability; 5 '-and 3' -untranslated regions for niRN stability and translation efficiency from highly-expressed genes like a-giobia or β-glohin; SV40 polyoma or igins of replication and CoIE l for proper episomal replication; int ernal ribosome iiidiiig sites C ESes efsaliie ultiply cloning sites; T7 and SP6 RNA romote s for m vitro transcription: of sense and ffitis iJS RMA; a ^suicide switch" or "saiei e gene" whic w m triggered causes cells carrying the- ector to die i¾g., HSV thymidine kinase, an inducible caspase such as iCasp9), and reporter gene for assessing expression of the cMmferic receptor. See section VI below. Suitable vectors mid methods for producing vectors containing transget.es are well known and available m the art Examples of the preparation of vectors for expression ofehrnierie receptors can be found, for example, is US20J 4/0106449, herein incorporated by reference in its entirety.

In some embodiments _* the chimeric receptor construct ot the nucleic acid encoding said chimeric receptor is a DiN A molecule, i some embedments, chimeric receptor construct or the nucleic add encoding said chimeric receptor is a DMA vector and may be eleclroparaied to immune cells (see, e.g.. Till, et ύ. Blood (2012) 19(17); 3940*3950). In some embodi ents, the nucleic add encoding the chimeric receptor is an RMA molecule, ^' which may e ^■ etectr.oporated to immune ceils.

An of the vectors comprising a nucleic acid seqaesee that encodes ¾ chimeric receptor ^' consfewct- des ribed herein i$ also ithin the scope of the present disclosure. Sach a vector may be delivered into host cells such as host immune cells by a suitable method. Methods o f delivering vectors to immune cells are well known in the art and may include DNA, R A, or transposes, eiectroporation, trausfectioa reagents such as liposomes or nanoparticles to deli very DNA, RNA, or tmnsposons; deli very of DMA, RNA, or transposons or protein by mechanical deformation (see, e.g., Sliarei et ah Proc, Natl Acad Set US4 (2013) 1 10(6): 2082-2087); or viral transduction, in some embodiments, the vectors lor •expression of the chimeric receptors are delivered to host cell s by viral transduction.

Exemplary viral methods for delivery include, hist are not limited to, recombinant retro viruses (see, e.g. _. . PCT Publication Nos. WO -90/07936; WO 94/03622; WO 93/25698; WO

93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S. Pat. Nos. 5,219,740 and 4777,1.27; GB Patent. No, 2,200,651 aud EP Patent No, 0 345 242), alphavims-based vectors, imd sdeno-associaled virus (AAV) vectors (see, -&g,,.PCT Publication ^' Nos, WO 94/12649, WO 93/03769; WO 93/19191; WO 94/2893¾ WO 95/119M and WO 93/®655).. in same enihodim ms, the vectors ibr expression, of the chimeric receptors are retroviruses.. IB-some embodiments, the vectors tor expression Of the chimeric receptors are ienttviroses, in some embodiments, the vectors for expression of me chimeric receptors ar adeao- associated viruses. la ex tples m which the e tors encoding chiMiefic ec^tcss are introduced to the: host cells n ft a viral vector, viral particles thai are capable of infecim the immune cells and cam' the vector tmy be produced b my method. kno n In the err and can be foirad,- foe example in POT Application Mo. WO 1991/002805A2, WO 1998/009271 Af and US. Patent 6,194,191. The viral particles are harvested from the cell culture supernatant and may be isolated and/or parified prior to contacting the viral particles with the iinmnne cells.

The methods of preparing host cells expressing any of the chimeric receptors described herein may comprise activating and/or expanding the immune cells ex vivo.

Activating a host cell means stimulating a host cell int an acti vate state w which, the cell .may be able to perfoma effector mnetions (eg,, cytotoxicity). Methods of activating a host ceil will depend em the type of host cell used for expression of the chimeric receptors.

Expanding host cells may involve my method that results in an increase in the namber of cells : expressiag chimeric receptors, for example, allowing the test cells to proliferate or stimulating the host cells to proliferate:. Methods for stimulating expansiofi of host cells will depend on the type of host cell used i r expression Of the chimeric receptors and will be _: evident to one of ski 11 in. the art. In some ernbodknen is, the host cells expressing any of ihe chimeric receptors described ^' herein are activated and/or expanded ex vim prior to dmisistration to a subject.

in some embodiffieais, the agents targeting a eeli-smfaee lineage-specific antigen is an a tibody-drug conjagate (ADC). As will be evident to one of ordinary skill m the art, the term "antibody-drag conjugate ^** can be used interchangeably with, "immanotoxin" and refers to a fusion molecule comprisin an antibody (or antigen-binding fragment thereof) conjugated to a toxi or dru molecule,. Binding of the antibody to the corresponding antigen allows for delivery of the toxin or drug molecule to. a cell that presents the antigen on. the its ceil surface (e.g., target cell), thereby resulting in death of the target cell.

In some embodiments, the agent is an antibody-drug conju ate, ¾ some

embodiments, the antibody-drug conjugate com rises an an en-bffidi«.g fragment and toxin, or drag that induces cytotoxicity in a target cell. I some eirtb dirneEts,: the antibody- drug coiiii!gate targets a type 2 antigen, hi some embodiments, the antibody-drug conjugate- targets CD33 or GDI ¾

in. some eKibodhneuts, ihe antigen-bind fiigrnent of the antibody-drag conjugate has the same heavy chain C0Rs as the heavy chain variable region provided by SEQ ID NO; 12 and the same tight chain CD S as the light chain variable region provided by SEQ ID NO: 13. In some embodiments, the antigen-bind fragment of the antibody-drag conjugate has the heavy chain va iable region provided fey: SEQ ID NO: 12 ami the sasie hght chain variable region provided by SEQ ID NC);

Toxins or drugs compatible for us in- antibody-drug epjrjngate are well know in the _: art and will be evident to one of ordinary skill in the art. See, e,g. , Peters et al Bmm, Rep Ol S) 35(4); e#02-25. in some embodiments^ the antibody-drag conjugate may further comprise a linker (eg., a peptide linker, such as a eieavabie linker) attaching the antibody and drag molecule.

An ADC described herein" may be used as a foliow-oa treatment to subjects ito have been unde gone fee combined therapy as described herein.

Hematopoietic Cells Deficient m a Lineage-Specific CeH-Sarfaee Antigen

The prese disclosure also provides hematopoietic cells such as HSCs thai have been genetically .modified to be deficient in a lineage-specific cell-surface antigen. In some embodiments, the hematopoietic cells are hematopoietic siem cells. Hematopoietic stem ceils (HSCs) a e capable of giving rise, to botli rnyeloid and lymphoid progenitor cells thai furihe give rise to myeloid cells (eg., nionecyi S;, macrophages^ neutrophils, basophils, dendritic cells, erythrocytes, platelets, etc): and lymphoid cells (e.g., T ceils, B cells, ^' K cells), respectively, HSGs are characterised b the expression p.fthe cell surface marker CD34 ( ^' e.g.- _* CJ>M ^' which can be used for die idemlfteation and/or isolation of HSCs, and absence of cell surface markers associated with commitment to a cell lineage.

In some embodiments, the HSCs are obtained front a subject, such, as a mammalian, subject, to- some mbodiments, the -mammalian, subject is a non-humaa primate, a rodent. ^■ {e.g. , mouse or fat), a bovine, a. porcine, an equine, or a- ^' domestic animal,. In some embodiments, the HSCs are obtained, from, a human patient _* such as a.humau patient ving a hematopoietic malignancy, to some ^• embodiments, the HSCs are obtained from a healthy draibr. In soffie empodimerits, the KSCs are bbtaffled from the subject to whom the immune ■cells expressing the chimeric receptors will be. subsequently administered, HSCs thai are tdmmistered to the same subject from which the cells were obtained, are referred, to as autologous ceils, whereas HSCs that are obtained if o a subject who is not the subject to whom the cells will.be administered are referred to a allogeneic eells,

HSCs may be obtained l¾m any suitable source using convention means known in tire art. in some embodiments, HSCs are obtained from a. sample from a subject, such as hone marrow sample or from a blood sample. Alternatively or in addition, HSCs may he obtained .from an ^• umbilical cord... hi some embodiments,, the HSCs are from bone marrow or

emhodimests, the BSC are expanded, after isolation o a desired, cell pepulata (s.g. ,

CD3 ^' Ci 33 ^r ) from a sample ebtained from a subjec and rior to genetic engineering. ¾ some embodiments, the HSC are expanded after genetic engineering, thereby selectively expanding cells that have undergone the genetic modification and are deficient in a liaeage- specifie cell-surface antigen, in some embodiments, a cell ("a clone") or several ceils having

sequence enco ng t e ce -surace neage-spec c antgen may e eete or one non-coding sei eiiceS;: sucirthat the hemafc jMe cell. & has substantially reduced expression of the antigen}.

In some erabodirneais, the eeli-surface lineage-specific antigen is€1333, T¾e predicted structure of CD33 includes two inn inog!obulin domains, an IgV domain and an IgC2 domain. In some embodiments, a portion of the immunoglobulin C domain of CD33 is deleted.

Any of the genetically engineering hematopoietic cells, such as SSCs, that are deficient in a cell-surface lineage-s$>eciik antigen can be prepared by a routine method or by a method described herein, in some embodiments, the genetic engint in is performed «sisag _. geno.Hje editing. As used herein, "genome editing" refers to a method of modifying the genome, including any protein-coding or non-coding nucle tide sequence, of an organism to knock out the expression of a target gene. In general, genome editing methods invol ve use of an endomicSease that is capable of cleaving the nucleic acid, of the genome, for example at a targeted nucleotide .sequence. Repair of the doohle-stranded breaks in the genome may be srepafred i troducing. lautsii.on$ and/orexdgeRbiiS nuoieic ¾cid,inay ¾ei»s ^ nto: the targeted site.

Genome editing methods are generally classified hased on the type ofendonactease that is involved in generating- double stranded br aks in the target nucleic acid. These methods include use of zinc linger n ucleases (ZFN), transcription activator-like effector- based ^■ .nuclease (TALEN), meganueleases. and CRiSPR/Cas systems..

DICsrfo et ^ Nmie d$ fe (20. 3} 7 _* *336; Hwang et al, Nat Bwt&chnoi (2013), 3:227); .Gratz et a.L Gmeti (201.3) ! 4:I029; Cong et ai, Sci ee (2013) «21 819 Mali et ύ,. Science )Π) 6121:823; Gho et ai. Nat. titotechnoi ' (2013) 3: 230; and Han et al, Nueleic Acids Mesearch (2013) 4l(20):el . Th present■disclosure utilizes tte system that ,foyk| i2 with: a target, sequence in a l neage specific an igen polynucleotide, where :t¾£:CRiSP¾ C¾siJ system comprises a Cas9 miclease ani an engineere crRNA irac EN . (or single guide RNA). €RISPR. as9 complex can bind to the lineage specific antigen polyaycleotide and allow- the cleavage of t e antigen |x>lynucieotide, thereby racdifyiag- the polynucleotide.

The CRISF Cas system, of the present disclosure ma bind to and/or cleave the region of interest within a cell-s urface teeage-spec c antigen m a coding or non-coding region, within or adjacent to the gene, such- s, to example, a leader sequence, trailer sequence or intron, or within a ^■ nori-traiiscribed region, either upstream or downstream of the coding region. The guide RNAs (gR As) used: in the present disclosure may he designed, such that the gRNA directs bindieg of the Cas9-gRNA complexes to a pre-deiemrined cleavage sites (target site) in& genome. The cleavage sites may be chosen .so as to release a .fragment that contains a region of unknown sequence, or a region containing SNF.

nucleotide insertion, nucleotide deletion, rearrangement etc.

Cleavage of a gene region may comprise cleaving one or two strands at the location of the target sequence by the Cas ers ynie. In. one embodiment., such, cleavage can resnlt in decreased transcription of a target gene, in another embodiment, the cleavage can further comprise repairing the cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein the repair results in an insertion, deletion, or substitution ^' of one or more nucleotides of the target polynucleotide.

The terms "gRNA/ ⁸ "guide RNA" and "CR1SP.R guide sequence" may he used, interchangeably throughout and refer to a nucleic acid comprising a sequence thai determines the specificity of a Cas DNA binding protein of a CMSFRCas system. A gRNA hybridises to (complementary to, partially or completely) a target nucleic acid sequence in the genome of a host, cell The gRN A or portion thereof (hat hybridizes to the target nucleic acid may be betwee 15-25 lueleotides, 18-22 nucleotides;, or 19-21 nucleotides in length. la some .embodiments, the gRNA sequence that hybridizes to the target ucleic acid is 15 _:; 1<L I.?, 1.8, 1 _: , 20, 21, 22, 21, .24, . or 25 nucleotides, i length. In some embodiments the g ^' RNA sequence that hybridizes to the target nucleic acid is between 10-3( , or between .15-25, nucleotides in length,

in, addition to a se ence that binds to a target nucleic acid, in some embodiments, the gRNA also comprises a scaffold sequence. Expression of a gRNA encoding both a sequence complementary to a target nucleic acid and scaffold sequence has the dual function of both binding (hybridizing) to the target .nucleic acid and recruiting the endonnciease to the target nucleic acid, which may result M site-specific CRISPR activity, In some embodiments, mc a chimeric gRNA may ¾e ί¾ί½π¾(! to as a tele guide N (sgRNA .

As used terete, a "scaffold sequence," also .refced to as a tracrRNA, refers to a nucleic acid sequence that recruits a Cas endonaciease to a. target nucleic acid bound

(hy rkfeed) to a compleraentaiy gRNA se uesce. Any scaffold sequence that com ises at least one stem loop structure and recruits an endofiuelease may be used in the genetic elements and vectors described herein. Exemplary scaffold sequences will be evident to one of skill in the art and can be found, for example, in Jinefc, a ai. Science (2012)

337(60 6};816-821 , Ran, ef alNp e Protocol? (2013) 8:2281-2308, PCT Application Ka WO2014/093694, and PCX Application No, WO2013/176772,

In some em odiments, the gRNA sequence does not comprises a. scaffold sequence and a scaffold sequence is expressed as a separate transcript, in such embodiments., the gRNA sequence further comprises an additional sequence tliat is. com lementary to a portion -.of the scaffold .sequence and functions to bind (hybridize) the scaffold sequence .and recrui.t the eridc¾;ueiease to ihe targei nucieie acid.

In some-em odiments, the gRNA sequence is at least 50%, 55%, 60%, 65%,: 70%, 75%, 80%, 85%, 90%, 95%, % , 97%, 98%, 99%, or at least. 1.00% complementary to a target nucleic acid (see also US Patent 8,697,359, which is incorporated b reference tor its teaching of coinpiernentarity of a gRNA sequence with, a target polynucleotide sequence), i has been demonstrated that mismatches between a CR1SPE guide sequence and the target nucleic acid near the 3' end of the target nucleic acid may abolish nuclease cleavage activity (Upadhyay, et at G es Gemnw ⁽ kmifes (2-013) 3 (12}:2233-223 B) . In some erobodimeots, the gRNA sequence is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 8 , 99%, or at least 100% complementa y to the 3' end of the target nucleic acid (e.g., the last 5, 6, 7, S, 9, or 10 nucleotides of the 3' end of the target nucleic acid).

The target nucleic acid is flanked on the 3' side by a protospacer ¾li¾ce«.t .motif

(PAM) that may Interne with the esdoiiuelease and he -farther involved, in. tar eting the endonuclease activity to the target micleic acid, It. is generall thought that the PAM sequence ftankmg the target nucleic acid depends on the endonuclease and the source Irani which: the eadonudease is derived, for example, lor Cas9 endottyefeases that are derived from Sirspiomecm yogenes, the PAM sequence is -NOG. For Cas9 endomtcieases derived, from Staphyhcoc s aureus, the PAM sequence is MGRRT, For Cas9 endortucleases that are derived from Neisseria meningitidis, the PAM sequence is NNNGATT. For Cas9 endonucieases derived from Streptococcus tkcrmophil s, the PAM sequence is NNA.GAA,

(CRiSPRi)). In som embodments, as s used to aa activator domain, s ch.

oc or identfyng ce s expressng the Gas endonuclease. Alternatively mm addition ifeCas ^'' endonaefease Is a G il nrselease. In sosrtt embodiments, t e ^' host call expresses a Cpfl Nucl ase ^' derived irm f'rovelelM spp. ox FrmciMi φμ In sorae enibaiiroents, the nucleotide seq ^' asace encoding the Cpil nuclease may be codon opini ed for expression in a host ceil

in some em odsmerits, the present disclosure provides compositions and methods for inhibiting a cell-surface imeags-specific aiiiigeri in hsnMopoistic ceils nsing a CRiSFR Cas') system, wherein pide RNA sequence hybridises to the nucleotide sequence encodkg; the ceil-surface lineage-specific antigen, In some embodiments, the celi-Sttrface iineage-specifie antigen is CD33 and the gR A hybridizes io a portion of the nucleotide sequence that encodes the CD33 (FIGURE 5), Examples of gRNAs that target. CD33 are provided in Table 4, although additional gRNAs m y be developed that hybricfoe to CD33 and can be used in the methods described herein.

Table 4 provides exen )!ary guide RM A. sequences tfist hybridi¾e or are predicted to bridise to a portion of€D3

Thus, in the case of GVBD. the treatment of the patient can involve ¾ following steps: (1) administering a therapeutically effective amount ofa I eell to the atient where the T cell comprises a nucleic acid sequence -encoding a. chimeric antigen receptor (CAR) targeting CD45 A lineage specific -antigen; and (2) infusing the patient with- hematopoietic- stem cells, where the- hematopoietic cells have reduced expression of C.D451A. lineage specific antigen

Additionally, the present disclosure provides, compositions and methods for the combined inhibition of bot CD33 and CD45RA lineage specific atittgens. Such tresipeih regimen ca -involve the following: steps: (1 ) a im erin -a ¾Jt e.«i cally effective amount of X cell to the patient where the ? cell comprises a nucleic acid sequence encoding a c meric- ntigen..receptor (CAR) ^■ targeting both CD33 and GB45RA lineage- specific antigens; and (2) infusing or retnfusiflg the patient with .hematopoietic stem cells, either autologous or allogeneic; where the hematopoietic cells have re uced e pr ssio o both the CD33 and CD 5RA lineage specifi antigens

hybridi¾e to exon 4 o r ex on 5 of human CD 5.

Table 5: Guide RNA sequences targeting CD45

Also provided herein are methods of _. prsduciug a cell tha is deficient m a cell-sutface. lineage-specific antigen kvo ^' lviag providmga ceil nd iritrod cmg into the cell eorspoBents of a CRI-SFR Cas system for .genome editing. In some mbodiments, a nucleic acid thai comprises a. CRlSFR-Css guide RNA (gRNA) that hybridises or is predicted, to h id se to a portion of the nucleotide sequence that encodes the . lineage-specific eelTsarfaee antigen Is introduced into the cell, in sorSe embodiments, the gRNA s introdneed Into the ceil on a Ys im. hi some ernhodimenis, a Cas srtdofiMciease. is i trodiiced into the cell some embeldments, the: Gas en o id-ease is iateodiieed inte the: cell as a OTcleic acid encoding s Cas eiidonuciease. In so me embodiments, the gRN A and a nucleotide sequence encoding a Cas eiidoaaelease are. mttodueed into the cell on the same nucleic acid (e.g., the same vector). IN some embedments, the Cas eMionuelea.se is introduced nto the ceil, in th form of a protein. ^' Is some embodieients, the Cas endonackase and the gRNA are pre-fonBed m vitro and are introduced to the ceil in as a -complex. The present disclosur ftrrlher provides engineered, s«¾i«aatui iy ^• ^Uixifl . ec^ts ^' and vecior systems, which can encode ose o mom compouerits of C:R!SI¾C s9 complex, wherein the vector comprises a polynucleotide encoding (i) a (GRISPR)~Gas system pide UNA that hybridizes to the lineage specific antigen sequence and (.«) a Cas endoRHcIease.

Vectors of the present disclosure can drive the expression of one or more sequences ia. mammalian ceils using a mam aliaa expression vector. Examples of mam aliaa expression vectors include pCDM (Seed, Nature (!9S7) 329; 840} and pMT2PC (£aafimn, et ai,

EMBOJ.- (W%7) 6; I S?). When used ia mamraalaa ceils, the expression . vector's control fimctions are typically provided by one or more regulatory elements. For example,

commonly used promoters are derived from polyoma* adenovirus 2, . cytomegalovirus, simian: vims 49, and others disclosed herein and known in fee art. For other suitable expression systems for both prokaryoiie and eirkaryotic cells see, <¾ Chapters 1.6 and 17 of Samhreok, ct al., MOLECULAR. CLONING: A LABORATORY MANUAL. 2nd eds,. Cold Spring Harbor ^■ Laboratory, ^' Cold Spring; Harbor Laboratory Press, Gold Spring Harbor, NX, 1989.

The vectors of the present disclosure are capable of directing expression of fee nii eic acid preferentially irs a particular cell type (e.g^ tissue- specific- regulatory ^' elements are used to express the nucleic acid). Such replatory elements mclnde promoters that may be tissue specific or cell specific. The term "tissue specific" as it apples to a promoter refers to a promoter that is capable of directin selective expression of a nucleotide sequence of interest to a specific type of tissue (e.g. , seeds) in the relative absence of expression of the same nucleotid sequence of interest in a different type of tissue. The term "cell type specific" as applied to a promoter refers to a promote that is capable of directing selective expression of a nucleotide sequence of interest in a speci fic type of cell in. the relative absence of expression of the same nucleotide sequence of interest in a diilereni type of ceil within the same tissue. The term "cell type specific" when applied to promoter als means a promoter capable of promo ing selective expression of a nucleotide- sequence of interest ia a region within, a single tissue, Cell type specificity of a promoter may be assessed usiag ruethods well too u iu the art, .&<g.., imrnnnohistocheinieal. staining.

Conventional viral a non-viral based gene transfer methods can. be nsed t introduce nucleic acids encoding CRJSPR Cas^ in mammalian cells or target tissues. Suc methods can fee used so administer nucleic acids encoding components of a CRISPR-Cas system t ceils in culture, or in a host organism. Non-viral vector delivery systems include DNA plasraids £NA {¾.,¾., a transcript of a vector described herein), naked nucleic acid, and nucleic acid com lexed with a delivery vehicle,. Viral vector delivery systems include DMA and -RNA vi tises. which have eilfeer ernsoroal o integrated genomes site: deliver to the cell for a review of gene her apy procedures,

Virat vectors can be administered; directly _. to pattenfe to manipulate cells in Vitro or ex vivo, where the mod fied cells ma be administered to patients, In one embodiment, the present disclosure utilkes viral based systems including, hut not limited to retroviral, lent ros, adenoviral. adeno~associaied and herpes simplex virus vectors For gene transfer. Furthermore, the present disclosure provides vectors capable of integration in the host genome, such, as retrovirus or lentMrus, Preferably, die vector used for the expression of a CRiSPR~Cas system ^' of the present disclosure is a iehtrvifal vector.

In one e nbodumeBt* the disclosure .provides for Introducing one or more vectors encoding CRISPR-Cas into eukatyotic cell. The cell can be a cancer ceil Alternatively, the tell is a hematopoietic cell, suc as a hematopoietic stem cell. Examples- of stem -cells include piuri ^' poient, niuitipoteut and umpoierit stem cells. Examples of phrtipoieni stem cells include enibryoaic siera cells, embryonic germ cells, eathryonic carcinoma ceils and induced ^" pkr poteni stem cells (iPSCs). ¾ a preferred embodiment, th disclosure provides

introducing CRISP -Gas^) into a .h matopoietic Steffi cell.

The vectors of the present disclos ure are delivered to the eukaryotic cell in. a subject Modification of the eukaryotic cells via CKISPR/Cas system cm _. takes place k a cell culture, where the method comprises isolating the eukar otie cell from a subject prior t the modifka ian. In some embodiments, the method further comprises returning said . eufeiryotie cell and/or cells derived therefrom, to the subject.

n re uce reecton o t e ost ce s as compare to a mustmton o non ^h omoo us Alternatively, the host cells are allogeneic ceils, i.e., th cells are obtained from a .first Subject, genetically engineered :iO: be dsficiarii for expression of the ceil-snriace lineage- specific antigen or for expression of the chimeric receptor constructs, and administered to a second subject that is different, from the first subject but of the same species. For example,

receptor) may be provided to the patient ai intervals of 15 days, .14, 13. _} 12 _s 11, 10, 9, 8, % (>, 5, 4, 3, or 2 days after the previous administration. More than one dose of the agent can be administered to tire subject per w ek, £¾ 2, 3, , or more administrations of ^' ire agent. The subject may receive more than, one doses of the agent an-immnne- ceil expressing a chimeric receptor) per week, followed by a week of no administration of the. agent, and Siiaily followed fey one o more a ditional doses of fee agent fe.g., more than one admiuistfation of immun cells expressing a chimeric receptor per week), The inffitme: cells expressing a chimeric receptor may be administered ever other day fo 3 administrations per week for two, three, four, five, six, seven, eight or more weeks.

In the context of the present disclosirr insofar as it relates to any of the disease conditions recited herein, the terms "tre t," ^ireatmeat ' and th like mean to relieve or •alleviate at least one symptom associated with such condition, or to slow or reverse the progression ofsach condition. Within the meaning of the present disclosure, the term "treat" also denotes to arrest, delay the onset (i.e., the period prior to clinical, manifestation of a disease) and/or reduce the risk, of developing or worsenin d isease. For example, in connection with cancer the term *'ireaf may mean eliminate or reduce a. patient's tumor burden, or prevent, delay or inhibi t metastasis, etc.

In some embodiments, an agent comprising m antigen-binding fragment that binds a cell-surface lineage-speeiie antigen and a population of hematopoietic ceils deficient in the cell-swface lineage-specific antigen. Accordingly, in. t ^rapeaiie- methods, .the agent reeopizes (binds) a target cel.! expressin the celi-surfece iineage-speeific antigen for targeting kilting. The ^' hematopoietic cells thai are deficient in the antigen allow for repopnJatiori of a cell type that is targeted by the agent. In some embodimen ts, the treatment of the patient can involve the .following steps: (1) administering a therapeutically effective amount of an agent targeting a cell -surface lineage-specific antigen to the patient and (2) infusing or resuftisiug the patient with ^' hematopoietic stent cells, either autologous or al logenic, where the hematopoietic cells have reduced expression of a lineage specific disease-associated antigen. In some embodiments, the torment of the patient can involve the following steps: (.1) administering a therape«ticaliy,efi¾ets _: ve amount of an immune cell expressing a chimeric receptor to the patient, wherein the immune ceil comprises a nucleic ac d sequence encoding a c meric receptor that binds a cell-surface iineage _^ speeifie, disease- associated antigen; and (2) infusing ar ieinfhsbig the patient with hematopoietic ceils hematopoietic ste : cells),, either atitologOMS or aUogeiuc _* where the hematopoietic ceils have reduced expression of a lineag specific disease-associated, antigen.

The efficacy of the therapeutic methods using a. an agen compfisiag im aeiigeu - feiiiding fragment tha hinds a cell-surface lineage-speeifk antigen and a population, of hematopoietic cells deficient in the cell-surface lineage-specific antigen m y he assessed by any method known in the art and would he evident to a skilled medical professional. For example, the efficac of the therapy may be assessed by survival of the subject or cancer b¾den m she sisbf ect or tissue or sam le thereei In some efttbesdiments _s the efficac cit ic therapy is assessed by quantifying me number of ceils belonging to a particular population or ineage of ceils, in some embodiments, the efficacy of ibe ; therapy is assessed. ^: y quantifying the number of cells presenting ihe cell-suriace lineage-specific antigen.

la some eoihodiaiemx, ihe agent comprising an. mtigea- kdmg fragment iliat iods to the celi-snrface iiaeage-specifk antigen and the pop ation of hematopoietic -cells- IS- ■administered, concomitantly.

In som embodiments, the agent comprisin m antigen-bindmg fragment that binds eeii-surfaee lineage-specific antigen (<?.g., immune cells expressing it cMimerie receptor s described herein) is administered prior to administration, of the hematopoietic cells. In some embodiments, me agent comprising an antigen-binding fragment that binds a ceii-surfece lineage-specific antigen (e.g., immune cells expressing a chimeric receptor as described herein) is administered, at least abont 1 day, 2 days. 3 days _. . 4 days, 5 days, 6 days, I week, 2 weeks, 3 -weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 3 months, 4 o ths, 5 months. ⁽ ■ months or more prior to adnAisiran n of the hematopoietic cells.:

in some embodiments, the hematopoietic ceils are administe ed prior to the agent comprising n antigen-binding fragment that binds a cell-surface lineage-specific antigen (e.g., immune ceils expressin a chimeric receptor as described herein). In some

embodiments, the population of hematopoietic cells is administered at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, i week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 3 months, 4 months, 5 months, 6 months or more prior to administration of the .agent comprisin an antigen-biriding fragment that binds to ike cell-surface lineage-spedSe antigen,

la some embodiments, the agent targeting the cell-surface lineage-specific antigen and the population of hematopoietic cells are administered at substantiahy fee same time,. In some er ^odimeni , agent targeting the cell-siuface lineage-specific antigen is administered, and the patient is assessed fo a period of time, after which: the population of hematopoietic cells is administered. In some embodiments, the population: of hematopoietic ceils is administered and. the patient is assessed, for a period of time, after which agent targe ting the cell-surface Imeage-specific antigen is administered,.

Also within the scope of me present disclosure are multiple administrations {e.g. , doses) of the agents and/or populations of hematopoietic cell s, in some embodiment?, the agents and/or populations .of hematopoietic cells are a mi istered to the subject once..: In

colon cancer, melanoma, pancreatic cancer, mammary cancer, prostate cancer, breast cancer, ovari.au cancer, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; beast cancer; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive: sysiefs; endom^niai cancer; esophageal caiicer; e cancer; cancer of the head and neck gaSirie canc&r; iiitr s-epithelial Oeopl asm; kidne cancer larynx cancer; liver dancer; ^' fibroma _* neuroblastoma; oral cavity cancer (e.g÷. Bp, tongas, month, and pharynx); ovarian cancer; pancreatic cancer; prostate caiicer: retinoblastoma;

rhabdomyosarcoma; rectal cancer; renal cancer; cancer of the respiratory system; sarcoma; skin cancer; stomach, cancer; testicular cancer; thyroid cancer; uterine cancer: cancer of the ■urinary system, as well as other carcinomas and sarcomas.

Carcinomas are cancers of epithelial origin. Carcinomas .atende . for treatment with, the methods of the present disclosure include, kit are not limited to, acina carcinoma, acinous carcinoma, alveolar adenocarcinoma (also called adeoocystic carcinoma, adenontyoepitheUohra, cribriform carcinoma and cyiinchxmm), carcinoma adeaom&iosum, adenocarcinoma, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell, carcinoma (also called b ouchiokr carcinoma, alveolar cell tumor and pulmonary adenomatosis), basal cell, carcinoma, carcinoma feasoceliuiare {also _: called basalo a, or basiloma, and hair matrix carcinoma):, basaloM eareinonm, basos narnons cell carcinoma, breast .carcinoma, broncbioal veotar carcinoma, bronc olar carclRoiM, bronchogenic caroinotna, eerehr.ifor . carcinoma, c&olangioceMular carcinoma (also called qholangioraa and cliolang-ioeardnoma}, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corps carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cuianeum, cylindrical carcinoma,, cylindrical cell carcinoma, duct carcinoma,. carcinoma durum, em ryonal carcinoma, eneephaloid

carcinoma, carcinoma mastitoides, carcinoma siedallare, .medullar carcinoma,

melanodes, melanotic carcinoma, m¾cino¾s carcinoma, carcinoma mneipsnrm, carcinoma moieocellulare, m cocpideraioid earcinoma, carcinoma mncesam, mucous carcinoma, carcinoma myxomatodes, nasopharyngeal carcinoma, carcinoma, nigrum, oat cell carcinoma, carcinoma ossificans,, osteoid carcinoma, ovarian carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prostate carcinoma, enal cell carcinoma of kidney (also called adeimcamhiotn of kidney and h ^egb^ oid ea mona),. reserve cell catdnotM, careinor sarcomatodes Scbebde iaH carcsiaroa, scirfhoits cardnonta, carcinoma Scroti, signet-ring cell ^' c cinooia* carcinoma simplex, small-cell carcinoma, soiano carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcmorna, carcinoma teJaagiectaiiciun, carcinorrsa telangiectodes, transitional, cell carcinoma, carcinoma tuberosum, tuberous carcinoma, .verrucous carcinoma, carcinoma vibstm in preferred embodiments, the -methods of the present disclosure are used to treat subjects having cancer of the breast, cervix, ovary, prostate, lung, colon and rectum, pancreas, -stomach or kidney.

ie i are aware o suc stan ar s, o care . u ects e ng treate ac-eor ng to

disclosure for a reftaetofy cancer therefore may- ve already been exposed to another treatment for their cancer. Alternatively, if the cancer is likely t be refractory f&g., given an analysis of the cancer ceils or history f the subject),, then the subject may not. have alread been exposed, to another treatment. ..Examples -of .refractory cancers include, but are not

Kits for Therapeutic Uses

Also it in the scope of the present disclosure are kits for use of the agents targeting cell-surface lineage-specific antigens ia combination with populations of hematopoietic cells that are deficient in the cell-surface lineage-specific antigen. Such kits may include one or more container comprising a first pharmaceutical composition that comprises any agent comprising an antigen-binding fragment that binds a cell-surface lineage-specific antigen (e.g. , immune cells expressing chimeric receptors described herein), and a pharmaceutically acceptable carrier, and a second pharmaceutical composition that comprises a population, of hematopoietic ceils that are deficient M the ceil-sar face ifficage-specffic antigen _; a hematopoietic stem ceil) and a pl armaceai&a!l acceptable carrier.

In some eraboduae»t% the kit can comprise instruc ioas .for use w arty of tie eietfeods described herein, the included insiractions can comprise a description of administration of the first and second phariaacetttical compositions to a subject to achieve the intended activity in a subject. The kit may fur&ef comprise a description of selecting a subject su table fox treatment based on identifying whether the subject is in need of the treatment. Is some embodiments, the instructions comprise a description, ofadmin eri g the first and second pharmaceutical compositions to a subject who is in need of the treatment.

The instructions relating to the use of the agents targeting c«lJ-surfacelmeage-specific antigens and the first and second pharmaceutical, compositions described herein generally include information as to dosage, dosing schedule, and route ofadministratioi. for the intended treatment. The containers may be unit doses., hulk packages muiti-dose packages) or sub-unit doses, !nstaictions supplied n the kits of the disclosure are typically written ittstmeiions on a label or package ^■ insert. The label or package insert indicates that the _: pharmaceutical composi tions are used i¾f- treating., delaying the onset, a&d/or alleviating a disease or disorder in a subject.

The kits provided herein are in suitable packaging. Suitable packaging includes, hut is not limited to, vials, bottles, jars, flexible packaging, and the like. Also contemplated are packages for ase hi combinstioa with a specific device, soeh as an inhaler, nasal

administratio device, or an infusion device. A kit may have a sterile access pott (for example, the container may be an intravenous solution bag or a vial having a stopper piereeable by a hypodern ic injection needle). Th container may also have a sterile access port. At least one active agent in the pharmaceutical composition is a chimeric receptor variants as described herein.

Kits optionally may provide additional components ^■ such as buffers and interpretive information.. omsally,,: the kit comprises a ^' container and a label or package Tnseft(s) on o associated with. the. container, la some embodiment, the discloshre -provides articles of manufacture comprising contents of the kits described: above.

General l chmqms

The pmc&e of the present disclosure will employ , unless otherwise indicated, conventional, techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are- within the skill of

Animal Cell Cailnte (R. 1 Ftesbney, ed. 1 87); troiictlon to Ceil and Tissue Culture (J. P. Mather and P _t E. Roberts, 1998) Plenum. Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, 1. S, Griffiths, a«d D. G. ewell, eds, 1993-8} 1. Wiley and Sons: Methods m Etymology (Academic Press, Inc.); Handbook of Experimental itnmunology <0, M. Weir and C> C Black eif eds.)'- ^' Gefte Transfer Vectors for Mammalian Cells (1 M, Miller and hi, P. Caios, eds.* 1987); Current Protocols in Molecular Biology ( , M. Atisu ^' l. et at eds. 1987); PGR: The Polymerase Chain Reaction, (Muliis, et at. eds. 1994); Current Protocols in Immunology (1 E, Coiigaa et al. _s eds... 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1 99); Immmiobiology (C. A. Jaueway and P. Travers, 1.997); . ^' Antibodies ^' (P. Finch, 1997); Antibodies; a practice ap mach (D. Catty;, ed., IRL Press, 1988-1989); Monoclonal antibodies; a practical approach (P. Shepherd and G. Dears, eds,, Oxford University Press, 2000) Using antibodies; a laboratory ffiatvuai (E _t Harlow and D, Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M, Zanetti and J. XX Capra, eds.. Harwood Academic Publishers, 1995); NA Oonmg: A practical Approach, Volumes I afid ΙΪ (D.N. Glover ed ^' \9 $y,Nmkic Acid Hybridization (B,D. Hames & SJ. Higgirss eds.(1.985»; Transcription and Translation (B..D. Hames & S.J Biggins, eds. (19$%: Animal Cell Culture (RJ. Ereshney, ed. (1 86»; JnmoMlhed Cells ami Enzymes (IEL Press, (1986»; and 8. PerbaL A practical Guide To Molecular Cloning (1984); F.M, Ausubel ettiL (eds.).

Without furfoer elaboration, it is believed, that one skilled in tfie art can, based on the above description, t ltxe (lie prese t disclosure to its fullest ^' extent The following speeiiic enibodinie s are, therel r , to b erMBto aoi;

■limitative of the remainder of the disclosure m any way whatsoever, All ^pu lications cited, herein are incorporated by ref rence for the purposes or subject matter .refepenced - heieui..

EXAMPLE in vMm Meifen of CD331» a Human Leukemic Cell Line

orde to test the ability of CRSPR-Cas system to target CD33 in vitro, -human leukemic cells K ^« 562 were co-transfected using eoti ^m (Thermo Fisher Scientific) with Cas9-GEP (PX458, X pyogenes) and a guide IMA containing NGG PAM seqiteace (FIGURE 4) where guide RNA was designed, to target hCD33 genomic sequence. 48 hours

system in human !etikemie cells,

EXAMPLE 2: In vitro Deletion of Cl) S iii H un? a a Leutornk Cell Lines

The CKISPR-C 9 system was used to target CD45RA in vim. Briefly, T3B-6? reticidnm cell sarcoma mouse .maerophage4ike cells were co«tr¾asfected asifig Neon™ reagent (Trsemjo Fisher Scientific) with Cas9-GPP (P.X45&, S, pyogews) and CRlSPRs gR As {contaioisg the " "NGG" PAM sequence) targeting hCD43RA geaomic sequence. 48 ^' hours post-tensfection, cells: expressing CRISRR~Cas9 system we e ideaiified and isolated using FAGS- sorting for GF . Cells were iners. inciAaied for 9 lours and tested for CD45RA expression (FIGURE 6), Flow cytometry plots asmg CD45.RA antibody show C04.5RA expression before (top plot) and after (bottom plot) delivery of Cas9 vector and. glide RMA, Similar to Example I, where CD33 expression was successfully reduced in leukemic cells, fmdiags in this Example indicate efficient targeting of CD45RA lising the CRI.SPR- Css9 system,

EX M ^' PLE 3: Targeting CeJ¼urface Linea e- pedfle CB33 is? Ac«te Myefojti

Leukemi (AML)

The presest example eacompasses targeting of the CD33 antigen, in. AML. The specific steps of ite example are ontlbed in Table ^(' .·.

4, emfcion of€B34¾D33 ^" ceils in to a

Pa&ni

III, Continued, treatment of a patient with a

CD33 antibody attached to a toxfe

provided below,.

Part I: Light chafe- linker -Heavy chafe (SEQ ID NO; 16): The Koxafc start site is showii in boldface. The peptide sipal Li is shown in italic. he a»ti~CD33 light chafe and heavy chain me shows itt bold imd italics,, separated, by a lioker.

ggtgfccgtgagc¾gcc¾ctgaactgGOC¾q^

CG G C mMmGCAQc mM cmcc GccTGG^

G&GCCCC&GGCTGCTGATCmCTGG^€CAGC&CC

cc&G€AGc GC G Gc

SGSCAGCACCA^

C G@ATC&&C&GACCCCCGG€C&&G&CCT&G& amp; C&GCACCA€€GCCfACA G€&Q€TGAG€&GCC GACCAG€

s&e is shows is. anderlme,

CW eostimulaiar doroaiii CSEQ I HC ⁾ : it)

GCGGCCGCAat.gaagttatgta -cc cctcctt¾cctaga!caatgagaag¾gc.atgg:a accattatccatgtgaaaggga acacctttgtGcaa

aagcccttttgggtgctggtggtggttggtggagtcctggcttgctata cttg tagta acagtggcctt attattttctgggtgaggag aagagga.gcaggctcctgcacagtgac acatgaacatgactccccgccgccccgggcCG^cccgcaagoattaccagccctatgcc ccaccacgcgacttGgcagcctatcgctccagagtgaagttcagcaggagcgcagacgcc ccc^egt-aocagcagggccagaadcagqtctataacgagctcaatctaggacgaagags tg gagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgaga aggaagaaocctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcc tacagtgagattgggatgaaaggGgagcgocggaggggcaaggggoacgatggcottta- o cagggtcfccagtacagacaccaaggacadctacgacgdccttcacatgcaggdc^tgcc c. cc cgcTA¾cgccc ^' ctptccc ccccccccectaa iCOS c stimuiatory doms n SEQ I ^' D NO: 18)

SCSGCC6CActatcaatttttgatcet:Cctcpt fctaaagtaaGtdtta<2aggagg<jfcat ttgG&tatttatg&a oacaactttg tgocagctgaagttctggttacccataggatgt gcagGctttgttgtagtctgca t tgggatgcatacttatttgttggcttacaaaaaag aagtattcatccagtgtgcacgaccctaacggtgaatacatgttcatgagagcagtgaac acagpcaaaa-aatctagactcacagatgtgaccctaagagtgaag tcagcaggagcgca gacgcccccgcgtaccagcagggccagaacGagctctataacgagctcaatctaggacga agagaggagL cgatgttttggacaagagacgtggccgggaccctgagatggggggaaag cogagaaggaagaaccctcaggaaggcc gtacaa gaactgcagaaaga &agatggcg gaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggc ctttaccagggtctcagtacagocaccaaggacacctacgBcgG Cttcaca gcaggcc Ptgccccct.pgcT¾ApgcccctctccctPCPcccccPctaa

Fusion (hybrid) GD 8 mi ICOS eosimylatory domain (SlQ ID NO: 19)

teeta ^■

In. the next: step, the h ng region, C.D2S domain (S!Q ID MO -3): sad a cytoplasmic component of TGI~C are cloned into the. Not! sad BamMl sties of pMIV-Zsgreen (already containing the peptide signal and tfee CD33 scFv, Alternatively. CD28 domain, can be substituted by ICOS domain (SEQ ID NO: 4).

la addition ID CD28 and ICOS domains, a fusion domain comprising fragments of CD28 and ICOS mfraceiiular sig aling domains will be .engineered (SEQ ^' ID NO: 5) and used to generate . ddiiioaal duffierie receptors. Such coafig raiion, where the . chimeric -nsceptor- comprises an antigefrbiridhig fragment, as anti-CD33 light chain variable region, a linker, an ¾nii-C033 heavy chain variable region, CD2S/i€ S hybrid, region (including a TM region of CD2% and signalin ; domain of TCR-C molecule.

Example ami no acid sequences of components that ma be used to generate the c imeric receptors are provided herein, such, as COM. omain (SEQ: W. NO; ICOS omaoi (SEQ ID Μ0: 7},.£Μ ΟΟ8 hybrid do∞! (SEQ 3D I O; H), and XCi-ξ .are pri d herein Alieraativeiy; the c merie receptor may be generated as well (Section B.)

B. Alternative Metkmifi>r (kmratmn fanti~€D33 CAM c(mstriw&

Schematics of example chimeric receptors are presented to ^' FIGURE- 7, panels A-I . The chimeric receptor will be generated using an extracellular humanized ^' scFv recognkkg the CD33 antigen, linked to an estraceilulat CDS hinge region,, a transmembrane and cytoplasmic signaling domain, and a CD3 ζ-sigr ing: chaiii (FIGURE 7, panel B). DMA encoding the anti-CD33 chiffieric receptor will be generated by using a temriiasd scF (Essand et at, J Intern MedJ2 } 273(2):166). Alternatives include a. CAR T cell that contains OX- 1 or ^' 4l*BB in place of CD28 or CD28 OX! or CD2S/4-1-B-B hybrids

(FIGURE 7, panels C and D),

la order to generate the anit-CD33 scFV seqaence, the coding regid»s of the heavy m& light chains of the variable regjoos of the anti~€D33 a ihody described above (SEQ ID NOs: 1 and 2) will be amp!; tied with specific primers and d

for ex ression in cells,. To evaluate the binding strength ^' of (he scFv (single c¾ variable fragments) to the target antigen, the scFv will be expressed in Hek293T cells. For this purpose, the vector (pBW-Zsgreen containing the coding a eas) will be t nsformed .into E _> mil ToplW bacteria and the plasmids prepared. The obtaine expression vectors that code for the ScFv antibodies will be introduced by tfimfection info 1 2 3T cells. After eulturing the tramfteled ceils fm fm dap, the sup r Mtani wB be reftiove ;aiid l m iibo ies p ftified..

The resulting antibodies can be h manized using l¾¾i» wo?"k su stiiofjoiis by protocols known ¼ the art See, for example, one such protocol is provided by BioAtia (San Diego), where synthetic CD encoding fragment libraries derived from a template antibod are tigated to huftifttt framework region, encoding fragments from a human framework pool limited to genuine ^' sequences ftom a ftmcUoaaiiy expressed antibodies

Cbi alkeo ^^ ^{^} ^

Affinity maturation may be performe in order to improve antigen binding affinity _* This can be accomplished using general techniques known in the art _* such as p age display (Sehier R., ,l Mai Bk>i(\9%), 263:551). The variants can be screened-fox their biological activity (<¾>,, binding affinity) using for example Biaeofe analysis. la order to identify hyjtervariafefe region residues which would be good candidates for modification, .alanine scanning mutagenesis can fee performed to identify hypervariable region residues -contributing signi!ieatnly to antigen binding. A4$tioa.aiiy ₅ com j«at0n¾}-.libi¾rie$ d s ribed -by- ea» afeo be used for improving the affinity of the antibodies ( ajpai et al _f! PNAS (2005) 102(24); 846f>). Alternatively, BioAtta has developed a platform: for the rapid and efficient, affinity maturation of antibodies, which, can also be u ilized for the purposes of antibody opti«*i¾atioa (hioatla om/applieations/fMciii)nal--iBaturation/).

(Q Assembly &fCAM e sim t

ext, the anti-CD33 scFv will be linked to an extracellttlar CD8 hinge region, a traiterrierrtbrane and cytoplasmic CP28.signaling domain, and a OD3 ζ-stgnaliog ^' chain... Briefly, printers specific for anii~C ^' D33 scFv sequence will be used, to amplify the scfy as described above. Plasmid (pONl DH). (ww,invivogeticom/pun.o-cd8a.) carrying the complete human CDS coding sequence will be used to amplify CDS hinge and

traftsmerftbrane domains {amino acids 135-205). C.D3 ζ fragment will be amplified -fro.© the Invivogen plasmid pORF9~hCD247a (httpii iny

hCD247a I0E26vO6.pdf) carrying the complete hitma ϋ03ζ coding, eqnenie. Finally, the Cf)28 (amino acids 133-220, corresponding to TM and signaling domains of CD28) will be ainpiii ed from cDN generated Msing R ^' NA -collected from- activated t cells by Trizol method. Fragments containing anti«CD33-scFv-CDS*hinp- FM-CD28-CD3C will be assembled using splice overlap extension (SQE) PCE.. The resulting PC fragment will then be cloned into pELPS ientivira! vector. pELPS is a deri vative of the tfeifd-genemtion leniivkat vecte Ri¾~SIM'CM^eOFP'WPRE ¾ which the CM V promoter wa apiaeed wit the EF-- i a promoter and the ^' central po!yparine tract of HIV was inserted.5' f the promoter ( ifone et aL Moirker. (2009) ( ): 1453, f et al, tf&iH. (2011) fS}; 725). All constructs will be verified by sequencing.

Alternatively., CAEs containing !COS, CD27, 4iBB, or OX-40 signalin domain instead of CD28 dora¾ia will be generated, introduced into T-cells and tested for the ability to eradicate CD33 positive ceils {FIGURE 7, pa te! C), The generation of ' ri-pBeraUot chimeric receptors are also contemplated (FIGURE 7, panel D), whic combine multiple signaling domains, such as CD¾~CD28~4iBB or CD3.¾-CD28-OX , to further augment otency (Sadelam et at, Cmcer Discov. (2013) 4:388),

CA S- enerated i section {1 ) of this Example will be transfected. into immortalized fetal renal 2S3T packaging cells together with Cells will be cultured with, high glucose

DMEM, including 10% FBS, 100 /ml penielHia and 00 pg mi streptomycin. 48-72 hours post-timtsfeetion the snpeniataat will be collected, and the recombinant lentivires concentrated .in DMEM without FB$„ Primary CPS ^*' T ceils will next be transduced at multiplicity of infection CM01) of*-5-.i in the presence of olybrene. Huma recombinant !L-S CR&B Systems) will be added ever other day (50 lU rnt), T cells will be catered for ■- ^■' 14 & s a fter stimulation Transdeetion efficiency of toman primary T cells will be assessed by expression of a Zs&reen reporter gene (Gloatech,: Mountain Vie , CA).

& ififttswti ttf CAM. eelh iffM a P ihmt

Prior to ti -iv, mfamm. of aniiCD33 CAR T cells into the patient, cells will be. washed with phosphate ^' buffered saline and concentrated. A cell processor such as a fiaeraonettcs CellSaver (Haemonetics Corporation, Braintree, MA), which provides a closed and sterile system, will be used for th washing and concentration steps before formulation.

expressing∞ti~CD33 chimeric receptors and immune cells expressing aoti~CP 7.RA.

chimeric receptors separately and infusmg the patient with both types of immune ceils separately, or 2) genefating imnume cells that target both CD33 andCD45RA simultaneously ( akarfe et al, C¾t¾?r (2): 151. {2014}}.

11.·. Aatotogtfes-ftematepoieffe Stem Ceil f ransplfmf (IISCT) Using€ί)34 ^' ¾®33

^' Ceils · · ·

It is understood that the protocols regarding stem eel! tsektior* from patients, conditioning regimens, as well as ittfusion of patients wife stem cells wy greatly depending on the patient's age, condition, treatment history, and. institution -where the treatment i$ conducted. Thus, the protocol described below is merely a .example and. is subject to routine optimization by a person having ordinary skill in the art.

.4 ImMkm of Hematopoietic Stem Ceils (Mag Peripheral leed- Stem Ceii

(PBSG) Mobilization- .following Adoptive Tmrnfer efen&O)33- Cdft T Cells

AML patient will be stimulated by i. v _t administration of granulocyte colony- stimulating factor (G-CSF) 1 mg kg per day, CD34 ceil, positive selection will be performed M immunomagnetk beads and an i muuomagnettc enrichiaent device. A 'mmh m of 2x !0° CD34 ^': cells/kg body weight are expected to be collected tssiiig a Fen all CS 3000+ cell separator { ^' Park et al,, -Ba M rr l ^iMlMim (¾)0¾ 32:88 ).

S* Conditi mg Regimm of a Pa&mt-

The eoBditio ing regimen for autologous peripheral. Mood stem, cell transplant (FBSCT): will be carried out asing etoposide (VP- 16) - c clophosp aiaMe (CY) + total body irradiation (TBI). Briefly, the regimen will consists of etoposide (VI 6) at 1.8g/rr iv. constartt infusion (civ.) over 26 h as a single dose followed by cyclophosphamide (C Y) at 60 mg ^' kg per din* f v over 2 ¾ for 3 ays,■ follo ed by total o it d¾ia TB .¾30 ^' .cCjy per day for the xi 3 days.

To calculate the -dose* ideal body weight or actss! body weight, wfeiehsver is iess,: will be used. As previously mentioned, factors such as the state of the cancer patient, patient's age, prior treatment, as. well as die type of institution where the procedure is conducted will all be take into eOBsideralion. when determining the precise conditioning regiroefL

I ⁾ . Tramfeetim ef€B34* ceih MSCs to generate€Β3 ^' 4* ^" €Β33 celk

Freshly isolated peripheral blood-derived CD34 ^'V cells ( ron} step 4) will be seeded at 1 ¥ Iff cells i n seram-ftee Cel!Gro SCGM Medium, in the presence of cells culture grade Stem Celt Factor (SCF) 300 ng/ml F.LT3-L 3IM ng mi Tteombopoieim (TPO) 100 ng and IL-3 60 «g ftil . Following 24 hoar, of pre-sthudation, CD34 HSCs will be tx nsfeeied with LestiCRiSPl v2 containing Cas9 and C ^' 033 g.R A. using- Amaxa Human 034 ceil. Nucteofector t (tW8> (#VPA» 1003) ( andal et at, Cetf Stem -Cell (2014) (S):643). 24- S hours po&Mransfeetion, CO ' C.D33 ^* cells are selected: with 1 ,2 pg/rol puroinyeiii.

Followiftg the pttroraycin selection,€B34 ⁺ CD 3 ^" cells will be maintained ^' ia pnrtraiyc-in-free media for. couple of days,

E Eeinf i t of€B34 ^' €B33 ^' ceils into the paitmt

€034' ce!kiran&fected ec vivo with CRlSF -eas9-CD33 (CD34¾D33 ^" ceils) are iirjiBediately rehrflssed throitgh a Hickman ca eter using a standard, blood, adntmis«ion.se.t witJtoat a flier {Bacehi-Bey Abiaa et ai. JAMA (2015} 313(15);i 551 ⁾ .

Generally, patients lu> have undergone the above outlined treatment protocol will be monitored for the reappearance of eircu mg blasts aid eyiopenias. Additionally, depending •on the underlying mechanism of AML in a specific patient he success of the treatment wil be monitored by. te ting for reappearance of an isf miaiiye i¾olecular or Mitogen tic mate or an hif o ti ve flow cytometry tte n . For ex mple, reemergence of a BCR-ABL signal •In Philadelphia: chreniosome-positive AML will be detected using. aoreseeat in situ hybridization (FISH) with probes lor BCR (on chromosome 22) and ABL (on. chromosome... ,%

To eyahsate the success of CFJ33 deletion via CRlSPR~Cas9 sysi^^ri heraihlood DW celts will be isolated from patients (post~tra«spla»t and assessed for the C 33 expression, for example using flow eytomen , Western blotting, or tomnnohistocherntsny.

As described herein, the HSCT described in this Example can be either autologous or allogeneic, and both approaches are suitable and can be incorporated in the methods described in the present disclosure. 111. Optional Step: Continued, treitttneitl of a aieiit itfe a€B33 antibody attached to a tmn

A. Treatment of Patients with C $3 fmmmwtoxin Gemtuz mah Ozpgumtein {GO}

Patients wil be treated, with 9 mg/nr of aati*€D33 antibody g&ntttHttaab ozogamiein (GO) as a 2-hotir intravenous iijiiis ii in 2 doses separated by 2 weeks (Larsoiiet at,€amer (2fM35) _? 104(7); 1442-52), GO is comprised ^' of a ^' kunanized monoclonal a»iibody against CD33 which is conjugated with a. cytostatic agent, calich amicin (FIGURE 8).

Alternatively, the anti~CD33 antibodies may be conjugated to different toxins, sae as diphtheria toxin,. P ewlomomis exoioxiB. A ( E% or ricin. toxin A chain (RTA) can be generate (Wayne et ah, tiod (2014} 12306): 2470), Similarly,: aijti-C S¾A antibodies siss be aiiacted to a toxin and induded; In lite treatiient regimen.

^: E¾MP!,E 4; : T cells mtl NK ceil 1mm ex ressing an ¾» I« P33 chimeric receptor i!iduce Cell death of target ce¾ expressiii CB33 iodiag- of chimeric receptors to€ 3

Chimeric receptors that bind CD33 (&&, CART! , CART2, CAR 3) were ge e ated usin convention, recombinant TJNA technologies and inserted into a pHTV-Zsgreen. vector (Addgeae; Cambridge, MA), The vectors containing the chimeric- receptors were used to genefaie tentivirai particles, which were used to transduce different ceil types, for example T cell ikes (e.g, _:! 293 T cells) and NK. ceil lines, (e.g., NK ^' 92 cells), Expression of the chimeric receptors was detected by Western blotting (FIGURE 9, panel A) and flow cytometry

(FIGURE 9, panel B),

Cells expressing the chimeric receptors were selected fey fluoteseence-activated ceil sorting (FACS) and assessed fot their ability to hind CE33, Briefly, ly sates of 293T ceils expressing the chimeric receptors were eoinctibated with CD3 or C0 -aliophycoGyan¾ (APG) conjugate. The samples were subjected o: protein electrophoresis and either stained with -Posceaa. pirotem siaftt ^' FIGURE 10, panel A.) or transferred: to a membrane and probed with as anti-CD3 primar antibody (FIGURE 10, panel B). In both cases, binding, between the chimeric receptors and their target, CD33,

K562 cells expressing the- chimeric receptors were also assessed for binding to CD33 by .flow cytometry using CD33 as a probe (FIGURE ii ^' K panel C). There was an. increase in the number of cells positive for expressio of the chimeric receptor (CART!„ C ARI2, or CART3) d C 33 Mnding as coni tred to cells caa Miiittg aa empty ¾eto control indiearittg the c nieric receptors bind to CD33.

Cytotoxicity i duced by cells expressing the clnmeric receptors

N -92 cells expressing the chimeric re<^ tewerel¾nctt¾«a criaracierked for the ab lity to induce cytotoxicity of target cells presenting CD33 on the ceil surface K562 are a i mm chroak myelogenous leukemia cell Ike that are G033*). To perforin the cytotoxicity assays, effector cells {immnse cells. sach as KK.-*)2 cells) ere infected with leabvirus particles encoding the chimeric receptors and ex anded. Seven da s post infection, cells expressing the chimeric receptors were selected by F ACS analysis by selecting for finoreseent markers also encoded by the chimeric receptor enco ing vector (e.g,, GFP+ or Red+). he selected cells that express the chimeric receptors were expanded for one week. Fourteen days post infection, the cytotoxicity assay was perlorraing involving staining the targes:, cells (cells expressing the- target cell-siirl¾ce liaeage-speciiic a igerr,€033) with earboyxflriotescem saecmiroidyl ester (CFSB) and cotmiing both the targe, eels arid cells expressing the .chimeric receptors, Piierent ratios of target cells and cei ls expressing the chimeric receptors were coiflciibated in round bottom 96-well plates for 4.5 hrs, after which 7-aftri5TOactirK myciri D (7-AAP) was added to stab nom viable cells. Flow cytometry was performed to enumerate the population of viable and non-viable target cells. As shown in FIGURE 11 , panels A id B _s N 92 cells expressing chimeric receptors CA. T1 , CA T2, or CART3 induced a substantial amotrnt of cell death of target 562 cells at each of the cell ratios assessed.

To determine that the cell death of K562 cells was dependent on specific targeting of the chimeric receptor to CD33, K.562 were genetically engineered to be deficient in C033 asrag a CRiSFE/Cas system. Briefly, a human codon-ojrti i¾ed Cas9 eudoiwctaase and a gRNA. mrgeiitig a portion of the IgC domain of C.D33 were expressed in the .562 cells, resulting in populations of Cl¾3-deficsem R562 cells. The cells were expanded and: co~ mcubated with NK.92 cells expressing the chimeric receptors, wad the cyiofoxi ty assay: was perfbrMed as described above. As shown in FIGURE 12 paael A _s the pooled€D33-deikiest K562 cells showed a modest reduction in cell death with co-incubated with the N 92 cells expressin the chimeric receptors. However, when single clones of 0)33-deikient 562 cells were isolated, expanded, and used to -perform the cytotoxicity assays, a more significant re uction in cytotoxicity was observed (FIGURE 12, panel B),

Genetic engineering; human hematopoietic stem cells

Several gR As were designed, to hybridize te the IgC domain of CF33 (see, far ©csmpie, Tabie 4 SEQ ID NO: 1 1 or 2 ;-3 i), Each ef the gR As were expressed along with a Cas? endoneuiease in 562 cells. Toe expression ofCD33 was assessed fey flow cytometry (FIGURE 15). As shown for Crispr 3 (SEQ ID M): 28) and CrisprS (SEQ ID NO: 29), a significant reduction, in CD33 was found in cells expressing the CD33-targefir¾ CRISPR/Cfcf system, as compared to control cells expressing CD33,

The CD33-deiIcient hematopoietic stem cells were also assessed for various characteristics, including proliferation, erythopoeiiic differ esi ation, and colony formation. Briefly, CD33-dei¾ient hematopoietic stem cells and control cells were induced to differentiate by exposin the cells to heroin, and CD7I, a marker of rythroid. precursors, was assessed by flow cytometry at different time points (FIGURE. 16, panels A and B), CD33- defieiem hematopoietic stem cells underwent eryt opoeitie differentiation and flo

.cytometric profiles appeared, similar to the control cells (CB33+). Th cells were als subjected to MIT assay to measure the metabolic activity of the C033-defIcient

heniato oieiic stem, cells. As shown in FIGURE 16, panel C, the CD33-defieieoi hematopoietic stem, cells performed comparably to the control cells. Finally, the ability of the cells to proliferate and form coloaies of cells was observed usin a microscopic colony formatioft assay. Again, the C033-deficient hematopoietic stern cells were able to form colonies to a similar extents as the control cells (FIGURE 18). These results indicate the ORiSPR/Cas _: de ion -of a $Mfa&- of CD33 does mt sipd ieandy impact the ability of the cells i» pr oii ferate, dii ¾i¾niial¾ of to colonies,

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined in toy

combination. Each feature disclosed in this spedfication may be replaced by m

alternative feature serving the same, equivalent, or simiSarpurpose. Titos, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or siniilar fetes,

From the above description,, one of skill in the art: can. easily ascertain the essential characteristics of tire present disclosure, and without, departing from the spirit and scope thereof, can make various changes and modiikaitorss of the disclosure to -adapt it to various usages and conditions. .Thus, _. other embodiments are also within the claims.

EQUIVALENTS

While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a vari ety of other means a»d,% structures for performing the taction and/or obtaining the .results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to h within the scope of the inventive embodiments described herein. More .generally, those skilled in the art will readily appreciate that ail. parameters* dimensions* materials, and.

configurations descnbed herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for whic the inventive teachings is/are used. Those skilled in the art will recognix-e, or be able to ascertain using no more than routine xperimentation, many eQuivaleats to the specific inventive embodiments described herem It is, ¾e¾J e, to be u derstood that tbe--fo«eg0i» :embod,ime»i$ are presented by way of example only and that, ifhin the sc0^. the¾p en4ed.claim¾ and .equivalents thereto, inventive embodiments- .may he practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual leature, system, article, material, Mt, and/or method described herein, in a diti n any combination of two or more such, features, systems, articles, materials, ki ts, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive .scope- of the present disclosure _*

have the same meaning as "and/of as defined above. For example, when separating items in a list, "or" or "and/of' shall be interpreted as being inclusive, le. _; the inclusion of at least one. hu t also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Onl terms clearly indicated to the contrary, such as "only one of or "exactly one of or, when used in the claims, "consisting of," will refer to the inclusion of exactly one e temeut of a number or list of elements. general, the term " r" as used herein shall, only be interpreted as indicating exclusive alternatives (ie, "one or the other hot not both''} when: preceded b terms of exclusivity, such as "either, ^* ' "one of ^' " "only one of? or "exactly one of." "Consisting essentially o T ^! when used in the claims, shall have its ordinary meaning as used in. the field of patent law.

As used hereta in the spciikation and in the elaims, the phrase "at least one," in reiereiice to a list of one or more el ements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessaril including at least one of each and every element specifically listed within the list of elements and not ' ^' exclsding any combinations of elements in the listof elsnteats. This definition also allows thai di^^Ls.j¾¾ >tio ily½ _: i¾^tefliia: ihaa ihe elements specifically ide ifk within the list of elements ^' to whic the phrase "at least ^' one ^* ' refers, whether related o isnfelated to those elements specifically identified, Thas, as a non-limiting example, "at least one of A an B" (or, equivalentiy, "at least one of A or B _? " or, equivalently "at least one of A and/or 6") cm refer, in .one embodiment, to at least one, optionally includi ng more than one, A, with o B ^' resent (and optionally including elements other than B); in another

embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); i yet another eftibodiftient, to at least one, optionally inckidiag more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It .should also be understood that, unless clearly indicated to the contrary, i». any .methods claimed herein that aielade more than one step or act, the orde of the steps or acts of the Method is not necessarily limit to the order in which the steps or acts of the method are recited.