NZ749647A - Pseudotyped oncolytic viral delivery of therapeutic polypeptides - Google Patents

Abstract

Described herein are pseudotyped oncolytic viruses comprising nucleic acids encoding an engager molecule. In some embodiments, the pseudotyped oncolytic viruses comprises nucleic acids encoding an engager molecule and one or more therapeutic molecules. Pharmaceutical compositions containing the pseudotyped oncolytic virus and methods of treating cancer using the pseudotyped oncolytic viruses are further provided herein. dotyped oncolytic virus and methods of treating cancer using the pseudotyped oncolytic viruses are further provided herein.

Description

PSEEDOTYPED ONCOLYTEC ViRAL DELIVERY OF THERAPEUTEC E’TIDES REFERENCE To RELATED APPLICATIONS {twill} This application claims ty to US. Provisional Application No. 62/357,195, filed lune 30, 20l6, the content of which is incorporated herein by reference in its en“P4-inHD«2 Descnierion or THE TEXT Fawn SrinMiTTEn ELEC'E‘RUNICALLY {9992} The contents of the text tiled submitted electronically herewith are incorporated herein by reference in their entirety: A er le format copy of the Sequence listing (tile name: ONCR_004_02W0_S'l"25.txt; date recorded: June 30, 20l‘7; file size: l 93 kilobytes).

OUND on THE INVENTION } Patients with certain hematologic and. solid tumors remain in need of new therapies. The use of bispecific dies to direct cytotoxic T cells to tumor cells, and chimeric antigen receptors (CARS) to engineer antigen specificity onto an immune effector cell are being demonstrated to provide a therapeutic . Also, oncolytic virus technologies are nsefiil additions to the t standard of care of solid tumors, expected to have a safety profile and the ability to , replicate in, and lyse tumor cells. l-lovvever, the antitnrnor efficacy of the bispecific antibodies, CARS and oncolytic virus are suboptimal, demonstrating the continued need for further advances of oncology, antibodies, and oncolytie virus therapy.

SUMMARY 0F 'i‘nit EON {9094} in some embodiments, the present in ention provides a pseudotyped oncolytic virus comprising a recombinant nucleic acid sing (i) a first nucleic acid sequence encoding an engager polypeptide, wherein the engager polypeptide comprises an activation domain specific for an antigen expressed on an effector cell and an antigen recognition domain specific for a cell—surface antigen expressed on a target cell. in some embodiments, the antigen recognition domain specifically binds to a tumor antigen. in some embodiments, turner antigen is selected from Table 2.

{MEGS} In some embodiments, the t invention provides a pseudotyped oncolytic virus comprising a recombinant nucleic acid comprising (i) a first nucleic acid ce ng an engager ptide, wherein the engager polypeptide comprises an activation domain specific for an antigen expressed on an effector cell and a therapeutic molecule domain that binds to an inhibitory antigen expressed on a cell surface. In some embodiments, the therapeutic mo ecule domain specifically binds to 9131, FULL or CD47. In some embodiments, the recombinant nucleic acid further comprises a second nucleic acid sequence encoding a eutic polypeptide. In some embodiments, the eutic ptide is an immune modulator polypeptide. In some embodiments, the immune modulator polypeptide is selected from a cytokine, a costimulatory le, an immune checkpoint polypeptide, an anti— angiogenesis factor, a matrix metalloprotease (Mb/Ill), or a nucleic acid. {@096} In some embodiments, the immune checkpoint polypeptide comprises (i) an inhibitor of PD—l, PDLul, CTLA—z‘l, LAG3, TIM3, neuropilin, or CCRA; (ii) an agonist of , OX~40, or CDZS, or (iii) a combination of (i) and, (ii). In some embodiments, the immune checkpoint polypeptide comprises an MMP, wherein the MM? is MMP‘). In some embodiments, the immune checkpoint polypeptide ses a cytoltine, wherein the cytokine is selected from lLUIS, IL~12, and CXCLlO. {9697} In. some embodiments, the effector cell engaged by the engage]? molecules herein is a. T cell, an NKT cell, an NK cell, or a macrophage, In some embodiments, the activation domain of the effector molecule specifically binds to CD3, CD4, CD5, CD8, (Die, (2928, CD40, 03134, CDl37, or NKGZD. {(3098} In some embodiments, the inant nucleic acid provideds herein are multicistronic sequences. In some embodiments, the istronic sequence is a bicistronic sequence or a tricistronic sequence. in some embodiments, the multicistronic sequence comprises a picornavimséZa—like sequence, and wherein the first and second nucleic acid sequences are expressed from a single promoter sequence present in the recombinant nucleic acid. {9009} In some embodiments, the t in /ention provides a pseudotyped oncolytic virus comprising a recombinant nucleic acid sequence comprising (i) a first nucleic acid sequence encoding an engager polypeptide, wherein the engager ptide comprises an activation domain specific for an antigen sed on an effector cell and an antigen recognition domain specific for a tumor cell antigen expressed on a target cell, n the antigen expressed on the effector cell is C793, and wherein the tumor cell antigen is CD19. In some embodiments, “the recombinant nucleic acid sequence encodes a polypeptide sequence that is at least 90% identical to SEQ ID NO: 44. in some embodiments, the recombinant nucleic acid sequence WO 06005 comprises SEQ ll) N0: 43. ln some embodiments, the recombinant nucleic acid sequence further comprises (ii) a second c acid sequence encoding a eutic molecule, wherein the eutic molecule is lL—lZ. In such embodiments, the recombinant c acid sequence encodes a polypeptide sequence that is at least 90% identical to SEQ ll) N0: 54. In some embodiments, the recombinant nucleic acid sequence further comprises (ii) a second nucleic acid sequence encoding a therapeutic molecule, wherein the therapeutic molecule is lL—ld. in such embodiments, the recombinant c acid sequence encodes a polypeptide sequence that is at least 90% identical to SEQ ll) NO: 53, In some embodiments, the recombinant nucleic acid sequence further comprises (ii) a second nucleic acid sequence encoding a therapeutic molecule, wherein the therapeutic molecule is CXCLlO. in such embodiments, the recombinant c acid sequence encodes a ptide sequence that is at least 90% identical to SEQ ll) NO: 55, in some embodiments, the recombinant nuc eic acid sequence r comprises (ii) a second nucleic acid sequence encoding a therapeutic molecule, n the therapeutic molecule is MMEX). [6018} In some embodiments, the present invention provides a pseudotyped oncolytic virus comprising a recombinant nucleic acid ce comprising (i) a first nucleic acid sequence ng an r polypeptide, wherein the engager polypeptide comprises an activation domain specific for an antigen expressed on an effector cell and an therapeutic molecule domain specific for an inhibitory antigen, wherein the antigen expressed on the effector cell is CD3, and wherein the inhibitory antigen is PDLl. In some embodiments, the recombinant nucleic acid sequence comprises a nucleic acid sequence encoding a polypeptide sequence that is at least 90% identical to SEQ ll) NO: 50. in some embodiments, the recombinant nucleic acid sequence comprises SEQ ID NO: 49. In some embodiments, the recombinant nucleic acid sequence further comprises (ii) a second nucleic acid sequence encoding a therapeutic molecule, wherein. the eutic molecule is Ill-12. in. some embodiments, the recombinant nucleic acid sequence encodes a polypeptide sequence that is at least 90% cal to SEQ ll) NO: 63. in some embodiments, the recombinant nucleic acid ce further comprises (ii) a second nucleic acid sequence encoding a therapeutic molecule, wherein the therapeutic molecule is lL— . in some embodiments, the recombinant nucleic acid sequence encodes a polypeptide sequence that is at least 90% identical to SEQ ll) NO: 62. In some embodiments, the recombinant nucleic acid sequence further comprises (ii) a second nucleic acid ce encoding a therapeutic molecule, n the therapeutic molecule is CXCLlO. in some ments, the recombinant nucleic acid sequence encodes a polypeptide sequence that is at least 90% identical to SEQ ll) NO: 64. in some embodiments, the recombinant nucleic acid sequence further comprises (ii) a second nucleic acid ce encoding a therapeutic molecule, wherein the therapeutic molecule is h’lMP‘). In some ments, the engager molecule further comprises a third binding domain. in some ments, the third binding domain comprises an iminunoglobulin Fe domain, in some embodiments, the recombinant nucleic acid sequence encodes a polypeptide sequence that is at least 90% identical to SEQ ll) NO: 52. in some embodiments, the recombinant nucleic acid sequence comprises SEQ ll) N0: 5 l. {can} in some embodiments, the present invention provides a pseudotyped oncolytie Virus comprising a recombinant nucleic acid sequence sing (i) a first nucleic acid sequence encoding an engager polypeptide, wherein the engager polypeptide comprises an activation domain specific for an antigen expressed on an effector cell and an therapeutic molecule domain specific for an inhibitory antigen, wherein the antigen sed on the effector cell is CD3, and wherein the inhibitory antigen is SlRPld. in some embodiments, the recombinant nucleic acid sequence comprises a nucleic acid sequence encoding a ptide sequence that is at least 9i % identical to SEQ ll) NO: 46 or 48 in some embodiments, the recombinant c acid sequence comprises SEQ ll) NC): 45 or 47. in some embodiments, the recombinant nucleic acid sequence r comprises (ii) a second nucleic acid sequence encoding a therapeutic molecule, wherein the therapeutic molecule is lL-lZ. in some embodiments, the recombinant nucleic acid sequence encodes a polypeptide ce that is at least 90% identical to SEQ ll) NO: 58 or 59, in some embodiments, the recombinant nucleic acid sequence further comprises (ii) a second nucleic acid ce encoding a therapeutic molecule, wherein the eutic molecule is lL—lS. in some embodiments, the inant nucleic acid sequence encodes a ptide sequence that is at least 90% identical to SEQ ll) NO: 56 or 57. in some embodiments, the recombinant c acid sequence further comprises (ii) a second nucleic acid sequence encoding a therapeutic molecule, wherein the therapeutic molecule is CXCLlO In some embodiments, the recombinant nucleic acid sequence encodes a polypeptide sequence that is at least 909/5 identical to SEQ ll") NO: 60 or til. ln some ments, the recombinant nucleic acid sequence fiirther comprises (ii) a second nucleic acid sequence encoding a eutic molecule, wherein the therapeutic le is MMPQ. in some embodiments, the inant nucleic acid sequence s a polypeptide sequence that is at east 90% identical to SEQ ll) NO: 65 or 66. in some embodiments, the recombinant nucleic acid sequence further comprises (ii) a second nucleic acid sequence encoding a therapeutic molecule, wherein the therapeutic molecule is an anti—PDl_,l scFV linked to an lgGl Fc , in some embodiments, the recombinant nucleic acid sequence encodes a polypeptide sequence that is at east 90% identical to SEQ ll) NO: 61% or 70. In some ments, the recombinant nucleic acid sequence comprises SEQ lD NO: 67 or 69. {9012} In some ments, the pseudotyped oncolytic s of the present invention are selected from adenovirus, herpes simplex virus l (I-ISV l), niyxoma virus, us, poliovirus, vesicular stomatitis virus (VSV), measles virus (MV), lassa virus (LASV), or Newcastle disease virus (NDV). In some ments, the pseudotyped oncolytic virus comprises a d neurotropism activity and/or neurotoxicity activity in a human subject as compared to a reference virus, In some ments, the reference virus is i) a non—pseudotyped oncolytic virus, or ii) a vaccinia virus. In some ments, the pseudotyped oncolytic virus is an attenuated tic virus. in some ments, the virus is not a vaccinia virus. {£110.13} In, some embodiments, the pseudotyped oncolytic viruses of the present invention comprise a single recombinant nuc eic acid. In some embodiments, the pseudotyped oncolytic viruses se a ity of recombinant c acids. in some embodiments, the oncolytic virus selectively infects a. target cell. In some embodiments, the target cell is a tumor cell and wherein the oncolytic virus is e of selectively replicating within the tumor cell. {0914} In some embodiments, the engager polypeptide is a bipartite polypeptide and is comprised of an antibody, an antibody domain, a human immunoglobulin heavy chain variable domain, a dual—variable~tloinai,n antibody (DVD—lg), a 'I'andab, a. diabody, a flexihody, a dock-and-loclc antibody, a Scorpion polypeptide, a single chain vanable fragment (scFv), a BiTlE, a y, an Fc—engineered IgG, an Fcab, a MabZ, or DART polypeptide, {0015} In some embodiments, the present invention provides a pharmaceutical composition comprising any of the pseudotyped oncolytic viruses described . in some embodiments, the pseudotyped oncolytic virus induces an immune response. In some embodiments, immune se is selectively cytotoxic to a target cell. In some embodiments, the target cell is a solid tumor cell or a hematologic cancer cell. In some embodiments, the target cell expresses one or more tumor antigens. in some embodiments, the one or more tumor antigens are selected from Table 2. were; In some embodiments, the present invention provides a method of treating a cancer in a subject in need. thereof, comprising administering a therapeutically effective amount of an oncolytic virus described herein or a pharmaceutical composition described herein. In some embodiments, the method further comprises administering one or more additional therapies to the subject in need thereof. in some embodiments, the one or more additional therapies comprise surgery, radiation, chemotherapy, immunotherapy, hormone therapy, or a combination thereof. {6&7} in some embodiments, the present invention provides a method of treating one or more tumors in a subject in need f comprising administering a therapeutically ellective amount of an tic Virus bed herein or a pharmaceutical composition described herein to a patient, wherein the one or more tumors express a tumor antigen. {0018} ln some embodiments, the present ion provides a method of selecting a patient for treatment comprising (a) determining the expression of a tumor antigen on one or more tumor cells derived from the t; and (b) stering an oneolytic Virus described herein or a pharmaceutical composition described herein if the tumor cells obtained from the patient express the one or more tumor antigens. in some embodiments, the one or more turnor antigens are selected from Table 2. in some embodiments, the present invention es a method of delivering an engager polypeptide and a therapeutic polypeptide to a tumor site comprising administering to a t in need thereof an oncolytic Virus described herein or a pharmaceutical composition described herein.

BRIEF Descarterion on THE DRAWINGS {0919} FIG. l rates an amino acid sequence of a {Big—CD3 ite ptide comprising a first single chain variable fragment (SCFV) directed against CDl9 linked, to a. second. ser directed against CD}. {302%} illustrates an amino acid sequence of a CDl‘B-CDB—lld 5 uct encoded by a bicistronic gene. The first gene encodes a bipartite polypeptide comprising a first scFv directed against C919 linked to a second scFv directed against CD3. A second gene encoding lL—lS is linked to the bipartite gene sequence by a TZA self-cleaving polypeptide linker. {(31}le PEG. 3 illustrates an arnino acid sequence of a CDl9—CD3nlLlZ construct encoded by a multicistronic gene. The first gene encodes a ite polypeptide comprising a first scFv directed against CDl‘? linked to a second scFy directed against CD13. A second gene encoding the p35 subunit of lL-lfi is linked to the bipartite gene sequence by a TEA selt‘ucleaving polypeptide linker and a third gene encoding the p40 subunit of anlZ is linked by a 'l‘ZA self— clcaving ptide linker. {($22} illustrates an amino acid sequence of a C9l9—CD3-CXCLlO construct encoded by a bicistronic gene. The first gene encodes a bipartite polypeptide comprising a first scli'y directed against CDl9 linked to a second. scFy directed against CD3. A second gene encoding CXCLlO is linked to the bipartite gene sequence by a TZA leaving polypeptide linker. {6&3} rates an amino acid sequence of a SlRPld—CD3 bipartite polypeptide comprising a first protein comprising the first 120 amino acids of SlRPld linked by a single amino acid linker to an scFV directed against CD3. [9024} FIG 6 illustrates an amino acid ce of a SlRPld-CDB-LL bipartite polypeptide comprising a first protein comprising the first 120 amino acids of SlRPio. linked by a G4S motif linker to an scFy directed against CD3. {$925} rates an antino acid sequence of a d—CDfi—lldfi construct encoded by a bicistronic gene The first gene encodes a. bipartite polypeptide comprising the first 120 amino acids of SlRPl a linked by a single amino acid linker to an scFv directed against CDE.

A second gene encoding lL-lS is linked to the bipartite gene sequence by a ’l‘ZA self~cleaving polypeptide linker. [9026} illustrates an amino acid sequence of a, SlRPld~CD3—lLl5—LL construct encoded by a bicisti’cnic gene. The first gene encodes a ite polypeptide comprising the first l2i’) amino acids of SlRlll Gt linked by a. G48 motif linker to an sclE'y directed against CD23. A second gene encoding lid—lo" is linked to the bipartite gene sequence by a TZA selfucleaying polypeptide linker. [3627} illustrates an amino acid ce of a SlRl’lcc-CDBJLlZ construct encoded by a inulticistronic gene. The first gene encodes a bipartite ptide comprising the first l20 amino acids of SIRP l. e: linked by a single amino acid linker to an scFV ed against CD3. A second gene encoding the p35 t of lL-lZ is linked to the bipartite gene sequence by a TZA leaving polypeptide linker and a third gene encoding the p40 subunit of lL—lZ is linked by a TEEA self—cleaving polypeptide linker {91328} FIG 10 illustrates an amino acid sequence of a SlRPlo-CDE—lLlZ—LL construct encoded by a niulticistronic gene. The first gene encodes a bipartite polypeptide comprising the first 120 amino acids of SlRPld linked by 3 G45; motif linker to an, SCFV directed against CD3: A second gene encoding the p35 subunit of ll.rl2 is linked to the bipartite gene sequence by a TZA self—cleaving polypeptide linker and a third gene encoding the p40 subunit of anlZ is linked by a ’l‘ZA self—cleaving polypeptide linker. {9029} FIG. ll illustrates an amino acid sequence of a. SlRllld—CDS—CXCMO construct encoded by a bicistronic gene. The first gene encodes a bipartite polypeptide comprising the first lZO amino acids of SlRPld linked by a single amino acid linker to an scFy directed. t CD3 A second gene encoding CXCLlO is linked to the bipartite gene sequence by a TEA self—cleaving polypeptide linker.

WO 06005 {sass} PIG. l2 illustrates an amino acid sequence of a SiRPlawCDS—CXCLlQ-LL construct encoded by a bicistronic gene. The first gene encodes a ite ptide comprising the first 120 amino acids of SIRPla linked by a 843 motif linker to an scFy directed against CD‘S. A second gene encoding CXCLlO is linked to the bipartite gene sequence by a TEA selfucleaving polypeptide linker. [0031} illustrates an amino acid sequence of a l’DLl-CDB bipartite ptide comprising a first scFV directed t PDLI linked to a second scli'y directed against CD3 {0032} illustrates an amino acid sequence of a PDLl-CD3—1Ll5 construct encoded by a bicistronic gene. The first gene encodes a bipartite ptide comprising a first scli'y directed against PDLl linked to a second scFV directed against CD3. A. second gene encoding llnlS is linked to the bipartite gene sequence by a TZA self‘cleaying polypeptide linker. @933} FIG. l5 illustrates an amino acid sequence of a 9i)Ll,—Cl)3mll_,l2 uct encoded by a multicistronic gene The first gene encodes a bipartite polypeptide comprising a first scFv directed against PDLl linked to a second scFy directed t CD3. A second gene encoding the p35 subunit of lL—lZ is linked to the bipartite gene sequence by a TZA self~cleaving polypeptide linker and a. third gene encoding the p40 t of lL—lZ is linked by a TBA, self— cleaving polypeptide linker. {0034} FIG. lo illustrates an amino acid sequence of a PDLl—CDE—CXCLlO construct encoded by a bicistronic gene The first gene encodes a bipartite polypeptide comprising a first scliv directed against PDLl linked to a second scliy directed against C133. A second gene encoding (IXCLlO is linked to the bipartite gene sequence by a TEA self-cleaving polypeptide linker. @035} FlG. 17 illustrates an amino acid sequence of a l’l)l.,l~CD3~Fc tripartite polypeptide comprising a first scFy directed against CD3, linked by a G48 motif linker to a second scFy directed against PDLl, which is in turn linked to the CHE—Cl-ES domain of human lgGl by an lgGl hinge. {3936} illustrates an amino acid sequence of a d—Clfi—lvll‘vll’lJ—Sln construct encoded by a ronic gene {Flt}. 18A) and an amino acid sequence of a SlRPld- CDB—MMP9—LL construct encoded by a bicistronic gene (3). {$637} FIG. l9A — l9C illustrate the binding of CDlQ—CD} Bi'l‘E constructs (A), SlRPld—CDB BiTE. constructs (FlG. lQB), and PDLl-CDB-Fc tripartite T cell engagers (FIG. l9C) CD3+ T cells, {6638} illustrates the cation of the T cell engager construct binding shown in FIG. l9. {9039} HG. 2i illustrates the CD3mspecitic binding of CDlQ~CD3 Bi'l‘E constructs ( A), -CDS‘ BiTE constructs (B), and PDLl-CD3—Fc tite T cell engagers (FIG. ZlC) through the use of an anti—CD3 antibody, OKT3. {904%} illustrates the specificity of the CD47—biriding SlRPld arm of a SIRE? l o—CD3 Bill-:3 construct. {$041} A — FlGr 23,8 illustrate the binding of CDlQ-CDB and SlRl’lot— CD3 Bill? constructs (FlG. 23A) to Rail cells (CDlVCDL’lT). % binding is quantified in 3. {6042} A HG 24B illustrate the binding of CDlg-CDE and SlRPld— CD?) Bi'l'E ucts (FlG. 24A) to U205 cells (CDlQ'CDél-T‘). % binding is quantified in FlG. 24B. {till-l3} A FlG. 258 illustrate the binding of CDlQ—CDB and Sllil’lot— CD3 Bi’l‘E constructs (FlG. 25A) to GBM30-luc cells (CDlQ'Cl‘fll-T). % binding is quantified in 3. {0044} A 8 rate the binding of CD3 and SlRPld~ CD3 Bi'l’E constructs (A) to U251 cells (CDlQI‘lMT‘). % g is quantified in .8. {hilt-£5} A — FlG. 27C illustrate the binding of PDLl—Fc-CD?) tripartite T cell engagers to U251 cells. The binding oi‘the PDLl-Fc-CD3 constructs (B) is compared to the binding of an anti-PDLl antibody (HG. 27A). Binding was not mediated by FcyRs. as UZSl cells do not express Fcle, Fez/RH, or Felell (FlG. 27C). {0046} rates CDl9uCD3 BiTE, SiRPla-CDS’ BiTE, and B— Fe tripartite T cell engagei‘wrnediated T cell—dependent cytotoxicity ('l‘DCC) chaji cells.

{Ml-4'7} illustrates Cl)19~CD3 Bi'l‘E and, l’l)Ll~Cl}3-l3c tite T cell engager—mediated TDCC of Tl-lPl cells. {9948} illustrates (IDl9uCD3 BiTE and PDLluCD3—Fc tripartite T cell engager-mediated TDCC of U25 l cells. {£1049} FlG. 3l illustrates —CD3 Bali—mediated TDCC of 293E cells compared to an osteopontinutusion control construct. {£3059} illustrates expression of SlRPloc—CD3 BiTE constructs from oncclytic—HS'V vectors. Expression of SlRPld~Cl)3 Bi’l‘E constructs with short linkers (Lanes l— 4 and ONCROSS in lanes 5-6; shown in FlG. 5) and SlRl’lo—CDS BiTE constructs with long linkers (ONCRllil’? in lanes 7—8, shown in are shown {9051} HG. 33 illustrates expression of PDLLCDBFC Bi’l’E constructs front oncolytic—l-lSV vectors. Purifed PDLl ~CD’3—Fc BiTlE protein is shown in lanes l—41 Concentrated viral supernatants are shown in lanes 5—6. [0052} A HS. 343 illustrates TDCC of UZSl cells by y produced SIRPld-CDR SiRPld~Cl)3-l_il., and Plfld—Clfi—Fc BiiF constructs. raphs of U25 l cell cultures after tion with the ted BiTE constructs and CD8+ T cells are shown in A. Activity of y produced BiTE constructs, measured by 0/0 of cell killing and quantified by flow cytometry is shown in FlG. 343. {$053} illustrates that Antieon ultrafiltration effectively removes virus from samples, as determined by Western blotting with polyclonal —lSV antibody, and indicated that BEE—killing is due to the BiTE and not viral infection. @954} rates a n representation of the tion of a. pseudotyped oncolvtic virus and a recombinant oncolvtic viius and infection of a target cell by the respective pseudotvped oncolytic virus and the recombinant oncolvtic virus. [3655} illustrates an amino acid sequence of a SlRl’ldnClJElnPDLlnli’c (Sh) construct encoded by a. bicistronic gene wherein the first gene encodes an anti—PDL l seFv linked. to an lgGl Fc domain and the second gene encodes a bipartite polypeptide comprising the first lZO amino acids of SlRPld linked by a single amino acid linker to an scFv directed against CD3. {(9956} FIG, 38 illustrates an amino acid sequence of a SlRPld—CD3~PDLl—Fc (LL) construct encoded by a bicistronic gene wherein the first gene encodes an anti-PDLI scFv linked to an lgGl Fc domain and the second gene encodes a bipartite polypeptide comprising the first 120 amino acids of SlR‘i’lu linked by 3 G48 motif linker to an. sel—‘v directed. against CD3. {3057} FlG. 39 illustrates a schematic of a SlRPlo—CD3-PDU—Fc expression plasmid Two plasmid constructs, one for duiCDS—PDLl —Fc (SL) and one for SlRPld~CDE— l’DLl—Fc (LL) were generated. {3058} illustrates purification of the SlRl—‘ld—CIB Bi'l‘F; (Sis), SlRPld- CD3 BiTE (LL), and the anti~PDLl—Fc compounds from supernatants of ected 293 T cells.

A shows purification of anti—PDLl—Fc compounds assessed by Coornassiei MG. 403 illustrates purification of dwClB BilF nds as assessed by Western Blot using an anti—l—lis detection antibody.

{QGSQ} PIG. 4i shows results of a PDl/PDLl blockade assay. A tic of the assay is shown in A B. The results of the PDl/PDLl blockade assay using the anti—PDLl—f'c compound produced from 293 cells transfected are shown in FIG. AllC Dn'rAiLsn Desperation or run INVENHON {$068} The present disclosure provides novel engineered, oncolytic viruses, in ular pseudotyped oncolytic viruses that produce artite polypeptides and/or other therapeutic polypeptides for the treatment of cancer including solid tumors (cg, advanced solid tumors) and hematologic malignancies. in some embodiments, the tic virus is engineered by pseudotyping or other recombinant technology in. order to modulate the tropism of the virus to result in a viral infection specific for tumor cells and/or surrounding tumor stroma and/or for other beneficial purposes as provided herein. ln some embodiments, the multipartite and/or therapeutic ptides produced by the tic viruses described herein mediate or enhance the umor effects of the oncolytic viruses, such as by or-cell mediated lysis of target cells (eg tumor cells). The lYllC viruses described herein may have multiple (cg. dual) modes of action? including effector cell—mediated sis of target cells as a result of the sion of multipartite polypeptides, and viral—mediated destruction of target cells. The present sure further provides therapeutic compositions comprising the engineered oncolytic viruses and s of use in the treatment of solid tumors and hematologic malignancies, Overview [0061} in some embodiments, the present invention provides pseudotyped oncolytic viruses, compositions thereof, and methods of use for the treatment of cancer. The pseudotyped oncolytic viruses provided herein comprise recombinant nucleic acids that encode engager polypeptides and/or other eutic molecules (cg, therapeutic polypeptides), lly, the engager polypeptides function as effector cell engagers and generally comprise a first domain directed against an activation molecule expressed on an effector cell (eg, an activation domain or an engager domain) and a second domain directed against a target cell antigen (e.g an antigen recognition domain) or other cell-surface molecule (eg, a therapeutic molecule domain). Also provided are bipartite, tripartite or multipartite polypeptides (cg, comprising one or multiple engager domains, one or multiple antigen. recognition domains, or one or multiple therapeutic molecule domains, and optionally one or multiple other functional domains). ltfile} Also provided are methods of treating cancer, comprising the step of ring to human subject in need f a therapeutically etlective amount of the tic s or ceutical compositions thereof provided herein. Such methods optionally include the step of delivering to the human subject an onal cancer therapy, such as surgery, radiation, chemotherapy, immunotherapy, hormone therapy, or a combination thereof. llefiniticus {(9963} As used herein, the singular forms "a,” ”an," or “the” include plural references unless the contest clearly dictates otherwise. {titled} Throughout this specification, unless the context es otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a. stated element or integer or group of elements or integers but not the ion of any other element or integer or group of elements or integers, moss; As used, in this application, the terms “about” and “approximately” are used as equivalents. Any numerals used in this application with or without about/approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art. in certain embodiments, the term ximately” or “about” refers to a range of values that fall within 30%, 25%, 20%, l9%, 13%, l7%, l6%, 15%, l4%, l3%, 12%, ll%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than} of the stated reference value unless otherwise stated or otherwise evident from the context t where such number would exceed 100% ot‘a possible value).

} As used herein the specification, "subject" or "subjects" or “individuals” include, but are not limited to, mammals such as humans or non-human mammals, ing domesticated, agricultural or wild, animals, as well as birds, and c animals. in some embodiments, subjects are livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like, and icated animals such as dogs and cats. In some embodiments (cg, particularly in research contexts) subjects are rodents {eg, mice, rats, hamsters), s, primates, or swine such as inbred pigs and the like. in particular embodiments, the subject. is a human. “Patients” are subjects suffering from or at risk of developing a disease, disorder, or condition or otherwise in need of the compositions and methods provided herein. None of the terms require or are limited to situations characterized by the supervision (tag. constant or intermittent) of a health care worker (eg. a doctor, a registered nurse, a nurse practitioner, a physician‘s assistant, an orderly or a hospice worker). {title'l’} As use , "treating” or "treatment” refers to any indicia of success in the treatment or an'ielioration ofa disease or condition, particularly cancer, ng or treatment may he med m vilro and/or in vivo, and may comprise delivering an oncolytic virus, or composition thereof, described herein to a patient or subject in need thereof. in some embodiments, treating includes, for example, reducing, delaying or alleviating the severity of one or more symptoms of the disease or condition, and/or reducing the frequency with which symptoms of a disease, de ,eet, disorder, or adverse condition are enced by a subject or patient. l-lerein, “treat or prevent” is used herein to refer to a method that results in some level of treatment or amelioration of the disease or condition, and contemplates a range of results directed to that end, including but not restricted to prevention of the condition entirely. 8} As used , "preventing“ refers to the prevention of a disease or condition, eg, tumor formation, in a patient or subjet and may also he refered to as “prophylactic treatment.” Prevention of disease development can refer to complete prevention of the symptoms of disease, a delay in e onset, or a lessening of the severity of the symptoms in a. subsequently developed disease. As a non-limiting illustrative example, if an individual at risk of developing a tumor or other form of cancer is treated with the methods of the present invention and does not later p the tumor or other form of cancer, then the e has been prevented, at least over a. period , in that dual. {3069} The terms "therapeutically ive amount” and “therapeutically effective dose” are used interchangeably herein and refer to the amount of an tic viruse or ition thereof that is sufficient to provide a beneficial effect or to otherwise reduce a detrimental non~heneiicial event (tag. an amount or dose sufficient to treat a disease). The exact amount or dose of an oncolytic virus comprised within a therapeutically effective amount or therapeutically eilective dose will depend on variety of factors including: the purpose of the treatment; the , sex, age, and general health of the subject or patient; the route of administration; the timing of administrations; and the nature of the disease to he treated. The therapeutically effective amount for a given subject or patient is ascertainahle by one skilled in the art using known techniques (see, eg. Lieberman, Pharmaceutical Dosage Forms (vols, 16, 1992); Lloyd, 2726 Art, Shier/ice and Technology (it"Phczrmacemic .Z Compounding (1999); and ?icl<ar, Dosage Calculations ( l999l). {9676} “l’seudotype” refers to a virus particle, wherein a portion of the virus particle (eg, the envelope or capsid) comprises heterologous ns, such as viral proteins derived from a heterologous virus or non—viral proteins. Non—viral proteins may include dies and antigenuhinding fragments thereof. Preterably, a pseudotyped virus is capable of i) altered tropism relative to non~pseudotyped virus, and/or ii) reduction or elimination of a non- beneficial effect. For example, in some embodiments a pseudotyped virus demonstrates reduced toxicity or reduced infection of norntumor cells or nonwtunior tissue as compared to a non" pseudotyped virus. {0071} The term “targeting moiety” refers herein to a heterologous protein linked to a virus particle that is capable of binding to a protein on the cell surface of a ed cell type in order to direct interaction between the virus paiticle and the selected cell type. The targeting moiety may be covalently or non-covalently linked and is generally linked to an envelope protein, 9g, El, ‘82, or E3. Representative targeting es include antibodies, antigen binding fragments thereof, and receptor ligands. A viral “envelope” protein, or “Env” protein, refers to any polypeptide sequence that s on the surface lipid bilayer of a virion and whose function is to mediate the adsorption to and the penetration of host cells susceptible to infection. {0072} The term “vector” is used herein to refer to a c acid molecule capable transferring or transporting r nucleic acid le. The transferred nucleic acid is generally linked to, cg. inserted into, the vector nucleic acid molecule. A vector may include sequences that direct mous replication in a cell, or may include sequences sufficient to allow integration into host cell DNA. ln some embodiments, the vector is a virus (219., a viral vector or tic viral vector) and the transferred nucleic acid sequence is a recombinant nucleic acid sequence encoding an engager molecule and/or a eutic molecule, A viral vector may sometimes be referred to as a “recombinant virus” or a “virus.” The terms “oncolytic virus” and “oncolytic vector” are used interchangeably herein. {(9973} “Nucleic acid genome” or “viral genome” refers to the nucleic acid component of a virus particle, which encodes the genome of the viius particle including any proteins required for replication and/or integration of the genome. in some embodiments, a viral genome acts as a viral vector and may comprise a logons gene operably linked to a promoter. The er may be either native or heterologons to the gene and may be viral or non—viral in origin. The viral genomes described herein may be based on any virus, may be an RNA or DNA genome, and may be either single stranded or double stranded. ably, the nucleic acid genome is from the family Rhabdoviridae.

{W74} “Retroviral vectors,” as used herein, refer to viral vectors based on s of the Retroviridae family. in their Wild—type (WT) form, iral s typically contain a nucleic acid genome. ed herein are pseudotyped retroviral vectors that also comprise a heterologous gene, such as a recombinant nucleic acid sequence described herein.

{ME} The term "antibody fragment or derivative f" includes polypeptide sequences containing at least one CDR and capable of specifically g to a target antigen.

The term further relates to single chain antibodies, or fragments thereof, synthetic antibodies, dy fragments, such as a, Camel lg, lg NARD Fab fragments; Fah‘ fragments? Rah)? nts, Ft’ah)? fragments, Fv, single chain Fy antibody ("scFV"), bis—scF‘v, (scFv)2, minihodyj y, triabody, tetrabodyi disull‘ide ized li'y protein (”dsFy“): and single— domain antibody (sdAh, nanohody), etc, or a. chemically modified derivative of any of these. ln some embodiments, antibodies or their ponding immunoglohulin chaint's) are further modified by using? for example, amino acid deletion(s), insertion(s), suhstitutiordsl, addition(sl, and/or reconrhinatiords) and/or any other ntoditicatior1(s) (cg posttranslational and chemical modifications, such. as glycosylation and phosphorylation), either alone or in combination Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence ofan immunoglohulin chain are well known to the person skilled in the art. {$976} The term "single—chain" as used. in accordance with the t. disclosure refers to the nt linkage of two or more polypeptide sequences, preferably in the form of a, co~linear amino acid sequence encoded by a single nucleic acid molecule. [3677} The terms "binding to” and “interacting with” are used hangeably herein and refer to the ction of at least two "antigen—interaction—sites” with each other. An "antigen~interaction-site” refers to a. motif of a. polypeptide (eg, an antibody or antigen binding fragment thereof) capable of specific interaction with an antigen or a group of antigens, The binding/interaction is also understood to define a "specific interaction" or “specific binding.” {@978} The terms fic binding" or “specific interaction” refer to an antigen- interaction-site that is capable of specifically interacting with and/or binding to at least two amino acids of a target molecule as defined . The term relates to the ability of the antigen" in.teraction~site to discriminate between the specific regions (eg. epitopes) of the target molecules defined herein such that it does not, or essentially does not, cross~react with polypeptides of similar structures in some embodiments, the epitopes are linear. In some ments, the epitopes are conformational epitopes, a structural e, or a discontinuous epitope consisting of two regions of the human target molecules or parts thereof. in context of this disclosure, a confonnational epitope is d by two or more discrete amino acid sequences separated in the primary ce which come together on the surface of the folded protein. Specificity and/or cross—reactivity of a panel of antigen bindings construct under investigation can he tested, for example, by assessing binding of the panel ofthe constructs to the polypeptide of interest as well as to a number of more or less (structurally and/or functionally) 2017/040354 closely related polypeptides under conventional conditions (see, eg, l-larlovv and Lane, dies: A Laboratory Manual, Cold Spring l—larbor Laboratory Press, l988 and Using dies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, l999). Only these constructs that bind to the polypeptide/protein of interest and do not, or essentially do not, bind to any of the other polypeptides are considered specific for the polypeptide/protein of interest.

Examples of specific interactions of an antigen—interaction-site with a specific antigen include the interaction of ligands which induce a signal upon binding to its specific receptor, the specificity of a ligand for its receptor, such as nes that bind to specific cytolcine receptors, and the binding of an antigen binding site of an dy to an antigenic epitope, among others. {tlfi79} in some instances, the c ction of the antigen-interaction—site with a specific antigen results in the initiation of a signal, eg. due to the induction of a change of the conformation of the antigen, oligoinerization of the antigen, etc. In some embodiments, c binding encompasses a "ltey—loclr—principle." Therefore in some embodiments, specific motifs in the amino acid sequence of the antigen—interaction—site interact with specific motifs in the antigen and bind to each other as a result of their primary, secondary or tertiary structure, or as the result of secondary modifications of said structure. in some embodiments, the specific interaction of the antigenninteraction—site with its specific antigen results in a simple binding of the site to the antigen.

Oncolytic Viruses {0089} Gncolytic viruses are able to infect, ate in, and ly‘se tumor cells, and are further capable of spreading to other tumor cells in successive rounds of replication. While past oncolytic virus therapy has shown promise in preclinical models and clinical studies, anti— turnor efficacy of these oncolytie virus, such as vaccinia, has been suboptimal. For example, these viruses demonstrated limited viral spread. throughout the tumor and/or d tion of anti—tumor T cell ses Within the tumor, Therefore, the present disclosure provides an oncolytic virus that l) facilitates tunior infiltration and activation of effector cells (cg, T cells), and 2) effectively lyses tumor cells that are not infected the virus (also known as bynstander killing).

{W81} in some embodiments, provided are viral vectors which have advantages ing one or more of the following properties: (i) the vectors are tic and have a particularly high oneolytic activity compared to other usly described oncolytic viral vectors; (ii) the vectors replicate preferentially in tumor cells and have a particularly high replication capability compared to other oncoly'tic viral vectors; (iii) the vectors infect actively dividing cells as well as g cells; (iv) the vectors induce a strong innate, humoral, and cellular immune response; (v) the vectors replicate purely cytoplasrnatically, i.e., as RNA s they cannot integrate into the host cell genome or recombine into replication—competent viruses; (vi) the vectors are easy to package; and/or (vii) the native viral rotein is interchangeable with a foreign envelope protein. {0682} Some embodiments of the invention relate to recombinant lar stomatitis viruses (VSV) and VSV vectors. The VSV genome includes five genes, l, m, n, p and g, which encode the proteins L, M, N, l) and G and are essential for the reproduction. of the virus.

N is a nucleoprotein which packages the VSV genomic RNA. The VSV genome is replicated as RNA-protein complex and L and P together form a polymerase complex which replicates the VS‘V genome and transcrihes the VSV mRNA. M is a matrix protein which provides stnictural support n the lipid pe and nucleocapsid and is important for particle sprouting at the cell membrane. G is the envelope protein which is incorporated in the viral envelope and is ial for the int‘ectivity and m ofthe virus. zjiped oncalytie viruses {0083} in some embodiments, the present invention provides oncolytic viruses that are capable of being pseudotyped or otherwise engineered. "‘Pseudotypetl viruses” refer to viruses in which one or more of the viral coat proteins (eg, envelope proteins) have been replaced or modified. In some embodiments, a pseudotvped virus is capable of infecting a cell or tissue type that the corresponding non—pseudotyped virus is not e of infecting. In some embodiments, a pseudotyped virus is capable of pert‘erentially infecting a cell, or tissue type compared to a non—pseudotyped virus, {0084} l'n general, s have natural host cell populations that they infect most efficiently. For example, retroviruses have limited natural host cell ranges, while adenoviruses and adeno—associated viruses are ahle to efficiently infect a relatively broader range of host cells, although some cell types are refractory to infection by these s. The proteins on the surface of a virus as g, en 'elope proteins or capsid proteins) meditate attachment to and entry into a susceptible host cell and thereby determine the tropism of the virus, [a , the ability of a particular virus to infect a particular cell or tissue type. In some embodiments, the oncolytic s bed herein comprise a single types of protein on the surface of the virus. For example, retroviruses and adenouassociated viruses have a single protein coating their membrane. ln some embodiments, the oncolytic viruses described herein comprise more than one type of protein on the surface of the virus. For example, adenoviruses are coated with both an envelope protein and fibers that extend away from the surface ofthe virus. {0085} The proteins on the surface of the virus can bind to cellusurface molecules such as heparin sulfate, thereby zing the virus to the surt’ace of the potential host cell. The ns on the e of the virus can also mediate interactions between the virus and specific protein receptors expressed on a host cell that induce structural s in the viral protein in order to mediate viral entry. Alternatively, interactions between the proteins on the surface of the virus and cell receptors can facilitate viral internalization into endosomes, wherein cation of the endosonial lumen induces refolding of the viral coat. in either case, viral. entry into ial host cells requires a, favorable interaction between at least one molecule on the surface of the virus and at least one molecule on the surface of the cell. {cuss} ln some ments, the oncolytic viruses described herein se a. viral coat (9. g. a viral envelop or viral capsid), wherein the proteins present on the surface ofthe viral coat (cg, viral envelop proteins or viral capsid proteins) modulate recognition of a potential target cell for viral entry. ln some instances, this process of determining a potential target cell for entry by a virus is referred to as host tropism. in some embodiments, the host tropism is cellular m, wherein viral recognition of a receptor occurs at a cellular level, or tissue tropism, wherein viral recognition of cellular receptors occurs at a tissue le el. in some instances, the viral coat ofa virus recognizes receptors present on a single type of cell. in other instances, the viral coat of a virus recognizes receptors present on multiple cell types (eg, 2, 3 4, 5, 6 or more different cell types). In some instances, the viral coat of a virus izes ar ors present on a single type of tissue. in other instances, the viral coat of a virus recognizes cellular receptors present on multiple tissue types (a g, 2, 3, 4, 5, 6 or more different tissue types). {6087} in some embodiments, the oncolytic viiuses described herein comprise a viral coat that has been ed to incorporate surface proteins from a ent virus in order to facilitate viral entry to a particular cell or tissue type. Such oncolytic viruses are rel’ered to herein as pseudotyped oncolytic viruses. in some embodiments, a pseudotyped oncolytie s comprises a viral coat n the viral coat of a first virus is exchanged with a viral coat of second, wherein the viral coat of the second virus is allows the pseudotyped oncolytic virus to infect a. paiticular cell or tissue type. in some embodiments, the viral coat ses a viral envelope. in some instances, the viral envelope comprises a phospholipid hilayer and proteins such as proteins obtained from a host membrane. in some embodiments, the viral envelope further comprises glycoproteins for recognition and attachment to a receptor expressed by a host cell. in some ments, the viral coat ses a capsid. In some instances, the capsid is led from oligomeric protein subunits termed protomers. in some embodiments, the capsid is assembled from one type of protomer or protein, or is assembled from two, three, four, or more types ot‘protomers or proteins. [0088} in some embodiments, it is ageous to limit or expand the range of cells susceptible to transduction by an oneolytie virus for the purpose of tic therapy. To this end, many viruses have been developed in which the endogenous viral coat proteins (cg, viral envelope or capsid proteins) proteins have been replaced by viral coat proteins from other viruses or by chimeric proteins. in some embodiments, the chimeric proteins are comprised of parts of a. viral protein necessary for incorporation into the virion, as well proteins or c acids designed to interact with specific host cell proteins, such as a targeting moiety. {0089} in some embodiments, the pseudotyped oncolytic viruses described herein are pseudotyped in order to limit or control the viral tropism (lie. to reduce the number of cell or tissue types that the pseudotyped oncolytic virus is capable of infecting). Most strategies adopted to limit tropism have used chimeric viral coat proteins (cg, envelope proteins) linked antibody fragments. These viruses Show great promise for the pment ol’oncoly/tic therapies. in some embodiments, the psendotyped oncolytic viruses described herein are typed in order to expand the viral tropism (22a, to se the number of cell or tissue types that the pseudotyped oncolytic virus is capable of infecting). One mechanism for expanding the ar tropism of viruses (ergo ped viruses) is through the formation of phenotypically mixed particles or pseudotypes, a process that commonly occurs during viral assembly in cells ed with two or more s. For example, human immunodeficiency virus type l (l-lanl). l-llVl infects cells that express CCR4 with an appropriate co-receptor. However, HlVl forms pseudotypes by the incorporation ofheterologous glycoproteins (Gill‘s) through plienotypic mixing, such. that the virus can infect cells that do not express the CD4 receptor and/or an appropriate co—receptor, thereby expanding the tropism of the virus. Several studies have demonstrated that wild type HIV—l produced in cells infected with xenotropic murine leukemia virus (ML-V), ampliotropic MLV, or herpes x virus gives rise to phenotypically mixed virions with an expanded host range, indicating that pseudotyped virions had been produced. Phenotypic mixing of viral GPs has also been shown to occur between HIV—1 and VSV in coinl’ected cell cultures. These early observations were key to the uent design of based lentiviral vectors bearing heterologous GPs. {fittlltl} There is an ever—growing list of alternative 69s for pseudotyping lentiviruses, each with specific advantages and disadvantages. The widespread use of VSV G'— proteins (VSV—G) to pseudotype lentiviruses has made this GP in effect the standard against which the usefulness of other viral GPs to form pseudotypes are compared. Addtional non— limiting examples of lentivirus pseudotypes include pseudotypes bearing lyssaviius-derived GPs, pseudotyped lentiviruses bearing lymphocytic meningitis virus Gl’s, lentivirus pseudotypes bearing alpliavinis GE’s (cg. lentiviral vectors typed with the REV and SFV GPs, iral vectors pseudotyped with sindbis viius GPs), pseudotypes bearing tilovinis GPs, and lentiviral vector pseudotypes ning the baculovirus GP64. {tltlfll} in some embodiments, the engineered re 3:, pseudotyped) viruses are capable of binding to a tumor and/or tumor cell, lly by binding to a protein, lipid, or carbohydrate expressed on a tumor cell. In such embodiments, the engineered viruses described herein may comprise a targeting moiety that s the virus to a particular host cell. In some instances, any cell surface biological material known in the art or yet to be identified that is differentially expressed or otherwise present on a particular cell or tissue type (eg, a tumor or tumor cell, or tumor associated stroma or stromal cell) may be used as a potential target for the oncolytic viruses the present invention. In ular embodiments, the cell surface material is a protein. in some embodiments, the targeting moiety binds cell surface antigens tive of a disease, such as a cancer (cg, breast, lung, ovarian, prostate, colon, lymphoma, leukemia, melanoma, and others); an autoimmune disease (eg. rnyasthenia grail/is, multiple sclerosis, systemic lupus erythymatosis, rheumatoid arthritis, diabetes mellitus, and others); an infectious disease, including ion by HlV, l-lCV, l-lBV, CMV, and HPV; and a genetic disease including sickle cell anemia, cystic fibrosis, Tay-Sachs, J3—thalassemia, neurolibromatosis, polycystie kidney disease, hemophilia, etc. in certain embodiments, the targeting moiety targets a cell surface antigen specific to a particular cell or tissue type, eg. cell—surface antigens present in neural, lung, kidney, muscle, vascular, thyroid, ocular, breast, ovarian, testis, or te . {0092} ary ns and cell e les for targeting include, eg P—glycoprotein, HerZ/Neu, erythropoietin (EPO), epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (\I’EGF—R), cadberin, carcinoembryonic antigen (CEA), C94. CD8, CDl‘). CD20, CD33, CD34, CD45, CDl l7 (c-kit), CD83, A, l-lLAB, HLA—C, chemolrine receptor 5 (CCRS), stem cell marker ABCGZ transporter, ovarian cancer antigen CAlZS, iniinunoglobulins, integrins, te specific antigen (EPSA), prostate stem cell n (PSCA), dendritic pecific intercellnlar adhesion molecule 3-grabbing nonintegrin (DC—SIGN), thyroglobulin, ocyte—macrophage colony stimulating factor (GM—CSF), myogenic differentiation promoting factor-l (MyoD—l), lieu—7 (CD57), , cell proliferation—associated human nuclear n defined by the monoclonal antibody Ki~67 (Ki— 67), Viral envelope proteins, HIV gplZO, transferrin or, etc. Additional antigens and cell surface molecules for targeting are shown in Table 2. {0093} En some embodiments, the pseudotyped oncolytic viruses provided herein are capable of selectively entering, replicating in, and/or lysing tumor cells. Such an embodiment is illustrated in, FlG. 36, wherein the pseudotyped oncolytic Virus gains entry to the target cell due to the incorporation of viral roteins derived from a different lie, logous) Virus that allow for entry of the pseudotyped oncolytic Virus into the target cell. in contrast, the non— pseudotyped tic virus is unable to gain entry into the target cell due to the non—permissive nature of the envelope proteins. In some instances, the ability of a pseudotyperl oncolytic Virus to selectively enter, replicate in, and/or lyse a tumor cells is due to a reduced or otherwise ineffective cellular interferon {lFN} response. In some embodiments, the pseudotyped oncolydic viruses produce an engager molecule and/or a therapeutic molecule, such as an immune modulating polypeptide, that eres or impairs the cellular lFN response, thereby enhancing the replication ofthe pse udotyped or engineered virus. [3694} The typed oncolytic Viruses described herein may be derived from a variety of Viruses, non~lintiting examples of which include ia Virus, itus, herpes simplex virus l (l—lSVl), niyxonia virus, reovirus, poliovinis, vesicular stomatitis virus (VSV), measles Virus (MV), lassa Vlqu (LASV) and Newcastle disease Vlqu (NDV). in some embodiments, the pseudotyped oncolytic s described herein can infect substantially any cell type, An exemplary iius for use in oncolytic therapy is Simian immunodeficiency Virus coated with the envelope ns, (ii-protein (GP), from VSV. In some instances, this Virus is referred to as VSV anseudotyped lentivirus, and is known to infect an almost universal set of cells. {@095} ln some embodiments, the pseudotyped oncolytic Viruses of the present invention are VSV Viruses pseudotyped against healthy brain cells, i.e., s and exhibit considerably reduced toxicity. Since neurotropism is a dose~liiniting factor in all applications of oncolytic VSV, the use of the vector according to some embodiments of the present invention is that they are used for all tumors types of solid tumors. {9696} In some embodiments, the pseudotyped VSV s have one or more key attributes including: (i) the VSV is not cell—toxic; (ii) the vectors are concentrated by ultracentrifugation without loss of infectivity, and (iii) the vectors show a tropism for tumor cells, whereas neurons and other non-tumor cells are infected inefficiently. To increase the safety during the use of replicable viruses in therapeutic: uses, some embodiments of the present invention provide a vector system which ensures that replication, oneolysis and the production of VS‘V viruses takes place only in cells which are infected by at least two replicatiorntletieieiit, mutually complementing vectors. {0097} in some embodiments, the genetic material (eg, the viral coat protein or the core genetic material) for generating a typed oneolytic virus is obtained from a DNA virus, an RNA virus, or from both virus types, in some embodiments, a DNA virus is a single— stranded (ss) DNA Virus, a clouble~5'traiided (cls) DNA virus, or a, DNA virus that contains both ss and els DNA regions. in some embodiments, an RNA virus is a single—stranded (gs) RNA virus or a double—stranded (ds) RNA virus. In some embodiments, an sleNA virus is further fied into a positive-sense RNA virus or a negative—sense RNA Vitus. {9098} in some instances, the genetic material for generating a pseudotyped oncolytic virus is obtained from a dsDNA virus of any one ofthe following families: Myoviridae, Podovii'idam Siphoviridae, rpesviridae, Herpesviridae, Malacoherpes‘vriridae, Lipotlnixviritiae, Rudiviriclae, Adenoviridae, Ampullaviridae, Ascoviridae, Asfaviridae, Beculoviriclae, Bicaudaviiidae, Clavaviridae, Corticoviridae, Fuselloviridae, Globuloviridae, Guttaviiidae, Hyti'osaviriclae, lricloviiidae, Marseilleviriclae, Mimiviritlae, Niniavii‘iclae, Pandoraviridae, 9apillomaviridae, navindae, flasmaviridae, Polydnavirnses Polyomaviiidae, Poxviridae, Sphaerolipoviiidae, or Tectiviridae. {0099} in some eases, the genetic al for generating a pseudotyped fiic virus is obtained from a ssDNA virus of any one of the following es: Anelloviridae, Baeillariodnaviii(lae, Bidnaviridae, Cireoviridae, viridae, lnoviridae, Microviridae, Nanoviridae, Parvoviridae, or Spiraviridae.

{M100} in some embodiments, the genetic material for generating a pSGtttithyptBCl oncolytic virus is obtained from a DNA virus that contains both ssl.)NA and dsDNA s. In some cases, the DNA virus is from the group pleolipoviiuses. in some cases, the pleolipovinises include l-laloarcnla hispanica pleoinorphic virus 1, Halogeometricnm pleoniorphic virus 1, Halornbmm pleomorphic virus 1, Halorubrum pleomorphic virus 2, Halorubi'nm pleomorphic virus 3 or Halonihmm pleomorphic virus 6‘ l} In some cases, the genetic material for generating a pseudotypetl oncolytie virus is obtained from a dsRNA virus of any one of the following es: Birnaviriclae, vindae, Cystoviridae, Endornaviiidae, Hypoviridae, Megaviriiaviriclae, Partitivii‘idae, Picobirnaviridae, Reoviridae, Rotavinis or Totiviridae.

F.) I‘J {@182} In some instances, the genetic material for generating a pseudotyped oneolytie virus is ed from a positiveusense ssRNA virus of any one of the following families: Alphaflexiviridae, Alphatetraviridae, aviridae, Arteriviridae, Astr‘oviridaee Barnaviridae, Betaflexiviridaey iiidae; Caiieiviridae Cannotetraviridae, Closteroviiidaea Coronaviridae, Dieistroviridae, Flaviviridae, exiviridae, lilaviridae, Leviviridae, Luteoviridae, l‘vlarnaviridae, Mesoniviridae, Nar‘naviridae, Nodaviridae, Permutotetraviridae, I’ioornaviridae, Potyviridae, Roniviridae, Seeoviridae, 'llogaviridae, usviridae, Tymoviiidaej or viridaei {06193} In some cases, the genetic material for generating a pseudotyped oneolytie virus is obtained from a negative—sense ssRNA virus of any one of the following Ianiilies: Bomaviridae, Filorririd.ae, Paramysoviridae: Rhabdoviridae, Nyamiviridae, Arenaviridae, Bunyaviridae, Ophioviridae, or yxoviridae‘ {001843 in some instances, the genetic al for generating a pseudotyped oneolytie virus is obtained from oneolvtic DNA viruses that comprise eapsid symmetry that. is isoeahedral or complex In some cases, isosahedral oneolytie DNA viruses are naked or comprise an envelope. Exemplary families of oncoiytie DNA viruses include the Adenovir’idae (for example, Adenovirus, having a genome size of 36—38kb), Herpesviridae (for example, HSVl, having a genome size of lZU—ZOO Hi), and Poxviridae (for example Vaeeiiiia virus and myxoma virus, having a genome size of Bil—230 kb). {00} 85E In some eases, the genetic material for generating a pseudotyped oneoljfiic virus is ed from oncolytic RNA viruses inehrde those having edral or helical capsid symmetry. In some cases, icosahedral oneolvtie viruses are naked without envelope and include Reoviridae (for example, Reovirns, having a genome of 22-27 kh) and Picornaviridae (for example, Poiiovirus, having a genome size of 7.2—8.4 kb}. In other cases, helical oncolytic RNA viruses arz enveloped and include Rhabdoviridae (for example, VSV.‘ having genome size of l3— l6 kh) and Paramyxoviridae (for example MV and NDV, having genome sizes of l 6—20 H3). {001%} In some instances, the genetic al for generating a pseudotyped oncolytic virus is obtained from a virus such as Abelson leukemia virus.( on nrurine ia virus, Abelson‘s i Acute laryngotraelieohronehiris virus? Adelaide River Vinis, Adeno asseeiated virus group Adenovinis, African horse sickness virus, African swine fever ( LEEDS virus? Aleutian mink disease irns, Alpharetrovinis, Alphavirtrs? ALV related virus, Aniapari vinis, .Aphthovinis, Aquareovinis, Arhovirus hrhovirns (5., arhovims group A, arbovims "J‘dF ronp B.) Arenavirus group? Argentine hemorrhagic fever virus, Argentine hemorrhagic fever virus, Arter’ivints, Astroviins, Ateline vints group, Airiezity's disease virus, Aura ,,,. 2017/040354 virus, Ausriuk disease virus, Australian bat iyssaviius, Aviadenovirus, avian erythrobiastusis virus, avian ious bronchitis virus, avian leuken’iia virus, avian ieulrosis virus, avian iyirrpliomatosis virus, avian niyeioblastosis virus, avian pararuvsovirus, avian pneumoerieepiialitis virus, avian reueuioendotireliosis virus, avian sarcoma virus, avian type C retrovirus groui, Avinepadnavirus, Avinoxvirus, B virus, Bl? virus, Babanki virus, baboon iierpesvirus, virus, Barman Forest virus, Bebaru virus, Berrimaii virus, Betaretrovirus, Bir‘iiaviius, Bittrrer virus, BK virus, Biaek Creek Canai virus, ngue virus, Boiivian hemorrhagic fever virus, Berna disease virus, border disease of sheep virus, borne, virus, bovine ainhalrerpesvirus 1, bovine heipesviius 2, bovine eoronavirus, bovine ephemeral fever virus, bovine immunodeficiency virus, bovine leukemia virus, bovine ieulrosis virus, bovine ruarirmiiiiris virus, bovine papiiiomavirus, bovine r stoniatitis virus, bovine parvovirus, bovine synevtiai virus, bovine type C oneovirus, buvine virai diarrhea. virus, Buggy Creek virus, bullet shaped virus group, Bunyarnwera virus supergroup, Bunyavirus, Burkitt's lymphoma virus, vaarir‘oa Fever, CA vi ms, Caiieiviius, Caiifornia encepiraiitis virus, ox virus, canaiypox virus, canid irerpesvirus, canine uoronavirus, canine distemper virus, canine herpesviius, canine minute virus, canine parvovirus, Cane Deigadito virus, re arthritis virus, earrrine encephalitis virus, Caprine Herpes Virus, Capripox virus, vinis, caviid herpesvirus 1, Cereopitireeid herpesvims i, r‘theeine iierpesvims i, Cereopitiiecine iierpesvirus 2.

{Tiiandipura virus, Ciianguinoia virus, ebanuei catfish virus, Charievilie virus, chickenpox virus, Cliilruiiguriva virus, chimpanzee herpesvirus, chub reovirus, chum salrnou virus, Cocai virus, Coho salmon reovirus, ooitai exauthema virus, Coioraelo tick fever virus, Coirivirus, Coiuiubia SK virus, common cold virus, ious eetnvma viius, contagious ar dermatitis virus, Coronavirus, {Iorripaita virus, eoryza virus, oowpes virus, eoxsaekie virus, CPV lasmic polyiredrosis , cricket paralysis virus, Crimean—Congo hemorrhagic fever virus, eroup assueiareri virus, Civptovirus, Cypovinis, Cvroniegaioviius, cytomegaioviius group, cytoplasmic polyhedrosis virus, deer papilioinavirus, deiraretrovirus, dengue virus, Deiisovirus, Depeiidovirus, Dhori virus, diploma virus, Drosophz‘i’a C virus, dunk hepatitis l3 virus, duck hepatitis virus l, duck hepatitis virus 2, riuovirus, Duvenhage virus, Beformed wing virus D‘v‘v’V, eastern equine encephalitis virus, eastern equine eiioepiiaiomveir’tis virus, FB virus, Ebola virus, Ebolalike virus, eeiiu virus, eeiiovirus, eeiiovirus it"), eeiiuvirus '23, eonuvirus 9, eetrorneiia virus, EEE virus, EiA virus, ElA virus. encephalitis virus, erreeplialoiriyoearditis group virus. eircepnalonivoearditis virus, Euteroviius, enzyme eievaring virus, enzyme elevating, virus {Ulli}, epidemic hemorrhagic fever virus, epizootie hemorrhagic disease virus, Epsteiii~Ba.rr virus, equid aiphaheipesviius l, equid aipliaheiriesvirus ii, equid liernesvirus 2, equine aboriiun virus, equine 44’3 arteritis virus, equine eneephalnsis virus, equine infeetious anemia virus, equine mnrhillivims, equine rliinopneurnnriiris virus, equine rl’iinnvirus, Eulrenarigu virus, European ell; papillomavinis, European swine fever virus, Everglades virus. Eyaeh virus l’elii‘l herpesvirus l. feline ealieivirus, feline fihrosarenma virus, feline herpesviius, feline innnunedefieieney virus, feline infectious peritonitis virus, feline leuken'ria’sareenra virus, feline leukemia virus, feline panleuliopenia virus, feline parvovirus, . a virus, feline srr’neytial virus. filovirus, Flanders virus, Flch‘v’ll’llS, foot and mouth disease virus, Fort Morgan virus, Four Cemers lianravirus, fowl adenuvirus l, fewlpux virus, Friend virus, Gaminareimvirus, GB heparitis virus, GB virus, German measles virus, Getah virus, gibbon ape leukemia virus, lar fever virus, geatpes virus, gelden shinner virus, Gorieinera virus, geese irus, granulesis virus, Gross' virus, ground squirrel hepatitis B virus, group A ai‘bovirus, Guanar‘iro virus, guinea pig; eytumegalevirus, guinea pig type C virus, l-laniaan virus, l-lanravirus, haril elam reevirus, hare lilirorna virus, e (human eyiemegalevirus), heinadseiprien virus 2, hernaggluiinating virus of Japan, heinm‘r‘liagie fever virus, henrlra vi ins, llenipaviiuses, Heparinavirus, heparitis A virus, hepatitis B virus group, hepatitis C virus, hepatitis l) virus, hepatitis delta virus, hepatitis E virus, hepatitis E virus, hepatitis G virus, l'repariris nonA nonB virus, hepatitis virus, hepatitis virus (nonhuman), hepatoeneeplialeinyeliris r‘eevirus 3, Hepatevirus, heron hepatitis B virus, herpes B virus, herpes simplex virus, herpes simplex virus l, herpes simplex virus 2, herpesvirus, herpesvirus ’7, l-lerpesvims areles l-lerpesvirus is, l-lerpesvirus infection, ltlerpesvirus saimiri, llerpesvirus suis, l—lerpesvirus varieellae, l—liglilands J virus, l—liranie rhabdevirus, ling a virus, human adenevinis 2., human alplraherpe svirus l, human alphaherpesvims 2, human alphaherpesvirus 3, human is lvniphetrepie virus, human lieraherpesvirus 5, human enrenavirus, human egaleviius group, liurnan foamy virus, hurnan garnniaheipesvirus cl, human gannnaherpesvirus 6, human hepatitis A virus, human lierpesvirus 1 group, human herpesvirus 2 group, human herpesvirus 3 group, human herpesvirus 4- group, human herpesvirus 6, human herpesvirus 8, human deficiency virus, human defieienev virus l, human inununodefieieney virus 2, human papillr‘rmaviius, human ”l" cell leukemia virus, human T cell leukemia virus l, human '1' cell leukemia virus ll, human T eell leukemia virus ill, human T eell ma virus l, human T cell lymphoma virus ll, human T cell lyinphnr‘mpie virus type l, human T cell lyinpherrnpie virus type 2 human T uiropie virus l, human T lympherropie virus ll, human 'l‘ lyinplierrepie virus lli, lchnovirus, irriiinrile enteritis virus, infectious bovine rhinorr‘aelieiris virus, infectious liaeinatnpoierie necrosis virus, infectious pancreatic nemesis virus, influenza. virus A, influenza virus B, nza virus C, influenza virus D, influenza virus prS, inseel iridescent virus, insect virus, irus, Japanese B virus, Japanese WO 06005 encephalitis virus, it? virus, ii virus, liapesi's sareoriia—ussoeiarted herpesvirus, Kemerovo virus, Kilharu's rat virus, lilaruaili virus, lioiorigo virus, Korean hemorrhagic fever virus, Rumba virus, Kysarrur forest disease virus. Kyzyiagaeli virus, La Crosse virus, lactic deliydrogeuase elevating virus, lactic delrvdrogeiiase virus, Lagos but virus, Luiigur virus, lapiiie parvovirus, Lassa fever virus, Lassa virus, latent rat virus, LCM virus, Leaky virus, Leritivirus, posvirus, leukemia virus, leukovirus, lumpy skin disease virus, lyiripliaclenopatliy ated virus, liyiiiplioeivptmiirus, iympliocvtie ehoriomeuirigitis virus, ivrnpheproliferative virus grriup, o virus, mud iteli virus, ian type B oneovirus ,‘ roup,.4 riiariimaliari type B retroviruses, imaliari type t) retrovirus group, rriarurrialiari type D retroviruses, mammary tumor virus, Mapuera virus, l‘vlarburg virus, l‘vlarburgwlilre virus, Mason l’lizer monkey virus, u'lastadeuovirus, Mayaro viius, Mir) virus, measles virus, Meriaugle virus, Meiigo virus, l‘vleiigovims, Middelburg virus, uiiikers nodule virus, uiiuk enteritis virus, minute virus of mice, MLV related virus, MM virus, Mokula virus, l‘vfilollusoiposvirus, Molluseuu'i cou‘tagiosum virus, moulrev B virus, morikeypux virus, R<luiiorieguv'irales, lVlorbiilivirus, Mount Elguri but virus, mouse evtoriiegaloviius, mouse eucephulomyelitis virus, mouse hepatitis virus, mouse K virus, mouse leukemia virus, mouse ruarurriary tumor virus, mouse i'riinute virus, rnouse prieui'uonia virus, mouse puliouiyelitis virus, muuse polyomavirus, mouse sarcoma virus, mousepos virus, lvluzaiiibique virus, Mucanilio virus, r‘nueosul disease virus, mumps virus, niurid bemherpesviurs l, d eytumegaluvirus 2, murme eytumegulovirus group, iriuriue eiiceplialorriyelitis virus, muriue tis virus, i'riuririe leukemia virus, iriurii're riuelule inducing virus, rnuriue polyomavirus, rnuriiie sarcoma virus, Muroruegaluvirus, Murray Valley encephalitis virus, mvxoma virus, Myxovirus, vaovirus iriuliiforme, rus puroritidis, Nairelii sheep disease virus, Nairovirus, Nariiriizuiirus, Nariva virus, Ndumo virus, Neethliug virus, Nelson Bay virus, rieuretropie virus, New Worltl r‘ireriavirus, newborn pueuruonitis virus, Newcastle disease virus, Nipah virus, uoneytopatliogeriio virus, ~J‘rlorivr-ill< virus, nuclear pulvhedrosis virus (NPV), nipple neck virus, O'iiyoiig’riyong virus, o virus, oneogeuie virus, uie viruslike particle, oncornavirus, ()rliiviius, {)rf virus, {)ropouelie virus, {lrtliolieputlriuvirus, Ortliomvsovirus, Girlioposvirus, Urtlioreovirus, Grunge. ovine papillomavirus, oviue lial lever virus, ovvl iiioulrev lierpesvirus, Palyam virus, ’i’apillomavirus, Papillomaviius sylvilagi, l’apmraviius, purairifiueiiza virus, parairifluerisa virus type i, parairrilueiiza virus type 2, pur‘aiui‘lueriza virus type 3, parairiflueiiza virus ‘rvpe Al, l-‘aramvsovirus, l’arapuxvirus, cemia virus, Parwrvirus.

Parvovirus Big, parvovirus gmup, Pestiviius, Plilebovirus, phoeme distemper virus, Pieotliiaviius, Pieoruavirus, pig oytorriegaloviruspigeoripox virus, ?irjv virus, l3ixuiia virus, pneurriunia virus of mice, ovirus, ptiliuruyelitis virus, poliovirus, Polyduavirus, polyhedral ,, . 2017/040354 virus, peljvoina viius, Foiyoinavirus, Foiyeniaviius bovis, Poiyoinavirus eei‘eopitheei, Polvomuvirus liOi’nii’liS 2, Foiyonizivir’us niaeeaeae i, Poiyornuvirus inuris i, Foivoinuvirus muris 2, Polyuinavirus papiunis l, navirus papiouis 2, l’uiyoniavirus syiviiagi, l’ongine iierpesvirus i, porcine epidemic diarrhea~ virus, porcine lieniagglutiuaung eneephaioniveliiis virus, porcine parvovirus, porcine nissibie gastroenieriris virus, porcine type C virus, pox virus, posvirus, pos‘virus varielae, Prospect Hill virus, Fi‘ovirus, pseudeeowpex virus, i‘abies virus, psinueinepox virus, quaiipov viius, rabbit fibreina virus, rabbit kidney vaeuieiaung virus, rabbit papiiioinuvirus, rubies viius, raccoon irus, raceoonpox virus, Ruiiiirliet virus, rat evron'iegairwirus, rar parvevirus, rat virus, Rauseher‘s virus, recombinant vaccinia. virus, recombinant virus, reovirus, reevirus l. reevirus 2., reovirus 3, reptilian type C virus, respiratory infection viius, respiratory synejvriai virus, respiratory virus, reticuleenderlieiiosis virus Rhabriuvirus, Rhabduviius earpia, Rhadinovirus, Rhinovirus, Ri’iizidiovirus, Rift Valiey fever virus, s virus, ririderpest virus, RNA tumor virus, Ross River virus, Ruiavirus, rougeole virus, Rous sarcoma viius, iubeila virus, nibeela viius, rus, Russian autumn eiieepliaiiiis virus, SA ii simian virus, SAZ virus, Sabin. virus, Sagivarna virus, Sziiinirine lierriesvinis i, ry gland virus, saiidfiv fever virus group, inba virus, SAKS virus, SDAV (sialodaervoacleniiis virus), sealpus: virus, Seniiiiri Forest Viius, Seoul virus, sneeppex virus, Siiope fibrninu virus, Siiope papiileaina virus, simian i‘oaniv virus, simian hepatitis A virus, simian human immunodeficiency virus, simian inunuuudefieienov virus, simian priruini'lueiiza virus, simian T oeil Brinphoirephie virus, simian virus, simian virus 40, Sinipiexvirus, Sin Nuinbre virus, is virus, sinaiipozi virus, South American heinerrliagic fever viruses, sparrewpex viius, Spuiriavirus, squirrei fibroinu virus, squirrel monkey re‘trovinis, SS‘V 1 virus group, STLV n T lynipiietropie virus) type i, STL‘V {simian T ivrriiilioirupie virus) type ii, STU-f (simian T urinpherrepie virus) type iii, stornatiris papulosa virus. submaxiilarv virus, suid aiphalieniesvinis l, suiri iierpesvirus 2, Suipexvirus, swamp fever virus, swinepox virus, Swiss nieuse leukemia virus, TAC viius, Taearibe complex virus, be virus, Trinapox virus, Tar-erases virus, Trench reoviius, Tlieiler’s eiieepi‘iaiornyelitis virus, 'l'neiler's virus, 'liiogore virus, 'i‘huuapaiayain virus, 'i‘ieli borne encephalitis virus, 'i‘iuinaii virus, ’i‘egaviius, 'l'rii'ovirus, tumor virus, "i‘upaia virus, turkey rliiiiori'aciieiris virus, turkevpox virus, type C retroviruses, type I} oiieovirus, type D retrovirus group, ulceraiive disease rhabdovinis, Una virus, Uukuiiieini virus group, vaeciuia virus, vaeuelaring virus, varicelia zosier virus. ceiioviius, Varieoia virus, a inajor virus, a virus, Vasin Gisliu disease vi ius, VEF virus, Venezueian equine aiiris virus, Venezuelan equine eneephaloniveiitis virus, Venezuelan hemorrhagic fever virus, vesiouiur stornu‘rilis virus, Vesieuluvirus, Viivuisir virus, 47’3 viper retrovinis, viral haemorrhagic septicemia virus, Visna Maedi virus, Visna virus, volepox virus, VSV (vesicular stomatitis virus}, Wallal virus, Warrego virus, wart virus, ‘vVEE. virus, West Nile virus. 'vvestern equine encephalitis virus, western equine eiiceplialoniyelitis virus. roa virus, Winter ng Virus, woodcliuclc hepatitis B virus, woolly monkey sarcoma. virus, wound tumor virus, “RSV virus, Ya‘oa monkey turner virus, Ya‘oa virus, ‘r’atanoxvir’us, yellow fever virus, and the "i’ug Bogtlanovac virus.

Afar]?ads ofproducing pseudraped oncolytic viruses {flfilll‘l’} in some instances, a pseudotvped oncolytic virus described herein is generated using methods well iniovvn in the art. in some instances, the methods involve one or more transfeetion steps and one or more infection steps. in some instances, a cell line such as a mammalian cell line, an insect cell line, or a plant cell line is infected with a, pseudotyped oncolytic virus bed herein to produce one or more viruses. Exemplary mammalian cell lines e: 293A cell line, 293?? cell line, 2’93F cells, 293 H cells, CEO DG-M cells, (THO—S cells, (ll-lG—Kl cells, Expi293FTM cells, Flp—lnlM T-REXTM 293 cell line, Flp—lnll‘l—293 cell line, Flp—lnTMu3T3 cell line, Flp—lriTM—Bl—EK cell line, FlpmlnlM—CHO cell line, FlpwlnlM—C‘le cell line, Flp—ln’anlurlrat cell line, FreeStyleTM 293-}: cells, yle'l‘M CHOnS cells, Grip'l‘iteTM 293 MSR, cell line, GS-CHO cell line, HepaRG'lM cells, 'l'—l{l£xTM lurkat cell line, PerCo cells, T— REle—Z93 cell line, T-REle—Cl-lO cell line, T*R.EXTM-l-lella cell line, 3T6, A549, A9, AtT—ZU, BALE/3T3, BHK—Zl, Bl-lL—lOO, BT, CacouZ, Chang, Clone 9, Clone lvl—3, COS—l, (3086, (308— 7, CRFK, (JV—l, [3-17, Daudi, Gill, Gl—l3, H9, l—laK, HGT—l5, Blip—2, HL—(SO, Ell—1080, Ell—29, HUVEC, l~lfl, lull-9, JEG—Z, , K4362, KB, KG-l, L2, LLC—WRC 256, McCoy, MCF7, VERD, Wl-38, WISH, XC, or Y-l. Exemplary insect cell lines include Drosophz‘ia 82 cells, Sty~ cells, StZl cells, High llivel”M cells, or expresSF+® cells. Exemplary plant cell lines include algae cells such as for example Phenom/stile panclzen‘z‘. lflOl {38} Any method known to one skilled in the art is used for large scale production of recombinant oncolytic vectors and vector constructs, such as pseudotyped oncolytic vectors. For e, master and working seed stoclrs can be prepared under lell’ ions in qualified. y Cli'Fs or by other methods. ln some instances, cells are plated on large e area flasks, grown to near confluency, and infected at selected Mill. "lire produced virus can then be purified. in some cases, cells are harvested and ellular virus is ed by mechanical disruption. in some embodiments, cell debris is d by large-pore deptli filtration and/or host cell DNA is digested with an endonuclease. in some cases, virus particles are subsequently purified and concentrated by tangential—flow filtration, followed by dial-iltration.

The resulting concentrated virus can ated by dilution with a buffer containing one or more stabilizers, filled into vials, and lyophilized. Compositions and formulations can be stored for later use. in some embodiments, a lyophilized virus is reconstituted by addition of one or more diluen ts, Engager Molecules {flfillllij in some embodiments, the oncolytic viral vectors provided herein are pseudotyped oncolytic s that are r engineered to include a polynucleotide sequence that encodes an engager molecule, eg, an engager polypeptide, The engager molecules of the present invention comprise at least two domains each capable of binding to a ent cell surface molecule. In some ments, engager polypeptides comprise an antigen ition domain and an activation domain that recognize particular cell surface proteins (cg, cell-surface receptors or s) expressed by target and elliector cells, respecti I/ely. As used herein, an en recognition domain” is a ptide that binds one or more molecules present on the cell surface of a target. cell (eg. a tumor antigen), and an “activation domain” is a, polypeptide that binds to one or more molecules present on the cell surface of an or cell (6.55., an tion molecule). An activation domain ma§l also be referred to as an “engager domain.” {Willi} in some embodiments, engager polypeptides comprise a therapeutic molecule domain and an activation domain. A therapeutic le domain is a polypeptide that binds to a particular cell e protein expressed on an efiector cell leg, cell—surface receptors or ligands) and that is distinct from the cell surface protein recognized by the activation . in particular embodiments, the therapeutic molecule domain binds to a cell surface protein that is a negative regulator of effector cell on (eg, an immune checkpoint molecule or other inhibitory molecule). Exemplary cellmsurface antigen for targeting by a therapeutic domain e CD47, Pill, PDLl, C'l'lJA/Ll, 'l‘lMZ, LAGT‘E, BRA, KIR, 'l'lGl'l‘, 0X40, Fl'l'R, CD27, SLAMF’I, and CD200. {00111} in some embodiments, binding of an activation domain to a molecule present on the surface of the effector cell results in activation of the etlector cell. in certain embodiments, binding of an activation domain to a le on an effector cell and binding of an antigen recognition domain to a molecule present on a target cell brings the effector cell in close proximity to the target cell and thereby facilitates the destruction of the target cell by the effector cell, in certain embodiments, binding of an activation domain to an activation molecule on an e fector cell and binding of a therapeutic molecule domain to an inhibitory molecule present on an effector cell enhances the activation of the ef‘ector cell and thereby facilitates the destruction of one or more der target cells by the effector cell. {9011.2} in certain embodiments, the engager molecule is a protein, eg, an engineered protein. in some embodiments, the engager molecule is a bipartite ptide. In some embodiments, the engager molecule is a tripartite or multipartite polypeptide. in such embodiments, the r molecule may comprise one or more activation domains and/or antigen recognition, domains, or other domains, including one or more co~stimulatory domains, one or more dimerization or trimerization domains, or other domain capable of binding a molecule expressed on the cell e. Alternatively, the one or more onal domains are optionally present on a separate polypeptide. in some embodiments, the engager mo ecule comprises an antibody or antibody fragment, In some embodiments, the engager molecule is a is atrifunctional antibody, an Fabz, a bi—specific scFv such as a bi~specific T—cell engager (BiTE), a bivalent minibody, a bispecific diabody, a DuoBody, or an MabZ. In certain embodiments, the r molecule is a bipartite T cell engager (Bill?) or a tripartite T cell engager (TiTE), [(30113] In some embodiments, the activation domain, the antigen recognition domain, and/or the therapeutic molecule domain of the engager molecule comprises an antibody or an antigen—binding fragment thereof, tag, a single chain variable fragment (SCFV), a monoclonal dy, FV, Fab minibody, diabody. in some embodiments, the activation domain, the antigen recognition domain, and/or the therapeutic molecule domain of the engager molecule ses a ligand, a peptide, a peptide that recognize and cts with a soluble TCR, or ations thereof. in some embodiments, these dynderived fragments or derivatives may be ed by al, biochemical, or molecular biological methods, Corresponding methods are known in the art and described, inter aim, in laboratory manuals (see Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd edition l989 and 3rd edition 2001; Gerhardt et at; bflethods for General and Molecular Bacteriology; ASM Press, l994l; liefnovits; immunology Methods Manual: The Comprehensive Sourcebool: of Techniques; Academic Press, 1997; Golemis, Protein—Protein Interactions: A Molecular Cloning Manual; Cold Spring Harbor Laboratory Press, 2902). in some instances, the polypeptides, antibodies, or n-binding fragments thereof used in the construction of the engager molecules described herein are humanized or deirnmunized constructs. Methods for the humanization and/or deimmunization of polypeptides and, in ular, antibody constructs are known to the person d in the art. in some embodiments, for any of the engagers described , the respective domains are in any order from N—terminus to C—terminus. For example, in some WO 06005 embodiments, the engas er molecule may comprise an N—terminal‘J‘v-r activation domain and a C‘- terminal antigen recognition domain. in some embodiments, the engager molecule may comprise an N—temrinal antigen recognition domain and a Cuterrninal activation domain. in some embodiments, the engager molecule may comprise an N-tenninal activation domain and a C— terminal therapeutic molecule domain. In some embodiments, the engager molecule may comprise an N—terminal therapeutic molecule domain and a C—terminal activation domain. in certain ments. T—cells are modified to secrete r molecules that have an antigen recognition domain or therapeutic le domain N—terminal to an activation domain. {06115} in particular embodiments, two or more of the domains of an engager molecule are linked by a linker. in some instances, the linker is of any suitable , and such a parameter is routinely optimized in the art. For example, linkers are of a length and sequence sufficient to ensure that each of the first and second domains can independently from one r, retain their differential binding specificities. The term "peptide linker” refers to an amino acid sequence by which the amino acid sequences of a ”first domain (eg. an activation domain) and a second domain (eg, an antigen recognition domain or therapeutic molecule domain) of a. defined construct are linked together. in some instance, one technical e of such peptide linker is that said peptide linker does not comprise any polymerization activity and/or does not e formation of secondary structures. Such peptide linkers are known in the art and described, for example, in Dall'Acqua et al, em. (1993) '37, 9266—9273), Cheadle et al.

(Mol lrnmunol (E992) 29, ZluSO); and Raag and Whitlow (FASEB (1995) 9(l), 73u80). in some embodiments, the peptide s of the present invention comprise less than 5 amino acids, less than 4 amino acids, less than ’3 amino acids, less than 2 amino acids, or 1 amino acid in some embodiments, the peptide linker is a single amino acid . In such embodiments, the single amino acid is typically a glycine (Gly). in some embodiments, peptide linkers that also do not promote any secondary structures are preferred. Methods for preparing fused, operatively—linked constructs and their expression in mammalian or ial cells are well—known in the art (Sec 8.3., l'nternational PCT Publication No. Wt) 99/134440; Ausubel, Current ols in Molecular Biology, Green Publishing Associates and Wiley lnterscience, NHY l989 and l994; and Sambrook et al, htlolecnlar Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory lJress, Cold Spring Harbor, New York, 200] ). 6} In some embodiments, the engager molecule is a single chain bi—speciiic antibody construct. The term "single chain bispecific antibody construct" refers to a construct comprising two antibody-derived g domains. One of the binding domains comprises variable s (or parts thereof) ofboth heavy chain (Vi-l) and light chain (VL) of an antibody 2017/040354 or antigen binding fragments or derivatives thereof, capable of specifically binding to/interacting with an tion molecule expressed on an effector cell (cg, CD3), The second binding domain comprises variable regions (or parts thereoi) of both heavy chain (VB) and light chain (Vii) of an antibody or n binding fragments or derivatives thereof, capable of specifically binding tel/interacting with a target antigen expressed on a Kirget cell (cg, CDl9) or an n expressed by and ellector cell (eg an inhibitor molecule). in particular ments, each of the two antibody or n binding fragments or derivatives comprise at least one complementary determining region (CUR), particularly a CUR}. In some embodiments, the single chain bi-speeific antibody construct is a bispecific scFV or diabody. ltltlllfl in specific embodiments, the single chain bispecific antibody construct is a single chain bispecifie scFV. An SCFV in general contains a VH and VL domain connected by a linker peptide. in some embodiments, a single chain bispecific scFV is sed of a signal peptide to allow for secretion from cells, followed by two scFvs connected by one or more linker peptides (La, ly, Lz). Bispecific single chain molecules are known in the art and are described in international PCT Publication No W'O 99/544140; Mack, l, lmmnnol, 0997), lSS, 3965—3970; Mack, PNAS, 0995), 92, 7021—7025; Kufer, Cancer lnimunol. lmmunother, U997), 45, 193— 197; Lottier, Blood, (2000), 95, 6, 2098—2l03; and , .l. immunol, (200B, 166, 2420-2426. {till} 18} in some embodiments, the molecular format of the polynncleotide encoding a. single chain bi-specific scFV polypeptide comprises nucleic acid sequence encoding a signal e (such as the signal sequences of SEQ ll) NO: 2 and 4} followed by two or more antibody—derived regions (eg, a first scFv and a second scFv). Each antibody—derived region (a;; scFy) comprises one Vl—l and one Via chain. in specific embodiments, the two or more antibody-derived regions are scFvs and are linked by a e linker to form a single chain bi— ic scFy construct. ln some ments, the bi—specitic scFy is a tandem binscFV or a diabody. Bispecific scFVS can be arranged in different formats including the following: VHO — lax—VLa—Ly—Vn —Lz-Vil3, Vta—LX—Vna~Ly-Vl-l ,l3, li"La—l_iX—Vri day—Vii ~Lz-Vl-l Vii: —l..x— Vi,a~Ly—VL 4L2» I’l-i Vii ~Lx—VL —Ly—VH —'L2uVi,a, Vr.a«Lx~VL ~Ly—Vl-l r Vii —'Lx-VH — , , LynVL aNLamL-x—VH mLwaL "LZ'VH VH ~Lx—Via—LynVn —LZnVL VL -LX-Vl.£t'-L§7~VH , , ~Lz—VH Vii -l_..x—VH -lly—‘iti'l_..a—l_iz—‘ti'l_, ,Vli —Lx-VH Joy—VlJa—Lz—V.1 {dill 19] in some embodiments, the engager molecule comprises multiple leg, 2, 3, 4, 5 or more) antigen binding domains to allow targeting of multiple ns. in some embodiments, the engager le comprises multiple (cg, 2, 3, 4, 5 or more) activation domains to activate effector cells. in some embodiments, the engager molecule comprises multiple (cg, 2, 3, 4, 5 or more) therapeutic molecule domains to activate effector cells. {seize} in specific embodiments of the disclosure, the engager molecule ses additional domains for the isolation and/or preparation of reconihinantly produced constructs, such as a tag or a label. The tag or label may be a short e sequence, such as a histidine tag {SEQ ll) NO: l2), or may e a "tag or label that is e of being imaged, such as scent or radioactive label. [001le in particular embodiments, the engager molecules of the present ion specifically hind to/interact with a. particular maticoal/structural epitopets) of a target antigen expressed on a target cell and an activation molecule sed on an effector cell (6g, an activation domain that specifically binds to one of the two regions of the human CD3 complex, or parts thereof). in particular embodiments, the engager molecules of the present invention specifically bind tor/interact with a particular coiifom1ational/structural epitopets) of an activation molecule expressed on an or cell and a different cell—surface protein expressed on an effector cell. Accordingly, city in some instances is determined experimentally by methods known in the art and methods as disclosed and described herein. Such methods comprise, but are not limited. to n blots, —linked immunosorhent assay (ELSA), radioimmunoassay (REA), radioimmunoprecipitation (RlP), electrochemiluminescence {:ECL), immunoradiometric assay {lRMA}, enzyme immunoassay (BIA), and peptide scans.

Activation dialecules and Target Cell Antigens [001 22E in some embodiments, binding of the activation domain of an r molecule to an activation molecule on the cell surface of an etl‘ector cell results in tion of the effector cell. As used herein, the term “effector cell” refers to any mammalian cell type that is capable of facilitating the death of a target cell. In particular embodiments, the or cells of the present invention are immune cells, such as a T cell, a B cell, an innate lymphocyte, a natural killer (MK) cell, a natural killer T cell (NET), a granulocyte (eg, a neutrophil, basophil, mast cell, or eosinophil), a macrophage, a monocyte, or a dendritic cell. Exemplary effector cell types include T cells, NK cells, NKT cells, and macrophages. {flOiZEE in some embodiments, activation of an effector cell may result in one or more of the following: (i) increased proliferation of the effector cell; (ii) changes in the expression or activity of one or more cell surface proteins of the effector cell; (iii) change in sion or activity of one or more intracellular proteins expressed by the effector cell; (iv) changes in the amount or nature of factors produced, and/or secreted by the effector cell, such as cytokines, chemokines or reactive oxygen species; (v) changes in the morphology of the ef‘ector cell; (vi) s in the chemotactic potential of the effector cell, such as through increased or decreased expression of one or more chemolcine ors; (vii) changes in the functional activity of the effector cell, such as increased cytolytic activity and/or sed phagocytic activity.

Activation of an eftector cell, or population of eil'ector cells, can be determined by any means known in the art. For example, changes in proliferation, protein expression, production, or secretion can be determined by tlow try, n blot, ELlSA, immunohistochemistry, immunoprecipitation, or immunofiuorescence and changes in cell morphology can be determined by us types of microscopy known in the art. [(99124] The skilled artisan will recognize that the nature of the activating molecule may vary according to the nature of the effector cell, although different groups of effector cells may share expression of certain types of activation molecules. For example, T cells express different surface receptors, ie. different activating receptors, than NK cells or macrophages. As an illustrative example, CD3 is an activating receptor expressed by T~cells that is not expressed by NK cells or macrophages, whereas CDl, CD16, NKGZD, and/or Nlipilll are activating receptors expressed by NK cells that are not expressed by T cells Therefore, in some instances, engager molecules that activate T-cells have a ent tion domain than engager molecules that activate Nli cells, hages, NliT cells, or other types of ellector cells.

Exemplary activation molecules are described below and shown in Table l. @6125] ln some embodiments, the effector cell is a T cell and. the activation domain of the engager molecule binds to an activation molecule expressed by the T cell. The T* cell repertoire is comprised of numerous sub—types of T cell, including NKT cells, cytotoxic T cells (Te or CTL), memory T cells, helper T cells (a g, Thl, T112, Tlil7, Tl’lg, and/or ThZZ cells), suppressor T cells (cg, regulator T cells )), l~associated invariant T cells, and “[5 T cells. In some instances, one or more surface receptors expressed by one T cell e are not expressed. by another T cell subtype. in some instances, one or more surface receptors expressed by one T cell subtype are expressed by at least one other T cell subtype. in some instances, one or more surface ors expressed by one T cell subtype are generally expressed by all, or most, T cell subtypes. For e, CD3 is a ing component of the T cell receptor (TCR) complex and is sed in multiple T cell subtypes. ary activation molecules expressed by T cells (cg. T cells, or helper T cell), include, but are not limited to one or , NKT, Tc, memory more components of CD3, (cg, CD3v, CD35, CD33 or C 35;), CD2, CD4, CD5, CD6, CD7, C08, CD25, CD27, CD28, CD30, CD38, CD40, CD57, CD69, CD70, CD73, CD81, CDSZ, CDl34-, CDl37, CDlSZ, or CD278. in some embodiments, the effector cell is an NKT~cell in such embodiments, the activation molecule includes, but is not limited to, CD3 or an invariant TCR.

WO 06005 {@126} In some embodiments? the effector cell is an NK cell and the activation domain of the r molecule binds to an activation molecule expressed by the NK cell.

Exemplary activation molecules sed by NK cells include, but are not limited to, CD316; CDQ4/NKGZ (gage, NKGZD); NKp3Q NKp44, NKp46, or killer activation receptore (KARE).

Table l: Exemplary Activation les CD3 or components thereof (6g, (3031/, CD35, CD33 or tone______________________________________ CDZ ' con: l..§£l.§________________________________________________ __ {3136 CDQL‘l/NKGQ {fig Naiozn) CD7 ‘ coir; c.1325 (3)27 cozs CD38 CD40 CD57 ............................................................................................................

CD69 icon gonzo / 134 loom CD”— lcozve [(99127] In some embodiments, binding of an engager molecule to a target cell and an effector cell (e. 3:, binding of an activation domain to a molecule on an etiector cell and binding of an antigen recognition domain to a le present on a target cell) brings the effector cell. in close proximity to the target cell and, thereby facilitates the destruction. of the target cell by the effector cell. As used herein? the term “target cell” refers to a mammalian cell that should be killed, attacked, destroyed, and/or controlled. in particular, target cells are cells that are in some way d compared to a normal cell ofthe same cell type, such as a cancerous cell, a bacterially~inlected cell, a yirally~inlected cell, a flingally~iiilected cell, and/or an autoimmune cell. In particular ments, the target cells of the present invention are cancerous cells (cg, tumor cells). Destmction (216., death) of a target cell can be determined by any means known in the art, such as flow cytometry (eg, by AnnexinV, ium iodide, or other means), cell counts, and/or microscopy to determine tl e cellular morphology of the target cells, } In some embodiments, the antigen recognition domain of an engager molecule brings a target cell (cg, tumor cell) into the vicinity of an ellector cell via ction between the n recognition domain and surface antigens expressed by the target cell (tag target cell ns). ln some embodiments, the -cell antigen is a tumor antigen. ln some embodiments, a tumor antigen is a tumor-specific antigen (TSA), and is expressed only by tumor cells. ln some embodiments, the target cell angien is a tumor—associated n (FAA), and. is expressed by tumor cells and one or more types of normal cells or non—tumor cells, In some cases, TSA is also present in one or more types of normal cells or non—tumor cells, but is predominantly expressed by tumor cells. In some instances, a tumor antigen (erg, "ISA or 'l‘AA) is present in one cancer type, in some instances, a tumor antigen is present in multiple cancer types In one embodiment, a tumor antigen is expressed on a blood cancer cell. ln another embodiment, a tumor antigen is expressed on a cell of a solid tumor. in some embodiments, the solid tumor is a glioblastoma, a non—small cell lung cancer, a lung cancer other than a non—small cell lung cancer, breast cancer, prostate cancer, pancreatic cancer, liver cancer, colon cancer, stomach , a cancer of the spleen, skin cancer, a brain cancer other than a glioblastoma, a kidney cancer, a thyroid cancer, or the lilre. in more specific embodiments, a tumor antigen is expressed by a tumor cell in. an individual.

{QOl 29] Exemplaiy tumor antigens (cg, TSAs or TAAs) include, but are not limited to, alphafetonrotein (AF-P), carcinoernbryonie antigen (CEA), (IA—lZS, epithelial tumor antigen (E’l‘A), tyrosinase, CD10 (also known as neprilysin, membrane metallomendopeptidase (MME), neutral endopeptidase {NEl’}, or common acute ’blastic leukemia antigen (CALLAD, CDlS, CDlQ, CD20, CD21, CD22, CD30, CD33, CD38, CD44, CD44Vo, CD44v7/8, CD7G, CDl23, CD138, CD171, ras, p53, y—ral‘ marine a viral oncogene homolog Bl (BRAF), calcium binding; tyrosine~(‘i”)~pltospliorylation regulated (CABYR), CYS’EClUG‘llCh secretory n 3 (CREPE), CSAG family, member 2 (CSAGL’), cancer/testis antigen 2 {CTAG2), dihydrotblate reductase (DE-{FR}, ferritin, heavy polypeptide l; testis— specific expression (FTi-H.Jl7), G antigen 1 , lactate dehydrogenase C , ma antigen i'ariiiiy A (MAGEA) l, i‘viAtli-EA3, b’lAGEr—M, (meianon'ra antigen faniiiy B, 6') MAGEBES, mitogennactivated protein kinase 1 {MAPKl}, MHC Class l poiypepticle~relatecl sequence A (MESA)? niacin (MUG) l, eeii surface associated {iii/SUCH); MUClfi NLR family pyrin domain containing 4 {NLRW}, New York esophageal squamous cell carcinoma l (NY— ESO~1), FDZ binding iiinase (PB), preferentially expressed antigen in melanoma (PEAR/Eli); sex ining region ‘Y-box (SGX)~2, SGX l0, SGKl in, sperm protein associated with the nucleus , K—iinked, family member Ai (SFANXAi)? synovi‘i sarcoma; X (58X) breakpoint 2 (835(2), SSX4, SSXSE testis specific, it} {TSGAlG}, testia-specitic seiirie kinase 6 iTSSKé}, tubby Bike protein {‘I'ULPZ), X antigen taming member 2 (XAGEZ)? zinc linger protein 165 fi). abeent in melanoma .Z (AlMZ), BMH poiycomb iing finger oncogene ii)? cyclooxygenase—2 {COX-2}, tyrosine related protein (TEE—l} TRF—Z? glycoprotein EGG {GPlOO}, epidermal growth factor receptor variant lll (EGFRVIH); enhancer of zeste hoinoiog 2. (Eli-{2), human Ll eeii adhesion molecule (liiCAM), Livin, ninitidmg resistance protein 3 (MRP-fi), Nestin, oiigodendroeyte transmigration factor {OLEGZ}, antigen recognized by T ceiis (.ART}~i, ART4, squamous ceil oma antigen recognized by T celis (SART)~L SARTZ, SART‘S, Encyclin, i — catenin, giiomavassociated oncogene noinlog 1 (Grill), caveoiinnl , cathepsin 3, cluster of differentiation (filth—M, iiai caicinm—dependent on erin), EFF? receptor A2 {prA2), EphAZ/epithelial kinase {EphAZ/Eckip fos~related antigen .1 {Fra-l/Fosi l)? Gangiioside/GDZ, GEE, aeetylgiiicosaminyltransferase~V {Girl—V, 151,644), hniiian epidermai growth factor receptor 2 (Herl/Neut nuciear erati0i1~associated antigen of fifiiibfidy Kit}? (Kim; human Kn beterodimer proteins subunits: (1370/89), interienkinJ'B receptor subunit alpha.— 2 (ii-lBRaZ), n'ieianoina n recognized by T cells (MARTJ :3, prospero homeobox protein 1 (PRUXl), prostate stein ceii antigen (955C231 vin, uroitinase~type plasminogen activator or ({ZiPAR}, Wiinis’ turner protein 1 (W'l‘llfn Folate receptor a, Glypican—TS, 5T4, 8H9, rape integrin, Eli-E3, Eli-36, CAEX, CA9? CSPG4, E01029 E63340, EpCAM, ERBB3, ERBB49 ErbB3/4, PAP, FAR FBP, fetal AchR, l-{LA—Al, HLAuAZ, ILuIRa, KDR, Lambda, Lewis—Y, MCSP, Mesothelin, NCAM, NKGZD ligands, PSCl, PSMA RORL 'l‘AG’72, TEMl, 8, VEGRRZ, HM‘W-MAA, VEGR 'VFGP ora P—glycoproteini erythropoietin (FPO), cadheiin, CD4, CD8, (ID/ii CDil’] (c—kitL C3133, HLA—A. l-‘ELA~B, HLA~C, chemokine or 5 (CCRS), stem cell marker ABCGZ transporter, immunogiobnlinsj integrlns, prostate specific antigen (PSA), prostate stein celi antigen (l’SCA), dendritic cell—specific interceliniar adhesion moieciiie B—grabbing nonintegrin (DC—SIGN), lobulin; granuloefiomacropliage colony stimulating factor (GM—CSF), myogenic entiation promoting factor—i (MyoD—l), Len—7 (CD57), LeuM~‘1, eel} proliferanon~associated human nucienr antigen defined by the onal antibody K967 (Ki—67): viral envelope proteins, HIV ngO, and transferrin receptor. Uther exernpiary tumor antigens are antigens that are present in the extraceliuar matrix of tumors, such as ta} variants of fibroneetin, ten again, or necrotic regions of tumors.

Tabie 2: ary Target Cell Antigens CABYR CAIX cathepsin B C 0 CD 1 17 CD 1 23 CD 1 33 CD 1 5 CD 1 7 i (name CD44v7/8 CGXuZ DEC—SIGN DEER E~cadhsrin EGFR EGE'RVEH EFCAM E .hAZ EuhAZfEci-g ErbBS/4 P REE!!- ervthro 01mm '.4 Folate Recs t0!" 3 Frwl/Posi i Hmw___________________________________________________ HLA A HLA B HLA C HLAuAZ 13.1-1ch kagpa light chain Lambda “le LECAM Livin MAGEA i MAGEAS MAGEA4 MAGEBG MAR"I‘~i, MCSP Mesetheiin M {113% MUC E MUFEE) or 5 M «31:31 F“-1/00 retain SPANXAI asst:F 353334 IEIIEIIIIIIIIIIIIIII If\_I__l_____ TEE/l8m tenascin thVroOlobulin errin reee tor rand IfifiIIIIIIIIIIIIIIII tactic ELP2.

Efiflflflfiflllllllllllllll ”Q80 UPAR VEGF VEGF Rece tois V[56332 ‘S-catenin In certain ments, the antigen recognition domain of an r molecule specifically binds a tumoruassociated antigen (TAA) or a tumor—specific antigen (TSA). in certain embodiments, the antigen recognition domain comprises an antibody or an antibody fragment or an antigenebinding fragment or portion thereof, such as for example, a monoclonal antibody, FV, a scFV, Fab, rninibody, or diahody that is specific for a. TAA or ISA. In certain embodiments, the antigen recognition domain of the engager is an scFV that is specific for a TAA or "ISA. in a specific embodiment, the ’l‘AA or TSA is expressed on a cancer cell. In one embodiment, the TAA or TSA is expressed on a blood cancer cell. In another embodiment, the TAA or TSA is expressed on a cell of a solid tumor. In more specific embodiments, the solid tumor is a glio‘olastoma, a nonmsmall cell lung cancer, a lung cancer other than a non—small cell lung cancer, breast cancer, prostate cancer, atic cancer, liver cancer, colon cancer, h cancer, a. cancer of the spleen, skin cancer, a brain cancer other than a, glioblastoma, a kidney cancer, a d cancer, or the like. In more ic ments, the TAA or TSA is expressed by a tumor cell in an individual. In some embodiments, the anti J en-recoqnition domain of theC‘ engager le is specific for one or more target cell antigens shown in Table 2.

E'nizA2 {9013M in some embodiments, EphAZ is referred to as EPH receptor AZ (ephrin type—A receptor 2; EPHAZ; ARCCZ; C’l‘PA, C’l'PPl; or ECK), which is a protein that in humans is d. by the EPHAZ gene in the ephrin or subfamily of the protein—tyrosine kinase family. Receptors in this ily generally comprise a single kinase domain and an ellular region comprising a Cys—rich domain and 2 iibronectin type lll repeats; embodiments of the antibodies of the disclosure target any of these domains. An exemplary human EphAZ nucleic sequence is in k® Accession No. Nl‘v’l_00443 l, and an exeniplaiy human EphAZ polypeptide sequence is in GenBank® Accession No. NP_004422, both of which sequences are incorporated herein in their entirety. An exemplary human EphAZ c sequence is in GenBanlc® Accession No Nl‘iy’l_0(‘i4/l-zl-8.2, and an exemplaiy human EphAZ polypeptide sequence is in GenBank® Accession No. NP_004439, both of which sequences are incorporated herein in their entirety.

The Eph family, the largest group among tyrosine kinase or families, is comprised. of the EphA (EphAl—lfl) or EphB (EphBl—é) subclasses of receptors classified as per their sequence homologies and their binding affinity for their ligands, lEphrins (Eph receptor interacting protein). The human EphAZ gene is located on chromosome l, encodes a receptor ne kinase of 976 amino acids with an apparent molecular weight of 130 kDa and has a 90% amino acid sequence homology to the mouse EphAZ. The Eph family contains an extracellular conserved N—terminal -binding domain folloi 'ed by a cysteine—rich domain with an epidermal growth rmlilre motif and two tihronectin typewlll repeats. The extracellular motif is tollowed by a membrane spanning region. and a cytoplasmic region that encompasses a juxtamenihrane , a tyrosine kinase domain, a sterile alpha niotif (SAM), and a post synaptic domain (disc large and zona occludens protein (PDZ) domain-binding motif}. EphAZ shows 25-35% sequence homologies with other Eph receptors, and the tyrosine residues are conserved Within the juxtamembrane and lcinase domain. {@133} EphAZ mRNA expression is observed in the skin, bone marrow, thymus, uterus, testis, prostate, urinary bladder, kidney, small intestine, colon, spleen, liver, lung and brain. EpliA2 expression in the colon, skin, kidney and lung was over ten—fold. relative to the hone marrow. EphAZ is also expressed during lation in the ectodermal cells and early enihryogenesis in the ping hind brain. in the skin, EphAZ is present in heratinocytes of WO 06005 epidermis and hair les but not in dermal cells (fibroblasts, vascular cells and inflammatory cells), EphAZ is also expressed in proliferating mammary glands in female mice at puberty and differentially expressed during the estrous cycle. Besides its expression in embryo and in normal adult tissues, EphAZ is overexpressed in several cancers, such as breast cancer, gastric cancer, melanoma, ovarian cancer, lunch cancer, gliomas, urinary bladder cancer, prostate cancer, esophageal, renal, colon and vulvar cancers. in particular, a high level of EphAZ is detected in malignant cancer-derived cell lines and advanced foims of cancer. in light of the thAZ overexpression in preclinical models and clinical specimens of many different types of cancer, the increased level of EphAZ expression is informative in both the prediction of cancer outcomes and in the clinical management of . The differential expression of EphAZ in normal cells compared to cancer cells also signifies its importance as a therapeutic target. militia-ll in some ments, HERE is referred to as human Epidermal Growth Factor or 2 (Non, Eth—Z, CD340, or pi 85}, which is a protein that in humans is encoded by the ERBBZ gene in the epidermal growth factor receptor (EFR/Elth) family. l-lERZ contains an extracellular ligand binding domain, a transmembrane domain, and an ellular domain that interacts with a multitude of signaling molecules HFRZ is a member of the epidermal growth factor receptor family having tyrosine hinase ty. Dimerization of the receptor results in the ospliorylation of tyrosine residues within the cytoplasmic domain of the receptors and tes a variety of signaling pathways leading to cell eration and tuniorigenesis.

Amplification or overexpression ofHERZ occurs in approximately 15—30% of breast cancers and -«30% of gastric/gastroesophageal cancers and serves as a prognostic and predictive biomarlrer, HERZ overexpression has also been seen in other s lilre ovary, endometrium, bladder, lung, colon, and head and neck. HERZ is overexpressed in 15—30% of invasive breast cancers, which has both prognostic and predictive implications. Overexpression of HERE protein, determined using ll-lC was found in 23% and gene amplification determined using FlSl-l in 279/5 of 200 resected tumors in a gastric cancer study. HERZ overexpression is directly correlated with poorer outcome in gastric cancer. in a study of 260 gastric cancers, HER}: overexpression was an independent ve prognostic factor and l-lERZ staining intensity was correlated with tumor size, serosal invasion, and lymph node metastases. thier studies also confirmed the negative impact ot‘HERZ pression in gastric . HEEL). overcxpression is reported in 0—839?) of esophageal cancers, with a tendency towards higher rates of positivity in arcinoma (l 0— 83%) compared to squamous cell carcinomas (ll»-56"?/£i). xpression ofl-lERZ is seen in 20-- % patients with ovarian cancer. in endometrial serous carcinoma, the reported rates of HERZ overexpression range between l4% and 80% with Ell—3R2 amplification (by fluorescence in situ hybridization lFlSHl) ranging from 21% to 47%. Embodiments of the antibodies of the disclosure target the extracellular ligand binding domain.

Distal?)gmmgz’iosit. e GD2 {fifill‘l'fij Disialoganglioside GDZ is a sialie acid—containing phingolipid expressed primarily on the cell e, The function of this carbohydrate antigen is not completely tood; however, it is thought to play an important role in the attachment of tumor cells to extracellular matrix proteins. GDZ expression in normal fetal and adult tissues is pnmarily restricted. to the l nervous system, peripheral nerves, and skin melanocytes, although GDZ expression has been described in the stronial component of some normal tissues and White pulp of the spleen. in malignant cells, GDZ is uniformly expressed in lastomas and most melanomas and to a. variable degree in a variety of other tumors, including bone and soft-tissue sarcomas, small cell lung , and brain tumors. GD?! is present and concentrated on cell surfaces, with the two hydrocarbon chains of the ceramide moiety embedded in the plasma membrane and the oligosaccharides located on the extracellular surface, where they present points of recognition for extracellular molecules or surfaces of neighboring cells.

Because of the relatively tumor-selective expression combined with its presence on the cell surface, GDZ is an attractive target for tumoruspecific dy therapy. ments of the antibodies of the disclosure target the extracellular domain.

Therapeutic 1W{decades {tltllfio} ln some embodiments, the pseudotyped oncolytic Virus comprises a c acid sequence that encodes an r molecule and one or more additional nucleic acid ces that encode one or more therapeutic molecules. As used herein, a peutic molecule” refers to a molecule that enhances the therapeutic efficacy of an oncolytic virus bed herein. ln general, the therapeutic ino ecules described herein are proteins, nucleic acids, or a combination thereof. Exemplary therapeutic molecules include cytokines, chemolrines, antibodies or antigen g fragments thereof, proteases, RNA polynucleotides, and DNA polynucleotides. {hill 37] ln some embodiments, the therapeutic molecule is capable of increasing or enhancing the therapeutic efficacy of an oncolytic Virus described herein by stimulating, or ting, a cellular immune response. In some embodiments, the therapeutic molecule is capable of increasing or enhancing the therapeutic efficacy of an oncolytic virus described herein by antagonizing a suppressive or regulatory immune response. In some ments, reduction of a suppressive immune response occurs in a tumor microenvironment. in some instances, reduction of a suppressive immune response by the therapeutic molecule enhances the oncolytic effects of a pseudotyped oncolytic virus described herein. in some embodiments, the therapeutic molecule further reduces immunoregulatory T cell activity in a subject treated with a pseudotyped oncolytic virus described herein, in some embodiments, the therapeutic molecule modulates or impairs the production level of a protein at a nucleic acid level or at a protein level, or disrupts a n on. {9&138} in some embodiments, a nucleic acid sequence encoding an engager molecule and a nucleic acid sequence encoding one or more therapeutic les are sed within the same vector, ln some embodiments, a nucleic acid sequence ng an engager molecule and a nucleic acid sequence encoding one or more therapeutic les are sed in different vectors~ ln some embodiments, the vector is a viral vector in some ces, a therapeutic molecule comprises a polypeptide or a c acid r, in some embodiments, the onal nucleic acid sequence is ed into a viral vector which allows higher expression levels and production of the therapeutic le. @6139} in some embodiments, the therapeutic molecule is a polypeptide. ln some instances, the polypeptide is an immune modulator polypeptide. in some cases, the immune modulator polypeptide is a cytohine, a co—stimulatory domain, a domain that inhibits negative regulatory les of T—cell tion (eg, an immune checkpoint inhibitor), or a combination thereof. {00140} In some embodiments, the immune modulator ptide modulates the activity of one or more cell types, such as regulatory T cells (”l‘regs), myeloid—derived suppressor cells (ls/EDSCs), dendritic cells, and/or '1' cells. Exemplary 'l'reg modulatory polypeptides include CCRd, l-lelios, TlGlT, GlTR, neuropilin, neuritin, CDl03, CTLA—Al, lCOS, and Swapfll Exemplary MDSC inodulatory polypeptides e TGRBRl, GM—CSF, lNFy, interleukins such as HAS, lL—lFZ, lL—o, anl0, anl2, IL—l3, lL—6, lid—elm, lL—é/anoR complex, ’I‘GFmpl, lyi— CSF, l’rostaglandin Ell/PGEZ, Prostaglandin E Syntliase 2, SlOOAS, and VEGF, Exemplary dendritic—cell directed modulatory polypeptides include GMuCSF and/or ill—l3, Exemplary T cell-directed modulatory ptides include lL—lZ, OX-n‘tfl, Gl'l‘R, (31792.8, or li—78, or an antibody that agonizes a pathway comprising ill—12, {Di—40, Gl'l'R, C1123, or ill—23. {90141} in other embodiments, the therapeutic polypeptides modulate the fibrotic strorna. Exemplary librotic stromal polypeptides include fibroblast activation protein—alpha (PAP). in some embodiments, the therapeutic polypeptide is a protease. ln particular embodiments, the protease is capable of altering the extracellular matrix, particularly the extracellular matrix within a tumor microeuvironmein. Exemplary proteases e matrixmetalloproteases (MMP), such as MMP"), collagenases, and elast‘ases. tines as Them eufic molecules {fillislfij in some cases, the immune modulator polypeptide is a cytokioe. Cyrokmes are a ry of small proteins between about 520 kDa that, are ed in cell signaling and e chemokines, interferons (ENF), eukins (1L), and tumor necrosis factors (TNF), among others. Chemokines play a role as a cheinoattractant to guide the migration of cells and are classified into four subfamilies: C‘XC, CC, CX3C, and XC. Exemplaiy chemokines include chemolcines from the CC subfamily, such as CCLl, CCU: {MCPJE}, CCL3, CCllLl. CCLS (RANTES), {3313146, CCU’, CCLS, CCU? {or CCLlO}, C(ZLl l, (ICU; CCLlS, (fluid, Gilli, 0:11.46, CCLH, 0:11.18, CClll‘), CCLZG, CCLZZE, €61,222, C0123, {Still/7.4, (18,25, some, CCLET’, and CCl.,28; tlie CXC subfamily, such as CXCLl, CXCLZ, CKCLB, t7XtTlA, CKCES, (IXCLo, (TXCLI CKCLEl, (IXCLQ, CXCthl, CXCLll, €XCLlZ. EXCLB, (IXCLM, CXCLlfi, CXCLlo, and CXCLN; the X1: subfamily, such as XCLl and XCLZ; and the CXS’C uly, such as CXTSCH. {90143} luterferous {lFNs} comprise Type l lFNs (cg. Ellie, lFN—B, lFN~e, lFl‘xlu EC and lFNwl. Type ll {FNS (cg. WNW), and Type Ell lFNs ln some embodiments, lFN—u is further classified into about 13 subtypes including lFNAl, lFNAZ, lFNAL’i, lE'NAfi, nuns, EFNAE EFNAS, lFNAl 0, lFNAl ‘3, lFNAlzl, lFNAlfi, . and IFNAZI. {00144} li'iter'leukins are a broad class of cytoltine that promote the development and differentiation of innnune cells, including 'l' and B cells, and other lieniatopoietic cells, Exemplary eukius include llrl, ill—2- l'l...—3, lip—4, llnfi, limo, lln7. liril {{TXCLS), ill»? ll...— ltl, llrl l, llrl 12,1133 '3, ll,~ l 4, lL~l ‘i, llnl c3, llrl 7 llrl 3 113‘ng 2t'l, lle i, H.932, lit—23. ll,— 24, 3'? ~25. ll .u'?6, ll 07, lLu‘Tiél, lLJQ, lLu30, lL~3l, lL~32, ELSE, lL~35. and lLu36.

{Midi-SE Tumor necrosis lactors ('l'NFs) are a group of cyrols’iues that modulate sis l'n some instances, there are about l9 members within the ’l'Nl: fiamily, including, not d to, TNFu, lyinphotoxin—alpha (LT—oi), lymphotoxin—beta (LT—E3), T cell antigen gpl‘i‘) {{TDJlUL), CD271, CDSGL, FASL, 4—lBBL, thils’le. and 'l'NF—related apoptusis inducing ligand ("ERA ll..-). in some ments, a pseudytoped oncolytic Virus comprises a nucleic acid sequence that encodes an engager and an additional nucleic acid ce that encodes a WO 06005 cytokine selected from cheniokine, interferon, interleukin, or tumor necrosis factor. in some embodiments, a pseudytoped oncolytic virus comprises a nucleic acid sequence that encodes an engager le and an additional nucleic acid sequence that encodesa chemokine, an interferon, an interleukin, and/or a tumor necrosis factor.

(”Jo—stimulator 7 domains as Them cutie molecules {00l47j in some embodiments, the immune modulator polypeptide is a co— stimulatory . in some cases, the (so—stimulatory domain es n—specific cytotoxicity. in some cases, the co-stimulatow domain further enhances cytokine tion. in some embodiments, the oer—stimulatory domain comprises CD27, CDZS, CD70, CD80, C983, (1386, CDl34 (OX—40), L (OK—40L), (1)137 (Ll lBB), CDl37L (41881;), or CDZZI-‘l. in some embodiments, a pseudjyrtoped oncolytie Virus ses a nucleic acid sequence that encodes an engager and an additional nucleic acid sequence that encodes a co— stiinulatmy domain, ln some embodiments, a pseudjyrtoped oncolytic Virus comprises a c acid ce that s an engager and an additional nucleic acid sequence that encodes a co— stimulatory domain selected from (ID27, CD28, CD80, CD83, (ID86, (ZDl34, CD134L, CD137, CD137L, or CD224.

Immzme check oint inhibitors as tic molecules [OOl 49E in some embodiments, the immune modulator polypeptide is an immune oint inhibitor polypeptide that inhibits a negative regulatory molecule of T-cell activation. lnimune checkpoint inhibitor bind to immune checkpoint molecules, which are a group of molecules on the cell surface of CD4 and (EDS T cells. in some instances, these molecules effectively serve as ”brakes“ to downnmodulate or inhibit an anti~tumor immune response. An immune checkpoint inhibitor refers to any molecule that modulates or inhibits the ty of an immune checkpoint molecule. in some instances, immune checkpoint inhibitors include antibodies, antibodyuderivatives (6g, Fab fragments, scFvs, minobodies, diabodies), antisense oligonucleotides, siRNA, aptamers, or peptides. {001%} Exemplary immune checkpoint molecules include, but are not limited to, mmed death—ligand l (PDLl, also known as B7—l—l l death l (EDD—l), , CD274), programmed PD-L2 (B7—DC, CD273), LAG3, TIMES, 284, AZaR, B7Hl, B7H3, B7H4, BTLA, CD2, CDlo', (TD/27, C1128, CD30, CD40, C7070, CD80, CDtlo, CD137, come, CD226, 00276, DR3, GAD), GlTR, l-lAVCRZ, HVEM, lDDl, IDQZ, inducible T cell costimulatory (ICDS), KlR, LAlRl, LlGl-lT, hage receptor with collageneous structure (MARCO), (Di-40, phosphatidylseiine (PS), SLAM, TIGHT, VISTA, and VTCNl. in some embodiments, an immune checkpoint inhibitor inhibits on or more of PDLl, PD—l, {FLA—4, PD—L2, LAGB, TlM3, 234, AZaR, B7Hi, B7H3, B784, B'l‘LA, CD2, CD27, CD28, CD30, C1140, CD70, CD80, CD86, CD137, CDléO, CD226, CD276, DR3, GALQ, GITR, HAVCR2, HVEM, lDOl, lDD2, ECDS, KlR, LAlRl, LlGl—lT, MARCO, OBI—40, PS, SLAM, TIGHT, VISTA, and VTCNl. [00l513 in some embodiments, a pseudytoped oncolytic Virus comprises a c acid sequence that encodes an engager molecule and an additional nucleic acid sequence that encodes an immune checkpoint inhibitor. in some ments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint molecules. in some embodiments, the immune checkpoint inhibitor reduces the interaction between an immune checkpoint molecule and its ligand (cg, reduced the interaction between PDml and l’DlJl), in some embodiments, a pseudytoped DECOlyllC virus comprises a nucleic acid sequence that encodes an engager and an additional nucleic acid sequence that encodes an immune checkpoint inhibitor that inhibits one or more of PDLl, PDd, CTLA—rl, PD—LZ, LAG3, TlMB, 2184, A‘ZaR, B7l-ll, Bid-D, B7H4, BTLA, CD2, CD27, CD23, CD30, CD40, CD70, CD80, C086, CD137, CDl60, CD226, CD276, DRE, GALQ, GITR, l—lAVCRZ, l-lVEM, lDDl, lDDZ, ICDS, KlR, LAlRl, LlGHl‘, MARCO, 0X40, PS, SLAM, ’l'lGHT, VISTA, and VTCN 1. {00152} in some embodiments, a pseudytoped oncolytic virus comprises a nucleic acid sequence that encodes an r molecule comprising an activation domain and a therapeutic molecule domain, n the therapeutic molecule domain is an immune checkpoint inhibitor. in some embodiments, a oped oncolytic virus comprises a c acid sequence that encodes an engager molecule comprising an tion domain and a therapeutic molecule domain, wherein the therapeutic molecule domain is an immune checkpoint inhibitor that inhibits one or more of PDLl, PD—l, CTLAJL, PD—LZ, LAGB, , 234, AZaR, B7Hl, B7H3, 3871-14, B’l‘LA, CD2, CD27, CD28, CD30, CD40, CD70, CDSG, CD86, CDl37, CD160, CD226, CD276, DR}, GAD), GITR, HAVCRZ, l-lVEM, lDOl, lDO2, lCOS, KlR, LAlRl, LiGlilT, MARCO, 0X40, PS, SLAM, TlGl-l‘T, VESTA, and VTCNl, a) PDL! Inhibitors {MESS} in some ments, the immune checkpoint inhibitor is an tor of l’DLl. In some embodiments, the immune checkpoint inhibitor is an antibody (cg. a onal antibody or antigen—binding fragments f, or a humanized. or chimeric dy or antigen— binding fragments thereof} against PDLl. in some embodiments, the inhibitor of PDLl reduces the expression or activity of PDLl. in some embodiments, the inhibitor of PDLl reduces the interaction between Will and PDLl. Exemplaiy tors of PDLE include DLl antibodies, RNAi molecules (cg), anti~PDLl RNAi), antisense molecules (eg an anti—PDLl antisense RNA), or dominant negative proteins (eg, a dominant negative PDLl protein}.

Exemplary anti—PDLl antibodies includes clone El-llZ; MPDESZSOA (Genentechi RG7446); ouse PDLl antibody Clone lOF.9GZ (BioXcell; Cat # BEOlOl); DLl monoclonal antibody MDX—llOS (EMS—936559 and EMS—935559 from Bristol-Meyers Squibb; MSBUOEOH 8C; mouse anti ~Pl_)l,l Clone '29E.2A3; and AstraZenecals MElHZl-l’llo. [($154] In some embodiments, the anti~PDLl antibody is an anti—PDLl antibody disclosed in international PCT Publication Nos. VVO 20l3/079l74; ‘vVO ZOE/036959; Wt) 56716; WG Zt‘itflfet‘iill’izl; W0 20l0/089411; ; ; WO 2006/133396; W0 208199906; W0 2012/l45493; WO 2013/l81634; US. Patent Application Publication No. 20:; 40294898; or Chinese Patent Application ation No. (1N lfll10464tl {00155l in some embodiments, the PDLl inhibitor is a c acid tor of PDlil sion. in some embodiments, the l’DLl inhibitor is one disclosed in international l3CT Publication Nos. VVO ZOl lil27l 30 or ‘WG 201l/00034l, in some embodiments, the PDLl inhibitor is rapamycin.

In some embodiments, a psetidytoped oncolytie virus comprises a nucleic acid sequence that encodes an i molecule comprising an activation domain that binds to CD3 (erg, an anti-CD3 scFv') and a. therapeutic le domain that binds to PDLl (cg, an anti—PDLl scFv). In such embodiments; the pseudytoped oneolytic virus may further comprise an additional nucleic acid sequence that s an additional therapeutic molecule. {£9957} ln some embodiments, a pseudytoped oncolytic virus ses a c acid sequence that encodes an engager molecule comprising an activation domain and a therapeutic molecule domain that binds to l’DLl. In some embodiments, a pseudytoped oncolytie virus comprises a nucleic acid, sequence that encodes an engager molecule comprising an activation domain and an antigen recognition domain? and an additional nucleic acid sequence that encodes a PDLl inhibitor. In some embodiments a pseudytoped oncolytic virus comprises a nucleic acid sequence that encodes an r and an additional nucleic acid sequence that encodes PDlil inhibitor selected from Elli; Genentecli’s MPDLSZERGA (RG7446); Anti—mouse lJDLl antibody Clone lOFQGZ (Cat # BEOlQl) from BioXcell; anti—PDlJ monoclonal antibody MEX—HOS 36559) and EMS-935559 from Bristol—Meyer‘s ; MSBOGlQ’ilSC; mouse anti—l’Dl..-l Clone 29E2A3; and AstraZeneca.’s MEN/41736. b) PD-L21nhz’bito/‘S {66°58} In some embodiments, the immune checkpoint inhibitor is an inhibitor of PDuLZ. In some embodiments, the inhibitor of PD~L2 is an antibody (cg, a monoclonal antibody or fragments, or a humanized or chimeric antibody or fragments thereol‘) against PD—LZ. In some embodiments, the inhibitor of PD—LZ reduces the expression or activity of EDD-142. In other embodiments, the inhibitor of PD—LZ s the interaction between PDul and PD-LZ.

Exemplary inhibitors of PD-LZ include antibodies (eg, an anti—PD—LZ antibody), RNAi molecules (eg, an anti—PD-lilZ RNAi), antisense molecules (cg, an anti—PD-LZ antisense RNA), or dominant ve proteins (eg, a dominant negative PD—LZ protein), {06159} In some embodiments, the PD—LZ inhibitor is GlaxoSmithKline’s AMP— 224 inmune). In some embodiments, the PIE—L2 inhibitor is 2B7. [$01.69] In some embodiments, a pseudytoped oncolytic Vitus comprises a nucleic acid sequence that encodes an engager molecule sing an tion domain and an antigen recognition domain, and an additional nucleic acid sequence that encodes a PD—LZ inhibitor. In some embodiments, a pseudytoped oncolytic Virus comprises a c acid. sequence that encodes an r and an additional nucleic acid sequence that encodes PD-LZ inhibitor selected from AMPu224 mmune) or rl-ilgM 12137.

In some embodiments, a pseudytoped oncolytic Virus comprises a nucleic acid sequence that encodes an engage-r le comprising an activation domain and a therapeutic molecule domain that binds to PDLE. In some embodiments, a pseudytoped oncolytic virus comprises a nucleic acid sequence that encodes an r molecule comprising an activation domain that binds to CD3 (tag, an anti—CD3 scFv) and a therapeutic le domain that binds to PD-IJZ (eg, an anti-P9142 sellv). in such ments, the pseudytoped oncolytic virus may further comprise an onal nucleic acid sequence that encodes an additional therapeutic molecule. 0) P04 [irzhibitors {00162} in some embodiments, the immune checkpoint tor is an inhibitor of FBI. in some embodiments, the inhibitor of PDLI is an antibody (eg a monoclonal antibody or fragments, or a humanized or chimeric antibody or fragments thereof) against it‘ll-l Exemplary antibodies against Pill include: anti—mouse PD~l dy Clone I43 (Cat # 2) from BioXceil; anti—mouse PD—l antibody Clone KMPl—lit (Cat ti BEOME) from BioXcell; mouse antimIlD—l antibody Clone EH12; Merck’s NEE-3475 ouse l’D—l antibody (Keytruda, penibrolizumab, iambrolizumab); and AnaptysBio’s anti-PD~l antibody, known as ANBOH; antibody MEX—l 106 (0N0u4538), Bristol-Myers Squibb’s human [2,64 monoclonal antibody nivolumab (Opdivofih, EMS—936558, MDXllOo); AstraZeneca’s AMP—5 14, and AMP~224; and Pidilizumah (CT-011), CureTech Ltd. {90163} in some ments, a pseudytoped oncolytic virus comprises a nucleic acid sequence that encodes an engager molecule sing an activation domain and an antigen recognition domain, and an additional nucleic acid ce that encodes a PDl inhibitor selected from ANBtll 1; antibody MEX-l 106 (GNU—4538); Bristol-Myers Squibb’s human lgG4 monoclonal, antibody nivoluinab (Opdivo®, EMS—936558, MDXllilo), AstraZeneca’s AMP— 514, and ARE-224; and Pidilizumab (CT—01 1), In some embodiments, a pseudytoped oncolytic virus comprises a nucleic acid sequence that encodes an engager and an additional nucleic acid sequence that s l’D-l inhibitor selected from ANBtlll, antibody MDX—l 106 {ONE} 4538), Biistol—Myers Squibbls human lgG‘il monoclonal antibody nivolumab (0pdivo®, BMS— 936553, MDXl 106), AstraZeneca’s AMP—5 14, and AMPQM; and Pidilizumab (CT—ill l ). 3 in some ments, a toped tic virus comprises a nucleic acid sequence that encodes an engager le sing an activation domain and an antigen recognition domain, and an additional nucleic acid sequence that encodes a PD—Ll inhibitor. in some embodiments, a pseudytoped tic virus comprises a nucleic acid sequence that encodes an engager molecule and an additional nucleic acid sequence that encodes PD~L2 inhibitor selected from Alvlll—Qlilzl- (Ainplimmune) or rngMl 2.87, {30165} in some embodiments, a pseudytoped oncolytic virus comprises a. nucleic acid sequence that encodes an engager molecule comprising an activation domain and a therapeutic molecule domain that binds to Fill. In some embodiments, a pseudytoped oncothic virus comprises a c acid sequence that encodes an engager molecule sing an activation domain that binds to (IDS (cg, an 3133 scFv) and a therapeutic le domain that binds to P131 (eg. an antinPDl scFv), in such embodiments, the pseudytoped oncoiytic virus may r comprise an additional nucleic acid sequence that encodes an additional therapeutic molecule. d) GEL/L4 Inhibitors [£10166] In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA—Ll, ln some embodiments, the an inhibitor of CTLA—Al is an antibody (eg a monoclonal antibody or fragments, or a humanized or chimeric antibody or fragments thereof) against — 4. in one embodiment, the anti—CTLAw-l antibody blocks the binding of C'l‘l_;A—/l- to CD80 (B7—l) and/or CD86 (37-2) expressed on antigen presenting cells. Exemplary antibodies against CTLA— 4 include ipilimtiniab (also known as Yewoyliil, O, EMS—7340l6 and MEX—101, Bristol Meyers ); anti—CTIA4 antibody clone 9l-ll0 from Millipore; ti‘emcliniuinab (GP-675,206, ticiliinumab, Pfizer); and antiuClLAél antibody clone BNB from Abcam. {00167} In some embodiments, the 'l‘LAw/l antibody is one disclosed in any of International PCT Publication Nos. l4424, , WO 86459, ; WI} 37504, ; ; , , W0 2006/029219, W0 2010/0979597, W0 2006/12168, US. Patent Application Publication No. 2005/0201994; or European Patent Application Publication No. 1313 1212422. Additional CTLA-4 dies are described in U .8. Patent Nos. ,811,097; 5,855,887; 318; 6,051,227, 6,682,736; 6,984,720, 7,109,003; 7,132,281; international PCT Publication Nos. W0 01/ 14424 and WO 00737504; and in US. Patent Application Publication Nos, 2002/0039581 and 2002/086014. ln some ments, the anti— CTLA~4 antibody is one disclosed in any of lntemational PCT Publication Nos. WO 1998/ 2752; US. Patent Nos. 6,682,736 and 6,207, 156, Hum/itz et al, Proc. Natl. Acad. Sci.

USA, 95(17): 10067411071 (1998), Camacho et al, I. Clin. Oncol, ): Abstract No. 2505 (2004) (antibody CP— 675206), h’loliyr et al, Cancer Res, 58:5301—5304 (1998).

} In some embodiments, the CTLA—4 inhibitor is a CTLA~4 ligand as disclosed in International PCT Publication No. WO 40915. {00169} In some embodiments, the C'I'liA—zl- inhibitor is a nucleic acid inhibitor of CTI..A~4 sion, such as an RNAi molecule. In some embodiments, anti—CTLA4 RNAi molecules take the fonn of those described in any of International PCT Publication Nos. W0 19997032619 and WG 2001/029058; US. Patent Application ation Nos. 2003/0051263, 2(“103/0055020, 2003/0056235, 2004/265839, 2005/0100913, 2006/0024798, 200810050342, 2008/00813’73, 2008/0248576, and 2008/055443; and/or US. Patent Nos 6,506,559; 7,282,564; 7,538,095; and. 7,560,438. In some instances, the anti—CTLA4 RNAi molecules are double stranded. RNAi molecules, such as those disclosed in. an Patent No. El) 1309726, In some instances, the anti—(£71,744 RNAi molecules are double stranded RNAi molecules, such as those described in US Patent Nos. 7,056,704 and 7,078, 196. In some embodiments, the CTLA4 inhibitor is an aptainei', such as those described in international PCT Publication No. W(I 2004.1’08K121, such as Del 60 or M914 del 55. Additionally, in some embodiments, the anti- CTLA4 RNAi molecules of “the present ion are RNA molecules, such as those described in US. Patent Nos. 5,898,031, 6,107,094, 7,432,249, and 7,432,250, and European Application No.

EP 0928290.

] In some ments, a psendjyttoped oncolytie Virus comprises a nucleic acid sequence that encodes an engager molecule comprising an activation domain and an antigen 2017/040354 recognition domain, and an additional nucleic acid sequence that encodes a CTLA—il inhibitor. in some embodiments, a pseudydoped tic virus comprises a nucleic acid sequence that encodes an r molecule and an additional nucleic acid ce that encodes a w/t inhibitor selected from umab (also known as Yervoy®, MDX—Olt’), EMS—734016 and MDXulOl), anti—CTLA4 Antibody, clone 9l-ll0 from ore; Pfizer’s tremelimumab (CP— 6'75,2()o, ticilimumab); and anti—C'l‘LALl antibody clone BN8 from Abcam. {flfil'I’lj in some embodiments, a pseudytoped oncolytic Virus comprises a nucleic acid sequence that encodes an engager molecule comprising an activation domain and a therapeutic molecule domain that binds to CTLA—Al, in some ments, a pseudytoped oncolytic Virus comprises a nucleic acid sequence that encodes an engagei‘ molecule comprising an. activation domain that binds to CDB (eg, an anti—CD3 scFV) and a therapeutic molecule domain that binds to CTLA—Al (eg, an TLA-él scli‘v). In such embodiments, the pseudytoped oncolytic Virus may further comprise an additional nucleic acid sequence that encodes an additional therapeutic molecule. e) LAG} Inhibitors ] In some embodiments, the immune oint inhibitor is an inhibitor of LAG3 (CD223). ln some embodiments, the inhibitor of lAGfi is an antibody (eg, a monoclonal antibody or fragments, or a humanized or chimeric antibody or fi'agments thereof.) t LAGS. in additional embodiments, an antibody against LAG3 blocks the interaction of LAG3 with major histocompatibility complex (hit-EC) class ll molecules. Exemplary antibodies against LAG3 include: anti—Lag—3 antibody clone eBir3C9B7W (€987W) from eBioscience; anti—[ag3 dy LS-B2237 from Lil’eSpan Biosciences; IMP321 (lmmuFact) from lmmuten; anti—LagB antibody BMS~9860l6; and the LAGn3 chimeric antibody A9812. in some embodiments, the anti—{AGE antibody is an AG3 antibody disclosed in International PCT Publication Nos.

W0 ZOlG/Ol 957i); l; or WU 2004/078928. {00173} l'n some ments, a pseudytoped oncolytic Virus comprises a nucleic acid sequence that encodes an engager molecule comprising an activation domain and an antigen recognition domain, and an additional nucleic acid sequence that encodes LAG3 inhibitor. In some embodiments, a pseudytoped oncolytic Virus comprises a, nucleic acid sequence that encodes an engager molecule and an additional nucleic acid sequence that encodes LAG3 tor selected from anti ~l'_.ag-3 antibody clone eBioC9B7‘W (CQBTW) from eBioscience; anti— LagB dy [43-82237 from Lichpan Biosciences; lMP32l (lmmuFact) from p; anti— Lag3 antibody BMsosceie; and the LAG—3 ic antibody A9Hl2. {@174} in some embodiments, a pseudytoped oncolytic Virus comprises a nucleic acid ce that encodes an engager molecule comprising an activation domain and a therapeutic molecule domain that binds to LAG3. in some embodiments, a pseudytoped oncolytic Virus comprises a nucleic acid sequence that encodes an engager molecule comprising an activation domain that binds to CD3 (eg, an anti—CD3 scFv) and a therapeutic molecule domain that binds to LAG3 (eg, an anti—LAG3 scFV). In such embodiments, the pseudydoped oncolytic Virus may further comprise an onal c acid sequence that encodes an additional therapeutic molecule. i) HMS Inhibitors {$0175} in some embodiments, the immune checkpoint tor is an. inhibitor of "l‘llvlll. in some embodiments, the inhibitor of Tilt/B is an antibody (tag a, onal antibody or fragments, or a humanized or chimeric antibody or nts thereof: against TlM3 (also known as HAVCRZ). ln additional embodiments, an antibody against 'l'lM3 blocks the interaction of TlMB with in-9 ((32119). In some embodiments, the anti—TIM3 antibody is an anti—TENS antibody disclosed in Interantional PCT Publication Nos. WO ZOE/006490; Wt) 201 1/55607; W0 201 1/159877; or W0 2001/1705? in another embodiment, a lB inhibitor is a 'l‘lMS inhibitor disclosed in international PCT Publication No. . {£10176} ln some embodiments, a pseudytoped oncolytic vims comprises a nucleic acid sequence that s an engager molecule comprising an activation domain and an antigen recognition domain, and an additional nucleic acid sequence that encodes TIM?) inhibitor. In some embodiments, a pseudytoped oncolytic Virus comprises a. nucleic acid sequence that encodes an engager molecule and an additional nucleic acid sequence that encodes lib/13 inhibitor such as an antibody against Tilt/l3 blocks the interaction of ’l‘ilvlil with galectinnél (Gall); lflOl 77E ln some embodiments, a pseudytoped oncolytic Vinis comprises a nucleic acid sequence that s an engager le comprising an activation domain and a therapeutic le domain that binds to Tilt/B. In some embodiments, a tooed tic virus comprises a nucleic acid sequence that encodes an engager molecule comprising an activation domain that binds to CD?) (a 3, an D3 scFV) and a therapeutic molecule domain that binds to LAGB (eg, an anti—'l'iM3 scFiI). in such ments, the pseudytoped oncolytic Virus may further comprise an onal nucleic acid sequence that encodes an additional therapeutic molecule. 2017/040354 g) B7113 Inhibitors {00178} In some embodiments, the immune checkpoint inhibitor is an inhibitor of B7nll3. in some embodiments, the inhibitor of BLED is an antibody (eg. a monoclonal antibody or fragments; or a humanized or chimeric dy or fragments thereot) again st B7—l-l3. ln some embodiments, the inhibitor of B7—l-l3 is MGA27l (MacroGemcsl. [001793 in some embodiments a pseudytoped oncoiytic virus comprises a nucleic acid sequence that encodes an engager molecule comprising an activation domain and an antigen recognition domain, and an onal nucleic acid sequence that encodes a B7—l—l3 inhibitor. in some embodiments, a pseudytoped oneolytic virus comprises a nucleic acid sequence that encodes an engager and an additional c acid sequence that encodes a Bil—HS; inhibitor such as MGAZH. {90189} in some embodiments, a pseudytoped oncolytic virus ses a nucleic acid sequence that encodes an r molecule comprising an activation domain and a therapeutic molecule domain that binds to B7—H3, In some embodiments a pseudytoped oncolytic virus comprises a nucleic acid sequence that encodes an engager molecule comprising an activation domain that binds to CD3 (cg, an antiuCD3 scFv) and a therapeutic molecule domain that binds to B7nH3 (erg an anti~B7nll3 scFv}. In such embodiments, the pseudytcped tic viius may further comprise an additional nucleic acid sequence that encodes an additional eutic molecule, l00l81l in certain other embodiments the engager molecule additionally comprises one or more other domains, eg one or more ofa cytokine, a co~stimulatory domain, a domain that ts negative regulatory les of T—cell activatiom or a combination thereof. in alternative embodiments, the engager is a first ptide provided within the pseudotyped oncolytic virus with a second polypeptide having one or more other domains, e. go, one or more of a cytolcine, a co~stimulatory domain, a domain that inhibits negative regulatory molecules of '3 cell activatiom or a combination thereof. in some embodiments? the first ptide and the second polypeptide are encoded in the same vector (cg, viral vector). in some embodiments, the first ptide and the second polypeptide are encoded in ditierent vectors (erg viral vectors). in c embodiments, the cytokine is ill—15, ill-2, and/or ill—7. in other specific embodiments, the co~stimulatory domain is CD27, CDSO, CD83, CD86, CDlle, or CD137” in other specific embodiments, the domain that inhibits negative tory molecules of T—cell activation is PD-ls PDLl or B7-H4.

, CTLA43 Anti—angio Ionic factors as Nzem )eulic M'tflec i! {@182} in some embodiments, the therapeutic molecule is a ptide such as an anti—angiogenic . Angiogenesis or neovascularization is the formation of new niicroyessels from an established vascular network. in some instances, the angiogenic s es communications from multiple cell types such as endothelial cells (EC) and circulating endothelial progenitor cells, pericytes, vascular smooth muscle cells, stromal cells, including stem cells, and parenchymal cells. These communications or interactions occur through secreted factors such as VEGF, fibroblast growth factor (FGF), et~derived growth factor (l’llGP‘), or angiopoietins. In some instances, an anti—angiogenic factor is a polypeptide that disrupts one or more of the interactions of the cell types: endothelial cells (EC) and circulating endothelial progenitor cells, pericytes, vascular smooth muscle cells, stronial cells, including stem cells, and parenchymal cells. in some instances, an anti—angiogenic factor is a polypeptide that disrupts one or more of the interactions of secreted factors such as VEGF, fibroblast growth factor (FOP), et-derived growth factor (PDGF) or angiopoietins. lflllillifil in other ments, provided are pseudotyped oncolytic viruses comprising nucleic acids that encode therapeutic polypeptides that modulate regulatory T cells. in some instances, regulatory T cells maintain the tolerance to self—antigens and in some instances abrogate autoimmune. in some cases, Treg supresses or do‘vvnregulates induction and proliferation of effector T cells, Exemplary 'l‘reg modulatory polypeptides include CCRAE, , TlGlT, GITR, neuropilin, neuritin, CD! 03, (TIA-4, ICOS, and Swap’ll). {0018M in other embodiments, ed are pseudotyped oncolytic s comprising nucleic acids that encode therapeutic polypeptides that modulate myeloicl—clerived suppressor cells (h’lDSCs). MDSCs are a lieterogenous population of immune cells from the niyeloid lineage (a cluster oi’dil'l’erent cell types that originate from bone marrow stem cells}, to which also includes rlendritic cells, macrophages and neutrophils. in some instances, myeloid cells interact with T cells to regulate the T cells function. Exemplary l‘vfllJSC niodulatoiy polypeptides include TGF—flRl, GM—CSF, lFN—y, eulcins (tag, lit-ll, llanZ, llro, ill—ll), lL—lZ, lL—lS, ll_.~6, lL—éRct, lLu6/lL—6R complex, TGF—fil, NLCSF, 'Prostaglanclin E2/PGEZ, l’rostaglandin E Synthase 2, S lOClAS, and VEGF. } in other emhodiments, provided are pseudotyped oncolytic viruses comprising nucleic acids that encode therapeutic polypeptides that modulate the fihrotic stroma. in some embodiments, fibrosis occurs in se to mation, either chronic or recurrent.

Over time, the repeated bouts of mation irritate and scar the tissue, g huildups of fibrous tissue. in some intances, if enough fibrous material develops, it turns into stromal fibrosis. Exemplary tibrotic stromal polypeptides include fibroblast tion protein~alplia (PAP).

Nucleic acid polymers as Them E‘ufiC iMiJKeatuir:,s {90} 86E in some embodiments, the therapeutic molecule is a nucleic acid polymer. in some instances, the nucleic acid polymer is a RNA polymer. In some instances, the RNA polymer is an antisense polymer those sequence is complementary to a NA (miRNA or mill) target sequence. In some ces, the RNA polymer is a microRNA polymer. in some embodiments, the RNA polymer comprises a DNA—directed RNAi (ddRNAi) sequence, which enables in yivo production of short hairpin RNAS (shRNAs). [$0187] in some embodiments, a. mieroRNA polymer is a. short non~eoding RNA that is expressed in different tissue and cell types which sses the expression of a target gene. For example, niiRNAs are transcribed by RNA polymerase ll as part of the capped and enylated primary transcripts (pfi'mlRNAS). ln some ces, the y transcript is cleaved by the Drosha ribonuclease lll enzyme to e an approximately 70—nt stem—loop precursor miRNA (preamiRNA), which is further cleaved by the cytoplasmic Dicer ribonuclease to generate the mature miRNA and antisense iniRNA star (iniRl‘lA’t) products. In some instances, the mature miRNA is orated into a RNA~induced ing complex (RJSC), which recognizes target niRNAs through imperfect base pairing with the niiRNA and in some instances results in translational inhibition or destabilization of the target inRNA. {00188} in some instances, dy‘sregulated microRNA expression is correlated with one or more types of cancer. ln some embodiments, the mioroRNA is referred to as an oncomiR.

In some instances, the ulated niieroRNA expression is an elevated expression. In some instances, the elevated expression level ofinicrolfi‘lA ates to one or more types of cancer.

For e, o‘v’erexpression of microRNA~155 (miR—lSS) has been observed in cancers such as Burkitt lymphoma, or laryngeal squamous cell carcinoma (LSCC) and overexpression of microlLNA—Zl (mill—2 l) has been observed in breast cancer. {60l89fl ln some embodiments, ary microlll‘lAs with an ed expression level include, but are not limited to, miR—lO family (cg lb), miR—l7, iniR—2l, iniR—lOfi family (cg, miR—l 06a), miR—lZS family (cg, niiR—lZSb), mill—MS, mill—146 family (cg niiR— l46a, mill—Hob), mill—155, miR—96, iniR-lSZ, miR—l83, mill—2.2L miR—ZZZ, and mill—12476;). {(116199} ln some instances, the nucleic acid polymer is an antisense polymer those sequence complements an R. ln some instances, the nucleic acid polymer is an antisense polymer those sequence complements an oncomiR that is characterized with an overexpression.

In some instances the nucleic acid polymer is an antisense polymer those sequence complements a inicroRNA target ce. In some instances, the nucleic acid polymer is an antisense polymer those sequence complements a microRNA target sequence that is characterized with an pression. In some ces the eutic molecule is an antisense polymer those sequence complements a microRNA target ce. In some instances, the therapeutic molecule is an antisense polymer those sequence complements a microRNA target sequence that is characterized with an overexpression in some instances, the overespression level is relative to the endogenous expression level of the microRNA. {flfilgll In some instances, the dysregulated microRNA sion is a reduced expression. in some instances the reduced expression level of mieroRNA correlates to one or more types of cancer, For example, a depleted level of 11,1er3 l. has been observed in both human and mouse metastatic breast cancer cell lines. {001923 in some embodiments, ary microRNAs with reduced expression levels e? but are not limited to, miR—3L mill—3.4 family (cg. iniRSAla, mill—34h and mill— 34c}, miR—lOl., miR—l26, miR—l45, miR—lgéa, and the mill—200 family. fl In some instances, the nucleic acid polymer is an oncomiR. In some instances, the oncomili is equivalent to an endogeous oncomiR wherein the endogeous oneomiR is characterized with a reduced. expression level. In some instances, the nucleic acid. polymer is a microRNA polymer. in some instances, the therapeutic molecule is a microRNA polymer. In some instances, the microRNA is equivalent to an ous niicroRNA polymer wherein the endogenous niicroRNA is characterized with a reduced expression level. {@9194} As described above, in some instances the RNA polymer comprises a DNA-directed RNAi (ddRi‘lAi) sequence In some instances, a ddRNAi construct encoding a shRNA is packaged into a viral vector such as a viral vector of a pseudotyped oncolytic virus described herein. in some instances upon entry into the target cell (eg. a. tumor cell), the viral genome is processed to produce the encoded shRNAsi The shRNAs are then sed by endogenous host systems and enter the RNAi y to modulate or e the desired gene target. In some instances, the gene target is a gene that is overexpresserl in a cancer type. in some instances, the gene target is a gene that is overexpresscd in a solid, tumor. in some instances, the gene target is a. gene that is overexpressed in a hematologic cancer. Exemplary genes that are overexpressed in cancer e but are not limited to, TP53, human mal growth factor receptor 2 (HERZ), mucin lacell e associated (Ml/Cl), human pituitary tumour— transforming gene l (hPPTGJL prostate and breast cancer overexpressed gene l protein (P30 V1), and the like. {6&95] in some instances, the nucleic acid polymer comprises a ddRNAi sequence. In some instances, the nucleic acid polymer is comprises a ddRNAi sequence which s a 0'ene that is pressed in a cancer. in some instances, the therapeutic molecule comprises a ddRNAi sequence. in some instances, the therapeutic molecule comprises a ddRNAi sequence which targets a gene that is overexpressed in a cancer.

EIxemplariv Engager JMOIecuies [(99196] In some embodiments, the engager molecules described herein comprise a iii-specific antibody construct sing an activation domain and an antigen recognition domain, in which the activation domain cts or binds to an effector cell surtace receptor shown in Table l; and the antigen recognition domain interacts or binds to a targetwcell antigen shown in Table 2. in some embodiments, the engager molecules bed herein comprise a bi— specific antibody uct comprising an activation domain and a therapeutic molecule domain, in which the activation domain interacts or binds to an ettector cell surface receptor shown in Table l; and the therapeutic molecule domain cts or binds to a cell surface antigen shown in Table 2.

In some embodiments, the engager molecules provided herein se an activation domain, wherein the activation domain comprises an anti~CD3 scFv. in some embodiments, the anti~CD3 scFv comprises a light chain variable fragment comprising an amino acid sequence that is at least 809/2», at least, 85%, at least 909/65, at least 95%, at least 96%, at least 97%, at least 98%, or at least 9' % identical to the amino acid sequence of SEQ ID NO: 20 and a heavy chain le fragment comprising an amino acid sequence "that is at least 80%, at least, 85%, at least 909/ , at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ll) NO: 22. in some embodiments, the anti-CD3 scFv comprises a light chain variable fragment comprising an amino acid. sequence that is £0094) identical to the amino acid sequence of SEQ ll) NO: 20 and a heavy chain le fragment "that is l0094> identical to the amino acid sequence of SEQ ll) NO: 22. In some embodiments, the anti— CD3 scFv comprises a light chain le fragment comprising the amino acid sequence oi‘SEQ ll.) NO: 20 and a heavy chain variable fragment comprising the amino acid ce of of SEQ ll) NO: 22. in some embodiments, the anti—CD3 scFv comprises a light chain variable fragment consisting of the amino acid sequence of SEQ ll) NO: 20 and a heavy chain le fragment consisting of the amino acid. sequence of of SEQ ll) NO: 2.2. in some ments, the engager molecules provided herein comprise an activation domain, wherein the tion domain comprises an anti—CD3 scFv, wherein the anti— 2017/040354 CD3 scFv comprises a light chain le fragment nucleic acid sequence that is at least 80%, at least, 85%, at least 909/, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ll) NO: l9 and a heavy chain variable fragment c acid sequence that is at least 809/6, at least, 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 9894:, or at least 999/6 identical to the c acid sequence of SEQ 11) NO: Zl. In some embodiments, the anti—CD3 scFv comprises a light chain variable fragment nucleic acid sequence that is 100% identical to the nucleic acid sequence of SFQ ll) NO: l9 and a heavy chain variable figment nucleic acid sequence that is “30% identical to the amino acid sequence of SEQ ll) N0: 2l. in some embodiments, the anti—CD3 seFv comprises a light chain variable fragment nucleic acid sequence comprising SEQ ll) NO: 19 and a heavy chain le fragment nucleic acid sequence comprising SEQ ll) NO: 2t. ln some embodiments, the anti—CD3 scFv comprises a light chain variable fragment nucleic acid sequence ting of SEQ ll) ND: l9 and a heavy chain variable fragment nucleic acid sequence consisting of SEQ ID NO: Zl. {flfilllllj ln some embodiments, the engager molecules provided herein comprise an antigen recognition domain, wherein the antigen ition domain comprises an anti—CDl9 scFv. in some embodiments, the anti—{3019 scFv comprises a light chain variable fragment sing an amino acid sequence that is at least 80%, at least, 85%, at least 90%, at least 95%, at. least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ll) NO: l6 and a heavy chain variable fragment comprising an amino acid sequence that is at least 39%, at least, 85%, at least 909/5, at least 9594:, at least 9694s, at least 97%, at least 98%, or at least 9‘ % identical to the amino acid sequence of SEQ lD NG: l8. in some embodiments, the anti-CDl9 scFV ses a light chain variable fragment comprising an amino acid ce that is 100% identical to the amino acid sequence of SEQ ID NO: l6 and a heavy chain variable fragment that is l00% identical to the amino acid sequence of SEQ ll) NO: l8. ln some ments, the anti~CD l9 scFv comprises a light chain. variable fragment comprising the amino acid sequence of SEQ ll) NO: l6 and a heavy chain variable fragment comprising the amino acid sequence of of SEQ ID NO: l8. ln some embodiments, the anti~CDl9 scFv comprises a light chain variable fragment consisting of the amino acid sequence of SEQ ll) NO: 16 and a heavy chain variable fragment consisting of the amino acid sequence of of SEQ ll} NO: } In some embodiments, the engager molecules provided herein se an antigen recognition domain, wherein the n recognition domain comprises an anti-CDl9 scFv, wherein the anti—CDl9 seFv comprises a light chain variable fragment nucleic acid sequence that is at least 80%, at least, 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ll) NO: l5 and a heavy chain variable fragment nucleic acid sequence that is at least 80%, at least, 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID N0: 1?. ln some embodiments, the anti‘CDl9 scFv comprises a light chain variable fragment nucleic acid sequence that is l00% identical to the nucleic acid sequence of SEQ ll) NO: l5 and a heavy chain variable fragment nucleic acid sequence that is 100% identical to the amino acid ce of SEQ ll) NO: l7. ln some ments, the anti— CDl9 scFv comprises a light chain variable fragment nucleic acid sequence comprising SEQ ll) NO: l5 and a heavy chain variable fragment nucleic acid sequence comprising SEQ ll) N0: l7. in some embodiments, the anti-CDl9 scFv comprises a light chain variable nt nucleic acid sequence consisting of SEQ ll) NO: l5 and a heavy chain variable fragment nucleic acid sequence consisting of SEQ ll) NO: l7, 3 in some embodiments, the engager les ed herein comprise a therapeutic molecule domain, wherein the therapeutic molecule domain ses an antiml-‘Dlll scFv. ln some embodiments, the anti-PDLl scFv comprises a light chain variable fragment comprising an amino acid sequence that is at least 80%, at least, 85%, at least 90%, at least 958/6, at least 9 %, at least 7%, at least 98%, or at least 99% identical to the amino acid sequence of SFQ ll) NO: 36 and a heavy chain variable fragment comprising an amino acid sequence that is at least 80%, at least, 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 93%, or at least 99% identical to the amino acid sequence of ElliQ ll) N0: 38. in some embodiments, the anti—PDLl scFv comprises a light chain variable fragment comprising an amino acid sequence that is l00% identical to the amino acid sequence of SEQ ID NO: 36 and a heavy chain variable fragment that is 100% identical to the amino acid sequence of SEQ lD NO: 38. In some ments, the anti—l’DLl scli-‘v comprises a light chain variable fragment sing the amino acid sequence of SEQ ll) NO: 36 and a heavy chain variable fragment comprising the amino acid sequence of of SEQ ll) NO: 38 In some embodiments, the anti-PDLl scllv comprises a light chain variable fragment ting ot‘the amino acid sequence of SEQ ID NO: 36 and a heavy chain le fragment consisting of the amino acid sequence ofofSEQ ll} NO: } In some embodiments, the engager les provided herein comprise a therapeutic molecule domain, wherein the therapeutic molecule domain comprises an anti—l’DLl scli'v, wherein the anti-PDLl scFv comprises a. light chain variable nt nucleic acid sequence that is at least 80%, at least, 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ll) NO: 35 and a heavy chain variable fragment nucleic acid sequence that is at least 30%, at least, 85%, at least 90%, at least 959/5, at least 96%, at least 9794i, at least 98%, or at least 99% identical to the c acid sequence of SEQ ll) N0: 37. In some embodiments, the antinl’DLl scFv comprises a light chain variable fragment nucleic acid sequence that is l00% identical to the c acid sequence of SEQ ll) NO: 35 and a heavy chain variable fragment nucleic acid sequence that is 100% identical to the amino acid ce of SEQ ll) NO: 37. in some embodiments, the anti— l’lflal scFv comprises a light chain variable fi'agment nucleic acid sequence sing SEQ ll) NO: 35 and a heavy chain variable fragment nucleic acid sequence comprising SEQ ll) N0: 37. in some embodiments, the anti-PDLl scFv comprises a light chain variable fragment nucleic acid sequence consisting of SEQ ll) NO: 35 and a heavy chain variable fragment nucleic acid sequence consisting of SEQ ll) N0: 37. } in some embodiments, the engager molecules provided herein comprise a therapeutic molecule , wherein the therapeutic molecule domain comprises a SlRPla polypeptide fragement. in some embodiments, the SlRl’la polypeptide fragement comprises an amino acid sequence that is at least 80%, at least, 35%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ll) N0: 32. in some embodiments, the SlRl’lu polypeptide fragement comprises an amino acid sequence that is 10l% identical to the amino acid sequence of SEQ ll) N0: 32. In some embodiments, the SlRl’la polypeptide fragement ses the amino acid sequence of SEQ ll) NO: 32. In some embodiments, the SlRPla polypeptide fragement consists of the amino acid sequence of SEQ ll) NO: 32. {(99284} in some embodiments, the engager molecules provided herein comprise a therapeutic molecule domain, wherein the therapeutic molecule domain comprises a SlRPld ptide li'agement, wherein the Sllil’lu polypeptide tragement comprises a nucleic acid sequence that is at least 80%, at least, 85%, at least 90%, at least 95%, at least 96%, at least 97%., at least 989/0, or at least 99% identical to the c acid sequence of SEQ ll) NO: 3i. in some embodiments, the SlRPla polypeptide fragement comprises a c acid sequence that is lOOllh identical to the c acid sequence of SEQ ll) NO: 31. in some embodiments, the u polypeptide fragement comprises the nucleic acid ce of SEQ ll) NO: 31. In some embodiments, the SlREla polypeptide frgement ts of the nucleic acid sequence of SEQ ll) ND: 31. {96295} ln some embodiments, the engager molecules comprise an activation domain comprising an scEv that binds to CD3 and an antigen recognition domain comprising an scFv that binds to (Big, referred to herein as a D3 BiTE, or a CDl9 BiTE. A schematic of an exemplary CDl9~CD3 BiTE is shown in FIG. I (SEQ ll) NO: 44). In such embodiments, the anti—CD3 scFv and the anti—CD” scFV are linked together by 3 G48 linker (SEQ ID NO: 6). in some embodiments, the oncolytic Viruses described herein comprise a bicistronic or miilticistronic c acid sequence, wherein a, first nucleic acid sequence encodes a CDlQ-CDB BiTE and a second c acid sequence encodes a therapeutic molecule such as lL-lS ( SEQ ID NO: 53;}; lL—IZ ( SEQ ID NO: 54), or CXCLIQ ( SEQ ID NO: 55). In such embodiments, the CDlQ-CDS Bi’IE (c. g, SEQ ID NO: 44) is linked to the therapeutic molecule, sag, Ill—15 (SEQ II) NO: 24), Ill—l2 p35 (SEQ ID NO: 23), IL—lZ p40 (SEQ ID NO: 26), and/or CXCLlO (SEQ ID N0: 30), by a T215: self—cleaving peptide linker (SEQ ED NO: 14). {tlfiZtIti} In some ments, the engager molecules comprise an activation domain comprising an sclr’v that binds to CD3 and a therapeutic molecule domain comprising a SIRPlu polypeptide fragement that binds to CD47 (SEQ ll) NO: 32), referred to herein as an -CDB BiTE or a SIRPlu BiTE. A schematic of an exemplary SlRPld-CDB BiTE is shown, in (SiRPlu—CIB (Sis), SEQ ll) N0: 4-6) and. (SIRI’lu—CDfi (LL), SEQ ID NO: 48). In some ments, the anti—CD3 scFV and the SlRPlo peptide fragment are linked together by a single amino acid linker, or a “short linker” (8L) (cg, t—CDS (SL) as shown in . in some embodiments, the anti~Cl33 scFv and the SIRI’Ict peptide fragment are linked together by G48 linker, or a “long linker” (LL) (cg. SlRI’lovCBS (Ills) as shown in FIG, 6). In some embodiments, the oncolytic Viruses described herein comprise a hicistronic or inulticistronic nucleic acid sequence, wherein a first nucleic acid ce encodes a SIRPlu— CD3 Bil‘E and a second nucleic acid sequence s a therapeutic molecule such as lL-lfi (, SEQ ID NO: 56 and PEG. 3, SEQ II) NO: 57), Ill—12 (SEQ ID NO: 58 and FIG.

It), SEQ ID NO: 59), or CXCLlO (FIG, ll, SEQ ID NO: 60 and , SEQ ID NO: 61} In such embodiments, the SlRlllu—CD3 Bi'I’E (cg. SEQ ID N0: 46 or SEQ ll) NO: 48) is linked. to the eutic molecule, cg ll..»l5 (SEQ ID N0: 24), llnl2 p35 (SEQ ID NO: 28), Ill—1?. p40 (SEQ ID NO: 26), and/or CKCLlO (SEQ ID NO: 30), by a. TZA self—cleaving e linlcer (SEQ ID NO: l4). {602973 in some embodiments, the oncolytic Viruses described herein se a bicistronic or multicistronic nucleic acid sequence, wherein a first nucleic acid sequence encodes a, SIRPlo—CD3 BiTE and a, second nucleic acid sequence encodes a, therapeutic molecule such as Mix/I119 (A, SEQ ID NO: 65 and B, SEQ ll) N0: 66). in such embodiments, the SIRl’lu—CD3 Bill?) (cg SEQ ll) NO: 65 or 66) is linked to the MM??? polypeptide (SEQ ll.) NO: 34) by a, TZA self—cleaving peptide linker (SEQ ID NO: 14). {@288} in some embodiments, the oncolytic Viruses described herein comprise a, hicistronic or multicistronic nucleic acid ce, wherein a first nucleic acid sequence encodes a Sllil’lo—CEB BiTE and a second nucleic acid sequence encodes a therapeutic molecule comprising an anti—PDLl scFV linked to an lgGl Fc domain (cg. comprises an lgGl Cl-lZ-Cl-lB— l-linge, SEQ ll) NO: 40), such as the SlRPla—CD3—PDLl—Fe (SL) construct shown in FlG. 3'7 (SEQ 1D NO: 68) or the Sllfl’la—CD3—PDLl—Fc (LL) construct show in (SEQ ll) NO: 70), [(99269] in some ments, the engager molecules comprise an tion domain comprising an scFV that binds to CD3 and a therapeutic molecule domain comprising an SCFV that binds to PDLl, referred to herein as an CE‘B i. or a PDLl Bi’l‘lil Exemplary l’lflsl—CIB Billis are shown in HS. l3 (SEQ ll.) N0: 50). in some embodiments, the anti—CD3 SCFV and the anti—PDLl scFV are linked together by G48 linker (SEQ 1D NO: 6), in some ments, the oncolytic viruses described herein comprise a bicistronic or multicistronic nucleic acid sequence, wherein a first nucleic acid sequence encodes a l’l)l_il~Cl')3 Bill} and a. second nucleic acid sequence encodes a therapeutic molecule such as lie—l5 (FlG. l4, SEQ 1D NO: 62), lLul2 (FIG. l5, SEQ ID NO: 63), or (IXCLlO {Flt}. 16,-, SEQ 110 N0: 64). in such ments, the SlRPianCDB Bi'l‘E (erg, SEQ ED NO: 50) is linked to the therapeutic molecule, cg, lL—lS (SEQ ll) NO: 24-), lie-l2 p35 (SEQ ll) NO: 28), llle p40 (SEQ ll) NO; 26), and/or CXCLI O (SEQ ID NO: 30), by a TZA leaving peptide linker (SE-Q ll) N0: 14). {0021M in some embodiments, the engager molecule is a tripartite engager molecule and cni‘nnnses an activation domain comprising an scFV that binds to CD3, a therapeutic le domain comprising an scFV that binds to PDLl, and a third domain comprising an lgGl Fc domain (cg, comprises an lgG-l Cl-lZ-Cl-B-l-linge, SEQ ID NO: 4G) and capable of binding to one or more Pct/Rs, referred to herein as an PDLlnCD3—Fc tripartite T cell engager, or 'l‘i'l‘E, or a PDLI Hill A schematic of an. exemplary l’l‘lliLCi’B-ll'c 'i'iTE is shown in FIG l7 (SEQ ll) NO: 52). {00211} The amino acid sequences of exemplary engager molecules and therapeutic molecules are shown in Table 3.

Table 3: Amino acid sequences of exemplarv r molecules and therapeutic les - — \. v: . JQPPKLLIYDASN’LVSG‘IPPRFSGt :WTD. 4..4N1HPVEKVDAATYHCQ‘Qo_ .4 I‘JPI‘II‘PGCJG'IKLF I KGG IGSC‘CJ'SCSGG JG‘ SSYWI‘INI‘J:7KQRPGQGLEWIGQ SVTJVISCKTSG Y'I'II“1‘12YTMHW‘7KQRPSQG..FD‘ISY INP..>R"Y"1‘NYI’DIxII‘KI‘KA'"' .-'I‘T1JK SSST YMIK‘II.SSLTSF’)SAV’YY"“RYYDDHVCLIJWGQG"1"I‘IJ’I'VSEVEGGSGGSSGS‘CC SCJCNIJIJIC1J'I"\SPAIMSASPGEK‘”I'M'I‘CII’ASSSVSYD’ND’V'QQKSG’I'SI WI YDTSKV a EASFVPYRFSGSGSCTSYSLTISSMZAEDAATYYCWQYSKNPLFGAGTFLEJKHAFHH 1 EH— ESIRPja— g FETDFLLLFVJLL»VFGSTGDFFELQIIQFDKIILVAAIETLKCTITSSTPVGPIC 46 CBS-SI. WFRGAGPCF‘VLIYNI‘IF‘QC.PFPRVTTVSDTTICRN‘CIIV'LI‘S IRIGNITPADAGTYYCI KPF1 KGSPDDVI'FKSGAGTELSVRA‘KPSASJIKLQDSGAELAPPGAS\7KMSCKTSGYTI FTP.Y TIV I-‘IWVKQ {PGQGIJEWIGYINPS {GYTNYNC‘KTK.ATIJ‘I‘TDKS.S ISTA“YWQ‘ _..5LTSED SA" . ARY‘. -IJI‘...' ’C'IJD YWG.._ .‘L'IVSSVIJ *' . . '.-. ’SGGVIJI). 'I" ‘AIE‘I’I SZ-YSPGEIC\.7T1\’ITC.J-\.SSSVSYM‘IIJYQQICSGT'SPKP. DTSKVASGVPYRFSGSC GTS YSLTISSIVIEAEDAATYYC‘QQWSSD PLTPG GTKLELICEHHHI—IH- SIRPlgz— EMTDTLLL’“'IVLLLWVPGSTG EEELQI IQPDKSVLVAACETATLRCTITS.JFPVG'PIQ 48 CBS-LL WFRGAGPGFVLIYN‘F‘Qx.PFPRVTTVSDTTICRN‘CIIV'LI‘S I RIGNI TYY"x.I KPR KGSPDDV KSGAGT. ELSVRAKPSASGGGGSDIIICLQQSGA.ELARPI".7ASVKI’L3CKTSGY TF’I‘EYTM. 7'J‘JKQRP’V‘ICILII‘WI" 7 I NI..E‘S.'."‘:Y’1‘1\".{I ’IICFKIJKAT T’I‘IJKS‘SSTAYMKLSS ‘-JI’‘RIJSAVYYCARYYJJJ I JIWGQCJ. I. J’I'VSSVECJGSCGSSGSGCSCJS'VDDICIIQ SPAIMSASPGEKVTIVITCRAS S S‘JSYMNWYQQKSGTSPK wIYDTSKVASG‘JPYRFSGS GSGTSYSLTISSMEAEDAATYYCQQWSSNPLTPGAG-KLII'LKHI- HHHH- PDLi—CDS .FGLS WVFI.‘.\7AIJPRG 7QC.‘ I . JQQSGAIiLA 3A...\7KMC . . GYTFTRY ’1‘1‘I’IHW\J’S“Q RPPG’QGLEWIGYINPSF‘”‘YTNYNQF‘FKDKATL'TTDKSSSTAYMQLSSI.-iSELSAVVYCA RY’YIDHYCLDY QGTTLTVSSVEGGSGGS3GSGGSG<3V IDIQLTQSPAIMBASPG‘EK 1‘11L7‘CRAS.SS‘VSYMNWYQQK. 1'J‘KRII‘T'V”)I‘SKVASC‘.X7PY"SS-ISGSGTSY SLTI...SS M. .'.A JI‘AAI’YYCQQWSSIIPI '1‘. SAG’I'KL‘. JKGGCGSLIQN’I‘I‘PSSJJSAJLJI. RWTI 17C; RASQDVS"-AVAINYQQKPGKAPKLL IYSJ— FLYSGVPSRPSGSGSGTDFTLTI S 7'- C'IQYLYHPnT EIK ' uSCGIS—F'SGCCGSEVQLVTSCG’“VQPGG‘ CAASGPTFSDSWIIIWVRQAPGK -EWVAWISPYGI "TYYADS-VKGRFTISADT , . .Y.JI’WWSIJRHEI“1‘A'\J’Y Y".AI€.:<.1-I’J.' ‘: JII‘IJ.N""("1‘._.\/I’VE?“*‘PH'I.”SH-- I’DLi—CDS— MTFCLSI/WFLVALPRGVQIJIKLQQSC‘AELAT‘PGAS‘7KMSCKT.S<3YTI FTRYTMHWVKQ Fe r1 I.” 'l'I'\/‘1’ . ‘GQGLEWIGYINP. {er.rrI7'YIYIYC\KT1KJjKA I... TDKSSSTAYIV' DL 4.7.1.) “LID.

.JJTVSSv’lJGC‘LJCJuSG"SC1.1thVDD ' I. '\.“ SSVS‘YW’NNYQQKSQTPKPWI.YDTSKVASJ-“JPYI ..SG:SSG‘"SYSLT E‘CEDAATI YYCQQWS"NTLTPCAGTKLELKSCCGSLIQMTQSPSSLSASVG'DRVTITI‘ RASQDVSTAVAWYQQKPGICAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTI S SLQ‘II-‘I IDEAYYC"r‘YI_.‘.I.-I'I'PA'I'II‘C"C‘I'I'ICJHIKRC-I’.‘ S_G(3C"? .GGGSFI’QLVII SC SLRI-SCAASGP'I‘ .‘S ‘JSW I.rII’G‘JPQAPCI GLEWVA'JI STYYADSVKGR F’I'ISADT SKNTAYLQ-V’INSLEI-I.EDTAVYYCARRI—1WPGGFDVWGQGTLVTVSAVDEAICSCDKTHTCP PCPAPELL GPSVFLFPI-‘YPKI‘TLI‘I'TSRT EVTC erDVSHEDPEVKFNWYVDGVEW-I PR. EQYNSTY'RV'VSVLTVLI-IQDWLNGKZYKCKVSNICATIPAP I EKTI SKAICGQP REI1")\I’Y'I‘ "EI. EL'I'KNQV’. .

. INIYKT'I‘PPVLDSD (Di 9-(73.53- “FFGLSIWFLIAL . ..aQSvJ..CUSYLIm 11.15 YQQIPCJQ SKIVS; . .. . .IIIPVFKVJA:YHCQQSTE DPWTFGGGTKJLIKCFGJSGCCPSJCGCSQVOLQQSPAKLVRFGS~VKISCKAGYAF SoYWIYWVKQHPGQSLEWIGQTWPGDGDTNYKGKPKGKATLTADSSSTAYMQLSSLA SEDSAVYFCARVNTTTVGRYVVAMDYWQGTTVTVSGGCGSDIKLQC7GAELARPCA JWKMsCKMFCYIFTRVTMHWVFn«UGQ©LFwIGYINFIFGYTNVMQFFKFKA"LTTDK SSSTAYIQLSSTDJubHVYYC n.LDDHYCLJ' SQETWTLFVSSVPIGSGGS( ' SGG DJIAJTO'PAIMSASPGEKYPV”CRASSSVSYMNWVQWKSGTSPKRW1YL.SKV ASVPYRPSGSGSGTSYSLTISSMPAFDAATYVCOQ‘SSNPSIIGAGTKLJLKHHHIH GRGSLLTCG‘VE IvfibflfLT‘AGTHVPI L JFSAG {TEANWVNV ' . SC\ITAIKC ELQWVISLESGDASIHDTV 1ND}. ILANNQIJDSNC.I\1 \l'.L. w/\/ EELFFKNIYLIIQ VHIVQM~INTS— CHM-CBS— WVFLVALRCQVQCDICLTWSPASLAVSI ‘ ATISCKASQSVDYDGDSYLNW 54 11.12 QPPY’T ' POPFSCS‘35:-..LNIHPVF*VDAA“Y‘0' DPWTFGGGTL :- CCGSIJQLQDGAELVR-GSSIKI SSYWMNWVKQR GQGLEWIGQIWPGJGDTNYNGKFKGKATLTADE SFWSA/YTR'RRLYTTVCRYYVAMTYu‘TTVTVSSGGCGSFTKLCQSSAELHRP1A VIS'CKTSGYTFTRYTIIHWVKQR'P’"QCLEWI‘JYINPSRGYTN'JNOKFWDKATLTTDK SSLTS DSAVYYFARY"DDHVCLD(WGCCTTLTVSSVFGGSGSGGSG SGGVDquLTQSFAfiuASFGhKVTVTFRASSSVYFJWVGQKSGTS‘TRNTYFTSKV ABCJPYRISCSGSGTSY'LTTSSFMAEDAATYICWQT”DNPLV‘QADYxLFLKHI HR}KRSQRGSL.TC;‘V"F akMV“PJSASPPDS1AAAT CPARSLLLVATLVLLDHLSLARNLRVATPDPCMFPCLdHSQNLL«AVSNMLOKARQTL EFYDGTSEEIDHHDTTKDYTSTVEACLDLELTKNZS LNSRETSFITTTTGSCLASRKTS FMMAACLSSTYFUD'MYDVDF TMNAKTLMJPKRQ1FLDQN1AVTFFLHQALNFVSE ”VPHKSFF?FYKTKTKLCTLTW“ ' "' . .- . . .. . VELD A DCITW'LDQSSHVLCSCKTLTIQVKEFGDACOYFYCGEVLSHSLLLLnKK EDG_NSTFIL<JQ(EPKNYTFLRCAKKVRGRFTCWWLTTICTDLTFSVKSSRGSDP AA'LSADRVPGDNTLYFYFVDCQEDSACPAAEESTUibvNVDAVHKUKYFNY TSSTFTRL11K3WRDKNLQLRL:KNSRGVFVSWFYFD”W‘T'HSYFSLTVQVQGK KF'EKKDRVFTDKTSATVIC(NASISVRAQVRYfkflquFQAVPCS— (tum—(rm— MEFGLSWVFLTALFRCV”CDTALTQSPASLAVSLCARAFLSCKASQSVDYDJDSYLF“ CXCLII} ‘"‘71"KL.1.1YLAST\TVS'L '. 'GSGTDR L TT.‘ TJVEKVT‘A. T‘YHCQQSI‘E DPWTFGCGTKLEIKGGGCSGCCCSGCGCSQVQLQQSCASLVRPGS*VKISC(AGYAF SSYWMNWVKQRPGQ- IGTTWPUDUDTNYNGKPKG&HfiLTAEESSSTAVMQLSSLA SEDSAVYFCARQ AMDVW30‘TTVT"SSGG’JSPTXLQF’CAELARPGA SVKMSCKTSGYTFTRVTMH’VKQ’”SQGLFWTSYTNPLRSYTNYNQKFKLKAFLTTDK SSSTAYTUJSSTSJUSAVYYQ‘LYYDDHVCLDVWGQ‘TTTFVSSVFGGSTGSGGSGG SGGVDDIQLTOSPAZMSAPCFKVTMTCRASSVSYMNWVQGKSCTPKRTYIYDTSKJ ASGVPYRFRGSGSSTSYSLTISSTFAFDAATY"CDQ'SSNPLT ”AGTKLELKHHHTH HRRKRDGRGSL’TCGDVEFMUGPMFQ”A1LTC’T1L”ISCIOGV" ' SVfT”NPRSLKLLT1PAQ”"‘RVRTTATMKKKC‘KxCLJPFS’ mabut ‘52l I'LJ. ' WV . _ PV’GPIQ WFRCACR'P/LIY'HPQ ..RRVTTVSD1LKRNQVFFSTR1QNTTPADASTYYCIKFR i {SL} KCSH-WVEFKSGATELUVRAKPSASDIKLQDGAELARRGASVKNSCKTSCYTFTRY TMHWVKQRPGQGLEW GYINPSRGYTNYNQKFKD(ATLTTTKSSSTAYMHZSSLTSED SAVY ARYYDDHY(LDYT(QG““L1JSS’T CSGGSGGSGGSGVDDIQLTQE IM LVTMPLEASSSVCYNNNYQ)KSCTSFKRVr-rv1-JFSKVAS‘YTVRFSCSSSGTS YSLP1SSMBALD IYYCCQWSSNP- 1 GT. MAHHHHH.LL: 1m r51/1 .JGRGSLL1 VEEMPGPMRI*KPHLRSISIGGVLCLLLNSHFLTLACIFVFILID0 "12! DJAGLPKTEANWV NVISD IQSMITATLYTESDVHPLCKVTAMKCFLLEJQVISLECGDASIKD TVEWLITDINNSLSSYGL7TSSGLKFCT JJDKNIKF I'HTV"MFNTSw SIRFEu— METTTLLLWVLLLWVPCFTGDEE"IQIIHPDKVLVA.GTTATLRCTITSLFPVGPIQ CD3JIJS WFT?A(JCPVLTYNQPQT‘FFRVTTVSD1. RNNMPFST{TGNTTPAFAGTYYGIKFR Tflj) EKGS:LJVFF’1GA" " " :4 .VQQuUMJMARPUMsVKMJCKmD I TFTRVTMHNvKQRUuQGLFW SYTNFSRSYTNYNQKPKDKAF"”DKSSTAYMQLSS LTS*”SAVVYCARY”“DHYCLDVWSRGTTLTVSSVFCCblGSGGSGGSuCVDD1QLTC SPAIMSAS G:\VTV¢CQASSSVSYMTWVCQKQGTCPKIVIYDTSKVASCVPRFSGS GLSTTS.STTSSM1AEJAAT'YC”SSNPLTRCAGJ JKHHHHHHRR REGRGSL TTCGDVEFNPGPMRTSKRHLTSTOLYLCLLLNSHFLT GTTVFLLC"FSAGLPKT bAANVWVlS'LKKIEDLTQSVHTQATTY‘HSDVHPSVKVTAMK FLLFTHV'TSLFSGD ASIHD VENLIILANNSLSSuCNVTESCC ECEELEEKNIKFFLQSFV VQMFINTS STRFlm—{B— MFTDTLLLQVLLLImpST'DFEELQIIQPDKVLVAAGETATLRCTITSLFRVGPIQ ILEZ (5L) WFRGAGPGRVLIYNQRQGPFPRJTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFR KGSPDUVFFKSFAGTFLSVRAKPSA-vIKLQQSrAELARPGASVK TFTRY *' KQRFGQGLEWTC ..' h. rEKSS AYMQSSLZ ED SAVYVCARVYDDHYLQYNGQSLPL ISSVEGCSGSSGGSGGSGGVDD'LTWSFAlfl SASEGEKVFMTCKASSSV”YNNTYQQKSCTSRKRHZYDTKVASGVPYRFSGSGSGTS YSLTISSVEAFTAATYYCQQWUNPLTFGAGTKLZLKPBHHHHRRKREGRGSLLT GD VEEXH-‘GPM‘. PPGSASQPPP 1I'JAAAT 9AART2‘VLQCR SMCPAR 1--'\/A“T_HVLT_1D HTULAnxLTVA D'SMFR 1.1 'CSQNLLRAVS fiLQ-KA JEFYPx EIDHEDTT KL‘K‘" -.".“LI'TELAC LT:‘T'.1;~.-11KT\FIJC“1N F'I‘""“I’T'T\(-‘SC“1A IK‘1‘S131’IJVTALC _SST. Y lT‘T.KT‘T YQVL1KTMNAKLLMDPKRQIFLDOkMLAVIDHLMGALNFNSETVPDKSSLTTPDFYKT KIKLCILLHAFRIRAVTIDRVMSYDNAURRKRJGRUS LTCG IVEENPGPPMCHQQLV 'F‘ SL1"'1"LASI‘IJVAIWLIKKD’YVVIJLFVJYPI“ rJ'JEMVVI1‘CD""FF15~ITWTIDQS LGSG \TLTIQ'RJKEFGDEAUQYTCI-IICUGE‘JLFY»IS.L.LL_IIIKKEGIWSTDILKDQ *- FLRCEAKNYSGRFTCWWT TISTDLTFS’VKSSRGSSDDF-GVTTGAATLSAERVR . KEYLYS‘J’I1’1Q...SACPAA JSLI? I ENE?’I‘J’DAVHKIJKYENY.'“'S.S17‘F.. EDI IKI—‘DI‘UK ':Q1JKPJK’QS«ZQVEVS1’1EYPDT‘NS ".‘1'.JI'.SYFSL’IFCVQ r\anKSKR._‘.KKDRVF"1‘1JKI'I.'1 "gum a?"' (29341.32 WF'RGAGIP'"P\JL1Y1\IQPQ11-‘E. E.'\J"I”I"JS1 . . ,. . a (LL) IKGSHDDWEEKSGA"ELSVRAKPSASGCCI‘SD' KLQQSGAL.-.RPLmSVYV’ISF‘KYQU' TFTR'V'TIYHAT/KQRDI—"‘L WIGYINPSRC—IYTNYNDKI‘FDKATLTTDKSS‘"'"AYIV:QLSS I TS * '7SAYVYLAW‘“'DDIIYCLD‘W’GQSTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQ .~\. . “(A.19 SVSYMW/‘VF‘QKSG'I‘S ‘KEWI YI‘TSK\/A~"‘VI‘YPFSGS . IVIEAF 1AA'1‘.’VM;1’1NSSNF1JT" LPIVIIJ‘J’PPGSASQPPPSPAZ- 1 LPVATPDPGMFPC‘LL .NTITIRA‘V’SNMT:QKARQ‘ ' , ”VIE. CIJPLE ‘S’1IJESRL‘TSF‘INGSCI.ASRK'I‘.

.L'.:HAF'l'-1IRAV'7‘IDKJMSY'NAS v-HJ—j .RL'J'GRGSLLTCC.VE."JNPG 913N111 ‘IQQLVI SWFSLV7LASPLVAIWELKKDVELVVETDWYPDAPGET‘IJVLTI CDTPEEDGIT’1 SSEVGSGK KKEDGIWSTDILK JQKEIFKNK'" " . .".L"l‘1r'-'; '\J’KSSRGS DIE‘QGVJ.‘CGAA'I‘LS AFTVPGDNKEYEYSVFCQEDSACPAAE‘L‘LPIE\J’M”DA‘LHKLKYL :YTSSFF‘IF’NI'IK "1‘1‘KN..IV1JKPIKESPQVFVSWE’YSI’I‘WS ’PHSY I‘S1J'I"""VQ‘\/Q :IlSKv<EKKDR\/F‘1‘11 KT ATVI S I SVRAQDRYYS SS"WSE'"IASVPCS — SIREN}— MF'TIJ'“IJ._ILWV’LIIWVIK'SSTGDE. "ELQIIC2131 ‘\/AAFn"AI‘LR (1'T‘I'1‘SLF'PV(’IPIQ 60 CD3: "' ‘ 'AGPGRVLIYNQRQ‘—PFDR‘JTTVSDTTKK'NNIVDFSIRILNITDADA viTY'YCIKFR i CXCLIQ DVEFKSGASTELQ\I’KAKPSASDIKLQQSGAELAEPGASVKI‘TSL"‘KTSGYTFTRY (SL) .

. VKQRPGOGIJFHAI NPSRGYTNYNQ\1"I.'\'I‘1A“rJIJ'I‘TDKSSS'IAVMQLS SI.-'I‘SED g SAVYYCARYYUDHYaI.UYNG’Y‘M’TTJ'NSE‘VEGGS‘GI’W‘CMS"’19-.-‘ "LTQSPAIM . C'ASPC-1EKV’IM’I'SRASSSVSYI‘I’NAIY’V‘I SGT‘SEKRWIVIL’I'SKV“nSGVPY. . SGSGSGTS YSLTI.3S1‘EAELAATYYF‘QQWSSNDLTTGAFTKLEE HI‘iHABHRrflKRE’RGSLLT ’GD <4’EENPGDY’YQTAILICCLIFLTLSGi QS‘TLSRTVRCTC’ ,SISNQPVNPRSLEKLEII :. .4 . V’E‘3'. IAI‘MKKKGE:.1?.('1JNI‘E.SKAIKNI...KA'\J’.SI\F(.SKRS I:‘-- 1 SIEME- BCETDTLLLN'ILLLIIC‘JPGST:DEELQIIQPDKSVLVAACETATLPCTITSLFPVGPIQ C113“ WERSAF'DGRVLIYEQRQPI‘DRVT’I“’"DT‘TKTN‘QMDFSIRI‘NITPADAGTYYF‘IKFR {7X CLIG KGI'PDDVF‘F‘KSGA"T lLSVRAI' " UQCSGAE‘LARI‘GAS\J’Ix‘Vlo(K ‘ ’ 'RY ’1‘1‘I”1-IW\J’KQR:<1?"aQfizLF‘WlS-I Q a (LL) 1 I TNYNQKFKDKATLI"".‘DKSESS’I‘A YIVIQIISS L:SEESAVVYF‘ARY‘“"DI-IYCLD"WG'AGT—‘LTVSSVEFGSGGSGGSGGSG VDDIQLTQ SPAII .SASPGE \V'I‘IYTCRA.SSSVSYV'NWVCQKSGTSPKRIIIYDTSI VASGV YRFSGS C” ' ‘SI‘I’IE'..'J'DAATYYCQOWSSNPLrI‘FY} . . .F : ERGSL MNQTAILI (1’31: .1' FL-rflu. ' ‘ FELT—(1113— . \Q y‘SGAF 1A -VKMS\ I.

[LES EFGQF:LWICYINPSPGYTNYNEKFKUKALTTDKSSSTAYEVLILI1SJDSAVYY“A R""DLEILLDYNGQCTTLTVSSJEG”SCSG"SGLSFG'DDIQLTQSPAIMSASPGE VTMTC“ASSVSYNNNYQQKSGTSTKRVIITSKVASGVPYLFSGSCSGTSYSLTISS"'1’ MEALuAAT”YLCQWSJSDLTTGAGTKLELYGGCGSDIQMTCSDSSLSASVGDRVTITC RASQDVSTAMYQQ.‘GKAPKIL1Y:ASFLYSGI‘SVESGJC GPDFTLTISSLQPF IFATYYC‘YLYHPAIFGQQFKVDIKKLGGGLC(GQSG"PGCEVQLVFLSG1LVDPGG SERLSCAASPETFSJSWIIWVRPADCKCEWVAWISPYG‘STYYADSVKGRFTISADT LHWNSLKA:DTAVYYEA?RHWDGGFDYWGQGTLVTVSAHHHHEHRWKREGR UVELKFGPMPISKPF-- ' 1 ZAGIHVEILGC ELF 1’77? £“YVVNVIS1LK , ,. . SIVHP’CKVTAWKCIILFI DAS1DTVFEI11LAYKSLSSNGNVTLSGCK‘CEJLDLKQIKFF'“SFVSIVIMFIN PETA—CD3— FFPIWVFLVALEC”Q“U1KLQQSQAE1ARPGASVKMSSKISGYTFTPVTMHWVKQ 63‘ ELIE WIPYIYTSRFYTNYNPKFKDKATLTIDKSSSTAVNQLSSLIDELSAVVYCA YYDDHYCLDYWGQGTTITVSSVLGGSGCSGGSGCSGVDDIQ.LTQSPAIMSASPGEK fTMT”TASSJVSYMNWYQCKSGJ19F”J DTS VASGV"VSISGS"SC”SYSTTISS ' IVYCAQWSSNPL”ECACI ;K(GGGSFIQVTSS I 1r I‘- ' 'r DFATYYCQQYLYHPATFGQGTKVEIKRGGGGSGGGGSEGGGSZVQLVESGGELVQPGG SLRLSCAASGFTFSDSWIHWVRQAPGnquWVAWISPYGub YYADSVKGRFTISADT 5K3 AYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSAHHHHEHRRKREGR SLLTCJUVEENPGPMWPPGSASQPPPSPAAATGIHPAARPVJLQCKJIMCPARSI’ ATLVLLDHLSLARJLPVATPDPGEFPCLHHSQNLLRAVSXELQKARQELEFYPCELES %ZDITXDKTSTVEACLPLELTKNESILNSRETSFITNGSCLASRKQ TMMALCJSS IYEDLmMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSHL—J EPDFYKTKIKLC {AFRIRAVTIDRVMSYLNASRRK EGR LLTCGDVEENPGP LJ' MCHQQLVISWFSLV.LASPLVAIWELKK7”YVVELDW{PJAPGEMVVLVCDVPEEDGZ TWTLDQSSFVLGSGKTI IQVKFFGDAGWV LSHS‘ ’?.f' LKDQKEPKNKTFL YSiiFTCW LSAERVRGBNKZYEYSVECQEDSACFAA.

I’DLiuCiBu . __ CKTSGYTFTRYTMHWVKQ 64 E CXCLIE} R7GQGLEWIGYINPSRGYTNYNQKFKDLATLTTDESSSTAYMQLSSLTSE GAVYYCA LTV?”VEGGSCTSGGSGGSGGVPFIA'TQSPAIMSASPGE] 4NWYQQTSL‘SPK" 'YDTSKVATGVPT GSGSGTSYSLTISS i QQWSSNPLTFGAGTKLELK GSDIQMTQSPSSLSASVGDRVTITC YQQKPGKAPKLLIYSAS GTDFTLTISSLQPE DEATYYCQQYLYHPATFGQGTKVEIR ‘ ' QLVCSGGELVQPGC R1) S CAASGF'V‘FSDSW IHWVRQAPG' .C V ' J» ' ' .._ YADSVF’C‘RF‘T I SADT 3INTAYLQMNSLRAEDTAVYYCARRHWé , . VSAHHHHHHRRKREGRG SLLTCGDVEENPGPMNQTAILECCLIFQ;LSGIQGVPLSRTVRCTHISISNQPVNPRS L KLEIIPASQFCPRVEIIATL’KKGEKRCLNPESKAIKILLKAVSKERSKRSP— 51?.le . TDTLLLWVLLLWVPGSTG EEELQIIQPDKS’LVAAGETATLRCTITSSFPVGPIQ 65 i CD3~MMP9 RVLIYNQRQGPFPPVTTVSDTTKRNNMDFSIRIGNITPAD.GTYYCIKFR (5L) VEEKS RAGTELSVRAKPSASD IKLQQSGAELAR SASVKMS C,K’i’SGYTF'1‘}?! TMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKBKATLTIDKSSSTAYMGZSSLTSED SAVY r‘15.\F<’..l"KDDHYCU.)':(.'§'.1C;‘:CJ:'L.‘TL'T'73S‘V’E‘JCL.SC"' :uSSGSGGVDDIQJTQS PAIM SASSGEKVTMTCAASSSVSYMNWYQQKSGTSPKRWEYDTSKVASGVPYRFSGSGSGTS YSLTISSMEAEDAATYYCQQWSSNPLTFG GTKLELKHHHHHHRRKREGRGSLLTCGD VEENPGPMSLWQPLVLVLLVLiCCFAAPRQRQSTLVLFPGDLRTNLTDRQSAEE YR VFYTRVAEMRGECKSLGPALLL:QKQLSi \ 1—: TSULDSATLLAMRT” ’aVPTLGhFQT ‘5. USELK . . 3N! I. TY'W' I QN'Y S ‘ . JPRAV II ,AFARAE‘AL . ‘PLT. ,. EADIV IQFGVAEHGDbYPFDGKDGLLAHAFPPGPGIQuuAHFDDDELWSLGKGVVVPTRFGNA DGAACHFPFIWEGRSYS‘CTTDGRSDGLPWCSTTANYDTDDRFGFC SYTRDGNA DGKPCQF SQSYSACTTJGRSDGYRWCATTANYDRT 'GF ADSTVMGG§ SAGELCVFPFTFLGKEYSTCTSEGRGWCRLWCATVSNFDSLRKWGFCJDQGVSLFLVA AHEFGHALGLDHSSVPEALMYE«YRFK 'PLHKDJVNGIKILYGPRL EPRPPTTT TP ETA?PTVCPTGPPTVHPSERPTAGPTGPPSaGPTGPPTAGPSTATTVPLSPVDDA CNVNIFDAIAEIGNQLYLFKDGKYWRFSEGRGSRPQGPFLIADKWPALPRKLDSVFEE PL: " L. E” E” “SGRQ‘V’WVY'I‘GASVLG PRRT..DKLGLGADVALVTGALR. ’{GKML 7' 1 5R. E L-WQED‘J’KAQIVI‘V'D PRS 13.8 EVL VPLDTHDVFQVRE.KAYFCQD k.E‘YWRVS S P.S“LN VDQVGYVTYDILQCPED- VAAG 1% m til WFRGAGPGRVLIYNQRQGPFPRVTTVSl.JKRNNMDFSIRIGNITPADAS. . i (LL) ................ KGSEDLVEFKSGAGTELSVRAKPSASGGGGSDIKLQQSGAELARPGASVKMSCKTSG" +....................................................................... MHWVKQRPGQGLEWIGYINPSFCYTNYNQKFKDKATLTTDKSSST.YMQLSLTSSUSAVYYCARYYDDHYCLDYRGQ ’LTVSSVEGESGRSGGSGGS SP IMSASP IKVTNTCRASSSVSYMNW \S W-.u' SSMEAEDAATYYCQQWSSNPLTFGA\LKLELKHHHHHHRRgREGRGSZ ETCGDVWWNPGPMSEWQPTVVVLLVTGCCFAAPRQRQSTLVLFPGDLRTNLTDRQLAE GYTRVAEMRGESKSLGPALLLLQKQLQLPETGELDSATLKAMRTPRCGVPDJ GRFQTLEE”. )L'. WHHHNT. TYW I QNYSRD E. P. 3A"! I.DDAFARAFALWSAVT : 'I‘E‘TRVYSR DADIVIQFGVAJHGJGYPFDGfDELLAHAFPPGPGXQGDAHFJDFELWSLGTGVVVPT RFGNADGAACHFPF; 'SYSACTTDGRSDGLPWCSTTANYDTDDRFG..PSERLYT DGNALG\PCQFPFIFQLQSYSACTTDGRSDGYRWCATTANYDRDKLFGFCPTRADST1) r-------- VMGGNSAGELCVFPPTFLGKEYSTCTSEG.GDGRLWCATTSNFDSDKKWGFCPDQGYS LFLVAAHEFGHALGLDHSSV ' PRPEPEPR 0“ ITPQPTAPPTVCPTGP ’ “JACNVNIFDAIAE'.GNQLYLjKDGKYWRFSE\— JIADKWPAL V\t‘RKLD j “ .1: PLSKKLFFFSGRQVWVYTGASVLGPiiK .

”RRLWRFDVKADMVDPRSASEVDRMFPGVPTL QVDQVGY‘\7‘FYDILQCPED Slkylu- R JLL'VLLLWVPGSTGDYPYDVPDYA\31“.l‘l—\ QPADDIQitemiVi L QSPSSLSAS’GDRVTIT 6S i CBS—Pl)“.- VSTAVAWYQQKPGKAPKLLIYEAS "LYSGVPSRFSGS4-1 ... A «m k: tax: i SSLQP Fc (SL) “mYYCQQYLYHPATFGQGT \‘IF‘. >ru.

A4. . \. i_._L\i. ' SGGGGSGGGGSCVQLVESGGGLVQPQ "I£LSCAAn nbuFTFCDSWIHWVEQAPJT . JEWVA7ISPYGGSTYYADSVKGRFTISAD TSKBTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSAVDIEAKSCDKTfiTC PPCPAPEL‘GGPSVFLFPPKPKDTLM SRTPEVTCVVVDVSHED EVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVL{QDWLNGKEYKCKVSNKALPAPIEKTISKAKG EREPQVYTIfiPSRDELTKNQVSLTCLVKGFYPiDIAVEWESNGQPEHNYKTTPPVLDS UGSFFLY JTWTKSRWQQGNVFSCSVMHEALHNHYTQ .SP..]DEQK1 “NRQKRWGRGSTLTCGDV «unseen *TDRLLEWVHKLWVEGSTGDE EELQIIQL LVAAGETATLRCTITSLFPVCPIQWFRGAGPGRVLIYNQRQGPFPDVTTVSDTTKRNN 1 LL!'“FSIRIGNITPADAGTY 9P“V“DKSGAGTEI’VRA/.LI J 1.3 J.‘ E’ .3‘OASDIKLQQSG AELARPGASVKMSCKTSGYT.TRY1MHWVKQRPGQCIEZIGYINPSEGYTNYNQFFKD DKSSSTAYMQLSSLTSEDSAVYV .—, - h. mm i a. , qv I": ' 4. \- . RYYDDHYCLDYWGQK}.' LiveSVE GD GGSGGSGGSGGVDDIQLTQSPAIM " 7\but:('I PGEKVTMTCRASSSVSYMNWYQQKS(’"T‘r'ant: KR WIY TSKVASGVPYRFSGSGS; “YSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKL ELKHHHHHH" srnei n METDRLLLWVLLLWVP ,.mG51 VPDYAGAQPABDIQMTQSPSSLSASVGDRVTIT 7O ens—emit- CRASQDVSTAVAWYQQKPGKAPKLLIYSAS"LYSGVPS.rouSREC” 4‘1 ... m rrrr\ be»: i SSLQP : Fe (LL) EDFATYYCQQYLYHPATFGQ/~<m'k7" \‘I‘F'fi\. i_._'L.\i. r‘r' SGGGGSGGG SIVQLVESJGGLVQPGmk: GSLRLSCAAn n *1 T CDSWIHWVRQAP :1. a. JEWVA’ISPYGGSTYYADSVKGRFTISAD TSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSAVDSAKSCDKTHTC PPCPAPELL(“l/‘1\Ju SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLT’'VLHQDWLNGKEY@CKVSNKALPAPIEKTISKAIGQ PRWDQVYTLPPSRDELTKNQVSLtTCLVKGFYPLDIAVEWESNGQPENNYhTTPPVLDS f" n n. v-.knbi‘ TVDKSRWQQGNVF'SCSVMHEALHNHYTQ .ioub"T”PGKVDEQKL VNRRKRVGRGSPETCGDVF”NPGPMFTDRLEEWVH11WVPGSTGDEEELQIIQ; JSDTTKRNN 9D.1. .,... ....lJ‘J ital? KSGAGT RAKPIASGGGfID A [YTFTR‘Y '“M ' SW"”" RPGQGLZWIGYINESRGV" v tag; i i i l - "rx-r 'ITLTr-Irrx-r 33811 .—.—. ‘AV \'r\ - v .—, Ju iYK. ,. . iJ. .‘J. , J .' t LDVWGQGTTJ.

EVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASEGEKVTMTCRASSSVDYMNWYQQKSG lTSPKRWIYDTSKVASGVPYRESGSGSGESYSLIISSJAAJD TYYCQQWSSNPLTFG AGTKL1-1 1-:1. HHH» } In some embodiments, the present invention provides recombinzmt nucleic acid sequences encoding an engager molecule and/or a therapeutic ino eeule. Exemplary recombinant nucleic acid sequences are shown in Table 4. {60213} ln some embodiments, the c acid sequences provided herein encode a eutic molecule, wherein the therapeutic le is. lL—lfii In some embodiments, the nucleic acid sequences provided herein encode an anlS therapeutic molecule comprising an amino acid sequence that is at least 80%, at least, 85%, at least 900/, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ll) N0: 24. in some embodiments, the nucleic acid sequences ed herein encode an lL—lS therapeutic molecule that is “30% identical to the amino acid sequence of SEQ ll) NO: 2.4. in some embodiments, the nucleic acid sequences provided herein encode an lL-lfi therapeutic molecule comprising the amino acid sequence of SEQ ED N0: 24. in some embodiments, the c acid sequences provided herein encode an IL—l5 therapeutic molecule ting of the amino acid sequence of SEQ lD NO: 24, In some embodiments, the nucleic acid sequences provided herein encode an ILnlS therapeutic molecule and comprise a sequence that is at least 80‘3"/,0 at least 85‘30, at less 90% atelast9,.5‘30, at least96/30, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 23. In some embodiments, the nucleic acid sequences provided herein encode an lL—lS eutic molecule and comprise the nucleic acid sequence of SFQ ID NO: 7.3. In some embodiments the nucleic acid sequences provided herein encode an lL—l5 therapeutic molecule and consist of the nucleic acid sequence of SEQ II) NO: 23 {(39214} In some embodiments, the nucleic acid sequences provided herein encode a therapeutic molecule, wherein the therapeutic moleculeis ll.—lZ (ie II.l2 p35 and/or Il_,—l.7_. p40). In some ments, the nucleic acid sequences provided herein encode an Il_3~l 2 therapeutic molecule comprising an amino acid sequence thatrs at least Si‘30,at a 5‘30, at least 90% at least935%,at. least 96%, at least 97%, at least 98%0, or at least 99% identical to the amino acid sequence of SEQ ID N0: 26. In some embodiments, the nucleic acid sequences provided herein encode an IL-l2 therapeutic molecule that is l00% identical to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the c acid sequences ed herein encode an IL—lZ therapeutic molecule comprising the amino acid sequence of SEQ ID NO: 26. In some ments, the nucleic acid ces provided herein encode an ll.3~l2 therapeutic molecule consisting, of the amino acid sequence of SEQ ID N0: 26. In some embodiments, the nucleic acid ces provided herein encode an IL-IZ therapeutic molecule and comprise a sequence thatis at least 80‘/'0 at least , at least 90%, at least 95%, at least 96%, at least 9796, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 25. In some embodiments, the nucleic acid sequences provided herein encode an IL—IZ therapeutic molecule and. se the nucleic acid sequence of SEQ ID NO: 25. In some embodiments, the nucleic acid sequences provided herein encode an llxl 2 therapeutic molecule and consist of the nucleic acid sequence Q ID NCD: 25. {602153 In some embodiments, the nucleic acid sequences provided herein encode an II- l7 therapeutic molecule comprising an amino acid ce thatis at. least 3030, at least, 85"30, at least lea 9%53,21tleast 9630, at least 97%, at least 9836, or atleast 99% identical to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the nucleic acid sequences provided herein encode an IL l2 therapeutic molecule that is 100"30 identical to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the nucleic acid sequences provided herein encode an lL~l 2 therapeutic molecule comprising the amino acid sequence of SEQ ll) NO: 28. In some embodiments, the nucleic acid sequences provided herein encode an ill—12 therapeutic molecule consisting of the amino acid sequence of SEQ ll) NO: 28. in some embodiments, the nucleic acid sequences provided herein encode an anlZ therapeutic molecule and comprise a sequence that is at least 80%, at least, 85%, at. least 90%, at least 95%, at least 96%, at least 97%, at least 989/5, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 27. in some embodiments, the nucleic acid sequences provided herein encode an lL—l2 therapeutic molecule and comprise the nucleic acid sequence of SEQ ll) NO: 27, in some embodiments, the nucleic acid ces provided herein encode an lL—l2 therapeutic molecule and consist of the nucleic acid sequence of SEQ ll) NO: 27. {99216} in some embodiments, the nucleic acid ces provided herein encode an llle therapeutic molecule comprising an amino acid sequence of SEQ ll) NO: 26 and 28 In some embodiments, the nucleic acid ces provided herein encode an llrll therapeutic molecule and comprise the nucleic acid sequences of SEQ ID N0: 25 and 27. {$9217} ln some embodiments, the nucleic acid sequences ed herein encode a. therapeutic le, n the eutic molecule is CXCLlO, ln some embodiments, the nucleic acid sequences provided herein encode a (ZXCLlO therapeutic molecule comprising an amino acid sequence that is at least 80%, at least, 85%, at least 90%, at least 959/ at least 96%, at. least 97%, at least 98%, or at least 99% identical to the amino acid. sequence of SEQ ll) NO: . In some embodiments, the nucleic acid sequences provided herein encode a CXCLlQ therapeutic le that is l0(l% identical to the amino acid sequence of SEQ ll) NO: 30. in some embodiments, the nucleic acid sequences provided herein encode a CXCLlO therapeutic molecule comprising the amino acid sequence of SEQ ll?) N0: 30. ln some embodiments, the nucleic acid sequences provided herein encode a CXCLlO therapeutic molecule consisting of the amino acid sequence of SEQ ll) NO: 30. in some embodiments, the nucleic acid sequences provided herein encode a CXCLlG therapeutic molecule and se a sequence that is at least 809/5, at least, 85%, at least 90%, at least 95%, at least 96%, at least. 97%, at least 98%, or at least 99% cal to the nucleic acid sequence of SEQ ll) NO: 29. in some embodiments, the nucleic acid sequences provided herein encode a CXCLlO therapeutic molecule and se the nucleic acid sequence of SFQ ll) NC: 29. in some embodiments, the nucleic acid. sequences provided herein encode a CXCLl 0 eutic molecule and consist. of the nucleic acid sequence of SEQ ll) ND: 29. {96218} ln some embodiments, the nucleic acid. sequences provided herein encode a therapeutic molecule, wherein the therapeutic molecule is MMP‘). in some embodiments, the nucleic acid ces provided herein encode an MMP‘) therapeutic molecule comprising an amino acid sequence that is at least 809/0, at least, 85%, at least 90%, at least 95%, at least 969/0, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID N3: 34. In some embodiments, the nucleic acid sequences provided herein encode an MMP9 therapeutic le that is l00% cal to the amino acid sequence of SEQ ll) N6: ’34. In some embodiments, the c acid sequences provided herein encode an MMP9 therapeutic molecule comprising the amino acid sequence of SEQ ll) NO: 34. In some embodiments, the nucleic acid sequences provided herein encode an Mlvllj’i) therapeutic molecule consisting of the amino acid sequence of SEQ ID N0: 34. In some ments, the nucleic acid sequences provided herein encode an MMP9 therapeutic molecule and comprise a sequence that is at least 80/0, at least, 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ll) NO: 33, In some embodiments, the nucleic acid sequences provided herein encode an MMP9 therapeutic le and comprise the nucleic acid sequence of SEQ ll) N0: 33. In some embodiments, the nucleic acid sequences provided herein encode an h4lh’lP9 therapeutic molecule and consist of the nucleic acid sequence of SEQ ID NO: 33, {09219} In some ments, the nucleic acid sequences provided herein encode a therapeutic molecule, wherein the therapeutic molecule comprises an antinl’DL-l scFv. in some embodiments, the nucleic acid sequences provided herein encode a therapeutic molecule comprising an antiJPDLl scFv, wherein the anti~l3DLl scFV comprises a light chain variable fragment comprising an amino acid sequence that is at least 80%, at least, 859/6, at least 90%, at least 959/ at least 96%, at least 97%, it least 989/ or it least 99% identical to the amino (cid , , sequence of SEQ ID NO: 36 and a heavy chain variable “ragment comprising an amino acid sequence that is at least 800/, at least, 85%, at least 90%;, at least 95%, at least 969/5, at least 97%, at least 98%, or at least 9 % identical to the amino acid sequence of SEQ ID NO: 38. In some embodiments, the nucleic acid sequences provided herein. encode a eutic molecule comprising an anti—PDLl scFv, wherein the DLl scliv comprises a light chain variable fragment comprising an amino acid sequence that is 100% identical to the amino acid sequence of SEQ II) N0: 36 and a heavy chain variable fragment that is 100% identical to the amino acid sequence of SEQ ll) N0: 38, In some embodiments, the nucleic acid sequences provided herein encode a eutic le sing an anti-PDLE scFv, wherein the anti-PDLl sth’ comprises a light chain variable fragment comprising the amino acid sequence of SEQ ll) NO: 36 and a heavy chain variable fragment, sing the amino acid ce of of SEQ ID NO: 38. In some embodiments, the c acid sequences provided herein encode a therapeutic molecule comprising an anti—PDLl scFV, wherein the anti—PDLl scFv comprises a light chain variable nt consisting of the amino acid sequence of SEQ ll) ND: 36 and a heavy chain variable fragment consisting of the amino acid ce of of SEQ ll) N0: 38, {90220} in some embodiments, the nucleic acid sequences provided herein encode a therapeutic molecule comprising an anti—PDLl 543%, wherein the anti-PDLl scFv comprises a light chain variable fragment nucleic acid sequence that is at least 89%, at least, 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ll) NO: 35 and a heavy chain variable fragment nucleic acid sequence that is at least 80%, at least, 35%, at least 90%, at least 95%, at least 96%, at least 97%, at least 93%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 37. in some ments, the nucleic acid sequences provided herein encode a therapeutic molecule comprising an antirPlfiLl scFv, n the anti—PDl_,l selr’v comprises a light chain variable fragment nucleic acid sequence that is lOO'il/ri identical to the nucleic acid sequence of SEQ ll) NO: 35 and a heavy chain variable fragment nucleic acid sequence that is l00% identical to the amino acid sequence of SEQ ll) N0: 37'~ in some ments, the nucleic acid. sequences provided herein encode a therapeutic molecule comprising an anti—PDLl scEv, wherein the anti— PDLl scEv comprises a light chain variable fragment nucleic acid sequence comprising SEQ lD NO; 35 and a heavy chain variable fragment nucleic acid sequence sing SEQ ll) NO: 37. ln some embodiments, the nucleic acid sequences provided. herein encode atherapeutic molecule comprising an antiJPDLl scFv, wherein the anti~l3DLl sclr‘v comprises a light chain variable fragment nucleic acid ce consisting of SEQ ll) NO: 35 and a heavy chain variable liagment nucleic , cid sequence consisting of SEQ ll) NO: 37. {£992le in some embodiments, the nucleic acid sequences provided herein encode a therapeutic molecule comprising an anti-PDLl scFv and an lgG-l Fc domain In some embodiments, the nucleic acid sequences provided herein encode a therapeutic molecule comprising an anti~PDLl scFv and an lgGl Fe domain, wherein the lgGl Fe domain comprises an amino acid sequence that is that is at least 809/6, at least 85%, at least 90%, at least 95%, at least 9 %, at least 97%, at least 98l?/li, or at least 99% identical to the amino acid sequence of SEQ ll) N0: 40, in some ments, the nucleic acid sequences provided herein encode a therapeutic molecule sing an anti-PDld scFv and an lgGl Fe domain, n the lgGl llc domain is l00% identical to the amino acid sequence of SEQ ll} NO: 40. in some embodiments, the c acid sequences provided herein encode a therapeutic mo ecule comprising an anti—EDLl scFv and an lgG l Fe domain, n the lgGl Fe domain comprises the amino acid sequence of SEQ ll) NO: 40, in some embodiments, the nucleic acid ces provided herein encode a therapeutic molecule comprising an anti-PDLl seEv and an lgGl Fe domain, wherein the lgGl Fc domain consists of the amino acid sequence of SEQ ll) N0: 40. in some embodiments, the nucleic acid ces provided herein encode a therapeutic molecule comprising an DLl scFv and an lgGl Fe domain, wherein the lgGl Fc domain nucleic acid sequence is at least 80%, at least, 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% cal to the nucleic acid sequence of SEQ ll) N0: 39. in some embodiments, the nucleic acid sequences provided herein encode a therapeutic molecule comprising an anti-Pfilsl scli'v and an lgGl Fc domain, wherein the lgGl Fe domain nucleic acid sequence comprises SEQ lD N0: 39. in some embodiments, the c acid sequences provided herein encode a therapeutic molecule comprising an DLl scFv and an lgGl Fc domain, wherein the lgGl Fc domain nucleic acid sequence ses SEQ ED N0: 39. in some embodiments, the nucleic acid ces provided, herein comprise a nucleic acid sequence selected from SEQ ll) NOs: 43, 45, 47, 49, til, 67, and 69. in some embodiments, the nucleic acid sequences provided herein are at least 30%, at least, 859/6, at least 90%, at least 95%, at least 96%, at least 97%, at least 9 %, or at least 9, % identical to a nucleic acid sequence selected from SEQ lD N05: 43, 45, 47, 49, Si, 67’, and 69. ln some embodiments, the nucleic acid sequences ed herein are lOO‘Vi-i identical to a nucleic acid sequence selected from SEQ lD N05: 43, 45, 47, 49, 51, 6’7, and 69. in some embodiments, the nucleic acid sequences provided herein consist of a nucleic acid ce selected from, SEQ ll.) NDS: 43, 45, 47. 49, Si, 67, and ($9. {00223E in some embodiments, the nucleic acid sequences provided herein encode an engager molecule and/or eutic molecule that is at least 80%, at least, 859/ at least 90%, at least 95%, at least 96%, at least 979/6, at least 99%, or at least 99% identical to an amino acid sequence selected from SEQ ID NOs: 44, 46, 48, 50, and 52, In some embodiments, the nucleic acid sequences provided herein encode an engager molecule protein that is 100% identical to an amino acid sequence selected. from SEQ ll) 'NOs: 44, 46, 48, 50, and 52 In some embodiments, the nucleic acid sequences provided herein encode an engager molecule protein comprising an amino acid sequence selected from SEQ lD NOS: 44, 46, 48, 5t), and 52. In some embodiments, the nucleic acid sequences provided herein encode an engager molecule protein consisting of an amino acid sequence selected from SFQ ll) N05: 44, 46, 48, 50, and :32, {99224} in some embodiments, the recombinant nucleic acid sequences ed herein encode an engager molecule and a therapeutic molecule. in some ments, the inant nucleic acid sequences encode an amino acid. sequence comprising an engager molecule and a therapeutic molecule, wherein the amino acid sequence is at least 80%, at least, 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 93%, or at least 99% identical to an amino acid sequence ed from SEQ ID N05: 53 — 66, 63 and 70. In some embodiments, the nucleic acid sequences encode an amino acid sequence sing an engager molecule and a therapeutic molecule, wherein the amine acid sequence is 100% identical to an amino acid sequences ed from SEQ ED NOS: 53 — 66, 68 and 70. In some ments, the nucleic acid sequences encode an amino acid sequence comprising an engager molecule and a therapeutic ine'ecnle, wherein the amino acid sequence censists of an amino acid sequence ed from SEQ ID N05: 53 — 66, 68 and 70.

Table 4: Nucieic sequences cf exemplary engager meiecuies (Dig-C133 TG’GAGTTCGGCCTGAG"C5 (3 GTTCCT GTGGCC TGTTCAGGGGCGTGCAGTGCG 43 ACATCCAGCTGACCCACJACC5C5C-C.CJC5C5AGC5CT’5’5’5 GA.’.5C5 5.CGGGCC5CAC5’5’5’5C5.

CATCAGC'. .AA 5GCC C5 ‘C5 5"C3‘TGC. ‘ . . . . .AC5C5‘15AC TACCAGCAG.‘CTCCCC 'GCCA‘ "CCCCAAGCTCCTCATCTACC—IZ‘CGCCA—CAACC G‘ TGAGCGGCATCCCCCCCA GTTCAGCGG‘CAGCGGCAGCGGC5 CC<3ACTTCACCCTGAA.

CATCCA5C5I’5C5G'I'GGACrAACJ 3"I‘GGAC5GC.(5’5’.5C_5AC-C.'IA’.5C_5. 5~C-’5AGC.AC:AC5C5 CC 5AG GAC‘C" 515GC3AC5’5TI55C5GC;GCGC:C5A’5C5AAC:C5'I"-5C-5ACATC5AAGGC:C5’5’5’5C-rG.‘G’5’5AGCG GCC GCC5CJC5AC1CC 5C 5C5C5‘. G’CCACJGTG’CAGCTGCAG . GC’5GC55 .A GCTGGTGZ‘aGG'CCC‘ CAGCAG'CG'TCAACATCAGCTGCAAGGCCAGCGGCTACGCCTTC AGCAGCTACTC"—GATGAACTGGGTGAAG"CAGA CCCGGCCAGGGCCTGGAGTGGATCG .5“CJA'I5’5'5‘C5GCC.C5’5C~5C5:ACG" 55AC5ACCAA 5AACGGCCAAGT‘I‘CAAGGC‘CAAGGI’.‘ C5‘A(5’5 15’15C5ACLCC3’5 ’5 CGACGACrAGCAGCAGCAC.CG (5...,’5"5AC.‘ATC5 ’5AGC".C"5- C.5 GA'5.C5'C5 CTGGCC AGC 5AGGACA GC CG"15C5TAC"“55C5"PGCGCC ,C5"C3" CCV5A 5.AC’5CAC' C a'.’"C5 5C5CAG’5'5.5 ACTACTACGCCATGGACTACTG 'GGCCAG'GGCACCACCGTGACCGTGAGCAGCGGCGG CGGCGG’CAGCGACA'TCAAGCTGCAGCAC‘AGCGGCGCCGAGCTGGCCAGGCC GGCG ’C AC5r5G'."GAAGA‘TGAG’ -ACC.AGC5"15A AC’5'5-5"5CAC.‘CAGG‘TACLACCA‘lGCACT C3-C5G'“C5AAGC. C5 C5 .‘AGG’5C'5C515GGAGT‘C5CA'TC.’5GC’5".15AC."' rI"5 .5‘AMCCCA C5 r5AG (.5.GGCTACACCAAC’I'AC . AG."I—‘CAACG"?r .‘C.A.AGGACA.AC?GC CACC’5.’5’15C5AC. "LACCGA .

AG‘CAGCAGCACCGCCTACATGC:GCTJAGCAGCCTGACCAGCGAGG.7-\C kGCG 'CG ‘ ACTACTGC‘GCCAG‘G'-ACTACG‘ACGACTCACTA“TGC'C'TGGACTACTGCGGCCAGGGCAC CIC"5A-’.5C ETGA’5’5AGCGT’5CAGGGCC.‘ GC. G”5C5C5GC2C5AC5C5GGC.C5G.’.5 1035C,. u (a:D"; C5C5G CGTCrGAC5‘GACA5‘ "l5CCAG CTGAC. C. .AGACJC- C. .CGC’5CA’IC7.TGAGCG CC.'5 5‘AGCC."C)GGCGAGAAGG‘TGACCATGACCTGCAGGGCCAGCAGCAGCGTC5GAGCTACATGAACTG *3“5CCAGCAGA.AGAGCGGCACCAGCCCCCAAG'AG(5JTGGATCTACGACACCAGCAAGC5:TG QC0CAGCCGCGTGC C5CTAC.AGGTTCAGCCGCAGCGCCA.GCGGCACCAGCTA.CAGCCTG‘A.

CJ Inc”, rC -‘x :CATM/s AGGCCGAC?GAC5GCCG.CCACC.5T (.5 711.1“ C‘T’5 ’5 5AG CA’5'55CrGACJ CAG ,.. . r3 ,"CC."CTGACC‘. :GC 5C5 CGC: CAC." C5AA 5CTGGA'C C’.'G.AAGCA C"ACCACICAC CAC." CACTAG SIRPla—CD3 CCACJAC-C.GA'5.5AC C5'C.T’5C’5" 5 -'"l"'i5 5''GGG”."TTTGC’5"l5'1"'C."I”.'TC‘G’;3'T5: 5"CAGGATCTACAG I15 (SA) GTGA’5 TGA AAGA . 5C5AGAT’5A"15C5C7.A’5C5.A"CSACAAA". . C‘TCGTG .. GG AGAGACCGCTACCC'“.1 'CAGATGTACCATCACTTCTCTCTTCCC'CGTTCCCCCCATCCAG CGAGGCGCAGGACCAGGACGAGTGCTTATTTACAATCAACGACAGGGCCCAT' TI’.5(.5C5‘AAC5..7«\C5"115A.C5AA’5AG'I'AT’5’5C"ATA C5’5AC5C5AA’5CG.r5AJATAA'55Al5GC5A’5'55".5‘15A“C5 CAT CGGC'AAC"AFAACAC5 C5CGC’I'C5A CCGGTAC."TAC"55.5 ."1"TG’I'A.555'15AAA’I""555C5GA AAGGGCTCACCAGACGACGTGGAATTTZ‘LAGTCAGGGGCCGGA-.CCGAZ‘1CTC-5CAGTTA -~AC’CA.AAACCTTCTCCTAGCC) A GCTG‘CAGC GAGCGGCGCCGAGCTGGCCAG GCCC GCGCCAGCGT. GAAGATGA<3CTGC5AA<3ACC5 G‘CGGCTACACCTTCACCAGGTAC ACC '.'I"GCA’.5.’15GG 5'I515AAC5CAGAGG .5.C‘-C'5’ .AGGC CT-5 .5.A.C5‘ATCA E ACCICC .C5C5'GGGGCPAC.”’5C5AAC.TA’5AAC-C.. GAACJ1 .CAAGCJAC.AAC 5C5CAC‘C5’5‘55CJAC.

CACCG‘ACAJAGAGCAGCAGCACCGCCTAC-ATGCAGCTGAGCAGCCTGACCAGCC3AGG AG'CGCCGTGTACTACTGCGCCAGGTACTACGACGACCACTACTGCCTGGACr51C i'GGG' 111........................_............... .......................................... GI- 1A..rI5IJI1ACAI” I.” I1T1ACCII55’51") I. ” ”‘ I 1CCC CA' 'CI1I1CI1I5I1AI5 I‘C .

CAGLC CCCCAGCCCCGCGTCwGACGACATCCAGC‘TJACCCAGAGC'CCCGCC5TCATG AGCGCCAC-,’C‘CC‘CGC5.1-CGAC‘AAGGTGACCATGACCTGC GG CCAGCAGCAGCGTGAGCT ACAT CTGI’1'13‘ACIAI5I‘1AI3AAI5AI1I‘I3I5I1AI1I‘ACCI1CI1AAI”AG““ .3GI.1A' ’CTT I‘1I1 AITAC CA 5I1AAGC33‘I5IJI. .AGC 5IJI-I1“I'CI‘CI”"‘AI‘AII53. ”’1"CAI~1I‘1I5I5I1A’1I‘ZGI5 AI1I'CI1CA “l”AI‘.‘A.I.-”r..CC..I‘iA .‘AI1I1AI5.A. :GAI5I5I . 1AI 5AI.I5I.I3I1CI1AI'1I‘1“.'AC'TAC“ ”GI‘. C. I5I1‘ AGTGGAGCAGCAACCCC -TGACCTTCGGCGCCGGCACCAAGC‘TC.5‘I.G:CC GAAG'CACC’A CCA‘TCATCACCACTGAG ....................-..................-....._.................._..........1......._............-....._......... SIRPlu-CDS ................................................................................................................ ATGGAGACC 5ATACCCCTC"1"1GTI. i ‘1 C .3 '3GG 5' - 'GCCAGGATCTACAG (LL) GTGATGAAGAAGAATTGCAGATCCLTCCAACCAGACAAATCCGTACTCGTGGCCGCAGG AGAGACCGCTACCCTACGATG CTTCT 3‘TC'3TCCCCGTTGGCCCCATCCAG '.‘“I.3I‘1I.1AGI5CI‘5‘I‘1AI;‘rI_-1ACI‘1A ..'“I”'.1311‘II_”.‘r : .“I'CAACGAITAGI5I5I.”CI‘.‘AT.1 ‘ 1AAI5‘AI1“ 3GACAA AGTA'l‘ I1AAI-5I4I1I.AA‘33A.A“"A“I'CIAI.TT“I'AI-‘ CAT TAGz-LATCCI“CAACATAACACCCCCTCACCCCCGTACA“3ACTATTGTATTAAATT ‘GA GGGCTCACC3AGA\GACGTI..GAATTTAAGTCA. G-GGCCGGAAC GAACTCTCAGTTA GAI3I‘AAAAI.I““I3TI"'13 5I.“I‘AI”CG 5I.I‘5‘I.‘~"I1I5II1I5I5I‘AI‘I”I‘AI.A.T"AAI5 I‘1“I3I.1C.AGCAI-1AGCI5G CI.” ”I45IA""""I1I5CI. 7/.” ”CCC 1"" 1C ..AGCC'“ ~1AAI‘5AT 5AI5I4 "‘AAGACCAGCGGC4“I‘AI.”.

AIC31‘A1AI'I“‘31‘JI-AI1“I'C 5I- GAAGCAGA 1GI‘.CI.”I5-I1CCAGI5'I5- I.“I'I.1GAI5 1" GGATCGGCTACATCAJ—ICCCCAGCAGGGGCTACACCAJ-LCTACA-ACCAI.—AAG'T'TCAAGGA CAAGGCCACCCTGACCACCGACAAG-.GCAGCAGCACCGCCT CATGCA.3CTGAGCAC—C ” ”GACCAGC4I‘5‘AI.1GAIZAG I1GCC5TG'I‘AC.‘TACTG I‘I.1 . ‘5‘GTAI""’A I‘1’1ACGACI‘1AI‘1TAI.”.“I.' I.-:ICTGGAI‘.‘13AI.”TI1 I” I” CAI-1 1' 3 3 ’ ' ‘ I5I1I5-I5I‘1AG I4I1I1I".. 1 51AI1I.I~CCI5I5I. GCIJI5I-I5I:I3..AI5I.”I5‘I5I. . -. .I ‘I1AI.'3 ..-1AI." T ‘1I‘.AI5I-.I1AI‘.CI.‘.AI1 AGCCCCGCCATCATGAGCGCCAGCCCCGGCGAC—AAGGTGAC ‘ATGACCTGCA'GGGCC‘A GC. GCAGCGTC‘AGCTACAT.GAACTCMGTA-CAGC‘AC.~ GA CGGC. 3CAGCCCC3AAGAG GTI3I1A I”.AI‘5‘I‘1GI1CI53’1’G I‘1I‘1I‘."I“AI." .‘5‘GTT .‘AGI‘1 vCAGC3x. 1/» r "‘15 . . L. .IJI- \..\. ..I-‘L I. I. CTG CI"AGCAG TIC-rI5AI- I. . . . . 1I‘1I3‘13I5AI- 5T GGA‘CCTCAI-XICCACCACACCA C'ACCACTAG .......................................................................-........................................................-................ FDLl—CDS ................................................................................................................................................ A‘TI‘I‘AI5'1‘“1"I.‘ 1I1C .'.3. I§AI1C1TI5I5I '“i‘ 1“"TC 5TI1I1TI1IC(”II'C' 'T'I1'CI"13I‘AI1 1G .1CI51I1C TGCG 49 -7\-CATCAAGCTGCAGCAGAGCGCCG C.”CGAGCTGGCC-AGGC 'CCGGCGC’CAGCGTG: aGA'T GAGCT<3C GC CCAGCGGCTACACCTTCACCAGGTACA3CATGC. CTGGGTGAAGCAG 'I1CC3I5GCI‘1AI5GI5CCTG.1AI5 GGATC.GGI‘.“ICAITA'.13IAAI""‘I”“.I1I‘1AI3 CACIZA AC3AI1'AACCAG I3“.““‘.3CAAI1'3 "AAIaIaCCAI”III-'1'I1AI1I”A” GA 1I7AI5I1AI1I1AI‘ I.”I1CI‘.'.13AI.”A'i'GCAG IT‘GAGC .I5 3GAC. GG ‘1 GI‘.‘.-1I.”I1I1“ .3G'I‘AI.I”PACT .-1C.I5I1I‘.

TACGA'CCCCACTACTGCCTGGACTACTGGGGC'CAGGGCACCACCCCT—ACCG TGAGCAGCGTGGAGC—GCGGCAG-CGGCGGC GCGGCGGCAGCGGCGGCAGC CCGGC2_71 GGAC 5ACA""CCCAGI.'LGACCCAGAGCI“ TCGCCATCA‘TGAG’TG I‘1’"‘CGI5I‘1C ' I.1313I5AC ‘17 ” .1I1 .3GI‘.i—.I5II5I1CC ' 5". Ij-AI1I3'13AI1A" TC AAC.‘ I5I1TA"I”AI5 AGAI‘ CI5I5I4AI‘.‘I‘1AI5I1I4I4CAAI5A'I5-I5TI1 .-1A".1‘I.'1AI‘1I.5AI”-AI4CAGCAAI1. 1“I'I.1GCI.”AI1C ‘ 1C. ' "' GCCLCTACAGGTTCAI.CJGCAGCGGCAGCGGCACCAGCTALACCC GACCATCAG'CAGC CCGAGGACGCCGCCACCTACTAC GCC GC. G-:C'GGAGCAGCAACCCC‘ CCTTC, ‘5I‘1I‘1.1chAI“I. I‘1AAGI”TGI‘5‘A n (“1 ’1‘GAAI‘I‘I3CGGI‘1I‘I‘"I5GI4AGI‘,.1“1’A“I3I“"AI‘A“J'.

.A" CAGAI.‘C I.”I-A“I"‘.I‘—I‘13I”"'CT . .. . ACT 1AI1I..ATTAI1AII:I‘.' AGAGCCT’CCCACAGA’CCGT‘TTCCACAGC GT 'G CTGGTATCA.C'CAAALAJ-ICCTGGTAAGG CGCLCCAA.GC“3TCTCATCTATTCAGC'CAG'i‘T' '1‘C'.'G‘i‘71"AGCG CGTT ‘CAGCCGA‘TT CTCTGGCTC".GGATCCGGCACGGAC'"TTAC“-‘T'"GACAAT'- _'CCTCTC"'TCAGCCCGAA GA“I.‘T'3-3313I5I“AAI1C‘313AI4'I‘AI1TI3 I1AA'TA" "‘I‘1“I3"'33AI1CA“I3""AGI.I‘1ACA’131‘CI‘5‘GAI7AI5G I1CAI.CA 73.. 5.3I1I1AAA‘13CAAA ‘ 3I-‘ " . .. . I:II I. I.-1"I5IJ'I5I5I5‘.““3.3CAI 5AGG GGGGTTC'C'GAAGTGCACACTLGTTCAAnGCCCI-ICC'AGG 5CTTGTCCAALCCTGGCGGG "CACTGC3GG“3TGAGCTGCGCCGCAAC—CC'GATTCAC3CT“3CTCAGACTCTTGGAT-CATT GGGTGCGCC. GGCTCCCG‘GAAAAGGCTTC—GAATGGGT’CGCT GGA‘T'TTCACCGT- ATG CGGTTCI‘ACATAC1‘AI1I5I‘1TI‘AAI5I‘1I3 “TA/15.1 TI.”.GA.“‘."I3CAI.‘I1..TI‘1TC'1GCAGATACTI.‘ AAAACACAGI‘ I3‘13A 1C I3“I"“I5A“I1AA'13AI51 1" I1A 5I5AI-AI‘I.‘I5I1I5I1 TT'13 . :1 A;“CTATTG~TGCCCGAAGACATTCGCCC 5GC'CGG‘TTTCGACTACTG GGGCZ-h-LGGTACGTT GGTC~ACTGTGAGCGCCCACCACCATCATCACCACTGA r------------- PIECE—CD3— ...................... :I 1I1C. .".”. G-AI1I‘1313I5I5 1“I'“.“‘C. 1TI1 1“I'I.1I.1I1I1‘I‘1“ .3G'T‘13 JAG 1G-1C. 5'1‘I1‘I1A.I.1‘13I5I.”I1 51 Fe CATCAAGCTI—LACAGAGCGGCGCCGAGCTCCCCACCCCCGGCGCLC 1GCGTLFHZ‘1GE‘T G GCTGC G C CCAGCGGCTAC..ACCTTCACCAGGTACAC'C'ATGCACTGGGTGAAGCAG AI‘5'GI1CC" I5GCI.AI.1I.1I5I4CTG ;1AI5’.1’I‘53I‘5‘ATI.1I5GI4“I'ACATCAAI‘1CC JAG I‘1A’.1-.1I5GI4TAI1AIJI7A. n1 IMGJI . I C.'C‘J'CC‘.‘1‘ACACI' JCAG .‘TC‘J' C3C‘.3 C5C‘C.’TC‘.AC ‘AGCC3AGCJAC-AC‘C‘5CCC“C‘Jr'C‘JTA‘CCI‘ACT .3C3C—5CC‘.

GGTACTACGAC""4.~.ATA1C'GCCTGGACTACTGGGGCCAGGGCAC'CACCCTGACCG TGAGCAGCGTCJ'IGGAIJGGCCJ‘GCAGCGGCGGC GC3GGCGGCAGCGGCGGCAGC GC3GG GT GGA". 5AC.‘.A"'I‘C‘.C‘1AGC.‘.’.ICJACCCAC‘ACJC‘C‘C‘C‘C‘CATC‘A‘I‘GAC‘C‘C‘ ICA.C‘J‘C‘1".C.‘.CC.‘. JC‘JCGAGAAC‘J GT 5AC1CA ACC I'GCAGC5C‘JCCAGCA' AGC‘ 5" CJA’3C‘TAC‘"'I'C3AAC‘I‘CJC‘J’I'AC3CAC 1 GA AGAGC‘C.GCJC.AC‘C1".AGCC.’.CCAAGACJG’I'C‘C‘APC.TACGA .AC1CA C-‘C.C‘.A.ACJ ‘J“I'GGCCAC‘J CGC.‘ CCJ'I' GCCLWTACAGGTTCAGCGGCAGCGGCAGCGGCALW'CAGCTAC SGCC GAC ’ATCAGCAGC C.C.'I'TC_‘GCJCCJC.C".TG’GAGGCCGAGGACGCCGCCACCTACTAC GCC GCAGT CAACCCCC GAJ3GCA".C.A‘AAGCTC‘JC‘J‘A - -C3GCGG ‘1C‘JC3C‘C5GCAGC‘. 5A‘I‘ATI'C‘1C3AGA'I‘ CTA C‘CC‘1A’I'CYA‘I‘C'I'C’3C3‘3-‘C‘T'C .AG’I‘C‘A I'. ..A‘I‘TAC.‘A'J-”3C ACl J - An J\ {- ‘1 nm uAFC.n - . .AAGAC.‘C"“I'“I"1‘C‘.CA . Klk LJC: “C L ‘ T“. C‘C. I" JG'J AACJCJ (“(1LJGCCCAAGCTTCTCATCTATTCAGC'ZGTTTTCTGTATAGCC JCGTTCCCAGCLGATT CTCTGGCTCTGGA CCGGCACGGAC3'I"I IAC‘-“I"I‘GAC‘-AA‘I’T‘I'CCI'C‘I’C‘I"I‘CAGC3 "'CGAA GA.'I‘T'TTCJC-AAC‘1.C."T C."I‘AC‘1'TG‘.“C‘ACJC‘AAl‘A’I’C‘1'.‘C‘.'T3 C‘C‘AG. 'I'I‘C‘LC‘J‘C‘2ACAC5C‘J ‘C‘AC-C.AAAC5’.‘C‘C‘JAAATC‘AAAAGA C3C‘IGGCCJ‘CJCA.G’ ‘C5"CJ C.C3 .3C‘. 5CJ'CJCJC‘S'f3'1‘C‘ACJ-CJAC3..‘ 3"GGGG TC“GAAIGTGCI-L—IC“"3CJTTCAAnGCC-CAJGAG'G J“C‘TGTCCAACCTGGCGIGG :‘C-‘ACTGCGGTTGAG- CTGC‘GCCCC-‘AAC'CCGATT"ACCT'TCTCA JACTCTTGGATCCATT' GGG’TGCGCCACJ‘GCTCCCGGAAAAGGCTTGGAATGGGT GCTTGGATTTCAC G. ATGG C.CJCJT'TC.‘C‘AC.A'I‘AC‘I‘AC.CJC‘1'TC‘A5A3C‘J‘C‘1.’.3’I“I‘AAC‘J‘C‘2I.TC‘C‘A’I‘TC‘AC‘".TC .‘TC‘G.‘AGATACT "“‘C‘WAAC‘AC‘ACJC‘C‘TA .‘C I'“I'C‘ACJATCJAATACJTT“'C3C‘3GCC 1 3C3AGC5AC.‘ C.‘AG CCJ‘C‘J‘CI'“I3'.I‘ ATTATTIGGCCCGAJJGACATTI—GCCGGCGGTTTCGZ‘JCTAC‘T"'G""‘CCAAGIT.-7\.C‘GTT GGTGACTGT JAGCGCCGTAGATCAAC'C1-.Z\.I-\ATTTGTGALZ‘JAAJ-XCC'CAT-ACC GCCCA CCATGCCCAGCCCCAGAACTTCTTGGCIG”ACC"TCTGTC‘TTCCTTTT'CCCTCCGAAJG CCAAGC‘A"‘AC‘CC."I‘CJA'I‘CJA'T.C.‘AGCCC‘AACICI". . -I A".‘G"C‘TI’3C3'I3G"'I‘C‘C3A'TG'I‘ TAGCC.‘ “I'-.AGCJA’I'C‘C““CJAAC3“I' C‘1AAATT“.'. .A...I3' .'A.TC53TAC‘J‘AC‘.‘5G‘TC I I'C‘CC5C AC‘.

AACCC.TAAJZ‘JACTAAGCCCAGGGAGCACCJaGTI-IZ—IACTCAJCCT JTCGCJTCGTATCTG 'I'GCI"I'ACCG I‘CCTGC’AI‘CAAGAC -‘GGC'-‘C'AATC—G'-‘.“A<3C‘JAA'-3.“.‘I'AAA’I'G'I'AA G‘I“'GAG TAACA‘AGGCACTGCC GCACCTATCGAAAAAAC‘CTA. CTCAA GGCGAAGGGACAGCCC AG AC‘C-C-C.ACG ‘C'I‘.T TAC‘I‘C.'I’C‘J‘C"‘AC‘C'I"‘ ‘1TCG 5GA'I‘C3AA’I I‘ AC‘C‘AACJAAC‘C‘AACJ 'I3‘1‘ACJ-C-C“I'C.‘ C‘A’"C‘J“I'C““C5CJ'I'CJA.AAC5CJTr .'“.""“AI‘C- C-AAG.C‘CJA'IA “ ‘JC3A .- AGC’I‘C‘J‘ TCGAC5’I‘CJ GTCCAATGGCCAACCTGAGAA’CAATT‘ATZ‘ JGACCAC-CCCACCL‘GTTC C‘GACAGCGI C GGATCCT'TTTTCCTGT‘ACTCAAJ-IACTCACT'GTCGA‘TAAATCAAGATCJGCAACAA GC’A AC‘Cr'l‘rI'T'I‘TAC‘ C""G'IAC3 C‘CJ'I'CJATC3 ‘J‘AAG C‘T"I”"A’I‘AA’"C‘AC"I‘ . . ACAC.AGAAGTC.

ACTCTC'.‘Cfi'I‘“I“'I3C.‘.TC.‘CAC‘C‘AC“ CCACC‘.A’..‘C 3“I'C".ACC.‘AC“ .‘C‘. 5mm a. TGGAAACCG‘ TACACTTCTGTTGTGGGTGCTGCTCCTGT. GGTCCC‘TLJCJTTC.AACAG 67 (rm—mm T’I‘A”"‘1C.C‘1'TACC‘A’I""“‘ ‘C‘C-C.CJA."TACJC.A."C‘C‘G.‘TCAG ICAC‘J‘CTG "IC‘JA'I‘A'TCCA Fe. (51.) C‘AI‘JACACAGAC‘JC‘1C C‘ATC‘ATCTCT’. ‘C5'I C“.' 3C‘3AACJ' C.’ “.I AGC5AC‘JACCGAJ'ICAC.CAI‘TACA TGCAGAIGCCTCC‘CAAGACGTTTCACAICAGTGGCCTGGTATCAGCAJ—XAZ‘EJCCTGGTA CC7 GJCTTCTCATCTATTCAG CACTTTT’3TGTAIACCGGCG CCCAGCCG ATTCTCTGGCTCTGGATCCGC~C CGGA 3TTTACTTTGAC3AA3-TTTCCTCTCTTCAGCCC GAA ATT. '.‘C‘J CAACCICI‘A C‘TA ..C. J.J «r. CA.«A -‘ " . G “..'C".AGC.‘AA“I‘A 3C“C C-‘JC 'I‘C‘C AGG ACCAAAC‘J“'CGAAA AAA. GA 5C3C‘15C5Cr GCGGCAC \/\ 33:7*1 ':k C ‘5 ‘3C-3GC5’I AGG'CGGI'G'3JGGTTCTI'G'3JAAGTGC: 7‘JCTCG'I‘TC.—AAAGC‘JC.G ITJGGAGGGCTTGTC'CAACCTGIGC GGGTCACTGCC‘JGTTGAGC-"TG‘CGCCCJGAAGCGGATTCACCTTCTCAC‘JACTCTTGGATCC TGCGCCAGGC “TC‘J'C‘J'C‘..C“I"1‘CC.A... I‘AC.’I'A.C.‘C‘C"’C‘J'A C‘AC‘ C‘G’"' 'AAC‘rGG’I'CC‘AT‘ ‘C‘A C‘1C‘1A'TC.‘..I‘C'.‘GC‘.AC" .T ACT.13C-AA.AAAAC-ACAC.3C‘C""AC CT’.‘‘ACJA'I‘GAA‘3A’3‘f3'3"I‘CJC-CJCC‘.C‘.C.C5ACJG. .C‘ACAC‘JC ATTGTGCCCTAAGACATTGGCCCGG 'GGTTT 3G. -C‘ ACTGGGC‘J'J'C‘IAGGTAC GTTCJGTGAC GTGA C“GCCGT..GATGAAGCAAAATCTTIGTGAC‘AA CCCATACC GC CC.“ C-CAT’SC."C_‘C‘ACJC‘"’_“C.‘ACJAA."T’.‘C"“T’" 5‘1‘A“ CCC'. C.“.I‘J'C‘TTC‘C‘" C‘C‘C‘I‘C‘"C‘1C‘1A AGCC‘. C.AAGC‘JATAC. ..C“'I‘C‘J‘A'TG‘ ATCAGC‘...C‘GAA'C1 C1C‘.C‘CC‘AC‘J‘GTAACIA.’ .‘GTG'IGC‘J .‘C‘ C‘ TCGA TC“I3“ACJC“C“A3.I‘CGAGGATC. ""CJAAC."I‘C‘AAA.'1'“‘.".‘‘AAC. "C3C5TA'3‘CI'ACACCJC‘J‘CI'C3'I‘TCJ'ACJC‘53'I3G CACAACGCTAA-AACTAAGI CCCAGG'GAGGA 'CA. JTALAA'TC: JCCTATCGCG‘ CGTAT CTGTGCTTACCGT '"TGCATC'3.1-\<.JAIC‘TGG"“CAA‘1GGTAAGGI-zZ-ITATAAAIr'GTAA."G-T AAC‘AAGCJCACTC‘C‘C‘ACJC‘A’3 ' . . . —AAAAAAC-C‘ A‘.‘C‘T‘CAAAGGC 3AAGC5GACAG .AGGGAACC GG‘I‘C'T TG . CC‘T'T. I‘GAACI3TGAC1CAA AAC .' AAGT GCCTTCGA A'Z“‘G3I‘CCI" 3C3' .; 3 . .~ .‘C’I‘ATC‘ICTAAC .‘1‘A’I‘ACJC ETC ‘“I'G GGAGTCCAZ‘JTGGCCAACCT'JAGAACAJAITTATPLAGAC'CACC CACCCGTTCTGG:.CAIGC GACGGATCCTTTTTCCTI-—TACT. CAAAACTCACTGTCGATAAA'TCAAGATGGCAACAAG ""333. C3'I3T‘T3I‘IACJC‘1TC313ACJCC‘T .3A‘1‘GC.AC‘1 5AAG".‘AC‘1T3‘ .‘AT‘AATCAC'I‘ATACACAGAA Amrc “I'C“1:’I“I3C' (“a WHICTT - «WA J\ C n ‘A7 A C’I 1...1‘311A5C:A’1C1AAC5AC:.-ICAAICi-GC.‘7.-I.(.-1 JCi-C1AC1CC’.1’1‘C.“3C1.A.’1.11‘L1C1’1C1’1C5AT 1“I'C1GAC5C1: AAAATCCGGGGCCTATGGAGACC JATACCC GCTC‘i‘TGTGGG' .TTr'r—w ..:nr1 l1 (“In-y.

JC. IC- A \JUI GCCAGGATCTACAGGTG1 T'I3AAGAAGA TTGCAGA"1. C. TCC CCAGACAAATCCGTA CT1’1TGGCCC5CACrL5AC5ACAC1C1GC'I‘A’1C1C111’CAC-A'1I1C C1A'1I1C.1.AC“I"I"1'11C113C“"I‘C1.C.1,C.1.C_1.C15 C1’11‘311C.1CAC1“I“‘15".11'I3“I3C1(~1A15C5C :C1AC-1‘ . (1C111'11A“I'""11AC1AA“I'C1.1‘ A;.' 1A.A.C5CA A CJCCATJC -5AC1“.C"1:AC , ‘1‘AC.1C.'AC.‘CAAC-C.C:C‘A.A‘11AA'" ATGGACTTTAGCATTAGAATCGGCAAC AACAC CCCT—ACGCCGGTAC.IAA1'CTATT.

CTATAAATTTCGAAAGGGCTCACCAGACGACG’TGCAATTTAAGTC'.1.G(.-.._5GCC‘GGAAI CG, 1x.- ‘T . AGAGCAA11.C.C 1GC.’“I‘A 5AC.‘A“I" AGC“.\."-1CAC1.CAGA 1C5C.

(- J 5GGCC_1.C.1C5C5C1G AGC" GCAAGA CA C1\ (C “TCAC C.AC1C1311AC1AC.C.'.-I.3,11C5C.1A 3C1C1C5’l C1C1IAC5AC:C:C1C1CC5I C..-I.(1.C 5CJ1C1“.C3’(1AC-511C1: GATCGG'CTACATCAACCCCAGCAC1.5GGG' 'TACACICI-I.A.CCCTAAACCAGE" -IGGE‘C4 AAGGCCACCCTGACCACCGACAAGAGCAGC. GCACCGCCTAC TCC‘.1.GCTGAC5Cz-\(.5CC 111C51AC1, ’.1AL5CC5AC1- rAC1AGC1C1-CCC51111TACTAC‘ I1C15‘C1C1 ’1 IA(1 15"I‘AC‘111ACLC15‘AC 11CCACTA TTG C1C311C5-C:AC1“ 1 C..‘.1L1: 1C1(.1’1C.1.AC1: 1C1C1AC .1AC'C1C1311C5ACC 1-: GAC5C.‘ C1C'C1311CJC5AGCC1C1 GC GGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACG.IACATJ CCAGCTGACCCLAA GC CCGCCATCATGAGCGCCAGCC C1CGGCGAGZ‘ aGGT- '.5A'CCATGACCTGCAGGGCCAG C GCAGCGTGAGCTACATGAACTGGT .1CACC‘.1.GAAGAC5C GCACCA-GCCCCAA-GAG 'LC5C5ATC1111ACC5ACC1AC5 CAAL5GTG’1C1C1AGC5‘C1-C1C1'111GCCCTAGG'I‘TCAGJGC- ’1 1 1:- C1C1A'C:C C1C1AC:C1“.“‘AC1AL:CTGACCA“,'C1AC5C.1AC:CA.'1.1C5CJA1GC1CC5AICC:AC1C1C1 5.: .

CTACTACTGCCIAGCAGTGGAGCAGCAJACCC 'CTGACICTCCGGCGCCGGCACCAAGCTG GAGCTGAAGCACCACCATCATCACCACTGA SIRE-‘10:— ATGC1AAACSI'Cg1A‘11ACAC I3“I"13115T? 1“'C-1 1 C".16 C1“ ‘C1CJC5“I'C.' C1C1311C.1IC5“I'“I3C1.A.AC1AL: J J1C1 C1“I'-. 69 CD3—PDL1- GCGATI TATCCCTACGATGTG 'CC A\ECAGGCGCTCAGCCAGCTGATGATATC’C'.IA FE (LL) GATGA.CAC G‘AGCCCATCATCTC GT “1GCAAGCGT GGAGACCG1 GTCACCATT'ACA .11GCACrAuCCTCCC1AAC5AC1C1311FTCCACAC5C."A.G“I‘I’1C;1 JCTGGTA1CAGCAAAAACCTGC." ' AC5CCL: CC1 '1AAC C“I.'“I3C1.31-1C.1A'I‘ C.‘“I'A'1-1311C.1AC1 1C1A15 “111111“I3’1'11CJ11A" T (1CC5C5'C11'I11‘C AT“ 'C1'1 1'11C5GC.'“I"1311CJL:AI3 1CC5C5C C'I‘TTA.’131.1".1'I‘GAC. . . . 1‘“I'3CC1“I‘C1.'11C.1I.

G.IAAGZ-‘3TTTTGCAACCTAC1- ITACTGTCAGCAATATJ C‘ C--T'ACCATC AGCCACA“1T1.GGI-IC AGG’GCACCAAAGTCGAAATCAAAAGIAGGC CC5C3GGCGGCAGTGGCGGCG<3GGGT'T'CAGG AC5GC 1C“5 59(11'I‘“I‘.’.1,'115AAGTC1C1AA 1TGAAAC AGGAGC5GC.‘‘“I"1'.1"C.1CAAC1’1'11C161C (1C1C5'l 1A C'TGC.C5C1: “I3“I'(17-. 5C1TC1CC1’1C.1.C5 AGC1. 5C5AT“."1.1C1 .1’11'I3C1“I"1AC5AC.“113.113.1 “I'CC AITTGGGTGCGCCAG'11C—C’1CC CI'C.'11—GAA./-I.A.GGCTTGGAATGG'GTTGCTTGGIATTIJITCAC’1CGTA TG :CGG' TCCACATACC1 CAGCGTTAAGGGTCI’C— 1 ITTCACCAT 3TC“.ICCm.-CAGAT CTTCAAAAAACACAGCC3TACCT“1C‘.1.(3ATGAAT1GTTTGC‘ GGACACA-GCG +3 ’1" 1CCC5C5CC I 'AT311A'111“I'15 IC1C1 JCTAAGACAI. '.'CGAC 1 "AC“ .‘G’1 1GGC.‘AA’1 1T. .C I?)‘.1-.131C5C1:“I3C1.A.C3.11C5"JGAGCC51-C1“I'.A.C;‘A‘.1 GAAC1 ’1 “..'C131-.1311C5'I L15AC.1AAAAC’ '1C1A3..1AC.1 mn mCACCATGCCCACCCCCI—ICAACTTCTTGGCGTACCCTCTGTCTTCCTTTTCCCTC GA CICAA.GGATACCCT- .5: TG'ATCAG'CGA_IACC'CCGGAC1.5GTZ‘sZ-‘ICIA1I'GTGTGG GGT ‘GIA TGTTAGCCATGAGGATCC3T<3AAG“.1C3A}.ATTTAIAC.1GGTATGTAGAC3GGTGTTG.1_GI3TG CACAACGCfI’AAAAC'.AAG’JC 3AC5GCA5GAC5CACT’.’/AACTICQ1.CC1“I‘ATC.1.G'G“I"1C1TAT -1 5TC1: CTT/AC1 C11“I3C1’1‘11CJC1A“I3C1A.ACJA . 1C’11C.1AA“ 1C 1311AAC1GAA3. 1AI‘AAATG’.11 1 \.:..

GAGTAACAAGGCACTGCCIAGI CACCTATCGAAELAJ-‘IACICATCT C .5AAGGG.IACAG CCCAGGG-1_A_CCCCAGG“1CTATACTCTGC3AA-CCTTCTCGGGATGAATTGACCAAC—AACC AAGTTAGCCTGA.CATGTC‘TGGTGAAAGGTTTCTA-TCCAAGCG TATAGCTGTCGAGTG GC5'AI’1'1I1C1C5AATC15G C1C1AAC1C1TC1ACAAC1AA‘1111'1ATAAC1 CCAC1’1’1C1ACCC111.T'11C'I'C1C15ACAC5C.‘ - A .C: 1’131‘11'13“I3“I"1’1‘.1CJ'I' C1““"1AAA.AC'I‘CAC1'11L1“."1C1AFAAA'I'CAACj-ATGGCAAC. AG GI‘CAACG TTTTAGCT "1.1-I.GCI'—TGATGCA'. GAAGC: 'CTTCAT:1‘aITCI-ICTATZ‘ICI-ICAGAA GTCACT 3TCTCTTTCTCCAGG.AJAAG5CGTTGACIGAACAGAAATTGA.TATCCGAGIC.5 T AA.TAI.’;1C;1AC5C15AAI.’;1AGAGAA 1AC5 C1 (1“ 1’11 ‘TC;1CGG CC5A'TG‘1.1C.1C5AGG AAAA'. .1.C.1.GGC5" . . “I."GGAG 11C . 1'I‘I.’ . .“I‘T '11PCTTT (1’1C1 "AC1GA':""1311 Cv:Cv:‘.1-1C5A“I‘GA G 5 EAGA.'I‘CA".11 AC1C1 ACAAAT CTCGTGCCCGCAGGAGA5ACCGCTPICCC-TCACATGTACCATCACTTCT CCCG C‘CATCCJCC5“-:TGGTTTCGAGGCGCAGI3ACCAGGACG G-=TGCTTATTTACAATC’A ACGACAGIC ~1..1C.1.CA“I3T’.1C.1C1AA1AG’115AC1AACAGT1.“I'CC 1A1'1AC3’1AC1C1AAC1C1C1AAJ'J—IJ-I.I. ’1 ’1 AGAATC1GC5CAAC1A AAC1A’1C1CG C1." "1AC1.GCC1C1-’.1TA .TACTA‘FI.‘ (1‘1-1AI I‘AAAT‘- ‘.1 CC1AAAC5C5C1: C.'“I'C1.AC.C1 AC1.7-I..’1.;1AC.1C5' If 1.1AA’l 'I I‘.-I.-‘I.7.A.G311C.1AC1: 1GGC1C.1L5L:AAC ICGIAAJCTCTI AG--TCJTA—AGC.IAA:AA'CCTTC CCTI—ICCGCCGCCJCCG'CAGCGACA'TCAAG CTGCAGCAGAGCGGCGCCGAGCTGGCCAGGCCCGuCGCCAGCGTGAAGATGAGCTGCA AG1 C1CAGC ;1GCTA C.1‘.1'I1CA’1’1 1 GCTACACC11TC ’1AC‘11GGC1TC1AAGAI..AC1C5CCL1’11 (x (I C1 (I 11/1. 1‘ ..ATnC1 - “I' A n - 2017/040354 CAGAAGTTCAAGGAC'TGGCJACCCTGACCACCGAC'fGAGCAGCAGCACCGCCTACA TFCAGCTGAGCAGCCTJACCAGCGAGGACAGCGC'GTGTACTACTGCGCCAGGTACTA CGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGAC AGC (lift3t}ik(}(}(§(it3{3t3it€§<i£§tl(I 3(3 IE3£§£T£7’}(3(§(§(TZ%{3(3(3(§(3(3Cl(31l(3(3(§(3(Yt3f7" 3i?(3(32l(3 l TCCAGCTGACCCAL (1 v - . 111 m , U .TGAGC TAGCCCQ \.. . :lfzznk- CAGlGCCA;\ TGCAGCGVGAGCEACATGAAuT"‘TACCTGCAGAAGAGCGGC lACCAGCCCCAAGAGGTGGATCTACGA‘ACCAGCAAGGTGLCCAGCGGCGTGCCCTACA AGC'GCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGC costar-inc y‘CCGPf”ACC’I‘ACTACTGCGAGCAG‘TQGAGCAGCA' . CCCCC'I'GACC'I‘TCGGC _______________________________ikkt‘uAA‘kN“(JMWZ‘JNALAUALHG {002253 Additional exemplarly embodiments of r molecules include engager molecules sing an activation domain comprising an anti—CD3 scEv (erg comprised of SEQ ll) N05: 20 and 22) and a therapeutic domain comprising an scFv that binds to a cell surface protein such as CTLAd, TIME, LAG3, BTLA, 1(le TlGlT, 0X40, or GlTR. In some embodiments, the oncclytic Viruses bed herein comprise a bicistronic or multicistronic nucleic acid sequence, WllCl’fle’l a first nucleic acid sequence encodes an engage-r les sing an activation domain comprising an anti~CD3 scFv (cg, comprised of SEQ ll) NOs: and 22) and a therapeutic domain comprising an scliv that binds to a cell surface protein such as , ’l'lM3, LAG'Sl B'l‘LA, KlR, 'l‘lGl’l", 0X40, CD47, or Gl'l'R, and a second nucleic acid sequence encoding a therapeutic molecule such as llx‘lfi (SEQ ll) NO: 24), Ill-l 2 (SEQ ll) NOs: 26 and 28), CXCLlO (SEQ ll) NO: 30}, or Nib/1P9 (SEQ ll) NO: 34). in such embodiments, the engager molecule is linked to the therapeutic molecule polypeptide by a TBA self~cleaving peptide linker {SEQ ll) NO: l4). {30226} Additional exemplarly embodiments of engager molecules include engager molecules comprising an activation domain comprising an anti—CD3 scFv (cg: comprised of SEQ ll) bills: 20 and 22) and an antigen recognition domain comprising an scEV that, binds to SLAMF?’ (also known as CD319) or CD27 (either the membrane bound form of (5027 or the soluble form ofCDZ7). in some embodiments, the oncolytic Viruses described herein comprise a hicistronic or multicistronic nucleic acid sequence, n a first nucleic acid sequence encodes an engager les comprising an activation domain comprising an anti—CD3 scEv (cg, comprised of SEQ ID NOs: 20 and 22) and an antigenu‘ecognition domain comprising an scliv that binds to a target cell antigen such as SLAMr? or CD27, and a second nucleic acid sequence encoding a therapeutic molecule such as lL~l5 (SEQ ll) NO: 24), lL~l2 (SEQ ll) NOs: 26 and 2%), CXCLll) (SEQ ll) N0: 30), or MMP9 (SEQ ll) N0: 34). ln such embodiments, the engager le is linked to the therapeutic molecule polypeptide by a TZA self—cleaving peptide linker (SEQ ID NO: 14). {00227} Additional eeii surface proteins that are suitable for target by the engager molecules described herein are shown beiow in Table 5. Additionai proteins that are suitahie for use as therapeutic nio1ecuies are Show heiow in Table 6.

Table 5: Cell—surface proteins ie for targeting by en gage-r moieeuies human NKGDZL NP 1 h—unianC'iI'LAé-_umanTIMB N—P005205.7N—1161713 human 11th3 NP 002277.4 humanBiIA (isofonn 13nd7 NF 0010125296 1 14-45,.3 ms teetiVeiv) hm — human TIGIT NP 2 human 0X40 NP 0033 1 8.1 human G1TR(isofi)rm 1., 2, 3 NP___004186.1; 83699.1; NP 683700 1 human (D27 NP 001233 1 human CD40 mis 1~5. respectiveiy) NP_001.241 1;NP_690593 1; NP___001289682.1; NP “001314193501 NP 00130935211 human NKGDZZL NP 079494.} human CD200 NP 005935.41 Tahie 6: Proteins suitable for use as therapeutic moieeuies human T‘Nth NP (1005852 human CXBCLl NP 0029871 human CCR4 NP 0054991 human CSF—l NP 000748.13 human TGFi NP 0006513 human 11.1""7 NP 000871.}, human GM—CSF NP 0007492 Therapeutic Uses of Oneoiytie Viruses {00228} In some embodiments; the present invention provides compositions and methods of use for the tion, treatment, and/or anieiioration of a eaheerous disease. in some embodiments. the methods described herein se administering an ei‘ieetive amount (eg. a therapeutieaiiy effective amount} of an onooiytie virus described herein to a subject in need thereof, wherein the virus expresses an r moieeuie or an engager moieeuie and a dierapeutie molecuie. {@229} In some embodiemnts, compositions and methods of the present invention are useful for all stages and types of cancer, including for minimal residual disease, early solid tumor, advanced solid tumor and/or metastatic solid tumor. in some embodiments, compositions and methods of the present invention are used to treat a variety of solid tumors associated with a number of different cancers. The term "solid tumors" refers to relapsed or refractory tumors as well as metastases ver locamd), other than atses observed in lymphatic cancer. {$9233} Exemplarly solid tumors include, but are not limited to, brain and other central nervous system tumors (cg. tumors of the meninges, brain, spinal cord, l nerves and other parts of l nervous , eg gliohlastomas or medulla hlastomas); head and/or neck cancer, breast tumors; atory system tumors (cg heart, mediastinum and , and other intratlioraeic , vascular tumors and turnor~associated vascular tissue); excretory system tumors (eg. kidney, renal pelvis, ureter, bladder, other and unspecified urinary ), gastrointestinal tract tumors (cg. oesophagus, stomach, small intestine, colon, colorectal, reetosigmoid junction, , anus and anal canal), tumors involving the liver and intrahepatic bile ducts, gall r, other and unspecified parts of biliary tract, pancreas, other and digestive organs), head and neck, oral cavity (lip, tongue, gum, floor of mouth, palate, and other parts of mouth, parotid gland, and other parts of the ry glands, tonsil, oropharynx, nasopharynx, pyrifonn sinus, liypopharynx, and other sites in the lip, oral cavity and, pharynx); reproductive system tumors (cg. vulva, vagina, Cervix uteri, Corpus uteri, uterus, ovary, and other sites associated with female genital organs, placenta, penis, prostate, testis, and other sites associated with male genital organs); respiratory tract tumors (cg. nasal cavity and middle ear, accessory s, , trachea, bronchus and lung, cg. small cell lung cancer or non~small cell lung cancer); al system tumors (cg. bone and articular cartilage ot‘limhs, bone articular cartilage and other ; skin tumors (cg: malignant melanoma of the shin, non—melanoma skin cancer, basal cell. carcinoma of skin, squamous cell carcinoma of skin, niesothelioma, 's sarcoma); and tumors involving other tissues including peripheral nerves and autonomic nervous system, connective and soft tissue, retroperitoneum and peritoneum, eye and adnexa, thyroid, l gland and other endocrine glands and related structures, secondary and unspecified malignant neoplasm of lymph nodes, secondary malignant neoplasm of respiratory and digestive systems and secondary malignant neoplasm of other sites, oligodendroglioma, oligoastrocytoma, astrocytoma, glioblastoma or medullohlastoma or other solid tumor. {(116231} in particular embodiments, the solid tumor is a brain tumor. in some instances, the brain tumor includes, but is not limited to, a glioma, in particular ependymoma, oligodendroglioma, oligoastrocytoma, astrocytoma, gliohlastoma, or a medullohlastoma. {@232} in some embodiments, compositions and methods of the present invention are used to treat a hematologic . The term “hematologic cancer” refers herein to a cancer of the blood system and includes relapsed or refractory hematologic cancer as well as a asized hematologic cancer {\s'lierever located). ln some instances, the hematologic cancer is a T—cell malignancy or a B—cell malignancy. Exemplary T—cell malignancies include, but are not limited to, peripheral ”f-cell lymphoma not otherwise specified (P'l‘CL-NOS), anaplastic large cell ma, angioimmunohlastic lymphoma, cutaneous T—cell lymphoma, adult T—cell leukemia/lymphoma (ATLL), hlastic Nchell lymphoma, enteropathy—type T—cell lymphoma, hematosplenic gamma—delta T-cell ma, lymphoblastic lymphoma, nasal NK/T-cell lymphomas, or treatment-related ”ll—cell lymphomas.

} Exemplary B-cell malignancies include, but are not limited to, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high risk CLL, a non— CLL/SLL lymphoma, prolymphocy/tic leukemia (PLL), follicular lymphoma (FL), diffuse large B~cell lymphoma (DLBCL), mantle cell lymphoma (MEL), W'aldcnstromk macroglohulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell ma, Burkitt’s lymphoma, non—Burkitt high grade B cell lymphoma, primary rnediastinal Bncell ma (PMBL), immunohlastic large cell ma, precursor Bwlymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma. cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell ma, primary effusion lymphoma, or lymphomatoid granulomatosis. in some cases, the hematologic cancer is a relapsed or reli‘actory hematologic cancer. In some cases, the hematologic cancer is a asized hematologic cancer. {00234} In some embodiments, the oncolytic virus is engineered to e a high level of expression of the engager molecule and/or the therapeutic ptide prior to the death ofthe virally~infected cell, eg, within l, 2. 3, I-‘l, 5., 6, 7', 8, 9, ll), ll, l2, l3, l4, l5, 16, l7, l8, l 9, 20, Zl, 22, 23 or 24 hours of infection, or Within 2, 3, 4, 5, or 6 days of infection, Expression of the engager molecule and/or the therapeutic polypeptide can be ined by methods known in the art, including Western blot, ELISA, precipitation, or electrophoresis, among others. ln l, a “high level of expression” in reference to a therapeutic molecule refers to a. level of expression that is greater than the basal level of expression of a corresponding polypeptide in a cell that is not infected with the oncolytic virus sitions and routes Qfodministmtizm {@235} in some embodiments, a therapeutically ive amount of an oncolytic virus or compositions thereof are administered to a subjectln ance with this disclosure, the term "pharmaceutical composition” relates to a composition for administration to an individual.

Administration of the compositions described herein can be local or ic and can be effected by different ways, eg, by intravenous, subcutaneous, intraperitoneal, intramuscular, topical or intradermal administration. in some embodiments, compositions disclosed herein are administered by any means known in the art. For example, the compositions described herein may be administered to a. subject intravenously, intratumorally, intradermally, intraarterially, intraperitoneally, esionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intrathecally, subcutaneously, subconjunctival, intrayesicularlly, mucosally, ericardially, intraumbilically, intraocularly, orally, locally, by inhalation, by ion, by infusion, by continuous infusion, by localized perfusion, via a catheter, Via a lavage, in a cream, or in a lipid composition ln particular embodiments, the composition is stered to the individual via. infusion or injection. in some embodiments, administration is parenteral, e.g, intravenous. in some embodiments, the oncolytic virus or composition thereof is administered directly to the target site, 60:, by tic ry to an al or external target site or by catheter to a site in an arteiy. in particular embodiments, the compositions described herein are administered subcutaneously or intravenously. in some embodiments, the oncolytic viiuses or compositions thereof described herein are administered intravenously or intraarteiially. {00236} in a preferred embodiment, the compositions described herein are formulated for a particular route of administration, for parenteral, transdermal, intraluminal, ultra-arterial, intrathecal, intravenous administration, or for direct injection into a cancer. In some embodiments, the itions tuither comprise a pharmaceutically acceptable carrier.

”Phannaceutically or pharmacologically acceptable" refer herein to molecular entities and compositions that do not e an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate, in some embodiments, the pharmaceutical compositions of the present disclosure r comprise a pharmaceutically acceptable carrier. A "phannaceutically acceptable carrier" includes any and all solvents, dispersion media, gs, buffer, stabilizing formulation, cterial and ngal agents, isotonic and tion delaying agents and the like. Examples ot‘suitable ceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions, etc. Compositions comprising such carriers are formulated by well—lrnown conventional methods. in some embodiments, Supplementary active ingredients are also incorporated into the itions. For human administration, the compositions described herein are met with sterility, pyrogenicity, and general safety and purity standards as ed by FDA Qilice ofBiologics standards. in some embodiments, the compositions described herein comprise a carrier such as a solvent or dispersion medium containing, for example, water, ethanol, polyol {for example, glycerol, propylene , and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity is maintained, for example, by the use of a. coating, such as lecithin, by the maintenance of the required le size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms is brought about by various antibacterial and antifungal agents known in the art. in many cases, it is preferable to e isotonic agents, for e, sugars or sodium chloride. in some embodiments, prolonged absorption of the inj ectable compositions is brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin [(30238] In some embodiments, the oncolytic viruses described herein are formulated into a composition in a neutral or salt form. ceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free yl groups are derived from inorganic bases such as, for example, , potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. {00239} Pharmaceutical forms le for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or sions. in some cases, the form is sterile and is fluid. in some cases, it is stable under the conditions of manufacture and certain storage ters (cg. refrigeration and freezing) and is preserved against the contaminating action of microorganisms, such as bacteria and fungi.

Aqueous compositions of some ments herein include an effective amount of a virus, nucleic acid, therapeutic protein, peptide, uct, stimulator, tor, and the like, dissolved or dispersed in a pharmaceutically acceptable r or aqueous medium. Aqueous compositions of vectors expressing any of the foregoing are also contemplated, [9024M in certain embodiments, biological material is extensively dialyzed to remove undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle, where appropriate. ln some embodiments, the active compounds or constructs are thrmulated for parenteral administration, eg, formulated for injection via the enous, intramuscular, sub—cutaneous, intralesional, intranasal or eritoneal routes. Any route used for vaccination or boost of a subject is used, The ation of an aqueous composition that contains an active component or ingredient is known to those of shill in the art in light of the present disclosure. lly, such compositions are ed as injectables, either as liquid solutions or suspensions; solid forms suitable for use in preparing solutions or sions upon the addition of a liquid prior to injection is also prepared; and the preparations are also emulsified. {tlfi24ll in some instances, the oncolytic virus is dispersed in a pharmaceutically able formulation. for injection, ln some embodiments, sterile injeetable solutions are prepared by incorporating the active compounds or constructs in the required amount in the appropriate solvent with any of the other ingredients enumerated above, as required, ed by filtered sterilization. [@0242] Upon formulation, the compositions described herein are administered in a manner compatible with disease to he treated and the dosage formulation and in such amount as is eutically ive, The formulations are easily administered in a variety of dosage forms, such as the type of iniefitable solutions described above, but also as slow release capsules or microparticles and microspheres and the li e. {00243E For parenteral administration in an aqueous solution, for example, the on is suitably buttered if ary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intratumorally, intramuscular, subcutaneous and intraperitoneal administration. In this context, sterile aqueous media that is ed is known to those of skill in the art in light of the present disclosure. For example, one dosage is dissolved in 1 rule of isotonic NaCl solution and either added to lOOO ml; of hypoderniolysis fluid or injected at the proposed site of infusion, {00244} in addition to the compounds formulated for parenteral administration, such as intravenous, intraturnorally, intradermal or intramuscular injection, other pharmaceutically acceptable forms include, eg. tablets or other solids for oral administration; liposomal ations; time release capsules, biodegradable and any other form currently use . {96245} In some embodiments, the viruses are encapsulated to inhibit immune recognition and placed at the site of a tumor. in some instances, ations for parenteral administration include sterile s or non—aqueous solutions, suspensions, and emulsions. Examples of non-aqueous sol 'ents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectahle organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered inedia. Parenteral es include sodium chloride solution, Ringer‘s dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. intravenous es include fluid and nutrient replenishes, electrolyte replenisliers {such as those based on Ringer‘s dextrose}, and the like. Preservatives and other additives are also present such as, for example, anti inicrohials, anti—oxidants, chelating agents, and inert gases and the like. in addition, the pharmaceutical composition of the t disclosure might comprise proteinaeeous rs, like, cg, serum albumin or irnmunoglohulin, preferably of human origin. it is envisaged that the pharmaceutical composition of the sure might comprise, in on to the proteinaceous hispecific single chain antibody ucts or c acid molecules or vectors encoding the same (as bed in this disclosure), further biologically active agents, depending on the intended use of the ceutical composition. @9247} in some embodiments, tumor—infiltrating virus~producing cells which continuously release s are formulated. for direct implantation into a tumor in order to se the viral oncolysis and the transfer efficiency of the therapeutic genes. lntranasal formulations are known in the art and are described in, for example, US. Patent Nos. 4,476,ll6, 5,ll6,8l7, and 6,39l,452, Formulations which are prepared according to these and other techniques wellshnown in the art are prepared as solutions in saline, employing henzyl alcohol or other suitable preservatives, lluorocarhons, and/or other solubilizing or dispersing agents known in the art. See, for example, Ansel, H. C. et al, Pharmaceutical Dosage Forms and Drug Delivery Systems, Sixth Ed. (1995). Preferably these compositions and formulations are prepared with suitable nontoxic phannaceutically acceptable ingredients. These ingredients are known to those skilled in the preparation of nasal dosage forms and. some of these are found in Remington: The Science and Practice of Pharmacy, 2i. st edition, 2005, a. standard reference in the field. The choice of suitable rs is highly dependent upon the exact nature of the nasal dosage form d, cg, solutions, suspensions, ointments, or gels.

Nasal dosage forms generally contain large amounts of water in addition to the active ingredient.

Minor amounts of other ingredients such as pH adjusters, emulsifiers or sing agents, preservatives, surfactants, gelling agents or ing and other izing and solubilizing agents are also t. The nasal dosage form is isotonic with nasal secretions. {$6249} For administration by inhalation, described herein is in a. form as an aerosol, a. mist or a. powder. Pharmaceutical compositions described herein are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nehulizer, with the use of a suitable propellant, cg, rodifluoromethane, trichlorotiuoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas in the case of a pressurized mrosol, the dosage unit is determined by providing a valve to deliver a metered amount.

Capsules and dges of, such as, by way of example only, gelatin for use in an inhaler or insuft‘lator is ated containing a powder mix of the compound described herein and a suitable powder base such as lactose or starch.

Therapeutically ive/lmriums and Ti:erapemic Regimens {06259} In some ments, the oncolytic viruses and compositions thereof described herein are stered to a subject at therapeutically effective amount. The therapeutically effective amount will. depend on the subject to be treated, the state (eg, general health) ofthe subject, the protection d, the disease to be treated, the route of administration, and/or the nature of the virus. in some embodiments, the person responsible for administration (eg, an attending; physician) will determine the appropriate dose for an individual. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, weight, body surtace area, age, sex, and l health, the particular compound to be administered, the particular disease to be treated, timing and route of administration, and other drugs being administered concun‘ently Therefore, it is expected that for each individual patient, even if the viruses that are administered to the population at large. each patient is monitored for the proper dosage for the individual, and such practices of monitoring a patient are routine in the art. {titllfil} in some embodiments, the therapeutically effective amount of an oncolytic virus described herein is administered in a. single dose, In some ments of the present invention, the pseudotyped oncolytic viruses or compositions thereof are administered to a subject at a dose ranging from about. liezli)+5 pfii to about leO'l” pfu {plaque forming , about lirlO'l'S pfu to about lxl 0H5 pfu, about lle+10 pfu to about lxlll'*‘15 pfu, or about lxlO“8 pfu to about lxll)“12 pfu. For example, in some embodiments, the pseudotyped oncolytic viruses or compositions thereof are administered to a subject at a dose ofabout 105, 106, 107, 103, 109, 1010, it)”, ll)”, 1013, it)”, or 10” pfu of virus. in some ments, the dose depends, on the age of the t to which a composition is being administered, For example, a lower dose may be required if the subject is juvenile, and a higher dose may be required if the subject is an adult human subje Tl. In certain embodiments, for e, a le subject es about lxl 0+8 pfu and about lxlO'HO pfu, while an adult human subject receives a dose between about lxlO"’1‘) pfu and about lirlO+12 phi. In some embodiments, the therapeutically effective amount of an 2017/040354 oncolytie Virus described herein is administered over the course of two or more doses, in some embodiments, the two or more doses are administered simultaneously (eg, on the same day or over a short period of time) or at appropriate intervals, for example as two, three, four or more sub-doses per day, E in some embodiments, the oncolytic Viruses or compositions thereof described herein are administered to a subject once. in some embodiments, the oncolytic Viruses or compositions thereof described herein are administered to a subject more than once. For example, a composition disclosed herein may be stered multiple times, including l, 2, 3, 4, , 6, or more times. in some embodiments, a ition disclosed herein may be administered to a subiect on a daily or weekly basis for a time period or on a monthly, bit/early, or yearly basis depending on need or exposure to a pathogenic organism or to a condition in the subject (cg. cancer). in particular embodiments, the oncolytic s and compositions thereof are formulated in such a way, and administered in such and amount and/or frequency, that they are retained by the subject for extended. periods of time. [(30253] In some embodiments, the pseudotyped oncolytic Viruses or compositions thereof are administered for therapeutic applications or is administered as a maintenance therapy, such as for example, for a patient in remission. In some embodiments, the pseudotyped tic viruses or compositions thereof are administered once every month, once every 2 months, once every 6 , once a year, twice a year, three times a year, once every two years, once every three years, or once every five years. {00254} lm some embodiments n a patient’s status does improve, the pseudotyped oncolytic Viruses or compositions thereof may e administered continuously upon the doctor’s discretion. in some embodiments, the dose composition is temporarily reduced and/or administration of the composition is temporarily ded for a certain length of time (ie, a “drug holiday”). in some embodiments, the length of the drug holiday varies between 2. days and l year, ing by way of example only, 2 days, 3 days, 4 days, .5 days, 6 days, 7' days, ll) days, l2 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, l80 days, 2% days, 2.50 days, 280 days, 30) days, 320 days, 350 days, or 365 days.

The dose reduction during a drug holiday is from ltl‘lti—l00%, including, by way of example only, %, 15 'iVi', 20 %, 25 'i/(i, 30 '34, 35 %, 4i") %, 45 %, 50 %, 55 %, 60 %, 65 '94», 70 'il/s, 75 (hi, 80 %, 85 %, 90 %, 95 A), or 100 %, 55} in some embodiments, once improvement of a patient's conditions has occurred, a maintenance dose may be administered if necessary. in some embodiments, the dosage and/or the frequency of administration of the composition is reduced, as a function of the WO 06005 symptoms, to a level at which the improved e, disorder or condition is retained, in some embodiments, patients may require intermittent treatment on a long—term basis upon any recurrence of symptoms. in some embodiments, toxicity and therapeutic efficacy of such therapeutic regimens are determined by standard pharmaceutical procedures in cell cultures or mental animals, including, but not limited to, the determination of the LDso (the close lethal to SC; % of the population) and the E050 (the dose eutically effective in 50 % of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index and it is expressed as the ratio between L350 and E950. Compounds exhibiting high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies are used in tomiulating a range of dosage for use in. human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the Ellis) with minimal ty” The dosage varies Within this range depending upon the dosage form employed and the route of administration utilized. {99257} ln some instances, tumor antigen expression levels are evaluated to assess the progress of treatment in a patient, to stratify a patient, and/or to modulate a therapeutic regimen. in some ii’istances, ment of antigen expression levels include the use of imniunohistochenustry {lilC} (including seniivquantitative or quantitative lilC) or other antihody~hasetl assays (Western blot, fluorescent immunoassay (PIA), fluorescence in situ hybridization (FlSl-l), radioiininunoassay {REA}, radioiinniunoprecipitation {Elli}, enzyme—linked inununosorbent assax (ELISA), immunoassay, imniunoradiometrie assajx, iluoroimmunoassay, chemiluminescent assay, bioluniinescen’r assay, gel electrophoresis} or indirectly by quantitating the transcripts for these genes (sag. lay in sin; hyhiidization, nuclease protection, Northern hlot, polymerase chain reaction (MIR) including reverse trzmscriptase PER {REESE}. in some instances, cells, for example, lymphocytes, are ed using FACs technology or paraffin embedded tumor sections using dies, } in some instances, antibodies are used to characterize the protein t of target cells h techniques such as ininiunohistochemistry, ELlSAs and Western blotting in some cases? this provides a screen eg. for the ce or absence of a t likely to respond favorably to oncolytic virus therapy and/or a need for co~adiniuistering an immune stimulating agent with an oneolytic virus» {96259} in some embodiments, iinniunohistochcniistry is performed on a sample of tissue from a biopsy. in some cases, the sample is ed fresh or honest. in some instaneea antibodies against antigens presented in the cell are added to the sample on a slide and the dies bind \N’l’lfii‘fiVifil‘ the ns are present. in some embodiments, excess antibody is then WO 06005 washed away, in some cases the antibodies that remain bound to the cell are further labeled by a secondary antibody for visualisation under a microscope. } in some embodiments, test samples are obtained from a subject such as for example, from tissue tag. tumor biopsy}, cerebrospinal fluid (CSF), lymph, blood, plasma, serum, peripheral blood mononuclear cells ), lymph fluid, lymphocytes, al fluid and urine. in particular embodiments, the test sample is obtained from CSF or tumor tissue, in other particular embodiments, the test sample is obtained from tumor tissue and eg the relative number of CD4+ and/or CD3" cells in the sample is determined and/or the level of one or more T111 andr’or ThZ cytoltines in the sample is measured rag. by immunofluorescent staining of fixed and abilized cells from the sample with antibodies against the 'l'hl and/or T112. cytolrines. in other particular emlrrodinients, the test sample is ed from blood and eg the level of one or more T111 and/or Th2 cytokines in the sample is measured by ELlSA.

Combination Therapy [@0261] In some embodiments, the viruses, expression constructs, nucleic acid molecules and/or vectors described herein are administered in combination with r therapeutic agent. in some embodiments, the oncolytie viruses and an adddtional thereapeutic agent are formulated in the same compositions in such embodiments, the composition may further comprise a pharmaceutically acceptable carrier or excipient. In some embodiments, the oncolytic viruses and an additional thereapentic agent are formulated in separate compositions tag, two or more compositions suitable for administration to t or subject). The disclosure further encompasses inistration protocols with other cancer therapies, cg. ific antibody constructs, targeted toxins or other compounds, including those which act via immune cells, including T—cell therapy. The c1inical regimen for co—administration of the inventive compositionts) encompass(es) co~administration at the same time, before and/or after the administration of the other component Particular combination therapies include chemotherapy, radiation, surgery, hormone therapy, and/or other types of immunotherapyln some embodiments, a therapeuticafly effective amount ofa pseudotyped onco1ytic virus is administered to a subject in need, f in combination with an additional therapeutic agent. In some instances, the additional therapeutic agent is a chemotherapeutic agent, a steroid, an immunotlierapeutic agent, a targeted therapy, or a combination thereof. {$6262} in some embodiments, pharmaceutical compositions are administered in conjuction with an adjuvant y, For examples, activating nt treatments are administered prior to, conten'iporaneons with, or after one or more administrations {c.g, intranimoral injection of the pseiidotyped rims). For example“, adiuvant therapy includes modulation of Tollulike receptor {TLR} ligands, such as ILRQ activation by DNA molecules comprising CpG sequences, or 'I'LRE?‘ activation (eg, by RNA ll(5‘?ands} Uther adjnvant treatments inciude agonizing antibodies or other polypeptidea (e.2; activation of CD40 or GITR by {3940 Ligand {CDLIIlL} or GITR Ligand (GITRLL respectively). Ftirtl'iei; provided are cyclic dlnneleotides (cg curlinGh/IP) that te STING. Another activating adjiivant includes interletikins such as H.133. [(99263] In some embodiments, the onal therapeutic agent comprises an agent selected from: bendamustine, omib, lenalidomide, idelalisih (GS-l 101), votinostat, everolimns, panobinostat? temsirolimns, psini VOIIHOSIEEI, flndarahine> cyclophosphamide, mitonantrone, pentostatine, prednisone, etopside, procatbazine, and thalidomide. {90264} In some embodiments? the additional therapeutic agent is a main—agent therapeutic regimen. In some embodiments the additional therapeutic agent comprises the HyperCVAD n (cyclophosphamide, stine, doxoruhicint dexamethasone altemating with rexate and cytarahine). In some embodiments, the I-lyperCVAD regimen is stered in combination with iituximah.

In some embodiments the additional therapeutic agent comprises the Rn CHOP regiment (ritiiximab, cyclophosphamidet ibicin, vincristine, and prednisone), {90266} In some embodiments the onal therapeutic agent comprises the FCR regimen (FCR (findarahine, cyclonhosphamide, ritnximah), {002657} In some embodiments the onal eutic agent ses the FCMR regimen (fludarahine cyclophosphamide, mitoxantronea rituximah) {00268} In some embodiments the additional therapeutic agent comprises. the Fit/IR regimen (tiudarahine, mitoxantrone, rituximah). [$0269] In some embodiments the additional therapeutic agent ses the PCR regimen (pentostatin? cyclophosphamidej rituxii’nab). {002703 In some embodiments the additional therapeutic agent comprises the PEPC regimen (prednisone, etoposide, procarhazine, cyclophosphaniide). {00271} In some embodiments the onal therapeutic agent. comprises radioimmunotherapy with 9“"iflihntnmoinah titixetan or 131I—tositnmomah. {(16272} In some embodiments, the additional therapeutic agent is an atttologous stem cell transplant.

In some embodiments, the additional eutic agent is selected from: nitrogen mustards such as for example, ustine, chlorarnbueil, chlormethine, eycluphosphamide, ifos’famide? alan, mustine, trnfesfamide; alkyl sulfenates like husulfan, mannosullan, treesulfan; ethylene imines like carhequone, ihiotepa, trlaziquone; nitrnseui'eas like cai’mustine, fotemustine, lemustine, mmustine, ranimustine, semustinei slreptczecin; epoxicles such as for example, etoglueid; other alkylating agents such as fer example dacarhazine, mitobi'cnitol, uman, lemide; folie acid analogues such as for example methotrexate, permetrexed, pralatiexate, rexecl; purine analogs such as for example cladnhine, clefarabine, ahine, mercaptepurine, nelarabine, tieguanine; pyrimidine analogs such as for example azaeitidine, capecitabine, eannofiir, cytarabine, decitahine, fluorcuracil, gemcitahine, tegafiir; Vinea alkaloids such as for example vinhlastine, stine, Vindesine, vinflunine, Vinorelbine; podophylletoxin derivatives such as fer e etopnsicle? teniposide; colehieine tives such as for example denieeoleine; taxanes such. as for example deeetaxel, paclitaxel, paelitaxel puliglumex; other plant alkaloids and natural products such as for example 'lrabeetedin; actincmycines such as for example dactinumyein; antracyehnes such as for example aelaiuhicin, elapnemhicini doxorubiein, ieini iclam‘oiciii, ntmne, piramhiein, valruhiciii, zombincin; other cytolexic antibiotics such as for example hleomyein, ixahepilone, mitumyein, plicamycin; platinum compounds such as. for example earliuplalin, eisplatin, exaliplatin, satraplatin; methylhydrazines such as for example pruearhazine; sensitizers such as fer example aminelevulimc acids efaproxiralfi methyl aminelevulinate, r “, 'lemoporfin; protein kinase inhibitors such as for example dasatinih, nih, everelimusg gefitinib, imatinih, lapatinih, nilotinih, nib, soi'afenib, sunitinih, ulimus; other antineoplastic agents such as for example ahtretinoin, alti'etaniine, anizacn'ne, anagrelide, arsenic triexide? asparaginase, bexaretene, hmtezomih, eeleecxih, denileukin diftitox, estramus'une, hydi'oxycarbamide, irinotecan, loniclamine, masoprocol, miltefesein, miteguazone, mitutane, uhlimersen, pegaspargase, tatin, toniidepsini sitimagene ceraelenovec, tiazofuiinei topotecani ti’etinoin, vomiostat; estrogens such as for e diethylstilbenoh ethinylestradioh fosfestrul, pelyestradinl phesphaie; progestogens such as fur example ngS‘lOHOI‘OflQ medroxyprogesterone, megestrel; gunadetropin releasing hormone analogs such as for example b’userelin, gosei'elm, leuprorelin, rehn; antimestrogens such as for example fulvesti'ant, tamoxifen, toremifene; aiiti—anilmgens such as for example bicalutaniide, flutamide, nilutamide, enzyme inhibitors, aminoglutethimide, anastrozele, exemestane, formestane, letrozole, le; other hormone antagonists such as for example aharehx, (legarelix; Inununostiniulahts such as for example histamine ochlonde, mifammtide, pidotimod, afer, requinimexi thymepentin; immunosuppressahls such as for example everelimus, gusperimus, lefiunemide; myccphehclic acid, sirelimus; calcineurin inhibitors such as for example eiclosporin, tacrelimus; other immunosuppressants such as for example azathiopiine, lenalidomide, rexate, thalidomide; and Radiopharmaceuticals such as for example, uane. {90274} in some embodiments, the additional therapeutic agent is selected from: interferons, interleukins, tumor necrosis factors, growth factors, or the like. {00275i in some ments, the onal therapeutic agent is selected from: aneestim, filgrastini, lenograstiin, inolgramostim, pegfilgrastim, sargramostim, lnterferons such as for example IFVNOE‘ natural, lFN d-Za, lFN Oil-2b, lFN n-l, lFN (let) i, lFN finatural, lFN 5— la, lFN fi-lh, IFN y, peginterferon a—Za, erferon a—Eb; interleukins such as for example aldesleuhin, oprelyeltin; other ostimulants such as for e BCG vaccine, glatiramei acetate, histamine dihydrochloride, imniunocyanin, an, melanoma vaccine, mit‘amuitide, pegadeniase, pidotiinod, plenxafor, poly LC, poly lCLC, i'oquiiiimex, tasoncnrnin, tliymopentinj, lmnnmosuppressants such as for example abatacept, abetimus, alefacept, antilymphoeyte immunoglobulin ), antithymocyte nnmunoglobulin (rabbit), eculizuniab, umah, everolimus, gusper’imus, leflnnomide, mummah—CD3, mycoplienolic acid, natalizumah, sirolimus, TNFo, inhibitors such as for e adaliniumab, afelimoniab, izumah pegol, e‘tanercept, golimumah, int‘lixiinah, eukin inhibitors such as for example anakinra, ximah, canahinumab, daelizumab, mepolizumah, i‘llonacept, tocilizumab, ustekinuniab; calcinennn inhibitors such as for example cielospor’in, taeroliinus; other iminunosuppressants such as for example azathioprine, lenalidornide, rexate, thalidomide. {00276E in some embodiments, the additional therapeutic agent is selected from: Adaliniumab, Alemtuzumab, Basiliximab, Bevaeizumab, Cetuximah, Certolizumab pegol, Daelizuma‘o, Eeulizumah, Efalizumah, Gemtuzumah, lbritumomah tiuxetan, lnfliximah, Muromonab-CDZ‘, zumab, Panitni'numab, Ranihizumab, Rituximah, Tositumoinab, 'l‘i'astuzumab, or the like, or a combination thereofi @0277] In some embodiments, the additional. therapeutic agent is selected from: monoclonal antibodies such as for example alemtuzumah, bevacizumab, cahnnaxomah, cetuximab, edrecolomab, gemtuzumab, nanitumumah, rituximab, trastuzumab; immunosupptessants, umah, efalizumah, muromaanD3, natalizuniah; TNF alpha inhibitors such as for example adalimumah, afolimomab, certolizumah pegol, golimumah, infiiximab; interleukin Inhibitors, hasiliximah, canakinumah, daolizuma‘o, mepolizuniah, tociiizuniab, ustelcinumab; Radiophai‘maceuticais, ihi‘itumomab tiuxetan, tositumoniab; additional monoclonal antibodies such as for example omah, adecatnmumah, alemtuznmah, anti—CD30 monoclonal antibody Xmah25l3, anti—MET monoclonal antibody Meth/lah, apolizumab, apomab, arcitumomah, hasiliximah, hispecific antibody 231, blinatnmomnb, brentnximab vedotin, eapromab pendetiole, oixutnninmnb, elandixiinab, conatumnmah, dacetnzurnab, denosnmah, eeuliznniab, epratnzumab, epratuzumah, ertnmaxornab, etataciznniab, tigituinninab, fresoliniuinab, galixiniab, ganituniab, geintuzuinab iein, glemhatumuniab, iomab, nmab ozogamiein, ipilimuniah, lexntninunia‘o, lintuznmah, lintuzuninb, luoatnmuniab, mapatumumn‘o, matnznrnab, inilatnzuinab, monoclonal antibody C(T49, neeitnmninab, niniotuznniab, oregovoinab, pertuzninab, i‘amaourimab, ranibizumab, siplizninab, sonepcizninab, tanezumal), tositninomab, trastuznniah, tremelimumab, tueotuzuniah eelmolenkin, veltuznma‘o, zuinab, voioeixiniab, zalutnmnmah {06278} In some embodiments, the additional therapeutic agent is seieeted from: agents that afieot the tumor micro—envirornent snoh as oeiluiar signaling k (eg: phosphatidylinositol 3—kinase (PBK) signaling pathway, signaling from the Ewell receptor and the lg}? receptor). in some ments, the additional therapeutic agent is a PBK signaling inhibitor or a sye kinase inhibitor. in one embodiment, the syk inhibitor is R788. in another embodiment is a i’KCy inhibitor such as by way of example only, enzastanrin. [(30279] Examples of agents that affect the tumor micro-environment include PIBK signaling inhibitor, sye kinase tor, protein kinase inhibitors such as for example dasatini‘e, erlotinib, everoiiinus, gefitinib, imatinib, lapatinib, nilotinib, pazonanib, nib, sunitinib, tenisitolintus; other enesis inhibitors such as for example (ET—ill, Jl—lfll, R1530; other kinase inhibitors such as for example ACZZO, AC480, ACE~O4L AMG 900, A?24534, Artyfiléi, AT75l9, ATQZBB, AV—QSI, axitinih, AZDllSZ, AZD7’762, AZDSGSS, AZD8931, bafetini‘o, BAY 73—4506, 361398, BGT226, Bl 812883, , BlBF £120, BEBW 2992, EMS—690154, EMS—777607, 3233, 13814461364, CAL-10L CEP-l lQSl, CYCl l6, DCC—2036, dinaoielib, dovitini‘o lactate, E7050, EMD 1214063, ENMD-2til76, atinil) disodinm, GSK2256098, GSl§690693, lNCBiSJlZZt, lNNQ—Jlfio, ENE-26483327, D9594, KX2—39l, linifanib, 1312603618, MGCDZéS, MKwGI-tfi’i’, MKlél-Qé, ML‘NSOM, MI.;N8237, lX/i'l’xi’i’i), NMS— ”16354, Nh/lS—1286937, 0N OlQlQNa, OSl-OZ’], 081—930, Btk tor, PF—(l056227l, PF— 02341066, 9303814735, PF~042l7903, 'PF«04554878, 91502, 58309, PHA— 739358, PLC3397, ipoietin, R547, R763, ramneininiab, regorafenib, 426, SARIGBiofi, SCH 727965, SGI—ll76, SGXSZB, SNS—314, EAR—593, TAK—QUL 'l‘KlZSS, ’l‘l_,N- 232, , XL147, X1228, XLZSlR05126766, XLMS, XL765. {(16283} In some embodiments, the additional therapeutic agent is selected from: inhibitors of mitogennetivated protein kinase signaling, eg '00126, P1398059, ESZ, PDOSZSQOl, ARRY—l42886, SB239063, SPGOOlZS, BAY 4343006, wortmannin, or 137294002; Syk inhibitors; mTOR inhibitors, and antibodies leg, rituxnn). {@281} In some embodiments; the additional therapeutic agent is seiected from: ZO—epi—i; 25 dihydroxyvitamin D3; S-ethynyiuracii; abiratei'one; aclambicin; acyifuivene; adeeypenoi; adozelesin; aidesieuitin; ALL—1K antagonists; altretamine; ambamustine; amidox; amifostine; aminoievuiinic acid; amnibiein; amsacrine; anagreiide; anastrozoie; andrographoiide; angiogenesis inhibitors; antagonist D; antagonist G; antareiix; oi'saiizing morphogenetic protein—1; drogen; prostatic oma; antiestrogeii; antineoplaston; aiitiserise oligonueieotides; apiiidicolin giycinate; apoptosis gene modulators; apoptosis regulators; ic acid; ara-CDP-DL—PTBA; argiiiine ase; asuiacrine; atamestane; atrimustine; axinastatin i; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatiri Hi derivatives; baianoi; batiinastat; BCR/ABL antagonists; benzociiioriiis; istaurosporine; beta iaetam derivatives; lethine; ainyein B; nic acid; bFGP‘ inhibitor; bicaititaniide; bisantrene; bisaziridinyispermine; bisnafide; histratene A; bizeiesin; breflate; bropirimine; biidotitane; nine suifoximine; caicipotnoi; ealpiiostin C; carnptothecin derivatives; pox IL—Z; capeeitabiiie; carboxainide~amino~triazoie; earboxyamidotriazoie; CaRest M3; CARN 1200; cartilage den e inhibitor; carzeiesin; casein kiiiase inhibitors (ECOS); eastanospennine; ceei'opiri B; eetroreiix; ciiiorlns; chioroquinoxaiine Sitifoiiarriide; cicaprost; cis—porphyrin; eiadi’ibine; eioniitene anaiogues; clotrimazoie; coiiismycin A; coilismyein B; combretastatin A4; combretastatiii analogue; coriageniii; crambeseidin 816; crisnatoi; eryptophyein 8; cryptophyciii A derivatives; curacin A; eyciopentamhraquinones; cycioplatam; cypeniycin; cytarabine octostate; cytoiytic factor; tin; daelixiinab; decitabine; odidemnin B; desiorelin; dexanietiiasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethyinorspemiine; dihydro—S~azaeytidirie; 9~ dioxamycin; dipiienyi spiromustine; doeosanoi; doiasetron; doxifluridine; di'oloxifene; dronabinoi; duoeatmycin SA; ehseien; tine; edeitosine; edrecoiomab; etiornithiiie; eieniene; emiteftir; epirubicin; epristeride; estrainustine analogue; estrogen agonists; en antagonists; etanidazoie; etoposide phosphate; exemestaiie; fadrozoie; fazarahine; nide; fiigrastim; finasteride; fiavopiridoi; fiezeiastine; i'one; fludarabine; fluorodaunomniein hydrochloride; forfenimex; fomiestane; fostriecin; foternustine; gadolinium texaphyrin; gaiiium nitrate; tabiiie; gaiiireiix; gelatinase inhibitors; gemcitabine; giutathione inhibitors; hepsuifam; iteregulin; hexamethyiene bisaeetamide; hypetic-in; ibaiidronio acid; idam‘oiein; idoxifene; idramantone; iimofosine; iioniastat; iinidazoaeridones; imiquimod; immuiiostiinuiant peptides; insulin—such as for example growth i receptor tor; inteiteron agonists; interferons; interioukins; io’benguane; xomhicin; ipomeanoi; 4—; iroplact; irsogladine; gazole; isoitomohaiicondrin B; itaseti'on; jasplakirioiide; kaiialaiide F; Eameiiarin—N triacetate; ianreotide; teinamycin; WO 06005 lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha eron; ieuprolide+estrogen-i-progesterone; leuprorelin; levamisole; liarozoie; linear polyarnine analogue; lipophiiic clisaccharicle peptide; lipophilic um compounds; lissoclinamide 7; lobaplatin; cine; lonie'trexol; lonidaniine; losoxantrone; lovasta‘tin; loxorihine; lurtotecan; lutetium texaphyrin; lysoijylline; lytic peptides; inaitansine; marmostatin A; inarimastat; masoprocol; inaspin; ysin inhibitors; matrix inetalloproteinase inhibitors; menogaril; inorbarone; nieterelin; inethioninase; nietoclopramitle; MlF inhibitor; inifepristone; miltefosine; mirirnostini; mismatched double ed RNA; mitoguazone; niitolactol; ycin analogues; rni‘tonafide; mitotoxin fibroblast growth factor—sapoiin; mitoxantrone; mofaroteiie; molgrainostini; monoclonal dy; human chorionic gonadotrophin; monophosphoryl lipid A+niyobacterium cell wall sk; inopidainol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1 —based therapy; mustard ncer agent; mycaperoxitle B; inycobacterial cell wall extract; inyriaporone; N—ace'tyldinaline; N—substituted benzamides; nafarelin; nagrestip; naloxone+pcntazocine; napavin; naphterpin; nartograstim; netlaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisaniycin; nitric oxide modulators; nitroxide antioxidant; ni‘trullyn; Oouberizylguanine; tide; okicenone; oligonucleotides; onapristone; ontlansetron; ondansetron; ; oral cytokiiie inducer; latin; osateronc; oxaliplatin; oxaunomycin; mine; palmitoylrhizoxin; pamidronio acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargasc; peldesine; pentosan polysulfate ; pen‘tostatin; pentrozole; perflubron; pert‘osi‘amide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; bicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinurn-triarnine complex; portimer sodium; porfirornycin; prednisone; propyl his—acritlone; prostaglandin 32; proteasome inhibitors; protein Anbasecl immune modulator; protein. kinase C inhibitor; protein kinase C inhibitors, niicroalgal; protein tyrosine phosphatase inhibitors; purine nuc eosicle phosphorylase inhibitors; puipurins; pyrazoloacridine; pyridoxylated hemoglobin poiyoxgy/ethylerie conjugate; raf antagonists; i'altitrexed; rainosetron; ras tainesyl protein traiisterase inhibitors; ras inhibitors; rasnGAl’ inhibitor; retelliptine deniethylated; rheniurn Re 186 etidronate; rhizoxin; ribozymes; RH retinamide; rogletiinitie; lcine; romurticle; i‘oquininiex; ruhiginone Bl; ruboxyl; safingol; saintopin; ; sarcoph'ytol A; sargi‘amostiin; Sdi l tics; semustine; senescence derived. inhibitor l; sense oligonucleotitles; signal transduction inhibitors; signal transduction modulators; single chain antigen-binding protein; sizofiran; xane; sodium ptazte; sodium phenylacetate; solverol; sornatomedin binding protein; sonermin; sparl‘osic acid; spicamycin D; spiroinustine; spienopentin; spongistatin 1; sqiiaiainine; Stein cell inhibitor; stern—cell division inhibitors; stipiamide; eiysin inhibitors; solfinosine; superaetive vasoactive intestinal peptide anh’t‘roiiist; snr‘adista; surainiii; swainsonine; synthetic giycosaininogiycans; taiiirniistine; tamoxifen methiodide; tauromustine; tazarotene; tecogaian sodium; tegafiir; teihirapyryiium; teiornerase inhibitors; teinoporfin; teinozoiornide; teninoside; tetrachlorodecaoxide; tetrazoniine; thaiihiastine; rztiine; throinhopoietin; thromhopoietin mimetic; thynraiihsin; thymopoietin receptor agonist; thymotrinan; thyroid, stimuiating hormone; tin ethyl etiopui'pinin; tirapazaniine; titaiiocene hichiiiiide; topsentin; toreinifene; totipotent stem cell factor; transiation inhibitors; tretinoin; triacetyhiridine; triciribine; trirnetrexate; triptoreiin; tropisetron; turosteride; tyrosine kinase tors; stins; UBC inhibitors; uheniniex; urogenital sinusnderived growth inhibitory ; ase receptor nists; vapi'eotide; vai'ioiin B vector system; erythrocyte gene therapy; veiai‘esoi; veraniine; verdins; verteporfin; vinoreibine; tine; n; vorozole; zanoterone; zenipiatin; ziiascorh; and zinostatin stimaiamer. {99282} in some embodiments, the additional therapeutic agent is seiecteri from: aikyiating agents, antinietaboiites, natured products, or hormones, sag, en mustards (6g, mechioroetharnine, cyclophosphamide; chlorambucii; etc); alkyd suifonates reg husnlthn), nitrosoureas (erg, carniustine, iomusitne, rate), or nes bazine; etc). Examples of antimotabohtes include but. are not limited to fohc arid analog (eg, rexate); or dine analogs (e, g; , Cytarahine), purine analogs (e. g, mereaptopuiine, anine, pentostatin). {00283E in some embodiments; phannacettticai compositions are administered in contention with an adios/tint y. For examines, activating adjiivaiit treatments are administered prior to, contemporaneous with, or after one or more minnnistratioiis (3.2;; intrittmiiorai injection of the psettdctyped virus). For e, adios/ant ti'ierapy iriehides modniation of 'I‘oii~iii<e receptor {'i‘LR) iigands, such as TIRE) activation by DNA inoiec‘iiies comprising; CpG sequences; or 31.15419 activation (9.5;, by RNA iigonds) Other adjuvant treatments ineinde agonizing antibodies or other polypeptides (e2; activation of {1840 or GETR by CD40 Ligand ittL} or GETR Ligand ); respectively); Further; provided are cyclic dinucieotides (cg. cwdhfiMi’) that modulate S'I'ENG, Another aetivating adjnvent inciodes interienkins such as ii.~33_ in some instances; the pharinaceiiticai compositions described herein are administered in eonjiietion with an Eighth/ant therapy, Kits in some embodiments, the present invention provides kits sing one or more cncoiytie Viruses as described herein, a nucleic acid sequence as described herein; a vector as described herein, and/or a host cell as described herein, In some embodiments, the kits comprise a pharmaceutical ition as described herein above, either alone or in combination with r therapeutic agents to be administered to an individual in need thereof. } lo some embodiments, the present invention provides kits for the use of vectors and tints-producing cells ing to the invention as drugs in therapeutic s. in particular, the vectors and virus producing cells according to some embodiments of the invention are used for the therapy or treatment of solid tumors in a subject. In some embodiments, the eutic effect is caused by the oncolytic properties of the recombinant vectors and viruses as well as by the use oftherapeutic genes. {9&286} in some embodiments, the present invention es kits for use with methods and compositions. Some embodiments concern kits having vaccine compositions of use to reduce onset of or treat ts having one or more solid tumors. Other embodiments concern kits for making and using molecular constnicts described herein. ln some instances, kits also include a suitable container, for example, vials, tubes, mini- or microfuge tubes, test tube, flask, bottle, syringe or other container. 'Where an additional ent or agent. is provided, the kit contains one or more onal containers into which this agent or component is placed, Kits herein also include a means for containing the constructs, vaccine compositions and any other reagent containers in close confinement for commercial sale, Such containers include injection or blow—molded plastic containers into which the desired vials. are retained. Optionally, one or more additional agents such as other anti—viral agents, anti—fungal or anti—bacterial agents are needed for compositions described, for example, for itions ol’use as a vaccine.

} All publications, patents, and patent applications mentioned in this specification are herein orated by reference to the same extent as if each individual publication, patent, or patent application. was specifically and individually indicated to be orated by reference.

EXAMPLES {60288} The examples below further illustrate the described embodiments Without limiting the scope of the invention, Example 1: Preparation of pseudotyped VSV—G {(116289} The following protocol was adopted to prepare an exemplary pseudotyped VSV—G, by combining ycoprotein (VSV—G?) with lllVl gag and rev proteins. {($293} Cell Citril‘lrli’e and ctirm: DNA of the following packaging plasmids was mixed and prepared for transfection into 293T cells: pMDLg/pRRE expressing HIV—l GAG/POL; pRSV/REV expressing l—lanl REV; and pMDZG 5 60 5.8 VSV glycoproteiri. The DNA mix was added to 500 ttL of pre-warrned Optimeni ll medium A g stock of polyethyleneimine tr’anstection reagent (PET) was prepared at l ug/nL in lXPBS, pH 4.5, and 88 oh of the working stock was added to the mixture maintaining a 4:1 V/w ratio of PElzDNA The mixture was vortexed briefly and left, for 5~l0 min at room temperature to form a FELDNA transfeetion complex. A total of 2.5 X l06 low e (less than P20) 293T cells were seeded per cm dish in l5 mL DMEM mented with 10% serum and l% Pen/Strep. 2 hours prior to transfection, the cell culture medium was aspirated and replaced with l5 mL offresh premwarmed growth medium (GM). The transfection complex. was then added drop—wise to each l5 cm plate, swirled briefly to mix and incubated for 8 hrs in 10% C02, 35° C. After 8 hours? the medium was replaced with 10 mL of fresh growth medium containing 25 rnl‘vl l-TEPES and l0% serum. The mixture was then incubated for 48 hrs ransfection. {60291} Virus collection: The medium from each dish was removed, pooled, and filtered through a 0.22 pm low protein binding/fast flow filter unit and stored at 4“ C. A 5 mL volume of fresh growth medium was added to each dish and incubated overnight at 4" C (605/2 hours post transfeetion). The second lot of medium from each dish was collected as in the previous step, and pooled with previous media harvest. The plasmid carryover is. removed by ion with DNASEJ (l mg/mL stock). A l ugme solution the viral supernatant, supplemented with l uL of 1M MgClz, was ted at room temperature for 30 min followed by 2-4 hrs at 4° C. The filtered supernatants can he used directly on cultured cells, or aliquoted and stored at —8{}“ C The pseduotyped VSV—G- viral atant can be optionally concentrated and purified. e 2: Construction of pseudotyped VSV~G expressing a CITES—CARS hispecifie antibody engager molecule {602923 l’seudotyped VSVnG is prepared as described in Example l and further processed to express a nucleic acid encoding an engager polypeptide sing an activation domain comprising an anti—CD28 molecule and an n recognition domain comprising an anti—CAMS molecule, and a nucleic acid encoding an anti-PDT immune tory peptide. The resulting oncolytic Virus is a pseudotyped onlcolytic VSV~G Virus encoding a —CAlZS engager molecule and an anti~PDl therapeutic molecule (CDEE—CAlZS—Hfl VSV—G).

Example 3: CDZS—CAl 25~PDl VSV—G activates human T cells and exhibits anti-tumor {90293} Human T cells are infected with the pseudotyped CDZS~CAl25wPDl VSV—G virus. 24 hrs to 48 hrs post viral ion, the T cell culture medium is collected and checked for the presence of prointlanirnatory cytokines. These results will show that T cells are activated by CDZS—CAES—l’fll VSV—G, as evidenced by presence ot‘proint‘lanirnatory nes such as lFN~l3 and ler in the cell culture supernatant of CDZS-CAlZS—PDl VSV’-G infected human T cells.

} EphAZ—overexpressing gastric cancer cells, from KATOS’ cell line, are infected with pseudotyped CDZS—CAlZS-PDl VSV—G or nonmpseudotyped CDZS—CAlZS-PDl VSV virus and the cell proliferation. is assessed. These results will show that cell proliferation is significantly reduced in cells KATO‘S cells infected with pseudotyped CD28~CAl25—PDl VSV— Ci compared to l§AT03 cells ed with non—pseudotyped (IDES-(315312513131 VSV virus.

Example 4: CDHLCIH, SlRl’lu—Cllfi, and PDlrl—Clfidl‘c engager molecules specifically hind to T~cells via CD3 ] The binding of bipartite (CDlngDS and SlRPld~CD3) and tripaite (PDLl—Clfil‘c) engager molecules to ”l" cells was assessed Briefly, 21000 ”l" cells were stimulated with 200 U/mL 114-2 for 12 days. After l2 days, T cell were incubated with varying concentrations of engager molecules (5005 1000, or 2000 rig/ml, for CDl9uCD3 and SlRPla— CD3; neat supernatant for C£)3~E‘c) for 20 minutes at room temperature in cate. Cells were then washed twice, followed by staining with an Xl-lis AFC antibody at 500 rig/ml, for an additional 20 minutes. Cells were washed again and treated with propidium iodide (Pl) to exclude dead cells from further is. Stained cells were ed by flow cytornetry on a El) 181% Fortesa cytorneter and the percentage of the cell. population positive for staining was set at 2% of the secondary only control. {002%} Results for (Z'Dl9—CD3 (FIG 19A): SlRPlu~€D3 (FlG. 19B}, and PDLl— CD3~FC (C) show that the CD3 binding moiety of each of these molecules functional binds to CBS—expressing 2937:“ T cells, as indicated by an se in the percentage of cells that are positive for the engager molecules compared to the secondary antibody alone. ln particular, a dose ent increase in the % positive cells is observed for CD19—CD3 (HG. 19A); while the SlRl’lu—CDTS construct demonstrated maximal binding at all trations. The amount of the neat PDLl—CD3-Fc supernatant used resulted in binding of the construct to the majority of T cells (FIG. l 9C) llglll {($297} The results of this experiment are quantified in HQ 20. in particular, all of the constructs demonstrated a cant se in the 9/5 positive '1' cells ed to samples where no engager molecule was added. [@0298] Additional expennients demonstrated that the binding of the CDlQ—CDBD SlRPla-CDE, and PDLl—CD3ch was mediated by interactions of the anti—CD3 domain of the engager molecules with CD3 expressed by the T cells. l’rior to exposure of'l‘ cells to the engager molecules, the T cells were incubated with an anti —Cl)3 monoclonal antibody (GK’l‘3). l3reincubation with the OKT'3 inhibited binding of the CDl9—CD3 engager, and substantially reduced binding of the PDLl—CD3-Fc engager, The lack of inhibition ot‘binding of the SlRPlot— CD3 engager by preincnhation with OKI‘S (F1G. ZlC) is likely due to an incomplete inhibition of CD3 by OK'l‘S in these samples.

Example 5: SlRPld-CBB constructs specifically bind to €347 {$9299} Experiments were performed to demrrnine the binding specificity of the SlRPld-CD3 engagei‘ constructs, Raji cells were preincuhated with SlRPld—CD3 engagers for 20. min at RT. Cells were then washed and incubated with a tluoreseently labelled anti—CD47 monoclonal antibody for 2.0 min at RT, alter which cells were washed and analyzed by flow try, Raji cells that were not pi‘eincnhated with the SlRPld—Cl'lfi engager showed significant g of the anti—CD47 monoclonal antibody (, lgG control histogram vs. the anti~CD47 histogram). Preineuhation of Rail cells with the SlRPld—CD3 engager blocked binding of the anti~CD47 monoclonal antibody (FlG. 22, anti—CD47 histogram vs. anti-CD47 +SlRPl (ii—CD3 liistogi‘aph).

Example 6: g of SlRl’ln—CDS and (731943113 r molecules to target cells [lltlilllll] Experiments were perfonned to determine the ability of SlRl’ld—CIB and CDlQ—CDS BiTEs to bind to Raji (CDIWCD—l’il, FIG? 23), U203 (CDlQ‘CDLlTl‘, FIG. '24)? u-luc (CDlQ‘CDL’lT, ), and U25l (CDlQ‘CDAf/fi ) target cell types For each target cell type, cells were d with 500 or 1000 ng/rnL of either (i) gged soluble SlRPld; (ii) SlRl’lct—Clfi Bi'l‘E; or (iii) or Cl)l9—Cl’)3 Bi’l‘li'. Cells were then d with a. cently labelled anti—His dy and analyzed by flow eytometry. {(16361} The results of SlRPld—CDB and CDl9—CD3 binding to CIl)l9*CDAl7+ Raji cells are shown in FlG. 2% Relative to the negative control lg (2° only), soluble SlRPlCL SlRPl (1693 BiTE, and CDlSl—CD3 BiTE were able to bind to Raji cells, as indicated by a shift WO 06005 s the right of the engager histograms ed to the lgG control histogram (FlG. 23A).

Quantitation of the g data showing percentage of BiTE positive cells is show in F16-. 233 {9030.2} The results of Sllil’loz—CEB and CDlQnCI‘B binding to CDlQ'CDZfil' UZOS cells are shown in FIG, 24. Relative to the negative control lg (2“ only), soluble SlRPlez, SIRPld-CDE BiTE were able to bind to UZCPS cells at all concentrations used, as indicated by a shift towards the right of the engager histograms compared to the lgG control histogram (A), CD19~CD3 Bi'l‘lis were unable to hind to U208 cells, which was expected based on the lack of CDl9 sion by U208 cells, Quantitation of these binding data showing percentage ot‘BiTE positive cells is show in 3. {tlfiiltlfi} The results of SlRl’loz—CEB and CDlQ—CIB binding to CDlQ'CD/l’i“ GBh<l30wlue cells are shown in FlG. 25. Relative to the negative control lg (2° only), SlRl’lor— CD3 BiTE were able to bind to GBMLEO—luc cells at all concentrations used, as indicated by a shift towards the right of the engager histograms compared to the lgG l histogram (A), in constast, CHEF—CBS Bi'l'Es were unable to bind to GBMSO—lne cells, which was ed based on the lack of CDl9 expression by GBM30—lue cells. Quantitation of these binding data showing tage of BiTE positive cells is show in PK}. 2513. [tltfilléll The results of ~CDB and CD19nCD3 binding to CD19‘CD47“ U251 cells are shown in FlG. 26. Relative to the negative control lg (2” only), o~Cl)3 BilE were able to bind to U251 cells at all concentrations used, as indicated by a shift towards the right of the engager histograms compared to the lgG control histogram (A). in constast, CDlQ— CD3 Bi'I'Es were unable to bind to U251 cells, which was expected based on the lack of €819 expression by U251 cells. Quantitation of these binding data showing percentage of Bill? positive cells is. show in 3, Example ”7: Binding of PDLl-Clfl-Fe ’l‘i’l‘Es to U251 cells is mediated by CD47, not FeyRs {£10385} As, the PDLl—CD3-Fc Till? construct comprises 2 domains that are capable of binding to target cells (the anti—PDLl and the Fc domain) experiments were performed to assess the g specificity ot‘these constructs. ClI‘lQ'CDél'i+ U251 cells were treated with 2 g/‘niL of a fluorescently labled ’l'flsl antibody, an isotype control, or PDLl —Cl}3—Fc ection stipeniant. Relative to negative l lg, the PDLl—CDS-Fc TiTE bound to UZSl cells (73), To assess whether this observed binding was due to interactions with CD47 or ll'cyRs expressed by UZSl cells, the li'cyR expression on U25l cells was determined. Cells were incubated with 2 L of throrophoreconjugated anti-CDlo/32 (recognizing Fclell/llclel) or anti-CD64 (recognizing Fcle) rnAbs for 20 min at RT. Cells were then washed and analyzed 2017/040354 by flow cytometry using a El) LSR Fortessa cytoineter. As shown in C, U251 cells do not express Few/RI; Fclel; or Fclell, indicating the binding ofthe PDLLCDB—Fc construct was mediated by interactions with CD47 and not Fey/Rs.

Example 8: CHE-CD3, SlRl’lelB, and Pfllil—ClB—F‘e constructs stimulate CD8+ T cell— mediated killing of target cells {393%} Experiments were performed to determine the afblity of CBlQ—CDTS, SlRPloc—CDBa and PDLl-CDS—Fc constructs to mediate killing> of target cells. Briefly; CD3+ T cells were stimulated for 8—l2 clays in the presence of 200 U/mL lL—Z and Dynabeads. Prior to co—culture with target cells, all Dynabeacls were removed by magnet and cells were washed to remove lL-Zt Raji (HG. '28), l’l (FlG. '29), U'ZSJ. (MG. 30), and 293F (FIG, 3.1) target cells were d with the fluorescent membrane dye PKl-lfi?’ green before plating CD‘fl+ effector T cells were then eo—cnltured with target cells at an effector to target ratio of lzl along with 1000 rig/ml; CDleCDE‘) Bi'I‘E, SlRPlo—CDS Bi’l'Es, or a l:3 dilution of l’lflsl—Clfilc transfection supernatant. Co—eultures of target and effector cells were incubated for 18 hours, afier which they were stained with 7—AAD arrdlivel'dead analysis was performed by flow try on 21 BD LSR a cytometer.

{W397} The results of these experiments indicate that the COW—GB, SlRl’lot— CD3 and PDLLCDB-Fc engager constructs were all capable of inducing or cellemediated death of Raji target cells (FlG. 28), The E030 for each of the CDlguClZPS’, SlRPlo—CD3 and l’DLl—CDB—Fe engager molecules on Rail cells are shown below in Table ’7 .I.

Table 7: ECsa of engage!" molecules on Raji cells Engager Mnleeule EEC-3r} ill) CD l9—CDS 0.6997 SlRPlu.—CD3 0.0l37 PDLlCDBPc 08007 [603983 The s of these ments r indicate that the PDLl—CD3-Fc engager constructs, but not the CDl,9—Cl)3 constructs, were capable of inducing or cell— mediated death of Tl-lPl target cells (). This is likely clue to the lack ofil’relatively low expression of CD19 by "ll-lPl cells. 1 O3 {@389} Further, the PlllJl—CD3-l7c engager constructs were capable of inducing or cellurnediated death of U25l target cells (FlG. 3(3), while the CDl9—CD3 constructs did not induce effector nediated death ofUZfil cells due to a lack of CDl9 expression by U25l cells. The ECso for each of the EB and PDLl-CDB—Fc constmc‘ts on UZSl cells are shown below in Table 8.

Table 8: ECso of engager molecules on “5325} cells Engager Molecule EEC-30 (riginiL) CDl9-CD3 i .47 Poiicnsn {0031lll Further, the SlRPla—CD3 r constructs were capable of inducing effector cell—mediated death of 293F target cells (HG. 3 l), indicated by the increase in cell death in SlRl’la—CDTS containing cultures compared to a control osteopontin—fusion protein (OPN l).

The ECs-a for SlRPla—CD3 engager les on 293F cells is shown below in Table 9.

Table 9: ECsu of SlRPla-CDl’i on 293E cells Engager Molecule ECsn L) SlRPla-CDB 00184 Example 9: l’DLluClB—Fc Bill?) enhances primary NK cell killing of {1251 cells [$6311] ments are performed to assess the ability of l’l3lJl-Cll3—Fc constructs to induce NK cell—mediated killing of target cells, Briefly, UZSl cells are lahleled with cell membrane dye FRI-{67 green, and then seeded and d to adhere to wells over night (). Primary NK cells (SteinCell Technologies, lnc.) are then added to each well at an or to target ratio of l:l, along with varying amounts of vitally produced PDLl—CDfi—Fc protein. Ellector/Earget cell (SO-Clllllll‘ii are incubated at 37" C for 6. hours prior to live/dead analysis by 7—AAD staining. Stained cells are analyzed by flow cytoinetiy on a Bl) LSR Fortesa cytorneter.

] These results will demonstrate that virally produced PDLl—CD3-Fc compounds are able to stimulate Nli cell—mediated death oftarget cells such as U25 l. l 04 Example ill: ollSV—infeeted Vero cells express SlRP—iwCDEE BiTEs {00313} To demonstrate that the oncolyric viruses described here are capable or producing the engager molecules, Vero cells were infected with OHSV sing SlRl’lu—CD3 BiTEs (FlG. 32) with either a short. linker (SL) (ONCE-O85; 2ASB SlRPld-CTB (8L) BiTE) or long linker (LL) (ON 7; ZASB SiRPl wCDS’ (LL) BYTE), or with ol-lSV expressing PDLl— e 'l‘i'l‘Es (ONCE—08% HQ. 33). Cells were infected for 3 days, after whichsupernatants front infected cells were passed. through a. 100K, MWCO ultrafiltration ne to remove any viral particles. The flowthrough was concentrated with a lth MWCQ ultrafiltration membrane.

Concentrated Vll‘éil supernatants and Hill ng, 59 ng, 25 ng, or 12.5 rig of purified SlRPloc—CD3 or l’DLl—CTB-Fc protein were then analyzed by PAGE followed by Western blotting with an anti" oxidis ion antibody in order to determine the amount of engager protein present in the Viral supernatants. {003143 The s demonstrate that cells infected with either ONCR—O85 or ONCR.~087 produced the SlRPlrt—CD3 (SL) and SlRllla—Cllfi (LL) protein? respectively (FIG. '32) r, cells infected with ONCR—089 produced the PDLl—CDTi—Fc protein (). The ability of the lOOK and lOK Arnicon filtration and concentration steps to remove remaining virus was assessed by n blot. The worktlow for clarifying Viral supernatants comprises low— speed centrifugation of the supernatants followed. by filtration through a 0.8 urn filter membrane) Supernatant filtrates are then ed through an Ainicon 100 klm filter to entrain the Virus? followed by passage ot‘the filtrate through an Arnicon l0 kDa filter to entrain remaining protein.

Aliquots of supernatants from Virally—infected cells were taken before and after processing with the Anticon filters and the presence of l-lSV was determined by blotting with an antisl-lSV polyclonal antibody These results show that the ultraliltration steps used to purify the engager ucts effectively removed virus ('). ore, any target cell killing ed in the presence of these engager ucts is due to the engager construct itself, and not a result of viral infection of the target cells.

Example ll: firstly—produced SlRl’lu-CDS and l’DLl—CDSuFC engager constructs induced effector—cell mediated killing of target cells {@315} Experiments were performed to assess the ability of yirally-produeed engager molecules (Sllfi’ld—CEB and l’DLl—CDS-Fc constructs) to mediate target cell killing.

Briefly, SlRFld—CIB (Sis), o—Cflfé (LL) and Cl)3—Fc proteins were prepared front Vero cellsas described in Example ll}. 50 tilt of the resulting SlRPlo—CD3 (8L), SlRPler-CDB (LL), and PDLl—CD3-Fc r proteins protein s were diluted lzl in tissue culture lt‘lS media ning 20% F38. The d engager proteins were then incubated with activated CD8+ effector T cells consultured with tluorescently labelled UZSl target cells at a target to or ratio of l:l for 18 hours. Cell death of U251 cells was assessed by flow cytometry on a BI) LSR Fortesa, cytoineter. l00316l The s of this ment demonstrate that virallyuproduced engager constructs direct ”ll—cell mediated killing of UZSE target cells (FlG. 34A). These results are quantified in FIG, 343.

Example 12: Expression of SiRPlu-CDSIPDLl-Fc compounds from 293 T cells ltltl3l7] Two expression plasmids encoding a SlRPlo-CD3 engager molecule and a PDLl —Fc therapeutic le were generated. One construct comprised a first gene encoding an l-lA—tagged c linked to a second gene encoding a. His—tagged SlRWu—CDS BiTE The SlRPlu amino acid sequence was linked to the anti—CD3 scFv by a single amino acid linl-rer (Le, a. short linker) (SEEP l o-CDB/PDLJ, —Fc (SL), MG. 37'), 'l'he other construct comprised a. first gene encoding a PDLl —Fc linked. to a second gene encoding a SlRPlo—CIB BiTE. The SlRPlo amino acid sequence was linlrerl to the D3 scFy by a G48 linl<er (216., a long linker) (SlRPld— CD3/PDL1—Fc (LL), ), The constructs were ed into a plasmid () and the resultant SlRleClfix’l’DlJl—Fc expression plasmids were transfected into 293 Free Style T cells. Four days after plasmid ection, e supernatants were collected {00318E Anti—PDLl—Fe compounds were purified from the culture supernatants using a Hill‘rap MabSelect SuRe Protein A column Hi'lrap column (GE Healthcare). Briefly, supernatants from 293 T cells transfected with either the SlRPla—CD3/PDLl—Fc (LL) or the SlRPltt—CDB/PDLl-Fc (LL) expression plasmids were loaded onto the column to purify the anti— l’DLlnFc compounds by binding of the HAntag to the . Flow through was collected for SlRl’lo—ClB BilE detection by Western Blot using an anti-His antibody (8). Columns were washed with wash buffer (20 told sodium phosphate, lSO mlvl NaCl, pH 7.4). Bound anti— PDLl—Fc protein was eluted with lgG elution buffer (pl-l 2.8, Pierce) and was immediately neutralized with a l M 'l‘rianCl buffer, pH 8. {90319} The anti—PDL l —Fc protein content of different n fractions then were visualized by Coomassie staning. Briefly, elution fractions were run on a 4% — 2% Bis-Tris Nul’AGE gel in MGPS buffer at 180 volts for 1 hour. Gels were stained for l hour in Simply Blue ain followed by destaining with water. Anti—l’chl —Fc protein t for each elution fraction is Show in A. After Coomassie analysis, elution fractions were combined and l 06 dialyzed against PBS at 4" C. Total anti~PDLl—l7c protein concentration was then determined by a, BCA assay.

Example 13: isolated PDLI—Fc proteins stimulate T cell-mediated death of target cells {0032M The ability of the antiuPDLl-Fc proteins to induce eileetor ediated death of target cells was assessed by a PDl/l’DLl blockade assay. A general schematic of the assay is Show in FlG. [ill A — 4H3. Briefly, {Elli}+ T cells were ltured with PDLl sing target cells (GHQ—Kl cells)” Varying concentrations of the anti-PDLl -Fc n isolated as described in Example 12 were then added to the culture. The t concentration of anti—PDLl— he used was 50 gig/nth 3a 2.5: fold serial dilutions were then performed to te the remainder of the anti~PDL l. ~Fe concentrations. Cell death was analyzed. by a Cytol‘ox—GloTM cytotoxicity assay in the presence (FEG. 4MB) and absence (FIG: MA) of the anti~PDLl—Fci s are fied in C. The ECso of the anti-PDLl—Fc is shown in Table ll}, These results demonstrate that the antivFDLl-Fc therapeutic les produced from the expression constructs described herein are capable of mediating or cell—mediated death oftarget cells.

Table ll}. ECse el antinPDLl-Fc compounds "i'fEi'iiEiiiiiiii""""""ECan antisPDL 1 -Fc 0‘45 ttgz’mls Example 13: ollSV—lnfected Vero cells express MMP9 and anti~?DLl—Fc therapeutic molecules @0321] In addition to producing tl e engager molecules as described in Example l 0, experiments are med to demonstrate that the oncolytie Viruses described here are capable or producing the MMP‘) and anti—PDLl «Fe therapeutic molecules, Vero cells are infected with OHSV expressing SlRPlamCDB/PDLLFC constructs Bi'fEs ( and ) or with OHSV expressing $lltPla.—Cl')3/lyil‘vil’9 constructs (FIG. lSA and. HG. l8B). Cells are infected for 3 days, after which supernatants from infected cells are passed through a 100K MWCO ultrafilttation membrane to remove any viral particles. The flcwthrough is trated with a lUK M‘WCO ltration membrane. MMP’Q and antiwl’DLLFC are purified from filtered, concentrated supernatants according to the protocol outlined in Example ll. Protein A~isolated MMP9 and anti—PDLl fractions are analyzed by PAGE followed by Coomassie staining. l 07 SlRPlu—CD3 BiTEs present in the Protein A rough are analyzed by Western blotting with an anti~6x His detection antibody. 2} The results will demonstrate that cells infected with oHSV vectors encoding either SlRPlu-CDB/PDLl-Fc constructs or SlRPlu—CDB/MMPQ constructs e the SlRFluuCD3 (SL) and SlRPloz-CDE (LL) Bill? protein, MMP9, and anti—PDLl—Fc.

Example 14: Virallymproduced SlRleCDfl/MMPl} and SlRPlonCDLl/PBIJ—Fc engager constructs induce effector—cell mediated killing of target cells {06323} Experiments are performed to assess the ability of ideally—produced engager molecules (SlRl’lu—CD3) and therapeutic molecules (MMP9 and anti-PDLl—Fc} to mediate target cell killing Briefly, SlR.Plu~Cl)3 (8L), SlRl’lu—CIB (LL), MMPQ, and anti— PDLl—Fc proteins are prepared from Vero cells as described in Example 13‘ 50 uL of the resulting protein samples are diluted in tissue culture media containing 20% PBS. The diluted proteins are then incubated with activated {798+ effector T cells or NK effector cells and are co— cultured with scently labelled target cells at a target to effector ratio of l:l for l8 hours.

Cell death of target cells is assessed by flow cytometry on a El) LSR Fortesa cytorneter.

The results of this experiment will trate that Virallynproduced SlRl’ln—CD3 engager constructs and eutic molecules MMPQ and anti—PDleL-‘c are able to direct T~cell and/or NK cell mediated killing oftarget cells. {00325E While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those d in the an: that such ments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art t departing from the invention It should be tood that various alternatives to the embodiments ofthe invention described herein may be employed in practicing the ion. it is intended that the following claims define the scope of the invention and that s and structures with in the scope of these claims and their equivalents be covered thereby l 03

Claims (18)

1. CLAiMS it A pseudotyped oncolytic virus comprising a recombinant nucleic acid comprising: i) a first nucleic acid sequence encoding an engager ptide, n the engager ptide comprises an activation domain specific for an antigen expressed on an effector cell and an antigen recognition domain specific for a cell—surface antigen expressed on a target cell.
2. 2. The pseudotjy'petl oncolstic Virus of claim L wherein the antigen recognition domain specifically binds to a tumor n.
3. 3‘ The pseudotyped oncolflic virus of claim 2, wherein tumor antigen is selected from Table
4. 4. A pseudotyped oncolytic Virus comprising a recombinant nucleic acid sing: i) a. first nucleic acid sequence encoding an engager polypeptide wherein, the engager ptide compn ses an activation domain specific for an antigen expressed on an or cell and a therapeutic molecule domain that binds to an inhibitory antigen expressed on a cell surface.
5. 5. The pseudotyped oncolytic Virus of claim 4-, wherein the therapeutic molecule domain specifically binds to l3Dl or CD47. , PDLl,
6. 6. The pseudotypetl oncolytic virus of any of the preceding claims wherein the recombinant nucleic acid further comprises a second nucleic acid sequence encoding a therapeutic polypeptide.
7. 7‘ The pseudotyped ‘tic virus of claim 6, wherein the recombinant nucleic acid is a i stroiiic sequences
8. 8. The typetl oncolytic Virus ofclaims '7, wherein the multicistronic sequence is a bicistronic sequence or atricistronic sequence.
9. 9‘ The oncolytic Virus of claim 7, wherein the multicistronic sequence comprises a piconiaviru.s—2a—like sequence, and wherein the first and second nucleic acid sequences are expressed from a, single promoter sequence present in the recombinant nucleic acid l 09
10. 10” The oiicolytic Virus of claim 6, wherein the therapeutic polypeptide is an immune modulator polypeptide.
11. l l. The oncolytic Virus of claim it), wherein the immune modulator polypeptide is selected from a cydoltine, a costiinulatory molecule, an immune checkpoint ptide, an anti— angiogenesis factor, a matrix metalloprotease (MMP), or a nucleic acid.
12. 12” The oncolytic Virus of claim l l, wherein the immune oint polypeptide comprises i) an inhibitor ot‘PD—l or CCR4; , PDL—l, CTLA—d, LAGS, TIMB, neuropilin, ii} an agonist of Gilli 0X40, or CD28; or iii) a combination of i) and ii).
13. 13. The oncolytic virus of claim l 1, wherein the Mh'll?‘ is Nth/1P9.
14. 14, The oncolytic Virus of claim l l, wherein the cytohine is selected from lL—l 5, , and (EXCLlO.
15. l5. The psendetyped oncolytic virus of any one of the preceding claims, wherein the effector cell is a T cell, an NKT cell, an NK cell, or a hage.
16. 16. The pseudotypecl oneolytic Virus of any one of the preceding claims, the activation domain specifically binds to CD3, CD4, CBS, CD8, CDlG, C928, CD40, CDll‘id, CDl 37, or NKGZD.
17. 17. A pseudotyped oncolytic virus comprising a recombinant nucleic acid sequence comprising i) a first nucleic acid ce ng an engager polypeptide, wherein the engagei' polypeptide comprises an activation domain c for an antigen expressed on an effector cell and an antigen recognition domain specific for a tumor cell n expressed on a target cell, wherein the antigen expressed on the effector cell is CD3, and wherein the tumor cell antigen is C019.
18. l8. The pseudotyped oncolytic virus of claim l1 wherein the recombinant nucleic acid sequence encodes a polypeptide sequence that is at least 90% identical to SEQ ll) N0: 44, lit)