NZ716064B2

NZ716064B2 - Multispecific antibodies, multispecific activatable antibodies and methods of using the same

Info

Publication number: NZ716064B2
Application number: NZ716064A
Authority: NZ
Inventors: Daniel Robert Hostetter; Bryan Allen Irving; Porte Sherry Lynn La; Henry Bernard Lowman; James William West; Chihunt Wong
Original assignee: Cytomx Therapeutics Inc
Priority date: 2013-07-25
Filing date: 2014-07-25
Publication date: 2022-06-28

Abstract

This invention relates to a multispecific activatable antibody that in an activated state binds two or more targets or two or more epitopes or a combination thereof, the multispecific activatable antibody comprising: at least an antibody (AB1) that specifically binds a first target or epitope, wherein AB1 comprises an IgG antibody comprising a heavy chain and a light chain; at least an scFv (AB2) that specifically binds a second target or epitope, wherein an AB2 is fused to the amino terminus of each heavy chain of AB1; at least a first masking moiety (MM1) coupled to the AB1 that inhibits the binding of the AB1 to its target when the multispecific activatable antibody is in an uncleaved state; at least a first cleavable moiety (CM1) coupled to the AB1, wherein the CM1 is a polypeptide that functions as a substrate for a first protease, and wherein at least a portion of the multispecific activatable antibody in an uncleaved state has a structural arrangement from N-terminus to C-terminus as follows: MM1-CM1-AB1; at least a second masking moiety (MM2) coupled to the AB2 that inhibits the binding of the AB2 to its target when the multispecific activatable antibody is in an uncleaved state; and at least a second cleavable moiety (CM2) coupled to the AB2, wherein the CM2 is a polypeptide that functions as a substrate for a second protease, and wherein at least a portion of the multispecific activatable antibody in an uncleaved state has a structural arrangement from N-terminus to C-terminus as follows: MM2-CM2-AB2. The invention enables targeting of multiple targets and/or multiple epitopes with a single molecule and also provides for increased selectivity for the intended targets and for a reduction of adverse effects upon administration. in AB1 comprises an IgG antibody comprising a heavy chain and a light chain; at least an scFv (AB2) that specifically binds a second target or epitope, wherein an AB2 is fused to the amino terminus of each heavy chain of AB1; at least a first masking moiety (MM1) coupled to the AB1 that inhibits the binding of the AB1 to its target when the multispecific activatable antibody is in an uncleaved state; at least a first cleavable moiety (CM1) coupled to the AB1, wherein the CM1 is a polypeptide that functions as a substrate for a first protease, and wherein at least a portion of the multispecific activatable antibody in an uncleaved state has a structural arrangement from N-terminus to C-terminus as follows: MM1-CM1-AB1; at least a second masking moiety (MM2) coupled to the AB2 that inhibits the binding of the AB2 to its target when the multispecific activatable antibody is in an uncleaved state; and at least a second cleavable moiety (CM2) coupled to the AB2, wherein the CM2 is a polypeptide that functions as a substrate for a second protease, and wherein at least a portion of the multispecific activatable antibody in an uncleaved state has a structural arrangement from N-terminus to C-terminus as follows: MM2-CM2-AB2. The invention enables targeting of multiple targets and/or multiple epitopes with a single molecule and also provides for increased selectivity for the intended targets and for a reduction of adverse effects upon administration.

Description

This invention relates to a multispecific activatable dy that in an activated state binds two or more targets or two or more epitopes or a combination thereof, the multispecific table antibody comprising: at least an dy (AB1) that specifically binds a first target or epitope, wherein AB1 comprises an IgG antibody comprising a heavy chain and a light chain; at least an scFv (AB2) that specifically binds a second target or epitope, n an AB2 is fused to the amino terminus of each heavy chain of AB1; at least a first masking moiety (MM1) d to the AB1 that inhibits the binding of the AB1 to its target when the multispecific activatable antibody is in an uncleaved state; at least a first cleavable moiety (CM1) coupled to the AB1, n the CM1 is a polypeptide that functions as a substrate for a first protease, and wherein at least a portion of the multispecific activatable antibody in an uncleaved state has a structural arrangement from inus to C-terminus as follows: MM1-CM1-AB1; at least a second masking moiety (MM2) coupled to the AB2 that inhibits the binding of the AB2 to its target when the multispecific activatable antibody is in an uncleaved state; and at least a second cleavable moiety (CM2) coupled to the AB2, wherein the CM2 is a polypeptide that ons as a substrate for a second protease, and wherein at least a portion of the multispecific activatable antibody in an uncleaved state has a structural arrangement from N-terminus to C-terminus as follows: MM2-CM2-AB2. The ion enables targeting of multiple targets and/or multiple epitopes with a single molecule and also provides for increased selectivity for the intended targets and for a reduction of adverse effects upon administration. 716064 B2 MUL'E‘ESPECIFIC AN’HBODIES, ‘ESE’ECEE‘IC AUi‘iVA'E‘ABLE ANIIBQDIES AM.) METHQDS 0F USING THE SAME RELATEB APPLICATEONS {(363631} This appiication claims the benefit of US. Provisional Appiication No. t3‘t/858A02. tiied i’niy 25, 2013, the contents of which are incorporated herein by reference in their entirety.

FIELD 0F THE INVENTIGN EtitBtBZ} The invention reiates generaiiy to rnnitispeciiic antibodies and to ninitispeciiie activatahie antibodies that speciticaiiy bind to two or more ent targets or epitopes, as weii as to methods of making and using these innitis eei’tie antibodies and/or rnuitispecitic activatahie antibodies in a variety of therapeutic, stic and prophylactic indications.

BACKGRQUNB OF THE INVVEN'E‘EQN {@093} onai antibodies have ernerged as nioieeuies for therapeutic intervention in a variety of disorders. However, targeting or iizing a single protein is not aiways sufﬁcient for effective treatment of certain disorders, which iirnits the therapeutic use ofrnonospeeiiic rnonocionai antibodies. Moreover, antibody—based therapies have proven e, fective treatments for some es but in some cases, toxicities due to broad target expression have limited their therapeutic effectiveness. in addition antihm’iyhased ti'ierapies have exhibited other iirnitations such as rapid clearance from the cireuiation foiiowing administration.

Etittttzt} Aeeordingiy, there exists a. need for antibodies and therapeutics that enahie ing of intiitipie targets and/or rnuitipie epitopes with a single rnoiecnie and aiso provide for sed seiectivity for the intended s and for a reduction of adverse effects upon administration.

SUMMARY 0F [HE INVEN'I'ION {@9635} The present disclosure provides rnnitispecitic dies and intiitispecitic aetivatahie an tihodies. The intdtispecitic dies provided herein are antibodies that recognize two or more different ns or epitopes. The multispecitic activatable antibodies provided herein are n'rultispeeific antibodies that include at least one masking moiety (MM) linked to at least one antigenv or e—binding domain of the multispeeific antibody such that coupling of the MM reduces the ability of the antigen-- or epitope-binding domain to bind its target. ln some embodiments, the MM is d to the antigen— or epitope~binding domain of the inultispecific antibody Via a cleayable moiety (CM) that functions as a. substrate for a protease. The activatable multispecific antibodies provided herein are stable in ation, activated at intended sites of therapy and/or diagnosis but not in normal, :7, 12., healthy tissue, and, when activated, exhibit binding to a target that is at least comparable to the eorrespon ding, unmodified multispecilic dy, {69%} hi some embodiments, the multispecillc antibodies and/or multispecific activatable dies are designed to engage immune effector cells, also referred to herein as immune~effeetor ng n'iultispeeific dies and/or immune—effector engaging pecific activatable dies. in some embodiments, the multispecific antibodies and/or rnultisnecitic activatable antibodies are designed to engage leukocytes, also referred to herein as leukocyte engaging multispeeilic antibodies and/or leukocyte engaging multispecitie activatahle antibodies. in some embodiments, the multispecific antibodies and/or multispecific activatable antibodies are designed to engage T cells, also referred to herein as T~cell engaging multisnecific antibodies and/or l—cell engaging rnultispecitic aetivatable antibodies. in some embodiments, the multispecitic antibodies and/or multispeeific activatable antibodies engage a surface antigen on a leukocyte, such as on a T cell, on a. natural killer (NK) cell, on a myeloid mononuclear cell, on a, hage, and/or on another immune or cell. in some embodiments, the immune effector cell is a leukocyte, in some embodiments, the immune effector cell is a T cell. in some embodiments, the immune effector cell is a NK cell. ln some embodiments, the immune effector cell is a mononuclear cell, such as a myeloid mononuclear cell. ln some embodiments, the n'rultispecific antibodies and/or multispecifie activatable antibodies are designed to bind or otherwise interact with more than one target an d/or more than one epitope, also referred to herein as inulti—antigen targeting antibodies and/or multi—antigen targeting activatable antibodies. As used herein, the terms t" and "antigen" are used hangeably. {6997} hi some embodiments, the immune effector cell engaging pecific antibodies include a targeting antibody or antigen—binding fragment thereof and an immune effector cell ng antibody or antigen—binding portion thereof in some embodiments, the immune or cell engaging multispecific antibodies include a cancer targeting antibody or antigen~binding fragment thereof and an immune effector cell engaging antibody or antigen-binding portion thereof. in some embodiments, the immune effector cell engaging multispecific antibodies include a cancer targeting lgG antibody or antigen~ binding fragment thereof and an immune effector cell engaging scFy. ln smne embodiments, the immune effector cell is a leukocyte. in some embodiments, the immune effector cell is a T cell. ln some embodiments, the immune effector cell is a NK cell. in some embodiments, the immune effector cell is a myeloid mononuclear cell. {lltltlll} In some embodiments, the T—eell engaging multispecitie antibodies include a targeting antibody or n-binding fragment thereof and a T—cell engaging dy or antigen—binding n thereof. in some embodiments, the T—cell engaging multispecific antibodies include a cancer targeting antibody or antigen—binding fragment thereof and a T- cell engaging antibody or antigen-binding n thereof. in some embodiments, the T-cell engaging multispeciflc antibodies include a cancer targeting lg6 antibody or antigen— binding fragment f and a T—cell engaging sefv, in some embodiments, the T—cell engaging pecific antibody includes an anti—CD3 n (CD38, also referred to herein as CD3e and CD3) scFy and a targeting antibody or antigen-binding fragment f. in some ments, the T—cell engaging multispecil‘ic antibody includes an anti—CD38 scFy and a cancer targeting antibody or an tigen—binding fragment thereof. in some embodiments, the 'l"~cell engaging multispeeific antibody includes an anti~Cf33tt scf'v and a cancer ing lgG antibody or antigen-binding fragment thereof. ln some embodiments, the T" cell engaging multispecifie antibody includes an anti~CD3 epsilon (CD38) scFV that is d from OKTS, ln some embodiments, the T—cell engaging multispeeific dy includes an anti-C'l‘LA-d scFV. ltlllll‘ﬁl in some embodiments, immune or cell engaging multispecific aetivatable antibodies of the disclosure include a targeting antibody or an ti gen—binding nt thereof and an immune effector cell engaging antibody or anti nding portion thereof, where at least one of the targeting antibody or antigen—binding fragment thereof and/or the immune effector cell engaging antibody or n—binding portion thereof is masked in some embodiments, the immune effector cell engaging antibody or antigen binding fragment thereof includes a first antibody or antigen-binding fragment thereof (ABl) that binds a first, immune effector cell engaging target, Where the ABl is attached to a masking moiety (MIX/ll) such that coupling of the Mix/ll reduces the y of the A81 to bind the first target! ln some embodiments, the targeting antibody or antigen ~binding fragment f includes a second antibody or fragment thereof that es a second antibody or antigen-binding fragment thereof (ABE) that binds a second target, Where the A82 is attached to a mashing moiety tl‘vllle) such that coupling of the MIX/l2 reduces the ability of the A82 to hind the second target. In some embodiments? the immune effector cell engaging antibody or antigen binding nt f includes a first antibody or n—binding fragment thereof (ABl) that binds a first, immune effector cell engaging target, where the ABl is attached to a masking moiety (MMl) such that coupling of the Ml‘vl‘t reduces the ability of the ABl to bind the first target, and the targeting antibody or antigen-binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen—binding fragment thereof (ABE) that binds a second target, where the ABE is attached to a mashing moiety (MMZ) such that coupling of the Mix/l2 reduces the ability of the ABE to bind the second target. ln some embodiments, the non—immune effector cell engaging dy is a cancer targeting antibody. in some embodiments the non—immune cell effector antibody is an lgG. in some embodiments the immune effector cell engaging dy is a scFy. ln some embodiments the targeting dy e.g., non-immune cell or antibody) is an lgG and the immune ef ‘ector cell engaging antibody is a scFy. ln some embodiments; the immune effector cell is a leukocyte. In some embodiments, the immune effector cell is a T cell. in some embodiments, the immune or cell is a NK cell, ln some embodiments, the immune ef ector cell is a myeloid mononuclear cell.

Eillllllll} ln some embodiments, T—cell engaging n'rultispecific activatable antibodies of the disclosure include a targeting antibody or n—binding fragment thereof and a "ll cell engaging antibody or antigen-binding portion thereof, Where at least one of the targeting antibody or antigen~binding fragment thereof and/or the T—cell ng antibody or antigen~binding portion thereof is d. in some en'rbm’lin'rents, the T~cell engaging antibody or antigen binding fragment thereof includes a first antibody or antigen~binding fragment thereof (AB t) that binds a first, T—cell engaging target, where the ABl is attached to a masking moiety (MMl) such that coupling of the MMl reduces the ability of the ABl to bind the first target In some embodiments, the ing antibody or antigenbinding fragment thereof includes a second antibody or fragment fthat includes a second antibody or antigen—binding fragment thereof (AB2) that binds a second target; where the A32 is attached to a masking moiety (MMZ) such that counting of the Mth s the ability of the A82 to bind the second target in some embodiments, the T—cell engaging antibody or antigen binding fragment thereof includes a first antibody or n—binding fragment thereof (AB i) that binds a first, 'l'-—cell engaging target, where the ABl is attached to a mashing moiety (Mb/ll i) such that coupling of the MMl reduces the y of the ABl to bind the first target, and the targeting antibody or antigen binding fragment thereof includes a second antibody or nt thereof that includes a second antibody or antige -- binding fragment f (A82) that binds a second target, where the A32 is attached to a masking moiety (MM2) such that coupling of the MMZ reduces the ability of the AB? to bind the second target, Etilitllll in some embodiments, the 'l‘--ceil engaging pecifrc activatable antibodies e a cancer targeting antibody or n—binding fragment thereof and a T— cell en gaging antibody or antigen—binding portion thereof, where at least one of the cancer targeting antibody or antigen-binding fragment f and/or the 'l‘uceil engaging dy or antigen~binding portion thereof is masked. in some embodiments, the T~cell engaging antibody or n binding fragment thereof includes a first antibody or antigen—binding fragment thereof (AB i) that binds a first, T~cell engaging target, where the A31 is ed to a mashing moiety (Mb/ll) such that coupling of the lViMl reduces the ability of the A31 to bind the first target. in some embodiments, the cancer ing antibody or antigen— binding fragment thereof includes a second antibody or tiagrnent thereof that includes a second antibody or antigen ing fragment thereof (ABE) that binds a second, cancer— related targe where the A82 is attached to a mashing moiety (Milt/l2) such that coupling of the MMZ s the ability of the ABE to bind the second, cancer—related target, in some embodiments, the T~cell engaging antibody or antigen binding fragment thereof includes a first antibody or antigen-binding fragment thereof (AB ii that binds a first, 'lT-ceil engaging target, where the ABl is attached to a mashing moiety (hills/ll) such that coupling of the MMl reduces the ability of the ABl to bind the first target, and the cancer targeting antibody or antigen—binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen—binding fragment thereof (AB2) that binds a , cancer-related target, where the ABE is attached to a mashing moiety (MMZ) such that coupling of the Mix/l2 reduces the ability of the A82 to bind the second, cancer-related target.

Etitltlll} in some embodiments, the T—cell ng rnultispecific activatable antibodies include a cancer targeting lgG antibody or antigen~binding fragment thereof and a 'l"~cell engaging scFv, where at least one of the cancer targeting lgG antibody or antigen— binding fragment thereof and/or the T—cell ng antibody or antigen-binding portion thereof is masked. in some embodiments, the T—cell engaging antibody or antigen binding fragment thereof includes a first antibody or antigen—binding fragment thereof (ABt) that binds a first, ’l'ncell ng , Where the A51 is attached to a masking moiety (Ml‘vll) such that coupling of the Mix/ll reduces the ability of the Mill to bind the first target. in some embodiments, the cancer targeting lgG antibody or an tigen—binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen- binding fragment thereof (A32) that binds a second, cancers-related target, where the A32 is attached to a masking moiety (MMZ) such that coupling of the MIX/l2 reduces the ability of the ABE to bind the second, cancer~related target, in some embodiments, the T—cell engaging antibody or antigen binding nt thereof includes a first dy or antigen-- binding fragment thereof (ABl ) that binds a first, T—cell engaging target, where the A81 is attached to a g moiety (MM l) such that coupling of the MMl s the y of the AB l to bind the first , and the cancer targeting lgG antibody or antigen—binding fragment thereof includes a second antibody or fragment thereofthat includes a second antibody or antigen—binding fragment thereof (ABE) that binds a second, cancer—related target, where the ABE is attached to a inaslring moiety (MMZ) such that coupling of the Mix/t2 s the ability of the ABE to bind the , cancer~related target. {tilllllj} in some embodiments of an immune effector cell ng multispecific actiyatable dy, one antigen is typically an antigen present on the surface of a tumor cell or other cell type associated with disease, such as, but not limited to, any target listed in Table 1, such as, but not d to, EGFR, erbB2, EpCAM, lagged, FED-Ll, 57H}, or CD7l (transferrin receptor), and another antigen is typically a stimulatory or inhibitory receptor present on the surface ofa T—cell, natural killer (NK) cell, niyeloid rnononuclear cell, macrophage, an dl’or other immune effector cell, such as, but not limited to, B7UH4, BTLA, CD3, CD4, CDX, CDl tia, CD25, CD27, CD28, CD32, CD56, CDl37, CTLA—t’l, GlTR, ll‘v’lEh/f, lCOS, LAGS, NKGZD, 0X40, PD—l, TlGlT, TIMB, or VISTA. in some embodiments, the antigen is a atory receptor t on the surface of a T cell or NK cell; examples of such stimulatory receptors include, but are not limited to, CD3, CD27, CD28, CDl3'7 (also referred to as 4—lBB), GlTR, H‘v’EM, lCOS, NKGZD, and 9X40. ln some embodiments, the n is an inhibitory receptor present on the surface of a ; examples of such inhibitory receptors include, but are not limited to, BTLA, , LAGS, P‘D—l, 'l'lGl'l", "fllVl3, and NK~expressed Kle, The antibody domain conferring specificity to the 'l‘mcell surface antigen may, also be substituted by a ligand or ligand domain that binds to a T~cell receptor, a NK—cell receptor, a macrophage receptor, and/or other immune effector cell receptor, such as, but not limited to, B7~l Pl) , 87—21, B7H3, l’f)~lﬂ,l, L2, or 'l'NFSlF‘).

Ellllllldl One ment of the disclosure is a multispecil‘ic activatable antibody that is table in a cancer rnicroenvironment and that includes an antibody, for example a lgG or scli‘v, ed to a tumor target and an agonist antibody, for e an lgG or scFv, directed to a co-stimulatory receptor sed on the surface of an ted T cell or NK cell, wherein at least one of the cancer target antibody and/or agonist antibody is masked.

Examples of(so—stimulatory receptors include, but are not limited to, (71327, CDlSTI’, GlTR, HVEM, NKGZD, and Gth). in this embodiment, the multispecific activatable dy, once activated by tumor~associated proteases, would effectively crosslinh and activate the T cell or NK cell expressed co—stirnulatory receptors in a turner-dependent manner to enhance the activity of T cells that are responding to any tumor antigen via their endogenous T cell antigen or NK-activating receptors, The activation-vdependent nature of these T cell or NK cell costimulatory receptors would focus the activity of the ted rnultispecitic activatable antibody to tumor—specific T cells, without activating all T cells independent of their antigen specificity, in one embodiment, at least the imulatory receptor antibody of the multispecific activatable antibody is masked to prevent activation of eactive T cells that may be present in tissues that also express the antigen recognized by the tumor tar et—directed antibody in the multis ecific activatable antibody, but whose activity is restricted by lack of co-—receptor engagement. idllllldl One embodiment of the disclosure is a multispecitic activatahle antibody that is activatable in a disease characterized by T cell overstimulation, such as, but not limited to, an autoimmune disease or inflammatory disease microenvironment. Such a niultispecitic activatable antibody includes an antibody, for example a lgG or scFv, directed to a target comprising a surface n expressed in a tissue targeted by a T cell in mune or inflammatory disease and an antibody, for example a lgG or scli‘v, directta' to an inhibitory receptor expressed on the surface of a T cell or NK cell, n at least one of the e tissue target antibody and/or T cell inhibitory receptor antibody is masked.

Examples of inhibitory receptors include, but are not limited to, BTLA, , LAG3, PD—l and NK-expressed Kle.

, TlGlT, TlM3, Examples ofa tissue antigen targeted by T cells in autoimmune disease include, but are not limited to, a e antigen sed on niyelin or nerve cells in multiple sclerosis or a surface antigen expressed on pancreatic islet cells in Type l diabetes. in this embodiment, the inultispecific activatable antibody when localized in the tissue under autoimmune attacl< or mation is activated and covengages the T cell or NK cell inhibitory receptor to suppress the activity of autoreactive T cells responding to any disease tissue—targeted antigens via their endogenous TCR or activating receptors. in one embodiment, at least one or multiple antibodies are masked to t supp ession of desired T cell responses in non—disease tissues where the target antigen may also be sed.

Ellllllltii in some embodiments, the T—cell engaging pecific activatable dy includes an anti—CD3 epsilon (CD38, also ed to herein as ClT33e and CD3) scFv and a targeting antibody or antigen-binding fragment f, where at least one of the anti-CD3s scFv and/or the ing dy or antigen—binding portion thereof is masked. In some embodiments, the CD38 scFv includes a tirst antibody or antigen—bim’ling fragment thereof (ABl) that binds CD38, Where the ABl is attach ed to a masking moiety (Mb/l l) such that coupling of the Ml‘vll reduces the ability of the ABl to bind CD38. ln some ments, the targeting antibody or antigen—binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen—binding tragment thereof (A82) that binds a second target, where the A32 is attached to a mashing moiety (MMZ) such that coupling of the MMZ reduces the ability of the A82. to bind the second target. ln some embodiments, the CD38 scFv includes a tirst antibody or antigen—bim’ling fragment thereof (ABl) that binds CD38, Where the ABl is attached to a maskin g moiety (Mitill) such that coupling of the Ml‘vll reduces the ability of the ABl to bind CD38, and the targeting antibody or antigen—binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or an tigen—binding nt thereof (A82) that binds a second target, Where the A82 is ed to a masking moiety (MMZZ) such that coupling of the Mth reduces the y of the A82 to bind the second target. {tilllll’ll in some embodiments, the T—cell engaging rnultispecitic activatable antibody includes an anti—CDSs schv and a cancer targeting antibody or anti gen—binding fragment thereof, Where at least one of the anti-CD38 scFv and/or the cancer targeting antibody or antigen—binding portion thereof is masked. In some embodiments, the CD38 scFv includes a first antibody or antigen~binding fragment f (A331) that binds CD38, where the A31 is attached to a mashing moiety (Misti) such that counting of the MM} reduces the abiiity of the A31 to bind CD38. in some embodiments, the cancer targeting antibody or antigen— binding fragment thereof inciudes a second antibody or fragment thereof that es a second antibody or antigen—binding nt f (A82) that binds a , cancer— related tar et, where the A32 is attached to a maskin g moiety (Mir/12) such that coupling of the MMZ reduces the ability ofthe A82 to bind the second, cancer-reiated . in some embodiments, the CD38 scEy inciudes a ﬁrst antibody or antigen~binding fragment f (A31) that binds CD}, a, where the AB} is attach ed to a masking moiety (MM 1) such that eouphng of the MM‘t reduces the ahiiity of the AB! to bind Cilia, and the cancer ing antibody or antigen-binding nt thereof inchrdes a second antibody or fragment thereof that inciudes a second antibody or antigen~binding fragment thereof (ABZ) that binds a second, cancer—related target, where the A32 is attached to a masking moiety (Mb/f2) such that coupiing of the MM2 reduces the abiiity of the A82 to bind the second, cancer—related target.

{Width} in some embodiments, the T—ceii engaging muitispecific tabie antibody inciudes an anti—CDSE scf'v and a cancer targeting igG antibody or antigenvbinding fragment thereof, where at least one of the anti-CDEs scFV and/or the cancer targeting igG antibody or antigen—binding portion thereofis mashed. in some embodiments, the CD38 scFV es a first dy or antigen-binding fragment thereof (AB} :3 that binds CD35, Where the A31 is attached to a masking moiety (MM 1) such that counting of the MMi reduces the abiiity of the A81 to bind CD38. in some embodiments, the cancer targeting ng antibody or antigen—binding fragment thereof includes a second antibody or fragment thereof that inciudes a second antibody or antigenvbinding fragment thereof (A82) that binds a second, cancer-related target, where the A82 is ed to a mashing moiety (MMZ) such that counting of the i‘s/iMZ reduces the ability of the ABE to bind the second, cancer-reiated target. in some embodiments, the CD38 scFv includes a first antibody or antigen~binding fragment thereof (A81) that binds CD38, Where the A31 is attached to a masking moiety (MME) such that coupling of the MM] reduces the abiiity of the A131 to bind CD33, and the cancer targeting antibody igG or n—binding fragment thereof es a second antibody or fragment thereof that inciudes a second antibody or antigen- binding fragment thereof (AB2) that binds a , cancer-reiated target, where the A32 is attached to a masking moiety (Mir/I2) such that coupling of the MMQ reduces the ability of the A32 to bind the second, cancer~retated target. with"); in some embodiments, the 'l‘—ceil engaging multispeciﬁc activatable antibody includes an anti-CD'S epsilon (CD38) scf‘v that is derived from OKTB, where at least one of the targeting antibody or antigen—binding fragment thereof and/or the OKT3 scFV or OKTZL deriyed scFy is . In some embodiments, the OKT3 scFV or OK'l‘S—derived scFV es a first antibody or antigen-binding fragment thereof (ABl) that binds CD38, Where the A81 is attached to a masking moiety (Midi) such that coupling of the MMi reduces the ability of the ABl to bind (2338. in some embodiments, the ing antibody or antigen— binding nt thereof includes a second antibody or fragment thereof that includes a second antibody or antigen-binding fragment f (ABE) that binds a second target, where the A332 is attached to a masking moiety (Mir/I2) such that coupling of the MMQ reduces the y of the ABE to bind the second target. In some embodiments, the OKT3 scFV or Oli'f3uderiyed scFV includes a first antibody or antigen-binding fragment f (ABl ) that binds CD38, where the ABl is attached to a masking moiety (MM 1) such that coupling of the MMI reduces the ability of the ABl to bind (2338, and the targeting dy or antigen~binding fragment thereof includes a second antibody or nt f that includes a second antibody or antigen-binding fragment thereof (ABZ) that binds a second target, where the A82 is attached to a g moiety (MMZ) such that coupling of the MMZ reduces the ability of the AB? to bind the second target.

} In some embodiments, the T—eell engaging multispecific activatable antibody includes an GKT3 scFv or OK'l‘BK-derived scFv and a cancer targeting antibody or antige -- binding fragment thereof, Where at least one of the OKT3 scFV or OKTS—derived scFV and/or the cancer targeting antibody or nbinding portion thereof is masked. In some embodiments, the 0K3?) scE'V or CRIB-derived scE'V includes a first dy or antigen-- binding fragment thereof (ABl) that binds CD38, where the A31 is attached to a masking moiety (MMl) such that coupling of the MMl reduces the ability of the ABI to bind CD38. in some ments, the cancer targeting antibody or antigen ~binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen— binding fragment thereof(ABE) that binds a second, cancer—related target, where the ABE is attach ed to a mashing moiety (MJVIZ) such that coupling of the MME reduces the ability of the A82 to bind the second, -related target. In some embodiments, the CECE?) scE'V or OKT3~deriyed scFy includes a first antibody or antigen—binding fragment thereof (AB I i) that binds CD38, where the AB} is ed to a g moiety (Mb/ll i) such that coupling of the Ml‘vfl reduces the ability of the ABi to bind (3338, and the cancer targeting antibody or antigen~binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen-binding fragment f (ABE) that binds a second, cancer—related target, where the A332 is attached to a masking moiety 2) such that coupling of the Mix/l2 s the ability of the ABE to bind the second, cancer~reiated target, liltiflill} in some embodiments, the f—celi engaging multispecifrc activatable antibody includes an OKT3 scFv or OKT3—deriyed scFv and a cancer targeting lgG antibody or antigen—binding fragment thereof, Where at least one of the OK'l'S scFy or OK’l‘3—deriyed scFy and/or the cancer ing lgG antibody or antigen-binding portion thereof is masked. ln some ments, the QKTEi scFv or OKTS-derived scFv includes a first antibody or antigen-binding fragment thereof (ABl) that binds CD38, Where the ABl is attached to a masking moiety (MMl) such that coupling of the MMl reduces the y of the A51 to bind CD343. ln some embodiments, the cancer ing lgG antibody or antigen—binding fragment thereof includes a second antibody or fragment f that includes a second antibody or antigen~binding fragment thereof (ABE) that binds a second, cancer~i'elated , Where the A82 is attached to a mashing moiety (MMZ) such that coupling of the MMZ reduces the ability of the A332 to bind the second, cancer—related target. in some embodiments, the QKT3 scliy or OKTS—deriyed scliy includes a first antibody or n— binding fragment thereof (A31) that binds C838, Where the ABl is attached to a mashing moiety {Mb/fly) such that coupling of the MMl reduces the ability of the A31 to bind CD38, and the cancer targeting antibody lgG or antigen~binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen—binding fragment thereof (ABE) that binds a second, cancer-related , where the A82 is attached to a mashing moiety (Mb/12) such that coupling of the MIX/f2 reduces the ability of the ABE to bind the second, cancer—related target" {@9922} In some embodiments, the T—cell engaging multispecific tabie antibody includes an anti—CTLA—L‘t scFv, where at least one of the targeting antibody or antigen~ binding fragment thereof and/or the anti—CTLA—il scFv is masked. in some embodiments, the anti—C'l'l'sﬁvd scFV includes a first antibody or antigen—bind ing fragment thereof (AB l) that binds C'l‘LA-4, where the ABl is attached to a masking moiety l) such that coupling of the Ml‘vll reduces the ability of the A131 to bind (FLA—4. in some embodiments, the targeting antibody or antigen—binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen~binding fragment thereof (ABE) that binds a second target, where the A82 is attached to a mashing moiety (MMZ) such that coupling of the MMZ reduces the ability of the A82 to bind the second target. in some ments, the anti~CTLA—4 scEy includes a first antibody or antigen~ binding fragment thereof (ABl) that binds CTLA '4, where the Afll is attached to a masking moiety (MMl) such that coupling of the MMl reduces the ability of the A51 to bind CTLA—L‘l, and the targeting dy or antigen—binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen—binding fragment thereof (AB2) that binds a second target, where the A32 is attached to a masking moiety (MME) such that coupling of the hilt/l2 reduces the ability of the A82 to bind the second target. {99923} in some embodiments, the T—cell engaging multispeeific table antibody includes an anti-Cl'LAnd scFy and a targeting lgG antibody or antigen-binding fragment thereof, where at least one of the anti—CTLA—L‘l scFy and/or the targeting lgG antibody or antigen—binding portion thereof is masked. in some embodiments, the TLA—él scFV includes a first antibody or antigen~binding fragment thereof (A81) that binds CTLA—Zl, Where the ABl is attached to a masking moiety (Mix/ll) such that coupling of the ll reduces the ability of the A131 to bind CTLA—d. in some ments, the targeting lgG antibody or n—binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen—binding fragment f (A82) that binds a second target, Where the A82 is attached to a masking moiety (MMZ) such that coupling of the Mth reduces the ability of the A82 to bind the second target in some embodiments, the anti"C‘TliA—dL scf'v includes a first antibody or antigen—binding fragment thereof (A81) that binds C'l‘LA-d, Where the ABl is attached to a mashing moiety ) such that coupling of the MMl reduces the ability of the A81 to bind CTLA—él, and the targeting antibody lgtfi or antigen—binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or n—binding fragment thereof (A82) that binds a second target, where the A82 is attached to a mashing moiety (MMZ) such that coupling of the Mth reduces the ability of the A82 to bind the second target, {$3824} in some ments, the multi—antigen targeting dies and/or mu lti— antigen targeting activatable dies include at least a first antibody or n-binding fragment thereof that binds a first target and/or first epitope and a second antibody or antigen—binding fragment thereof that binds a second target and/or a second epitope. in some embodiments, the multi—antigen targeting antibodies an d/or multi—antigen targeting activatable antibodies bind two or more different targets, in some embodiments, the multi— antigen targeting antibodies and/or multi-antigen targeting activatable antibodies bind two or more different epitopes on the same target. ln some embodiments, the mnlti—antigen targeting antibodies and/or mnlti—antigen ing activatable antibodies bind a combination of two or more different targets and two or more different epitopes on the same EilllllZE} Various embodiments of multispecific activatable antibodies of the disclosure are shown in Figures 3A, and 5—9, in some ments, a, mnltispecii‘ic activatable antibody comprising an lgG has the lgG variable domains mashed. in some embodiments, a pecil‘ic activatable antibody comprising a scFv has the scFv domains maslted. in some en'ibt’xlin'ients, a multispecific activatable antibody has both lgG variable domains and schv domains, where at least one of the lgG le domains is coupled to a masking . in some embodiments, a multispecilic activatable antibody has both lgG le domains and scFv domains, where at least one of the scFv domains is coupled to a masking moiety: in some embodiments, a mnltispecilic activatable antibody has both lgG variable s and schv domains, where at least one of the lgG variable domains is coupled to a mashing moiety and at least one of the scFy domains is coupled to a mashing moiety in some embodiments, a maltispecillc table antibody has both lgG variable domains and scl3y domains, where each of the lgG variable s and the sch‘v domains is coupled to its own masking moiety. in some ments, one antibody domain of a. maltispecific activatable antibody has specificity for a target antigen and another antibody domain has icity for a ll surface antigen, in some embodiments, one antibody domain of a mnltispecific activatable antibody has icity for a target antigen and another dy domain has city for another target antigen. in some embodiments, one antibody domain of a m ultispecilic activatable antibody has speciﬁcity for an epitope of a target antigen and another antibody domain has speciﬁcity for r epitope of the target antigen.

Eillllthi} in a n'niltispecitic activatable antibody, a scFy can be fused to the carboxyl terminus of the heavy chain of an lgG actiyatable antibody, to the earboxyl terminus of the light chain of an lgG activatable antibody, or to the carbonyl termini of both the heavy and light chains of an lgG activatable antibody. in a mtritispeciiic activatable antibody, a scFV ma. 1,49 can be fused to the amino terminus of the heavy Chain of an igG aetivatable antibody, to the amino terminus oi‘the Eight chain of an igG activatahie dy, or to the amino termini of both the heayy and Eight ehains of an lgG aetiyata‘oie antibody. in a mnitispeeifie aetivatahie antibody, a son can be fused to any combination of one or more carbonyl termini and one or more amino termini of an igG aetivatable antibody. in some embodiments, a masking moiety (MM) iinked to a eieavabie moiety (CM) is attached to and masks an antigen binding domain of the igG. in some embodiments, a masking moiety (MM) linked to a eleavahie moiety (CM) is attached to and masks an antigen binding domain of at ieast one scFV. in some embodiments, a masking moiety (MM) linked to a eieavabie moiety (CM) is attached to and masks an antigen g domain of an igG and a masking moiety (MM) linked to a eieavahle moiety (CM) is attached to and masks an antigen binding domain of at least one SCFV. {99927} The disclosure provides exarnpies ot‘ i'nuitispecitie aetiva‘tahle antibody stmetui'es which include, but are not iitnited to, the foliowing: (\I’L-—CL)2:(VH-CH1CHE-- L4~Vi—i*i3 —VL*—L2~CM—Li —MM)3; (314)} —Ci-i i —Ci-i2—Ci-i3 —L4~VL*—L3— VH3" ‘y’i—i... i —MM)Z; (MM—L i -t:fh/i—i_..2—V"L—Ci_i)3 : (VHCH} {HZ—CH3 —i_..4—VH*-i_.3 _ ig; (MM—L i,~CM~L2Wings);(Vii—CHl H3 ~i..r4—VL*—i..r3—VH*)2; (Vb MM--Li x-CM-VLZuVL*--L3-—VH* -vL4--VH-CH1-VCH2--CH3)2; (V L-CLbIM‘i‘s/i-LiCM L2—VH*—L3—VL*~L4—VH~CH l ~CH2~CH3)2; (MM—L l ~CM—LZ—VL—CL)?:(VL’B—L3—VHKL4— Vii—CH1~Cli»i2—CIHS)2; (MM—L l ~CM—LZ—Vi_..—CL)2:(VH’k-LS—VL*—L4—VH—CH 1 (H?— CH3)3 ; (V’LuCLuLZt-N’Hgk--L3 --Vif x-LZ-{TMnLi -I\di\li)2i(VH--CH1--CHZ--CH3)3; (VL-CL-L4u \I’L*~L3—VH*~L2—CM—Li~h/ih'i:)2:(Vi-i—CE-ii—CE—i2—CE— 3h; (MM—Li—CM—L2—VL*~L3—VH*— i..4—‘v"L—CL)2:(VH{1H i -CH2—CH3)2; (MM—Li —Ci\’i—i_..2—VH* ~13 —VL* —i_J4—Vi_i—CL)2:(VH— CH 1 UCHE—CH3)2; (VLvCL—i/l—VHWLS~Vl.i*—i_/2vCM—L l $4M); (MM—L i ~CMUlJ2—X/Lit— L3--VH*~L4--VH~CH1~CH2-CH3)2; (VL—CL--L4-—VB?"--L3--VL*--L2~CM-Ll"Evin/Eb: (MM-- L i —Ci\/’l,— L2~Vi-i*—L3 —VL*—L4~Vi—i—Ci-i l —CE—i2—CE—i3 )2; (VL—CL~L4—VL*—L3 —VH*—L2—Ci‘vi— L i —MM)2: (MM—Li ~CM—LZ—‘V’L* —Lf§—VH*~LL‘i—VH—CHE ~CHZ—CH3 )3 ; —CL—i_..4—V"L*—LS — VHii‘x-LZ-Ci‘vinhi-MM)22 (MM-Li--CM--L2x-VFW-LE--VL*--L4-VH—-CHl--CHZ--CH3)2; (VL- CL—Léi—V’i-ﬁ—LES—VLﬁg: (Mix/iii~CM~L2—VL*—L3—VH*~L4—VH~CH i~CH2~Ci-i3)z; (Vb Ci...—Léi—‘v’i»i*—L3-VL*)g: (MM—Li ~CiVCt—L‘2—VH’1‘~13,—‘v’L*-L4—VH—CHiCHZ-C’HPih; (VL— Ci'..r—i/l—VL*~L3 —VH*)3: (MMUL l —Cl\/l~i..2—VL*~L3 —VH*~i..r4—VH~CH i, H3)2; (Vb CL--L4—VL*-—L3--VH*)22 (Mix/i-le-{Th/t-LEUVH*--L3~VL*-—L4--VH-—CH1-—CH2-—CH3)2; (VLCL —Léi—VH*—L3—VL*—L2~Ch/1~Ll—MM)22 (Vie/’1‘—L3—‘v’E-i*—L4~Vi-i—CE—ii—CHZ—Ci—i3)2; (VL— CiL—Lél—Vl-ﬁ—LES—VL*~L2—CM—LI~Mi‘s/llg: (VH*~L3—VL*—L4—VH~CH1~CH2—Cl-l3)2; (Vb Cl.;—Ld—VL*—L3—VH*—L‘2—Ch’i—Ll $4M); (VL’l‘~L3"Viv-LAVH—CH l-Cl’lZ-i:li’l3)g; or (Vb CL—i/l—VIJ‘41.3—VH*—l_/2wChI—I.II ans/n2; (VH*as v‘t/Ii‘Jed—VH~CH l, ~CH2~CH3 )2, wherein: VL and VB represent the light and heavy variable domains of the first specificity, contained in the igG; VL’Z‘ and Vl—i* ent the variable domains of the second specificity, contained in the scFv; LI is a linlrer peptide connecting the inaslring moiety (MM) and the cleavable moiety (CM); L2. is a linker peptide connecting the cleavable moiety (CM), and the antibody; L3 is a iinlrer peptide connecting the variable domains of the scFv; L4 is a linker peptide connecting the antibody of the first specificity to the antibody of the second specificity; CL is the light-chain constant domain; and CH1, CH2, CH3 are the heavy chain constant domains. The iirst and second specificities may be toward any antigen or e. {99928} in some embodiments of a T—ceIl engaging rnnltispecii'ic activatabie antibody, one antigen is typically an antigen present on the surface of a tumor cell or other cell type associated with disease, such as, but not limited to, any target listed in Table 1, such as, but not limited to, EGFR, erbBZ, EpCAM, .Iagged, PD—Ll, B7l’I3, or CIJ7l (transferrin re "eptorl, and another antigen is typically a stimulatory (also re e "ed to herein as activating) or inhibitory receptor present on the surface ofa Tnceil, l killer (NK) cell, myeloid nclear cell, macrophage, and/or other immune effector cell, such as, but not limited to, 87—H4, BTLA, CD3, C134, CITES, CDI 6a, CUES, (2927, (2928, C2332, (2356, CHI 37 (also referred to as 'I'NFRSF9), CTLA '4, GITR, HVEh/l, ICOS, LAGS, NKGZD, 0X40, PD-i, ‘, 'l'lM3, or VlS’l‘A. The antibody domain conferring specificity to the T—celI surface antigen may also be substituted by a ligand or ligand domain that binds to a T—cell receptor, a l receptor, a macrophage receptor, and/or other immune effector cell receptor, such as, but not limited to, B7-—l, 37-2, B7H3, PD-Ll, Pill-LE, or lNFSFQ. in some embodiments of a multi—antigen targeting activatable antibody, one n is ed from the group of s listed in Table l, and another antigen is selected from the group of targets listed in Table It Etllltlllgl in some ments, the targeting dy is an GFR antibody. in some ernbodirnents, the targeting antibody is CZZSVS, which is specific for binding to EGFR in some embodiments, the targeting antibody is C225, which is speciﬁc for binding to EGFR. in some embodiments, the targeting antibody is C225V4, which is c for binding to EGFR. in some embodiments, the targeting antibody is 7225x165, which is speciﬁc for binding to EGFR. in some embodiments, the targeting dy is an anti~ Jagged antibody. in some embodiments, the targeting antibody is 43l l, which is specific for binding to human and mouse lagged l, and lagged 2, in some embodiments, the targeting antibody is élDl lv2, which is specific for binding to human and mouse lagged l and Jagged 2. {99938} In some embodiments, the targeting antibody can be in the form an activatable antibody. in some embodiments, the ) can be in the form of a Pro-scFV (see, e.g., {tilltliil} in some embodiments, the scFv is ic for binding CD38, and is or is derived from an antibody or fragment thereof that binds €338, erg, CH2527, EN l8, HL‘ZC, OKTB, 2C1 l, UCHTl, or V9. in some embodiments, the scFV is specific for binding CTLA—ll (also referred to herein as CTLA and CTLAd).

{MERE} in some embodiments, the anti~C33s seFv includes a sequence selected from the group consisting of those ces shown in Table 7 and/or in Example 5. in some embodiments, the anti—C333 scFV includes an amino acid ce that is at least 90%, 9 %, 9 %, 939/0, 94%, 95%, 96%, 97%, 98%, 9 % or more identical to an amino acid sequence selected from the group consisting of those sequences shown in Table 7 and/or in Example 5,, {@9933} in some embodiments, the scFV is specific for binding one or more 'l‘--cells, one or more NK—cells and/or one or more macrophages. in some embodiments, the scFV is specilic for binding a target selected from the group consisting ot‘BillZl, BTLA, CD3, 034-, C38, (33162:, C325, C327, C328, C332, C356, C3l37, C'i‘lsAth , HVEM, lCOS, LAG3, NKGZD, OX4tl, l’D-Vl, 'l‘lGlT, Hit/l3, or VlSTA. {96363343 in some embodiments, the multispeciﬁc antibodies, multispeciiic activatable antibodies, conjugated multispecific antibodies and/or conjugated iniiltispeci’ric activatable antibodies provided herein e at least a ﬁrst antibody or antigen g fragment thereof (A131) that speciﬁcally binds a Jagged target, e.g., Jagged 1 and/or lagged 2, and that contains a combination of a Vii CDRl sequence, a Vii CDR2 sequence, and a Vll CURB sequence, wherein at least one of the Vii CDRl sequence, the VH (DRE sequence, and the 7H CDR3 sequence is selected from a VB CDRl that sequence includes at least the amino acid sequence SYAMS (SEQ ll) N3: 6); a Vl-l (332 sequence that es at least the amino acid sequence SIDPEGRQTYYA3$VKG (SEQ ID NO: 7); a VH C393 sequence that es at least the amino acid sequence DIGGRSAFDY (SEQ ll) NO: 8), and combinations thereof. {$9935} in some embodiments, the ntultispecii‘ic antibodies, pecific activatahle antibodies, ated multispecitic antibodies and/or conjugated multispecific activatable antibodies provided herein include at least a iirst antibody or antigen binding fragment f (Alli 7; that specifically binds a lagged , eg lagged l and/or lagged 2, and that contains a combination of a VL CDRl sequence, a VL CDRB ce, and a VL CDREl sequence, wherein at least one of the VL CDRl sequence, the VL CDR2 sequence, and the VL (IDES sequence is selected from a VL ClTJRl sequence that includes at least the amino acid sequence RASQSISSY (SFQ ll.) N0: 9); a VL CDRZ sequence that, includes at least the amino acid sequence AASSLQS (SEQ ll) NO: ill); a VL CDR3 sequence that includes at least the amino acid sequence QQTVVAPPL (SEQ ll) NO: ll), and combinations thereof. l9llll3ti} in some embodiments, the multispecitic antibodies, pecific activatable antibodies, conjugated rnultispeciiic antibodies and/or coniugated multispecilic activatable antibodies provided herein include at least a ﬁrst antibody or antigen g fragment thereof (ABl) that speciﬁcally binds a lagged target, e.g., lagged l and/or lagged 2, and that contains a ation of a VB CDRl sequence, a VB CDRZ sequence, and a Vll CURB sequence, wherein at least one of the Vl-l CDRl sequence, the Vl-l CURE sequence, and the Vll CURE ce is selected from a Vll CDRl sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence SYAMS (SEQ lD NO: 6); a Vll CDZ sequence that includes a sequence that is at least 90%, 9l%, 92%, 93%, 94%), 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence Sllﬁl’FGRQTYY/aDSVKG (SFQ ll.) NO: 7); a Vii CDRB sequence that includes a sequence that is at least 99%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9992; or more identical to the amino acid sequence DlGGRSAFDY (SEQ ll) NO: 8.), and combinations thereof. {99937} In some embodiments, the inultispecitic antibodies, inultispecitic activatahle antibodies, conjugated rnultispeciiic antibodies and/or coniugated multispecilic activatable antibodies ed herein include at least a ﬁrst antibody or antigen g fragment thereof (ABl) that speciﬁcally binds a lagged target, e.g., lagged l and/or lagged 2, and that contains a combination of a VL CDRl sequence, a VL CDRZ sequence, and a VL CDRS sequence, n at least one of the VL CDRl sequence, the VL (DDR2 sequence, and the VL CERES sequence is selected from a VL CDRl ce that includes a sequence that is at least 90945, 9l%, 92"34;, 93%, 94‘36, 95% ,96‘34;,97‘3/o, 98/0,99% or mere identical to the amino acid sequence RASQSlSSY (SEQ ll) NO: 9); a Vl3 CUR}: sequence that includes a sequence thatis at least 99%, 9l%, 92932;, 93%, 94%, 95%, 9%, 9‘33'0, 98%, 99% or more identical to the amino acid sequence AII‘tSSLQStSEQ ll) NO: l0); and a VL CDR3 sequence that includes a sequence that is at least ‘3/6, 9.7‘)34), 93%, 940/4), 95%, 96%, 979/0, 98"32,, 9 % or more identical to the amino acid sequence VAPPL {SEQ 1D NO: ll), and combinations thereof.

E99938} ln some embodiments, the niultispecific antibodies, multispecilic activatahle antibodies, conjugated multispecific antibodies and/oi~ conjugated multispecilic activatahle antibodies provided herein include at least a first antibody or antigen binding fragment thereof (ABl) that speciﬁcally binds a lagged target, eg, lagged l and/or‘ia{IQ030 _ed37,and that contains a combination eta Vll CDRl sequence, a Vll CDRZ ce, a Vll CDR3 sequence, a VL CDRl ce, a, VL CDRZ sequence, and a VL CDR3 sequence, wherein the ‘vl-lCDRl sequence includes at least the amino acid sequence S‘rAMS (SEQ ll) NO: 6);, the Vl’l CDZ sequence includes at least the amino acid ce Sll)l’EGRQ"l‘YYADSVKG (SEQ ll) NO: 7); the Vll CDR3 sequence includes at least the amino acid sequence AEDY (SEQ ll) NO: 8,); the VL CDRl sequence includes at least the amino acid sequence SSY (SEQ ll) NO: 9); the VL CDRZ sequence es at least the amino acid sequence AASSLQS (SEQ ll) NO: l0); and the VL CDR3 sequence includes at least the amino acid sequenceVQQl‘\IVAl’Pl (SEQ ll) NO: l l3 {99939} in some embodiments, the inultispecitic antibodies, multispecific activatable antibodies, conjugated specitic antibodies and/or conjugated multispecillc activatable antibodies ed herein include at least a first dy or antigen binding fragment thereof (A81) that specifically binds a lagged target, e.g., lagged 1 and/or lagged 2, and that contains a combination of a VH CDRl sequence, a VB CDRZ sequence, a VB CDR3 sequence, a VL ClTJRl sequence, a VLtDRZ sequence, and a \I’l. CURE~ sequence, wherein the Vll CDR 1 sequence includes a ce thatis at least 99%, 9 l‘3/o, 92‘3"3o, 93%, 94%, 95%, 9692;, 97‘3"3'0, 98"30, 99943 or more identical to the amino acid ce SYAMS (SEQ ll) NO: 6); the Vii CD2 sequence includes a sequence that is at least 9094), 9l%,92°"/0, 93%, 9I—"3l3’0, 95%, 96%, 97"/o, 98‘3"3o, 99% or more identical to the amino acid sequenee SIDPEGRQTYYADSVKG (SEQ lD NO: 7); the Vll CDR3 sequence includes a sequence thatis at least 90%, 9i ‘34), 92%, 93%, 94%, 9594;, 96%, 97%, 98%, 99% or more identical to the amino acid sequence AEDY (SEQ 11:) N13: 83; the VL {DDR1 ce includes a sequence that is at 1east 909/11,, 91%92m, 93%, 94%, 95%1969/11,979/0, 98%,99% or more identicai to the amino acid sequence RASQS1SSY (SEQ 11) NO: 93; the VI... CDRZ ce includes a sequence thatis at 1east 91390, 91",0, 929/121, 93%, 9' %, 959/11, 969/21, 97%, 989/0 9 % or more 1dentica1 to the amino acid sequence AASSLQS (SEQ 11) NO: 103; and the VL C13R3 sequence inciudes a sequence that is at 1east 91190,919/o,112%,93%, 94%, 959/0,9(1‘721 9790,98",0, 999/121 or rnore identicai to the amino acid sequence QQ'i‘V‘v’APPL (SEQ 113 NO: 11). {111111411} 111 some embodiments, the ntultispecific antibodies, muitispecific activatab1e antibodies, conjugated multispecific antibodies and/or conjugated muitispecitic activatahle dies provided herein include at 1east a first antibody or n binding fragment the1eot ("1813 that speL11iLa11y binds Epidermal Growth Facto1 Receptor(EGFR): and that contains a combination of a ‘\.I11 CIDRi sequence, a 3.711 CIDR2 ce, and a Vii CIDR3 sequence, wherein at 1east one of the 31711 CDRi sequence, the VB CDR2 sequence, and the Vii CDR3 sequence is seiected from a V11 {DDR1 sequence that includes at least the amino acid sequence NYCi‘v111SEQ 11) NO:123, a V11 (1)2 sequence that includes at 1east the amino acid sequence 3/191"SC:GN'i'D‘r’N"1‘RE"1‘S (SEQ 11) NO: 133; a V11 C19R3 sethence that includes at 1east the amino acid sequence AL'LYYD‘YEEAY (SEQ 11) NO: 14); and combinations thereof. {91194111 111 some einbt'idiinents, the tnultispecitic antibodies, intdtispecific activatab1e antibodies, conjugated muitispecific antibodies and/or ated muitispecitic activatabie antibodies provided herein include at least a first antibody or antigen binding fragment f (A1313 that specit1cal1y binds EGER and that contains a ccnnbination of a ‘x.IL CDR1 sequence,a V1 CDR2 sequence, and a VL C‘DR3 SIequence, wherein at least one of the V1,.

CDR1 sequence the VL CDR2 ce and the VL CDR3 ce is ed from a VL CDR1 sequence that inciudes at least the amino acid sequence RASQSKtTNii-1(SEQ 11) NO:153, a V1... CDR2 sequence that inc1udes at 1east the amino acid sequence RYANiISiS (SEQ 11) NO:163; and a VL C13R3 SIequencetthat inciudes at least the amino acid sequence QQNNNWPTTLSEQ 113 NO: 1 ’3, and combinations theieot {11111142.} 111 some embodiments, the specific antibodies, pecific activatab1e antibodies, conjugated pIecittc antibodies and/or conjugated muitispecitic activatahle antibodies provided herein include at 1east a first antibody or antigen binding fragment t ("1813 that speL11iLa11y binds ECSFR and that contains a combination ot a V11CDR1 ce, a Vl—l CDR2 sequence, and a Vl—l CURB ce, wherein at least one of the V11 CDRl sequence, the V711 CDRZ sequence, and the VH CUR?) sequence is se1ected from a 9'11 CDRl sequence that includes a sequence that is at least 99%,919/11, 9.7911,9396, 949/11, , 96%, 97%, 989A1, 99/c or more cal to the amino acid sequence NYGVH (SEQ 11) NO: 12); a ‘17l1 CD2 ce that includes a sequence that is at least 909/151, 919’/c4 929lo, 93%, 949/11, 95% 9690, 97969894199911 or more identical to the amino acid sequence VlWSGGN'l’DYNTPFTS (SEQ 11) NO: 13); a VB CDR3 sequence that includes a sequence that is at least 90921, 919’/c4 9 %, 939/151, 94911, 95%, 96%, 97'1/21, 989/114 99% or more ide1=1tica1 to the amino acid sequence A1.TYYD‘YEFAY (91.7171 11) NO: 14); and combinations thereof, {111111431 111 some embodiments, the niultispecilic antibodies, multispecil‘ic activatahie antibodies, conjugated nrultispeciiic antibodies and/or conjugated multispecitic activatahle antibodies provided herein include at least a first dy o1 antigen binding lraginent thereof (ABl) that ically binds EGFR and that contains a combination of a VL CDRl sequence, a VL CDR2 sequence, and a VL (DDR3 sequence, wherein at least one of the VL CD111 sequence, the VL CD112 sequence, and the VL CDR3 sequence is selected from a VL CDRl sequence that inclut es a seqt ence that is at least 90%, 91%,929/11, 931 9,6 94%, 95911, 969',o 97%, 989721 999c or more identical to the amino acid sequence 1L’XSQSIG’1‘N1H (SEQ l1) N9: 15); a VL CDR2 ce that includes a sequence thatis at 1east 9119/o, 91%, 92%,939’/'o, 94%, 959/11, 969’97%, , 99% or more identical to the amino acid sequence KYAhhE119 (SEQ 11) NO:16); and a \11 CUR} sequencethat includes a sequence thatis at least 9119',o 91%, 97911 93%, 94%, 959.21, 96%, 979o, 98%, 999/11 or more identical to the amino acid sequence QQNNNWPTT (SEQ ID NO: 17), and ations thereof. {11111144} in some embodiments, the niultispeeilie antibodies, pecific activatahle antibodies, conjugated multispeciﬁe antibodies and/or conjugated multispecific activatahle antibodies provided herein include at least a ﬁrst antibody or antigen g fragment thereoi‘ (A‘Bl) that iical ly binds FGFR and that contains a combination ot a 1711 ClTJR1 ce, a V11 CURE sequence, a V711 CDRS sequence, a V11 CDRl sequence, alt/1.. CDR2 sequence, and a VL CERES sequence, wherein the V11 CD111 sequence includes at least the amino acid sequence NYGV1:1 (SEQ 11) NO: 12); the V711 CD2 sequence includes at least the amino acid sequence V1WSGGN'l‘D'YN'l'PF'l'S (SFQ 11.) NO: 13); the V111 CURB sequence includes at least the amino acid sequence AL’l‘Y‘i’DYEFAY {SEQ 1D NO: 141); the VL CDR1 sequence includes at least the amino acid sequence RASQSlGTNlE-i (SEQ 1D NO: l5); the VL CDRZ sequence includes at least the amino acid sequence KYASESlS (SEQ ll) NO: 16); and the VL CDRB sequence includes at least the amino acid sequence QQNNNWP'IT (SEQ ll) N0: l7). {999453 in some ments, the niultispecitic antibodies, multispecific activatable antibodies, conjugated ntultispecilic antibodies and/or conjugated inultisnecitic activatable antibodies provided herein include at l1.'1st 21 ﬁrst antibody oi antigen binding fragment thereof (ABl) that specifically binds EGFR and that contains a combination of a Vii CDRl sequence, a Vl-l CDR2 sequence, a Vl-l CDR3 sequence, a VL CDRl sequence, a VL {DDR2 sequence, and a VL CDR3 sequence, wherein the I’ll CDRl ce includes a ce thatis at least 999/, 91%, 1,93./o,949/11,959/13, 96%, 979/11, 98%, 99% or more identical to the amino acid sequence NYGVH (SEQ lD NO: l2); the Vii CDZ sequence includes a sequence thatis at least 90911, 9l911, 92%, 93%, 94%, 959/21, 96%, 97%, 989/11 99% or more identical to the airline acid sequence V1WSGGNTDYNTPTT":(SEQ ll") NO: l 33 the Vll £3913 sequence includes a sequence thatis at least 99%,9l9/11, 97911 93%, 94911, 95%, 96%, 97%, 98921, 99% or more identical to the amino acid sequence ALTYYDYEFAY (SEQ ll) NO: l4); the VL ClTJRl ce includes a sequence that is at least 90%, 9t%, 92/219391/11, 9/49l/,t1 959/, 96%, 97911,1 989111, 999/ or more identical to the amino atid sequenec RASQSlG’l‘NlH (SEQ ll) NO: 15); the ‘VL CDRZ sequence includes a sequence that is at least 90921, 9l%, , 93911, 94%, 959111, 96%,97'V15, 989%, 99916 or more identical to the amino acid sequence KYASESlS (SEQ ll) N0: l6); and the VL CUR?) sequence includes a sequence that is at least 909/,11 9l9’111, 92%, 93%, 94%, 959«{1,9t:19/£1,9 '96, 989’111, 99% or more identical to the amino acid sequence QQNNN WPiT (SEQ ll) NQ: l7). {99946} in some embodiments, the n'iultispecific antibodies, rnultispecillc activatable dies, conjugated innltispecitic antibodies and/or conjugated niultispecitic activatable dies ed herein include at least a heavy chain amino acid sequence selected from the group consisting of those sequences shown in Table 7 and/or in Example 5 including Table ll therein, ln some embodiments, the rnultispecilic antibodies, multispecil‘ic activatable dies, conjugated niultispecitic antibodies and/or conjugated innltispecitic activatable dies provided herein e at least a light chain amino acid sequence selected from the group consisting of those sequencessshown in Table 7 and/or in e , including Table ll therein, in some embodiments, the niultisiecitic dies, niultispecific activatable antibodies, conjugated ntnltispecitic antibodies and/or ated multisnecitic activatalile antibodies provided herein include at least a heavy chain amino acid sequence seleCted 11om thegroup Consisting 01 those sequenCes shown111 Table? and,01 in Examples1., . inCluding TablC l1 , n, and a light Chain amino aCid quuC=nCe seieCted from the group ting of those sequenees shown in Table 7 and/or in Example , including Table 11 t11e1e1n. {91111471 in some embodiments, the multispecitic antibodies, multispecitic aCtivatable antihodi1.2se,onjugated ninltispCCifie antibodies and/0r conjugated multispeeifie aetivatabie dies provided herein include at 1e21st a heavy, Chain amino acid sequence that is at 1e21st 911,41, 9194:, 92%, 93°A), 94.41, 95°71), 96%, 97%, 98941, 99% or more identical to an arnino aCid sequence seleCted from the group ting of those sequencessshown'in Table 7 1111 Example 5, including Table 1 1 therein. In some embodiments, the peeifie antibodies, multispecifie aCtivatahle antibodies, conjugated mnitispeeitie antibodies and/or conjugated multispecitic aCtivatable antibodies provided herein inClude at least a light Chain amino acid seunnee that is at least 91°/0, 91°,0, 92°41, 93%, 94%o, 95°4, 96%, 97%, 98,41, 99°76 or more identiCal to an amino acid sequence selected from the group consisting of those sequenCes shown in Table 7 and/or in Example 5, including Table l l therein. in some embodiments, the 1111.1ltispCeiiic antibodies, multispeCilie activatable dies, conjugated multispeeitio antibodies and’or conjugated pCCitie aetivatabie antibodies provided herein include 211 least a heavy Chain amino aCid Ce thatis at least 90% 91°/0, 92°,0, 93°41, 94%, 95%, 96%,’ "7.94)/ 98°, 0, 99921 or more identiCai to an amino aCid sequenCe ed from the group consisting of those sequences shown in Table 7 and/012111 Exampie 5, including Table ll therein and a light Chain arnino 21Cid sequenee that‘15 at least 90°41. 91%, 92%, 93%, 94941, 95%, 96°:41 97°/0, 98°,0, 999/41 or more identiCal to an amino acid sequence seleCted from the group Consisting of those sequences shown in Table 7 and/or in Example 5, including Table 11 therein. {111111481 ln some embodiments, the rnultispeciﬁC antibody and/or inultispeeifie aetivatable antibody also includes an agent conjugated to the AB. in some ments, the agent is a apeutie agent. in some embodiments,the agent is an antineoplastie agent in some embodiments, the agent is a toxin or fragment thereof, in some embodiments, the agent is coniugated to the multispecitic via a linker 111 some embodiments the linker1s a non-Cleavable linker. 111 some e1'nbodi1'ne11ts, the agent is a nrierotubnle inhibitor. in some embodiments, the agent is a nucleic 21Cid ng agent. such as a DNA alhylator or DNA interCalator, or other DNA damaging agent. in some embodiments, the linker is a Cleavabie linker. 111 some embodiments, the agent is an agent seleCted from the group listed in Table 4. In some embodiments, the agent is a dolastatin. in some embodiments, the agent is an atin or derivative thereof. in some embodiments, the agent is anristatin E or a derivative the eof. in some embodiments, the agent is monoinethyl auristatin F (MMAE), in some embodiments, the agent is monomethyl auristatin D (Mb/IAD). in some embodiments, the agent is a nsinoid or maytansinoid derivative. in some embodiments, the agent is Dix/ll or Dir/ill, in some embodiments, the agent is a duocarmycin or derivative f. in some ments, the agent is a calicheamicin or derivative thereof. in some embodiments, the agent is a pyrrolobenzodiazepine.

Eilllll49} In some embodiments, the rnultispecific antibody and/or inultispecitic activatable antibody also includes a detectable moiety, in some embodiments, the detectable moiety is a diagnostic agent.

Eddhﬁtli in some embodiments, the pecitic antibody and/or multispecitlc activatable dy naturally contains one or more dis ultide bonds, in some embodiments, the multispecil‘ic antibody and/or rnultispecitic activatable antibody can he engineered to include one or more disultide bonds.

Eiltltiﬁl} The disclosure also provides an isolated nucleic acid molecule encoding a multispeciiic antibody and/or niultispeciiic activatahle antibody described herein, as well as vectors that include these isolated nucleic acid sequences. The disclosure provides methods of producing a pecitic dy by culturing a cell under conditions that lead to expression of the antibody, wherein the cell comprises such a nucleic acid molecule. in some embodiments, the cell comprises such a vector, iddttﬁl} The disclosure also provides multispecitic table antibodies and/or multispecilic actiyatable antibody conrpositirms that include at least a tirst antibody or antigen—binding fragment thereof (ABl) that s1, ecitically binds a ﬁrst target or first epitope and a second antibody or antigen-hiding fragment thereof (ABE) that binds a second target or a second epitope, where at least AB} is coupled or otherwise attached to a masking moiety (MMl ), such that coupling of the MMi reduces the ability of ABl to bind its target in some embodiments, the Ml‘vil is coupled to ABl via a first ble moiety (CMl) sequence that includes a substrate for a protease, for example, a protease that is aiized with the target of ABl at a ent site or a diagnostic site in a subject, The mnltispeciiic activatable antibodies provided herein are stable in circulation, activated at intended sites of therapy and/or sis but not in normal, Le, healthy tissue, and, when activated, exhibit binding to the target of ABE that is at least comparable to the corresponding, unmodified niultispecific antibody {$3853} ln some embodiments, the multispecific activatahle antibody ses a g peptide between the MMl and the CMl.

Ellllllﬁdl in some embodiments, the multispecitic activatable antibody comprises a linking peptide between the CM} and the ABE. {tillllﬁﬁi in some embodiments, the activatahle antibody comprises a first linking peptide (LPl) and a second g peptide (LPQ), and at least a portion of the rnultispecific actiyatable antibody has the structural arrangement from N—terminus to C—terminus as follows in the uncleayed state: Ml‘vl‘t —LFl—Cl\v’ll—Ll)2—Al3l or ABl—i..i’2~Ch/,ll~l..l’l hilt/EL in some embodiments, the two linking peptides need not be identical to each other. {999563 in some embodiments, at least one ol'iLPl or Ll?2 includes an amino acid sequence selected from the group consisting ottGS)", , (GSGG'S)11 (SEQ. ll) N0: l8) and (GGGS)n (SEQ ll) N0: l9), Where n is an integer of at least one. in some embodiments, at least one of LPl or LPZ includes an amino acid sequence selected from the group consisting of GGSG (SEQ ll) NO: 20), (36886 (SEQ ii) NO: 21), GSGSG (SEQ ll?) NO: 22), GSGGG (SEQ ii) N0: 23), GGGSG (SEQ ll) NO: 24), and GSSSG (SFQ ll) NO: 25). {999573 in some embodiments, the multispecitic table antibody includes at least a lirst antibody or antigen—binding fragment thereof (ABl) that specifically binds a ﬁrst target or ﬁrst e and a second antibody or antigen ng fragment thereof (ABE) that ically binds a second target or second epitope. in some embodiments, each of the AB in the multispecilic activatabie antibody is independently selected from the group consisting of a monoclonal antibody, domain antibody, single chain, Fab fragment, a h nt, a scFV, a scAh, a dAh, a single domain heavy chain antibody, and a single domain light chain antibody. in some ments, each of the AB in the mnltispecil‘ic activatable antibody is a rodent (cg, mouse or rat), chimeric, humanized or fully human onal antibody.

Etlllllﬁtil in some embodiments, each of the AB in the rnultispecilic activatable antibody has an equilibrium dissociation constant of about lGG anl or less for binding to its corresponding target or epitope.

EiltltlSQ} in some embodiments, MMl has an equilibrium dissociation constant for binding to its ponding AB that is greater than the equilibrium dissociation constant of the AB to its corresponding target or epitope. gasses; in some embodiments, Mir/ll has an equilibrium dissociation constant for binding to its corresponding AB that is no more than the equilibrium dissociation constant of the AB to its corresponding target or epitopel ltil} in some embodiments, MMl does not interfere or compete with its corresponding AB for binding to the corresponding target or epitope when the multispecil‘ic activatable antibody is in a cleaved state. {lllllle} in some ments, Mlvll is a polypeptide of about 2 to 40 amino acids in . in some embodiments, each of the MM in the multispeciflc activatable antibody is a, polypeptide of no more than 40 amino acids in length. {99963} in some ments, MMl has a polypeptide sequence that is different from that ol‘target of the corresponding AB.

Eiltltlhd} in some embodiments, MMl has a polypeptide sequence that is no more than 50% identical to any l binding partner of the corresponding AB. in some embodiments, Mix/ll has a polypeptide sequence that is no more than 25% identical to any natural binding partner of the corresponding AB. in some embodiments, Ml‘vll has a polypeptide sequence that is no more than ltl‘E/o identical to any natural binding partner of the corresponding AB.

} In some embodiments, the coupling of Mir/ll reduces the ability of the ponding AB to bind its target or epitope such that the dissociation constant (lid) of the AB when coupled to the Mix/ll towards its corresponding target or epitope is at least 20 times greater than the Kd of the AB when not coupled to the Mir/ll towards its corresponding target or epitope.

Million; in some embodiments, the ng of l‘s/lMl s the ability of the ct‘rrresponding AB to bind its target or epitope such that the dissociation constant (Kid) of the AB when coupled to the Mir/ll towards its corresponding target or epitope is at least 4t) times greater than the lid of the AB when not coupled to the Mix/ll towards its con‘esponding target or epitope. {lllllld’l} in some ments, the ng of MMl reduces the ability of the corresponding AB to bind its target or epitope such that the dissociation constant (Kid) of the AB when d to the MMl towards its corresponding target or epitope is at least lOG 7’ ‘2 .r.i./ times greater than the lid of the AB when not coupled to the Mix/ll towards its con‘esponding target or epitope. {stress} in some embodiments, the coupling of Mh/ll educes the ability of the corresponding AB to bind its target or epitope such that the dissociation constant (lg) of the AB when coupled to the MMl towards its corresponding target or epitope is at least lOGG times greater than the Kd of the AB when not coupled to the Mia/ll towards its corresponding target or epitope.

Eiltltldéﬁ in some embodiments, the ng of MMl s the ability of the conesponding AB to bind its target or epitope such that the dissociation constant (lid) of the AB when d to the Mh’ll towards its corresponding target or epitope is at least lQﬂOO times greater than the Kd of the AB when not coupled to the MMl towards its corresponding target or epitope. {99978} In some embodiments, Mir/ll is an amino acid sequence selected from a MM shown in the Examples provided herein.

Elitltl’i’ll in some embodiments, the inultispeeilic activatahle antibody includes at least a second masking moiety (MME) that inhibits the binding of the A82 to its target when the multispecilie activatahle antibody is in an ve' state, and a second cleavahle moiety (CMZ) coupled to the A32, wherein the CM}: is a polypeptide that functions as a substrate for a second protease. in some embodiments, Cit/l2 is a polypeptide of no more than 15 amino acids long. in some embodiments, the second protease is co—localized with the second target or epitope in a tissue, and n the second protease s the CM2 in the multispecific activatable antibody when the multispecitic activatable antibody is exposed to the second protease. in some embodiments, the ﬁrst protease and the second protease are alized with the first target or e and the second target or cpitope in a tissue. in some embodiments, the first protease and the second protease are the same protease. in some embodiments, Civil and CMZ are ent substrates for the same se. in some embodiments, the protease is selected from the group consisting of those shown in Table 3. in some embodiments, the first protease and the second protease are different proteases, ln some embodiments, the ﬁrst protease and the second protease are different proteases selected from the group consisting of those shown in Table 3. {$3872} in some embodiments, each of the MM in the multispecifie activatable antibody, eg MM} and at least MME, has an equilibrium iation constant for binding to its corresponding AB that is greater than the equiiibrinm dissociation constant of the AB to its corresponding target or epitope {$3873} in some embodiments, each of the MM in the muitispeeiﬁc activatahie antibody has an equiiibriurn dissociation constant for binding to its corresponding AB that is no more than the equilibrium dissociation constant of the AB to its corresponding target or epitope.

EQtBtBM} in some embodiments, each of the MM in the muitispeciiic activatabie antibody does not interfere or compete with its corresponding AB for binding to the con‘esponding target or epitope when the rnuitispeciiie activatabie antibody is in a cieaved state: {8963753 in some ments, each of the MM in the rnuitispeciiic activatahie antibody is a polypeptide of about 2 to 40 amino acids in length. in some embodiments, each of the MM in the rnuitispecitic tabie antibody is a polypeptide of no more than 4() amino acids in iength.

Etititi’i’di In some embodiments, each of the MM in the ni'uitispeciiic activatabie antibody has a poiypeptide sequence that is different from that of target of the corresponding AB 73 in some embodiments, each of the MM in the speciiic activatahie antibody has a polypeptide sequence that is no more than 50% identicai to any naturai binding partner of the corresponding AB. in some embodiments, each of the MM in the muttispecitie aetivatabie dy has a polypeptide sequence that is no more than 25% identical to any naturai binding partner of the corresponding AB. in some embodiments, each of the MM in the ispecifie activatabie antibody has a poiypeptide sequence that is no more than 19% identicai to any naturai binding partner of the corresponding AB. {@9978} in some embodiments, the ng of each of the MM reduces the ahiiity of the corresponding AB to bind its target or e such that the dissociation constant (Kd) of the AB when coupled to the MM towards its corresponding target or epitope is at ieast 20 times greater than the rid of the AB when not conpied to the MM towards its corresponding target or e.

{BMW} In some embodiments, the coupiing of each of the MM s the ability of the corresponding AB to bind its target or epitope such that the dissociation constant (Kd) of the AB when coupled to the MM s its corresponding target or epitope is at ieast 4i) times greater than the lid of the AB when not d to the MM towards its corresponding target or epitope. ltlil} in some embodiments, the coupling of each of the MM reduces the ability of the corresponding AB to bind its target or epitope such that the dissociation constant (Kd) of the AB when coupled to the MM towards its corresponding target or epitope is at least 100 times greater than the Kd of the AB when not d to the MM towards its corresponding target or epitope.

Eiltitittl} In some embodiments, the ng of each of the MM s the ability of the corresponding A3 to bind its target or epitope such that the dissociation constant (Kl) of the AB when coupled to the Mh'l towards its corresponding target or epitope is at least ltltltl times greater than the Kd of the AB when not d to the MM towards its corresponding target or epitope, {@9982} In some embodiments, the coupling of each of the MM reduces the ability of the corresponding AB to bind its target or epitope such that the dissociation constant (lid) of the AB when coupled to the MM towards its corresponding target or epitope is at least lbﬁﬁtl times greater than the Kd of the AB when not coupled to the MM towards its corresponding target or epitope. {899833 in some embodiments, each of the MM is an amino acid sequence selected from a MM shown in the Examples provided herein. {99984} in some embodiments, the protease that cleaves the lirst eleayable moiety (CMl) sequence is co-—localized with the target of the ABl in the multispecitic activatable antibody in a tissue, and the protease cleaves the Civil in the multispeciﬁc activatahle antibody when the niultispeeit‘ic actiyatable antibody is exposed to the protease. {lllllltloi} in some embodiments, the niultispeeiﬁc activatable antibody es more than one cleavable moiety sequence, and the protease that cleaves at least one cleayable rnoiety sequence is co—localized with the target of at least one of the AB regions in the rnultispecitic activatahle dy in a tissue, and the protease s the CM in the multispecitic activatahie antibody when the inultis eci’tic actiyata‘oie antibody is e 'posed to the protease.

Etitltlliti} In some embodiments, each CM, e.g., CMl and at least CM2, is positioned in the mnltispeeitic table antibody such that in the uncleayed state, binding of the multispecilic activatahle dy to a target of one of the AB s is reduced to occur with an brium dissociation constant that is at least 20~told greater than the equilibrium iation constant of an unmodiﬁed AB binding to its target, and whereas in the cleaved state, the AB binds its target. lil’ll ln some embodiments, each CM is positioned in the pecitic activatahle antibody such that in the uncleaved state, binding of the multispeeiﬁc activatahle antibody to a target of one of the AB s is reduced to occur with an equilibrium dissociation constant that is at least zl-(lvfold greater than the equilibrium dissociation constant of an unmodified AB binding to its target, and whereas in the d state, the AB binds its target.

Eiltltlilii} in some embodiments, each CM is positioned in the multispeciﬁc activatable antibody such that in the uncleaved state, binding of the multispeciﬁc table antibody to a target of one of the AB regions is reduced to occur with an equilibrium dissociation constant that is at least 50—fold greater than the equilibrium dissociation constant of an urnnodilied AB binding to its target, and Whereas in the cleaved state, the AB binds its target, Etltltléiél} In some ments, each CM is positioned in the niultispecitlc activatable antibody such that in the uneleaved state, binding of the multispecitic activatable dy to a target of one of the AB regions is reduced to occur with an brium dissociation constant that is at least ltltlufold greater than the equilibrium dissociation constant of an ilied AB g to its target, and whereas in the d state, the AB binds its target, {@9998} In some embodiments, each CM is positioned in the inultispecii‘ic activatable antibody such that in the uncleaved state, binding of the multispecitic aetivatahle antibody to a target of one of the AB regions is reduced to occur with an equilibrium dissociation constant that is at least mil—fold greater than the equilibrium dissociation constant of an unmodified AB binding to its target, and Whereas in the cleaved state, the AB binds its target. {999%} in some embodiments, each CM in the multispecitic activatable antibody is a polypeptide of up to t5 amino acids in length. lilll} In some embodiments, at least one (M in the inultispecilic activatable antibody includes the amino acid sequence LSGRSDNH (SEQ ll?) NO: 26). in some embodiments, at least one cleavahle moiety is selected for use with a ic protease, for example a protease that is known to be co-iocalized with at least one target of the inultisnecitic activatable antibody. For example, suitable cleavable moieties for use in the nmltispecilie activatable antibodies of the disclosure are cleaved by at least a protease such as ase, ain, and/or rnatriptase (also refen‘ed to herein as MT-SPI or MTSEl ).

In some embodiments a suitable eleavable moiety includes at least one of the following sequences: ’l‘GRGPSWV (SEQ lD NO: 27); SARGPSRW (SEQ ID NO: 28); 'I'ARGPSFK (SEQ ll) N0: 29:); LSGRSDNE—I (SEQ ID NO: 26); RN (SEQ ID NO: 30); H’l‘GRSGAL (SEQ II) NO: 3 l); Pljl‘GRSGG (SEQ. ll) NO: 32); AIH (SEQ II) NO: 33); RGI’AE‘NI’M (SEQ Ii) NO: 34); SSRGI’AYL (SEQ ID NO: 35); RGI’A’I‘I’IM (SEQ ID NO: 36); RGPA (SEQ ID NO: 37); Gili'QPsili'h/l‘th’ili'ii’ (SEQ ID NO: 38); FERPLGITGL (SEQ II) N0: 39‘); FLGP (SEQ ll?) NO: 40); SPLTGRSG (SEQ II) NO: Ill); SAGFSLPA (SEQ ll) NO; 42); IAAPIerIsQRR (SEQ ll) NO: 43); SGGI’LGVR (SEQ ID NO: 44); and/or l’LGL (SEQ ID NO: 45). {9999333 In some ments, each Cit/l, in the mnitispeciﬁc aetivatahle antibody is a substrate for a protease selected from the group consisting of those shown in Table 3. In some ments the protease is selected from the group consisting of nPA, legumain, MT—Sl’l, ADAMW, Bh/lP—l, TMI’RSS3, TMPRSSL‘l, nentrophii se, MMRI MMP—9, MMP—l 2, Mix/IRIS, and MMILI 4. In some embodiments, the protease is a eathepsin, such as, but not limited to, cathepsin S. In some embodiments each CM in the inultispecific activatable antibody is a substrate for a protease selected from the group consisting of nPA (nrokinase piasminogen activator), legnmain and MT—SEl (inatriptase). In some embodiments, the protease comprises tiPA. In some en'ibodiments, the protease comprises in. In some embodiments, the protease comprises lVl’IlSPli In some embodiments, the protease comprises a matrix metalloproteinase (ls/IMP). lga’l} In some embodiments, at least one CM in the rnnltispecitic aetivatabie antibody is a substrate for at least two proteases. In some embodiments eaeli protease is selected from the group consisting of those shown in Table 3. In some embodiments at least one CM in the nmltispecilie activatable antibody is a substrate for at least two proteases, wherein one of the proteases is selected from the group consisting oi’uPA, legumain and IVI’I‘—SI)l and the other protease is selected from the group consisting of those shown in Table 3. in some embodiments, at least one CM in the mnitispeciﬁc aetivatahle antibody is a ate for at least two proteases selected from the group consisting of uPA, legumain and ’l. {899953 in some embodiments, the multispeciﬁc activatable dy includes at least a ﬁrst CM (CMI i) and a second CM (CMZ). In some embodiments, Civil and CMQ are part ofa single cleavable linlter that joins an MM to an AB. in some embodiments, CMl is part ofa cleavable r thatjt’iins MMl to ABl and CM2 is part of a separate cleavahle linker that joins an MM2 to ABE. in some embodiments, a nniltispecific activatable antibody comprises more than two CMs. in some embodiments, such a specific activatable antibody comprises more than two CMs and more than two MMs. in some embodiments, CMl and CM‘Z are each polypeptides of no more than 15 arnino acids long. In some embodiments, at least one of the first CM and the second CM is a polypeptide that functions as a substrate for a protease selected from the group consisting ofthose listed in Table 3. in some embodiments, at least one of the first CM and the second CM is a polypeptide that ons as a substrate for a protease selec cd from the group consisting of ul’A, legumain, and M'li-Sl‘ll. in some embodiments, the first CM is cleaved by a first cleaving agent selected from the group consisting of ul?A, leguinain, and MT—SPl in a target tissue and the second CM is cleaved by a second cleaving agent in a target tissue, ln some ments, the other protease is selected from the group consisting ofthose shown in Table 3. in some embodiments, the first cleaving agent and the second cleaving agent are the same protease selected from the group consisting of those listed in Table 3, and the first CM and the second CM are different substrates for the enzyme, in some embodiments, the first cleaving agent and the second cleaving agent are the same se selected from the group consisting oful’A, ain, and MT—SPl and the ﬁrst CM and the second CM are dill‘erent ates for the enzyme. in some embodiments, the first cleaving agent and the second cleaving agent are the same protease selected from the group listed in Table 3 and the first CM and the second CM are the same substrate. in some embodiments, the first ng agent and the second cleaving agent are different proteases, in some embodiments, the first cleaving agent and the second cleaving agent are different ses selected from the group consisting of those shown in Table 3. in some embodiments, the first cleaving agent and the second cleaving agent are co—localized in the target tissue. in some ments, the first CM and the second CM are cleaved by at least one cleaving agent in the target tissue. liltlllfld} in some embodiments, the inultispecific activatahle antibody is exposed to and cleaved by a protease such that, in the activated or cleaved state, the activated niultispecilic activatahle antibody includes a light chain amino acid sequence that includes at least a portion of L92 and/or CM sequence after the protease has cleaved the CM.

Etltltig’l’} in some embodiments, the rnultispeeilic activatable antibody also includes a signal peptide. in some embodiments, the signal peptide is conjugated to the pecitic activatable antibody via a spacer. in some embodiments, the spacer is conjugated to the multisbecific activatable antibody in the absence of a signal peptide. in some embodiments, the spacer is joined directly to at least one of the MM of the multispecitic actiyatabie antibody. {tillmd} in some embodiments, the mnltispecitic activatable antibody in an uncleaved state comprises a spacer that is joined directly to a tirst MM and has the structural ement from N—terminus to (fl—terminus of —MMl SL in some embodiments, the spacer includes at least the amino acid sequence QGQSGQ (SEQ. ll) NO: 46). {dilllggl in some embodiments, the serum half—lite of the multispecil’ic activatabie antibody is longer than that of the corresponding multispecitic antibody; e.g., the {iii of the mnltispecitic activatable antibody is longer than that of the ponding mnltispecific antibody. in some embodiments, the serum half—life of the ninltispecil‘ic table antibody is similar to that of the corresponding nmltispecitie antibody. in some ments, the serum half—life of the innltispeeitie activatable antibody is at least l5 days when administered to an organism. in some embodiments, the serum half-life of the multisnecitic activatable antibody is at least l2 days when administered to an organism. in some embodiments, the serum hall'liite of the multispecitie activatable antibody is at least l l days when ste ed to an organism, in some ments, the serum half—life of the mnltispecitic table antibody is at least l0 days when administered to an organism. in some embodiments, the serum haltllit‘e of the multispecitic activatable antibody is at least 9 days when administered to an organism. in some embodiments, the serum half~life of the multisbecific activatable antibody is at least 8 days when administered to an organism. in some embodiments, the serum hall‘llife of the mnltispecitic activatable antibody is at least 7 days when administered to an organism. in some embodiments, the serum half—life of the iniiltispeei’rie activatable antibody is at least 6 days when administered to an organism. in some embodiments, the serum half—life of the multispecitic activatable antibody is at least 5 days when stered to an organism. in some embodiments, the serum half—life of the maltispecifie activatable antibody is at least 4 days when administered to an sm. in some embodiments, the serum half-life of the mnltispecitic table antibody is at least 3 days when administered to an organism. ln some embodiments, the serum haililife of the mnltispecilie actiy'atabie antibody is at least 2 days when administered to an organism. in some ments, the serum haltllife of the innltispecitic activatable antibody is at least 24 hours when administered to an organism. in some embodiments, the serum i "e of the peeific activatable antibody is at least 2.0 hours when administered to an organism. in some embodiments, the serum half—life oi‘the pecitic activatable antibody is at least l8 hours when administered to an organism. in some embodiments, the serum half— li ‘e of the multispecifie activatable antibody is at least l6 hours when administered to an organism. in some embodiments, the serum half—lite of the multispecilic activatable dy is at least l4 hours when administered to an organism. in some embodiments, the serum half—life of the inititispeciiic aetivatable antibody is at least 12 hours when administered to an organism. in some embodiments, the serum half-iife of the multispecifie aetivatable antibody is at least it) hours when administered to an organism. in some embodiments, the serum halillitl: of the multispeciiic aetiyatable dy is at least 8 hours when administered to an organism. in some embodiments, the serum half-life of the mnltispecilie actiyatable antibody is at least 6 hours when administered to an organism. in some embodiments, the serum haltllife of the inultispecitic activatable antibody is at least 4 hours when administered to an organism. in some embodimentsﬂ the serum half—life of the mnitispecific activatable antibody is at least 3 hours when administered to an organism. {ddiillliil The disclosure also provides compositions and methods that include a innltispceitic activatable antibody that es at least a iirst antibody or antibody llagment (ABl) that speciﬁcally binds a target and a second antibody or antibody fragment (AB2) where at least the first AB in the mnltispecitic aetiyatabie antibody is coupled to a mashing moiety (MMl) that decreases the ability ofABl to bind its target. in some embodin'ients, each AB is coupled to a MM that decreases the ability of its corresponding AB to each target. for e, in bispecific activatable dy ments, ABl is coupled to a, lirst mashing moiety (Mix/ll) that decreases the ability ot‘ABl to bind its target, and A132 is coupled to a second mashing moiety (MMZ) that decreases the ability of ABE to bind its target. in some embodiments, the multispeeiﬁe activatable antibody comprises more than two AB regions; in such embodiments, ABl is coupled to a lirst mashing moiety (Mix/ll) that decreases the ability of ABl to bind its target, A32 is coupled to a second mashing moiety (Mb/l2) that decreases the ability of A82 to bind its target, A83 is coupled to a third mashing moiety l3) that ses the ability of AB3 to bind its target, and so on for each AB in the pecilie activatable antibody. (,4 1,49 liltltlltll} in some embodiments, the specitic actiyatable antibody turther es at least one cleavable moiety (CM) that is a substrate for a protease, where the CM links a MM to an AB. For example, in some embodiments, the mnltispecific activatable antibody includes at least a first antibody or antibody fragment (A31) that specifically binds a targe and a second antibody or antibody fragment (AB2), where at least the tirst AB in the multispecific table antibody is couple l via a first cle' vable moiety (CMl) to a masking moiety (MMl) that decreases the ability of A81 to bind its target. in some bispecilic tabie antibody embodiments, ABl is coupled via CMl to MMl, and A82 is coupled via a second cleavable moiety (CM?) to a second mashing moiety (MMZ) that decreases the ability of A32 to bind its target. In some embodiments, the multispecific activatable antibody comprises more than two AB regions; in some of these embodiments, ABl is coupled via CMl to l, A82 is coupled Via Cit/l2 to MMZ, and A33 is coupled via a third cleayable moiety (CM3) to a third mashing moiety (MMS) that decreases the ability of A83 to bind its target, and so on for each AB in the multispecitic activatable antibody. lithium} The compositions and methods provided herein enable the attachment of one or more agents to one or more cysteine es in any of the AB s without compromising the activity (eg, the masking, activating or binding activity) of the multispecitic activatable dy. in some embodiments, the compositions and methods provided herein enable the attachment of one or more agents to one or more ne residues in any of the AB regions without reducing or oth em'ise disturbing one or more disulfide bonds Within any of the MM. The compositions and methods provided herein produce a nniltispecitic aetivatable antibody that is conjugated to one or more agents, ex ., any of a variety of therapeutic, diagnostic and/or prophylactic agents, preferably without any of the ageiit(s) being conjugated to any of the MM of the multispecitic table antibody. The compositions and methods provided herein produce conjugated inultispecitic activatable antibodies in which each of the MM retains the ability to effectively and efficiently mash its corresponding AB of the niultispecifie table antibody in an ved state. The compositions and methods ed herein produce conjugated multispecitic activatable antibodies in which the activatable antibody is still activated, lien, cleaved, in the ce of a protease that can cleave the CM. {999193} The multispecific activatable antibodies have at least one point of ation for an agent, but in the methods and compositions provided herein less than all possible points of conjugation are ble for conjugation to an agent. In some embodiments, the one or more points of conjugation are sulfur atoms involved in disullide bonds. ln some ments, the one or more points of conjugation are sulfur atoms involved in interchain disul’lide bonds. in some embodiments, the one or more points of conjugation are sulfur atoms: involved in interchain sulﬁde bonds, but not sulfur atoms involved in hain disuliide bonds, in some embodiments, the one or more points of conjugation are sulfur atoms of cysteine or other amino acid residues containing a sulfur atom. Such residues may occur naturally in the antibody structure or may be incorporated into the an tihody by site—directed mutagenesis, cheinical conversion, or niis~ineorporation of non~natural amino acids, {0630194} Also provided are methods of preparing a ate of a multispecitic activatahle antibody having one or more hain dis uliide bonds in one or more of the AB and one or more intrachain dis ulfide bonds in the corresponding MM, and a drug reactive with free thiols is provided. The method generally includes partially reducing interchain disullide bonds in the activatahle antibody with a reducing agent, such as, for example, TCEP; and conjugating the drug reactive with free thiols to the partially reduced activatable antibody. As used herein, the term partial reduction refers to situations where a multispecific activatahle antibody is ted with a, reducing agent and less than all disultide bonds, cg, less than all possible sites ofconjugation are reduced. in some embodiments, less than 99%, 989/5, 97%, 96%, 95%, 909/6, 85%, 80%, 759/5, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, l5%, l0% or less than 5% of all possible sites of conjugation are reduced. lillllllllS} in yet other embodiments, a method of reducing and conjugating an agent, eg, a drug, to a multispecitic activatable antibody ing in selectivity in the placement of the agent is provided. The method generally includes partially reducing the multispecific activatahle antibody with a reducing agent such that any ation sites in any of the masking es or other non—AB portion of the activatable antibody are not d, and conjugating the agent to interchain tliiols in one or more of the AB regions of the multispecilic activatahle antibody. The conjugation site(s) are selected so as to allow desired placement of an agent to allow conjugation to oce ur at a desired site. The reducing agent is, for example, 'l'CEl’, The reduction reaction ions such as, for example, the ratio of ng agent to activatable antibody, the length of incubation, the temperature during the incubation, the pH of the reducing reaction on, etc, are determined by identifying the conditions that produce a eoniugated aetiyatab1e antibody in which the MM retains the abi1ity to et‘t‘eetiye1y and efﬁcientiy mask the AB of the aetiyatab1e antibody in an une1eayed state, The ratio of reduction agent to peeitie aetiyatab1e dy wi11 vary depending on the aetiyatab1e antibody. in some embodiments, the ratio ofredueing agent to inu1tispeeiftc aetiyatah1e antibody W111 be in a range from about 20:1 to 1 :1, from about 111:1 to 1:1, from about 9:1 to 1:1, from about 8:1 to 1:1, from about 7:1 to 1:1, from about 6:1 to 1:1, from about 5:1 to 1:1, from about 4:1 to 1:1, from about 3:1 to 1:1, from about 2:1 to 1:1, from about 20:1 to 1:15, from about 10:1 to 121.5, from about 9:1 to 1:15, from about 8:1 to 1:15, from about 7:1 to 1 :1.5, from about 6:1 to 1:1.5, from about 5:1 to 1:15, from about 41:1 to 1:15, from about 3:1 to 1:15, from about 2:1 to 1:15, from about 1.521 to 121.5, or from about 1:1 to 1:15. 1n some embodiments, the ratio is in a range of from about 5:1 to 1:1. 1n some embodiments, the ratio is in a range oft‘rom about 5:1 to 1.5: 1. in some embodiments, the ratio is in a range of from about 4:1 to 1:1. 1n some embodiments, the ratio is in a range from about 4:1 to 1.5: 1. In some embodiments, the ratio is in a range from about 8:1 to about 1:1. 1n some embodiments, the ratio is in a ran{70 A of from about 2.5:1 to 1:1. {1111111116} 111 some embodiments, a method of reducing interehain disu1tide bonds in one or more of the AB regions of a niu1tispeeifie aetiyatab1e antibody and conjugating an agent, e.g., a containing agent such as a drug, to the resu1ting interchain thio1s to se1ectiye1y 1oeate agent(s) on the AB is provided. The method genera11y ine1udes partia11y redueing one or more of the AB regions with a reducing agent to form at 1east two interehain thio1s Without forming a11 possib1e interehain thiois in the aetiyatab1e antibody; and ating the agent to the hain thio13 of the partia11y reduced AB. For exarnp1e, one or more of the A13 regions of the muitispeeiﬁe aetivatab1e antibody is partia11y reduced for about 1 hour at about 370C at a desired ratio of reducing otivatabie antibody. 1n some embodiments, the ratio of reducing agent to aetiyatab1e dy wi11 he in a range from about 20:1 to 1:1, from about 10:1 to 1:1, from about 9:1 to 1:1, from about 8:1 to 1:1, from about 7:1 to 1:1, from about 6:1 to 1:1, from about 5:1 to 1:1, from about 4:1 to 1 :1, from about 3:1 to 1:1, from about 2:1 to 1:1, from about 211:1 to 1:15, from about 10:1 to 1:15, from about 9:1 to 1:1.5, from about 8:1 to 1:15, from about 7:1 to 1:15, from about 6:1 to 1:15, from about 5:1 to 115, from about 4:1 to 1:1.5, from about 3:1 to 1:1.5, from about 2:1 to 115, from about 1.5:1 to 111.5, or from about 1:1 to 1:15. 1n some embodiments, the ratio is in a range of from about 5:1 to 1:1. 1n some embodiments, the ratio is in a range of from about 5:l to l5: l. In some embodiments, the ratio is in a range ot‘from about 4:l to l: l. in some ments, the ratio is in a range from about 4M to l.5:l. ln some embodiments, the ratio is in a range from about 8:l to about lzl. ln some embodiments, the ratio is in a range ol‘from about 2.521 to l:l. {dtltllll‘l’i The thiol—eontaining reagent can be, for example, cysteine or N—acetyl cysteine. "l‘he reducing agent can be, for example, 'l‘ClELP. in some embodiments, the reduced activatable antibody can be purified prior to conjugation, using for example, column tography, dialysis, or diai’iltration. atively, the reduced antibody is not purified alter partial reduction and prior to conjugation. {tlllhliltl} The disclosure also provides partially reduced peeit‘ic activatable antibodies in which at least one interchain disulfide bond in the multispecitic activatable antibody has been reduced with a reducing agent without disturbing any intrachain disuitide bonds in the rnuitispecilic table antibody, wherein the pecitic activatable antibody includes at least a first antibody or an antigen binding fragment thereof (A81) that speciﬁcally binds to a target, a lirst masking moiety (MM 1) that inhibits the binding of the ABl of the multispecilic activatable dy in an uncleaved state to the target, a ﬁrst eleavable moiety ) coupled to the ABl, wherein the Civil is a polypeptide that functions as a substrate for a protease, and a second dy or an antigen binding fragment thereof (ABE) that speciﬁcally binds to a second target. ln some embodiments, the MMl is coupled to the ABl via the Civil. ln some embodiments, one or more intrachain disultide bondt’s) of the multispecitic activatable antibody is not disturbed by the reducing agent. ln some embodiments, one or more intrachain disultide bonds) of the MMl Within the multispecilic table antibody is not disturbed by the reducing agent. in some embodiments, reducing agent is "l‘CEl’. {9639199} ln some embodiments, the multispecitic antibodies and/or multispecific activatable antibodies bed herein are used in conjunction with one or more additional agents or a combination of onal agents. Suitable additional agents include current pharmaceutical and/or surgical therapies for an intended ation, such as, for example, cancer. For example, the nrultispeeitie dies and/or multispecil‘ic aetivatable antibodies can be used in conjunction with an additional chemotherapeutic or anti— neoplastic agent. {titlillltl} ln some embodiments, the multispecitic antibody and/or multispecific activatable antibody and onal agent are formulated into a single therapeutic composition, and the multispecilic dy and/or multispecilic activatahle antibody and additional agent are administered simultaneously. Alternatively, the tnultispecitic antibr’idy and/or multispecitic table antibody and additional agent are separate from each other, e.g, each is formulated into a separate therapeutic composition, and the multispecific antibody and/or multispecilic table antibody and the additional agent are administered simultaneously, or the multispecific antibody and/or niultispeciﬁc activatahle antibody and the additional agent are administered at different times during a treatment regimen. For example, the rnultispecitic antibody and/or multispecilic activatable antibody is administered prior to the administration of the additional agent, the rnultispecilic antibody and/or mu ltis c activatable antibody is administered subsequent to the administration of the additional agent, or the multispecitic dy and/or multispecific activatable antibody and the additional agent are administered in an alternating fashion. As described herein, the anti— rnultispeeilic antibody and/or tnnltispecitic activatable antibody and additional agent are administered in single doses or in multiple doses.

Ellllllllll The disclosure also provides an ed nucleic acid molecule encoding a multispecilie antibody and/or multispecilic activatable antibody bed , as well as vectors that include these isolated nucleic acid sequences. The disclosure provides s of producing a multispecilic dy and/or multispecilic activatable dy by culturing a cell under conditions that lead to expression of the multispecilic antibody and/or i'nnltispecitic activatable antibody, wherein the cell ses such a nucleic acid molecule, in some embodiments, the cell ses such a vector.

{Milli 12} The disclosure also provides a method of cturing multispecific antibodies of the disclosure and/or rnnltispecitic activatable antibodies of the diselos are by (a) culturing a cell comprising a nucleic acid construct that encodes the multistecifie antibody and/or multispecific activatable antibody under conditions that lead to expression of the multispecitic antibody and/or multispecil‘ic activatable, and (b) recovering the innltispecitic antibody and/or rnultispecilic activatable antibody {$89113} The present disclosure also provides methods of ng, preventing, delaying the progression of or otherwise ameliorating a symptom of one or more pathologies or alleviating a symptom associated with such pathologies, by administering a multispecitic antibody and/or multispecitic activatable dy of the disclosure to a subject in which such treatment or prevention is desired. The t to he treated is, eg, human or other mammal. in some embodiments, the t is a non—human mammal, such as a non—human primate, companion animai (cg, cat, dog, horse), farm animal, worlt animal, or zoo animal in some embodiments, the subject is a rodent, {tidbit-4} The present disclosure also es methods to induce target—dependent T— cell activation and kiiling of a target cell by administering a multispecitic activatable antibody of the disclosure to a subiect in which such induction is desired, wherein when the iniiitispettiﬁe table dy is in the cleaved state, e.g,, each masking moiety in the muitispecific table antibody is no longer attached or otherwise associated with the corresponding AB , target~dependent T—cell activation and killing of the target cell occurs, and when the multispecitic table antibody is in the uncleaved state, eg. at least one masking moiety of the innitispecitie activatable antibody is attached or otherwise associated with the corresponding AB domain, target-dependent T-vceli activation and killing of the target cell is reduced or otherwise inhibited. Any of the niultispecitic activatable antibodies bed herein are suitable for use in such methods. The subject to be treated is, eg, human or other mammal. in some embodiments, the subject is a non-- human mammal, such as a non—human primate, companion animal (cg, cat, dog, horse), farm animal, work animal, or zoo animal. in some embodiments, the subject is a rodent, {$883.15} A multispecitic antibody and/or multispeciiic activatabie antibody used in any of the embodiments of these methods and uses can be administered at any stage of the disease and/or any stage in which l—ceil activation and g ofa target cell is desired. For example, such a multispecitic antibody and/or multispeciiic table antibody can be administered to a patient suffering cancer of any stage, from early to atic, The terms subject and patient are used interchangeably herein. A multispecific dy and/or multispecitic activatable antibody used in any of the embodiments of these methods and uses can be used in a treatment regimen comprising neoadjuvant therapy, A multispecitic antibody and/or muitispecific activatabie antibody used in any of the embodiments of these methods and uses can be administered either alone or in ation with one or more additional agents, inciuding smail molecule inhibitors, other antibody—based therapies, polypeptide or peptide—based therapies, nucleic acid—based therapies and/or other ics Etititiiiti} The disclosure also provides methods and kits for using the peciﬁc antibody and/or muitispeciiic activatable antibodies in a variety of diagnostic and/or prophylactic tions For example, the sure provides methods and kits for detecting presence or absence of a cleaving agent and a target of interest in a subject or a. sample by (i) contacting a subiect or sample with a inultispecilic activatable antibody that includes at least a ﬁrst masking moiety (MIX/ll), a lirst cleavable moiety (Ch/ll) that is cleaved by the cleaving agent, and at least a ﬁrst n binding domain or fragment thereof (ABl) that specifically binds the target of interest and a second antigen binding domain or fragment thereof (A32) that ically binds a second target and/or a second epitope, (a) wherein the i‘vllvll is a peptide that inhibits binding of the ABl to the target, and wherein the Mir/ll does not have an amino acid sequence of a naturally occurring hin ding partner of the ABl and is not a modiﬁed form of a natural binding partner of the A81; and (b) wherein, in an ved, tivated state, the i‘vllvll interferes with specilic binding of the ABl to the , and in a cleaved, activated state the MMl does not interfere or compete with specific binding of the ABl to the target; and (ii) measuring a level of ted multispecitic activatable antibody in the subject or sample, wherein a. detectable level of activated multispecilic activatable antibody in the t or sample tes that the cleaving agent and the target are present in the subject or sample and wherein no detectable level of activated multispecific activatable antibody in the t or sample indicates that the cleaving agent, the target or both the cleaving agent and the target are absent and/or not sufﬁciently present in the sub} ect or . {$388117} ln some embodiments, the activatable multispecitic activatable antibody is an activatable multispecific activatable antibody to which a eutic agent is conjugated. in some embodiments, the activatable multispecilic activatable antibody is not coniugated to an agent. in some embodiments, the activatable rnultispecil'ic table antibody comprises a de ectable label. in some embodiments, the detectable label is positioned on the A31. in some embodiments, measuring the level of activatable multispecitic activatable antibody in the subject or sample is accomplished using a secondary reagent that specifically binds to the activa ,ed multispecific activatahle antibody, wherein the reagent comprises a detectable label. in some embodiments, the ary reagent is an antibody comprising a detectable label. ltllllll ill} in some embodiments of these methods and hits, the activatable multispecil‘ic activatahle antibody includes a detectable label. In some embodiments of these methods and hits, the able label includes an imaging agent, a sting agent, an , a ﬂuorescent label, a chrornophore, a dye, one or more metal ions, or a ligandbased label. in some embodiments of these methods and hits, the imaging agent comprises a radioisotope, in some embodiments of these methods and hits, the radioisotope is indium or technetium. in some embodiments of these methods and hits, the contrasting agent comprises iodine, gadolinium or iron oxide. in some embodiments of these methods and hits, the enzyme comprises horseradish peroxidase, alkaline atase, or li—galactosidase. in some embodiments of these methods and hits, the cent label comprises yellow fluorescent protein (Y’Fl’), cyan ﬂuorescent protein (CFP‘ ﬂuorescent protein (GEP), modiﬁed , green red lluorescent protein (mRFP), red cent protein tdimeer (RF? tdiiner2), D, or a europium derivative. ln some embodiments of these methods and hits, the scent label comprises an N— acrydium derivative. ln some embodiments of these methods, the label comprises an Alexa Fluor® label, such as Alex Fluor® 680 or Alexa Fluor® 750. ln some embodiments of these methods and hits, the ligand~based label comprises biotin, avidin, streptayidin or one or more haptens. {999119} ln some embodiments of these methods and hits, the subject is a mammal. in some embodiments of these methods and hits, the t is a human. in some ments, the subject is a non—human mammal, such as a man primate, companion animal (eg, cat, dog, horse), farm animal, work animal, or zoo animal. in some ments, the subject is a rodent. liltilllle} in some embodiments of these methods, the method is an in viva method. in some embodiments of these methods, the method is an in site method. in some embodiments of these methods, the method is an ex vivo method. in some ments of these methods, the method is an in viz‘ro method. {999121} in some embodiments of the methods and hits, the method or hit is used to identify or otherwise reline a patient population suitable for treatment with a multispecilic activatable antibody of the disclosure. for example, patients that test positive for both the target and a protease that cleaves the ate in the first cleavable moiety (Civil :3 of the multispecilie table antibody being tested in these methods are identified as suitable ates for treatment with such a, multispecific activatable antibody comprising such a CMl. se, patients that test negative tor both the target and the protease that cleaves the substrate in the Civil in the peciiic activatable antibody being tested using these methods might be identili ed as suitable candidates for another form of therapy, liltitllZZ} in some embodiments, a method or hit is used to identity or otherwise rel’ine a patient population suitable for treatment with a multispecilie activatable antibody and/or eonjugated multispecitic aetivatable antibody (eg, multispeeifie activatable antibody to which a therapeutic agent is conjugated) of the disclosure, followed by treatment by administering that multispecil’ic antibody and/or multispecitic activatable antibody and/or conjugated multispecitic activatable dy to a subject in need thereof. For example, patients that test positive for both the target and a protease that cleaves the substrate in the first cleavable moiety (Ch/ll) of the niultispecitic activatahle antibody and/or conjugated pecific activatable antibody being tested in these methods are identiﬁed as suitable candidates for treatment with a inultispecilic activatable antibody comprising such a Civil and/or ated multispecific activatahle antibody sing such a Chill, and the t is then administered a therapeutically effective amount of the multispecific activatable antibody and/or coniugated inultispecitic activatahle dy that was tested. likewise, patients that test negative for either or both of the target and the protease that cleaves the substrate in the Civil in the niultispecitic activatable antibody and/or conjugated multispecific activatable antibody eing tested using these methods might be identified as suitable candidates for another form of therapy. {999123} in some embodiments, such patients can he tested with other multispecifie antibodies and/or niultispecific activatable antibodies and/or conjugated multispecific activatable antibodies until a suitable multispeciiic activatable dy and/or conjugated multispecifie activatahle antibody for treatment is identiﬁed, eg, a multispecitic table antibody and/or conjugated niultispeciﬁc activatable antibody comprising a CM that is cleaved by the t at the site of disease. in some embodiments, the patient is then administered a therapeutically effective amount of the multispecific activatable antibody and/or conjugated rnultispecil'ic activatahle dy for which the patient tested positive. {earn 24} Pharmaceutical compositions according to the sure can include a multispecific antibody and/or a. multispecitic tahle antibody of the disclosure and a carrier. These pharmaceutical crnnpositions can be included in ltiiS, such as, for example, diagnostic hits, {899125} One skilled in the art will appreciate that the antibodies of the disclosure have a variety ot‘uses. F "ir example, the proteins of the disclosure are used as therapeutic agents for a variety of disorders. The antibodies of the disclosure are also used as reagents in diagnostic kits or as diagnostic tools, or these antibodies can be used in competition assays to generate therapeutic ts.

BRlEF DESCREPTHEN OF GS Etitltliiﬁti} Figure l is an illustration depicting various il‘ic antibody forrnats t d from Chan and Carter, Nat. Rev, lmmunol. auto).

EiititBEZ’Z’} Figures 2A~2C are a series of iiiustrations ing various muitispecil‘ic antibody formats suitable for use in the compositions and methods of the disciosure. in a bispecific antibody, a scFv can be fused to the carboxyi terminus of the heavy chain (He) of a igG antibody (Figure 2A); to the carboxyi-terminus of the Eight chain (Le) (Figure 2B); or to the yl us ot‘both the heavy and tight chains (Figure 2C). Figure 23 is a diagram of a singie genetic construct for the expression of muitispecitic antibody fusions" {ﬂitting} Figure 3A is a series oi‘iiiustrations ing various multispeeitic activatable antibody formats suitable for use in the itions and methods of the disclosure Figure 33 is a diagram of c constructs for the expression of n'ruitispecitic activatabie antibody fusions {8639129} Figure 4,», Panels Aid, are a series of schematic diagrams of a seiected set of the possible permutations oi‘muitispecitic antibodies of the disclosure. haded boxes cover the conﬁgurations represented in Figure 2 and are included here for con'rpleteness and comparison.

Etitititfﬁb} Figure 5, Panels A—J, are a series of schematic diagrams of a ed set of the possibie permutations of muitispecitic activatable antibodies of the disclosure. Gray— shaded boxes cover the conﬁgurations represented in Figure 3 and are inchided here for teness and comparison. in particuiar, this ﬁgure shows muitispecific activatable antibodies in which the primary antigen binding site is masked (i.e., activatable) and the additional antigen-binding domaint’s) is not masked {$630131} Figure 6;, Panels A—L are a series of schematic diagrams of an array of muitispecific activatable dies in which ail antigen-binding domains are masked. {iiiiiiBE} Figure 7, Panels A J, are a series of schematic ms of an array of muitisteciiic activatable antibodies in which the secondary antigen—binding domain is masked and the onai antigen-binding domaint’s) is not masked.

Eddﬂifidi Figure 8, Panels Ad, are a series of schematic diagrams of an array of rnuitispecii‘ic activatable antibodies in which the majority/9 but not ali of the n—binding domains are masked and at least one additional antigen—binding domain(s) is not mashed.

EiititilM} Figure 9, Panels A—D, are a series of schematic diagrams of an array of multispeciiie activatable antibodies in which the primary antigen—binding domain and another anti nding domain are masked, and the remaining antigen~binding doniain(s) is not masked. #-1,..9 Figure 10 is a photograph depicting PAGE analysis of multispecific antibodies and pecific activatable antibodies demonstrating the molecular weights of the scFv fused heavy chains, samples 1-8, as compared to the respective monospecific antibodies or activatable antibodies, samples 9-11.

Figure 11 is a graph depicting the ability of pecific antibodies to bind specifically to their cognate ns.

Figures 12A-12B are a series of graphs depicting the ability of pecific antibodies and multispecific activatable antibodies that include anti-human CTLA-4 scFvs to bind specifically to (A) mouse and (B) human CTLA-4.

Figure 13 is a graph depicting the ability of the OKT3-containing multispecific activatable antibodies ed to herein as anti-EGFR multispecific activatable antibody 3954C225v5—OKT3 and anti-Jagged multispecific activatable dy 53424D11v2—OKT3 to specifically bind human CD3ε in ELISA binding experiments.

Figure 14 is a series of photographs ing PAGE analysis of uPA digests demonstrating activation of multispecific activatable antibodies without cleavage of heavy chain scFv fusions.

Figure 15 is a series of graphs depicting the ability of activated pecific activatable antibodies to bind various s.

Figure 16A is a graph depicting binding of multispecific antibodies of the disclosure to CD3-positive Jurkat T cells.

Figure 16B is a graph ing co-binding of multispecific antibodies of the disclosure to CD3-positive Jurkat T cells and EGFR.

Figure 17 is a series of graphs depicting EGFR-dependent tion of Jurkat T cells by multispecific antibodies of the disclosure.

Figure 18A is a graph depicting EGFR-dependent activation of primary CD8+ T cells by multispecific antibodies of the disclosure.

Figure 18B is a graph depicting EGFR-dependent killing of target cells by multispecific antibodies of the disclosure. 6] Figure 18C is a series of graphs depicting CD69 tion of primary T cells by multispecific antibody C225v5-OKT3m-H-N in EGFR-expressing SW480 cells compared to minimal activation in EGFR-negative U266 cells.

Etititildﬂ Figure lbD is a series of graphs depicting EGFR—dependent g of target cells by rnuitispeeitic antibody CZZSVS—OKTﬁ'n—PLN in EGFR—expressing SWdSG cells compared to negligible killing in EGFR~negative U266 cells. 8} Figure l9 is a series of graphs depicting priinaiy 'l‘ ceil lysis of a panel of EGFR—expressing cell lines by nrultispecitie antibody €225V5—0KT3ﬂl—H~N compared to there being no lysis of the ROPE—negative U266 cell line.

EGtBtBMQE Figure 2tiA and Figure 205 are a series of graphs depicting decreased binding of inultispecifie activatable antibody 204—(3225V5—Ol{T3ni~H—N to EGFR~ expressing cells cornpared to EGFR binding by ntultispeeit‘ie antibody CZZSVS—OKUrnJE— N Furthermore, Figures 29A and 2GB demonstrate that protease activation of specitic activatable antibody 3954-12tl4-{7225VS-vOK'l‘3rn--ii--N by rnatriptase (also referred to herein as l‘s/lTSPl or MT—SPI ) restores EGFR binding to a ievel equivalent to that exhibited by rntdtispecitic antibody C22SVS—OKT3m-PLN. Figures 20A and ZOB also demonstrate that such niultispecific activatable antibody and rnuitispeciiic antibody bind EGFR in a manner sirniiar to that of the tive pecitic activatable antibody and inonospecilﬁic antibody. {bilillﬁtl} Figure LiiA and Figure 218 are a series of graphs depicting binding of rnultispecific activatable antibody 3954"lZiitil-{jZZSVS-'Oi ElliiiiiS3} Figure 233 is a graph depicting sed EGFR—denendent killing oftai‘get cells by rntiitispecilic activatable antibody 3954—i 25VS-OKTSm—H—N compared to g by rnnltispecific antibody C225v5—0K'T3m—H~N, rmore, the figure demonstrates that protease activation of niultisnecific table antibody 3954- CQZSvS—OK'Hmd-l—N by iptase restores EGFR~denendent target cell killing to a level equivalent to that exhibited by mnitispecilie antibody C225v§ —0i The sure also includes nrultispecific activatable dies that se another immune effector cell engaging antibody or fragment thereof, such as one that binds a surface antigen of a natural killer (NK) cell, a mononuclear cell, such as a myeloid mononuclear cell, a macrophage, and/or other immune or cells. ll 633 in some embodiments, the pecific activatable antibody or fragment thereof of the disclosure includes at least ti) two T-cell ng antibodies or antibody fragments thereof that engage one or more T cell activating receptors, such as, for e, T~cell ng scFv fragments, including but not limited to, 0X4tl/GlTR, CDl3'7/ GITR, CDlS7/OX40, CD27/NKGZD, and additional combination of activating receptors, where the A31 of one of the "ll—cell engaging antibody fragments is attached to a masking moiety (MM) such that coupling of the MM reduces the ability of that A81 to bind its respective T-- cell engaging target, and 4: ii) one ~binding antibody such that the T—cell engaging scFv fragments are linl Eilllllldﬁ} In some ments, the inultispeeific activatahle antibody or nt thereof of the disclosure includes at least (i) a first arm comprising an antigen—binding fragment of a 'l"~ceil engaging dy that includes a first antibody or antigen~binding nt thereof (AB if) that binds a first, 'l'-—cell engaging target, where the A81 is ed to a mashing moiety (MM) such that coupling of the MM reduces the ability of the ABl to bind the lirst target and (ii) a second arm comprising an antigen—binding fragment of a target binding antibody that includes a, second antibody or n—binding fragment f (ABE) that binds a second . in some embodiments, the first and second targets are the same antigen. in some embodiments, the first and second targets are different antigens, in some embodii ents, the first and second tar ets are different epitopes on the same antigen. In some embodiments, the 'l"--cell engaging antibodies or fragments f engage one or more T cell inhibitory receptors d of one or more T cell activating receptors. ltllllll as} in some embodiments, mnltispecilic actiyatable antibodies or fragments thereof of the disclosure include at least (i) a 'l‘—cell engaging dy or f'agment thereof that includes a first antibody or antigen—binding fragment thereof (ABl ) that binds a first, T— cell engaging target that is a surface antigen on a T cell and (ii) a second antibody or fragment thereof that includes a second antibody or n~binding fragment thereof (AB 2) that binds a second target, where the A32 is attached to a masking moiety (lvilvl) such that coupling of the MM reduces the ability of the A132 to bind the second target. in some ments, the first and second targets are the same antigen. In some embodiments, the first and second targets are difterent antigens In some embodiments, the first and second targets are different epitopes on the same antigen. In some iII’IISI’IISg the 'I'—eeii engaging antibody or fragment thereof is attached the iinns of the heayy chain of the target—specific antibody. In some ments, the T~ceIi engaging antibody or fragment thereof is attached the C—terrninus of the heavy chain of the target~speeific antibody. In some embodiments, the 'f—ceii engaging antibody or fragment thereof is attached the N- terminns of the light chain of the —specific antibody. In some embodiments, the T~ceii engaging antibody or fragment thereof is attached the inns of the light chain of the tar et—specifie antibody: In some embodiments, the muitispeeitie activatabie antibody comprises 'I'x-ceii engaging antibodies or fragments thereof attached to a combination of one or more N~termini and/or one or more C—termini of the mnitispecifrc activatabie antibody.

The diseiosnre aiso inehtdes rnuitispecitic activatabIe antibodies that comprise r immune ef ‘ector ceII engaging antibody or fragment thereof, such as one that binds a surface antigen ofa natural kiiier (NK) ceii, a mononuciear ceii, such as a myeioid mononnciear ceii, a macrophage, and/or other immune effector eeIIs. {$883.67} In some embodiments, the niuitispecifie activatabie antibody or fragment thereof of the disciosure inchides at Ieast (i) two 'i‘uceii engaging antibodies or antibody fragments thereof that engage one or more I ceiI activating receptors, such as, for e, T~ceii engaging scFV fragments, incinding but not limited to, OXdﬂ/GITR, CUISW GITR, CD} G, CI327/NKGZI), and additional combination of activating ors and (ii) one target-binding dy that es a second antibody or antigen-binding fragment thereof (ABE) that binds a second target, where the A82 is attached to a niaskin g moiety (MM) such that coupling of the MM reduces the abiiity of the ABE to bind the second target, Where the i engaging scfy' fragments are iinhed to both arms of the target g antibody. in some embodiments, the necifrc antibody or fragment thereof of the diseiosure inehtdes two T—ceii engaging antibodies or fragments thereof that engage one or more 'I' ceii inhibitory receptors, and one target~binding antibody such that the Tweed engaging scFy fragments are iinked to both arms of the target binding antibody. In some embodiments, the two T—ceii engaging antibody fragments bind the same T-ceii engaging target. In some embodiments, the two "I‘vceii engaging antibody fragments bind different '1‘— ceII engaging targets. In some embodiments, the two 'f—ceii engaging antibody fragments bind different es on the same I—ceii engaging target. In some embodiments, the T— ceii engaging antibodies or fragments thereof engage one or more T ceii inhibitory receptors instead of one or more T ceii ting receptors. {$883.68} In some embodiments, the ntuitispecific activatabie antibody or fragment thereof of the disciosnre inchides at ieast (i) a first arm comprising an antigen-binding nt of a T—celi engaging antibody and (ii) a second arm comprising an antigen~binding fragment of a target binding antibody that inchides a, second antibody or antigenbinding fragment thereof (A32) that binds a. second target, where the ABE. is attached to a mashing moiety (MM) such that counting of the MM reduces the ability of the A32 to bind the second target. in some embodiments, the first and second targets are the same antigen. in some embodiments, the first and second targets are ent antigens. in some embodiments, the first and second targets are different epitopes on the same n. in some embodiments, the T—ceii engaging antibodies or nts f engage one or more T cell inhibitory receptors instead of one or more T cell activating receptors, EddﬁldQ} in some embodiments, ecific activatabie dies or fragments thereof of the disciosure inchide at least (i) a T—ceii engaging antibody or fragment f that includes a first antibody or antigen~binding fragment thereof (ABE) that binds a first, T— ceii engaging target that is a surface n on a T ceii, where the ABi is attached to a mashing moiety (MMI) such that coupling of the MM} s the abiiity of the A81 to bind the first target and (ii) a second antibody or fragment thereof that incindes a second antibody or antigen—binding fragment thereof (ABE) that binds a second target, Where the A82 is attached to a masking moiety (MMZZ) such that conpiing of the MM2 reduces the abiiity of the ABE. to bind the second target. in some embodiments, the first and second targets are the same antigen. In some embodiments, the first and second targets are diffe ent antigens. in some embodiments, the first and second tar ets are different epitopes on the same antigen. in some embodiments, the feed ng antibody or fragment thereof is attached the N—termintis of the heavy chain of the target—specific antibody. in some embodiments, the T—ceii engaging antibody or fragment thereof is attached the C— termimis of the heayy chain of the target—specific antibody. in some ments, the T— ceii engaging antibody or fragment thereofis attached the N~terminns of the tight chain of the target~specific antibody. In some embodiments, the T-ceii engaging antibody or nt f is attached the C~terminns of the light chain of the target~specitic antibody. in some embodiments, the mnitispecific tabie antibody comprises Tnceii engaging antibodies or fragments thereof attached to a combination of one or more N—termini and/or 11: 1,49 one or more C—termini of the multispecil‘ic aetivatahle antibody. The disclosure also includes niultispecific activatahle dies that comprise another imrnune effector cell engaging dy or fragment thereoﬂ such as one that binds a surface antigen of a natural killer (NK) cell, a clear cell, such as a rnyeloid mononuclear cell, a macrophage, and/or other immune effector cells. {earn 7%} In some embodiments, the m ecific activatable antibody or fragment thereof of the disclosure includes at least (i) two ’l'--cell engaging antibodies or antibody fragments thereof that engage one or more T cell ting receptors, such as, for example, T—cell engaging scFy fragments, including but not limited to, Ode/GlTR, CDl 37/ GlTR, CDlSWOXdG, CDZWNKGED, and additional combination of ting receptorsg where the ABl of one of the T-cell engaging antibody fragments is attached to a ing moiety (MM) such that coupling of the MM reduces the ability of that ABl to bind its respective T~ cell engaging target, and (ii) one target—binding antibody that includes a second antibody or antigen-binding nt thereof (ABE) that binds a second targe where the ABE is attached to a masking moiety (Mix/l2) such that ng of the Mth reduces the ability of the A82 to bind the second target, where that the T—cell engaging scFy fragments are linked to both arms of the target g antibody in some embodiments, the multispeeific antibody or fragment thereof of the disclosure includes two Tucell engaging antibodies or fragments thereof that engage one or more T cell tory receptors, and one target— binding antibody such that the T—cell engaging scFy' fragments are linked to both arms of the target binding antibody. in some embodiments, the two 'l'~cell engaging antibody fragments bind the same "f-cell engaging target. in some ments, the two T-cell engaging antibody fragments bind different T—cell engaging targets, in some embodiments, the two T—cell engaging antibody fragments hind different epitopes on the same T—cell ng target. In some embodiments, the T's-cell engaging antibodies or fragments thereof engage one or more T cell inhibitory receptors instead of one or more T cell activating receptors. {$630171} In some embodiments, the m ultispecific activatable antibody or fragment thereof of the sure includes at least (i) two T—cell engaging antibodies or antibody fragments thereof that en gage one or more T cell activating receptors, such as, for example, T—cell engaging scFy fragments, ing but not limited tog OX40/Gl'l‘R, Clitl 37/ Gl’l‘R, CDlB 7/GX4Q CD‘Z7NKGZD, and additional combination of activating receptors, where each of Mill of the T—cell engaging antibody fragments is attached to its own mashing moiety (MMl) such that coupling of each h/lMl to its respective ABl reduces the y of that ABl to bind its respective T—cell engaging target, and (ii) one target—binding antibody that includes a second antibody or antigen—binding fragment thereof (A821) that binds a second target, Where the A52 is attached to a masking moiety (MMZ) such that coupling of the MMZ reduces the ability of the A82 to bind the second target, Where the T—cell engaging scFy fragments are linked to both arms of the target binding antibody, in some embodiments, the multispecific antibody or fragment thereof of the disclosure includes two T~cell engaging dies or nts thereofthat engage one or more T cell inhibitory receptors, and one target-binding antibody such that the T~cell engaging scFV fragments are linked to both arms of the target binding antibody, in some embodiments, the two 'i'~cell engaging antibody fragments bind the same 'f--cell engaging target. ln some embodiments, the two T—cell engaging antibody iragments bind different T—cell engaging s. In some embodiments, the two T—cell engaging antibody fragments bind different epitopes on the same Tncell ng . in some embodiments, the T—cell engaging antibodies or fragments thereof engage one or more T cell inhibitory ors instead of one or more T cell activating receptors. {$388172} in some embodiments, the multispecific activatable antibody or fragment thereof of the disclosure includes at least (i) a first arm comprising an antigen-binding fragment ofa T—cell engaging antibody that es a first antibody or antigen—binding fragment thereof (ABl) that binds a first, T—cell engaging target, where the ABl is attached to a masking moiety (Mb/ll) such that ng of the MM reduces the ability of the ABl to bind the first target and (ii) a second arm comprising an antigen-binding fragment of a target binding dy that includes a second antibody or antigen—binding nt thereof (A32) that binds a second target, where the ABS? is attached to a mashing moiety 2) such that coupling of the MM reduces the ability of the A52 to bind the second . in some embodiments, the first and second targets are the same n. in some embodiments, the first and second targets are different antigens. in some embodiments, the ﬁrst and second targets are different epitopes on the same antigen, in some embodiments, the 'l‘—cell engaging antibodies or fragments thereof engage one or more T cell inhibitory receptors instead of one or more T cell activating receptors. {$388173} in some embodiments, the target antigen is an antigen that is highly expressed on both normal, healthy tissue and on ed tissue. in some embodiments, the target antigen is an antigen from Table l that is highly expressed on both normal, healthy tissue and on ed tissue {$388174} in some embodiments, the target antigen is an antigen that is highly expressed on diseased tissue, but is not highly sed on normal, healthy tissue. In some embodiments, the target antigen is an antigen from Table l that is highly expressed on diseased tissue, but is not, highly expressed on normal, healthy tissue, The target antigen may e expressed on normal, healthy , but it is not highly or otherwise overexpressed on the normal, healthy tissue.

Elllllll’FS} in some embodiments, the g moiety (MM) is coupled to the antibody or anti ding fragment thereof (A l3) Via a eleayable moiety (Chit) that functions as a substrate for a protease. Suitable proteases for use in the multispeeifie aetiyatahle antibodies of the disclosure are determined based on the protease expression at the intended site of treatment and/or diagnosis, in some embodiments, the protease is u—type plasminogen activator (11PA, also referred to as na.se), in, and/or rnatriptase (also referred to as MT—SPl or MTSPl). ln some embodiments, the protease is a matrix nietalloprotease (MMP). {$33176} in some embodiments, the multispeeiﬁe aetivatable antibodies are engineered to include a masking moiety (MM) that is coupled to an antibody or antigen-- binding fragment thereof (AB) via a non—cleayable linlrer. For example, in some ments, the multispeeitie aetiyatable antibody is a T—eell engaging niultispeeifie aetiyatable antibody that includes a targeting antibody or antigen—binding fragment thereof and a T-veell engaging antibody or antigen-binding portion thereof, Where the feed engaging antibody or antigen—binding fragment thereof es a lirst dy or antigenbinding fragment thereof (ABl) that binds a first, "f—eell engaging , where the A81 is attached Via non-eleayable linker to a masking moiety (MMl) such that coupling of the MM reduces the ability of the Mill to bind the first target, and the targeting antibody or antigen— binding fragment thereof is not masked. {$630177} In some embodiments, the multispeeiﬁe aetiyatable antibody is a ’l‘—eell engaging peeilie actiy'atable antibody that includes a ing antibody or n g fragment thereof and a T-eell engaging antibody or antigen—binding portion thereof, Where the "l‘veell engaging dy or antigen~binding fragment thereof ineludes a first antibody or antigen-binding fragment thereof (ABl) that binds a first, 'l‘weell engaging target, Where the ABl is attached Via non—cleayable linker to a masking moiety (MMl ) such that coupling of the MM reduces the ability of the ABl to bind the ﬁrst target, and the targeting antibody or antigen~binding fragment thereof includes a second antibody or antigen—binding nt thereof (AB2) that binds a second target, where the A82 is attached via a cleayable linker to a mashing moiety (MMZ) such that ng of the MM reduces the ability of the A32 to bind the second target. {$630178} The general concept of bispecitic antibodies was ﬁrst uced at least 50 years ago {Nisonofﬁ A. and Mandy, Wat, Nature l94, 355-359 (1962,), as cited in Chan, AC. and Carter, Pd}, Nature s linmunol. ltl, Still—3 lo (2tlltl)). lillllll’FS‘} A variety ot’bispecitic platforms have been described (see 3.3., Figure l; l_.,iu, M.A., et al., Proc. Natl. Acad. Sci. USA 82, 8648—8652 (1985); ed by Kroesen, Bl et al. Adv. Drug Delivery Rev. 3 l, 9 (1998,); Marvin, 3.8. and Zhu, 2., Acta l’harm.

Sinica 26, 649—658 (2005); Chan and Carter, Nat. Rev. lmmunol. 20H); Fitzgerald and Lugovslty, MAbs. 3(3):299-3G9 (lel l); Riethmuller, (3., Cancer ty l2, l2~l8 (2012.)). The general t for construction of bispecific antibodies is to linlt together protein~binding domains, y based on multiple immunoglohulin s, in order to construct a molecule that is capable of binding to two or more target antigens and demonstrates lte physiological distribution, pharinacoltinetics, and effector function The latter may include antibody-dependent cytotoxicity tADCC), complement-dependent cytotoxicity (CDC), T~cell recruitment (Bi'fEsTM) (see eg, Baeuerle, PA. and rdt, (3., Cancer Res. 69, 494l—4944 (2009,), and Tandui‘ibsTM (see Cochlovius et al., Cancer Res. (it), 433643341 (2000)), and/or delivery of a cytotoxic payload in the form of a chemically conjugated moiety such as a microtuhule inhibitor, a DNA all On the other hand, the DVD format (Wu, C, et al., Nature Biotechnol. 25, l29(3--1297 (2007)) provides for the engagement ottwo copies of antigen A in addition to two copies of antigen B. The spacing and relative Orientation of each of the g domains may also be important for engagement of le antigens in such a way as to confer the intended activity—for exarnple, cross~linl Acad. Sci! USA lllTI’, l2605—l 26% (20mg). Thus, there remains a need for effective antibody—hased th erapeuties that, exhibit improved safety, ego reduced toxicity: {999182} lvlultispecitic activatable antibodies of the disclosure are safer than multispecific reg, hispeciﬁc) antibodies because multispecil‘ie activatahle dy activity / is restricted to a localized disease environment. In some embodiments, a multispecific activatable antibody is an immune or cell engaging multispecific activatable antibody.

In some embodiments, a multispecific table dy is a T-cell engaging multispecific activatable antibody. In some ments, a multispecific activatable dy izes two or more targets. In some embodiments, a multispecific activatable antibody comprises an IgG-scFv format confers the long-half-life properties of an IgG. In some embodiments, such a multispecific activatable antibody is further modulated through the use of Fc mutations in the FcRn binding site (Petkova, S. B. et al., Intl. Immunol. 18, 1759-1769 ); Deng, R. et al., mAbs 4, 101-109 (2012); Olafson, T Methods Mol.

Biol. 907, 537-556 (2012). In some embodiments, such a multispecific activatable antibody includes mutations in the Fc domain, such as an N297A mutation (Lund, J. et al., Mol.

Immunol. 29, 53-39 ) that reduces IgG effector functions (ADCC and CDC) in order to reduce off-target toxicities.

Multispecific activatable antibodies leverage the up-regulation of protease activity widely recognized as a hallmark of multiple e states to achieve disease-tissuespecific targeting of such therapeutics. The activatable antibody is based on the use of an IgG antibody, or fragment thereof, such as a scFv region, Fab region, single VH or VL domain that has been modified to include a masking moiety (MM), linked to the antibody through a cleavable moiety (CM), such as a se-cleavable moiety (see, e.g., PCT International Publication Number WO 25846, hed February 26, 2009; PCT International Publication Number Alternatively, a non-antibody protein domain (see e.g., PCT International Publication Number 286, 41273-41285 (2011)) may be used to achieve one or more binding specificities. A multispecific antibody, like conventional IgG, can be used as the starting point to produce a multispecific activatable antibody. Such a multispecific activatable antibody would allow the high-affinity targeting of all antigens recognized by the parental multispecific dy, but with tissue-specificity ed by the ively activated cleavable linker. Depending upon the multispecific format used, the MM and CM might be placed at the N- or C- terminus of the appropriate .

A multispecific activatable antibody comprises at least one IgG-derived domain with specificity towards a first antigen, linked synthetically or biosynthetically to another domain (which may be derived from an IgG or other protein) with specificity for binding to a second antigen. Additional specificities may be added h linking such a multispecific table antibody to one or more additional domains conferring additional g specificities for the first antigen, the second antigen, or additional antigens. In some embodiments, a pecific activatable antibody has one or more of these domains masked by an appropriate masking moiety (MM). Each of the multispecific formats described in Figure 1 can potentially be converted into a multispecific activatable antibody by masking one or more of the antigen binding moieties of one or more of the immunoglobulin domains. Examples of suitable multispecific table antibody formats are shown in Figures 3A, and 5-9.

The use of a scFv domain to confer specificity to a specific antigen allows for a modular construction of multispecific antibodies and multispecific activatable dies. The use of scFv domain(s) fused to the terminus of IgG heavy or light chains for construction of bispecific dies has been previously described (see e.g., Orcutt, K.D. et al., Prot. Eng. Design Select. 23, 221-228 (2010)); Dong et al., (2011). This format ("IgG-scFv") allows a conventional IgG to be converted into a bispecific antibody wherein a first specificity is encoded in the variable domains of the IgG and a second specificity is encoded in the scFv domains ed through a flexible linker . Variations of this format include fusing scFv domains at the N- or C-termini of the heavy or light chains; the scFvs may have the same or differing antigen-binding specificities (Spangler, J. B. et al., J.

Mol. Biol. 422, 532-544 (2012)). In addition, through the use of chain heterodimers (for example, using knob-hole or similar constructs), scFvs of differing specificities may be attached to the N- or C-terminus of each heavy chain.

In some embodiments, a multispecific activatable antibody has the IgG variable s masked. In some ments, a multispecific activatable dy has the scFv domains masked. In some embodiments, a multispecific activatable antibody has both the IgG variable domains and the scFv domains masked. In some embodiments, one antibody domain of a multispecific table dy has specificity for a target antigen and another antibody domain has specificity for a T-cell surface antigen. In some embodiments, one antibody domain of a multispecific activatable antibody has specificity for a target antigen and r antibody domain has specificity for another target antigen.

In some embodiments, one antibody domain of a multispecific activatable antibody has specificity for an epitope of a target antigen and another dy domain has specificity for another epitope of the target antigen.

Etititiihﬂ The disc1ostire provides examples of niuitispecitic tah1e antibody structures which include, hut are not iii'nited to, the foiiowing: (Vi...—CL)2:(VPLCPH~CH‘2— CH3dA—VHi‘~13 —V1..r* —1.12~C1\It~11; 1 —MM); ; (Vie—CL); : (Vii—CH i UCHZUCHS —i.i4—'VI.J* —LS ~ ‘VH’k M-L 1 --iv1t‘vt)2; (MM-L i --Ct‘Vi—L2—-VL--CL)2 :(VH-CHI --CH2~CH3 ~L4--VH*-—L3 -- VB" )2; (MM~L1 —CM—L2—VL~CL)2:(Vi—i—CH1—Ci—12—Ci-13 *~L3 —Vi—i*)2; (VL— Ciﬁgt’hdh/i—M —CM—L‘2—V11*:113—VH*—1_/1~VH—CH1 —CH2~CH3)2; (VL—CL);:(MMvL1 —CM— L2-VH* --L3 -- VL*--L4-VH--CH1--CH2-—CH3)2; (MM-Li "CM-12WL-CL)22(VL*~L3 --VH*-—L4-- Vii—C111 ~CHZ~CH3)3; (MM—L 1~CM~L2—VL—CL)2 :tVi—i*—L3—VL*~L4—VH~CH 1 ~CH’L CHE)2; (\714-6314"IJ4-‘\7E{*"143 —Vi_..* ~LZ—Ci‘y’hici ~Mh/i)2:(VH—CH1~Ci’12—Ci’13iz; (VL~Ci_..—L¢i— V1.1*~1..3 —VH* ~1_/2—CM—Li,~h/ih1)2:(\/’H—CHI—CH2—CH3)2; (Mix/t~11; 1 —Ci\«i—LZ~VL*—i_/3 "VH2"— L4--VL--CL)2 : H i --CH2~CH3)2; (MM-Li _- CM-va2--VH* ~L3 --VL* "144--VrL--CL)QI(‘V7H~ CH1 ~CHZ—CHS)2; (Vi...~CL—i_..4—V"H*-L3 "VL*~L2~C1\A—Li hiMh: (ix/{hill ~CM~LZ~VL*- i433 Lr-t—VH—CHE—CH2—CH3)3; (VL—CLJA—VHi‘~13—ViJ*—i..y2—CM—i_livMMh: (MM~ L 1 *’ --L4-- VH-CH1 --CH2--CH3)3 _- CM-vaZn \«’H* ~L3 --VL ; (Vic-CL-Léi-VL" ~L3 --VH* --L2--CM-- L1 ~MM)3 : (MM—L i —C1\/i—L2—VL*—L3 ~Vi-i*—L4~V1—i—Ci—i 1 {ii-124313 )2; (VL—CL—Lit—VL’RLZ — L2—Ci‘vi~i_..i ~MM)2: (h’ih’hLi-Ch/i—EQ—VH "3-1.3"V14*~14~VH~CH 1 (312431392; (Vb Cier'i/i—VHKLS ); (MMwL 1 ~1..2—VL*~L3 —VH*~1..4—VH~CH i ~CH2~CH3)2; (Vb CL--L4~VH*--L3-—\I’L*)gi (MM-L1"CM-12.W‘H‘i‘"LB~VL*-—L4--VH-—CHi--CH2-—CH3)2; (VL- CL—L4—VL*—L3~VH* )2 : (MM~L1—CM—L2~VL*—L3—Vi—i*—L4~VH—CHi —Ci-i2—Ci-i3)2; (VL— CL—EA—Viﬁdﬁ—VH*)2: (MMJJ~CiVi~L2~Vii>t{.3—VL*aL4-VH*CHi"CHZaCi’Bﬁ; (VL— CL~L4--VH*-—L3--VL*-vL2--CM--L1midis/1):: (VU‘--L3--VH*--L4-—VH--CHt--CH2--CH3)2; (Vic-- CL—Ld—Vi—H—LEﬁ—VL*~L2—CM—L 1 ~h/i1‘s/i)2 : (\71i*~L3 —VL*—L4—VH~CH 1—CHZ—Ci—i3h; (Vb Ci.;—ifi—VL*—L3—VH*—L‘Z—CNI—L1 $4M}: L3"Vii*-1,/4*Vii—CH i—t?i»i2-t:3i'i3)2; or (Vb Chi/13:71.1*~1..3 —VH*—1_/2wCM—1.11 ~MM)2: (VH* —i43 UV1* ~1..4—VH~CH i ~CH2~CH3 )29 wherein: VL and VH represent the 1ight and heavy vai‘iahie domains of the first specificity, contained in the igG; VL" and VH* represent the variahie domains of the second speciﬁcity, coinained in the scFv; 141 is a linker peptide ting the masking moiety (MM) and the eieavahie moiety (CM); 1.2 is a tinker peptide connecting the cieavahie moiety (CM), and the antibody; L3 is a iinitei" peptide ting the variahie domains of the scFv; L4 is a 1inker peptide connecting the antibody of the ﬁrst specificity to the antibody of the second speciﬁcity; CL is the iight-chain constant domain; and CH1, CH2, CH3 are the heavy chain constant domains. The first and second speciﬁeities may he toward any antigen or epitope. Additionei ures inchide, but are not iiinited to, the following: (\I’L—CL—Lll—Vl-ltk—L3~VL*—L2—CI\/l—Ll—l\«ilvl)g:(‘v’I-l—CI-li—Cl—lZ—Cl‘lfllz; (VL—CL— L* ~13 -Vl»l*—l_42~CM—L l ~MM); : (VIi—CII l —CII2-Cli3 )2; (MM—L l -CM-I_.2~VL—CL—Lzl— Vli" —I,3 ~Vli*)3 : (VH~CH l ~CH2~CH3 )2; (MM—L l ~ClVi—L2—VL—CL~—i_/l~VI,*~L3—VH*T; :(VH~ CH1 CHE.--CH3)2 ; (V'LnCL-Ld-N’Hl‘ x-L3 --VL* -vL2--CM--L l --MM)2 2 (Vll-Cll l --Cll2-—Cil3 --L4-- VI-l *—L3 —VL*—L2~Clvl~L l —MM)3; (VL—CL—Lél—VLPk—L?) ~Vl-l*—L2~CM~L l —lVIl\«i)g:(_'\/’H~CH l ~ 3 ~i_/l-—Vif" ~13 —VH*JAE—CM—I, i ~MM)2; (bin/LL l 2~VL—Cinli4—VH$41.3 — V’L*)2:(VH--CH1--CH2--Cil3lz; or (MM-Ll--(WI-L2--\/"L--CL----L4UVU‘--L3--Vll*)2:(‘y’ll--Clll-- Cl-lZ—Cl-lZl )2.

Elllllllliﬁ In some embodiments, a multispecii‘ie aetivatabie antibody comprising an igG ltas tlte igG variable domains masked, In some embodiments, a miiltispecific actiyatable antibody comprising a scFy has the scFy domains masked. In some embodiments, a moitispecii‘ic activatabie antibody has both igG variable domains and scFy doi'nains, where at least one oftlne lgG variable s is coupled to a masking moiety, In some embodiments, a pecii‘ic activatable dy has both lgG variable domains and scFV domains, Where at least one oftbe scFy domains is coupled to a masking moiety. in some ments, a mnltispeeitic actiya‘table antibody has both lgG variable s and scFy domains, Where at least one of the lgG variable domains is d to a masking moiety and at least one of the scE’y domains is coupled to a masking moiety. in some embodiments, a moitispecii‘ic tabie antibody has both igG variable domains and scFy domains, where each of the IgG variable domains and the ser domains is coupled to its own masl H 1,49 llllllllglll} in some embodiments, the targeting dy can he in the form an activatahle antibody. ln sorne en'rhodin'rents, the scl?y(s) can be in the form of a Pro~scFV (see, e.g., WO LEW/025846, WC EOlO/(llll l73). {899193} in some embodiments, the schV is specific for g CD38, e.g., oars. in some embodiments, the scFy is specilic for binding CTLA—él (also referred to herein as C'l‘l'..A and C'l'l'..A-4). {tilllllgld} in some embodiments, the scFV is specific for binding one or more 'l‘ucells, one or more NK—ceils and/or one or more macrophages. in some embodiments, the scFV is speciﬁc for binding a target selected from the group consisting ld, BTLA, CD3, (i134, C138, Cl'Jloa, CDZS, Cl) .7, CDZS, C332, €856, CDl37, Cl‘llAa-l, GlTR, H\/’El\/l, lCOS, LAG3, NléG‘ZD, OXdll, l’D-vl, 'l‘lGlT, 3, or VlSTA, and combinations thereof.

Definitions {lllllll o5} Unless otherwise d, scientific and technical terms used in connection with the present disclosure sh all have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall e the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, rno ecular biology, and n and oligo-v or polynucleotide chemistry and ization described herein are those well—known and commonly used in the art. Standard techniques are used for recon'rhinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e. g., oporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's ications or as commonly accomplished in the art or as described herein. The ing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more speciﬁc nces that are cited and discussed throughout the present speciﬁcation. See cg, Sambrook at of. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor l...ahoratory Press, Cold Spring l’larbor, NY (1989)). The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry bed herein are those well—known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings.

As used herein, the term "antibody" refers to immunoglobulin molecules and logically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. By "specifically bind" or "immunoreacts with" or "immunospecifically bind" is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides or binds at much lower affinity (Kd > 10-6). Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, fully human, domain antibody, single chain, Fab, and F(ab')2 nts, scFvs, and an Fab expression library.

The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids ily responsible for antigen recognition. The y-terminal portion of each chain defines a constant region primarily responsible for effector function. In general, dy molecules obtained from humans relate to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule.

Certain classes have subclasses as well, such as IgG1, IgG2, IgG3, IgG4, and .

Furthermore, in , the light chain may be a kappa chain or a lambda chain.

The term "monoclonal antibody" (mAb) or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene t. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the les of the population. MAbs n an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.

The term en-binding site" or "binding portion" refers to the part of the immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent stretches within the V s of the heavy and light chains, referred to as "hypervariable regions," are interposed between more conserved ng stretches known as "framework regions," or "FRs". Thus, the term "FR" refers to amino acid sequences that are naturally found between, and adjacent to, hypeiyariahle regions in irnmunoglobulins. in an antibody le, the three hypervariahle regions of a light chain and the three hypervariahle regions of a heavy chain are disposed ve to each other in three dimensional space to form an antigen-binding surface. The antigen~hinding surface is complementary to the three—dimensional e of a hound antigen, and the three hypetvariahle regions of each of the heavy and light chains are re ‘erred to as ementarity--deterniining regions," or "CDRs." The assignment of amino acids to each domain is in ance with the deﬁnitions of Rabat Sequences of Proteins ofimmunological interest (National lnstitutes of Health, Bethesda, Md U98? and l99l)), or a 84: {KESR J. Mol, Biol, l96:9tll—9l7 (l 987), Chothia er al. Nature 342:878— 883 (1989). lilillllell As used herein, the term "epitope" includes any protein determinant capable of specific binding to an irnrnunoglohulin, an scFv, or a T—cell receptor. The term "epitope" includes any protein determinant e of specific binding to an immunoglohulin or 'l'x- cell receptor. Epitopic inants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as speciﬁc charge characteristics. For example, antibodies may be raised against N-terminal or inal peptides of a polypeptide. An antibody is said to speciﬁcally hind an antigen when the dissociation constant is _<1 l old; for example, in some embodiments _<_ lth anl and in some enihodirnents i: it} nh'l, iﬁiiiiltlll As used herein, the terms "specific binding," "immunological binding," and "immunological binding properties" refer to the non-covalent interactions of the type that occur hetv'een an immunoglohulin molecule and an anti en for which the immunoglohulin is specific. The strength, or affinity of immunological binding interactions can he expressed in terms of the iation constant (Kd) of the interaction, wherein a smaller Kit represents a greater affinity. lnimunologieal binding properties of selected polypeptides can be quantified using methods well known in the art. One such method s ing the rates of antigen—binding site/antigen x formation and dissociation, wherein those rates depend on the trations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions, Thus, both the "on rate constant" (Ken) and the "off rate constant" (Km?) can be determined by calculation of the concentrations and the actual rates of ation and dissociation. (See Nature 36l : l 86—87 (l 993)). The ratio of lief/Km enables the cancellation of all parameters not related to allinity, and is equal to the dissociation constant Kd, (See, ﬁy, Davies et al. 0990) Annual Rev Biochem 59:459—473), An antibody of the present sure is said to speciﬁcally bind to EGFR, when the equilibrium binding constant (Ks) is El old, for example in some embodiments _<. lot) his/l, in some embodiments < l0 anl, and in some embodiments S ltltl pM to about l phi, as measured by assays such as radioligand binding assays or similar assays known to those slrilled in the art.

{Milli-2%} The term "isolated polynucleotide" as used herein shall mean a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the "isolated cleotide" (l) is not associated with all or a n of a polynucleotide in which the "isolated polynucleotide" is found in nature, (‘2) is operably linked to a polynucleotide that it is not linlted to in nature, or (3) does not occur in nature as part of a larger sequence. Polynucleotides in accordance with the disclosure include the nucleic acid molecules encoding the heavy chain globulin molecules shown herein, and nucleic acid molecules encoding the light chain immunoglobulin molecules shown {$3thle The term "isolated protein" ed to herein 1 cans a protein of eBNA, recombinant RNA, or synthetic origin or some combination thereof, which by virtue of its origin, or source of tion, the "isolated protein" (l) is not associated with proteins found in nature, (2,") is free of other proteins from the same source, cg, tree of rnurine proteins, (3) is expressed by a cell from a different species, or (ll) does not occur in nature {Milli-2%} The term eptide" is used herein as a generic term to refer to native n, fagments, or analogs of a polypeptide sequence. Hence, native protein fragments, and analogs are species of the polypeptide genus. Polypeptides in accordance with the disclosure comprise the heavy chain globulin molecules shown herein, and the light chain immunoglobulin molecules shown herein, as well as antibody molecules formed by combinations comprising the heavy chain imrnunoglobulin molecules with light chain immunoglohulin molecules, such as kappa light chain oglohulin molecules, and Vice versa, as well as fragments and analogs thereof. {snares} The term ally—occurring" as used herein as applied to an object refers to the fact that an object can be found in nature, For example, a poly eptide or polynucleotide sequence that is present in an organism (including viruses) that can he isolated from a source in nature and that has not been intentionally modified by man in the laboratory or ise is naturally—occurring. {tlllllZil’i’} The term "operably linked" as used herein refers to ons of compmrents so described are in a relationship ting them to function in their intended manner. A control sequence "onerably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is ed under conditions compatible with the control sequences.

Eiltltllltl8} The term "control sequence" as used herein refers to polynucleotide sequences that are necessary to effect the expression and processing ofcoding sequences to which they are ligated. The nature of such control sequences differs epending upon the host organism in prolraryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term "control ces" is intended to include, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences The term "polynucleotide" as ed to herein means nucleotides of at least ll) bases in length, either rihonucleotides or deoxynucleotides or a modified form of either type of nucleotide.

The term es single and double stranded forms of DNA. {ﬁlltthWl The term ucleotide ed to herein includes naturally occurring, and modified nucleotides linked together by lly occurrin i, and nmi—naturally occurring oligonucleotide linkages. Gligonucleotides are a cleotide subset generally comprising a length of200 bases or fewer! in some embodiments, ucleotides are ll) to fit) bases in length, for example in some embodii ents l2, l3, l4, l5, l6, l7, l8, l9, or 29 to 40 bases in length. Oligonucleotides are usually single stranded, cg, for probes, although oligonucleotides may be double stranded, eg, for use in the construction ofa gene mutant. Oligonucleotides of the disclosure are either sense or an tisense oligonucleotides. ltltlllzlfl} The term ally occurring nucleotides" referred to herein includes deoxyrihonucleotides and ribonucleotides. The term "modified nucleotides" referred to herein includes nucleotides with modified or substituted sugar groups and the like. The term "oligonucleotide linkages" referred to herein includes ueleotide es such as phosphorothioate, phosphorodithioate, phosphoroselerloate, orodiselenoate, phosphoroanilothioate, pl'toshoraniladate, phtisnhoroitniiclate, and the like. Sec 6.3:, LaPlanche at a]. Nuci. Acids Res. 14291181 (1986); Stec e: of. 1. Am. Chem. Soc. 106261177 (1984), Stein et ai. Nuci. Acids Res. 16:3209 , Zon at til. Anti Cancer Drug Design 6:539 (1991); Zen er HZ, Oligonucieotides and Anaiogues: A Practicai Approach, pp. 877—108 (F. Echstein, Ed, Oxford University Press, Oxford Engiand (1991),); Stec er 622’. US. Patent No. 5,151,510; Uhlmann and Peyrnan Chernicai Reviews 90:543 (1990). An oiigonucleotide can incinde a label for detection, if desired. restrain As used herein, the twenty tionai amino acids and their abhre 'iations t‘oiiow conventional usage. See immunology — A Synthesis (an n, ES. o and DR. Gren, Eds, Sinauer Associates, Sunderiand?’ Mass. (19% ,1). Stereoisomers (eg. 1)— amino acids) of the twenty conventional amino acids, rai amino acids such as d—, d— disuhstituted amino acids, Nt-ailqu amino acids, 1actic acid, and other unconventionai amino acids may aiso be suitable components for polypeptides of the present disclosure. Examples of unconventional amino acids include: 4 hydroxyproiine, y-carhoxygiutamate, a—N,N,N— triniethyilysine, i: ~N-acetyiiysine, phoserine, N- serine, N-forrnyimethionine, 3K-niethyihistidine, roxylysine, o-N--methyiarginine, and other simiiar amino acids and imino acids (eg 4— hydroxvproiine). in the poiypeptide notation used herein, the 1eft—hand direction is the amino terminal direction and the right-hand ion is the carhoxy-terntinai direction, in accordance with standard usage and convention.

{WHERE} riy, unless specified oti'ierwise, the 1eft—hand end ot‘singie— stranded poiynucleotide sequences is the 5' end the and direction of double-stranded poiynucleotide sequences is referred to as the 5' direction. The direction of 5‘ t"; w addition .\1.) of nascent RNA transcripts is referred to as the transcription direction sequence regions on the DNA strand having the same sequence as the RNA and that are 5' to the 5’ end of the RNA transcript are referred to as reani sequences", sequence regions on the DNA strand having the same sequence as the RNA and that are 3‘ to the 3‘ end of the RNA transcript are ed to as "downstream sequences". {913132131 As appiied to polypeptides, the term "substantial identity" means that two peptide sequences, when optirnaiiy a1igned, such as by the programs GAP or BESTFiT using defauit gap weights, share at 1east 80 percent sequence identity, for e in some embodiments, at least 90 percent sequence identity, in some embodiments at least 95 percent sequence identity, and in some embodiments at least 99 percent sequence identity.

Eiiiiiils': 14} in some embodiments, residue positions that are not cai differ by conservative amino acid substitutions.

Ellllllll 15} As discussed herein, minor variations in the amino acid sequences of antibodies or irnm unoglohulin rnolectrl es are contemplated as being encompassed by the present disclosure, providing that the variations in the amino acid sequence maintain at least 75%, for e in some embodiments at least 80%, 90%., 95%, and in some embodiments 99%. In particular, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into families: (l) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginine, ine; (3) non~polar amino acids are alanine, valine, leucine, isoletrcine, proline, phenylalanine, methionine, tryptophan, and (-4) unchar ed polar amino acids are e, asparagine, glutamine, cysteine, , threonine, ne. The hydrophilic amino acids include arginine, asparagine, aspartate, gltrtamine, glutamate, histidine, lysine, serine, and threonine.

The hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine. Gther families of amino acids include (i) serine and threonine, which are the aliphatic~hydroxy family; (ii) asparagine and glutarnine, which are the amide ning family; (iii) alanine, , leucine and cine, which are the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine, which are the aromatic family. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an ate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding or properties of the resulting molecule, especially if the replacement does not involve an amino acid within a, framework site. ‘Nhe‘ther an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the polypeptir e r erivative. Assays are described in detail herein. fragments or analogs of antibodies or glohulin les can be readily prepared by those of ordinary skill in the art. ln some embodiments, arnino— and carhoxy~termini of fragments or analogs occur near boundaries of onal domains. Structural and functional domains can be identified by ison of the nucleotide and/or amino acid sequence data to public or proprietary sequence ses.

Computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or on.

Methods to identify protein sequences that fold into a known three-dimensional structure are known. Bowie at a]. Science 253:164 (l99l). Thus, the foregoing examples demonstrate that those of slrill in the art can ize sequence motifs and structural mations that may be used to deﬁne structural and timctional domains in ance with the disclosure. {899216} ln some embodiments, amino acid substitutions are those that: (_ 1) reduce susceptibility to proteolysis, (:2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter g afﬁnities, and (4) confer or modify othe physicochemical or functional properties of such analogs. Analogs can include various nruteins of a sequence other than the naturally—occurring peptide sequence. For e, single or multiple amino acid substitutions (for example, conservative amino acid substitutions) may be made in the natural ly~ occurring sequence (for example, in the n of the polypeptide outside the domainiis) ‘orming intermolecular contacts. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (tag, a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of art—recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed, W. ll.

Freeman and Company, New Y'orl'x' 098-4)); introduction to Protein Structure (C. Branden and .l. e, eds, d Publishing, New York, NY @991»; and Thornton et at.

Nature 3541ltl5 (l99l).

{GWEN} The term "polypeptide fragment" as used herein refers to a polypeptide that has an amino al and/or carboxy—terminal deletion and/or one or more internal deletionts), but Where the ing amino acid ce is identical to the corresponding positions in the naturally—occurring sequence deduced, for example, from a full length cl)NA sequence. Fragments typically are at least 5, 6, 8 or l0 amino acids long, for example in some embodiments at least l4 amino acids long, in some embodiments at least amino acids long, usually at least 50 amino acids long, and in some embodiments at least 70 amino acids long. The term "analog" as used herein reters to polypeptides that are comprised of a segment of at least 215 amino acids that has substantial identity to a portion ot‘a deduced amino acid sequence and that has speciﬁc binding to EGFR, under suitable binding conditions. Typically, ptide analogs comprise a conservative amino acid substitution (or addition or deletion) with respect to the lly— occurring sequence.

Analogs typically are at least 2% amino acids long, for example in some embodiments at least 50 amino acids long or longer, and can often be as long as a full~length naturally~ occurring polypeptide, {$388218} The term "agent" is used herein to denote a chernical nd, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials. 19} As used herein, the terms "label" or "labeled" refers to incorporation of a detectable , egg, by oration of a radiolabeled amino acid or attachment to a polypeptide ot‘biotinyl es that can be detected by marked avidin (cg, streptayidin containing a ﬂuorescent marker or tic activity that can be detected by optical or calorimetric methods), ln "ertain situations, the label or marker can also be therapeutic.

Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not d to, the tollowing: radioisoto. - - . , 2 1 5 , 35a 91799 W t1ln 1 257 13,17 es or radionuclides (c. 14L. a ("Y £31:, "ll l\ n lc \ 9 7 7 7 9 i, ll, fluorescent 7 , ,y labels (6.53., a fluorophore, rhodamine, lanthanide phosphors), enzymatic labels (rag, horseradish peroxidase, p—galactosidase, luciferase, alkaline phosphatase), uminescent, yl groups, predetermined polypeptide epitopes recognized by a secondary reporter (cg, leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). ln some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric nce. The term "pharmaceutical agent or drug" as used herein refers to a chemical compound or composition capable of ng a desired thera eutic effect wh en properly administered to a patient. {tillllZle} As used , "substantially pure" means an object species is the predominant species present (126., on a molar basis it is more abundant than any other individual species in the composition), and a substantially purified fraction is a ition wherein the object s comprises at least about 50 percent (on a molar basis) of all rnacromolec ular species present. {$630221} Generally, a substantially pure composition will comprise more than about lit) percent of all macromolecular species present in the composition, for example, in some embodiments, more than about 85%, 90%, 95945, and 99%. In some ments, the object species is puriﬁed to essential homogeneity (contaminant species cannot be de ,ecte' in the composition by conventional detection methods) wherein the composition consists essentially ofa single macromolecular species.

EiititiZZZ} The term patient includes human and veterinary subj ects.

HERMES} Other chemistry terms herein are used according to conventional usage in the art, as exenipiiiied by The McGraw—Hiii Dictionary of Citemicai Terms (Parker, 8.. Ed.

McGraW’x-Hiii, San Francisco (1985)).

Militis _ ecific dies and Muitis eciﬁe /table Antihndies {9992243 Exempiary multispeeiiie antibodies and/or multispeeitic aetivatabie antibodies of the diseiosui‘e ineiudeﬁ for e. those shown in the Examples provided herein, and variants thereof.

EiititiZZS} in some non—limiting embodiments, at least one ofthe AB in the multispeeiiie antibody is a binding partner for any target listed in Table i.

Tabie i: Exempiery "i‘argets Aipha—d inteorin alpha4beta i inte_rin beta’i DSGi {E'Nbeta 'i'GFbeta etiti________________________________________________________________________________________________________________________________________________________________________________________________________________ AHMWIWFWRW _______________Esf:____________________________________ Apeiin 3 Endotneiin B igE Receptor receptor receptor {FeeRi} F CAM EPHA2 Mucin— 1 6 (MUCI e, CA~ t 25 " C3117 \ta/K ase Keutrophii coin lenient se CD132 F protein of (ii..—2RG‘) RSV CD} 3 3 CA 1 9—9 CD} 3 7 1L 121349 Lewis a Rece )tors Carbonic C3138 FGFEi iL—12R, Notch 1 TNFRSiZ anhydrase 9 IL-- 1 ZRbetai A [ CD: CD 166 FGFRJ 11-13 Notch 21 TRAii..—R1 ODS CD172A FGFRE II..13‘R. Notch 3 TRAH..—R21 FGFR3 Noun 4 ei1in FGFR4 1iansfei1in reee tor GA138T1 1L27x’1L27R PDGRBB VCAM— 1 1wsx1 ) Co11agen ‘ 11329 PDGFR3113113, VEGF Cripto GLL111 1L-—3 1R eta \I’EGE'x-A CSFR GL1114 1L3 1/1133 1R 1311-1 VEGF13 CSFR—i GM-CSF ' ' veefx: CTLAJE- GM-CSFR VEGFI) CTGF GP 1113/1113 11341{ atidyL- VEGFR1 serine CXC1 1G ‘ 1, 116 11611 P1GF VEGFRZ CXC1 13 _ , n PSCA VEGFRS Receitor CXCR1 " _ 1iagged 1’ SMA V1STA mne 1 1111033112111: {1111112261 111 some non-1imiting embodiments, at 1:22:51 one of the AB ofthe muhispeciﬁc antibody is: or is derived from a sequence set forth in TabEe 7 in the Exammes provided herein. 1111111227} In some non~1irniting embodiments, at 16:21:11 one 011111: AB of the muhispeciﬁc antibody is or is derived from an antibody 1isted in 131316 ‘2 "17211118 2: Exempiar‘y sources for ABS Antibody Trade Name (antibody name) Target Avastinm 1’bewracizumab" VEGF 1311013111151" 1’ran ibizumab‘) VEGF Erbituxm ('cetuximam FGFR Vectibixm 1’ xnab‘) FGFR Remicadem "inﬂiximam '1‘1‘115'1‘1 Humiram (3111111m 13mab) '1‘1‘115'1‘1 ’1‘Vsabi'11M (n ataﬁzumab) 11111) Y11110141- Simu'iectm ’basﬂiximab" 11.12111, Soliris™ (eculizumab) Complement C5 a™ (efalizumab) CD11a Bexxar™ (tositumomab) CD20 Zevalin™ (ibritumomab tiuxetan) CD20 Rituxan™ (rituximab) CD20 Ocrelizumab CD20 Arzerra™ (ofatumumab) CD20 Obinutuzumab CD20 Zenapax™ (daclizumab) CD25 Adcentris™ (brentuximab vedotin) CD30 Mylotarg™ (gemtuzumab) CD33 Mylotarg™ (gemtuzumab ozogamicin) CD33 Campath™ (alemtuzumab) CD52 ReoPro™ (abciximab) Glycoprotein receptor Ia Xolair™ (omalizumab) IgE Herceptin™ uzumab) Her2 Kadcyla™ (trastuzumab ine) Her2 Synagis™ (palivizumab) F protein of RSV (ipilimumab) CTLA-4 (tremelimumab) CTLA-4 Hu5c8 CD40L (pertuzumab) Her2-neu (ertumaxomab) CD3/Her2-neu Orencia™ (abatacept) CTLA-4 (tanezumab) NGF (bavituximab) Phosphatidylserine (zalutumumab) EGFR (mapatumumab) EGFR (matuzumab) EGFR (nimotuzumab) EGFR ICR62 EGFR mAb 528 EGFR CH806 EGFR MDX-447 EGFR/CD64 (edrecolomab) EpCAM RAV12 RAAG12 huJ591 PSMA Enbrel™ (etanercept) TNF-R e™ (alefacept) 1LFA-3 Antril™, Kineret™ (anakinra) IL-1Ra GC1008 TGFbeta Notch, e.g., Notch 1 Jagged 1 or Jagged 2 (adecatumumab) EpCAM (figitumumab) IGF1R (tocilizumab) IL-6 receptor Stelara™ (ustekinumab) IL-12/IL-23 ™ (denosumab) RANKL Etltltlﬂd} Also included in the disclosure are niultispecii'ic antibodies and/or niuitispecitie actiyatable antibodies that bind to the same epitope as the niuitispecilie antibodies and/or speeific aetivatabie antibodies bed herein. {8639229} Those skilled in the art will ize that it is possible to determine, without undue experimentation, if a specilic antibody and/or a rnultispecitic activatable antibody has the same or similar speciﬁcity as a niultispecitic antibody and/or inultispecitie activatable antibody of the disclosure by aining whether the forrner ts the latter from binding to a target. it the rnultispecilic antibody and/or a niultispecii‘ic activatable antibody being tested competes with the tn ultispecil'ic antibody and/or a niultispeeifie actiyatable antibody of the disclosure, as shown by a decrease in binding by the ninltispecific dy and/or a niultispecific activatable antibody of the disclosure, then the two inultispecilic antibodies and/or ntultispecilic activatable dies bind to the same, or a closely related, epitopei {Milli-2’38} One embodiment for determining r a, inultispeciflc antibody and/or a niultispecitie table antibody has the same or ar specificity as a niultispecilic antibody and/or a inultispeeitic actiyatable antibody of the disclosure is to pro—incubate the niultispecitic antibody and/or a niultispecitic actiyatable antibody of the disclosure with soluble target with which it is normally reactive, and then add the niultispecitic antibody and/or a ntultispecitie activatable antibody being tested to determine if the speeitic antibody and/or a rnultispecitic aetivatabie dy being tested is inhibited in its ability to bind the target. If the specifie antibody and/or a niultispeeitic aetiyatable antibody being tested is inhibited then, in all likelihood, it has the same, or functionally equivalent, epitopic specificity as the rnnltispecitic antibody and/or a tnultispecitic aetivatabie antibody of the disclosure {999231} A niultispecitic antibody and/or a niultispecilic activatable antibody is generated, for example, using the procedures described in the les provided below. A rnultispecitic antibody and/or a tnultispecitic aetivatable antibody can also be generated using any of a number of art—recognized techniques "or antibody production and/or puriﬁcation.

Eiltltl232} Antibody fragments, such as by, F(ab’)g and Fab, for use in a niultispeeitic antibody and/or a speeitic aetiyatable antibody may be prepared by cleavage of the intact protein, eg by protease or chemical cleavage. Alternatively, a truncated gene is designed. For example, a chimeric gene encoding a n of the Ftab’)2 fragment would include DNA sequences encoding the (fl-ll domain and hinge region of the El chain, follmved by a translational stop codon to yield the truncated n'iolecule. {$388233} sion vectors include plasmids, retroviruses, YACs, EBV derived episomes, and the like. A convenient vector is one that encodes a ﬁtnctionally complete human (fl-l, or CL irnmunoglohulin sequence, with riate restriction sites engineered so that any VH or VL sequence can be easily inserted and expressed. in such vectors, splicing usually occurs between the splice donor site in the inserted l region and the splice acceptor site preceding the human C region, and also at the splice regions that occur Within the human CH exons. Polyadenylation and ription termination occur at native somal sites ream of the coding regions. The resulting antibody may be joined to any strong promoter, including retroviral L'l‘lis, cg, SV-dl) early promoter, (Okayama er a]. l‘v‘lol. Cell. Bio. 3:280 6983):), Rous a virus LTR (Gorman er (2!.

PNAS. 79:6??? (l 982)), and moloney rnurine leukemia virus LTR (Grosschedl er a]. Cell 4l2885 (1985)). Also, as will be appreciated, native lg promoters and the like may he used. lllllél} Further, multispecilic antibodies and/or inultispecific activatable antibodies can be generated through display type technologies, including, without tion, phage display, retroviral display, ribosornal display, and other techniques, using techniques well known in the art and the resulting molecules can he subjected to onal maturation, such as allinity maturation, as such techniques are well known in the art. Wright of a]. Crit, Reviews in lmrnunol. l2l2S—l 68 (l 992), llanes and Pliickthun PNAS USA 94:4937-4942 (l 99?) (rihosornal display), Parmley and Smith Gene 73:305—3 l8 (l 988) (phage display), Scott, 'l‘lBS, vol. 17:24lK-245 (1992), Cwirla er a1. l’NAS USA 87163786382 , Russel er a]. Nucl. Acids Research 2i :ltllll—ltlgﬁ (1993), l’loganhoorn er a]. lmmunol. Reviews l30:43—68 0992), Chiswell and h'lcCafferty "l‘llﬁ’l‘ECH; l0:8l)~SA 0992), and US. Patent No. 5,733,743.

EllllllZflSl it can be desirable to modify the rnultispecitic antibody and/or multispecilic activatahle dy of the disclosure with respect to or function, so as to enhance or reduce such function to improve the effectiveness of the antibody in treating diseases and disorders. For example, ne residue(s) can be introduced into the Fc region, thereby allowing interchain disullide bond formation in this region. The meric antibody thus generated can have improved internalization capability and/or increased complement— ed cell killing and antibody-dependent cellular cytotoxicity (ADCC). {See Caron et al., J. Exp Med, l7o: llSll—ll95 (1992) and Shopes, J. immunol, l48: 922 0992)). ’7mII Alternatively, an antibody can be engineered that has dual Ec regions and can thereby have enhanced cornp1ement lysis and ADCC capabilities, (See Stevenson et at, Anti—Cancer Urn0 Design, 3: 219230 ), In some embodiments Fe mutations are made to remove ’V {:3 Ly 9 , , , ‘lycosyiation sites thereby reducing E'c function. 4 .. ) , U Mnltis eeit’ic Aetivatable Antibodies {000236} The inultispecitic activatab1e antibodies and rna1tispeci1ic aetivatable dy itions provided herein contain at least a ﬁrst antibody or antibody fragment thereof (collectively referred to as ABl throughout the disclosure) that specifically binds a ﬁrst target and/or a first epitope and a second antibody or antibody fragment thereof (collectively ed to as A132 throughout the sure) that speciﬁcally binds a. second target and/or a, second epitope, wherein at least one of the AB is modiﬁed by a masking moiety (MM). in some embodiments, each AB in a mnltispecitic activatabie antibody is tnoditied by its own masking moiety. 7} When at least one of the AB in a multispecilie actiyatahle antibody is modified with a MM and is in the presence of its target, specific binding of the AB to its target is reduced or inhibited, as compared to the specific g of the AB not modiﬁed with an MM or the speciﬁc binding of the parental AB to the target. {000238} '1'heK.d of the AB modiﬁed with a MM towards the target is at least 5, 10, , 25, 40, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 0, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or greater, or between 510, 10—100, 10— 1,000, 10—10000, 10100000, 101,000,000, 1,000,000,000, 100—1000, l00~l0,000, l00~ 100,000, ,000,000, 100--10,000,000, l,000--10,000, 1,000--100,000, 1,000--1,000,000, 1000~10,000,000, 10,000—100,000, 10,000—l,000,000, 10,000—10,000,000, 100,000 1,000,000, or l00,000—l0,000,000 times greater than the Rid of the AB not modiﬁed with an MM or of the al AB towards the target. Conversely, the binding afﬁnity of the AB modified with a MM towards the target is at least 5, 10, 2.0, ‘25, 40, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 000, 10,000,000, ,000 or greater, or n 5—10, 10—100, 101,000, 00, 10—100,000, 10— 1,000,000, ,000,000, 1001,000, 100-10,000, 100~100,000, l00-—1,000,000, 100- ,000,000, l,000~10,000, 1,001.1—100,000, 1,000—1,000,000, 1000—10,000,000, 10,000— l00,000, 10,000~1,000,000, 10,000-10,000,000, 100,000~l,000,000, or 10000040000000 times lower than the binding affinity of the AB not modified with an MM or of the parental AB s the target. {000239} The iation nt (K33) of the MM towards at least one the AB in the specifie actiyatabie antibody is generahy greater than the Kid of the AB towards the tar et. The Kd of the MM towards the AB can be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, l,000,000 or even 10,000,000 tirnes greater than the Kd of the AB towards the target. Converseiy, the g afﬁnity of the MM towards the AB is gene "ally lower than the binding affinity of the AB towards the target, The binding ty ofMM towards the AB can be at least 5, 10,25, 50 100, 250, 500, l ,0002,500, ,000, l0,000, 100,000, 1,000,000 or even l0,000,000 tirnes‘ lower than the binding afﬁnity of the AB towards the target. {000240} When at ieast one of the AB in the ninitispeciﬁe aetiyatabie antibody is modified with a MM and is in the presence of the target, specific binding of the AB to its target is reduced or inhibited, as compared to the specific binding of the AB not modiﬁed with an MM or the specific binding of the parentai AB to the target When ied to the binding of the AB not modified with an MM or the binding of the parental AB to the target, the ABS ability to bind the target when modiﬁed with an MM can be reduced by at least 50%, 60‘343, 700/43 h %, 90%, 92‘Vi3, 93%, 94%, 95943, 960/ 97"m, 98%, 99‘V43 and O3!en 10(‘3' for at ieast 2, 4, 6, 8, 1,2, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or l80 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more when measured in viva or in an in vitro assay. {000241} The MM inhibits the binding of at ieas‘t one of the AB in the rnnltispeoitic aetivatabie antibody to its target, The MM binds the antigen binding domain of the AB and inhibits binding of the AB to its target. The MM can stericaiiy t the binding of the AB to the target The MM eanaaliostericaliy inhibit the binding of the AB to its target in these ments when the AB is modified or conpied to a Mix/1 and in the presence of target, there is no binding or snbstantialiy no binding of the AB to the , or no more than 0001%, 001%, 0 1%,1‘343,2%, 3% 6‘343, 717:3, 89/o 9°M), 10343, 1513/13, 20%, 25%, 30043 35%, 40%, or50% binding of the AB to the target, as compared to the binding of the AB not modiﬁed with an MM, the parentai AB, or the AB not d to an MM to the target, for at least 2, 4, 6, 8 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, l5, 30, 45, 60, 90, 120, l50, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or ionger when measured in vivo or in an in vitro assay. {00024-2} When at least one of the AB in a muitispecitie activatable antibody is coup1ed to or modiﬁed by a MM, the MM ‘masks’ or reduces or otherwise inhibits the c binding of the AB to its target. When at least one of the AB in a multispeeilie activatable a1'1tibody is coupled to or r1'1oditied by a MM, such cotipling or modiﬁcation can effect a structural change that reduces oi inhibits the y of the A8 to specifically hind its targe {0002433 in a multispecil‘ie aetivatable antibody, when at least one AB is coupled to or modified with an MM, at least a portion of the multispeeiﬁe aetiyatahle antibody can be represented by the following formulae (in order from an amino (N )1 terminal region to earboxyl (C) terminal region: (MM)—(AB) (AB)~(h When compared to the binding of the parental A8 or the binding of an AB not modiﬁed with an MM and a CM to its target, the AB’S ability to bind the target when modiﬁed with an MM and a CM can be 1educed by at least 50",o, 60%, 700//,0 80/0, 90%, 92% 93%, 94",o, 95""/o 96%, 979-11, 98‘76, 99% and even 0100/0101 at least",4 0, 8 1128,2430, 10,48, 60, 72, 84, or 96 hours or 5, ll}, 15, 30, 45, 6t), 90, thi, 150, or 180 days, or 1, 2, 3, 4, 5, 6, '7, 8, 9, it), l l, or 12 months or longer when measured in vivo or in an in vitro assay. {@82le As used , the term cleaved state refers to the condition of the niultispecific activatahle antibodies following modification of the CM by a protease. The term uncleaved state, as used herein, refers to the condition of the rnultispecil‘ic activatable antibodies in the absence of cleavage of the CM by a protease, As discussed above, the term "inultispecitic activatable antibodies" is used herein to refer to a. inultispecitic activatable antibody in both its uncleaved (native) state, as well as in its cleaved state. it will be apparent to the ordinarily skilled artisan that in some en'ibodin'ients, a cleaved niultispecilie activatable antibody may lack an MM due to cleavage of the CM by protease, resulting in release of at least the MM (cg, where the MM is not joined to the rnultispecilic activatable antibodies by a covalent bond tag, a disultide bond between cysteine residues). {999251} By activatable or able is meant that the m ultispecilic aetivatable antibody exhibits a first le *el of g to a target when in a ted, masked or ved state (to, a first conformation), and a second level of binding to the target, in the uninhibited, unrnaslred and/or cleaved state (126., a second mation), where the second level of target binding is greater than the first level of binding. in general, the access of target to the corresponding AB of the inultispecitic activatahle antibody is greater in the presence of a ng agent capable ol‘cleaving the CM than in the absence of such a cleaving agent. Thus, when the nniltispecitic activatable antibody is in the uncleaved state, at least one AB is inhibited front target binding and can be masked from target binding (to, the first conformation is such the AB cannot bind the target), and in the cleaved state the AB is not inhibited or is unmasked to target g. $th52} The CM and AB of the multispeciiic table antibodies are selected so that the first AB represents a binding moiety for a first target and/or epitope, and the CM represents a substrate for a protease that is ctr—localized with the target at a treatment site or diagnostic site in a subject. The nniltispecitic activatable dies disclosed herein tind particular use where, for example, a protease capable of cleaving a site in the CM is present at relatively higher levels in target—containing tissue of a treatment site or diagnostic site than in tissue of non—treatment sites (for example in healthy tissue). $th53} ln some embodiments, pecific aetivatable antibodies provide for reduced ty and/or adverse side effects that could otherwise result from binding of the first AB at non—treatment sites if the AB were not masked or ise ted from g its target. {$388254} ln general, a multispecific activatable antibody can be designed by selecting at first AB of interest and constructing the remainder of the activatable antibody so that, when conformationally constrained, the MM provides for masking of the AB or ion of binding of the AB to its target. Structural design criteria, can be to be taken into account to provide for this functional feature, EtltltBZSS} Mnltispecitic activatable antibodies exhibiting a switchable phenotype of a desired dynamic range for target binding in an inhibited versus an uninhibited contbrmation are ed. Dynamic range generally refers to a ratio of (a) a maximum de ,ected level of a parameter under a first set of conditions to (b) a minimum detected value of that parameter under a second set ot‘conditions. For example, in the context of a multispecific table antibr’ldy, the dynamic range refers to the ratio otta) a maximum detected level of target n, binding to a multispecific activatable antibody in the presence of protease capable ofcleaving the CM of the activatable antibodies to (b) a minimum detected level of target protein, binding to a multispecil'ic activatable dy in the absence ofthe protease. The dynamic range of a mnltispecilic activatable antibody can be ated as the ratio of the equilibrium dissociation constant of a multispecific activatable antibody cleaving agent (rag, enzyme) treatment to the brium dissociation constant of the activatable dies ng agent ent. The greater the dynamic range of a multispecilic activatable antibody, the better the switchable phenotype of the activatable antibody.

Activatable antibodies having relatively higher dynamic range values (cg, greater than l) exhibit more desirable switching phenotypes such that target protein binding by the activatable antibodies occurs to a greater extent (egg, predominantly occurs) in the presence of a cleaving agent (c.g, enzyme) capable of cleaving the CM of the activatable dies than in the absence of a cleaving agent. 6} Multispecitic activa‘table antibodies can be provided in a variety of structural configurations. Exemplary formulae for at least a portion of a multispecific aetivatable antibody are provided below. it is specifically contemplated that the N— to C~terininal order of the first AB, the corresponding MM and CM may be reversed within an activatable antibody. it is also specifically contemplated that the CM and MM may overlap in amino acid sequence, cg, such that the CM is contained within the MM. 09‘1,..9 EiltltlZS’Z’} For example, at least a portion of the multispecitie aetivatable antibodies can be represented by the following formula (in order from an amino (N3 terminal region to earboxyl (C) terminal region: (MM)"(tilt/li-(AB) (AB)~(Ch/l)~(h/ll\/l3 {000258} Where MM is a masking moiety, CM is a eleayable , and AB is a first antibody or fragment thereof. lt should be noted that although MM and CM are indicated as distinet components in the formulae above, in all exemplary embodiments (including torniul ae 3 disclosed hereinit is contemplated that the amino acid sequences of the MM and the CM could overlap, erg, sueh that the CM is eompletely or partially contained within the MM. ln addition, the formulae aboye provide for additional amino acid sequences that may be positioned N~terminal or C—terminal to the aetiyatable antibodies elements. 3} in eertain embodiments, the MM is not a natural binding partner of the AB. in some embodiments, the MM contains no or substantially no homology to any natural binding r of the AB. ln other embodiments the MM is no more than 55%;, l0%, l5%, ,0, 25%, 30% So 40%, 45%, 50%, SS%, 6094), 650/6, 70%, 7594, ir80‘3/r)Slimilai to any l binding partner of the AB. ln some embodiments, the MM is no more than 5%, l0%, 15%, 20%, 25"7o 30%,35%, 40%, 450/,5.( ‘io,:>55%,660%, 65%, 5% or 800A; identical to any natural binding partner of the AB. ln some embodiments, the MM is no more than 50% identical to any natural binding partner of the AB. in some embodiments, the MMis no mote than 250/0 cal to any natural binding partner of the AB. ln some ments, the MM is no more than 20% identical to any l binding partner of the AB. in some embodiments, the MM is no more than l0% identical to any natural binding partner ofthe AB {000260} hi many embodiments it may be desirable to insert one or more linkers, rag, flexible linkers, into the multispeeil‘ie aetivatable antibody uct so as to provide for flexibility at one or more of the h/lh’l—Clvljnnetion, the (TM-AB on, or both. For example, the AB, MM, and/or CM may not contain a sufﬁcient number of residues (egg, Gly, Ser, Asp, Asn, ally Gly and Set, particularly Gly) to e the desired ﬂexibility. As sueh, the switchable phenotype of sueh rnultispeeilie aetivatable antibody eonstiuets may benefit from introductimi of one or more amino acids to provide for a flexible linker in addition, as described below, Where the mnltispee fie activatable antibody is provided as a eonl‘ormationally constrained construct, a flexible linker can be operably inserted to tate formation and nance of a cyclic structure in the uncleaved multispecific activatable antibody. 1] For example, in certain embodiments a multispecific activatable antibody comprises one of the following formulae (where the formula below represent an amino acid sequence in either N- to C-terminal direction or C- to N-terminal direction): (MM)-L1-(CM)-(AB) (MM)-(CM)-L2-(AB) (MM)-L1-(CM)-L2-(AB) wherein MM, CM, and AB are as defined above; wherein L1 and L2 are each independently and optionally present or absent, are the same or different flexible linkers that include at least 1 flexible amino acid (e.g., Gly). In addition, the formulae above e for additional amino acid sequences that may be positioned N-terminal or C- terminal to the multispecific activatable antibodies elements. Examples include, but are not limited to, targeting moieties (e.g., a ligand for a or of a cell present in a target tissue) and serum half-life extending moieties (e.g., polypeptides that bind serum ns, such as immunoglobulin (e.g., IgG) or serum n (e.g., human serum albumin (HSA)).

In some non-limiting embodiments, at least one of the AB in the multispecific activatable antibody is a binding partner for any target listed in Table 1.

In some non-limiting embodiments, at least one of the AB in the multispecific activatable antibody comprises, is or is derived from a sequence set forth in Table 7 in the Examples provided herein.

In some non-limiting embodiments, at least one of the AB in the multispecific table antibody comprises, is or is derived from a sequence set forth in Example 5 in the Examples provided herein. In some non-limiting embodiments, at least one of the AB in the multispecific activatable dy comprises, is or is derived from a sequence set forth in Table 11 in the Examples provided herein.

In some non-limiting embodiments, at least one of the AB in the multispecific activatable antibody is or is d from an antibody listed in Table 2.

In some embodiments, the masking moiety is selected for use with a specific antibody or antibody fragment. For example, suitable g es for use with antibodies that bind EGFR include MMs that include the ce CISPRG (SEQ ID NO: 75). By way of non-limiting examples, the MM can include a sequence such as CISPRGC (SEQ ID NO: 339), CG (SEQ ID NO: 76); CISPRGCPDGPYVMY (SEQ ID NO: 77); CISPRGCEDGPYVM (SEQ ED NO: 78), CESERGCEPGTYVPT (SEQ 110 NO: 79) Mid CISERGCPGQENPWE (SEQ ID NO: 80). Other suitable g moieties ineiw’ie any ofthe EGER,—speeiﬁc masks disdesed in PCT i’ublieatim N0. by way if non-iimiting cxampia HNMGAESC (SEQ 113 NO: 81); CISERGCGGSSASQSGQGSE—KILIPENMGAPSC (SEQ ID NO: 82); CNHHYEYTCGCISPRGCEG (SEQ ID NO: 83); ADHVEW’GSYGCISPRGCPG (SEQ 11) NO: 84); CHHVYWGHCGCISPRGCEG (SEQ ID NO: 85); CPHETTTSCGCISPRGCPG (SEQ ID NO: 86); CNHE-iYE-IYYCG(ESPRGCPG (SEQ ID NO: 87); CPHV’SEGSCGCISERGCEG (SEQ if) NO: 88); CEYYTESYCQCISPRGCPQ (SEQ ID NO: 89); CNHVYEGTCQCISPRGCEQ (SEQ 11') NO: 91)); CNHE"€111"?CGCISPRQCPG (SEQ 11) NO: 91); CHHEI‘LT'E'CGCISERGCPG (SEQ 1D NO: 92.); YNPCATPMCCISERGCPG (SEQ ID NO: 93); ‘x’EYTCGﬂSPRGCG (SEQ ID NO: 94); HYYCGCESPRQCG (SEQ if) N0: 95); CNHVYEGTCGUSPRGCG (SEQ if) NO: 96); CHEVY WGHCGQSERGCG (SEQ 11) NO: 97); CPHETTTSCGCiSPRGCG (SEQ ID NO: 98); CN1—1ETLTTCGCISPRGCG (SEQ 113 NO: 99); CHHETLTTCGCESERQCG (SEQ 11) NO: 100); CP‘YYTLSYCQCISPRGCG (SEQ 11?) NO: 1111); CPHVSEGSCQCISERGCG (SEQ 11) NO: 102); ADHVEWQSYGCISPRGCG (SEQ 1D NO: 103); YNPCATPMCCISPRGCG (SEQ ID NO: 104); CHE-1VYWGHCGCISPRGCG (SEQ ID NO: 105); C(N/P)H(1>i/V/E)(Y/T)(E/VV/T/L)(Y/G,I’T/l )(T/S/V/H)CGCISPRGCG (SEQ 11) NO: 106); C1Si’RGCGQE’11)SVK (SEQ 11') NO: 107'); CISER.GC'1‘QEYHVSR (SEQ 11) N0: 198); and/er CISERGCNAVSGLGS ( SEQ ID NO: 109).

HERMES} Suitab1e masking es for use with antibodies that bind a. Jagged target; eg" Jagged 1 and/or Jagged 2; include, by way of non—limiting exampieﬂ masking moieties that inciude a sequence such as QGQSGQCNiWEVGGDCRGWQG (SEQ 113 NC): 338); QC:QSGQGQQQWCNHV1NGGDCRGWNG (SEQ ID NO: 1 1(1); PWCMQRQDELRCPQE (SEQ 11) N0: 1 1 1); QEQLPAYMCTFECLR (SEQ 11) NO: 1 12); CNLWVSGGDCGGLQG (SEQ 11) NO; 113); 1'SGSCI.PHSE (SEQ 11) NO: 1 14); YCLQLH—WMQAMCGR (SEQ ID NO: 115); CELYSCTDVSYWENT (SEQ ID N0: 116); PEVCMQRQDYLRCEQP (SEQ 11) NO: 117); CNL‘WISGGDCRGLAG (SEQ ID NO: 118); CNLWVSGGDCRGVQG (SEQ 11) NO: 1 19); GQDCRGLRG (SEQ ID NO: 12(1); CNLW1SGGDCRGLPG (SEQ 1D NO: 121); CNLWVSGGDCRDAEW (SEQ ID NO: 122); CNLWVSGGDCRDLLG (SEQ 110 NO: 123); CNLWVSGGDCRGLQG (SEQ 113 NO: 124); CNLWLHGGDCRGWQG (SEQ ID NO: 125); CN1WLVGGDCRGWQG (SEQ 1121 NO: 126); CTTWECGGDCGVMRG (SEQ 11) NO: 127); CN1V’V"G1)SVQCGALLQ (SEQ 11) NO: 128); CNIWVNGGDCRSEEG (SEQ 11') NO: 129); YCLNLPR‘YMQDMCWA (SEQ 1131 NO: 13(1); PHYl‘AQADCAR (SEQ ID NO: 131); GDVSYWQSA (SEQ ID NO: 132); CYLYSCTDSAEWNNR (SEQ ID NO: 133); NDVSYWSNT (SEQ 11.) N0: 134-); CELYSCTDVSYW (SEQ 11) NO; 135); CPL/YSC'I‘DVAYW’NSA (SEQ 11) NO: 136); CELY SCTDVSYWGDT (SEQ 111 NO: 137); TDVSYWGNS (SEQ 11) NO: 131%); CELYSCTDVAYWNNT (SEQ 113 NO: 139); CELYSCGQVSYWGNEGLS (SEQ ID NO: 141)); CELYSCTDV’AYWSQ1., (SEQ 112) NO: 141); CYLYSC’1‘DQSYWNS'1' (SEQ 11') NO: 1112); CELYSCSDVSYWGN1 (SEQ 11) NO: 143); CELYSCTDVAYW (SEQ ID NO: 144); CELYSCTDVS‘YWGST (SEQ 1131 NO: 145); CELYSCTDVAYWGDT (SEQ 11) NO: 146); {1'CN1WLNGGDCRGWVDPLQG (SEQ 11) N0: 147); GCNENLVGGDCRQW18111118 (SEQ 11) NO: 148); GCN1WLVGGDCRGW1EDSNG (SEQ 11) NO: 149); GCN'1WANGGDCRGW1DN11316 (SEQ 113 NO: 150); GCN1WLVGGDCRGWLGEAVG (SEQ 1L1 NQ: 151); GCN1W1...VGGDCRGWEEEAVCs (SEQ 11) NO: 152); GGPALCN1WLNGGQCRGWSG (SEQ 11) NO: 153); GAEVECN1W1JNGGDCEGWMG (SEQ 11) NO: 154); GQQQWCNENINGGDCRGWNG (SEQ ID NO: 155); GKSEECN1WLNGGDCRGWIG (SEQ 1D NQ: 156); GTPGGCNEWANGGDCRGWE13 (SEQ 113‘ NO: 157); GASQYCNI..W1NGGDCRGWRG (SEQ ID NO: 158); GCN1WLVGGDCREWW’EGG (SEQ 11') N0: 159); GCN1WAVGGDCREEVDGQ (SEQ 11) NO: 160); GCNENLNGGBCEAW (DIG (SEQ 111110: 161); GEN1W1VGGDCREE1NDG (SEQ 11) NO: 162); CsCN1WLNGGDCRPVVEGG (SEQ 112) NO: 163); GCN1W1;SGGDCRMEMNEG (SEQ 11.) NO: 1134); GCNIWVNGGDCRSEVYSQ (SEQ 11') NO: 165); GCNIWLNGGDCRGWEASG (SEQ 111 NO: 166); (ECNEWA11GGDCRGEEEPG (SEQ 1D NQ: 167); Néli'iiiﬂCRTEVASG (SEQ ID NO: 168); GCN1WA1’1GGDCRGE1EEG (SEQ 11") NO: 169); GELENCNHVLNGQDCRTG (SEQ 11') N0: 170); G1YENCN1W15NGGDCRMG (SEQ 11') N0: 171); and/or QIEDNCN1W1NGGDCRYG (SEQ 11) N0: 172).

ESMZES} Suita131e g 1110163116311 1131113162 with antibmiies 1112111311111 an 1n1€2r1euk1n 6 , 6%.; interbukin 6 recaptm' (115—611.), inc1m1e, by way of 1103—1117011,ng example; 11191311:ng moieties that 1nc1ude a sequence such as QGQSGQYGSCSWNYVH1E1VEDC (SEQ 113 NO: 174); QGQSGQGDED1,1’ERA1—1W‘v’1’11‘ (SEQ 11) NQ: 175); QQQSGQMGVEAGCVWNYAIIIEIVIDC (SEQ ID NO: 176); YRSCNWNYVSIELDC (SEQ ID NO: 177); PGAEDIFEFAI‘IW‘VFNT (SEQ II) N0: 178); FSSCVWNYVIIIYIVIDC (SEQ II) NQ: I79); YEGCKWNYDRIEIIDC (SEQ II) NO: 180); YR'I’CSWNYVGIELDC (SEQ ID NO: 181); ‘YGSCSWNY VIIIFMDC (SEQ II) NO: 182); YGSCSV‘MNYVI—IIELDC (SEQ ID NI): 183); YGSCNWNYVIIIFLDC (SEQ ID NO: 184); YTSCNWNYVIIIFMIXi (SEQ II) NO: 185); YEQCKW‘NYIJEIFLIJC (SFQ II) N0: 186); WRSCNWNYAIIIELDC (SEQ II) NO: 187); NYVHIELDC (SEQ II) NO: 188); DRSCTWNYVRISYDC (SEQ ID NO: 189); SGSCKWDYVI—IIELDC (SEQ ID NO: 190); SRSCIWNYAHIIIIDC (SEQ ID NO: 191); SMSCYWQYERIEIJDC (SEQ II) NO: 192); YRSCNWNYVSIFLI)C (SEQ II) NO: I93); YGSCSVVNYVIIIEE/IIJC (SFQ II) NO: 194); SGSCSWDYVHIELDC (SEQ II) NO: 195); YESCHWDYVIIIFLDC (SEQ ID NI): 1%); YQSCTVII’NYVI-IIFIVIEC (SEQ ID N0: 197:); ESSCNWNYVHIELDC (SEQ ID NO: I98); WRSCN‘IVN‘YAIIIFEDC (SEQ II) NO: 199); NYV’IIIELDC (SEQ ID NO: 2&0); YRSCNWNYVHIELDC (SEQ ID NO: 291,); NMSCHWDYVIIIFLDC (SEQ ID NO: 202); FGPCTWNYARISWDC (SEQ ID NO: 203); XXSCXWXYVIIIEXCIC (SEQ II) NO: 204); CVWNYAIIIFMDC (SEQ ID NO: 205); RDTQGQCRWDYVHIFMDC (SEQ II) NO: 206); AGVEAGC'I‘WNYVIIIFMEC (SFQ II) NO: 207); VGVI’NGCVWNYAHIEMEC (SEQ II) NO: 2&8); DGGFAGCSWINYVIIIEMEC (SEQ II) NO: 209); AVGPAGCWWNYVI—IIEIVIEC (SEQ II) NO: 210); CTVVNYVIIIEIVIDCGEGEGF (SEQ II) NO: 21 I); CsGVEEGCTWNYAIIIEMEC (SEQ II) NO: 212); AEVFAQCWWNYVIIIEMEC (SEQ II) NO: 213); AGVI’AGC’IWNY VIIIFMEC (SEQ ID NO: 214); SGASGGCI‘QWNYVIIIEIVII)C (SEQ II) NO: 215); MGVEAGCVWNYAHIFMDC (SEQ ID NO: 216); TEGCRWNYVHIEIVIECEAL (SEQ II) NO: 217); VGVPNGCVWNYAIIIFMEC (SEQ II) NO: 218); PGAEDII’FEAIIW «’I’NI' (SEQ II) NI): 2.19); I’EIELXIIWIPN’I (SEQ ID NO: 220); QGDEDIEEI’AI—IV‘IWEIT (SEQ ID N0: 221:); XGaIDIEEPAHWVEnT (SEQ ID NO: 222); RGDQNDSIJIEFFAIIVRWERI (SEQ II) NO: 223); SGVGEDRDII)FEAIIW'VPETI‘ (SEQ ID NO: 224); WAQGNI)CHIPFI’AIIWIPN’I‘ (SEQ ID N0: 225); X/IDVDIPFPAIIWVEVT (SEQ ID NO: 226); AGSGNDSDIPEPAI‘IWVFRT (SEQ II) NO: 227); ESRSGYADIFEPAIIWVFRT (SEQ II) NO: 228); and/or RFCGRCGIHFFPAIIWVFR’I’ (SEQ II) NO: 173)" RENEE); In some embodiments, the eieavable moiety (CM) of the muitispeciﬁc aeiivatabie antibedy ineiudes an amino acid sequence that can serve as a subsirate for a protease, usually an extracellular protease. The CM may be selected based on a protease that is co-localized in tissue with the desired target of at least one AB of the multispecific activatable antibody. A variety of different conditions are known in which a target of interest is co-localized with a protease, where the substrate of the protease is known in the art. In the example of cancer, the target tissue can be a cancerous tissue, particularly cancerous tissue of a solid tumor. There are reports in the ture of increased levels of proteases having known substrates in a number of cancers, e.g., solid tumors. See, e.g., La Rocca et al, (2004) British J. of Cancer 90(7): 1414-1421. Non-liming examples of disease include: all types of cancers (breast, lung, colorectal, prostate, melanomas, head and neck, pancreatic, etc.), rheumatoid arthritis, Crohn’s disease, SLE, cardiovascular damage, ischemia, etc. For e, indications would include leukemias, including T-cell acute lymphoblastic leukemia (T-ALL), lymphoblastic diseases including multiple myeloma, and solid tumors, including lung, colorectal, te, pancreatic and , including triple negative breast . For example, indications include bone disease or metastasis in cancer, regardless of primary tumor ; breast cancer, including by way of non-limiting example, ER/PR+ breast cancer, Her2+ breast cancer, triple-negative breast cancer; colorectal cancer; endometrial cancer; gastric cancer; glioblastoma; head and neck cancer, such as esophageal cancer; lung cancer, such as by way of non-limiting e, all cell lung cancer; le myeloma ovarian cancer; pancreatic cancer; prostate cancer; sarcoma, such as osteosarcoma; renal , such as by way of nonlimiting example, renal cell carcinoma; and/or skin cancer, such as by way of nonlimiting example, squamous cell cancer, basal cell carcinoma, or melanoma. In some embodiments, the cancer is a squamous cell . In some embodiments, the cancer is a skin squamous cell carcinoma.

In some embodiments, the cancer is an esophageal squamous cell carcinoma. In some embodiments, the cancer is a head and neck squamous cell carcinoma. In some embodiments, the cancer is a lung squamous cell carcinoma.

The CM is specifically cleaved by an enzyme at a rate of about 1500 x 104 M-1S-1 or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500 x 104 M-1S-1.

For specific cleavage by an enzyme, contact between the enzyme and CM is made. When the pecific activatable antibody comprising at least a first AB coupled to a MM and a CM is in the presence of target and ient enzyme activity, the CM can be cleaved. ient enzyme activity can refer to the ability of the enzyme to make contact with the CM and effect ge. it can readily be envisioned that an enzyme may be in the vicinity of the CM but nnsbie to Cleave because of other eeiiniar factors or protein modiﬁcation of the enzyme" {8639273} Exeinplaiy substrates ineiude but are not limited to substrates eieavabie by one or more of the following enzymes or proteases in Table 3: Table 3: Exempiary ses andI’or Enzym es ADAMS, ADAMTS, 9.1g. Cysteine proteinases, age, Sei'ine pretenses, eg, ADAMS szipain activated protein C ADAM?) Legumain Cathepsin A ADAMEG Gotham—2 Cathepsin G ADAM i 2 se ADAME 5 KLKS, ag eoaguiation factor proteases ADAM '7/TACE KLKA’E (egg F‘V’Ha, FIXa, FXa, FXIa, ADAMDEC} KLKS *Xﬁa) ADAMTSI nuns ADAMTSA KLK? ADAMTSS KIKS KLKie Asparta’te pretenses, eg, KLKH BACE KLKis » Benin KLKM Marapsin ............................................................................1....

NEE/AA ______________________________________________________________________ Aspartie esthepsins, e.g., Metalh’) n-ages, egg, PACE4 Cathepsin D Meprin Plasmin "1meNspriiysin PSA PSMA tPA Caspases, egg , TEMP—i_________________________________________________________ ‘nin Caspase 1 Tryptase _____________________________________________________________________________ Caspase 2 M‘yiPs, erg, uPA Caspase 3 MMPI Caspase 4 MVH’Z '1"pr ﬂ Transmmnbrane Caspase 5 MMPE Sei'ine Proteases (TTSPS), 6.53., Caspase 6 MM’P?’ onset Caspase 7 M‘s/1P8 Dee—4 Caspase 8 MVP?) FA? Caspase 9 EVER/{PEG i-iepsin e it) MMPM Matripmseﬂ Cas Base 14 M‘viPtZ MT—SPUManiptase M‘viPtES TMPRSSZ Cysteine eatnepsins, ag, M‘s/1PM TMPRSS3 Cathepsin B MM?! 5 'I'MPRSS4 Cathepsin C MMPio Cathepsin K MMPE'Z Cathepsin i... MM???) Cathepsin S MMPZG sin V/LZ MMFB Cathe sin X/Z/i) MMI’ZI—‘I MMP26 strum? {3%,274} For exampIe, in some embodiments, the substrate is cieavabie by one or more of the toiiowing enzymes or pretenses: uFA, iegumain, MT—SPI, ADAMIZ BMP—I, TMPRSS3, TMPRSSd, MMP~9, MMP~IZ, MMRI 3, and/or MMP~I 4. In some embodiments, the protease is seieeted from the group of uFA, Iegumain, and MT-SPI. In some embodiments, the se is: a matrix metaIioproteinase. In some embodiments, the protease comprises uPA. In some ernbi‘idirnents, the protease comprises Iegumain. In some embodiments, the protease comprises M'I'—SI’I. genitive} In some embodiments, the CM is ed for use with a, ic protease. In some embodiments, the CM is a substrate for at ieast one protease selected from the group consisting of an ADAM 17, a EMF—I, a cysteine se such as a eathepsin, a I’ItrAi, a Iegumain, a matriptase (MI-SIN), a matrix metaiioprotease (Mb/IF), a neutrophii eiastase, a TMPRSS, such as TMPRSSZI or TMP RSS4, a in, and a n—type piasminogen activator (uPA, also referred to as urokinase). {$630276} In some embodiments, the CM is a substrate for an ADAMN, In some embodiments, the CM is a substrate fora EMF-I. In some embodiments, the CM is a, ate for a cathepsin. In some embodiments, the CM is a substrate for a cysteine protease. In some embodiments, the CM is a substrate for a I’ItrAI. In some embodiments, the CM is a substra ,e for a iegumain. In some embodiments, the CM is a substrate fora MT! SI’I. In some embodiments, the CM is a ate for a MMP. In some embodiments, the CM is a substrate for a neutrophii etastase. In some embodiments, the CM is a substrate for a thrombin, In some embodiments, the CM is a substrate for a "i‘MPRSS. In some ments, the CM is a substrate for "I‘MPRSSB. In some embodiments, the CM is a substrate for TMPRSSL‘t. in some embodiments, the CM is a substrate for uPA.

IdhttZ’F’f} In some embodiments, the eieavabie moiety is seteeted for use with a specific protease, for exampie a protease that is known to be (so—hteaiized with the target of the activatabie dy. For example, suitabie cieavabie moieties for use in the activatabie antibodies of the disetosure inetude the sequence TGRGPSWV (SEQ ID NO: 27); RW (SFQ II.) N0: 218); 'I'AIIGPSFK (SEQ. ID NO: 29); IJSGRSIJNH (SEQ ID NO: ‘26); GRN (SEQ ID NO: 33); HTGRSGAL (SEQ III NO: 3 i); FLTGRSGG (SEQ ID NO: 32); AARGPAii-i (SEQ 153‘ NO: 33); RGPAENEM (SEQ 11) N0: 34); SSRGPAYL (SEQ 1):? NO: 35); RGPATPEM (SEQ ii) N0: 36); RGPA (SEQ H) NO: 37); GGQPSGNEWGW (SEQ 11') N0: 38); EPRPLGITGL (SEQ 11) NO: 39); VHMEEGEEGP (SEQ 1E? NO: 4%); SEL’i‘GRSG (SEQ 1D NO: 41); SAGESLPA (SEQ 11) NO: 42); LAPLGLQRR (SEQ 13 NO: 43); SGGPLGVR (SEQ 11) NO: 44); and/or PLGL (SEQ ID NO: 45), {WEBER} in some embodiments, the CM is a, ate for at 1east one matrix oprotease (MME). Examines of MMPs include MMEI; MMP2; MMP3; Nib/1P7; MMPS; MMPQ; MM? 1 0; MM? 1 i; MMPE 2; MM? 1 3; MM? 1 4; MMP1 5; MM? i 6; MMPi 7; MM?) 9; Mitt/EEK); MMP23; MMPZ4; Mix/{1326; and MMP27, In some embodiments, the CM is a, substrate for MMP9, MMEM, MMi’i, MMP3, Mix/£1313, MME17, MMPi 1, and Mix/1H9. in some embodiments, the CM is a substrate for MMPT in some embodiments, the CM is a substrate for MMEE). in some en'ibodin'ients, the CM is a substrate for MMPM, 1n some ments, the CM is a substrate for two or more MMi’s. in some embodiments, the CM is a substrate for at ieast MMP‘) and MMPM. in some embodiments, the CM comprises two or more substrates for the same MME. in some embodiments, the CM comprises at least two or more MMEQ substrates in some embodiments, the CM comprises at least two or more Mix/11314 substrates.

EGWZWE in some embodiments, the CM is a substrate for an MM? and includes the sequence iSSGLESS (SEQ ii) NO: 316); MA (SEQ 1):? N0: 317); AQNMCMV (SEQ 11') N0: 318); S'1‘E1’EGME (SEQ 1E) NO: 319); 1))!GYTSSL (SEQ 11) N0: 329); DWLY V’i’GI (SEQ H) NO: 321); MiAi’VAYR (SEQ 1E3 NC): 322); Ri’SPh/ft‘y’VAY (SEQ it) NO: 21); WATEREMR (SEQ 11") NO: 323); ERLLDWQW (SEQ ID NO: 324); i..i Computational Chem, lll73—l42 (1992)). Exemplary tlexihle linkers include, but are not limited to Gly—Gly—Ser—Gly (SEQ ll) NO: 29), Gly~Gly—Ser—Gly—Gly (SEQ ll) NO: 2i), (Sly—Ser~Gly—Ser—Gly (SEQ lD NO: 22), GlynSer‘~Gly—Gly—Gly (SEQ lD N0: 23), lyn Gly-Ser—Gly (SEQ if) NO: 24), Gly—Ser~$er-Ser—Gly (SEQ ll") NO: 25), and the like, The ordinarily skilled artisan will recognize that design of an tahie antibodies can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less llexible structure to provide for a desired pecilic activatable antibodies structure. $be83} in addition to the elements described above, the multispecit‘ic aetivatable antibodies can contain additional elements such as, for e, amino acid sequence N-- or C—terrninal of the multisnecitic activatable antibodies. l:or example, multispecilic activatable antibodies can include a targeting moiety to facilitate delivery to a cell or tissue of interest. Multispecitic activatable antibodies can be ated to an agent, such as a therapeutic agent, an antineoplastic agent, a toxin or liragrnent thereof, a detectable moiety or a stic agent. Examples of agents are disclosed herein. {b88284} The multispecifie activatable antibodies can also include any of the conjugated agents, linkers and other components described herein in conjunction with a necitic antibody ol’tlie disclosure, including by way ofnon—limiting example, any of the agents listed in Table 4 and/or any of the linlters listed in Table 5 and/or Table 6.

Con treated Multiso ecitic Antibodies and Con .u rated Multis ecifie Aﬁiva’table Antibodies liltitlleS} The disclosure also pertains to imrnunoconjugates comprising a specitic antibody and/or a multispeciiic activatable dy conjugated to a cytotoxic agent such as a toxin (9.33., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (Le, a radioconiugate). le cytotoxic agents include, for e, dolastatins and derivatives thereoltag. atin E, APP, Mix/EAT), b/lh/lAF, MMAE). For example, the xic agent is monomethyl auristatin l? (MMAE). in some embodiments, the agent is monometliyl atin D (MMAD). in some embodiments, the agent is an agent selected from the group listed in Table 4. in some embodiments, the agent is a dolastatin. in some embodiments, the agent is an auristatin or derivative the eof. in some embodiments, the agent is auristatin if or a derivative thereof. in some embodiments, the agent is rnonometliyl auristatin E (MMAE). in some ments, the agent is a tnaytansinoid or maytansinoid derivative. in some embodiments, the agent is DM l or Div/T4, in some embodiments, the agent is a duocarmycin or tive thereof. in some embodiments, the agent is a calicheanticin or derivative thereof. in some embodiments, the agent is a nyrrolobenzodiazepine. liltillletl} Enzyrna'tically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active nts of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alnha~sarcin, Aleurites fordii ns, dianthin proteins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis tor, gelonin, mitogellin, restrictocin, ycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, 64Cu, 125I, 131I, 131In, 99mTc, 90Y, 186Re, and 89Zr.

Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl(2-pyridyldithiol) propionate (SPDP), hiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), ido compounds (such as bis (p-azidobenzoyl) hexanediamine), bisdiazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), yanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro- 2,4-dinitrobenzene). For example, a ricin immunotoxin can be ed as described in Vitetta et al., Science 238: 1098 (1987). Carbonlabeled 1-isothiocyanatobenzyl methyldiethylene triaminepentaacetic acid (MX-DTPA) is an ary chelating agent for conjugation of radionucleotide to the dy. (See WO94/11026).

Table 4 lists some of the exemplary pharmaceutical agents that may be employed in the herein described disclosure but in no way is meant to be an exhaustive list.

Table 4: ary Pharmaceutical Agents for Conjugation CYTOTOXIC AGENTS Auristatins Turbostatin Auristatin E Phenstatins Monomethyl auristatin D (MMAD) Hydroxyphenstatin Monomethyl auristatin E (MMAE) Spongistatin 5 hyl auristatin E (DMAE) Spongistatin 7 Auristatin F Halistatin 1 thyl auristatin F (MMAF) Halistatin 2 Desmethyl auristatin F (DMAF) Halistatin 3 Auristatin derivatives, e.g., amides thereof Modified Bryostatins Auristatin tyramine Halocomstatins Auristatin quinoline Pyrrolobenzimidazoles (PBI) Dolastatins Cibrostatin6 Dolastatin derivatives Doxaliform Dolastatin 16 DmJ Anthracycline analogues Dolastatin 16 Dpv sinoids, e.g. DM-1; DM-4 Maytansinoid derivatives Cemadotin analogue (CemCH2-SH) Duocarmycin Pseudomonas toxin A (PE38) variant Duocarmycin derivatives monas toxin A (ZZ-PE38) variant Alpha-amanitin ZJ-101 Anthracyclines OSW-1 Doxorubicin 4-Nitrobenzyloxycarbonyl tives of O6-Benzylguanine Daunorubicin Topoisomerase inhibitors Bryostatins Hemiasterlin Camptothecin Cephalotaxine Camptothecin derivatives Homoharringtonine 7-substituted Camptothecin obenzodiazepine dimers (PBDs) , 11- Functionalized pyrrolobenzodiazepine Difluoromethylenedioxycamptothecin Combretastatins Calicheamicins Debromoaplysiatoxin Podophyllotoxins Kahalalide-F Taxanes Discodermolide Vinca alkaloids Ecteinascidins CONJUGATABLE ION ANTIVIRALS Fluorescein and derivatives thereof Acyclovir scein isothiocyanate (FITC) Vira A Symmetrel RADIOPHARMACEUTICALS ANTIFUNGALS 131I Nystatin 89Zr 111In ADDITIONAL ANTI-NEOPLASTICS 123I Adriamycin 131I Cerubidine 99mTc Bleomycin 201Tl Alkeran 133Xe Velban 11C Oncovin 62Cu Fluorouracil 18F Methotrexate 68Ga pa 13N Bisantrene 15O Novantrone 38K Thioguanine 82Rb Procarbazine 99mTc (Technetium) Cytarabine HEAVY METALS ACTERIALS Barium Aminoglycosides Gold Streptomycin Platinum Neomycin Kanamycin ANTI-MYCOPLASMALS Amikacin Tylosin Gentamicin Spectinomycin Tobramycin Streptomycin B Spectinomycin Ampicillin Sulfanilamide Polymyxin Chloramphenicol Those of ordinary skill in the art will recognize that a large y of possible moieties can be coupled to the resultant multispecific antibodies and/or multispecific activatable antibodies of the disclosure. (See, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds), Carger Press, New York, (1989)), the entire contents of which are incorporated herein by reference).

Coupling may be accomplished by any chemical reaction that will bind the two molecules so long as the dy and the other moiety retain their respective activities.

This linkage can include many chemical mechanisms, for instance covalent binding, affinity g, intercalation, coordinate binding and complexation. In some ments, the preferred binding is, however, covalent g. Covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external ng molecules. Many bivalent or lent linking agents are useful in coupling protein molecules, such as the antibodies of the present disclosure, to other molecules. For example, representative coupling agents can include c compounds such as thioesters, carbodiimides, succinimide esters, yanates, glutaraldehyde, diazobenzenes and hexamethylene diamines. This listing is not intended to be exhaustive of the s classes of coupling agents known in the art but, rather, is exemplary of the more common coupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987)).

In some embodiments, in addition to the compositions and methods ed herein, the conjugated activatable antibody can also be modified for pecific conjugation through modified amino acid sequences inserted or otherwise included in the activatable antibody sequence. These modified amino acid sequences are designed to allow for controlled placement and/or dosage of the conjugated agent within a conjugated table antibody. For example, the activatable antibody can be engineered to include cysteine substitutions at ons on light and heavy chains that provide reactive thiol groups and do not negatively impact n folding and assembly, nor alter antigen binding. In some embodiments, the activatable antibody can be ered to include or otherwise introduce one or more non-natural amino acid residues within the activatable antibody to provide suitable sites for conjugation. In some embodiments, the activatable antibody can be engineered to include or ise introduce enzymatically activatable peptide ces within the activatable antibody sequence.

Suitable linkers are described in the literature. (See, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use of MBS (M- maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Patent No. 5,030,719, describing use of halogenated acetyl hydrazide derivative coupled to an antibody by way of an oligopeptide linker. Particularly suitable linkers e: (i) SMPT (4- succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene e Chem. Co., Cat. (21558G); (ii) SPDP (succinimidyl-6 [3-(2-pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat #21651G); and (iii) Sulfo-LC-SPDP (sulfosuccinimidyl 6 [3-(2- pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat. #2165-G. Additional s include, but are not limited to, SMCC, sulfo-SMCC, SPDB, or sulfo-SPDB.

The linkers described above contain components that have different attributes, thus leading to conjugates with differing physio-chemical properties. For example, the linker SMPT contains a sterically hindered disulfide bond, and can form conjugates with increased stability. Disulfide linkages, are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less conjugate available.

The t EDC yl(3-dimethylaminopropyl))carbodiimide hloride is useful to create a carboxamide starting with a carboxylic acid and a y or secondary amine. Thus, EDC may be used to link lysine residues in an antibody with a carboxylic acid in a linker or toxin, or to link aspartate or glutamate residues in an antibody with an amine in a linker or toxin. Such conjugation reactions utilizing EDC may be ed by addition of NHS (N-hydroxysuccinimide) or sulfo-NHS (N-hydroxy oxysulfonylsuccinimide). Addition of NHS or sulfo-NHS to such conjugation reactions may enhance the rate, completeness, selectivity, and/or reproducibility of the ation reactions.

In some embodiments, the s are cleavable. In some ments, the linkers are non-cleavable. In some embodiments, two or more linkers are present. The two or more linhers are all the same, 6g, cleavable or non—cleavable, or the two or more linlrers are different, eg, at least one cleavable and at least one non—cleavable. {$388296} The present sure utilizes l methods for attaching agents to Abs of the multispecific antibodies and/or multispecific activatable antibodies: (a) attachment to the ydrate moieties of the AB, or (b) attachment to sullhydryl groups of the AB, or (c) attachment to amino groups of the AB, or (d) ment to carboxylate groups of the AB. According to the disclosure, ABs may be covalently attached to an agent through an intermediate linlrer having at least two reactive groups, one to react with AB and one to react with the agent. The linlrer, which may include any compatible organic compound, can be chosen such that the n with AB (or a cut) does not adversely affect AB reactivity and selectivity. Furthermore, the attachment of linker to agent might not destroy the activity of the agent. Suitable linkers for reaction with oxidized antibodies or oxidized antibody fragments include those containing an amine selected item the group consisting ol’priinary amine, secondary aniine, hydrazine, hydrazide, ylamine, phenylhydrazine, semicarbazide and thiosemicarbazide groups. Such ve functional groups may exist as part of the structure of the linlrer, or may be introduced by suitable chemical inodil'ication of linkers not containing such groups. {899297} According to the present disclosure, suitable linkers for attachment to reduced ABS of the multispecitic antibodies and/or rnultispecific activatable antibodies include those having certain reactive groups capable of reaction with a sull'hydiyl group of a reduced antibody or fragment. Such reactive groups include, but are not limited to: reactive haloalkyl groups 6 including, for example, haloacetyl groups), curibenzoate groups and groups capable ofMichael—type addition reactions (including, for example, maleimides and groups of the type described by Mitra and , l979, J. Amer. Chem. Soc. lGl : 3697’— 3 l l0).

{Gllllzglll According to the present disclosure, le linlrers for attachment to neither oxidized nor reduced ABs of the mtdtispecitic dies and/or n'iultispecific activatable antibodies e those having certain ﬁrnctional groups capable of reaction with the primary amino groups present in unmodiﬁed lysine residues in the AB. Such reactive groups e, but are not limited to, Nl’lS car‘boxylic or carbonic esters, NHS carboxylic or carbonic esters, 4—nitrophenyl cai'boxylic or carbonic , pentatluorophenyl carboxylic or carbonic esters, acyl imidazoles, isocyanates. and isothiocyanates.

Etltlt3299} According to the present disclosure, suitable linkers for attachment to neither oxidized not reduced ABs include those having certain functional groups capable of reaction with the earboxylic acid groups present in aspartate or glutamate residues in the AB, which have been activated with suitable ts. Suitable activating reagents include EDC, with or without added Nl-lS or sulfo—Nl-lS, and other ating agents utilized for carhoxamide ion. In these instances, the functional groups present in the suitable linkers would include primary and secondary amines, hydrazines, hydroxylamines, and hydrazides. lllllllilllli} The agent may be attached to the linker before or after the linker is attached to the AB. in certain applications it may be desirable to first produce an AB—linker intermediate in which the linker is free of an associated agent. ing upon the ular application, a speciﬁc agent may then be covalently attached to the linker. ln other embodiments the AB is first attached to the MM, CM and ated linkers and then attached to the linker for conjugation purposes. llltll} Branched Linkers: in specific embodiments, branched linkers that have multiple sites for 'nnent of agents are utilized. For multiple site linkers, a sing e covalent attachment to an AB would result in an ker intermediate capable of binding an agent at a number of sites. The sites may be aldehyde or sullhydryl groups or any chemical site to which agents can be attached. {999MB} Alternatively, higher ic activity (or higher ratio of agents to AB) can be achieved by attachment of a single site linker at a plurality of sites on the AB. This plurality of sites may be introduced into the AB by either of two methods. first, one may generate le aldehyde groups and/or sullhydryl groups in the same AB. Second, one may attach to an aldehyde or sulthydryl of the A8 a "branched linker" having le functional sites for subsequent attachment to linkers. The functional sites of the branched linker or multiple site linker maybe aldehyde or sulthydryl groups, or may be any al site to which linkers may be attached. Still higher speeilic activities may be obtained by combining these two approaches, that is, ing multiple site linkers at several sites on the AB.

EtitltlStB} Clem/able Linkers: Peptide linkers that are susceptible to cleavage by enzymes of the complement system, such as but not limited to urokinase, tissue plasminogen activator. tiypsin, in, or another enzyme haying proteolytic activity may be used in one embodiment of the present disclosure. According to one method of the present sure, an agent is attached via a linker susceptible to cleavage by complement.

The antibody is selected from a Class that can activate complement. The antibody—agent eonji,tgate, thus, activates the complement cascade and releases the agent at the target site: ' Etititi3tl4} Non—liming examples ofeleavahle linker sequences are provided in Table 5.

Table 57: Exemplary Linker Sequence-s for Conjugation Tvaes of Cieavahle Se uenees Amino Acid Se uenee Plasmin eleavahle set uences oltinase PREnnoo (SEQ no no: 47) ennnnoo (SEQ in no: as) TGFB ssnninmto (SEQ in no: 49) Piasntinogen RKSSHIRMRBVVL (SEQ lD no: 50) Staphyloltinase SSSEDnonYnnGDoA (SEQ in no: 51 l SSSFDKGKYKRGDDA (SEQ in no; 52 ) Factor Xa eleavahle se uenees llEGR (SEQ in no: 53) IDGR (SEQ in no: 54) Gosmon (SEQ in no: 55) MM? hle setuences Gelatinase A PLGLWA (SEQ to no: 56) Collaoenase eleavabie se uenees Calf shin collagen (til ('1) chain) GPQGIAGQ (SEQ ID no: 57) Calfskin collagen (0.24:1) chain) GPQGLLGA (SEQ in no: 58) Bovine eaitilage collagen (nl (ll) chain) GIAGQ (SEQ in no; 59) l-ltinian liver eoliagen (oi (ill) chain) oPLoiAGi (SEQ in no: 60) Human nigh/l GPEGLRVG (SEQ to no; 6}) Human PZP YGAGLGVV (SEQ in no: 62) AoLoWEn (SEQ to no: 63) ST (SEQ to no: (341) Rat a1M EEQAEAMS (SEQ ID no: 65) QALAMSAi (SEQ to no: (56) Rat nigh/l AAYHLVSQ (SEQ to no: 67) ninAEEEss (SEQ in no: 68) Rat 011125) ESLPVVAV (SEQ in no: 69) Rat 27J} SAPAVESE (SEQ in no: 70) Human ﬁbroblast eollagenase DVAQEVLT (SEQ in no: 71‘) tantoivtie eleava tes VAQFVLTE (SEQ in no: 72) AQE‘VL’l‘EG (SEQ no no: 73) EVQEIGPQ ’SEQ in no: 74) liltitlStPS} in addition, agents may be attached Via disuliide bonds (for example, the disult‘ide bonds on a cysteine molecule) to the AB. Since many tumors naturally release high levels of glutathione (a reducing agent) this can reduce the disultide bonds with uent release of the agent at the site of delivery. in certain ic embodiments the reducing agent that would modify a CM would also modily the linlter of the conjugated activatahle antibody, {ﬁlilBllti} Spacers and Cleavable ts: in still another embodiment, it may he necessary to construct the linker in such away as to optimize the spacing between the agent and the AB of the activatable antibody, This may be accomplished by use ofa linker of the general structure: W (CHﬁn Q wherein W is either CHg—— or "Clip; Q is an amino acid, peptide; and n is an integer from t) to 20. lilillliiil’fl in still other embodiments, the linlrer may comprise a spacer t and a cleavable element. The spacer t serves to position the cleavahle element away from the core of the AB such that the cleavable element is more accessible to the enzyme responsible for cleavage. Certain of the branched linkers described above may serve as spacer ts, {$89388} Throughout this discussion, it should be understood that the ment of linker to agent (or of spacer element to cleavable element, or cleavable element to agent) need not he particular rnode of attachment or reaction Any reaction providing a product of suitable stability and biological compatibility is acceptable. 9} Serum Conrapl’emenr and Selection nfLinkers: According to one method of the present disclosure, when release of an agent is desired, an AB that is an antibody of a class that can activate complement is used. The resulting conjugate s both the ability to hind antigen and activate the complement cascade. Thus, according to this embodiment of the present disclosure, an agent is joined to one end of the cleayalile linlter or cleavahle element and the other end of the linker group is attached to a specch si e on the AB, For example, if the agent has an hydroxy group or an amino group, it may be attach ed to the carboxy terminus of a peptide, amino acid or other suitably chosen linker Via an ester or amide bond, tively. For example, such agents may be attached to the linker peptide via a Carbodiimide reaction. If the agent contains functional groups that would interfere with attachment to the , these interfering functional groups can be blocked before attachment and deblocked once the product ate or intermediate is made. The opposite or amino terminus of the linker is then used either ly or after further cation for binding to an AB that is capable of activating complement. 0] Linkers (or spacer elements of linkers) may be of any desired length, one end of which can be covalently attached to specific sites on the AB of the activatable antibody.

The other end of the linker or spacer element may be attached to an amino acid or peptide linker.

Thus when these conjugates bind to antigen in the ce of complement the amide or ester bond that attaches the agent to the linker will be cleaved, resulting in release of the agent in its active form. These ates, when administered to a subject, will accomplish delivery and release of the agent at the target site, and are particularly effective for the in vivo delivery of pharmaceutical agents, antibiotics, antimetabolites, antiproliferative agents and the like as presented in but not limited to those in Table 4.

Linkers for Release without Complement Activation: In yet another application of targeted delivery, release of the agent without complement activation is desired since activation of the complement e will ultimately lyse the target cell.

Hence, this ch is useful when delivery and release of the agent should be accomplished without killing the target cell. Such is the goal when delivery of cell mediators such as hormones, enzymes, osteroids, neurotransmitters, genes or enzymes to target cells is desired. These conjugates may be prepared by attaching the agent to an AB that is not capable of activating complement via a linker that is mildly tible to cleavage by serum proteases. When this conjugate is administered to an individual, antigenantibody complexes will form quickly whereas cleavage of the agent will occur slowly, thus resulting in release of the compound at the target site.

Biochemical Cross Linkers: In other embodiments, the activatable antibody may be conjugated to one or more therapeutic agents using certain biochemical crosslinkers. linking reagents form lar bridges that tie together functional groups of two different molecules. To link two different proteins in a step-wise , heterobifunctional cross-linkers can be used that eliminate unwanted homopolymer formation.

Peptidyl linkers cleavable by lysosomal proteases are also , for example, Val-Cit, Val-Ala or other dipeptides. In addition, acid-labile linkers cleavable in the low—pH nment ol‘the lysesome may be used, for example: bis~siaiyi ether. Either suitabie linkers include eathepsin—la‘niie substrates, particularly those that Show optimal function at an acidic pH. tenants; Exemplary hetero--bifunetinnai crass-linkers are referenced in Table 6.

Table 6: Exemplary itletem—Bifunetional Cross Linkers HETERG—BIFBNC'E‘KQNAL CRO§$—LlNKERS Spacer Arm Length after cross-l ink ing Linker Reactive 'i‘eward (Angstroi’ns) Primary amines Greater stab il ity dryls SPDP y amines Tliiolation Sulfhydiyls Cleayable eross~lmkmg LC~SPDP Primary amines Extended spacer arm ydryls Sulfe—LC-SPDP Prii’narj amines Extender spacer aim Sulf‘tiydryls soluble SMCC Primary amines Stable inaleiinide reactive group Sult‘hydryls Enzyme-antibody eoniugatien Hapten-eairier protein conjugation Suifo-SMCC Primary amines Stable inaleiinide reactive 11.6 A group Sult‘hydryls Water-soluble Enzyi’ne—antibedy conj Ligation MBS Primary amines Enzyme~anti body eonjugation Sulfhydiyls —earrier protein eunjugation Sulfa-MB S Primary amines Water-soluble Sulihydryls SEAR Primary amines Enzymeanti kindly conjugation Sulfhydiyls Suifo—SIAB y amines Watensoluble Sult‘hydryls SMPB Prii’nary amines Extended spacer arm Sulitiyrlryla Enzyme~antibody conjugation Suife-SMPB Primary amines Extended spacer arm Sulfhydryls water—soluble lfo-NHS Primary amines Hapten-Carrier conjugation Carboxyl groups ABH Carbohydrates Reacts with sugar groups nselective lllllllli 16} Non—Cieavuble Linkers or Direct minister/tent: in still other embodiments of the disclosure, the conjugate may he designed so that the agent is delivered to the target but not released. This may be lished by attaching an agent to an AB either directly or via a non-cleavable linker. ltlllllfil‘l’l These non—cleavahle linkers may include amino acids, peptides, D~amino acids or other organic compounds that may he modified to include functional groups that can subsequently be utilized in attachment to ABS hy the methods described herein. A- l a for such an c linker could be W — (Cllﬁn — Q W is either ----Nll-—-—Cllg-—-— or (ii, Q is an amino acid, peptide; and n is an integer from t) to 29. {ﬁlllB 18} 2--Cieamble Conjugates: Alternatively, a compound may be attached to ABs that do not activate complement. When using ABs that are incapable ofcomplement activation, this attachment may be accomplished using linkers that are susceptible to cleavage by activated complement or using linkers that are not susceptihle to cleavage by activated complement. {9993193 The antibodies disclosed herein can also he formulated as irnnrunoliposomes.

Liposomes containing the antihody are prepared hy methods known in the art, such as described in Epstein et al,, Proci Natl, Acad. Sci, USA, 821: 3688 0985); llwang et al, l’roc.

Natl Acad. Sci. USA, 77: 4tl3t) 0980); and US. Pat. Nos. 4,485,045 and 4,544,545.

Liposomes with enhanced circulation time are sed in US. Patent Ne 5,tll3,556. {$33328} cularly useful liposomes can be generated by the reverse—phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG~derivatized phosp’ha‘tidylethanolarnine (PEG—PE). Liposomes are extruded h ﬁlters of defined pore size to yield liposomes with the desired diameter. Fah’ fragments of the antibody of the present disclosure can he conjugated to the liposomes as described in Martin et al., J. Biol. Chem, 257: 286—288 ( l 982) via a ide—interchange reaction.

Multispecific Activatable Antibodies Having Non-Binding Steric es or Binding Partners for Non-Binding Steric Moieties The disclosure also provides multispecific activatable antibodies that include non-binding steric moieties (NB) or binding partners (NB) for non-binding steric moieties, where the BP recruits or otherwise ts the NB to the multispecific table antibody.

The multispecific activatable antibodies provided herein include, for example, a multispecific activatable antibody that includes a non-binding steric moiety (NB), a cleavable linker (CL) and at least a first antibody or antibody fragment (AB1) that binds a first target or epitope; a multispecific activatable antibody that es a binding partner for a non-binding steric moiety (NB), a CL and an AB1; and a multispecific activatable dy that includes a BP to which an NB has been recruited, a CL and AB1 that binds a first target or epitope. Multispecific activatable dies in which the NB is covalently linked to the CL and AB1 or is ated by interaction with a BP that is covalently linked to the CL and AB1 are referred to herein as "NB-containing multispecific activatable antibodies." By activatable or switchable is meant that the activatable antibody exhibits a first level of binding to a target when the activatable antibody is in an inhibited, masked or ved state (i.e., a first conformation), and a second level of binding to the target when the activatable antibody is in an uninhibited, unmasked and/or cleaved state (i.e., a second conformation, i.e., activated antibody), where the second level of target g is greater than the first level of target binding. The multispecific table antibody compositions can exhibit increased bioavailability and more favorable biodistribution compared to conventional antibody therapeutics.

In some embodiments, multispecific activatable antibodies provide for reduced toxicity and/or adverse side effects that could otherwise result from binding of the multispecific activatable antibody at non-treatment sites and/or non-diagnostic sites if the pecific activatable antibody were not masked or otherwise inhibited from binding to such a site.

In one embodiment, the multispecific activatable antibody includes a nonbinding steric moiety (NB); a cleavable linker (CL); and at least a first antibody or antibody fragment (AB1) that binds specifically to a first target or epitope, wherein the NB is a polypeptide that does not bind specifically to the AB1; the CL is a ptide that includes a substrate (S) for an enzyme; the CL is oned such that in an uncleaved state, the NB interferes with binding of the AB1 to its target and in a cleaved state, the NB does not interfere with binding of the ABl to its target; and the NB does not inhibit cleavage of the Cl... by the enzyme. As used herein and throughout, the term polypeptide refers to any polypeptide that includes at least two amino acid residues, including larger polypeptides, full-length proteins and fragments thereof, and the term polypeptide is not limited to single- chain polypeptides and can include multi—unit, 6g, multi—chain, polypeptides. ln cases Where the polypeptide is of a shorter , for example, less than 5t) amino acids total, the terms peptide and polypeptide are used interchangeably herein, and in cases Where the ptide is ofa longer length, for e, 50 amino acids or greater, the terms polypeptide and n are used interchangeably herein, {$388324} ln one embodiment, the multispecific activatable antibody includes a non binding steric moiety (NB); a cleavable linker (CL); and at least a first antibody or antibody fragment (ABl) that binds specifically to a tirst target or e, wherein (i) the NB includes a polypeptide that does not bind specifically to the A31; (ii) CL is a polypeptide of up to 50 amino acids in length that includes a substrate (S) for an enzyme; (iii) the CL is positioned such that in an nncleaved state, the NB interferes with binding of the ABl to its target and in a cleaved state, the NB does not ere with binding of the ABl to its target; and (iv) the NB does not inhibit cleavage of the CL by the enzyme. For e, the Cl, has a, length of up to 15 amino acids, at length of up to 20 amino acids, a, length of up to 25 amino acids, a length of up to 30 amino acids, a length of up to 35 amino acids, a length of up to 40 arnino acids, at length of up to 45 amino acids, a length of up to 50 amino acids, at length in the range of lO—St) amino acids, a length in the range of lS—SO amino acids, a length in the range of 20-50 amino acids, a length in the range of 25-50 amino acids, a length in the range of 30—50 amino acids, a length in the range of 35—50 arnino acids, at length in the range of 4069 amino acids, a length in the range of 45—50 amino acids, a length in the range of Hindi} amino acids, a. length in the range of l5~40 amino acids, a, length in the range {dill—40 amino acids, a length in the range onS—L‘ltl arnino acids, a length in the range lll amino acids, a length in the range of 35—40 amino acids, a length in the range of lO—St) amino acids, a length in the range of l5—3O amino acids, a length in the range of 20~30 amino acids, a length in the range of25—3G amino acids, a length in the range of l 0—20 amino acids, or a length in the range of lQ—lS amino acids! {$388325} ln one embodiment, the multispecific activatable antibody es a non binding steric moiety (NB); a cleavable linker (CL); and at least a first antibody or antibody fragment (ABl) that binds specifically to a tirst target or epitope, wherein (i) the NB includes a pnlypeptide that does not bind specifically to the ABl; (ii) the CL is a polypeptide that includes a substrate (S) for an enzyme; (iii) the CL is positioned such that in an nneleayed state, the NB interferes with binding of the ABl to its target and in a cleaved state, the NB does not interfere with binding of the ABl to its target; (iv) the NB does not t cleavage of the CL by the enzyme; and (y) at least a portion of the mnltispeeitie activatable antibody has the structural arrangement from N—terminus to C— terminus as follows in the nneleayed state: NB—CL-ABl or L,x-NB. liltitlllild} in one embodiment, the multispecil’ic actiyatable antibody includes a non~ binding steric moiety (NB); a cleavable linker (CL); and at least a lirst antibody or antibody fragment (AB l) that binds speciﬁcally to a first target or e, wherein (i) the NB includes a ptide that does not bind specifically to the A81; (ii) the CL is a ptide that includes a substrate (S) i‘nr an enzyme; (iii) the CL is positioned such that in an uncleaved state, the NB interferes with binding of the ABl to its target and in a cleaved state, the NB does not interfere with binding of the ABl to its target, and wherein the NB in the uncleayed activatable antibody reduces the ability of the ABl to bind its target by at least 50%, for example, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 96%, by at least 97%, by at least 98%, by at least 99%, by at least 100% as compared to the ability at the cleaved ABl to bind its target; and (iv) the NB does not inhibit cleavage of the CL by the enzyme. The reduction in the ability of the AB to bind its target is deteri'nined, for example, using an assay as described herein or an m vitm target displacement assay such as, for example, the assay described in PCT Publication Nos. W0 2t)(39/'025846 and W0 zeta/ear l73. {b88327} in one embodiment, the innltispeeitie activatable antibody includes a binding partner (BP) for a nding steric moiety (NB); a ble linlrer (CL); and at least a first antibody or antibody nent (ABl) that binds specifically to a lirst target and/or e, wherein the BP is a ptide that binds to the NB when exposed thereto; the NB does not bind specifically to the AB 1; the CL is a pclypeptide that includes a ate (S) for an enzyme; the CL is positioned such that in an nncleayed state in the presence of the NB, the NB interferes with binding of the AB to the target and in a cleaved state, the NB does not interfere with binding of the AB to the target and the Bl) does not interfere with g of the AB to the target; and the NB and the Bl?l do not inhibit cleavage of the CL by the enzyme. in some examples of this einbndiinent, the B? of the actiyatable antibody is optionally bound to the NB. in one embodiment, the NB is recruited by the BF ol',‘ the actiyatable antibody in viva, {$388328} in some examples of any of these moltispecitie activatable antibody embodiments, the mnltispecitic activatable antibody is formulated as a composition. ln some of these embodiments, the composition also includes the NB, where the NB is co— formulated with the moltispeciﬁe activatable antibody that includes the 8?, the CL, and the AB. ln some examples of this embodiment, the BP is selected from the group consisting of an albumin binding peptide, a librinogen binding peptide, a libronectin g e, a obin binding peptide, a trai'isl'errin binding peptide, an imm unoglohulin domain binding peptide, and other serum protein g peptides. {999329} in some examples of any of these multispecitic table antibody embodiments, the NB is a soluble, globular n. in some es of any of these specitic activatable dy embodiments, the NB is a protein that circulates in the tream. ln some examples of any of these mnltispecitic activatahle antibody embodiments, the NB is selected from the group consisting of albumin, ogen, tihroi'iectin, hemoglobin, transferrin, an iminnnoglobulin domain, and other serum proteins, {llhllBStl} in some examples of any of these moltispecitie table antibody embodiments, the CL is a polypeptide that includes a substrate (S) for a protease. ln some examples of any of these mnltispecitic activatable antibody embodiments, the protease is cr‘rlocalized with its target in a tissue, and the protease cleaves the CL in the multispecitic activatable antibody when the tnttltispecitie activatable antibody is exposed to the protease, in some examples of any of these mnltispecific actiyatable antibody ments, the CL is a polypeptide of up to 50 amino acids in length in some examples of any of these mnltispeeilic activatahle antibody embodiments, the CL is a polypeptide that includes a ate (S) having a length of up to l5 amino acids, eg, 3 amino acids long, 4 amino acids long, 5 amino acids long, 6 amino acids long, '7 amino acids long, 8 amino acids long, 9 amino acids long, ll) amino acids long, l l amino acids long, l2 amino acids long, l3 amino acids long, l4 amino acids long, or l5 amino acids long, liltillSSl} In some examples of any of these mnltispecitic activatable antibody embodiments, at least a portion of the multispecilic activatahle antibody has the ural arrangement from N—terniinus to C—terniinus as follows in the uncleaved state: NB—CLUAB, ABEL-NB, Bil-CL-AB or ABuCLK-Bl). ln embodiments Where the mnltispecilic activatable antibody includes a Bl" and the mnltispecilie activatable antibody is in the presence of the corresponding NB, at Ieast a portion of the muitispecitic activatabie antibody has a structurai arrangen'ient from N~terminns to C~terrninus as foiiows in the ved state: NB:BI’~Ch BBeZE In some exampies of any of these mnitispecifie tabie antibody embodiments, the muitispeeiiic tabte antibody inciudes an antibody or antigen g fragment thereof that specificaiiy binds Its target and is a monocIonai antibody, domain dy, single chain, Fab fragment, a Rabi); fragment, a scFv, a scab, a dAb, a single domain heavy chain antibody, and a singie domain hght ehain antibody. In some embodii ents, such an antibody or immunologieaiiy active nt thereof that binds its target is a. mouse, chimeric, humanized or fuiiy human monoclonal antibody.

{GWESE In some exampies of any of these mnitispecifie activatabie antibody embodiments, the innitispeeitic activatabie antibody aiso inciudes an agent conjugated to the AB. In some embodiments, the agent is a therapeutic agent. In some embodiments, the agent is an antineopiastic agent. In some embodiments, the agent is a toxin or fragment thereof. In some en'ibodin'ients, the agent is conjugated to the AB via a iinher. In some embodiments, the iinker is a eieavabie tinker. In some embodiments, the agent is an agent selected from the group Iisted in 'I'abIe 4. in some embodiments, the agent is a doiastatin.

In some embodiments, the agent is an attristatin or derivative thereof. In some embodiments, the agent is anristatin E or a derivative f. In some embodiments, the agent is monomethyi antistatin E (It/IMAIEL), In some embodiments, the agent is monomethyi auristatin D (IVIMAD). In some embodiments, the agent is a. maytansinoid or maytansinoid derivative. In some embodiments, the agent is ITEM} or DIX/I4. In some embodii ents, the agent is a duocarmycin or tive thereof. In some embodiments, the agent is a caiicheamicin or derivative thereof. In some embodiments, the agent is a pyrroiobenzodiazenine. {999334} In some es of any of these multispecifie activatabie antibody embodiments, the muitispeeiiic activatabte antibody aiso inctudes a abie moiety. In some embodiments, the detectabie moiety is a diagnostic agent.

} In some exainpies of any of these mnItispecitic activatabie antibody embodiments, the muitispecit‘ie aetivatabie antibody aiso ineiudes a spacer. In some exampies of any of these muItispecitic activatabie antibody embodiments, the pecific tabie antibody aiso includes a signed peptide. In some embodiments, the signai peptide is ated to the mnitispeeitie activatabie antibody Via a spacer. in some examples of any of these multispeeifie aetiyatable dy embodiments, the spacer is joined direetiy to the MM of the ispeeitie aetivatabie antibody. {8639336} in some ments, the serum half-dire of the mttitispeeit‘ic aetiyatahie antibody is longer than that of the corresponding specitie antibody; e. g, the pit: of the iniiltispeeiﬁe aetivatable antibody is longer than that of the corresponding muttispeeitie antibody. in some embodiments, the serum half-life of the mnltispeeiﬁe aetivatable antibody is sirniiar to that of the corresponding speeiiic antibody. in some embodiments, the serum haif—iife of the mtdtispeeitie aetiyatahle antibody is at least 15 days when administered to an organism. in some embodiments, the serum half—life of the mnitispeeifie aetiyatahle antibody is at least 12 days when stered to an organism. in some embodiments, the serum hail‘liii‘e of the multisbeeiiie activatable antibody is at least i i days when administered to an organism. in some embodiments, the serum l'ialf—iite of the moitispeeifie aetiyatable antibody is at ieast 10 days when administered to an organism. in some embodiments, the serum haif—iife of the innitispecitie aetivatabie antibody is at ieast 9 days when administered to an organism. in some embodiments, the serum hail'liite of the ninitispeeitie aetiyatable antibody is at least 8 days when administered to an organism. in some ments, the serum haif—iife of the mnltispeeitie activatabie antibody is at toast 7 days when administered to an organism. in some embodiments, the serum half—Eife of the inidtispecitie aetivatahle antibody is at least 6 days when administered to an organism. in some examples of any of these mnltispeeitie aetivatable antibody embodiments, the serum haif—iife of the nmitispecifle aetiyatahle antibody is at least 5 days when administered to an organism. in some embodiments, the serum haitliit‘e of the mnitispeeitie aetivatabie antibody is at least 4 days when administered to an sm. in some embodiments, the serum half-life of the mnltispeeitie tabie dy is at ieast 3 days when administered to an organism. in some embodiments, the serum half—life of the mnitispeeiiic aetiyatabie antibody is at ieast 2 days when administered to an organism. in some en'lbodin'ients, the serum half—iife of the multispeeiﬁe table antibody is at ieast 214- hours when administered to an sm. in some embodiments, the serum hail‘liii‘e of the multisbeeifie aetiyatabie antibody is at ieast 20 hours when stered to an organism. in some embodiments, the serum bait—life of the intiitispeeiﬁe aetiyatabie antibody is at least i8 hours when administered to an organism. in some embodiments, the serum half-life of the innitisnecitie aetiyatabie antibody is at least in hours when administered to an organism. in some ments, the serum half—life of the multispecitic activatable antibody is at least l4 hours when administered to an organism, In some ments, the serum haltllit‘e of the mnltispeeiﬁc activatahle antibody is at least l2 hours when administered to an organism, in some embodiments, the serum half-life of the multispeciﬁc activatable antibody is at least l0 hours when administered to an organism. in some embodiments, the serum half—life of the inultispecific activatahle antibody is at least 8 hours when administered to an organism. ln some embodiments, the serum half-life of the pecific activatable antibody is at least 6 hours when administered to an organism. in some embodiments, the serum half—life of the rnultispecil'ic activatable antibody is at least 4 hours when administered to an organism, in some embodiments, the serum half~life of the multispecilic table antibody is at least 3 hours when stered to an organism. 73 The disclosure also provides an isolated nucleic acid molecule encoding any of these moltispecitic activatahle antibodies, as well as vectors that include these isolated nucleic acid sequences. The disclosure provides methods of producing a innltispecitic activatahle antibody by culturing a cell under conditions that lead to expression of the nialtispecilie aetivatahle antibody, wherein the cell comprises such a nucleic acid sequence. in some embodiments, the cell ses such a vector, {000338} The dissociation constant (Kid) of the NEE-containing mnltispecitic activatable dy toward the target is greater than the lid of the AB towards the target when it is not associated with the NB or NBBF. The dissociation constant (K4) of the NB~containing peciflc activatable antibody toward the target is r than the Kd of the parental AB towards the target. For example, the Kd of the NB~containing innltispeciﬁc activatahle antibody toward the target is at least 5, l0, 25, 50, l00, 250, 500, l,000, 2,500, 5,000, l0,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or greater, or between 5--l0, l0-vl00, 000, l0--l0,000, l0--l00,000, 000,000, l0-—l0,000,000, l00—l,000, ,000, l00—l00,000, l00—l,000,000, l00—l0,000,000, l,000—l0,000, L000— l00,000, l,000-l,000,000, 0,000,000, l0,000—l00,000, l0,000—l,000,000, l0,000e l0,000,000, l00,000—l,000,000, or l00,000~l0,000,000 times greater than the lid of the AB when it is not associated with the NB or N828? or the Kd of the parental AB towards the target. Conversely, the g, afﬁnity of the NB—containing n'iultispeciﬁc tahle antibody towards the target is lower than the binding afﬁnity of the AB when it is not associated with the NB or NBBP or lower than the binding afﬁnity of the parental AB towards the target. For example, the binding afﬁnity of the NB~containing nrultispecilio activatabie antibody toward the target is at 1east S, 10, 25, 50, 100, 250, 500, 1,000, 2,500, ,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or greater, orhetween 5—10, 10—100, 10—1,,000, 1010,0011, 10~100,000, 100,000,000, 10- ,000,000, 100--1,000, 100--10,000, 100-v100,000, 100-1,000,000, 100--10,000,000, 1,000- ,000, 1,01,11,1—100,000, 1,000—1,000,000, 1000—10,000,1.100, 10000—100000, — 1,000,000, 10,000v10,000,000, 100,000—1,000,000, or 0—10,000,000 tinies iower than the binding afﬁnity of the AB when it is not associated with the NB or N528? or iower than the binding afﬁnity of the parentai AB towards the target. {000339} When the NB~containing n'in1tispecifie actiyatabie antibody is in the ce of1ts target, specific binding of the A13 to its target is d or inhibited, as compared to the c binding of the AB when it is not associated with the NB or N831). When the NB~containing mn1tispeci1ic activatab1e antibody is in the presence of its target, speciﬁc binding of the AB to 1ts target is reduced or inhibited, as compared tn the specific binding of the parenta1 AB to 1ts target. When compared to the binding of the AB not associated with an NB or NB:BP or the binding of the parentai AB to its target, the abi1ity of the NB— containing pecitic activatab1e antibody to bind its target is reduced, for examp1e, by at 1east 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% for at 1east 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 00, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, ti, '7, 8, 9, 10, 11, or 12 months or 1onger when meas tired in viiro and/or in viva. 1000340} When the aining in ii1tispecific activatab1c dy is in the presence of Its target but not in the presence of a. modifying agent (for exampie a protease or other enzyme), speciﬁc binding of the AB to 1ts target is reduced or inhibited, as compared to the speciﬁc binding of the AB when it is not associated with the NB or NB:BP. When the NB" containing nnthispecitic activatah1e antibody is in the presence of Its target but not in the presence of a ing agent (for exarnpie a protease, other enzyme, ion agent, or 1igh‘t), speciﬁc binding of the AB to its target is d or inhibited, as compared to the specific binding of the parental A13 to Its target. When compared to the binding of the AB not ated with an NB or N828? or the binding of the parentai AB to its target, the abiiity of the intaining muhispeciiic activatah1e antibody to bind its target is reduced, for exainp1e, by at 1east 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 970/8, 98%, 9 %, or even 100%.» for at 1east 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, it}, 15, 3t), 45, 6t), 90, 120, 150, or Hit) days, or i, 2, 3, it, 5, 6, 7, 8, 9, it), it, or t2 months or longer when measured in vitro and/or in Vii/0. {$388341} in some exampies of any of these mnitispeoitie aetivatabie antibody embodiments, the mnitispecitic aotivatabie antibody incindes an agent ated to the AB to produce a mnitispeeifie activatabie antibody conjugate. in some embodiments of the mnitispeoifie tabie antibody conjugate, the agent is a therapeutic agent. in some embodiments, the agent is a diagnostic agent. in some embodiments, the agent is a detectable marker. in some ments of the mnitispeeifie activatabie antibody conjugate, the agent is an antineopiastie agent. in some embodiments of the n'niitispeeii‘ie aetivatabie antibody eonjngate, the a ent is a toxin or nt f. in some embodiments of the mnitispecific tabie antibody conjugate, the agent is conjugated to the AB via a linker. in some embodiments of the mahispeeitie activatabie antibody oonj agate, the linker is a eieavahie tinker. in some embodiments, the agent is an agent selected from the group iisted in 'i‘abie 4. in some embodiments, the agent is a doiastatin. in some embodiments, the agent is an auristatin or derivative thereof. in some embodiments, the agent is atin E or a tive thereof. in some embodiments, the agent is monomethyi aui‘istatin E (MMAE). In some embodiments, the agent is monomethyt antistatin D (MMAD). in some embodiments, the agent is a sinoid or maytansinoid tive. in some embodiments, the agent is DMi or DMd. in some embodiments, the agent is a dnoeari'nyein or derivative thereof. in some en'ihodinients, the agent is a caiioheamioin or derivative thereof in some embodiments, the agent is a obenzodiazepine. {titBtB'n’iE} in some exainpies ot‘any of these muttispecitio aetivatabie antibody embodii ents, the maitispeeifie aetivatabie antibodies are dnai~target binding maitispeeifie aetivatabie antibodies. Such dnai target binding mnitispeoifie activatabte antibodies contain two Abs that may bind the same or different targets. in specific embodiments, d’nai— targeting niuitispeeifie aetivatabie antibodies contain hispeeitie antibodies or antibody fragments.

Etititi343} Dnai target g multisbecitio aetivatabie antibodies are designed so as to have a CL eieavahte by a cleaving agent that is eo~ioeaiized in a target tissue with one or both of the targets e of binding to the ABs of the muitispeeifie aotivatabie antibodies.

Dual target binding mnitispecific aotivatabie antibodies with more than one AB to the same or different targets can be designed so as to have more than one CL, wherein the first CL is cleayable by a cleaving agent in a first target tissue and wherein the second CL is ble by a cleaving agent in a second target tissue, with one or more of the targets binding to the ABs of the niultispeeiiie activatable antibodies. in one embodiment, the first and second target s are spatially separated, for example, at different sites in the organism. ln one embodiment, the first and second target tissues are the same tissue temporally separated, for example the same tissue at two different points in time, for example the ﬁrst time point is when the tissue is an early stage tumor, and the second time point is when the tissue is a late stage tumor.

{Milli-44} The disclosure also provides nucleic acid molecules encoding the niultispeeitic activatable antibodies bed herein. The disclosure also provides vectors that include these nucleic acids. The multispecific activatable antibodies described herein are produced by culturing a cell under conditions that lead to expression of the multispecific activatahle antibody, wherein the cell es these nucleic acid molecules or vectors. liltltl345} The sure also provides methods of manufacturing pecific activatable dies. in one embodiment, the method includes the steps of (a) culturing a cell that includes a nucleic acid construct that encodes the rnultispecitic activatable antibody under conditions that lead to expression of the multispecilic activatable antibody, wherein the multispecific activatable antibody includes (i) a non-binding steric moiety (NB); (ii) a cleavable linl See also Baldrick l3. "Pharmaceutical excipient development: the need for preclinical guidance." Regul. 'l'oxicol Pharmacol. 32(2):.2lO-8 (letltl), Wang W. "Lyophilization and development of solid protein pharmaceuticals." lnt. J. Pharm. 203(12): l—ot) (2000), Charrnan \«VN s, lipophilic drugs, and oral drug ry~some emerging concepts." J Pharm Sci.89(8):967—78 (2090), Powell et a]. "Compendium of exeipients for parenteral formulations" FDA J Pharm Sci ’l‘echnol. 52:23 8-3 ll G998) and the citations therein for additional ation related to formulations, excipients and carriers well known to pharmaceutical chemists. {ﬁllledl ln one embodiment, a multispeciflc dy and/or a. pecilic activatahle antibody of the sure may he used as therapeutic agents. Such agents will generally he employed to diagnose, prognose, monitor, treat, ate, and/or prevent a disease or pathology in a subject. A therapeutic regimen is carried out by identifying a subject, 9.33.. a human t or other mammal ing from (or at rislr of developing) a l l 6 disorder using standard methods. A multispecitic antibody and/or a multispecil‘ic activatable antibody preparation, for example in some embodiments, one having high specificity and high affinity for its two or more target antigens, is administered to the subject and Will generally ha 7e an effect due to its g with the targets. Administration of the multispecitic antibody and/or a multispecilic table antibody may abrogate or inhibit or interfere with the signaling function of one or more of the s. stration of the multispecific antibody and/or a. multispecitic activatable antibody may abrogate or inhibit or interfere with the binding of one or more of the s with an endogenous ligand to which it naturally binds. {$388349} lly, alleviation or treatment of a disease or disorder involves the lessening of one or more symptoms or medical problems associated with the disease or disorder. For example, in the case ofcancer, the therapeutically effective amount of the drug can accomplish one or a combination of the following: reduce the number ofcancer cells; reduce the tumor size; inhibit (216* to decrease to some extent and/or stop) cancer cell ration into peripheral ; inhibit tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms ated with the cancer. in some embodiments, a composition of this disclosure can be used to prevent the onset or reoccurrence of the disease or disorder in a subject, eg, a human or other mammal, such as a non—human primate, companion animal (6g, cat, dog, horse), farm animal, work animal, or zoo animal. The terms subject and patient are used interchangeably herein. lblllliiﬁfll A therapeutically effective amount of a inultispeeitie antibody and/or a, multispeeifie activatable antibody of the disclosure relates generally to the amount needed to achieve a therapeutic objective. As noted above, this maybe a binding interaction betw’ en the peciﬁc antibody and/or a, mu ltis eeifie activatable antibody and its target antigens that, in certain cases, interferes with the oning of the targets. The amount required to be administered will furthermore depend on the binding affinity of the pecitic antibody and/or a rnultispecil'ic aetivatable antibody for its speciﬁc n, and will also depend on the rate at which an administered multispeeific antibody an d/or a multispecil‘le activatable antibody is depleted from the free volume other subject to which it is administered. Common ranges for therapeutically effective dosing ofa multispeeific antibody and/or antibody fragment and/or a multispeeifie table antibody of the disclosure may be, by way of nonlimiting example, from about (ll mg/lrg body weight to about 50 mg/hg body weight. Common dosing frequencies may range, for example, from twice daily to once a week. {$88351} Efticaciousness of treatment is determined in association with any known method for diagnosing or treating the particular disorder. Methods for the screening of multispecitic antibodies and/or rnultispecitic activatable antibodies that possess the desired specificity include, but are not limited to, enzyme linked immunosorbent assay (ELlSA) and other logically mediated techniques known Within the art. l352} in another embodiment, a pecific antibody and/or a multispecilic actiyatable dy directed two or more targets are used in methods leiown within the art reiating to the localization and/or ouantitation of the targets (e. g, for use in measuring levels of one or more of the targets Within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). in a given embodiment, a multispecitic antibody and/or a multispecilic table antibody directed two or more targets, or a derivative, fragment, analog or homolog thereof, that contain the antibody derived antigen binding domain, are utilized as pharinacologically active compounds (referred to hereinafter as "‘Tlierapeutics"), {$88353} in another embodiment, a multispecific antibody and/or a multispecifie activatable antibody directed two or more targets is used to isolate one or more of the targets by standard techniques, such as irnmunoaftinity, chron’tatography or immunoprecipitationi A multispecific antibody and/or a multispecitic actiyatable antibody directed two or more targets (or a fragment f) are used diagnostically to monitor protein levels in tissue as part of a clinical testing ure, 8.33., to determine the efficacy ot‘a given treatment regimen lTJetection can be facilitated by coupling (£12., physically linking) the antibody to a de e substance es of able substan "es e various enzymes, etic groups, fluorescent materials, luminescent materials, bioluininescent materials, and radioactive als. Examples of suitable enzymes include horseradish peroxidase, alkaline pl'rosphatase, li—galactosidase, or acetylcholinesterase; examples of le prosthetic group complexes include streptayidin/biotin and avidin/biotin; examples of suitable tluorescent materials include umbelliferone, lluorescein, tluorescein isothiocyanate, mine, dichlorotriazinylamine fhn’lrescein, dansyl chloride or rythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials e luciferase, luciferin, and aequorin, and examples of " ~ suitable radioactive material include 1"l, Li i . . . 7r , ll , l, "E; or ’l-l. ~ liltltlllﬁdl In yet another embodiment, a inultispecitic dy and/or a multispeciiic actiyatable antibody ed two or more targets can be used as an agent for detecting the presence of one or more of the targets (or a fragment thereof) in a sample. In some ments, the antibody contains a detectable label. Antibodies are polyclonal, or in some embodiments, monoclonal. An intact dy, or a nt thereof (eg, Fab, scFy, or Franz} is used The term "labeled", with regard to the probe or antibody, is ed to encompass direct labeling of the probe or antibody by coupling (27.9., physically linking) a, detectable substance to the probe or antibody, as well as indirect labeling ot‘the probe or antibody by reactivity with another reagent that is directly labeled, es of indirect labeling include detection of a primary antibody using a lluorescently~labeled secondary antibody and end-labeling of an antibody with biotin such that it can be detected with lluorescently—labeled streptayidin. The term gical sample" is intended to include tissues, cells and biological fluids ed from a subject, as well as tissues, cells and fluids present Within a subject. included within the usage of the term "biological sample", therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph, That is, the detection method of the disclosure can be used to detect a protein in a biological sample in vitro as well as in vii-'0, For example, in Vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELTSAs), Western blots, immunoprecipitations, and iminunoiluorescence. ures for conducting immunoassays are described, for example in "ELTSA: Theory and Practice: Methods in Molecular Biology", Vol. 421, l. R. Crowtber (Ed) Human Press, "l‘otowa, NJ, l995; "immunoassay", E, Diainandis and T. opoulus, Academic Press, inc, San Diego, CA, D996; and "Practice and Theory of Enzyme lrnniunoassays", P. n, er Science Publishers, Ams ordain, l985. Furthermore, in vii-‘0 techniques for detection of an analyte protein include introducing into a subject a labeled anti-analyte protein antibody For example, the antibody can be labeled with a ctive inarlt'er Whose presence and location in a subject can be detected by standard imaging techniques. {$630355} The multispeeitic antibodies and/or multispecilic actiyatable antibodies of the disclosure are also useful in a variety of diagnostic and lactic formulations. in one ment, a inultispecii‘ic antibody and/or multispecilic activatable antibody is administered to patients that are at rislr of d eyeloping one or more of the aforementione disorders. A patient’s or s predisposition to one or more of the disorders can be determined using genoty pic, serological or biochemical markers’ Vii" I L 7.‘ ’, ‘ l' in ‘7 . J _,, Etltltlllﬁti} in another embodiment of the disclosure, a rnultispecitic antibody and/or multispecilic activatable antibody is administered to human individuals sed with a clinical indication associated with one or more of the aforementioned disorders. Upon diagnosis, a multispecitic antibody and/or multispecilic activatahle antibody is administered to mitigate or e the effects of the clinical indication. 57} Multispeciiie antibodies and/or peciﬁc aetivatahle antibodies are also useful in the detection of one or more targets in t samples and accordingly are useful as diagnostics. l:or example, the multispecilic antibodies and/or rnultispecitic actiyatable dies of the disclosure are used in in vitro assays, rag, ElJlSA, to detect one or more tar et levels in a patient sainple. {999358} in one embodiment, a multispecific antibody and/or multispecific activatahle antibody is immobilized on a solid support (eg, the welds) of a microtiter plate). The immobilized antibody and/or activatable antibody serves as a capture antibody for any target(s) that may be t in a test sample. Prior to contacting the immobilized multispecilic antibody and/or immobilized m'ultispecilic table antibody with a patient sample, the solid support is rinsed and treated with a blocking agent such as milk protein or albumin to prevent nonspecific adsorption of the analyte. {899359} Subsequently the wells are treated with a test sample suspected of containing the antigen, or with a solution containing a standard amount of the n. Such a sample is, eg, a serum sample from a subject suspected of having levels of circulating n considered to be diagnostic of a ogy. After rinsing away the test sample or standard, the solid support is treated with a second antibody that is detectably labeled. The labeled second antibody serves as at detecting antibody. The level of able label is measured, and the concentration of target anti en(s) in the test sample is determi ed by comparison with a standard curve developed from the standard samples. ltltltlfit’illl It will be appreciated that based on the results obtained using the inultispecitic antibody and/or rnultispecilic activatable antibody in an in vitro stic assay, it is possible to stage a e in a subject based on expression levels of the target antigen(s). For a given disease, samples of blood are taken from subjects diagnosed as being at various stages in the progression of the disease, and/or at various points in the therapeutic ent of the disease. Using a population of samples that provides tically significant results for each stage of progression or therapy, a range of concentrations of the antigen that may be considered characteristic of each stage is designated. {dilllildl} Multispecific antibodies and/or multispecit‘ic aetivatable antibodies can also be used in diagnostic and/or imaging methods. in some embodiments, such methods are in vitro methods, in some embodiments, such methods are in viva methods. ln some embodiments, such methods are in site methods. in some embodiments, such methods are ex vivo s. for example, pecitic antibodies and/or multispecific activatable antibodies having an enzymatically cleavable CM can be used to detect the presence or absence of an enzyme that is capable of cleaving the CM, Such nniltispecitic antibodies and/or multis eci’ric activatable antibodies can be used in diagnostics, which can e in viva detection tag, qualitative or quantitative) of enzyme activity (or, in some embodiments, an nment of increased reduction ial such as that which can provide for reduction of a disullide bond) h measured accumulation of multispecitic activated antibodies (ie, antibodies ing from cleavage of a multispecific activatable dy) in a given cell or tissue of a given host organism. Such accumulation of activated multispecilic dies indicates not only that the tissue expresses enzymatic activity (or an increased reduction potential depending on the nature of the CM) but also that the tissue expresses at least one target to which the activated antibody binds. {(3thth For example, the CM can be selected to be a protease substrate for a se found at the site of a tumor, at the site ol‘a viral or bacterial infection at a biologically confined site (egg, such as in an abscessﬂ in an organ, and the like), and the like, At least one of the AB can be one that binds a target antigen. Using methods familiar to one skilled in the art, a detectable label tag, a ﬂuorescent label or ctive label or radiotracer) can be conjugated to an AB or other region of a multispecific antibody and/or multispecitic activatable antibody. Suitable detectable labels are sed in the context of the above screening methods and additional specitic examples are provided below. Using at least one AB specific to a protein or peptide of the disease state, along with a protease whose activity is elevated in the disease tissue of interest, activatable antibodies will exhibit an sed rate ol‘binding to disease tissue relative to tissues where the CM specitic enzyme is not present at a able level or is present at a lower level than in disease tissue or is inactive (egu in zymogen form or in complex with an inhibitor), Since small proteins and peptides are rapidly cleared from the blood by the renal ﬁltration system, and because the enzyme speciﬁc for the CM is not t at a detectable level (or is present at lower levels in non— disease tissues or is present in inactive conformation), lation of ted niultispeeillc dies in the disease tissue is enhanced relative to non-disease tissues {@8363} in another example, activatable inultispecitic antibodies can be used to detect the presence or absence of a, cleaving agent in a sample. For example, where the tnultispecilic activatable dies contain a CM susceptible to cleavage by an enzyme, the multi specific activatable antibodies can be used to detect r qualitatively or tatively) the presence of an enzyme in the sample. in another example, where the niultispecilic table antibodies contain a CM susceptible to cleavage by reducing agent, the specilic activatable antibodies can be used to detect (either qualitatively or quantitatively) the presence of reducing conditions in a sample. To facilitate analysis in these methods, the multispecific activatable antibodies can be detectably labeled, and can be bound to a support (eg, a solid support, such as a slide or bead). The detectable label can be positioned on a portion of the activatable antibody that is not released following cleavage, for e, the de ectable label can be a quenched ﬂuorescent label or other label that is not detectable until cleavage has occurred. The assay can be conducted by, for example, contacting the immobilized, detectably labeled specil'ic activatable antibodies with a sample suspected of containing an enzyme and/or reducing agent for a time sufﬁcient for cleavage to occur, then washing to remove excess sample and contaminants. The presence or absence of the cleaving agent (cg, enzyme or reducing agent) in the sample is then assessed by a change in detectable signal of the niultispecil‘ic activatable antibodies prior to contacting with the sample (g, the presence of and/or an increase in de ,ectable signal due to cleavage of the inultispecil‘ic activatahle antibody by the cleaving agent in the sample. {lllllle-tt} Such detection 1 ethods can be adapted to also provide for detection of the ce or absence of a target that is e of binding at least one AB of the tnultispecilﬁic activatable antibodies when cleaved. Thus, the assays can be adapted to assess the presence or e of a cleaving agent and the presence or absence of a target of interest, The presence or absence of the ng agent can be detected by the presence of and/or an increase in detectable label of the tnultispecitic activatable antibodies as described above, and the ce or e of the target can be detected by detection of a target—AB complex eg by use of a detectably labeled anti "target antibody. {899365} lvlultispecific activatable antibodies are also useful in in, size; imaging for the validation of activatable antibody activation, cg, by protease cleavage, and binding to a particular target. In Sim imaging is a technique that enables localization ot‘proteolytic activity and target in biological samples such as cell cultures or tissue sections. Using this que it is possible to conﬁrm both binding to a given target and proteolytic activity based on the presence of a detectable label (eg a fluorescent label). lililllBt’itil These techniques are useful with any frozen cells or tissue derived from a disease site (erg tumor tissue) or healthy tissues: These techniques are also useful with fresh cell or tissue samples Etltltllldﬂ ln these techniques, an activatahle antibody is labeled with a detectable label.

The detectable label maybe a scent dye, (eg. a lluorophore, Fluorescein lsothiocyanate (E'l’l‘C), Rliodamine lsothiocyanate ("l‘lll'l'CL an Alexa Fluor® label), a car infrared (NIR) dye (eg Qdot® nanocrystals), a colloidal metal, a hapten, a radioactive marker, biotin and an ampliﬁcation reagent such as streptayidin, or an enzyme (cg. horseradish peroxidase or ne pl’iospliatase,. ltlllll368} Detection of the label in a sample that has been incubated with the d, multispecillc activatable antibody indicates that the sample contains the target and contains a protease that is specific for the CM ol‘ the specil'ic activatable an tibody. in some embodiments, the presence of the protease can be confirmed using broad um protease tors such as those described herein, and/or by using an agent that is specific for the protease, for e, an antibody such as Al l, which is specific for the protease ptase (MT—SH) and inhibits the proteolytic activity ol’MTSPl; see eg international Publication Number W0 2tll (ll/l29609, published ll November 2G"). The same ch of using broad spectrum se inhibitors such as those described , and/or by using a more selective inhibitory agent can be used to identity a protease or class of proteases specific for the CM of the activatable antibody: in some embodiments, the pr 2sence of the target can be conﬁrmed using an agent that is specific for the target or the detectable label can be competed with unlabeled target. in some ments, unlabeled activatable antibody could be used, with detection by a labeled secondary antibody or more complex detection system Etltltllldéll Similar techniques are also useful for in viva imaging where detection of the ﬂuorescent signal in a subject, eg, a mammal, including a human, indicates that the disease site contains the target and contains a se that is speciﬁc for the CM of the multispecific activatable antibody.

Etltltlfi’l’ll} These ques are also useful in ltits and/or as reagents for the detection, identiﬁcation or characterization of protease ty in a variety ot’cells, tissues, and organisms hased on the proteasevspecilic CM in the multispecific aetiyatahle antibody, Therapeutic Administration and Formulations of Multispeciﬁe Antibodies and/or h'lultispeeil‘ic tahle Antibodies 1} it will he appreciated that administration of therapeutic entities in accordance with the disclosure will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the lilte. A multitude of appropriate formulations can he found in the formulary known to all pharmaceutical chemists: Remington’s Pharmaceutical Sciences (lSth ed, Mach Publishing Company, Easton, PA 0975)), ularly Chapter 87 by Blaug, r, therein. 'l‘hese formulations include, for e, powders, pastes, ointments, jellies, waxes, oils, , lipid (cationic or anionic) containing vesicles (such as Lipol’fectinTM), DNA conjugates, anhydrous tion pastes, oil—invwater and water~in—oil mis, emulsions carhowax (polyethylene glycols of various molecular weights), semi-solid gels and semi—solid mixtures containing cathowax. Any of the foregoing mixtures ma§l be appropriate in treatments and therapies in accordance with the present sure, provided that the active ingredient in the formulation is not inactivated hy the formulation and the formulation is physiologically compatible and tolerable with the route of administration.

See also ck P. "Pharmaceutical excipient development: the need for preclinical guidance," Regul. col Pharmacol. 32(2):}3lO—8 (2900), Vang W. "Lyophilization and development of solid protein pharmaceuticals." lnt. J. Pharm. 2t)3€_ 1-2.): lot) (2099), Charman WN "Lipids, lipophilic drugs, and oral drug delivery—some emerging concepts." J Pharm Sci.89(8):967—78 (2000), Powell at oi, "Compendium of excipients for parenteral formulations" FDA l Pharm Sci l, 52:23 8—3 ll (l 998) and the citations therein for additional information d to formulations, excipients and carriers well known to pl’iarmaceutical chemists. {$630372} in some embodiments, the multispecitic antibodies, the multispecific activatahle antibodies and/or the conjugated multispeciiic activatahle antibody compositions are stered in conjunction with one or more additional agents, or a combination of additional agents, Suitable additional agents include current pharmaceutical and/or surgical ies for an intended application, For e, the multispecitic antibodies, the multispecific activatahle antibodies and/or the conjugated multispecitic activatahle antibody compositions can be used in conjunction with an onal chemotherapeutic or antineoplastic agent. For e, the multispecific antibodies, the multispecific activatable antibodies and/or the ated multispecific activatable antibody compositions and additional agent are formulated into a single therapeutic composition, and the multispecific antibodies, the multispecific activatable antibodies and/or the ated multispecific activatable antibody compositions and additional agent are administered simultaneously. atively, the multispecific antibodies, the multispecific activatable antibodies and/or the conjugated multispecific activatable antibody itions and additional agent are separate from each other, e.g., each is formulated into a separate therapeutic composition, and the multispecific antibodies, the multispecific activatable antibodies and/or the ated multispecific activatable antibody compositions and the additional agent are administered aneously, or the multispecific antibodies, the multispecific activatable antibodies and/or the conjugated multispecific activatable antibody compositions and the onal agent are administered at different times during a treatment regimen. For example, the multispecific antibodies, the multispecific activatable antibodies and/or the conjugated multispecific activatable antibody compositions is administered prior to the administration of the additional agent, the multispecific antibodies, the multispecific activatable antibodies and/or the conjugated multispecific activatable antibody compositions is administered subsequent to the stration of the additional agent, or the multispecific antibodies, the multispecific activatable antibodies and/or the conjugated multispecific activatable antibody compositions and the additional agent are administered in an alternating fashion. As described herein, the multispecific dies, the multispecific activatable antibodies and/or the conjugated multispecific activatable antibody compositions and additional agent are administered in single doses or in multiple doses.

In some embodiments, the additional agent is coupled or otherwise attached to the multispecific antibodies, the multispecific activatable antibodies and/or the ated multispecific activatable antibody compositions.

Suitable additional agents are selected according to the e of the intended application (i.e., killing, prevention of cell proliferation, hormone y or gene therapy). Such agents may e but is not limited to, for example, pharmaceutical agents, toxins, fragments of toxins, alkylating agents, enzymes, antibiotics, antimetabolites, oliferative , hormones, neurotransmitters, DNA, RNA, siRNA, oligonucleotides, antisense RNA, aptamers, stics, radiopaque dyes, radioactive isotopes, iluorogenic nds, magnetic , nanoparticles, marlrer compounds, lectins, compounds that alter cell membrane pern'reability, photochemical compounds, small molecules, liposomes, micelles, gene therapy vectors, viral vectors, and the like. Finally, combinations of agents or combinations ol‘dil‘ferent classes of agents may be used. lB‘ll'Sl The multispecilic antibodies, the rnultispecitic activatable antibodies and/or the conjugated multispecitic table antibody compositions of the disclosure (also referred to herein as "active compounds"), and derivatives, fragments, analogs and homologs thereof, can be incorporated into ceutical itions suitable for adininis'tratit‘in. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington’s Pharmaceutical Sciences: The Science And Practice Of Pharmacy l‘9th ed. (Alfonso R.

Gennaro, et al., editors) Maclr Pub. Co, Easton, Pa. : l995; Drug Absorption Enhancement : Concepts, Possibilities, Limitations, And Trends, llarwood Academic Publishers, rne, Pa, 1994; and Peptide And l’rotein Drug Delivery (Advances ln Parenteral Sciences, Vol. 4), l99l, i‘vl. r, New Yorlr. ldllllii’ltl} Such compositions typically se the multispecilic antibody and/or the multispecilic activatable antibody and a pharmaceutically able carrier, Where a pecific antibody and/or a multispecific table antibody includes a nt of the AB domain, the smallest tragment of the AB that speciﬁcally binds to the binding domain of the target protein can be used. For example, based upon the variable—region sequences of an dy, e molecules can be designed that retain the ability of the AB to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. (See, rag, Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889~7893 0993)). {999377} As used herein, the term "pharmaceutically acceptable carrier" is ed to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remingtonls Pharmaceutical Sciences, a standard reference text in the ﬁeld, which is incorporated herein by reference. Suitable examples of such carriers or diluents include, but are not limited to, water, saline, ringer’s solutions, dextrose solution, and 5% human serum albumin.

Liposomes and non-aqueous vehicles such as ﬁxed oils may also he used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use l’in the compositions is contemplated. {$388378} 'l'he formulations to he used to ' in Viva administration must be e, This is readily lished by filtration through sterile filtration membranes. {(399379} A pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, cg, intravenous, intraderrnal, subcutaneous, oral (cg, tion), transdermal (La, topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium hisull‘ite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the ment of tonicity such as sodium chloride or dextrose. The pl-l can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral ation can be enclosed in ampoules, disposable syringes or multiple dose Vials made of glass or plastic. lllllllillllll Pharmaceutical compositions suitable for injectahle use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the externporaneous preparation of sterile inj e ons or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ.) or phosphate buffered saline (PBS). in all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists, it must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

The r can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of sion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various cterial and antifungal , for example, parabens, chlorobutanol, phenol, ic acid, thimerosal, and the like. ln many cases, it will be suitable to include isotonic , for example, sugars, cohols such as manitol, l2',7 ol, sodium chloride in the composition. Prolonged absorption of the injectahle compositions can he brought about by ing in the composition an agent, that delays absorption, for example, aluminum monostearate and gelatin. {899381} Sterile iniectable solutions can be prepared by incorporating the active compound in the ed amount in an riate solvent with one or a combination of ingredients enumerated above, as ed, followed by lilte ed sterilization. Ge erally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. in the case of sterile powders for the preparation of sterile inj ectable solutions, methods of preparation are vacuum drying and freeze—drying that yields a powder of the active ingredient plus any additional d ingredient from a previously sterile-- tiltered solution f. {999382} Oral compositions lly include an inert diluent or an edible carrier.

They can be enclosed in gelatin es or compressed into tablets. for the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the cmnpound in the flui d carrier is applied orally and ed and expectorated or swallowed. aceutically compatible g agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the lilte can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacantli or n; an excipient such as starch or lactose, a egrating agent such as alginic acid, Prirnogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring, {llllllfiilill For administration by inhalation, the compounds are red in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, ag, a gas such as carbon dioxide, or a nebulizer. lllllllliéld} Systemic stration can also be by transmucosal or erinal means.

For transmucosal or transderrnal administration, penetran ts appropriate to the barrier to he permeated are used in the ftn‘rnulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. l: or transdermal administration, the active compounds are t‘orn'rulated into ointments, salyes, gels, or creams as generally ltnotvn in the art. {$33385} The compounds can also be prepared in the form of suppositories tag with tional suppository bases such as cocoa butter and other glycerides) or retention eneinas for rectal delivery. 6} in one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as sustained/controlled release formulations, including implants and ncapsulated delivery systems. Biodegradable, hioci‘impatihle polymers can he used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the {999387} For example, the active ingredients can he entrapped in rnicrocaps ales ed, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxyrnethylcellulose or nnnicrocapsules and poly—t:methylmethacrylate) niicrocapstiles, tively, in colloidal drug delivery systems (for example, mes, albumin sphcres, microemulsions, nano—particles, and nanocapsules) or in macroemnlsions. {9993883 Sustained~r‘elease preparations can he prepared. Suitable examples of ned—release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, rag, films, or microcapsules. Examples of ned-release matrices include polyesters, hydrogels (for example, poly(2~hydroxyethyl—rnethaeiylate), or pi‘ily(yinylalcohol», poiylactides (US Pat. No 3,773,9l9), copolymers of let—glutamic acid and y ethyl~L~ ate, gradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT TM (injectahle inicrospheres composed of lactic acid—glyeolic acid mer and leuprolide acetate), and poly—D—(—)—3~l'1ydroxyhutyric acid, While rs such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over lllll days, certain hydrogels release ns for shorter time periods. lilllllillllll The materials can also he obtained commercially from Alza Corporation and Nova Pharmaceuticals, lnc, lsiposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) and can also be used as pharmaceutically acceptable carriers. These can be prepared according to s known to those skilled in the art, for example, as described in US. Patent No 4,522,8l l. $thth lt is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as y dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical r. The specitication for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique teristics of the active con'ipound and the particular therapeutic e feet to he achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

Etlllllfigll The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. lhhtlBQ’Z} The formulation can also n more than one active compound as necessary for the particular indication being d, for example, those with mentary activities that do not adversely affect each other. Alternatively, or in addition, the composition can comprise an agent that enhances its on, such as, for example, a cytotoxic agent, cytolrine, chemotherapeutic agent, or growth-inhibitory agent. Such les are suitably present in combination in amounts that are effective for the purpose intended. {$90393} In one ment, the active compounds are administered in combination therapy, i.€., combined with other agents, eg, therapeutic agents, that are useful for treating pathological conditions or disorders, such as autoin‘imune disorders and intl ry diseases. The term "in combination" in this context means that the agents are given substantially contemporaneously, either simultaneously or sequentially, lf given sequentially, at the onset of stration of the second nd, the first of the two compounds is still detectable at effective concentrations at the site of treatment. {$90394} For example, the cmnbination therapy can include one or more antibodies of the sure eoformulated with, and/or coadministered with, one or more additional therapeutic agents, rag, one or more cytolrine and growth factor inl'iihitors, immunosuppressants, antiwinflammatory agents, metabolic inhibitors, enzyme inhibitors, and/or cytotoxic or cytostatic agents, as described in more detail below. Furthermore, one or more dies described herein may he used in combination with two or more of the therapeutic agents bed herein. Such combination therapies rnay advantageously utilize lower dosages of the administered tl'ierapeutic agen ts, thus avoiding possible toxicities or complications associated with the various ntonotherapies. {@9395} hi other embodiments, one or more antibodies of the disclosure can be coli‘orniulated with, and/or coadniinistered with, one or rnore anti~inilarnniatory drugs, irnniunosuppressants, or metabolic or tic inhibitors. Nonlirniting examples of the drugs or inhibitors that can be used in combination with the antibodies bed herein, include, but are not d to, one or more of: nonsteroidal anti—inﬂammatory drugts) (NSAle), 6.5;, ibuprofen, tenidap, naproxen, rneloxicani, piroxicarn, dich‘il‘enac, and indmnethacin; sulfasalazine; corticosteroids such. as prednisolone; cytoltine ssive anti-inﬂammatory drug(s) (CSAle); inhibitors of nucleotide biosynthesis, egg, inhibitors of purine biosynthesis, folate antagonists tag, trexate (N—H—[ﬁZﬁ—diamino—o— pteridinylﬁ'nethyl] rnethylaniinol benzoyil~L—giutarnic acid); and inhibitors of pyrimidine biosynthesis, rag, oorotate dehydrogenase (DHODH) inhibitors. Suitable therapeutic agents for use in combination with the antibodies of the disclosure include NSAle, (ISAIDS, (Di‘lODl‘l) inhibitors (tag, lellunornide), and lolate antagonists tag, niethotrexate). {@9396} Examples of additional inhibitors include one or more of: corticosteroids (oral, inhaled and local injection); irnniunosuppressants, 6.53., cyclosporin, tacrolinius (EK~ 506,"); and rnTOR inhibitors, 6,51, rnus (raparnycin — RAPAh/EUNETM or rapaniycin derivatives, tag, soluble rapainycin derivatives (eg ester rapaniycin derivatives, eg (ECL- 779); agents that interfere with signaling by proinllanunatory cytoltines such as TNEa or ll_..—l tag. lRAK, NIK, lKK, p38 or MAP ltinase inhibitors); COX? inhibitors, 6.2g, xib, rofeeoxib, and variants the eof; phosphodiesterase inhibitors, cg, l (phosphodiesterase Type l‘v’ inhibitor); phospholipase inhibitors, 6.33., inhibitors of lic phospholipase 2 (cPLA2) , trilluoroniethyl ltetone analogs); inhibitors ot‘vascular endothelial cell growth factor or growth factor receptor, sag, VEGF tor and/or VEGR R inhibitor; and inhibitors of angiogenesis, Suitable eutic agents for use in cornbination with the antibodies of the disclosure are irnniunosuppressants, cg, cyclosporin, tacrolirnus (FK~506); rnTOR inhibitors, sag, sirolirnus trapanrycin) or rapanivcin derivatives, egL, e rapaniycin derivatives (age ester rapainyein derivatives, tag, 031- mm I I 9); COKE. inhibitors, cg, celecoXib and variants thereof; and phospholipase inhibitors, e.g., inhibitors of cytosolic phospholipase 2 (cPLA2), e.g., oromethyl ketone analogs. 7] Additional examples of therapeutic agents that can be combined with an dy of the disclosure include one or more of: 6-mercaptopurines (6-MP); azathioprine sulphasalazine; mesalazine; olsalazine; chloroquine/ hydroxychloroquine (PLAQUENIL®); penicillamine (intramuscular and oral); azathioprine; colchicine; beta-2 adrenoreceptor agonists (salbutamol, terbutaline, salmeterol); xanthines (theophylline, aminophylline); cromoglycate; nedocromil; ketotifen; ipratropium and oxitropium; mycophenolate mofetil; adenosine agonists; antithrombotic agents; complement inhibitors; and adrenergic agents.

All publications and patent documents cited herein are orated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of ations and patent documents is not ed as an admission that any is pertinent prior art, nor does it constitute any admission as to the contents or date of the same. The invention having now been bed by way of written description, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and that the foregoing description and examples below are for purposes of illustration and not limitation of the claims that follow.

EXAMPLES The following sequences shown in Table 7 include sequences that were used throughout the Examples provided herein: Table 7: Exemplary ces Anti—human CTLA-4 scFv Antibody: GGAGGTGGATCTGGAGGTGGCGGTTCAGGCTCTGGCGGAGGCTCAGGTGGTGGAGGATCAG GTGAAATTGTGTTGACACAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAG CCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAG CAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCA TCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACT GGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCGCTCACTTTC GGCGGAGGGACCAAGGTGGAAATCAAACGTTCCGGAGGGTCGACCATAACTTCGTATAATG TATACTATACGAAGTTATCC' C11A1~L,GL5TA1CCAL1CTC’AACCTGGTL5C1G1 CGTGG' C'CAACTGCGACGTCCC 'G..A CCTCTC’AACCC CTC5GA11LJCACCTTTL1GC.

TATGCC GAGLJTGGG C'CGCCAGGCTCCAGGGALGGGGC" GGAGTGCGTA‘TCACCTA TTAG GGT11G1GG1GG'’7‘AG 'A’ACAT'A_CTA1CL5CAGACT CCG' GAACC5GCCL'GTTCA CCATC CAGAGACAATTCLJAACEL17‘C7‘CGC.‘TATCTCAAATAA7‘C1ACAC.TGAGA1GC,CGAGA1ACL_CG TATTACTGTGCGCAAAC' CCCTTTACTGCTACTTCCATCTCTGGGGCCGTCGCA 1'v'L,' {:111'TC1A1CiLJ1LL1'kTTL,AL3'L,TI-XG'L,"/3 1 r‘iﬁmmmr‘1/‘17r‘ﬁ1‘r (131111 1— T x f ‘ 1 '1 IGRATLSCRACQS'V/) \SSSY AmQ SR EE‘EDFA1‘1A'A1YCQQYCSS PLTF 'v1wL31'1Q n,\1 O n,\1 O \Q 3g3 agoagjtjfngacccf'Ppcpcatpcqo£9399343q3pcadOdacqagg+cagc ctLJLCLCLCLCLCLaagqﬂLLLLALLccauadvafc qLOOJPLuo~osgc3a:g cggagdacaa;:gks’ggaccgpctcucj:g‘jg3P'PLLPuTuLccttc1t cc1ctacagaauctcaccgfagCgiOdOCﬁg_iJ’:3gcqgjgaacgtcttctca+'h r:\ r‘.\1 O r—r\ LL) Q; rr ('\TéCOdUGCtCtg3L33(uaupsc:‘gc;g333gcc:c,ccch+ctcccg qtiai (SEQ ID NO: 237) SGPGLVQLGGSVR'SCAASG"TISSY1MCWWRAEMLGLP"\SGLDPLRC'1Y YA LSVRGRFTISRDNSRNT YQVD,4...SIRA'DmA" UIGGRCAFDYWGQTVLVSQAOl KGPSVFPLAPSQKQTSCGALCC'JSDYFRER"TVLWNSGAL10CLHTEPAVLQESGL’" LSSVVIVRSSSLGTQm‘inNVLIK SNTLVLYK"LLKSCL'I1IPPCPAPELLGGPbVFL FPPRPRLTLMIQRTPEWIFVW‘3""1UREVKLVWVVBGVL" ’PREEQYNS 'lYRV\7 S'7LI‘LL11QLJEL‘: NCKEYKC‘ \‘w’SlefLEAP TEi1‘1SKLI:KG’H‘REPC‘VYTLPPSK'LV"'il"£\IC""LSt‘j L1CL":r\’"-E‘YPSDIAVLﬂi/‘ESNGQ ENI{YRTTLPVLDSLGSFFLYSRLTVDKS'NQ"GN"PS" Ant;_Jnged Alvaa ChaLn cgchgttctgacat ccalzjatgacccag agagtcacbatcacttgccgggcaagtLagbg01ttagcagc"a"Lga cagggaaa.gccc0t3agctcctgatutitg _ cocaicaaggttcagtggLag"ggachgggicigigLLcacﬂc"cacbat01gcagtctg gccaagggacuaaogtggaaafcaaacgtacggtggctgcacca,ch,CLLcaLCLLccc gccatctga:ngC1gttga3atctggaauLgchchLLngLCCLLCCLgiuLaacLLc tatcccagagaggccaaagtacagtggaaggtggutaacgccctucaatcgggtaaCEccc agcacactq:cuLuaugcaggacagcaagguLuqc1cctacaccbtbagcaaoua.ccctgac 11’: "JACJCchdC‘CL Iagaa r717:acaaaq 17.172 '17. .chccttjcgedot1,d1:c1;ctcagggc ctcde;COPCCCL1(canagaactL-ddcaquQA (SEC ID NO: 239) mm,,{LJQLDL3LJLA1I("1,111 1I1-'T'Zl'd_Ll\JlG LJDC P17133131.171:1D7DEL.71:I- Jab 3GTTTI 1FT¢11,1~1(1~1 «,1 T ,1..:111.

' S 3P11.‘L1‘NHL:L.L:S«,1 TQMTQSP‘SSLSASVGC RVTTTI-RASQST. CCKPGKZ PKT LTYAASSLCSGV P SGCGSGTDFTLTTSSL ‘EPECFATTICQQTVVAT’R IE'GQC T'KVLTLKTVAAPSVT TI- k‘PSCbQLTS JTAS.7‘7’717-T-NT.

YPLEZKVQE7T.VDT ALQSGN S1."TSVTEQDSKDSTYSTSSTLT XKADYEKHKVYACEVTHQG L LSSPVTKSF’DIRGEC - L 17.70: 77740) Anti—Jan:G 4D11v2 — anti—CD3 OKT3 Mul'i speciﬁic H:’.a\7y AGTGC' CTGTTCLIAG 'GGGGG GGCTTG 3T?.CAGCCT1.I.13C3GTCCCGTAG "CT CC 'GT'GC CCTCTCGATTCACCTTTACAGCTAT 3CCA' GAGCT13GTCCGCC-GGC' CC 2’1x3'-:\-I27&2‘1L37:1,1,.1r1 1 -L:IbL3L, _.(JL31’.1111 11,.T1 7""1""‘""""‘"‘ TL3'3L3TL.T1.2\-2& 777Z' mm - 1. ' 71TT'713AL, 1, ,1,1,1,1r11~\1r1.E-L3'3-r*1[73-'1;:\-ITC IL,AC3A 7SAT ATT'Z-1.'.3'1.A11,1 m {1.151.

A77lCCG’77'GZ‘121GG7-7G’7GGT'7'.7"CCAT'CTCCAGAGACAAT'7CC/AGZ‘12».C 7GG7T7GTA'TC' 7'GC Z31AA'77GZ71AC217IC AC ZIGGKT. ’-77T7A7T7A '17 TGCGA1227‘‘17GAGA 7GGGCGG~ [7313M 7r7T1r1.1 'L,IL,L'- TL-IL;I273.L-.T-r»‘1t.,T'n'-1~I1r1.T1mr1» ,1111» r1.»1r1..1 GCCAbL-ILLA ’7.-271CCGI7L7777L77CC’7'7'C7Z "7.177%" ’731.\I1.1.1.Lr‘ ~r1r~= ~11 I-rIArLUGI'C,ITI.-IIr~ T1.n1.1.r{1. T10 r1, _1~ILIiL-IGTGAK-LA3TW1.1.1.T11, 1 (1,1,1 "IT/1,1... .1 1n 1111(1,1r11.T11,1r1r1I..1.r1I..1.1» ~I1r1~ '11 11,111,1r11» I LLZTLIAGL-TTGL3'wLT/73CL.1.Zb1L,L'ZA-2 {1.1 TL. TL3-I--r1.:\-1.\-I .»11»» Ar1~> 1»,I 1 »1,1~,1»nr1 -CZ1./»-'1/3-'1.'C.Z AG -IL- GAK.-A2»\.L32»TZ»‘12»-'1-» ‘1 1 1 1 1T1 1»1 '1 ,1» », .21 ‘1- .K- .21 r1» -1.1.1.2«GCCLLGA2TL.TL.I-,1..1.,1I..1.1 1 11 11m 1,. ,1111r1rw T'TCCCCCG"7‘2"71E'5GGAAGCZ3I’2’CCC7TC271’7-7‘717£T7GT’7..7G ACCCCTGA T7‘K'.7AGATGCG GI-I T" G(: «CACGTLJZH CK7.7Z3.1"CA/771132CCCTGZ\GG' ,-.r1rq1 I .7731: »7-: 1.71 VH17.16.:’l J. f1f1f:.:1[,':{-w’HJM‘J \J\JAL\\ GGT GC7I7Z‘1'7 7Z\2"\.732'GCK-77Z3Z2-1GACAAAGCC’ ’77GGIG2-GKGIZ3K3 7271G'717Z3CZ’3I1C77I71GC,CGT ACK-777"7T" Z31G'C77G'7f'1,[’7 C 71'.7Z\KI-C7K.7-—7I'2{7K-C717’7CAC n7’3C7E7GGC7E771 2-1Z3Z37E.7"7GGC2 Z—G’1I71L371'27KC2A Z\K.7-—7I'2{7K.7-—7IC '7I7CT "CAHCALAGC,C" CCA."C"CCC 1'1::\1,L\1,'1 111 T1 .T1II11 1 1,1 \,.'.2 Z' 1127\.,'T ' 27\IL\L3L,‘Z.2-\..Z\‘231‘23LZ\1L1\1,LI\1,'1_113 2730.721AL \/£7\C27'7\Cj71371,.1- (171." _ K77-—7I'2{723 7ACCC77’wCC77CC77C2A71'.,C7.7GK.7-7I G171"GAGA '7]2'GIZ‘1K.(,7"\7\L3Z\11C77 31GG'7E7C’A "17:71.7 GACCT G K127C777 GK77-7I'2{7K-Z3.12Z71Z31GGC '[7 7] .'Z’-\.7T'KI27CK.7Z3K.7-—7I CKZK777377’17GC77C14717L773GA $.17 :IKSLII’XLIIDIC.C!1D1 CGCI7‘1(~CC77G777\L3ZAC?3C7'[AAAGACCAK.G7C777C77C77' GC77’wG’V7777CK7G2-CK} 7K7.7'2{7CC'71.7'2 7'C'7.[7'7].' "7’7'[’7'[7IZ‘1(77,Z\L3(,Z3K3 C7E7CACC’G77’wG71,-.AL3AL73CAGK3'227KEGCAGC77I711~GGG2KZ1 %7E.7 7.7,E.7TC'2 7CZ31'77GC Mar. 1.- m \1,'1_ \J. 73.3.713L,\. er‘terr f . 17512773J Vf’ﬁl:V.[/’\:I..y’\I/"Z\’lk/ \J7\_ AL\\_ \KI72'2'772-1C2-7"CGCAC AAGG17,(,777C,777(,K77C777 7717C LL/KJLJ G717 Z\IZ‘712-’\G(27AGGTGK"Z3 7E.7C'7.'1GAGL 7CGG7T'777CAGK73 C777’777’7G177G’wIZ‘1L3G177733.AK: 17E7GG'2 76G!" ATCAGGC TJGAGGTCA.GGT' ’CAGCTGC GCATLGALJCGGTG 7TGATACTCGCATCGTCJCGGG 'GCA AGCGTTAAAATGAGCTGTAA/TGCAAGCG TJT TTALTTJ,[:4 CTTTACGCTTATTACCJATGCA' 'GGG TTAAAJLAGLJTJTGC'GGTCACTJG GTJATATCJGATT JLJTTATATLITATCLJTJALJL'CGTLJLJTTA CTACTATACCALJAAAT' CAAAGATAATAGCAACGCTGZ CCACJCCJ'ATAAAAAGCACGCGC ACCGCCTATATGCAGCTGACCAJJLJCTGTCCJTCJAGAGL»ATAGGGGATGTT TTACT»J'GCT7C LJLJTATTATJATGATCACTATTGCC' GTJATTTATTGGGLJTCAGGGGATCCCCCTGACCGTTT G J! CAGGGGTGGTGGTGCTAG' ’LJLTTGGGG TJTGGTTGAGGCGG' GGCGGTJTTJCCAGATTGTTC G ACCCT7 GAGT ATTA 'GTAGCGCAAT JGGCLJTGAAAAGTTALJCJTTGACCTC' AGCG CCAJJCAGCATGCGTTAGCTATATAA 'TCJCJTATCAGCLAG7‘LAAGGLJGLJA CGCCGAACCG 'T * JJ r‘I'L31T1Z-‘TTTT 23TiLLrauAJLACL. ALJ'LJAAA3- mm m I" «#37 Jrﬁ"‘T'J'LJ‘CTAT LJLJGLJTLJJL"In J m ﬁ'xrvﬁﬂ‘rr‘'LJ'LJ'LJ'LJ‘LT mr‘imrﬂ m JL’JTA'LJ'J'LJ'L’J‘TﬁJT‘IﬂMﬂ".

\V-TTi i i iC-Gi I7"LJTAC"TCA717ATAJGCCTJGACCAT "TAG ""-‘GTII"\TTG"LZATT73TGCAT' IGTIJIT "CAGCAZTCCT AT T 1311 TUTLJTACJ7‘J’1r1r\r1r~7‘ ~~J~\ J-Jrﬂ ~r~r}1/\*.JJTTrJ‘Tu'LTJJJTTA7r~r~v 7:-Jr~.r~.~xwr1rrwn Ax E'IIIK «IN 1 Z: ' TC; - T LTLJ'L3i i LT»: .ETJT T CL3'L3Il L‘V'TLLJ.S GGGLVQPG 7S R\ ' SCAASGIII‘TISS‘T'AIVISW‘JI’P DPLCKCTV YAT "\TKL' 3 L311" Y QDL JRA"DII‘337YYC1 D .JrvssA<11 "(V-J ‘7 I )Y'FPEPW'I‘VSWT C"TALTSC37E1TE‘PAVLQS‘7’7 YS C"J'LLJ 'LIVTTT LT U) U; "’3< (\J I"U U1' CI) U1 t-4 Y TCNVATKPSNTLVLKKVL‘PKSCL' TITTCKT. PPCPAI‘ELLGGPSI 7FL I:UP"PNATLMTSPTPTI‘YTC"’7 VDIJJ7SI'TLL1PTI'VKT‘T‘/1""LL: ‘\ 'ITL'QYNS IlYRW 7 SI7LTIJJ7LITQL17J/vL7M’KLVKCBVNIIALPAPTEKI' ' KG’JI‘REPQVYTLPPSK'LV'TYNC'VS LIl CLV::7"-CT‘YPSDTZ33J7ITﬂIJAYESNGQPI'L7-NTLI 'T'LLTTI PVLDS TCSTEJLYSILLTVDKSIWQQ"‘GN‘J."ESC GSGSGGGSJGGCC GQJJ7QLQQSGALLA PGAT SI7K§CSCJ [Al/\KEJTK/‘x zfX‘LJ'.1.1J«r‘1r\r r\{‘-‘ "1;. I 1 11 (ALC Z‘l'L,11 (11.11».'L,.CJ7'\ JZ‘1x .- JZ‘.1 J-[1J1-Im - «r11 ‘1"I'I'I'nf‘f\l' ,n, -- r'HTI 11 —1 11 erf‘ 1m 1-1 1 . GLJC;C‘JJ'L H'LJLJ .[7L7'L3 JJZ‘xi JJ‘LJCZC‘ ’I‘J W‘JC. LJKTJL Z‘l'xJCj r11 ,A ,—. r\l «m \3 \J \J U IGAG::G3t .AK ,1L,-1 ,A,L. ,A 1 'f"7"‘7(' HWI 3 ’lk/IICLKJMKJ ,LL,JL‘L:JC:I; G3 '11-,1'1-‘r1r-er(-\JIA.U37\_1CI\ J :‘II'UIT'ICG]r(1«J- 2‘17: .fx',‘ :1;f‘L/IA; \JC: 'LJ'LJLJI-VLJIL:IArum—Ln .m 7LLJLL7J.*1LJ.*1LL.'LJ 7\ vﬂmm/‘V‘Ar‘ 7‘. ,v-L—H—me 7m m 'LJATLJALJI-VLJ'AGmL :LCLJPLLLJ vm‘nl "ALMA/Ab.JTLJLJLJ CLJTLJLLIJCJAJLLCLGL AFLJ'LLJI-L'LJ mm , .m ITI J", Am IV- m. mm‘ﬂr‘m ‘Vf‘fwl‘m‘ "\ﬁm "I mm mum: LLJ~~V*r‘ .nrﬂmr‘i «h AILJ JAJTI. CCACJ'AL L.A.L J. LJLJLJLJIVJAALJLL'ALJAGLALLA‘ILJI ALLLAJAATTGCJTAT LLJ~~V7. ,"W ALLA]-AIV- LJALJGLJZ‘*1JPL.*17\GLJ LJLJLJTAAIJLJ7 rv‘r 7 r7," 7I "FIN/7 r7". "\ LJC' GALLCLL’ALLm IT! mam J'LJ'LJ'LJLJf-‘Lf—‘LTLJIVJAJLG L L LGLJAAALJTLJ'JLJLJ*er ﬁn «m pmmm In m. .rvr-‘rw '1.

I'x/‘I'NnLJ'LJLJFL .r‘7"AnmmLLCh JLLJLJ'LJZ‘L'LJmr',"/‘I7\rv .mﬁn'IL'JLJz-LTC' ELLJPLLLJPLTTTC AIV m L, mmm ‘n .. r7 .

\V L CJALCC Tmmnn7r‘../‘ILJPLLJLJJI-‘L'LL LJ IL': .n C‘JAACC' GAAATTTTGCAATTACTACTGJAALJAGACLJTTLJTLJGLJJCJ'CTCCCJTTAT' CC.

CJCCAACLJ GAJCCJ'-"AS TC"LAATCAAACLJ'TACGCJTCJGCJ' CGCCCATCTGTCTTCAT’7T 'CCC CT JATGAGCAGT 'CAAA' CTGGAACJ'TGCCTC 'GTTG' GTGCCTCJ'CTCJAATALCJTTC TA 'CCCAGAGACJCCCA LAAILJSTACAG' J LJJJJAGCJTCJCAT7‘ICCJCCC' CCLJCTAAC 'CCC ALJLle-LLJz-I'.LJ L LJLImLJ'ALJ'AGA'LJ' JA' J'LJf-‘LIV-f-‘LILLJALSLGLJALJ'AGL.LSLJ'LJTA LJL-LLJII-V7m—L1—L mm .«m-rrwn—L —, ~7\ «7‘ «mamJ'LLJ GCALJLJ''LJan .F m'LJ- I V- 7‘." .n-rJAIN LJ'AJLALJLJELLJPLLJTJI-‘LLJ' JALJZ-IL ALJALJ'AJAJLAG Lw—L mnmm..7J—m—L «AAA—L a ‘7\»"~(Irm"‘:/‘V .n—Ln LJ'LJ LJ' L A J'LJ LJ'LJ IL'JLJIL'JEELALJL LJ'AC "1m" Z\17L'LJALJ'LJ LJIVr‘: «FLA C' "GAGCTCC7"7I'7'7"7ACA/ .2;th7.1.7.321 .‘ '9ITIjITIr:g/\I:r:I LSCG -725IJ 7ST.

VAL; JIC.» .m. LH A». Lama,'-G ~\ L 7. ~ 1 m~~ -AG«Am/Ln -- -I7AALJC LJ'II".A2IA2I .~. Jr . -IAJ»_A.. 7CGTT«CC'5I’I‘C25IJ'I"i"-7 GG'T'I'ZVI I‘CCGGJTCAGGGTC ‘LVJ‘LAT7C775I’77‘TGJ7'I‘TALZITCAA' "CCGZ‘'LGC 7’7T’7GTJ2 m1 LEAN VALVLJICL. ZC L G L ISL.mm L.L. L L L.L"JVAII’I IGLJ- G mm »J~ m'yn-LJLL"Z JLJIJL 7’7\ f"‘T‘ 7\ J \.l -L1 ,1 1J I "J I J C:-’I CC GGLVCAA'I‘TZITGAI’"7’7 ALA/VA .

ACCAT' GACC'I‘G'I‘AGCGCCZLGCAGCAGCG’I‘TAG". TZITA' "GA JCACCAGCCCGM"GTT‘CAIITPTC"" an». ~\ r 7" I 'LJ'WVL‘LZX A1r11*7m‘cm L VA"CmCIm9:LGC‘r"A"" ATTAGCG 7TZ LIAGC25IJACCTATTI GCA5 71' GGCTCART-CARTC CGT'P'. CCTTTGG zzIICIIwc. (SEQ ID NO: 245) ﬁr;QC’Irv"LJ'LJLJL, I7IILI’I7J‘KJ'I31].}C7R'f7’;WCC (ESSGG SGGE GI:7' CPSI.‘NH«G’wSIJILIIVI'I‘Q‘ PSS ISA SV'GI) RV"TI Ih.\LQL ‘ O \7 IL) .1. ‘NWY"JKP(KAPLLiVA' 'ISCVP‘WLI‘SLJ'SLJ'S T I} I T 1'..- T I S S L 1.2.. [.VV (a QTY"7‘7; ‘VF I'JI: 'P P S I) L.L SGTAS‘V 7'I’L7I INIL A, .rhIhmnv YPIuJZJJIﬁ/QIIJ{CDVI JQ-f' G 7SUBS v-.S.ILJSS'I'L-.- L . L (K. 25. I‘.)’I: 7 ‘JK."\/"1.’Z3.L,"7EV'I HII’7G 17.5".E7E‘L7’I'ELSII "AGE LIL-7 WLEJLJLLI’JLTSC‘SC‘GL LJLJ‘GISGJGIJCIJ‘VIJ]’ 7SLJAVJIJZIILLJrG5Z3S\'IxIJIISCKAa L ~ V: \r w w "(x - «J: -L r' L, 771-wa L J ~ v w SGY’I' I'LY'I‘IVI 7G7: INP\I\I3.L'.7"\I"N’ "]?‘7‘77’7S’3"\ " 7‘17VI7ARV717I7HH71V77x) .L ’YINIJ'QGL LLJLVE) JrL, ~rv1n-n-1Tv70 - ' \I _I_ L, JJL ’1‘J'LJIJLJIJ'LJLJL-(«p f\(\4,’\lr' pl, J'LJ.L 1‘ "BPS ’"M’J'LSSSSZSYV'NWYQ‘IKSLJ’I'SPK'PSLIY" 7‘7KIJZ‘.SG‘V'PZ5I.".E-I I‘RI'7SISSC'ISY I "]7' -.-I)25I....AT.Y‘fCI') I7 I) NC) : 2 4 6 ) AGTCC CTCTTCLJALJ 'GCGCG CTACACCCTLJLJ J'»CCCTCCCCTAC ’C'T CC 'CT'CC CCTCTCLJATTCACCTTTAJ'CACCTAJT JCCA' GAGCT-J'CTCC,CCCC.CJC' CC ACGCALJ-JLJCL,TCCAJCTC.JLJTLJTCAACTATTGACCC"LJC1AAGC' CATATTACCCA C/-LC" CCGTCJAALJCLJLL.JLJTT"CCATCTCCACAGATAT' CCAACJAACAJCCCTCTTA" C' GC AAA GAACAGLJL.—\ AJerI-L mrL IJI—L/wJ—xrw—rn—L—‘v-L 1‘1 m7\win—I7. .

LJA J -LJ 'LJLJ'LJA‘JLJALJAC .UCJK.CHGJJI1JJJHT\LTTAC GCLJAJAAGACA(:1 ,IJ—erI—LI/w—LnL, 'LJLJ'LJ LJ‘J IJ—L.LALJ‘.'2T :V-LJ.JLJC'mmmnmm. 7/‘1 mm YmmIA‘VFV rJ—xnm r1 "Irv-1 . m. L -LJJ-Lr. _'. JLLJAL..TA'LJT'LJanII—Lmv—LJ‘JJL’J‘CLJALJGLJ."AA.L LJL..J'L J CA‘LJ‘JiC-ic Ji CAGL. .7\rVIJ~JAJ'L p AALJ‘.JLJ\-VLJCJJTLJLJJTLJ;-, «,L—Lﬂ—L ~.,L(—. (""1" «Vary—.»L. «n—L CC- mm .mrI—1C .AALJ—L «m-Jmam-Jam-J». ,L m LJ'LJLJ'LJLJ LJ‘JLJ L -LJL-J \-LJiLJiLJGGLJGLJALJ LJ‘.':Ir‘: Ham—Ln J i C .. 1..:(:I,:Cr:1 :CJ/Cry1rLIfL.TlrL7\ 7» .3er»"XrNr. 2L L, 21L,_‘.Ir~.x1»2r.1 ‘1ch 3’72"JACCCC CACCCJ’CJ’T‘L,V"V"VCA/\’VT’VACC \JrN... r .111chA. r: TCCT C2»"JCCACTCTAC‘ "CC L_.JI1r‘2. r".Z\\,L'\L.J‘CJ'ArL LC2»‘JL1 t CL. J LG'LJIJJLJZALLJLALJAL,CC 'A‘:AlJ~ L. .1 "mm. VLJLJL ,JJ,.,J~J. J L .... mC' "(V17L3I,rJ[\L,1.(V.

CCC.A»L,-‘AL /-'L,,AALJCJCLJCAACAAACTTC-‘ﬂﬁ/‘V7 m"\ﬂ "‘ m ‘ﬂﬁ‘ﬂ7 "V..rﬁ‘rrﬁ" 1.1L,‘CL,‘AA. '7 mmmmﬂ #17 f"‘""\JL..JJAILJAA CC’VCACCSTCV’VCCACC ‘.CTCAAJ GGGZACCLI~11». I'w..JAFHIK‘K‘IIHC1f‘2r1r.C .»..J1I.J~.rLI.J~..L LI.J~. V2»JAAA‘,» 1 1. 1,~.~1».‘.,AJ»_'L3I.,1.r»‘I,AXL,'L,L,TC2»‘JJL'JL'JA 1J, ,rLImrLr L1. ,1».A.1,Lm,L~JLJLJ—L».‘.,'L.JIJJ'LJJ/:\_L,LJ'L,L,i '21;va ‘,AL, Al'LJI,'LJIJ~rLIJ~rLJ » L» ,1 («hf-L.

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C222\(:2GIC22CC'2\CCTCIJ1T "PCT GAGA.C2". C".I.‘CJ".1.‘CJJACCCAC’V’V"V‘C"V‘L,"V""".V‘ ".2.‘C"CC}‘JC(:C AA.LL:.LL:L,‘ CC‘,""V C’:C’VC, VTCJ2-C21 V"V"V:IT'[A(:CJCLVLALJCTVCT". ACCC'." .’3.C,IC_ 2‘\(:L,2’\C2\.2\.J‘.L,V..,.. ‘("' .L rm ,IC_ AGCL2.. :"ICCV1CH--n.[.Cr . -.J_L,L,mm V '1 ‘3 1L,L AGIJ' 1‘,Z3.1.JLV.I.JLL,AL,7'er f‘f‘r" r‘ '.["C(:2CATIC VC,ACZ‘3.C,‘Z‘3.C AT' "CACTCCCA ATCCCTCTC"C: Ann. "I‘C2".C"."C".1.‘C2—"CCV’3"1"VAL:L, ACAC'T‘CCACC‘I2' 5AACA'['T'['T’:L,JC'T‘CTV TT."C"V‘C"V"VAC.C2’\’V.'.[‘"V‘CL3"1'2"\CC,‘ ,‘A CI’VJCI’VJTI’Z'V‘\K/MKJM \ A\ CT "I CCCCC2—.CCC2-1CJCAA"VC'.[C’:.L2.2"V‘L,AIJCCTTCLXVCAC’:C"V‘L,L:‘,V\L,L,T2"1AC'['TCC'[.L 'T"AATI A"V‘ 3 CAACTTA"].' CC'T‘ CCACCCV V"V‘ i: -.rL-JK/ZAJGLL/LLJCji.1‘_/.5\.\J/Ax\_/m1. Lm LIJ—. ,JL-,. 2'11 ,JLI- ,L 1.7km; 'LJLJALJ'LJLJLJJJLJLJ LJ‘,AL‘S LJL,(«rL- rLIJ- rLIJ :1. ~er LIJ—L LrLr I'JLIJLJL-\J‘J.l,'\\jl\,LI, V‘L'3TCA‘.SAC —L.:v.[rLIr~.r1Ir-L,L .L: 3C2"\CC,L,"V‘L,"V‘C‘3"7CC23.C( TTAGCAC TATGCCALGAGTCGCTCCCSCCAGGCTCCA3GCAZGGGGCTGGAJTGGGTCTC TAGTGGTAGLCGTGGTACCACATACTAC3GAGACTCCGTGAACSGGCC,GGT'C/CC A'C' C.AGAG.CAATTCCLAGAA,ACGCTCTATCTGCCAATGZACAGCCTGACAGCCCCG ACAICGCCL'ALALLACTCTCCGACAACik.CJLLLAC CCTACTLJJALCLCLCCLLVC7/11r11v1ﬁ IrI'I I"'I‘7‘l"'H‘TI r1 f‘ rI'Nrﬁ‘r r‘ir‘I mmmm7ﬁ1ﬂ".1 r1 '1 r11 rI'I rI'IrIﬁrI ﬁﬁrﬁ TC'EA.'CI GGLCACLGT'TIVTTCACC..r' "x m IrI'I mm r3 r3 "\7\ "‘ 1,.‘L‘ T1D NO, 14/)z- vr-l (SLJ '_ EVHLLFSCCGLVQRCCSLPLSCA7SCFT SSYAMSWVRQAEGKGLLVVSSLDEEGRGTYYA DSVKGRFTTGRDWCRJTLYLOCLLRAEDTA‘ YCAKDZGGRSAFDYWGQGLLVTVSSAST KGPS‘FFT.PCCLCTSGC AALGCLVKDYFPEPVLVSWNSGAL TPSS,LCTQTVZGNVGHKPSGTLVDCKVSPKSCLKTH wUEWENVTLMLLJTCGWLTYWLJVC1DPEV N'TCVNSTYR‘" L/7L'1QD1/I1L1V’KLV1"BLI’SLMLLE’ZLPTEKTTS{AKS’LI‘REPQVYTLPPSKLLVT1"NC'VS LchvJur"93D:AVLWESNGQPEN1 \1 LETTL I‘V’LDS Liiut LYSELTV’DKSR1!Q7G 1‘»’FSC »1,\-waC-CSCGG~QGG-LLVLL‘or-TLSLS «(x «A - » '1 V ~ 1.» ,. 1’1h(\1’}rV. 1- 1 C\ LR’LTLSCRZSQSV.‘ ILLS 'rjv"vm:"~n~rVYLLALSQSGTQV’L/ I 7\ 777,477r1'" CE~6V]QPGRSL-~hq» » 1 - .. - , . . 3 w . . - - . i , , LA TSRUNSKI; NO: 213‘) J'T LO LM ('1) —antiC"'LLwé‘ c AntiZbody: Light ChaLn '7/LC11 'CCZLGZLT‘IA’YCZ_GL’71CCA1TCF'77ACT"?CTGCATCTGLGGZLGZLCLGZLC‘TCACCA TCACT‘ '1'GCC -GGCZ ACT’YLZLGCA'TTZLGC.In"TATTTTA/LT" 7'GGTZ=LTCA 3CAGZLZ-7‘CC‘lLGC \_. ..1\(~CC7 CC3’1Z'AZL3ICTCC'Z'C’A \Jlix .GCL3CCL71.‘ 37‘ZI3Z'"[ '1.7'Z C17 1Z1. .1LL3’1'CCL3GTC7‘CA'Z CZLAGG uni/<1,[1.'\_1LL'\ GCIEAGT ‘.3 1111K, '\3'\3\J7'\.\/7'\.\L7'\o'\‘4'7\|'I'I/’\I' Mar," r1 r: 11-nrnx1--IJ.‘»ZZL‘JZ'L‘I 11.1 nCZLCLZ3,«p -1V1\,.ZI\\J‘.3Z.‘LL3r1111-n-m11 14- r1f‘tr1lr‘ (J. ‘3C1‘L.L\_,L . QZLZAC, Z3."'1.7'Z[7"17"7‘GC AZ— CT'Z AC I {"1131", '1 I"Ir‘1 .LC 'GZI-LJZLCALZL‘Z(1‘31\ r- \ r r71 -1r~,'1r~,w1r .f\{‘-f\r'1I1IZ_\ 11Ir'1 J. LCDilkaCKJAAK/('1 V r1 (1 r1 f‘ ,1. 1,1.G‘3LI3LC CC \31 . CG 7177,1713? r~(‘<-1'73r~;"\r‘JG r.1 xA7367] £ij I LACK.A: M r111. Crf. ILCZZL r.1cr.1111~1C~r~ :CZ "CTGAT GALr~ 7177117113"F 13‘;Ammmtjr‘7»GILLL'C GCCr\.1.‘—r~l‘~. 3pm«mm/"11AM"«a.LAC" r:Gib: IZLZL LZLLC1 m 1 r'n-u 1111 n I-rlrwrxrwy r1» 1 r1 Ck,» I [L 1 .1 LU' Lj' U‘Jx. L3 " I I . .315L033" 1L i‘14.1.1.1; «Jx.» ILC:"C:71\Z11L\LJDIA CA Z‘1A.GG'Z CGZ‘TAZ—ICGC7‘CTC3AAL'CCC’11'AZIZ3’1Z'CZ3 (,CZI 7 AI’GAG’ZGT 7 .‘J3CAZ1GC7 Z‘CACC3TA 32LG7‘C'1 ACCZ-. IZ3CC3TL3 LCG"TGZ1151C GCZ-LGZ’LC,"J[ZLCZC1ICILACACAZLA( CC,(3'77‘I7‘CILAG""CA CZfCC71LTC2IGL3 77‘CTCA’7C' "CGC CAZIACZHC71 .L‘CZLACACCGC’LCAC 11GI3ZLI3I’7' 1’71CZ’L1"717L3C1ICL3’1'I3 3C 3G1"TCAGG Z'3\3'_r' rm 1L31LL3G‘.’11-12 «.11 Apr 1r‘1r\r.r:lr'.'L ‘3 .LIJ'C. .'\_1I3. 11\\1C:I 711111." 11—1 1r rv111-|--111|--111|r11 V'n 1 1r - » C’I‘J/LIGC1 NOV/\JL—1-, r1» J. L3J. L3J. ..1-(1LCZLC C171.k1.

CCAGGCACCCTCJTCTTTLJ CTCCAGGJCJAAJACAGCLJ'AILJCCTCTCCTJ'CAJGGCJCACGTCAGA.

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JL.- 3 1GL .LJ . (31:.-JLIJ; rL,Z"ILJ L L: .fT.AG T" G G'TG' G.""' G Ir r 2 '2‘.=.G'"" C1 G '22 1.1G‘JLLJL31'V 11 —1IJ~JLIJ--.v1IJ-JLIJ--J: '1 h- '11 K3 '21 1L. .1 13,151 LJLJL -1 JL,C2-1G2—122 1' L. IT'T'CTC Z31CIC2'CZ3 ’\G'"(,23G3CZ1Z1"]."2["Z1'2'GZ—VL: \71Vr:1CI:GL‘J-~ qty/5' _..IZLZ'1..2Z\JIZ'1'L. _ L I 2".1' Z1C3C 11 [TI 2-1'J. "L2JZ'. Cv 1 JVL1TZ3 JV. JL - J .I21CZ1C1C23 3"."‘22'Z1GCT Z1'2[".Z1'2' GZ3IZ1'2["TGGTZ3 ("CH2" V’133‘ZZLGI2312Z'1Z31JIZ'1L3: CL2JCJ (L223IZXL23CZ'1 V_II::C,-LIJ1-Z\ 'L. 1,13. \LJLJ AIL ("f\ TTGG.’ TTTLLT TJ'ATALJCAGCAA.’ VGTGGCACLAJ'CGGTCJT 'CCGGC: CLLTTTT GTGGTAGCGG' mTATAG'.IL,TG-m V..rVVr TTLAGL,'LGG_A_GL»3AAGL,'AL»JAAGLL=LG ACJ,'AACCTATTVVV\ .m V m m VIKIVVV VVV\ rv-VVV AT 'GTVGAG",‘AGTGGTCAAGCAATCCGTTTACLJTTT JGTAGTG'GCACC ACTGCJAAATTAA 'C'G (SEL, TD NC: 27:-) C SGT‘ .»JLV PSQSLL'G' ’CT‘V/SG ST TNYGVHW‘VZRQSPGKGLETjTT G ’TWSLJGNTTYET,4 TETSRLSTTTTDNSKSQ‘LFEKTV’TTT.>LQC.Q TATYYGARALTYY'YD’EF"5 GJGTLVTVV.7SAAST KGPSVEPTAPSC. LCJTCJGG AALGC VT. YFTEPVTVSWN GALTSGVI‘TTFPAT TLJS GETS JTVJPSSSLGTQTV':C1JVJ \TJVTKPST1T: VD CKVEPKSCDKTETCPPCP :::VVV:"VL A FPPT‘VT'bLLLTTVTTCVT‘LVt’VJTCVVJV‘PJSE F‘J'I"‘DGVEVENLLETEE‘REEQYNL.TYRV/‘VJ S33 ’L'T‘7LTVTQTJWLNGT7KEYKC 3L",I’SNTV’LALPAPTEKTT SKAKGGTREPCV' ITLPPSATL‘EL'TKN("'37S L'T CL":VVIL "-GT'YPSDTAVET'ESNGQPENTI'\‘Y'LL'TTL PVLDS LCS-ELYSKL‘TWDKSIWQC‘GN‘VI’ESC S..7. 3GG’3SGSGGGSGGGGSGGGQ‘VQLQQGGALLA PGA SITKTV’TSCKAS 33V’TTTRY'TT‘I’TTTTI‘VTV'QRPG GLETIT‘TGYTLVTTSRGY'TNYNJOKEKDKATLT'T‘KSSS TAY"'7WLSGL""SIV"38'"\I’I"TYCARYY DDTVTYCLDYWGQGTTLTVSSGCGGSG73GGSG’TGGSQTV' TQSPAT TKV'TM"'C SAS 5' SVS YMNWYQQKSGTS TKRWTY TJTSKLAS GVL ATTERG SG- 3.3";SYSL'T‘TSGT‘I’TTAEDAA'TYYCQQJWIS. ._ ispecific 2373' CELIA (:11?erfrVr. 113(3ch2—:3 (‘2:.V m»"A./\.J_/\J\u\_;\'i(VI Arman. VrV:C" iVAmm/Vm/VIA "MW-LI 1,7 ..,'L3' [\GCGCLICLIC VrVIIVrV» » nun 3V _, .3 Z313 .GTVVTDZI‘JVV«.TJKVIVVTKVIIVH T TTA G'T'GCC 3C"-JGAGCCAGAGC/T'T'-3 GL32IVLCCAA’3AT 'TL‘AT" 3'GGTATCZLGCZ CAA rVrV.IVrV7puck/P3 VIIV.CC'CYL/L'J‘LLIJ.V ~rV VerVmvaVJTVGVZI‘JTTZTAZTTATGr1r1*V\ 7 'V\r1V m VA/VIA ""VVIV1I3V‘._,L31I3_ru2/I_'L3VC, r3YT7. V, Jr GCL'I VILATT‘GV». VAN mm'L/Vny (Vamp/V.VVZI‘JVVLIVJGL, 'TTTAGCG 3CA'3L.’G3 23AZIL73CGAAG ALTA. . G7: GG... 'IT'. IGC’CAGCAGAACAZ'3Vi'ij-3L3'I"3GCC(V'AC'’3 2"VC‘I.3'T."T"T".I;J3 3C 3CGGGCALC LCGTALCGC'T 73’3CTGI CC.AT',3'.[I' G'T'C'II' 37‘.TCT'"C‘I3G"CATTC. I'GA'IT' [AZ-3. 5. LL;.GVJZ5LLZ'3'L.r11 Vrn I"? rV.rwr-rV/V- ‘h‘l hVIrV1 Vrn VrV1 IVVrV. er rVr 1C 7\ V\ r'1"lI'V1T1'I""II'I'T'7\I'I'If’ﬁf‘if’",f\' rV .'3L,'L_. _.J.‘L3J'. TTCVI. C33. GK, G'TJL. VGA-LAT .‘\2I53 .‘..,.'LL’3.'LV.,'L.V.,Z5.LVJZVL3 Z5LV3'L3L,'L_. J'3.-27\LZ\‘L337'3" rV rV-‘D Vh‘l'n VV VrV1 V rV er.T' L:r\ J'. 2-3L2-3L31. GVJZTLZ'3sz ,I3'.[' A2—VCGCCCTCSAP3'.ICLVIG Z, 3A. I.3 CVI,3Z.LG 3A. I2 VJxxVLA‘Cj‘LJZ‘.,Z5.‘.3 Ll'3/333L3Z'3LA\\"\/£,\(/‘ - a V VV .. , rV VII'I'A V-n a 2V3 1V3G‘., L3.I—‘ erV. Z'.3.'L3I.V\.,Z\\JL. A\\":3C":._L ‘L3Z\‘ G‘., f‘f’" rVP-IL m. amp-,3 .L \J!x\_J\Z\1.Z'\AIL\1 L373’\73’\73'[AC.G2VGI3L2-3L3IALAAAL I.’\(,G(,(3'II"I3CA233L3I13L3JAC'CC'A’I‘C2VGL'3 3'3L3'I'GA’3C' I'CLVIC CZ5.Z'3': 3.’V\LL:IZV3VI Tie: G‘3 ..‘.3'L3.' .'LJVJzVV- V-n "T3 ".1 I ..-.,V WVIL- . .‘LJGHG‘LJJ. ‘.3 1‘:1\_J[ .'L.L.-. V-n Vn. V,IV.- WV- . rV.. .3301. C33AGGL,r' r-VrV ‘32 I'VITI‘* )rVrV. Wr‘<,’Vr[er'.'LVVJT VnLVLjuJI.A V I—‘ -V ‘., LV. n|(- ('\ G‘J/LLJL-,V L_.J'3'\3V.3.r‘-‘ r' rVranL-I L. .L J'. \‘L3L,.GL, Z5.LVJL.Z' ['3wa rV1 In]5"\f‘iZ\ GG'. 'GCAGA2—VCTGGC2—3L3G'. CCGGC'TGCAAG 3'T'TAZ'32-3Z'V'I.AGC' GTA2VJAGL,I3ZHCGG 'IT'T'A.'I.[3'A (V 1 I rm-rJ-nJ-n-n VrV-, .erV ,.....,L,- -.- ,..

L.V.,.$1 AL. C‘3G1 .L "V3 CZ5. .'VIVL,Z\']. 033131mm-n H3C3G‘.,V- AC.nary-pl,- VmI—JmI—V 327'3Z5LI erV .'L.V.,'\3V:n'\3'].' ‘3Z\‘L3L3‘L3J'. ‘..,.'L‘.3 _ 'VVJAT.

T.3C3TTATATC.AAT-CCC/CCCCTI-3C3TTATACAACJLAI’TAACCACAA TTCAAACAT7‘AAC'A- ACCC 'CACC’AC’C 3AATAAAT-C3.,AC3CACC..CCCCT7TT7"C3I.,AC3CTC3AC3C CTCACN 'CAC ACC'C3’ATACCC3CACTTTATTA 'CCA 3TATTATCA‘ CATC: CT TTC3CC,_CC3ATTATT CCC' CAC.3C3CACC'ACCC’ CACCCTTACJCA»CCCTCCT.3C3 CC3TAC3TC3C3TC3CCCC' CCTTCA CCCCCTC.3C.3C3TACC'CCA'C3ATTCTTCTC3ACCCACACTCCCCCAATTATCACCCCAAC CCCC3 C' CA: AAAETTACJCALCACC,‘ CTAC3C .3CCCACCCACA‘3I,C3TTACCTATATC..ATTCC A' CA "C3CACAAAC3CC3C3C,-CCA.C3CCCCAAAC-3TTC3C3ALTLATCATAC'CACCAAACTCCCAACCCC' C' TCCC3CCA"CATTTTCC 'C3CTAC3CC3CTAC3CCCCACCJTCATATACCJCTCACCATTACCCCTA 'CCA-ACCCA3AAC1CCACCAACCTATT..TTC3TCAC3C,'ACTCCTCAA ACATCCCL TACCTT TCC' ACTCCCACCAAAIIT.3C3AAATTAATCC (SEQ TC- \TC): 22227.5) I5/ H Mb I—T :r, U) \ EVTLEV L3 ECERVSL ‘ r U) SC'T‘LJTTLST \TL3T7ESTETJTP-LY21CQ‘ I.\TT-\TN'2T’TETT ETC-AC TELE A@ '3TI2'TCITATT‘’TVCL LINT-\T'EYT‘EE’L"VQWTTTV’DNALQSCNSQE'ISTVTTECDSLCDS'T‘YSLSSTLTLSTZ AL YEKT:TK‘I.I/IL'ACEVTHQC \/'TKSLITI-TRG_J 3 1.

CAL.3LATE.E’GASTv'KTV‘TL3 CKASIC Y'1TL 'TRY T'MHWTV'KQ T EC C‘C2 ("TAT T2 CYT NEST C 212'LTT\TYT-\TQKT:TKDKA S S'T‘AYTV‘TQL ’3 S TUV MCI-J 'ARY" D2TTT‘1'C23L2YT1TCQCT'TTL'TTTV’TSS C CSCC,3 ,3,3l,.~,3.3,\» ,-33 Q3" 3 . 3 3.3, ~.I3,,33~,,:3 Chg. QC}, 3 2 ,v 033,33LSEA. INLLTJTSELASC3 3 m TC. 3,3 3 «,3 C3II.,3 -I\;.-L3QTI.2LIQLIIEAITTTSASL’I._CSALISLI‘JLI1E1NTI‘xTLQxQKo Actlatable Pntiordy 3954w Light Chain TJ CLG CDC-.23 (3'21qu.-. ».,33~,.T, 3,3 ,3». (3,3 3% 1C2..._ACCTC.CC3\ C‘GCC ‘2AL'I'2AL’TG'TTCZTLTGT 21C3, 3,333,: ,~. 'Cv2‘. 'LI L-JCJ .I-CJLJ "4-1.1LID,~. CC". 3.3,, 1f‘f‘7‘f"‘f"‘ '1CLCC’LTCCA’LTCCCCTCTC2CCGCTCCC’TT'T'CCCA'T-AA 'I'CJA7 3.~r3 ﬁlls-Griﬂ’n Gf'qur1|T[/'\r‘(fwr‘(L/'\LD_\ri1rNr.)'E’Tb1Crl1r:(2 G2I._ 'C C32LC3CC'2TCACCI3 mmr, an 3A. ~ 3 r' r1." rv'LC,\.1I.:I'\.1C,'\.12.‘\(,L-1 .L‘J-L‘JCIJL3.3-, «m ,v’\_r‘t.'1r'.r1-T. T 12,-" r11 33‘ICGA ‘JAC21(23CA'TTTTTCCCACCZA2-ICA'2T"T.TL‘JA [T'T' CC'2T2T‘1'2T'CCI2LAL3CI C2—CCA2ACCCCAC" CI233‘ ICCT3‘ CAT'TAZATATC'T2G233‘"CAAACCZT2' [DawaC-IC2.r r1; C‘C‘Z r'1hl'('l V I "1 xxx/1r» '..'1r ‘1 r C‘.’ _‘Z ‘1 -3 V {-3} "IKV,’\I/‘ {x r\{1-f\'l\hlll'?1hlll'pz '1 ‘4 ("In-1;"? {\fV I. 3. ~1WCTBZTCTIJCK/L-K/ [T T2L\C1C,C1C:II,2\L3C‘ TJMKJL-K/AK/‘x 0124.1 J. .I. A‘ CT-,-'CC72uJ'C .C,2T\'\_1C,'\_1J.LJC:IA23T.,G2£'1.2TAIJ2TAC'12’-"TTC3', CIT-11?. L 12-31,,...Z Ifrv' ,3_-, 3. -3 r1? .'1' .v-..- {\P- «3 rprrx-nrrxr Im ,v'\_r‘t/'\- r\r1-Z - m 3 —3 Vr1lr1r\_r‘tr\lr' ,1. '1 I-3C Ida/"UK 2T\L-1,.Z\2-‘.CALLV TEAL. I-3C C,\12LT\ CCACCTTTTTT'T'LJCQ‘ CGIS 3C2ICL‘AA23C GAA(I2 AAACCI'TTACGC'L23C'2TCI23,T\I2TC A'22'C'2C'2 C'T'T' CA'T'C'TT'C‘ I23C3II-'T2CATZ'TCZ'I’TCAGCACT'T'LJA T'T'C'T'CIJAACT.T’wCC‘ 22T'T' T'T'T'TC'TTG'TTCCCTT’TC' 'C .I2-,J.L3J.. QC, C: 2%xf'1...T2LT1I2’-".C,T.3. .-I.I3 - r3.-3,3-,--3,3-,-n a -3 (I, 2'3 r3? rv- {3/3 I MAJ-21C."31C! GTE-MALE1C- 3mm 3.- wm -,I J. J. 2-\. .L CC‘J CIA/Aqu23CI-3C1CC - r1r\u.r:llr"1 .12LT\.Trn‘ 2.‘\ 321C‘ C..C KAI/Tub1.2-3 m1 «n-I .rxlr" r1 I:I . ,3, 3C32,]A.AC- CII2ACG22-"3G2"A‘ TT CTCI"ACAC..3" (23(32-ICCAIAC, C2—ACC2 CACCACUTz-C ‘2TC'TTCAC.CAC C -CCC' CACJCTCnCLAA/CCAHAJTACJAJAAACHCAFAG'CTACGCCTGCGAAGTC/CC[:4 LJATA‘JG .JCC' LJGA JJ LJLJLJLJLJLJ"V rV‘r mCV ‘7‘ ("vm/"VrV/"w."V/"1"V "V7\"V LJALJAJJALJAGC T7 JCAALJ/JLJLJLJ‘JAJv7 ("V "Vb"Vr: C73JJJ J JLJCALJLJ r1,"V ."VKVJ.

J LJLJAT ,»"V. ,"Vrﬁ‘rrﬁﬁm "Jr"V rpm/"V '"JrV ‘:1LJJ' LJGLJLJGJ iL.ALJLJC LJ\:1LJ "x LJLJCJCA ’JLJTLJLJT‘JLJALJJJJ1LJAGLJCLJLJFJLJ JTLJAvm v ,1"V. ,;"Vr' r:"V7\ Am Chf'7\f‘;’"n ."V"\ GCTGCAGLAGJGCGC 'GCAGAACTGGCACGTCCGGG 'GCAAGCJTTAAAATQLGC TGTAAAGCA GCGGTATACCTTTACCCJJJAJACCA"CCATTHC>TTHAACASCG"CCGG G' CACGCTTTCCAGC"TTLJLJTTJATATCATCCGATCCCTGC TATACCAACJACAACCA AAATTA?JAGATAAAGCAACCC 'GACCACCJATAAA/GCAGCAGCEJCCJCCTA'A‘GCAG C' CACC-CLCTC7CVJCAGACCATACCJCECTTTTTTAC L?TGCACCJCTATTATGATGATC ACTATTCCCTGGATTATTLTGC LJAGJGCACACCJTL7LCmTT'AGCGGTGCTJC CG TALTCGTGGCGGTGJTTC/CSCGC CJLJGJAGCCACAT"GTTC ACLCAGTG' CCCGCA AS71171(731-713 71321JGTCCGCGTGPQAPQAAGT‘TAC’7291'1‘'7Z‘JCCl3'1AG- (3713 7291 73’ CL. /_G7 7G "TA 7A11SGCTATCAGCAGAAAATCGGCZCCAGCCCSAAACcilb7LTTTA1CA RC ACC/AACTCCLAAGCGGTGT"CC o7ﬁ7ﬁTTTTCG'CC A" AGCCTCACS731 rmCCJiJACCT11CC7’17CC3CCAAKCTCGLAATTAATCTT (SEQ IL NO: 2751 ,LJ, Lz-JQQ 1SPJL- P1113:YVMJ be:.CLJLJC»fJJ JJSCBSIJR111,3; 71Q1JJ1JQCJ‘J1119233 , D 7m, LJ 1 ,L L,‘ 1,: JJ 7 LJ J LN, »»»»» TWLQL J 1; m SCW7SR10CS7SGTLFTLST NSVESL‘ TADYYCQQJNNWPTT'IC RTJAACSWC1CPPCUQL NNCYERVZ‘V'NYVLMALSCRSCVSVTJQDSTLSTYLaolLlLS1"LK1KVYAC?V" HQGJSQEV1J‘LJRFLJCHCSC\»» nlr‘V’ "(1 \rr'1T. "x \ rur'er/‘V(1.~ 1bGSGb-S-G-GbuuuQvQLQQSGAJJARBDASvLug"1 v17 ~~r~~r~ «A » a LJLAJL', L7,, , r "x L» TLT 7 (L7 (~1\'*r'~rl;(w CKZSGYTETC"TM1WVYQRPCCGJEWTT1TNPSRCYTNYNCTC7UVTLTiLKCSSl1YMO LSSQTSEDSAVJYCARYYDLHYCJDYWGQGTTLTVS86GB"SCCCCC7CCCCQT"L1QSE7 TSKLﬂSbMPAP.T 77GSGTK FTﬂR (SEQ ID N": C76) E 4 Mult Chain C23CTGFRbLmGHACA.CICCGCTCTGGG-CWGGTGCACCTCKC'LNGN7"CTGACCPTTA rer1 P- 1wa '\ rV xxrV ,-.rL- ,v-m J-L Lm L —L —L L 1- ,1 (Ln-p LL, \J'L. AL. CLJJLL ’waCTFTA ' 7J\3\/\_ \JK/,l"\J .1.ij Ji LGL,C Z71. .A.L:\_,'L_,LJ L:.\J'L:\,z.JA2" LJLJC‘(1,1L,- rL- - 1".1f‘f\f‘ir1r‘f\r‘f\r‘ﬂl'lJG"3A r.1 "LJL . L:\JL:\,'L:1_LJLZ\.1 {JPKSCCCCGCL‘JLNLNUG72"1T 723. 17"LAACC 712',2’-\.CCABC"G"C17’11-"1’7"7Z\1""[nACAJKpG/"L'I‘fslJLCCAAJLLAC'7C2-\GG’1'13'I‘ ‘WTTFTTZRZLZLEL AACAnLmeCAAGCCFSCAJKC" 37M? "C"C"MC'7"7'7"T \J'x. \JL. \JL, "IL, "IL, "1‘ 1‘3.\K/M"C'FQTTP TGAATTTGCGTATTGGGGCCAGGJ'CACLJCTGJG’G’ CCC. +3]GAGCJCCCLTZ‘CJ/CC AALJ‘JLJpLJC-ALLLJLJGLCLTLJLJLJLJLJ'7 «mam—L mac 1.11.1LJ—1J1LJ—1—L.—L,~—LLJ\.1LJ CC- «11.11 vmrIﬂm 1 —L / -L -A‘.-LJL J L CL J'L L.L:AzA'h :m "11"erLCJGGLJGC.AL: -LJLJLJ‘J«In—Ln Ara—LT «n—LLJLJLJ'TLJ‘JLJCL GLJ LJLLJGL'LJA/-LJ'LJ.Am mmmn 17 Arm—1mm m A_LJ'L L -LJL. LJLJAAC JLJLJ"~11 "AF—L. LJA.LJ‘.:'LJLL«71mm Am LJ'L' LJ .JLLJGAALTLJA'TLJ‘J CGCCC' GAACCAGCGGCG CC 'TCCGCCTGTLJLJTACAC CCTCAGGACTCTACJ CC.

Im "\7\ "F‘ 1 r1 IT! AA #1 "ﬂ": 1-1 LJA.LJ\-AGLJ .JLLJGL GALJLJLJ J-LJ\-LJ LJCALGLJA JL.J'L LGLJLJLJ/-LJM.LJA.LJmm. I‘er'x "A"? m *1/37 An LJ LJC-L ALJA LJLJAA LJ\.:'mm. .ﬂJﬂLJAAT\.-ACALALJC JLJALJL.AA.LJ.r1‘r "1 ("mm/ﬁr" 1'11 "F‘A‘W "1 ‘7‘ / --LJ\..AAILA_J 1‘7 .JLrT'GLJACAALLJAAA.LJTT\.J-n\JLLJCLJ l-TII‘JI-THTIK1 r17r'1‘11LLJ'L L LJ AAL.AA.

AACJ'TCAC CATGCCJAC‘JCLJ'GCCCAGCACCT AAACTCJC CTGGG CAJ TL.T' CC 'C TTLJM.LJM.LJCALAALACLJC'7\A'JLJA'LJA\.-LJ\.-TCALLLJA LLJJ'LLLCJLJLJLJALJ-L.. "AAA" 1‘1 ﬁr17 r'1‘rr1'1r1 . m mmmr‘v IIW AﬂjﬂmLGALGLJLL C.AJATLJLJLJTLJ‘Jlr11 Arm." TLJLJ' LJ‘JACLJLL LJA JLLJJALJ'JAALJ.A -LJ‘-TGALGLJ LCAAA"Irv-1 1 r'11‘1 7 r1 r1 r1 1r"! ‘7‘ ﬁm #17'LJAAC "mm '3 LJ\.1L LJ .JL :GALJr1 ﬁrw'xru .ﬂr‘ﬂ JJ'L JLJ' JLJ'TLJLJA_ GG G'CATAATGCLJAAGEAGAAAGCCGCJGGGA 7» C‘FT'IKI".rN.~ 1111:L.L.LL.I11\ Aux.GL‘L-L..lL3-._J.r»1-.-L.I1'L3«ITI'1/xr1r-xr11» (Wm-A ~r‘7‘1C' vaC~r'1 1’ .r»1~3L.\- .1"may"..- .- L .t- -LL.L.»11L.C1-».1,LJLI,I ."r‘/ .7".va -(3 L.’AIL. L.".J'LL3L.\.L.L.C.J- L.‘.JL3"L3 _[1.3.r»1.»-CCC5"Jan 1.1». J LIN 1 ~rLIJ1rLIJ1 L». ,1I1~.JLI~JL LJL" »» L ,1 «ACCT TCCTGG'T‘CAZ.23GL—C 1' '1C1ATCCCAG’TGACATCGCCG1GGI. 1T —7 G’-‘.13.’-‘;»-GCA;»-TG L C‘I'N'Z‘-, vL3I'1C-JI3.3.1L». /\'n L313'L.I1'L3L.L. CTCCG13CGGjTC"T1"11 r‘n411k,fNr‘. GCA’ GG .GG--A»‘L1r «ITI wr-xr17» ,~f\ 1-1Cv.5»\I'1L.»n 1 ~1;r11(:r11IT1r1. (11» >1.~r~ " .L'"".A L3L.

LJCJL CL11: er1 er1 ~I~CrI1r~r 1Irx.» 1Ir‘(rwI."r‘‘. L. _,L3\-. r-x. 1 ~r'1r~ «7 r11rxr1r-«r11» (1.1-1; ~~n1q~ \ r1r‘r}1r~f\r}f\r'~<' ,~f\ L3 'vLLJrLJJAL LL3|3.A'3"1Z'.L3 vL.L L.JI1‘C3LJLLJ'JLKJLJI'-"1\.:E' .r»‘1"'L.ID1L3\3"L.Mr vL3I'1L.vL.vL'»'.A./:\.'L LL31'L311'1 L'\3I1C.5»‘J.5»Jum 1-1 Grnru » ">1 ~ 1r r1 f ‘1 »n » r‘r}1/‘1r}1/\ '11» (1.1x r1 er-Arxrq :r'1 Nr'111'1r11rVr11/Nr Jr‘er f‘;"‘f‘ 3'. .1 .‘L-.r»‘1|.3'1"-.11L.L.L.LvL 1L L LL31 'L.".J.r'1~3L3\3 ~ ‘1 I 1'1'1I1'1r1rx.1r-xr17 ~r~ A 1 ‘r11 ' mr117 In 3r r1 -1GCCILCC:£-=--' L3"-1' -L3L..zIL.J'I'1L3CLI'1‘._LI 3L."_J'L' r.-L..1GL.»1GA».1\L£-7" f‘{‘@ *1 1 , J. CCLI1GCvL:LJ.ILL.11- 1::1'1'1C1r11/x»' .I»1-LJC21»1GCAJUKJL153£ALJ1,11 J L.LJLJLJ.» ,L Z GGCA'TCCCAGA'JI7 I‘"GG‘TCTGJGACAGAL"TC"CTCT‘ AGACIGGAGCA’TGAZ‘ZT'T'T'TGCI1T 1"‘I 'LI1Lr1"\r,'1 vL3L "~»1,1I»1»n I’17‘.JL‘L.J r1ImrLIm..J'L.'3'L.'L"\.I1:"-‘\ J. /‘1 C'T1"1CC "CGGAGGG‘ G‘AAI'= 13A»? nrxr1IJ-Lr L.L-.. . ._, 3131' GAL.L.LL...T.I.I,J..»...IJLJLx : 1-1 L-J TAATG" SATE-CTI' mACGAAG'T‘"TI’3 ']Z Z'C'[CG.ZXGC13 CCCAGG -rL-3C1... L3 1«[1. LT L IZ—LALJALJ ['LJLJLJ,JL-,1 I’L.- ,JL, 1"CACGCGTGGTC3A1JCC'Z GGGAC’ ‘-'~1DI'I( {"Ier _v r J. LJL . Z1C2"\G‘IZJCJTCJTGZ‘TC’CAC’Z‘ : T'T.’3GLZAC CT.ZXT1‘ 'ZC‘GJ'LGCT’1G .CGC‘IZAG ‘-..3CA"GG.\Z.3LGGGGCZE‘GGI "I'I'If‘If‘ CT. (:ror‘ 3L \J'LJ‘J _A.’]Z"Z[".ZXG’1'LSG'I‘A13'ZC‘GCI"""3'IXGLJ.CATAC C‘CJ-ALJALJL ,JL-L I'Lr ,LIJ:-,Lm -,1 1 L —L L. 1’" {up . GAALJ 1‘:J3L.- 113['J]A L-[Imn1IJ~ J AT (Z -rLZ\J ALGA‘JGAXCA L- r11I-I-1CIm A1103:.xL.\LIn-Z Ir" 'vLJL‘LJLA-m-I-u-.1-n| -1AAJZ' z..\L\‘LJLJ‘JL‘15b J'\(:/'\:Z 1 ml —~ v1 1» .r\.0 .LM\/'\J\Lj\-,1 JL- ,LIJ- r1 1.-.1-n.- m -1 rrx ,v'xI(3 IAI(3 {1 Ir- r; 3 . _CG L A. 3LC'Z C'[GC"C’\LJI..Z C?" "'73 ""1"?" --:I V-A11I':'1II ."L A . J'EL . ./ .L \J'L. \J'L..\L/'\_ TGLJCA"’"CJ'Z‘GG'ZZ‘"CTG'V‘TCH. Z'ICTIXGC (SEQ ID NO: 277) QV'QLK38G}? ALA/1Z3? S Z[ "TCT‘L 1 G3.ZHW‘L’RQS PGKCIZJCLWL1GvZ1: .1 EGGN1‘)‘J’ZNT ._ .."Z["SRZ[JSIZ=.L=Z'~.\1NQRSQV"<=V'NS ]Z.QSQIZIT2"\. .. iJI3PZ’3ZLTYYLE.ZXZ’WL {JV-1"»’SAAJA"."]Z' KCPS‘IJ’TFPTAPSS LCTSCG L‘I/TKDYFRER‘JT‘JSWNSG7LTC-.VHTFPAVT LJSS'3L1S LSSTL.TTI/TTTIJPSSSLCTQTVTCNTL \TErIKPSiTTTVDKKTL/3PKSGDKTL TC,‘PE‘CE‘APT3LTG'3RSTL7FL FRET"PKL 113M: SR.:33TL7TCTx/T‘L7‘I/fx/Sr13DPEVTKFNT/I.71TTI/TDGTL7ETL7EN..T.TT w {21LJ4 3QYNSTYR‘I/TV STL7LT rIQD.....]CK3Y"LC. VSNKA, LT‘ATTEKTTS{AKG‘ERREPQVLTLPPSR D3LTKNCJ‘I/TS LTGL‘I/TT'iGFY S ITAVEWESNC"PCI‘LAYKTTPPVLDSDGSFFLYSKT TTTDTSRTJQQ">.NT\/TFS..x.

STL7T1HEALHTTIHT1TTQKSLSLSPCI.1 .GGSGGGGSGSG3GSG"3GGSGG3ET‘TLTQSPG LSLST‘: T3RATLSGR..S’L. SVSL CLYLAWYUCJKP..QAPRLLTYGASSRATG:PDRFSGSGSGTD-"'" LT :TS FA Y1LCITQQ1‘TGL Lsr/LTF‘GGC 'KVEJJ.KKSGGS’.L'--T.L'S71TN7\7YT1TTKTSSS GTQTL7Q IVJTG GCw‘IJTVQPGRST SGSTFSSY 1 .1 4J4wGIE7 I"- C) 371 C) AI ‘1 vLI (J.\I CI [1y K {J .. 1 . '3 .U) J. isU L/1 (J r"3:(J r"L. fC7). r1] H :SRDNSKTTTTT1LQMNSLRA3DTA‘I/TYYCAT1TISL1W.FD ITJCRGTLVT 7SSAS (STQ D NO: 273‘) AntLNEI’ZFR (.14 EVE-"Anti"CTLA" ChaLn AC"D fJVr. m». VmJV .LJVJV CGL I /\G,CCCIw-T-.JVJDJVJV. ~JV TTA7 C'lGCLGGGG’7 G77’7AGAGC7TT:23 -7T-GC>AAGCAA’7.

.JVrVnJrVTLUCK/PA LCVCGLJLJ‘.J 1JV. V ~J .JJVmJVJVerT r1r1*~\* IJTDAT ThAhiAi "\r‘.‘ m V~.JV» JVJJJVJJL 7.» JVJV> ﬁrm J. VJ. V~.~ mmJVJVJVJ JVJVJVJVJV 'I u". -‘.., -1»\..r3L./»-L3I.,.rAl ThbGL-J ADA; TL, .HAHLJ‘G'LJL, w JJV- VJ, J JV J JVJVJVJJJV. JVJVJLJ, m... T "l T'AGCG .LAGLGGGA'IJLAJ'LJEAC'C'I--.-»> :1 .__.LiI»:V:LJ 7T) I;I": C) .7..I'.’I.)7} ’.\I I4 I—j .J (T. '5‘.V- CI (3 1 I-J. C) \ 'l ::=MyV \ a(:1 ('7‘J a(:1>5) 7711 T'G‘C'‘Ir \r‘: V’1 r'II'rI ITH’TI {171‘ ‘4 > \r‘t’ -.GGAL 1A. .Al'TGCCAG' 731'"[AZ;C.Z;£7A‘J.:I7AZ*EL.»-~ > ~> ' >nmr71..GI.7.L.(T1 ."-« ‘ V GLLl'xJ‘CJLACLJ‘\ "‘-r‘ II ‘AIIW’IFI'H_ 1C ._ l-"I (I) . c (T.C (J (7 IIIC/ml. (ml. Cri. II‘CZZA rICICC/«LJf‘ (an.LA1.3.1:173']: mﬂﬁm1AnrJ . I,L .jﬁmLLICaCi'I.m...mm~mnmnm,. JV 7 L:J, V.J.L...L.I. .J\.‘., IA'QIA (,.... "I_.1 AC77~CPAAG'"AC. ‘G 77.7.7 3GAA771GT ACHGAGC‘TV D GLJACAGLZJ112'/T\\JLT\\.J-K/’fJ17 J7 VJV 7» J.JV D JV ACCLJ' -GACUCTGI;L){..»i_.tf’ALL1 V~.«.>. JJVJw VTJVV> J D ACTA’GZ>.GAA.TInG2-GZ>-ZAGT7171CT7C7’T77GAA{.17’7AGCGAT'7C"7T7G7GG'17GZL77," C'GC (VJVJ- V V-JL «n. ‘.‘1 1‘: 1‘3? .LJ..V VJV. . 7J\I\..AI'T:_.i AGG’:G7\G2\ 7TGTGG7Z3.I3G' .7GGZ-\ _ .‘LJGZ'ECJ‘JJ.J-V J -J,VJV ‘4 w- _"\J'_ GL3I.‘\‘VTJG‘.J >V.-.-.‘.J Z3LIJ'C. .UIJT nwaJV.» wJVJVr[.J:J- -.L.:-..- V—VG‘3/3LIGC1JV.» TAL\\ZI\;AL\\r'Ihl*le‘hlrh AC Z31}: ‘C —V STILLW." "I .'L J. ‘0'._ ‘VJ'._ .1. .

CCAGGCACC3C'17GTE7C'1 17G17CTCAG7:GGAIIZ>GZ>ICIJTJZALCGC'I'C'. (,77II3’7'GLJIIGI GA :27.

:LJIIGTZ"C .CT’ 7ZLGCC'I77.(’7'"L,I.Z>I GAAA7777'[G7:C\GGT3’7'JT7CT7.LGGC' 'CC’T' [,JVrT‘7TT'GLJJ. SCI-1.L ‘JJC XXL: .L\J'L:\:.'.GAG}. GLJC MIL ‘JJC‘JJ/3LIJ/3LG1’3L3'L-. "ml V-n.-.-.. V .JJJV ,JVJ- ,VIJJJV- Vm J-V V-n.-.-. V—V ,JV-, ,JV- ,V, '.E‘CAGTGGCL?"G" .7GGG 1.77:7? GGwACAGAII37."?7 777ACCA'T'77JLGLJIIGIICTG7Z‘1I3GC'T‘GA.L‘L(T:Z‘L'77’1_".T7’7'GL, 2-3G777G17A'17TAL777 . .:L,Z-\rx Vrv 1G‘:1:T'-1'" .m- ,JV-,,JVJ./’\7 ,v'\ r\ ’l /L\..z’\ :I‘VJC:IZ‘C’I‘ C‘J.'L‘J\:.L\.J.z73.\.. rw'VI-‘fxlrﬂ. IrDI -V ‘FU V. 7!.f‘ f\7‘l 7.777 r\ «r: V(-"1DI..‘VTJG‘J.'L‘C/(JKJMK/IXKJ\T7\/"I"I.‘1,v’\.r‘4/’\lf\lr r\ . \J» \J\, 'L: ..‘LT3CZ-\G‘J., C;.C. chm/3. .'L_L,.73L, f‘.’:‘ ITII’V1I1'2II:... ‘JG‘J.JZ3L3‘ C.-V '3LT.'C1LJ'LA.1.r‘ r- Vr -f‘ ALjZ‘J TCCACGCAAGGCECTGCACTC CIGTCTCACTATTACTGGTAGTGTL'GT GCACATACTAC.

CC"C"CTCL..-"SAAGGCCCCCTTCACCAT:‘TCCAGATAAAT 'CC,‘AAGAACACGC’ 'CTATC TCCAAATGAACAGC.CTC1AC1AC1CI..GAGGACACG’}CCGTATATTA. TGCGAL.IAA'-C' CCC' TTACTGLVJA... . ,W Try-Ir111‘ 111/1iCL’JAﬁ'T'L,'T'3.1L:‘-.L:\V-L,Gi:JLJL.IA3L.IL,'L.I.L'3'3.1TWTC'CTMCGLITAKJL, .r1,.r""r1"‘r1"1 r1 "Im‘n 1‘1 mmrxm 'C m I". ,7 KW" 'TNT HWY?,‘RTTTCSP DU -( "1 !"4 W )- :w C!) G C!) CO C] 1 "L L ,_ ADYFKHKVTATE‘ THQCLSSPVTKSFTIRGGCGGCGGCGC. CSCIC 3C1GGGGSGGGTI "VT-T053 ""L'QC‘KI‘GC‘A.RLLT"3I2"\.SST‘.'".’:'G‘TELRI1'SCSGS TKRSCCSTLTSY‘JVVV'TKLSSS I1}Q‘(\/QL\\7(Q!1’IG :II11\7\JQEARQ0 » V'RQAP" AL‘SVI CAT'S- S{DNSKN T' CATNS LM'KG LA!»1.» 1 rV. I111AG: CTGGCLA11,111.11. 1,11 ,1 'GIJATL.TCACC'LG1 1., .11. 111 111». -- 1.11. C:GCCI~1L1AILIIILIAILC(»:I 'CZA'I'GT \.:I'3. CCC’. 'Lm L1G 16bLJ‘IJIXJLAJL'.(1,1,1... .1 1C". "Tl "‘f‘TC 1 I1 .'.LJLL']11,3171" 17 r71r‘r1/‘xr‘r1rxr. r1» 17 >1 'J. 'GIALLAIJ'LJDTL, '01:.GCCYC]1U.1I11r~r~1.-Inr1mr37.I. IA,'L.511' 31A. l'nA7 1.."r‘ 1-.AG: .\.Gf,G1 ~3117I r~r1r~Gl11I/1\mr1m/v\Afr‘r'"T'CI urn/II :3 /\r1/\* (1» (\rwf‘trﬂfl:f:rl1cy’l\ )‘1’1r.1Cr}1r_/n\G CGTGA‘GTIC CCCCGCG''"CGTGTGz G‘CT'T'TA J'l'GCC 3CGC’7213’7CCAGA-GCA' "1"?IG3C2".3C"3 "-T'T"AT 'T CAGC GCG TA: L'r‘«(1.1.111.Aim/L: ILAGL-GG L,.t -1.1".1 11.1 ,1. 3 V1, "1,1,1," r1I11r311,1111,111111 11,111.T.,11 r‘r" 1-..11 LA-‘ ,T l "i i'- il3£,L.£,.I.1.r»‘11.1L,;-T 1 1'53A1L, 1L,'3.1 410651253"‘Z ."71,- I11 II 11LGA1/5313/5'].A].'[L:', (3ij-,I ,13-I .11- .I-..- pp. .31 r'1lTl 'anlr - I 11 I 73 73 ,1 131-31 11- rw-Z 1:1,«,1V~,11I,- , . [LJL k/Akj'x11'351;\Z'\.CZ\.LL\'J..511'3(, LJL L,312L‘1 ]""[""GG3'3‘ "GGI 1L,ACCAZ\.3. GA.AC A.AACCITAC(1C'GCTG3'3ACC53TC'G' CT'T CA".["C".["TC CGL’.'CATCTGZ'."GAGCAC'I'TLmA 5TCTGGAAC'T’1CC r3111"Ix1lr71G.., G'.GI. GL,Vr71 Vr71 errVr1IrVr1r1rx .GL, G 1'53A1T A1AL, ['1 ' r V. 'V - rm‘ 1x11 --1 x11 '13 ‘4 {1-1 ,I'\ ,I'V," ,I'\ r r1 r" Z3 1x11 71 ‘1' Vr71 7‘ r71 7 '1 Vr71 14,11 ".r1 IrrV J. L, J. 21 :CL, 151531531GL 2‘11,\.1.Z\LJ7\ J. AC1-HQI GEM/52‘,3 1L.f; ,1:.- 1r: If V. '3.12L‘1,'. .‘\ACL5C‘I, (,J.C LARA1 L,'3.1L:I («:1\3'.] AALr- \LLL V Plz ,- r171 1. 'H‘ihl 13 «111 wan («,11 ,1 .1 1’1 1’1 Tran/V 1:71:12131GILA L"’I:1 I-UJG. L3.‘33:2L,2-I. (334.2'CI'XCII'IJC11.LI.Z\1\..L/AL— CAI’2'2-1CGCC3'1'GCG :AGCAAAGCAL’V—ICT'XCGG.2-3A2-332"3CA'3AAG "'TCA mm :1 1.3111."~11: 325‘ CAA. AGA"SIC' ,1'53A1CZ‘1311..I1L.IZ‘C.111C: (3,1.G‘..1.53L7L1.'., ,3,- pm 1m 1m 13 .LL,L1.L,LL,3.1' WNW-"C":52/55].

IrV,.v1Ir-rV'_ rv- DIIZ" U.--3C "mm3.1L:I,z/\.L:'3.1.I. L111. ‘1 1I.'3.1\JL(V rliﬁj. .1L:I',GLJI.‘\L51 ‘I.,.L ‘I.,}5,"L..,.I,1. .31 1. "II'I'I '7/‘V'1r‘ ,13. .r:lIr' 73 I1. 1. —1 LI J ’l 1 3.11.7. 1.1L5C:IZ\..I_L31\‘1I.,‘ 13 ,13-,I,1VII.I1I,-- . 133/5313L1'1J3.1.Z‘1 211:1"):1- : :. 1Ir71G-{I.CLZ'X 14733 V711IrV ‘C’CAG:10; ‘IC"‘G"‘C' 1r‘Ir1/‘tr1' 1 1- "A I ,. '0']. 3,L,,z'.L:L1r\«-I 1L525XZ'825,3—31 , A.GCC2’5.CCC',['L,C r 7,1'1 I11 L,._3 «CAGGGCCAGTECAGAG".‘G' ."AGC2-G'AKCT'"'["T"5.CCC'G'l'ACAGCGAAACC'’3.

GCCAGGCTCCCACGCTCGTCATCTATGG-T.ATCCAGCAGG~C~TCTGG ATCCCAGACAC ,4 GTTCTGTGGCAGTGCGTC’GGGACAGACTTCACTCTC/GCA CAGCAGACTGTAGCC 'GAA GATTTTCCAGTGTATTACTGTCAGCATT TGGTAGCTCACCGC' ACTTTCGGLGG-GGGA GCAAGGTGGAAATCA/CCTTCCGG_GGCGACCATAACTTCHTATAATTTATACTTAC GAAGTTATCCTCGAGCGTACCCTGGTGCAGCTCGTGCAG-.TwCCGGAGGCGTGG CCAG hm .nﬁn vm mﬂ7ﬁ h ﬂ": "m ﬁrmmmm 7Mwﬂ’ﬂﬁmﬂm v «NW1 CC GTAGGTCCCTGAACAACTCT~C' GTGCAGCLTLTLTA CCACCTTTAGCAGCTATGCCA_) mﬂ’ﬁ":TLJAG'L G JGT. UK. Gk. CAL1JL'T'LT"m m ﬂﬁﬁ ﬁﬁnﬁnﬁLT'CA'L'T‘\T'1'L'T‘ATAGL3L'JGLTL'JGATJTLﬁﬁ mmﬁ 7ﬂ.ﬂ*n.ﬂm mnhvn Lu 'L'T‘G Ammmm L, TCTAG ,1 T AT T A'JT'JL'T'TAG TGG' GGTAGCACATACTACGCAHA~TCCTTGAACGGCCGC CACCATCTCC GAGCAATW 'CCAAGACACGCTCTATCTGL’"’TGAACACLCTGACACCAGGACACGGCGTATATT ALTT\'1L,.GTTCTAAZ\1CATKLTGTTTACTHLTTTACT TCGATCTLTTLJLTGLTC TT'JTGG7h m NM * Tnﬁm mmv mmv h ﬂﬁnﬁﬂﬁ -Ak.ka.vTvT'L2mmvmbhmmnvm LT- C- TGTCTCT’TCPTGCU‘TGC (SEQ ID NO: 281) TGQSGCCTSPRGCLLCCYVV‘GCSCGSGGSGGECCCRSDAICSS'EQILLQQC\1LSVS PGEDVSFSCRASQSTGTNTHW’C’RTGGSPR_LTLYASES’SG"‘SRTSCSSG‘‘LETLST NSVESEDTA YES NNWAT'EG2GTL'KATVAATSVETEPPCDEQGLGTTASVVCLT NNFYPREAI(VQNYVL[ALQSCRS’LSXTEQDSKTCT SLSSTLTLSKADVLKLKVYACTJT HQGLSSEVTTLVACLCGGGSGCGCS7SGGCSCG‘GSSGGL;"LTQSPGW.S:SP‘LAATT SCRASCSVSSSYLAWYQTKPGQAPR'LTYGASSRZTGTPDRESGS-"TDFTLTTSRL3"E DFAVYYCQQTASSPLTFGGGTKVETKRSJGSTITGLNVYYTRL seewove"G‘GCG’VO PGRSTDTSCAACGC"SSTIGS‘vSAPGV"T‘WVSATSGSGGST XUQVKFRHTIRDN SKNTTYTQMNSLPALLT’YYCAT1S="NVLLLWPCTLVTVSSAS (SEA ID NO; EXAI‘V’EFLE i: l’roparation of muiﬁspeeiﬁe dies {ﬁﬂﬂlﬁbﬁ} This Example demonstrates the construction, sion and purifieation of anti-Jagged CD3, agged i, anﬂ-ﬁGFR CD3 and anti-EGER CTLAV-A’E muitispeoiﬁe dies.

{MAME} Vectors were used to express the enti-To.eggel (41")? TV».) heavv chain the 5342129441)! 1V2. 1i ght chaﬁng the anti—EGFR CZZSVS heavy chain? and the 3954,1304- 3225V5 iighi chain sequences shown above. Such vectors are described in eo~pending appiieations 2013/G38540, filed April 26 2013 (entitled "Activambie Anhbodies That Bind Epiderma} Growth Faetor Receptor And Methods Of Use Thereof") and PCT/USZOI3/O471G9, filed June 2L 2913 (entitled "Anti-Jagged dies, Aetivatabie Anti—lagged Antibodies and Methods of Use Thereof"), the contents of each ofwhieh are hereby orated by reference in th eir entirety. tsunami Vectors were digested with restrietion enzymes Nliel and Noti and the veetor fragment isolated by gel electrophoresis. lnserts were prepared as s. Human igG CH2Cl-l3 traginent was ampliﬁed in reaction l, from Pop Hygro 4Dl lVZ using primer HCEoerie (Table 89 SEQ ll) N0: 3i and primer HCReVOL (Table 89 SEQ. ll) N0: ll)? C'l‘LA-4 scEV was ampliﬁed in reaction 2," from C'l‘LA-4 seEV CDNA (SEQ ll) NO: 229) using primer CTReVNot (Table 8, SEQ lD N0: 1) and primer CTEorOL (Table 8, SEQ lD NO: 2), OKT3 seFv was amplified in reaction 3, from OKT3 seEv CDNA (SEQ ll) NO: 23 l) using primer QKReVNot (Table 8, SEQ ll.) NO: 5) and primer CTForQL (Table 8, SEQ ll) NO: 2). Human lgG, CH2CH3/C'l‘LA-4 seFV fusions were prepared by combining lllll/E; ofreaetion l, l(l% ol'reaetion 2, and amplifying with primers, HCEoerie and CTReVNot.

Table 8. Erimer ces Primer Nucleic acid sequence SEQ ll) C'l‘lievNot 'l‘CGAGCGGCCGC"l‘CAAC'l‘AGCTGAAGAGACAGTG SEQ ll) CTEorOL GCCC'l'C’l‘AGAC'l‘CGATCTAGCTAGCTGAAGAGAC SEQ ll) AGTGACCAGG NO: 2 l-lCEorNhe CTCAGCTAGCACCAGGGCCCATCGGTC SEQ ll) NO: 3 VOL CTTTACCCGGAGACAGGGAGAGGCTCTTCTGC SEQ ll) NO: 4 OKReVNot CTCGAGCGGC:(::{i’(:T(:f ACGATTAATTTCCAGTTTG ________________________________________________________________________________________________________________________________________________________________________________________________________________ {$630483} Following ication? tire resulting DNA was digested with restriction s Nilel and Notl and the CHTZCHS/Cl'LA-A scEV fusion DNA isolated by gel ophoresis. Human lgG, Cl-iZCl-i3/0KT3 scEV fusions were prepared by combining "3% ofreaetion l, lO‘Z/o of reaction 3, and amplifying with primers, i’iCFOi‘NllQ and C’i‘RevNotl Following ampliﬁcation, the resulting DNA was digested with restriction enzymes Nhei and Noti and the CHQCE-iS/ClLA—d seFv fusion DNA isolated by gel electrophoresis. {$388484} To insert the CH2CH3/seFv fragments into expression vectors? the following combinations shown in Table 9 were ligated overnight with T4 DNA Ligase (lnvitrogen ind, Carlsbad, CA). Following ligation, the DNA was transformed into E. coli strain MCl on and selected for ainpiciilin resistance, DNA sequeneing identiﬁed clones containing DNA inserts encoding the t speeitic antibodies or innltispeeifie activatahle antibodies, and DNA was prepared for ian cell transfection.

Table 5‘}: Ligation reactions 1 til Nhe/Notl digested anti-Jagged (4D! lv2y) it} Edi Nhe/Notl digested human lgG, heavy chain Cl-iZCi-lEi/CTLA scFv n DNA 1 nl Nhe/N otl digested anti-Jagged (4131 lvl) 10 iii Nhe/Noti digested human lgG, heavy chain Cl—iQCI—B/OKT?) seFv fusion DNA 1 gal Nhe/Notl digested 5342—1204—4Dl iv? 10 ui Nhe/Noti digested human IgG, heavy chain ‘lS/CTLA scFv fusion DNA l til tl digested 5342—1204413} iv? 10 gal Nhe/Noti digested hurnan IgG, heavy chain CHECH3/OKT3 scFv fusion DNA 1 gal Nhe/Notl digested anti—EGER (C2256) it) ui Nhe/Noti digested human IgG, heavy chain CliZCi‘lS/CTLA scFv fusion DNA l til Nhe/Notl digested anti—EGFR (C225v5) 10 gal Nhe/Noti ed hurnan IgG, heavy chain CHECH3/OKT3 scFv fusion DNA "rinnssinsi'a;s;is;{at"3'as2t:r:5ait:i5::":"§;¥§""" "i'53"iiiiiiiéﬂiéiiﬁlééiiéiiiiiiiiiéiiiigii""""""" CH2CH3/C'1‘LA seFv fusion DNA "'i"'ﬂi'ﬁii'élﬁ§iiiiialsotartan1204(136 "'iii'ii,i'iiiiEEili75ii'iiigééiéii"iiﬁiii§iiii§i§""""""" CH2CH3/0K’i‘3 SCFV fusion DNA EXAI‘VD’LE 2: i’rndnetion of Mnltispeciﬁe Antibodies and Multispeciﬁc Activatahle Antibodies Eiititidﬂﬁ} Fully human lgGs were expressed from ently tr‘ansi‘eeted HEK~293 cells: nsfeetion with distinct heavy chain and ii ght chain expression vectors, shown in Table 10 enabled the expression of ntultispeeitie activatable antibodies.

Table 10 'l‘ransfection Light chain vector Heavy chain vector number GFR CZZSvS LC C225v5-{3KT3 BC, 2 2Q4—CZZSVS LC CZESVS—OK'I‘S HC .....................................................................................................................................................................................................§ C225v5 LC CZZSVS—CTLA no IZZSVS—CTLA HC anti—Jagged ilDl lv2 LC ilDl ﬁlT3 HC 204-4Dl lv‘Z LC dill lel-OK'I‘S HC """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""2i inhabitant" ....................................................................................................................................................................................................4 + , 5342—l 204l~4ll2§l lv2 LC le lVZ—CTLA llC : Ellillldtltl} Multispeeifle antibodies and n'rultispeeifie aetivatable antibodies expressed in HEKQQS cells were puriﬁed by Protein A chromatography. {999497} BirzdingAssays: As shown in Figure ll, ELlSA-vbinding experiments ed that anti—Jagged»»»»»»CTLA—él and anti~lagged»»»»»»QKTES multispeeilie antibodies and intiltispeeiﬁe table antibodies bound human lagged l9 and anti—EGFR—C’l’lA4 and anti-EGFR------Oléfl‘?) niultispeeiﬁe antibodies and multispeeitic activatable antibodies speciﬁcally bound human EGFR.

Ellillldtm} Human Jagged l—Fe (nan Systems; Cat # l277—l64350) and human EGFR (nan Systems? Cat # 344—EvalﬁG) were adsorbed to different wells of a 96~well ELlSA plate. Purified anti-Jagged------CTLA and anti-lagged-------OKT’}, anti-EGE'R------C'l‘LA or anti-- »»»»OKT3 antibodies were applied to the plate and allowed to bind. Bound antibody was visualized with an anti—buntan lgG~HRP ate (Fab speciﬁc, Sigma, St Louis, MO; Cat #- All293--llvlL) and developed with the ehromogenie substrate 'l'MB.

Elllllléllhlj As shown in Figures 12A and l2B, ELISA—binding experiments revealed that antidagged—CTLA—ll and GFR—‘TTLAd speciﬁcally bind both human and mouse C'l‘LA—zl- (the anti—human C'l'l'..A~-4 antibody crossreaets with both mouse and human CTLA-d): Either huinan C'l‘LA-d (R851) Systems; Cat # 325--C'l‘~200/CF) or mouse C’l'LAn 4 (R841) Systems, Cat # 434—CT—200/{3F} were adsorbed to the wells of separate l ELISA . Puriﬁed anti—Jagged 4Dl lVZ—CTLA—d, antidagged aetivatable antibody 5342—1201l—r-l1'3llv2—CTLAJL anti~EGFR C225V5—CTLA -4, or aetivatable antibody 3954~1204—(3225v5 »»»»»»CTLA—L‘l were applied to the plate and allowed to bind. Bound antibody was visualized with an anti~lruinan lgG—HRP conjugate (Fab speciﬁc, Sigma, St Louis, MO; Cat # A0293-li‘vil...) and developed with the ebrornogenie substrate TMB.

Speciﬁcity of binding was demonstrated by the in ability of antibody—OKT3 fusions? specific for human CD38, to bind.

EXAMPLE 3: Cliff» binding to CD38 {ddtldltl} As shown in Figure l3, binding ments revealed that anti—EGFR intiltispeeiﬁe activatable antibody 3954—1204~C225v5—OKT3 and anti—Jagged inultispeeifie activatable antibody 5342-l204-4Dl ly‘Z-------OKTB specifically bind human CD38. Human CD38 (Novol’rotein, Can‘t {3578) was adsorbed to the wells of a 96~weli ELISA plate. Puriﬁed anti—EGFR innitispeciiic aotivatable dy 3954—l Edd—CZZSVS— CTLA—d, anti—EGFR multis eeitie table antibody 3954—1204~C225v5—OKT3, anti— Jagged multispeciﬁc aetivatable antibody 5342n12tl4-4Dl lVZ------C'llA-d, or anti-Jagged multisnecitic aetiva‘table antibody 5342—1204—le 1V2»»»»»»QKTES was applied to the plate and allowed to bind. Bound antibody was Visualized with an anti—human lgG—HRP conjugate (Fab specific: Sigma, St Louis" MO; Cat # At)293--lML) and developed with the elu‘omogenie ate TMB.

EXAMPLE 4: se activation of inultisneeifie aetivatable antibodies.

Activation {Mile-til} peeifie antibodies and niuitispeeiﬁc aetivatable antibodies were d in PBS to a final concentration of 0.8 ing/ini. Recombinant human uPA (R851) Systems, Catalog l31tl~SE) was added to a final conceinration off/’00 nM and incubated at 37%? for ~29 be Digest aliquots were removed and prepared for SDS—PAGE analysis as described below, and diluted to ltltl nM in PBS; 015% 'l‘weenZO and 10 nig/rnL bovine serum in for analysis in binding ELISA assays.

{Mill-412} For PAGE, the samples were denatured at 70°C for it) min in in LBS sample buffer and TH??? was added to a final concentration of 40 mM before ophoresis. Six gig of antibody was loaded onto a. Nul’AGE 10% Bis-fins gel (lnyitrogen) and proteins were separated by size using the MOPS electrophoresis buffer.

Following electrophoresis the gel was stain with Coomassie blue and the results are shown in Figure l4. The change in mobility of anti-EGFR activatabie antibody 3954 , anti—EGER inultispeeiiic activatable dy 3954~1204—(3225V5 »»»»»»(ILA—4, anti— Jagged aotivatable antibody 5342—l 204—4Dl lVZ, and anti—Jagged innitispecii‘ic aotivatable antibody 534242044131va»»»»»»CTLA~4 light chains, in the presence ofuPA, demonstrate protei‘ilytic activation of the inuitispeeitic activatable antibodies, The lack of any change in the mobility of the heavy chain fusions demonstrates the resistance to se ge, Bindin ‘ Assa s {titltldlﬂii Panel A in Figure l5 shows that GFR multispecil‘ic activatahle antiht’idy 3954—l204~€3225v5—CTi_JA—4 bound to EGFR, hy ELiSA, with lower affinity (Kd = 12.8 nM) as compared to C225v§ (3 nM) or to C225v5—CTIA -4, (9.33 nM). However, once activated by uPA, anti-EGFR multispecific activatable antibody 3954-—12tl4--CZZSVS ------- ‘l bound to EGFR with similar affinity (0.45 nM) as compared to % (0.21 nM) and (IZZSVS—CTLAd (0.33 . importantly uPA did not cleave the anti—CTRAd scFv fused to the carbonyl terminus of the heavy chain: As rated in panel B of Figure 15, uPA ent had no effect ofbinding to human CTLAd. {9994M} Similarly, anti—Jagged multispecific activatahle antibody 53424204- 4135} le—C'l'LAA bound to human Jagged l, by ELISA, with lower affinity (15 nM) as compared to 4101 NZ. (0.44 nM) or -------CTLA4 (0.77 nM). Once activated, antit- Jagged multispecifie activatahle antibody 5342—1204—4131 iv?»»»»»»CTLA~4 bound to human Jagged l, with similar affinity as 4131 iv]: {0.5-4 nM) and 41M lv2—CTLA4 (0.92 nM) (Figure 15, panel C). uPA ent had no effect on C'l’LAd binding (Figure 15, panel D).

ELISA binding measurements were performed as follows. Human Jagged l—Fc (R8410 Systems; Cat it 127748—050), human EGFR (R85) Systems, Catit344—ER—05tl) or human C'i‘LAr-t (R8513 Systems; Cat # 325—C’f‘—ZQO/CF) were adsorbed onto wells of a 96—well ELISA plate. Three-fold dilutions, starting at Hit} nlvl, of untreated and uPA treated antibodies, multispecific antibodies or muitispeciﬁc activatahle antibodies were applied to the plate and allowed to associate with plate—hound antigen for l hour. Following binding, the bound antibody was visualized with an anti—human lgGvHR 3 conjugate (Fab specific, Sigma, St Louis, MO; Cat ti Atl293~lML) and developed with the ogenic substrate EXAMPLE 5: Additional multispecific antibody and multispecific activatahle antibody sequences {hhhdiﬁ} This e es the sequences of additional multispecitic antibodies and niultispecific activatabie antibodies. dy QKTBm SCFV ""1NO AC.3113 SEQLEII12E(2:; "VALAPS5‘1)» 1I31A1 :‘I1I.1A.RFG'A.SV7I~C1‘I11 CICASCYTFTRVTMEWVLCILI‘GQI. I..1F1WIGYTLII'SRGYTNYN1 LI1111Dru7 77‘1'r1'r mm1 E1.1.1 SS .iTIEDS. .VY717C1A1R7X7YI1‘DIIY S I..1D1:} G GGGGSGGG 35,1T‘7I1TCSFATV1SASPGF.1K'\17TIVITCSASSSE’7SYMNIWYI.‘IQKSGTSPKRWTYI‘ TSET.1A1 GVFAEFRGSG1 GTS7.7'3T1TTS GMEA.1F1DA1A.1T7‘X7YCQQWSSFNFT' FGSI’TKLFTNR (SEQ ID EC): :06} NUCLEIJT.1..

CA’113TTCAGI‘T G'CAAA1'\/‘:7A7"» IK‘JCZ' .JL/AGAA.1K11'1.IL7"rpm »ﬁm'\ .1A—1," ,-1. ,«r— ,«r—m U-"mmT'nT'nFTIN’A :1 11A1L,\3T\-\A1L111 I3CA1AI.1.3 ,IL71'1. .1.Ahdh. L71". "3CT'13TAAAGI.1.1A.G'C 13TTATACITTTACCCIL3TT1A1T1A1CCA' CI1‘TTI3GI3TT1.1A1A1CA3C3TCC‘El ;'\r-','\r1.,'\7\,'\( m m "r7\11r,'\1.r717\m7\mﬁ7 mm a,«7\,r\r'vr\/".1/" mm’nm’n "—1 7k..\7. \/1L/’\.I\7JI.3 7 . :IIL71.1ATL117J1T 3L7.""23 J. "AAA. 1c 7d 7L,\/'1.1.T'1.1. 7i. i211.' tick/1A1.

GAGAAATTCAA.1AG'ATAAAAGCAZ‘CCCTC.31.A.1CCAC'L"GATA ACI‘AaCAGL11C'L31'C'.AIA’GI {-17,-1vf-1—‘1r. Z71 -'\mmm7 ’\ «..'M.

Hub.7r"\r1'\"\'\([L7A’L7I1711A1I3CK1TIL71A1K1'L1TL1.\'131.L‘1'1.1.’L7A. AGI: (SICK/191L711.ﬁmﬁﬁ’ r". w .. Wm mm". .7'13A'.r'7 .'\"\IL71".

... TAIL CAL. L7. GI...-1A1I1..-GIL.-1LA.J..1.A'7 ‘IAJA1GCCT .'G1A.1T'7.7'AT .'GGGG'. CAGGGCACC’AC’CCTGACCGTIIL"1I.3CA.G'C.1'L3'." ,«r1_.—\~._~—11—11.—1m.—1r1.I\7I.3' I3GC .’A—1L’A'L713'L/13'L7i. GI.3CIL7G'..ﬁr‘ ,HV- I‘m "177-" .q—1yq—1_.r1~.a1:—17/1717. [\7. I7.- I31..1I: C AJJA {FLA/L713111—1 .nﬁ 1 _’1'\/‘:'\/-‘ 7&3 .A'7. LIIL7'13T'. 1.Ab IVA ,n—xv.__.—-\_CAZ‘LI .A. 1I3CIL7CA.1..1IL.7I—1.—\ ijrtfﬁr'l"7'13'L7. [\7A1L‘1AAAGTTACKHA1I."1:z.\'11'L1.'\7\7 m .m—1_ {-17 {‘1'\1_'\._L7. 1A1I.3CI.3CI:.1.LA.IL7—1 —1 V" 7A.'\:z\/Aa f";'\",4'\/‘"‘IT L71, 7K1'L7. .1 TAGCTATATGAAT_GC AA 1 .GC7 GAAA GCGGCAC’TL'3CCCGAA C-1TTGGATTTAT13AT A11111A111A7\/7r\7\r‘r‘7MA CTG1.1 GLCAAGGG3‘ CTTC11-1L1CACA1T1T'3GTGGTAIwC-1GTAGGCACCTCA TAGCCTGACC-T1AGC"3GTATGGAJAGCAL3A'AGATGCA77/771.1AA'11- 1A1 1A1 L"\r‘m mm mmﬁmrx 1"\/"7 rv1nmug-1'13 L'! L3' CAAGCAAT"'TCTTTALNTTTI~11T 3TGGCA3CAAACTGGAAATTA1ATCGT (SEQ ID NO : 3 O 7 ) Antibody QKT3m scFv Lv AM]NO ACID SEC7)UENCI7'. i; I‘v777.] T'QS '.C'SAS E S‘1'S’]\’I\I‘~I‘I"Q " "n 0 f1\ r'r\" 3111\3 V." ., TGSGSE, SGM I7'AI7'DZA11L‘L'IY :{CEjQIAS TCSC'IﬁLE: H1 (SEQ ID NO: N C 7.1EO’T 1" -,A7A 1 1 1:C‘JAIJAIJ.V 1‘ ,L\-, ,n.- zrxlr- f\r' r"\’\ T3CGC’1 ..‘11G'7L1'C'IG173GTG2\Z " I7GT'IACCA \L/l 1C1: .. L,'1_L7'_7L,Z-\11..

TGAIC'IGTAGCGC"AGCA.GCAGI G'I '"G73’1'151T51TI3Z[TGC'IC2\G7‘7\L3ZAAACCGCCPxI CAGIC-ICALXACGTTGI‘A"I‘TTAT(3A'7I1L - ~‘1—1]11Z13.1\L1L_\L,/311L\17L,17 7«mp/3n, 1'1- err' Vrp «Em 11.003‘1 7:".."u'1.I I CZ-‘7 7 C/31'I.«7 xv, I. .I 1111L\1I13I,I131-1 1..-1 ‘11 _ 7 1 3C 1' ".TAGCC 1 . . GG’I'A’I'GGAAI AG: 'I‘ I" 'I‘ 11303!) 3 «1 «pp;1—1 m 1123‘: vrx/‘Vv/ﬁ. fry-1a. n1 I -'ij-V_Zl-L'L, Al iiii iLJTCJAL’J'VJf-IIJTI'JCmmmmmn. ml ..

I LAZALJCAATC171p mm 1mmﬁmnﬂ1~1 12*1/‘17-1 J J'VJTTTAL/IV i i iGGiZﬁLJJLLJ'VJCJI_LJ'VJZI-‘1Zl-\_ ACTIJC_AATTJAI1Z‘1TCGT (SE. D\.1 NO: dy CDKTBm SCFV HV AMINO AC,VTIJ SECLTEI CE IIDVQr1." SICJZ‘ JARPGASVKITS CKASGY'I' .‘T'PYII'IVIPII‘VKI'I1P’:QC:IL1EN3 GYTI‘IIQCSRGYI‘IxIYI1I ILIVJ_IJ"1I’I'1,.I 7 V/ 141""- . Y1'IL‘Z1IQYYI: 1. II'\7853 N C DEC." LEIICE'J 1r1- V «11-. V-n ,.V-, ppm». , r11 . —..-.-1—. V ,VrV. 1pm- 1I. J'_I3C.2-IGI.,Z'I1\JZI1\J'L \J . k/Z" C31,2-\G2—AI 1G 1ILI .I1C,L1 .L.C,'1_ CG': ZI1Z-I1ZI12-I1'III'GZI.

.Z\I12IVI 3321GI‘GI'I' 'II.‘Z31'IIZI1CC: II'III’I'ZICI3CGT Z3 1CCDE .11-1J.I._11I._1V «111m 5.121 21113—17 f\f‘i,v’\|‘ .rxlr‘.,Z1\J'C\J.\,1_ GGG'. ’I’VIGGII‘CIIIG "GGLA'I I.‘GG'."I I'ZII'ZII'I 1.1'IC'."CGZ1CCVCC 3G.' 122'J. 2-\\CILrm1-rn11-rn VIZ—I 'nr‘t. 1-, {‘7VII ZI12L\'1_1', CZI1GZ-\.Z‘1Z-\ I'IIIC,2\.Z. 251GII1'I'1-1Z1AC CZI1Z‘1CV CC: ’IJGJAII: ‘G2I1'II'Z‘1Z31AZ1GCV1I11:C,2\ VII.3Z\C3 CG CIL"I .I'IGCZ‘ AGC'. ’I’\’I.VI\GC,I 'II'G "’VIICVALII1C:CII' 1GTIII'I I'IITZI1C'. "C'."I VII.3Z\C3 VI3'IIII'II'III’IIG.. .1 2-3 .1. Z1 ITCACZ T'Z-\.T'2I\. VCCIIIGGZ‘IIIIIIIA'I".ICV:‘GC:G'.IIC2"\1GGIL‘ ..1IL‘C2"1CCC'I"V Z‘1CV CGT'IIIJAGC,2\ (SEQ ID NO: 31. i]. ) Antibody GETSm SCEIV w/ long Iinker t0 aIIOW attachment to the C—terminus of Ciﬂfaer the henw chain (BC) or ﬁght chain (LC) of an antibody or activatabie antibody to produce a, multispeciﬁc dy 01‘ activatable antibody, respectively "VIE'D Z-\C..y-< ICEI.\ICE GIJKJSKJG'-1,«rV, 1, ,V,- V.V QGIJ. —V .-.’H.J\3\.JE,\.J\3\.JDI/ 'QI,.V_,«,.V V,.V, ,V_, -,, -v~,«.,CNIQE‘.G2YIII .'1JZI1I‘9. L’IJZ'I1J\/Jﬁ..'1‘/IEJIV" - V-,.V L. V1111 .. -n (1 V r11 ; 7 11.. .SGJ. 1. 1'.I\Y.I'.I'I.YIIIVVIK CDRPGCDGIJEIII I: G IISRCY‘ZI'.I'II.\I YI\"D :I:\_Z"1 A ITI' .1 I'IDK S S SI.‘.Z-\.Y. IQIJS E‘. JT SE IDSZI1VYYLIVA IV‘Y' ].SY IJ CJG'I'II' .I‘I'ISGC:C’:SC: 1G 1E GG 1GSC‘ IVI "IIICDS PA I.I.V.IE\IJAE\IPGI K‘v’ "III" "Ci-SEAS KIWI."Ci). [VTODKRIIYLISVv- Tr‘xﬁf‘xZI—I..7171r1r‘.I:l‘;\CJC.i.G.L P1.,Q1L‘J'C,J"1A 1'11’0'13. T1;..1"AI: Cm1’1 m W113 —1 '\r"7’\ "r‘ﬁr‘r‘u Em m3 ﬁr';'\r-'. .' I ZL‘1 JZ‘1I: CA TJCAJL 'G.37 mm '"r .'I. .717 . 1 P1 J'C,L,'\.. r‘xl—G‘ p.7r1—Vr1r7r..'1_.CI.:JGI.:JJLKJP1.L1C 1T ,T—V ﬁmﬁa .—V .1," "A _,rV,-17—1_r.,-jCJJ.‘ZI—\. 1mm "—me r« m . ﬂr—Vqrm 7—1 .'13. 321i. I :IIJJL l 11.11% .'C,.‘ZI—\.T'1_IL,I.:JZA.1I.JCI:GI GGJLTA .'A"1—1 ,' .‘ TAAAGCHCCCTGAC,'CZACCGZATZAAIAGCAGCAEGACTGIC TATATJCAGCTGAGCATJCGTGACGTCACACC-TA JGGCJL_TTTATTACTCTCLAGLJLJTAT TATCJATCJATCACTATACTJC ATTGGGGTCAJGGGLCCTTCCTGCCJGTTAGCAGCG LJTC. ,«m—FC,‘Z'L’J‘ 'T‘ JLJTLJ'LJLJ'LJLJA V—L Aha LJ‘Jan IT! i LJALJLJC LJG1mﬁr— 'T‘ T—Y—J T—‘ry 7\ «rum mm Tmm. T—J «Tn—‘7‘ JLJLJLJ'LJ ALJMV JALTJAi JL.L TLJALJLJLJA GAL-’7JTLJMVLJ -JLCAA i JLA LJ LJLJLJLJAF""\ﬂ" mm mr‘ rxm‘x/‘wr a. *x LJi G."AAAAALJTTALLle‘liLT-JACLJi (‘_J.JLJLJLJLJLJ/ LJ‘\-V'T‘ ‘7‘ 17m .. r‘ﬁﬁmp ""11 IT! an"V/‘V "F‘ GTTALJ’CTATATGATTIJLJTACAGLTALJAAAAGTJGGCACLJAGCCJ'CGAAACLJT 'GGA TTTATGATAC TJ'AGCAAACJ' GC .JGTGTTCCJ'GGCJL-L'JATTTTCJ' .JT GLGTAGCJ' JGTALTG'LTCf7) GL-CCTIV.7 TATAGCCCTJALJCATTAGCG .JT TGGAACJCAGA‘L_GATGCALJCA CCTATTAT' GT G L-GIV.AGT J TJ'TCJ'AAJCAATCGGTTTACCTTTGG 1 AGT'TJ J'TJLCCAAACTGG AAT1AA'L CTTr. 1 Antibody OKTBm SCFV with," t Einker t0 allaw attachment to the N—tetminus ﬁfths: E-iC of an antibody 01" mbie antibody to produce a. multispeciﬁc antibOdy or activatabie antibody respectively AMTITC‘ ACTD SEQUENCE LRYTM I'NVKCRPGQGLENTGYTE PSRG 7TTNYN .‘3A7L771’YCJ'ARYYDDHYSJLDYWG’QGTTL 'VSL’GGG LJCHJLJ‘JLJLD LJGLJGSQJ XVTMTCSA-SSSVSYETJIVYQQI S JTCSPKRVTYD TSELASGVPAIIFRCECSJTSYSLTTTJGMEAEDAAT1Y'TJL"EWSSUPFTFGSGTKTITNRGGG JTT E SEQUENCE CAGGTTVAGCTGCAGCAGAGGGGTGGAGJTACTCLJTJ- »GGGGGTGCAAGCCTTALAATG7 TACCTT T17"CCC "TTAT7CC Jmmm»Lsz’ IJLJ JiC. 1J1 J1 ~er mm -GAA'|J'LJ1\'T'71, . JLJL J.V" m» m J1, J .TTJMJ‘J my"; J~ "mm mm JVT, J may J" D J~.

IV-I 'L3\Ji' 1131131 CANAL .anAxVJLTLJ'L-J G - 1 1111A .LJAALJTACAJLJLJ J" n, J J D J J" J» JV" DIJJJLIJJTVJD A, JVMJ J» 7» ppm 131%31XAAL C11A11L31111»11»‘J1»_L3LJ111‘1LVLJLV1 L311LJ 11GbVD .T.» -1»111»_1»‘J;_11\;I‘LV1‘1L3 .T.» J11 AGCTI‘ CAGCCTGACC‘ "TATAG 3117171»;7" -7JC JCAGTTTL "7 TCAGATA GGAT T1\'T'77GIC-7IGIC-7I'C1"\T -7JGGC71CC.19TC -'77G(‘.-T' GG: "AG-7 ‘GGTG -7JC-7GTGG'7 '7 CACGCGCT7GCG7GT71GCCAGA'TTGTT T’T TAT7CAGACTCCCG CAAT TAT TATCGTAAGT CCGGGTGAAAAAGT"1‘1" C‘ATLJAT7TH".JTALJGC TC T’AG’7AGCAGCGT '7717‘GC: "ATAT TAATTTGTATCAGL’7AL7-7I1‘JA17‘J1»TGC’7GTAT.CAGCCLTGAAAC TTTT7GAT T 'TA"GAT ALJLJACI‘LJL‘uuh» an? pp." » J Am 1‘1'L31V'L3‘JVJTJ-(Jw-(J‘rwwnnlran ’1 \V'I'T lGLJL3L3LJI1LJAi 1 1 1 ~JJ ' 7 m J ~J~T (3«"017» JJJJJJJJDH-EV1»‘J‘V'L.Jl'LJ1‘1T i C3L3LJ1 r~J.TJr:J.Ter—<. JArwi 'L3Uf'1‘1L3LLJGLA J ~ «n LATHLJLJLJn.~ JGD ALL141 JACJ'LJ‘VJLJlJ "mm? J‘ x J.~r\*x D » WT WT 'L3\VI'1XL‘1L3CJ11L311AC3A17 «7 1'}erer JVJD , ﬁrm» mm D "1"." «ma-LJ_r»‘Jn'L.J1‘11XL/LVJ1»‘JL iAi YCJYLJAC(J_. L)a(1-) JJTLJAL‘ L3LJ1‘XALC‘VL31 1 11"(71‘ C 'T'T'7 7GGTAGTGGCACC7"AAC’ "GIG/AA’T‘TAATTGTTGAGG "GIG-Tf‘l’ :ra7 >~\ .»~r~ ~\ ['4 ‘1 ’1/‘(I’Y7‘ITH'Y7‘ 7 GGA 'CC (SE ID NG: 293) Multispeciﬁc antibody CZZSVS OKT3m—IEN ha:Ivy chain (antibody OKTBm attached to this N~terminus of antibody C225V5 EEC) LD’LEDO ACID CE CQS EAET ARDGASVKD’ESCKASGYTFTPYTME'NVKCRPCQCLEDLGYINPSRGYTNYL QKFKCKATT . TAYD’E‘DLSSLTSEDSAVYYCARYYDDHYSLDYWGQGTTL '"ZS. GGG IV/LTC $1.123-VL\7" WTKMWlr1§l.ACI/7\ (‘00 0-30 1 L7l77 GD’EE77'Z7‘3L 7 YEYCQQW SNPE'TE'GSGTKLE'IN TIA-(AV,...LICK:Gx GSCVCLI‘LQS GPGL"ZE’QSQSL8.7.7'ZLC7 7x773 ALGL'I'NYI’\IDWVEQSPC TE Y NIE7777ESSLSL7L7YD317KS Q7327E7E7I SLQ’J'WD" EY‘L’C2RAL717YYEAYE7I7E'Z93L JPN/SAL 3LG7C7JVKD‘'7E'IL'I‘V7177\I7SEI‘17D177GGALTS "VEE'I'FL 293‘\,I7Z..’7:S 8 GL VTVPSSQLGTWTYICIVEES FLEEDKDLDLLMLSRTLLV‘CVVVL ‘D vvvaLVLDQDWLDQKQYKCK‘5LI.L\..: r: «373 ,«rV.J/kajy big G'Z 7CZ-\.AZ‘3G2C29.Z3777CIA7J177E ACC717T ]":G'[A3G71'G 7C3I77C29 "77.777:CZ\.A3Z\.71'7.77Z\7777'I7G777GGZ33GC 1AV rm- 1 . GUI GGZ‘3'ZE7CC77 C27IGG'3'GCI73G Z7GZ33Z3.Z33CZ3GZ7’3GI777.3GGCI7JCIT3 1GCC'Z 'GC GCAILG773CGZ3GCI7JZ3AGZ33GC r\.- p -\A. \_j.£7'\3 GCAZ‘3'7I ]..Z\CI77.377Z'GC293 CA77 77.E.7'GIZ3.GCGG'C77 7'29\.GI.7.773C7.E.77 293CI7JAZ3C7.7Z37]Z'C:2GC77 G7E7GI7.377AH G AG'Z 'GC ,nAV rx \JL \/ 2’3 GZ73GC77 CC77GG 1I7 ..293Z3G71 7JC7.7’7:GZ33Z‘3'7'GC:2C7.E7GG7 I7747E7 4293'?71.7'Z GCAGCGG77GGC2IZ 7JCGZ71.7'Z[' Z‘3'7.[' Z73.Z3A\,.Z7-3.\,r\ rNr' V n AV f‘f‘i/"f‘if‘r r\' r ' CCIJCC. \J.£7'\A\J» I' 2-3 AI7J A293AGZ3 ‘7‘ 17.3273"\ .3 . C2\.Z\2\.Z\GC7ZI 17 'Z[7 Z' T TTAAAATGAACJAGCCTCJCA.7AAGCCAG.JA.JJACC LJCJATTTATTATTGLJ .JCJ .JC CGCGCTCJ: C TATTATGATTATJAATTTGCGTATTG GCC-7‘GGGGAGCCT'GCJTG.CCJ -JTGAGCGCGCJLJ ACJ GC JCA1 CGGTCTTCCC CJTGGCJA CCC‘ 'CC' CCAAA GCACCTCTCGCJGCJCJA C -GCGGC CJTGGGCTGCGTLJCTGAAGJJA .TACTTCCGCGAJAG'GLJGTLJACJ .JGTGTCG' GCAA GTCAGGC .JLCJCCJTGAC GCGCJLJLJTJJLJALJAG'GTTC'CCGCJG GTCCT CJAGTCCTC’-GGAC 'C TAC 'I 1 A A A A J I CCCTCJAGCALJCG I J GCJ 'CJACCGTGCJC JTCJCAGCACG‘JT I I J 'GGGLJALJCLJAGACLJTALJA LJT "I 7 f"7I "‘1- CCAz-‘LJHTGJ1ATLJP.C'AAL- JLJ'LJ/-'L’J‘L-1'-\_LJI"\n 1‘1"" ‘7‘ rvH'x/‘J'x "mp; "\ CCJ1Z‘1 ﬁrJ—Ir" 7\/‘J7 '5 ‘rﬂ'ﬁmrﬂ JL’JJLL’J 31-1I-‘1'7J1-‘1/-'L3A1‘-‘11"‘_L3i TGJAJLGCC CAAA 7.‘ 'LJT'T‘: J. .n GACAAAALJTLJALJACJATLJJLJLJLJA1LJLJTGLJCLJAGLJJALJCJTCJAACA A .1 .1 . J «rum/3. mmrx .. 7 LJ- 1 Gﬁr‘mp GGGACJJ L.JGJTLJJALJ"#1 J/‘J7r' JrJ .JJTTLJLC-TTAF‘ r1 m "I" mm 7/3»'~1/‘1‘r"\" "cmm‘ﬂm' 7J ’LJ-LJLJG"\ﬂﬁrﬁ ' LJALJGL-TLJJALJATmC‘‘7‘ r1. "77/77, = CJCLJLJCJLJJAAAALJ'JLJAALJCJAACACLJ .LJJJLJAJ JJA GCGTGGTGLJ-GGACJG1CAC'GCACGAAGACCJCTGAGGTCAACJTTCA CJTLJGTAC'LTGCAGCC Irerr. r , *\ ~r1~~ m7 r‘ rT’ 1*" ‘\ 'L.'3 \ L31‘1'IL3 1i vL-EZEJL 11"yIuJL/‘JI’AIJLJA.:111Y19‘1C3CJL-J9 ‘V‘ﬁ 7r r77X EC m (7:77-— 7I’7J-IrJC7 (777p. (777:7I7r77 n 'L3 L;.3 ETC. .JJ. 3T".J'LJ YCJL311L3L31A7~J~ A, 1‘113'L;'IT'IL. TLCAALJA1‘.AGLAJLJELJ-JCAGLJCC7 J7 ..7 ~ A 7 7J7r7r7. ..~1I r777. 7~J "'T‘r’ (37,771 7» 7 r7I..~.7r-.r7m 777»11TLJECJ 111.11G7 7/7777» J1J‘.- 1J\..rJ‘JJJ--rJJa_J 17;: ,7r7r7r7I77r77 7 W7 "71/77 5 (JG) .»1_EJ/9‘ LJILJ'ILJI-L3LECLJ7 7/73,? 7 JCI'7/7AJTJ LJ'L.‘.J'L313L31‘1IL31-J1J‘.C31J.i7r7r7J7r7I~7I7r7 ,1 7» ,7»J»77\ 77,7».1J\.A1J-rJ‘JJJ-'L.A_J1JY\J7 (77» '11L; J7r77 r7 'L3.J'LJLJ'LJ\-J'1J G1JL-J.11L..’J .J 7 I- 11 I- -1(J .

(«I ‘."D:"r‘r- .l C-'777,707.".71,' -‘.1-CLCAGCGACAELJG J7 7 7 7» 7» .J7~ 7A'C77,77 "77,777 7 7 7. ..7» 'L3 LJLl'L-Ik/ - 1G -1\. - EGGGAGA -3111:1 TCA9’» TGCT‘ CC J‘JTLJ-‘JI/"1'T'GCJJ-'TGAGG" -CTC"GC1‘JGAAGCAC TAGAGCJGPGAAGEJGCG'E‘GNTCCC'E GT Muitispecihc a111ib0dyC22515QKTBm—H—C heavy chain (antibody QKTEEm ed to EEK: Lterminus of antibody C225/5 EEC) A. E9 E9) I I9] ELL) AC II'IEEEIEI CF92 EJKIQSGIPGIEIJYZQJ"S9 J E9 '9EI'C'E‘J’SGE‘CL'E'YIIII GY’ "IE/"E\RIECS1 PL} IEIJEEEWEJ JV 9EJAE‘13GE‘E'9I'DYEYE'9E" KIDNEJKEJ("I’EILIK'V' .ESEJQ. Si,3I.ETZ.\9E9 II‘17C2‘1IELAE9J'ETYY MJVW ;J":)G JZI'9I'EYVSAA S'9E J.I_JI‘.’ KLJC.I..PSJYI’JE' PE9JJZ.9 EJGC 9J\JI’ KID‘.’E9'E9‘I9‘ 9‘\/’I'."/’Lj‘1/JEE‘ESGI’-\I 9.E.SJSI\/YI"9"‘\JI’ 93E EJA'E.‘ ‘TYEE‘EY’. EE"PSEL'E'K‘\J’EIKK‘IL"‘PKSJCDJE'E-ETE‘CPIPCGIPAPEEE JCCPLS‘LI’I'I‘I‘EJ 1":"[)/[)IIL)I'EV MT (3‘. TYPE-'1‘ZI’EE'TV‘Vn . .0 .

L\_ DPI'I‘9J9‘JZE\E"\E"‘EVZUC‘L’T.\Z'EELIAKTKPPE "'EY IRYI’IV JY"[J[\J]JJ] )DEAELJ-EGE’EI‘YKCIK‘7SJNKAEJPAP 1I"."E9'EJQPPEJE9 Irv—HI.n—LD . -, m n31, .. «ﬂq.—LAJ,1I-J7JmL3 -ALAL3iAiGL3'L3L3 L3\-AATIGLJGIGL3«pH—L «33-3 3—1 n 7m ‘-L3'In -L3'L3L3\-I.—L3—J—L3‘ L3'L3 T‘.‘:L3AL3CLJim‘LJAGLJGL’uT-I‘T -L3‘\-L3 L3'L2L3\-AAAGLJCL3 i GLJAA"Vr' "F‘ ‘\ "I "HT! "‘7‘ J ‘x ﬁn "F‘"m3—, "\F‘ L3'L2L3 L3\.'.'L3\-L3 i rpmrJ—Ir‘ In i I LJGA JL3'L2L3 'L3L3\.'.'L3A.ALCALCL3 -JA.i I A AAL3("‘(Ir'"V/‘V {*an mmmn ‘ L.3 CCGTTTACCAGCC "ALJLAITTAACL. AGAIAA.’AC"TLAAACCCACGTCTTTTTT‘AAA Jr"r‘\"\7\"‘ mﬁ/‘VH’K r11" mmm‘nmrJ—I7 .mﬁ "\n m .7.. ."\ 'L AAL3AL3CL3T Jlﬂ‘ﬂLJAAALJ'L3L3 LJIJATA‘-’L3 -JL3 -JAL i i iAi iATTLJLJﬁ.:'L3\.:'L3 -JL3 -JL3 -JCIGA'L3L3 ATTA.

ATTATCJL.ATTTCJGTATTSGCICCCALJCJJ'CACLJCTGGTGACCGTGAGC.JCGGCTAG"TZ-CC Ir\J~r~I.—~.C7JITCGG J-rJ CIICLJLL~JJTJ3W » L: 3x» I.»-3L»3 3L~J,1»J~J,1»J»7'L3L3L3L3L «31 «3‘» 3x» ..‘L.3 L-JJ. >-L3L.2IL3L.3iL.3i 3«3LI,.-.3L 'i..l\.2i..l\3"-/\3‘J' "I'rllr‘g\ 7. (3r: T’ICr aflcriwrn NCCI’[__ IL TGTC LITAGIXAL3x.113‘[\GCJ It" (1 3‘: F‘. 1~J3 ‘ CC L'J'u‘u' :Cr. '1." JL-3CL-3 T CCTCAGGAC'I‘ CTAC’ ’IL.2IVL./J'CJIG J'I"L. I..~.D 3L L3 J 7 32 ISL-3'3 L3L»J3L3~. L.‘L."I"L.'C2\.V1L.AGL’ 2 IGG|1LZIL.L.L.2«L32IL.C'2IC2IIL... .- L 3 33mm. L L3L L I..~.3LI..~.D 317 LJJJ MC. 7 L3 7(3-.‘2IA'L.J-JI3L3L 'I'GAATI‘CACA" CA «.G -VJT -VJG2I'."2IAGAAAG 2‘2\.2‘IT AA 'TC2 CA QCAVC TC'T‘GZ "-3 Tr Ir‘trNIr". ‘5‘" ‘1 '\3‘-3'\3‘-3'\3 CCCIC2".’I‘2Ir. .. .£-L,.5L1;_"L.'L."L.'L.- -VJ2.GL'3’I '."2’ICA‘IGC‘G' T G25; -JfG'I‘T."2IAV I3 I3~.3L3 3~. .3'L.L32III'L. GA J2’52'5I2II2I‘L-L 7 7 313‘" .»J3D.»J3.2\._[J_, L L3L «3‘» 3L3D3~J3DI3~.3L ‘ 3L.2‘12I2DIGCCAAIIGULJD 3 'LI_I\3l‘-/I\-2l‘-/\J\:J' 7» 3L3 DI3.3L32IC2ILn LGJTJ «JIJJJ 3 33 I L»JJJ1»J~J3~J~3L"’L3L32I'L3x32Ix32IL3L»JJ 3L3L7» 3L3 21x32I2L-L. 'L3J -rL3 I2IL.2-‘L.I.3 'L. IL-L3L.L3L.C2-- L3‘.3~3LI3~.3LI3~. .JII.~»D ~JL1JL32‘1L3CJ2I D 3-1" *n 3x317 3L3~J.»13~J» I 'AA .‘L-2I‘J3'L3I'L.2IL3L3I3~3L 1 ~DI3~.3~r.~JI~J~JrI13~J 'L.» OLA-'9‘. L-3JC, UL:NITKI’17.- ‘J.3 J‘VI7» 3L3~<3ur1 CCCZ L3‘JL.AL-L3L. JL32I Ark-2 ‘.'2-2IC'I(.22-!Ir‘ 3&3: CZICG I~JCTCCCGJIJGKJl\EnIZXIv"W J«3:3I1.113"erCT .1L3LI3~.D 3 L317 31 /~J*r>\7r*'*/> CC. 'Cl'. 2\C"J'IL3L3‘AII-I‘CWCACCGIGCL32.J‘IL31‘IL3C25IG‘3 "f' {1733'231-3 3 r‘ Vrn VrVJI'I'erJ- .3'17‘ rer3\ J'L.1\.32L\.7'L. J\L1\..JL:1L:I'A"I‘L3 1 .LL3.\.32IL_1L3 '[CC'G' ’7"I’I'L'1‘C25' SAGGC’L,"' CCVJ'LCVJZ‘LAUIL"I""C"C"’VIC1AAG.C.'3(."‘C."‘C3"‘C"‘‘C'ICCGC i'fImebl ‘TC'V'LJJ’. JG’Z'GG’I'TV '5IGG VT’V'V'CCVC’1JLL1L1.'3’I'.,JIK" VT1G'[CC.AL1L.

C'JZIigG'C'GL1"'ILJGTC 1GT’I‘CLZ" ’EC'I' GC'JZIGC25G2511V (TI'JGK'SJ' 'GC’P ’_1JZI2’\-CTGGC?CI} 3C3:Ir‘ ,'\J- "A In- 2‘\JLJ-IJ \J\_ A\ 73 f\ I31- rm ..A.2Z. ZIJFII- 'A 1 m 12-I LI—JJ-I-m‘.3.'L\.7'.'[ A/IAL1L322\L1L3L3L:I-3 3 313\ ~rnJ-I-J -JJ .11 a —L m '.‘I AI. L3'L. \Jl{"rNIr"f\l'f1hI'l'l\J.Z'3J.Z'\C‘:_/]J1I-J-JJ '1}In".1\.32II'."[\.7'L_1\.:J3L3 .JI 3L3 3L .[ UJ'x. \J .'L 5'JIC'[ I J{\GQ V"‘C"C1IV"17CAGG 3 GAAT G (3Ar" SGT'I" TA" CA.ZITCCIGA V'.‘CG'IGGTTJZI '[AC'CAJZ‘LC"’I'2\C.ACC?IGAAA''[""]‘C'JZI2’\-JZIGZLII'T‘ZI252‘I3CA ‘ICCC’ LACCVJZ‘L(.LjJZIr'5'IIIZIGCJJCCAGC TAICf-Cr-CJFZ\rV1LZ\r:1_I~I:-_:ZAI3Cr1Ir;IZ\3-CICZ_\r:II3:-r\:r[.G-I_\\CI-:1r »-~JVAL‘I‘JZI‘J'LJA. "r1 Z51.'L_1L3'L_1L32-‘L:JIJ_.L213 \‘A ~rV1hIIrVJZ rV1 CT/wwr3LZ_\C 31J3ZLJ-1'1J - 2‘51 ITI m V'nl' 'L3L3 I . .A.’_._ LJJAI 1A . J33.cm A.[AGO-Cf 16521.".1‘.‘ 3 I‘SGTCLZ‘ GIG-(3L,"25CC72IC'3CC :JACCG'I‘TJAG L1G"‘C1 253."11".‘"1 GIC’1Ci1GmGL'1": r" V CZT’1CCVCL3’I'25GC'fCAGA'i "'1I"]"'["L,"".

.I"1L,'L,C-7 n—Vﬂ'r r31 ‘TAGiC‘xz‘L’JCSIC2 m "ICC" 1:4 1.ATTA AA -JTCLCCGCT'TAAAAAC 1.. CCATC1ACC' C'7‘ACCC CCACaCACCACCCTTACCTATAT’.TAATTCCTATCAII) ) ’T J‘ 3 3 ,ACAAAACCCCCACCACCCCC'-A'-CC T"CCATTT..TTATCCACCAA-TCCCA1ACCCC1L1T'CCCCCAC.ATTTTCGT :C' ACCCC' ACCCaCCAC-CTT'ATATACCCTCACC,ATTACCCGTATGLTAACCACAACAT C LLTCTATT ATTC CACTACTGTTCAACCAATCCCTTTAC,‘TTT JCTACT'CCCACC CTGCAAATTAA Muhispaciﬁc body (IZZSVS—OKTﬂmL—C Tight chain ody OKT3m attached to £116 inus of antibody (3225115 LC) AMINO AC TTJ SEQUENCE DTELTCSP‘V’TLSVS'LR‘I’S75T‘TCRASQSTCTTTTNY"R'TT‘TCSEFLLTE‘T'AST'T‘STSCTLTSR 10111111.. >0w1— J Db DTADYY CQQTTNNW; rT‘T‘.ETCAC'TKLL T'T'Y PRTC2D1KT‘L7Q‘Z‘ZT\\/TJTTZAL C‘ :’\7 CAT S ’71 S VT;T" "3 DST1 TJSE":(T!) t-u (2’) (J) k—E', ‘7;F: {A U) ,2: :11ThJ-J (Vfwy‘xyrh'rQVTﬂkrrV’11":'VrV «Ac/«rw-Jr‘cnorl.—— v ‘-‘ 'J‘JCIVJU‘CJ~»J,\ «A «A 7-,\ "0.40.322. \I TT‘LC‘L‘ T\.'1 'Q‘J (J '.J "J‘JDQUC‘\.3": 1.) 1‘3 I\/ IL "/10I\ L: CAELARPCAS‘v’T/T‘TCCT/Z‘SCY'TETTR'T’'TMTTWVK ‘RL CQC 'J TIJ’TCY TN ‘SRC‘T’TNYNC2K TLTTJKSSCTAA‘T2.11:1...TSE'. mm- W, (W 1". VJ C Hm. -11 C. ~,~ WWW . 1" .21111YDLT11S31YT» "Aapmm .

-J\ «1 TLixbeJGCGCSCwC11J: YL‘ 'T'SKLASC NUC LEO." CE \JZ"1L,.Z".T,n-, p 'TT‘G‘--:x1|1321 'V"T"!V73 rV IAI'\’VF"T r1 '. "V rV1I1I1>WI11 "2 ' " f‘ p. «20.1!me \/\ \J .‘JZ. .‘ ‘., 111‘ )‘ h" Af‘f‘f‘F‘\JZ"1L:I\_C\_C.1‘3C.1‘.C1Z‘Z r1r' "'TT'T'2Z‘1GC'. CTCLJC,‘CC .ZCCACACCDTTCTUACCZAP'CA"""’WA"' :Z\‘T1C.I'1G‘.,'L31L,Z".1\.,'C 21.2" 1.— an," {\f‘iz - C \.,J 0.1! 0.3 1 . \.:n . .-_ CCCCT27‘1(:C("CCCCCC'T‘ITCTC:A'T ].A22Z‘1AT'2‘1’1'L3C‘Z‘CCC ‘A1/‘1CC2Z\'T]..A.CCT"3CCC2‘1'T TCC ACCCCC T..'T'T :.\_ \.:.‘3CZ'\.1‘.,G‘TT11 ----- pr ,\ - ‘2 '1 12-"I1L_\.,L3\.J'C2L‘\\ L,'\3.2L‘1.‘.AJ nAJ ,ZVV. V A rVJ- Z rm1-Irm1-I "n3 VJ '1 «m‘.,C.:C1Z‘1L_\.,Z"] T ’".(,Z'\.C:If‘ n ~ - 12-1 - Vm pap-,1 J. G.‘T1 "1\.JC\.J/12L‘\\J- .13C‘3C1Z"..L\.1'C1C'CJ\Jl{ ~71 T"'" .'C1\.,'CJ.2‘wwan ,erT1L,Z".Z\.:'\..r111 -- w "n CV'n' r‘ {’1/‘4. rx 7_\frV/V V am am VJ "(‘th AJ A JV. 1 . ‘. «In-Um \/"\_ ,.‘. ..‘G‘3C‘.1'CJ\J'C1\J'CJ2L._J- - "V \ f‘CZ‘12Z‘1.Z-\.L,T'C‘:2.\2-\.CT.G2‘.‘112-1'C13T'A'CC1':rVr r71 2" f‘if\l' rVrVr' 1C" ‘f‘1-'u1l.‘3C2‘J1CZ‘L-V (-1 r71 V. 1rVV1/V2'..'1rVELL] 1. C.Z-\.JT‘.:J.hl(''H1IrI1 V V ('1 1rVr1rV V r1 .. C‘T:CJ.3CCZ‘1".'CT.'C:.2Z‘1.

\JZ"1\.:.\2‘1'13..‘.{V rVr- r" .rV1 V' V "'V ' C12".Z\\.I'1'.1 "n I11( :1 V 'V ‘., T ‘.1L3Z‘1Z"1\., {"I‘ .r;r'- rVr .TL,T rV rV1.‘ VrI1x1I "IT! "IT!".1.1 ‘.1'.1 ‘T1CC VrVn' .errV: 32L‘1.2’".'1_Z'\.2.\‘...C.:.111-11 'sx1lrI1 VrV1 .rV (V. 1. \J'C CJC'C Z‘1'LT12Z‘1'13.

ACCCTC222T12-CTTXCZ": TJCD3!x\\f ] TC’\]'2‘\.2TT"TT‘CCTJCAZ ‘CCCC'TT‘A2—CTC,‘ C2‘1GK3Z‘1GZ‘1G'T'C'T' \.,Z".1L,Z"\....Z"\.:.‘.\‘JG’TCCid/11L3\.J/"1L,2,"\.:'n p r: p -J~ 1.— ,1 AA- ,1 er‘ ICT\CAICZTCACCAGCJCIthaC3CTGILAGCAAA CL 2Z‘1\_.2m J . 2,:.CCAK 21Z1C/‘1C/‘LAZ.CTC'T'Z.IT3CCT3C‘TTGT‘G2‘1Z‘1G'T 'CZ‘LC'CT CA...‘T3ACC ‘3 C C I‘1rr » .[T‘JC jr'rv:\U'x anrVr 71 «pmK/Z,".'2\,Z‘..\Z'XGZAG‘C.‘1 J—m 11m A rVJ- rVJ- Jm—u-n ,.,.[.7’\f" 1.- .pr.[CII‘J 2.\‘T1C:I.‘ G‘J C‘J'CT CZ".1\.:I'\3."2 Vrn V'Vr' 1.- .n rVJ- C'C \.:n .1. 'CJZ‘1Z"1\.,Z"1\...1L:'\12\‘32‘\‘31 ‘.1."T1G2'1 1\.1.[\J'L32L‘1.

C'TC'TC":CL:C2\CSCTT‘C2‘1CC'T'CC TC‘CCJ’V'TCACKCC TA.CG'T "CAL—7C TTC2‘HGT .—1 V n r‘ 2'1 1y" ‘3 ALCACACI; C T TCAC2‘.AC.3TK3 TC‘ .‘1‘7 "(V/"C r‘.’ --.1 1-- 'n1- ‘7‘ 1 - Ck/ L3\.jx_. . ..Z\‘T1C‘T1. 2'1Z".Z‘1.T‘:JZ.1C:I.Z"IT'2‘.2.Z12‘\.CCT3Z‘.1C"’TC' T‘Z‘.

I\,CTTTACCCGTTATACCA'GCATTGGG==ATACAGJTCCGGGTCWAGGTCTCGAA'GGAT TGGTTATATCA-TCCJGAGCGCTGGTTATACAACJTAAACCAGAAT .77] TTCJ AAGATA‘ LTAGCA ACCC ACJC 3ATA/TTA7"GCAGCAGCATCCJGCCTATA ’GCAGCT GCA CTGAGCTC7 G A‘CAL«GA.TAGCC3CAT3TTTATTAL, 'GCA 3.TATTAT3A" CATTACCCTCGATTT' GC«G' CAGT3C3CATC TGACGCTTAGJCACCGTGCTGGTG.TAGTCGTGCLCG GGTTCA GGCGGTL3T3L,T3GTAGCJAGAT'LTTTTGATCL,AGAGTCCGGCAATTATGAG CJAAL3TCCGG G' GAAAAA73TTAACJCATCACC' CTA3«L,-3L,CTTL3L,AGCA»,L3TTAGCTATATGAATTCG A' CA M"CAC.AA_AAGCL3L3L,ACJCA'73CL7‘TCGATAACC3TTGCATTTA—r —r LT«ATATCAGCAAACTGGCAAGGGG TTTCGGCATCATTTTTCTTT3TAGCGGTATGCJGGCACCJTCATATAGCJCTGATCCJATTAGCGGTA 'GGAAGC GAACT3ATGCAGCAACCTA T'TATTCTCA 3L,AC«TC«LGTCAATGCJAATTCJCGTTTACCTT CG«-"CACCZAC' 'GGZTAATTAATCTTTAG (8777‘: IT D30:.3 29LE)'\ Muitispecihc a11tib0dyL35225v5£N297Q)LDKTT3111HN heavy chain AAA]. IPL'C' ACIHIIIII SEC7)UEINC,IE «VA A, 3A Am V-n V 7-, V v 7 IWVK ’73'IRIP ‘'Vgg '32)?)le 3 vaw -n-.--- 3I\3.' ELI-35,31 ITiJCE) (A: GIT 3'_ _ _LR II "x' 2"."1':LIL) KA'r v '1 r'1'V' r'1h'l 'x' ' L 31x: I=I I131 3. 1.35SS'I"[AY7[.LSSL’] ISA" ‘7L717 YCAIPIY‘1’I IDIDHYS 1.119117%3 ,AchY- pr\JQL:A_1L3\_17>E)LJC:ILJC:ILJ©_.1.‘4 A ‘5. \"1 3mm L V L V3 V 73 I 73'SPAI I]I INSIASSP ."v T'L MiLI,33ASSSV’QEYLAENXWQSGT.-.-1—‘ 17SKLASCTP-1I'L-1}7.7[\L77‘7GSCTYS ]I3"]I'I[S 'I[II1A}[DA LTYYCCQSSA PPTE SGT’ I].I[\IIP.GGG GSQVQLKUSGP773VCPSQSLST'Q’ SG. 7T7GVHWVPLQSPCKGLELIGVTISGGNT1.’ <4 NTPPTSRLSTNKDNSKSQVPPKMNSICESQIDTATTYYCJAIPAL TYYDVT'7EPA7VT'7TA7GL72GTLVTVSAA P "xTPPI3APSS TSGG'. AAI_.C«GT37KIDVPTETT"L7§:73‘7v‘d71\£S’GAI.T SGVHT PP VLQSS’GL YSLL S-VVTV. SS7 SLGT3. QTYIICJNVNHK L KvPKKTEPKS’GII.TIITCPPCPAPELLGGPSV _ SNT PLPPPK¢KDTLMT T E‘v7r~,..V——7V—r.V—T.,——h H .'L, \3’\I \3’; VSFIEDPE VKPL ,— m T—vv’YViD’GV'rAIFIT-AKTKP—V— «WA—7." 7 Y mv A. R SCSVEVIII EAT:7317: TfiCKSLSLSP (SEQ TD NC: 300) NUCI3T3LJT1I SEQUENCE AL:L.1.7.7L/2"G(I.-JCAGCA 3A jK,L:L.17\,'\/'1r1..,'\7\ HTIW‘ 4'\1J&,4'\r",'\/"r.r' ,'\7\ "mm ,4'\/‘ .Ir‘,'\r' ,ﬁrm .I.EIan—I\n.u'\rlrr17\ 7\7\7\r1.,'\"\ . :LDHGHATK/iJGCPTLJ'L: .L,L,L.1.'L3L.1.3'_ .JCHIM.L.:1 L71".

GCJ'JGTAAAGLIATAGC «ATTATA’GCTTTA.CCCGTT.ATAC,CJA'. ’GCI‘TTGGGTT,TAAL73A'73CJ'ETC/C GC. ’G'. CAGC.3 3T ,T GGATT3GTTATATCAATCCJGAECJ TGGTL ATAA. J7AAC,’ JACAAC "7‘TLTGAAATTCJAAAGATAAAGGAACCTTGTTCL,T,L.CGATAAAAGCI AT .CJGCJCTATATGC TAGCTGAGCATGCJCTGAL.CTCAGAGGATAC3CCCA3TTT7‘TTA'GATGCTCCC3TATT_TGATGA TCTACTATACCL,TCGATTTTGGGG' CAC3GGCCACJACJ'7‘TC 'GT7 CCGTTATGCJAGCGGTGC«TL« G' GGTAG' GTTGGCCGTGC" GGGGGTGGCGCTAGGGATATTGTTCTGACCCAGLG"GCGG GAATTATGAGCGCAAJTGCGGG CAAAAAGTTTCCA"CATC CTAGGJLCAGGAGGAGCGT TATGATTGG5—]A" G/TGC: GAAAACGLIGCJJCJACGTGCGCGAAACJ TITTCGATTTATAT ACGAGCTAACTGGAACGCG GTTCCJCCACATTTTGCTGGTAGCTGTAGGGCAGC"CAT ATAGGCTGACLTTTAJCv'TATGG’AG"A A. i5 LGATGGGGAAGTTATTATTGTG TGIGAG‘7‘. Im/ 7\r\r:r‘I mm mmﬁmm/ I‘Vr‘v‘nrv .r3 I, G G' GAAGGAATGGGTTTAGGTTTGGTAGTGL"AJJAAAC"GGAAATTATATCGTGGAGGTGG K2 A. GLG C3 JTLICJAJC AGAGAGCGGGGGJJGGTGCTGGAGCLTAJGCAGTGCG' GA GGATTAGCTGLTJGTGAC’TCCTTTAJCCTGACCAACTATGGCGTGCATTGGG'GCGCGA TGCGCGJCJCAAAGGCCTCGLA' GGGTCJ CGTGATTTGCCGJJCGGLACACCGATTAT 7\/ {‘7‘ 1"‘F‘I‘ "’71 _\/l-\_\/"V\/ Tji In]'TACGAGGCGCGT? GAGCATTAACAAAGATAAGTGLAAA C.AGCTGTTTT TTTAATTGA'HCT‘CCTGGAAAGCCAGGATACCGC"TTTATTTTT7A7C73GCGCGCT "11mTG’"’I"T"AT‘2-111"GTG AT‘T JGGG I I J MTG GG‘T‘GA ‘CTGT .TTMAAAmmnAnMn.TGJG.L.ATGGGTG mGG671AﬁALU LIFT«J ILT N'MTI'MTI‘K‘rNr‘r-xr‘rﬁbI'TT/TL'VTL DLIDLIDLL‘.

LTGGGGTGGGTWTAIAmAnmI TGITGLL-GAAGGG-TGACGGT GT GTmmﬂﬁmnﬁIInmnwmnIﬂ IMIWFAI GZTA ‘ICCG'TGA'mTGCGC I.TI~I~II~IIV~II~I I~ TLL-L GIIwLT'L-TTL G1Tppm}i~II\».n»mI~ImI~I» IVWI‘HI‘A-TL .T .TJTGIJT'TIJTG :T"uCICLJAIITKI'1/‘1’n "(I'm 7» r GAAAGTTGA7CCCAAT’TG’K‘TTr ATACATGCGC/CCC "GCGZG"""CTGTAC'CCTCGCGGGACCG'CAGTC TIT (:Cr'L,.rNr..IIICCCCC «AIVHI‘mmL'LIT .1JTTGTJ‘TTLII7» :1 «I1 LLGIIL:13LLLITﬁrx. ~.p».I I. ‘mc A».VII1J\TL;I|TI'TIT:TrJ‘TIT:T/JTTIII » :3: I GGT‘ :Gm AIAIMI GGA.GTGAGGCACG TGHC T INT T TAG"i'1 I \'1 G) H (J I;L’; I If) A' G I G. l-—_; (NC. :5 4 l (.- '5‘:.J CT) 'an TI :3'7': . 'I‘lP. K1‘ LLlLJ‘\ ‘1':1 NALI'ZA' [7 ZTTL'QL. J'TLZ'\VLLA\JLAIX[ALJCLJU'm7 HAITKK‘ ‘Ir 7" ‘\ " UV G 1.~r\r.r~/:X~~ILCIrTL 'T,TL'T,t.JIIx'III-In'n L'JT'KT,(II L:LZJT ILAGCJALITLITJLITLITJTATG GL1'31J‘TIT «AI G mIITI‘n-II I TIT T L:\.:n‘ I T,LL‘\TL:LLIa." I~r~II~. «ALLI1J‘TTL: LI1J‘TWI mI G’n I‘m 7»-1J‘TTrJTTJTTrJTIT,G1‘«TrT'LIL1'31J‘T137» (TINA r‘T' TA" I,. II r-x (w r". r‘.’ ’1’ "AAA, by» "(WIT I -.r~r~.,1IJI > LIL, LL'-. GLLTL:L:LL I/V’w/an'rl'wrxrwr'kr5."*\ ‘TT'JL‘T, L:IIKJTLTLnT-it‘T TT313L, .1J\TAITITrJ‘TTJTLIKT,TJTL'i IvnIIA-Zc'TLT'3K3LK_ TJIVIILI- fNIr"I1'I «m w I" V'Wf‘,’\.f\f:l{'r'1[1'l('1l'rl'n LIL. UL? L:I'.ZT'V \JCT ;CCJ .LIT T13 ‘4 7‘r\r~r\ r".

T,£AIIUJL \JAGA TjLI'IT:CTJ'T-.'JG1"*I§-- m-I «:er,:J\_.TZ\3\"A\x» "2 IA? L’ILLr.Tz'x \JK3C:II,1\LJLJG'IBATkJ/IAJ'TLZ'Tz-TLf"/’\' 1.er VII—‘ /\f\r 'ZTXKTFXITAGALL ACJ *ILLIIA-Z\ VII—T wr‘lr‘trwlr‘terP-rMTIT '1le m V" v (warn. fx .LL,'\_ \TjLIIIT'JLlECTLLL’IALKJLLTL CTT'CT 7CTCTZMPICAAGCTCACCGTGG.C?AIACCCCICGCAGUAGAGGATCGTC'TC {\1"CT'CC "'7'AA: I’JCA 'GZ‘TGGCT"'[GUALI]ATCICZ"TA("GCAGA.GA "‘CTCTCCCT’ erI-I erI-I D'I'ATK.LK3\:I\3T1:.\1-\.I .,., V‘GSFCKCLEITGVTWTGGNTDYNT «FF MUQLCSQDTATYTJAPCALTYYDYEFAYINGQGTLVTVSAAST KCF‘STV7FF‘TIAPSE. ICTCCC AALGCLVKDYFTJETJ‘JT‘JCsINNSG‘LTGI1VT'1T F I.SSTI171/TT1/PSSSLCTQTT'ICNT \IrIKP mIITIVDKKVILU TI TCPF‘CF‘AFELTCGFSTJFL FFF‘FF‘KL TLMTSRTt’Ii1T7TCVT1712JVSFIEDPEVTKFNIIT.YVDGVEVF NIIITIE'REEQI QSTYRVV ST7LT rIQD11C111CKE7Y.C, VSNKA, EKTIS{AKC‘EFREF‘QVITLFPSRI LT-J Lu 1:»h}1-3 '1‘,L.‘41 (W U) LTCLVKGFY S 1TAVEWESNC"PGI‘1I\.Y1 I'M/-1: ECLI‘IIII‘IYIQKSLSLSPCI1IISGLTCGC1GGG71GGC'CGSICLTQVQGQQCCnELARF’134 ST17KI1’ISCI 21S .1YIFTRYTI7IHV‘1IT7IQRPGQGLEWTGYINI SRCYTITYTKQKFKDKn'TGTTD SSS TAYMQLSSLTSEDSAVYYCARYYDD1YCGDYTPJGQGTTTTT17SST..1GGS .1LC1GC1CGGGI QIIVL TQSPI FCEKVIMTCS7-SSSTI7S 1[VIIIWYQQKGCTSUIIRWIYDTSILASG‘JTJAHFR. SG SCTSYETLTISGMZAZDAATYYYCQQTJWTSSITPFTFGSGTKTETNE‘k (SEQ ID NO: 302) NUTLI'C'ITIJE CEQUENCE C21Gr‘v I."'r\r1.r\r‘=* ’n /~1' r-x' («r1I~r~Ir~1r1Ir-. ~»1 ‘AV‘H’YW 11T11Ir~1 :1 ~ 1/1111;1r' «r11» 1r1'~\ .-- 1.1C22‘1GC'ﬁrm 712721\ICI1G2-GCG1.1CCC 1G 1CC1 GC ICC21GC C1-12 .1C‘271IGI'IC1-C1G2-1C-»"I I21.("(rer‘tf\71\f:/~1 CC"CCP1CCG’7721CCC7-‘C'I'1I/\GCC'7GT2'1CC7C'T‘71'I‘C’I7‘77 'GC.7' 7T ‘T‘CTCC""77/177/1G7‘CC ,1, :C'I 1»1 1~111Ir11f1»11 11 11 11 111 11 111» r1» {1/1 GG'G -1ID121'1GC71 GGG2'121'G'C1C71,1r1r1.1.1r1.I:r1I.~11»1»111111111111"G\G'1GC"IG21I .LJI. 'GAIT1r1r1f1r1I~rv111'11'1 12"IC1I'I 1C '12'111211212'-L.12:1GC CCGT"I '1.71CCT’21GCC7-‘CC'I'G2»G.C271T‘II71271C21I7 "121’7271'I‘27121C21CC2»-.712‘1.7 GC .IIC C’I‘7"I‘II"T‘ '17'1' 172-7171 I7121I1G2‘1GC C’I‘GC271212717-‘CCI'1G7571' .71CG 1C -‘127\I'I‘III'I‘271'I‘II'21’7'"C-‘1C 1CGC 'IC71CC' 7271’21171 271"PT'I1'I'C‘27\I‘T'I‘II'Gf'77‘I7‘17'I73G177GTCC2'1G -‘1GC‘’TCCII'GC'I'CACGCT127\I 1C 1CGGC1'21CC21GC 'T‘r‘FT‘ C 'TIf'T'TI' 1Ir~1r1Ir~1Ir1Ir~111I~ 1.": x "‘IITI ' GGJ. I./.L '\.‘ '\.‘./'G 'GI 'i'G'ZXCCI.’ ..I CCTCTGGG‘GCLCLGCGC 1r"TG'ILLlG'_1_ i11 r~=rr1r1 1A1/1I1r1IJLI./ 77’ ‘1"er IJZXACE'1 7C2- r‘r‘r‘-r‘r‘--’"111G21C'21GCGC11 "(1,1 1,1,1.»1,1r1» 1 'GG'GC'G .GIG'GAIGAIGCI..11 1r1r 111C'CC 'IGCI 'I'GCII1'G21G11,1 1,1111,1n1r1r1.»1» ,1»1..1.»1~11mCIICII'IC1C21CGI'21'G11 11 r11T1r1.»_r1.1I..1r1 {1.lGL‘G‘G/P»~ r1, 1C1 1'"m r1|T1r17 11 1,1r1r1I,1r1I..1»1 ~11 111111 '17 111,111,1r11» ~1 f1.».r1I.‘1r1I.‘1.-1I\J‘1 r" 11 {1,1 G _1_ G12 «.IG'G‘ 'J'G‘G'G 21'1C21GCIIGGQCA'G‘G'G2 '21 C1721I 21 .1' 1., "I:\ T17(:1 , r ,1 1 212D1I'G21CI'I2'1', »1,1'1,1»11r1r11»» r1», I—1r1f 1 1 111 1»1 11,1» L, .21 12'-C2:1CC2' 21G '11- 121G2' .7‘21'C'1 GAG 21C2‘1ETCI‘ 1.l LI‘ My1% r1r1,1.»1,1I..1r1I,11 1DI'GIGCIG21G lT‘CVLLGICHLJ.C1 1~111Ir1 1 1f1r1.»1 C . '1" .‘C'CC'CC'CAAAZjC"21I1GC2‘IC’\7"'7"[ ATE ..1I1.CII.7CCCGC.I. 3C"CTC2CGTCpr:"az WV‘31: 3C7CL"7'G}HC"ALIS/711.1177"7"TG271IC1G'IIJ271GII I‘CZ'TIAC 71.712 f\r~.,’\1‘ ,1’\Ir :G 1,.1C.3GII7GG21C' 11JCG— IG—GA GG'ICC'A"1271’I'GCC'71271G2-71C71/17111CC'G7C1GC121CI‘12'TG ‘271G 1‘"ICCIxI'111L3C21CG1’I'I71C"G'I‘GI .‘GG'I'C 2' 1r1Ir —1 1. 13,1111 (1(121 11.71.1'" r C111.,11G'G 17113, "CTCGCT11Ir" . \/'\_ Z1I3'1I2-"A‘C2MHZ1IJ'I' "‘C 71111.7(; . 777271217112.21GCCI3CIIIC"C 71CCCCC C2’1"]I'CG271G2712'127121I'J ‘ 7.1'I'C7" CC2‘12’\-7‘1GCC321271.717: 1":1CI71GCCCC G 111.1711D1KIG27-1C2-‘ICIL3I1'1 11 P-rN' 1-21 .-1 1111.’1C'D1C\.,C1 IrerIrerIr' r1» r1» 21111I- ‘1’" 111(1,’ '1" 1 1- 1«-1111 1»—1 Apr. 1/1- r"r\ . :‘K/x. G'G C2-‘IJII'1.3CI.,GI3 '1'1\3\.J/IG:I\131'1II3.,C2"I.71I131.7121CII .113'L3. '1 CAGG ICII' CC."'3CTGC1. 371777'77'"31’7.1136 \C/ T'LI3CCI3 rer‘ir1Ir-erru .L \J'\. \/'\_ .GGAGII‘GGCI1C71CC21I' .". 1r‘\J GGCI‘111CC'C"'\G}I711C27.".C"I'21C2‘12’-\IC‘CTNC'CI'TC"CK"' 'GCI CII'CCI3G271CG C‘TCCT'IC'I] CC'I‘C'IAC'AGCNZHCI'TCACCC .‘71C.’\7‘-.2\G2\GC271CG'IIGCC2TICC'I'111CGCAZXI 1T ‘.TTC' (21‘ "(3C "ECG" 'G7'\_1.II7C2"I11211-Gk. .11.":GC'I'C"I7" CC'111'[1AC(.321C.3'I"271C271CGC7‘ 7117\7‘77‘'CTCTCT'CI'C 7CI7CCGG ‘GTAAACJ~AG.JTLJCATTTLGAIAILJ LJ'GGCG'TCJAJGGCT'TJTGGCJGCJALJGCT'AGGTGCTLJLJ -GCJ C -JLJALJLJTTJAJGCTTCJAGCT:JL, GCAJGJAJGCJCJGTCTJLGAAC T GCAJCGTCC GCJICJTGCA’_ AL'LJL‘JLJTTAAJJAALJALJC_'J1TAA/ J'LJ\VAA'LJum" mﬁmﬁr mm 7 "‘ﬂ‘rﬂﬁ -LT3 Ji iAiALJCTTT/JLJMV'LJ .Ji iJAJiJACCAALJLLJ'LJATmm‘n er ..... "IF‘WF‘F‘H'V'! WA V7J WT‘I"'LJ1'LJLJ TTAAAC-AGCJJTCCJCJGG C.-GGG' C' GGAJATCJGATJGLJTTATATCJJATCGJJAJGC'TTTLJLJTTA TACC-ACTCA.AJTJA.JAATTCAAAGATAAAC.AACJTJC 'GACCACJCCJATAAJAAJAJGCAG'TJLGCLi) ACCGCCTATATAGCTGAGCAJJCJCTGAJCTJTCA JAGL»ATAGCGCA.GTT TTAC ’ 'TJ'GCAL.3 LJ.,TATTAAGTLJJAJJCJACTATAGCC G.JA.TTATJGGGLJTCJACJGCJCAJCCJACJCCJT JACJCJGTTCJJ:3 / 'LJV'LJLJTFJLJ"‘r‘ﬁrﬁ "Iﬁrﬂf‘ IT: ,I"VJ .,I"Vr' hﬁﬂ" m rpm/"V1 ﬂ KV"V. («w/A "ITV'IA ‘7‘ ﬁ‘n 'LJ l. ,"V. Jr:V I JLTJALJTLJ'LJTHLJV'LJ .JiGLJi JL "JLJG'LJ'LJ'LJ LJLJLJLJLJLJAGCCAJLJAJ LJTTLJ ACCCC GAGT CJGCJCAATTATGAGCGCAA. JTLCCJCLJ A7AAAAGTTACJCAJTCJAACJCTG' AGCCJ GCAGCAGCAJGCJGTTJA.GCTATATCJAAATTGGTAATCJAJ J CJAGAAAACJCLJGL,AL,CALJCCCG7-A7-CG f lJr JibAJALJLJ/JLJJIAJ/JALJLV11:1"7 n17 ’ "’ "Vr 7" ’ r~r1r {Tr‘ 7&rx IIH'THIIi1CK1I:lK1rV.I|pAr:Cr:G.‘I CCCTC/CCAT:T T"CCTGT\JTC‘ TWAGCAGAAGAT TCAGCAATCTATT Al LJ A7‘.’1r‘.r\r1r~7‘ ~~JJ\ ’VITK NIVFI'l/V’.J»__r»‘JI.1'LJJ/»_ATf‘l‘" 7.". r~.r~.~J1JrJ1J1JV: A: JITJrUIIJ "Tr‘r'.

' JCJ - J LJL/L3: i LJJ‘J CLJ'L3'1AxV1Jr'V .EVJ J AAACTGGZA/-T'TAA Muitispecihc antibody C5225V5(1\2N297(2){3KT3m—L—C light chain ""1 [\C‘ ACID SHQTNE'.

I'ETJTJTQS PV JT.‘}’f"FSCRASQ‘S" PTNGSPRTJT r ’GL ’GL GTDFTTJS TNS‘7HSH:J‘ TAP{TC/3,JT='INT/AJFTTFGAGTV..JFT.JKRT\J7AALPS'7FTFPPSF EQLKSG .ASV‘7C...TJN\]YpRFAKVLJWKVDN. TJJLJD{LS r TLTTSK A:)3LF."MJYACFJVT CTJSSPVTT.L 'FNRGFJC J':'3 J'S J'GCJGLSLJLSLJLJ JbSGCJGLJLJLJC3QVQLLJLJS L1)AFJL.7‘AFPGASLS7KMSCKASCYTTT'CJ’TTHT."".77KQR:».":J JGTF‘JTGY TN. S..<.GY T=.IYNQKFK:J‘K.A. :—3 ‘74TTTYV'T‘QOFTAXJILJLLLJA A ..JL. SLTSFJDLAV LJ :I‘J'LJQ'LJ'LJ'LJLJL..—L_,-..C_,- «(-p.

SGGGSQTVLTQSFATMLSAPCJJTJT’LVTI.TTCCASSSVSYMTVTYQCI S GTSPITF‘TFJTYDTSTTJASCJ ’GCGCGTSYSTJTTSGMFJAFJT‘AATYYSQQWSSNPFTFGSGTKLFJTNR* (SEQ TD NU’CTJFJIC:TTDFJ SFHFNCF ("T/\1_lf‘lr‘lmJALJATLJ .i.GL.-J'_LJJALJ ,LJA JAwmn—Jn JJATynTynr-WAFJI—‘J-rj—J(LAT.mm Jib/ALJ JLJ.a;"\.1"\7\l'\/" ,«r m—J A 'C." .r""7\r‘,'\.

. J'L, LJL..LJ'L: J'_ J LJ'LJ GL:1ch GA.1.ALL/G l.'L3.'L3."1'L3\/.

TTJ.‘L3LJ. 'LSEuCCJCG}J-A. A AA—J . . J‘LJPJLJ'LJLJALJALJ'LJ J.‘..JL—J Arum—LAT Ar . _~.—L.——x . «JD—mm Ar,q—L,q,-.7—\ . .er.J.LLJCAPJ "A. . LJATTLJLVJAJLJELLJCAGLJ'LJLJALJLA‘A CCJ’CJCJACJC.C(JCLJCJLJT3C, JGATTAAATATGCGAL:'3C,GALAA AGLG.3CATT,‘TCJALJCCGCElli) TTTA’LJ \V-LJ .JJLCALTJ.LmGLJLJA'ALJ'1J'LJ1'JLJA\V-'LJLJAi i i iACLJCiGA'LJ TATTAALJ.. "‘F‘T‘ mrxrva/‘V‘r ﬂwrﬁ‘r H‘V'IF"! m 7 GCGTGCJJAAGL,LJA_AJJ JATATTHLJ .JLJAi iJJi. mm‘nrﬁm rv»"V/"V‘rr~"V"\"‘ : ~7‘I"V:-."I:"Vr'"V/"V"‘7\ "VF‘A A: mm JL’JFLJ'LJ'LJ'LJ'LJLJAL-'TCﬁ/"Vﬁr'ﬁ/"V‘rﬂ JL ATT'LJ' J'LJALJ'LJA'LJAJAJCJJALJAALJJL'J'LJ1 J'LJ'LJA'LJ\-V AJCLJL i i CAAAC' GGAJACJTGAJAJAC'TJ ’ACGGTGGCTGCJALJCJAJJCJTCTCTTCAT .TTCJC.LC,GCJCATCTGA' J\V-"V "‘ﬂ‘rh .m . mmmr‘rv7’JLJAA,I'II.JT'LJMV'LJ"F‘ﬁ- 'LJiLji1LJ1LJ1LJLJLJTLJLJTHFXA1A91C11C151LJCJLJA\L1"\m Imrﬂﬁmr‘mr‘rI'm ‘V/‘I F‘HT! VITI‘7‘lTI/‘V r37rv n Us 1GAALAJLLJL \.:I AGGCGAAAGTACAGTGGA‘AGJG 'GGATAA.CGCC"CT AA 'CGGG' CTCJCCAGGA'AGGT LJALJC..GLJ7\ CAGL..AALJ JALJALJIV-ACL 1ACALJCL. JLLJALJLJAIJLJALJCLJ-(1 1111—. m 7m3 11 111 m 1r. Amp. ,11,am CLJCJJLJJALJJ JAAA..171," 11 GGAGACTACJAJAAACAAAAG GTACCJCCTGCGAAT1ACCCAT CCTG-GC 'CGC GCGTCACA..ALJAGT1CAAG7-GGGIGAGA JTGTGGA J‘1'GGATCJTG AGGTGGCJGGT' GALJG G' C' GGC .JC GGCTCJ- GG' ’GJG GGAG-JATCAGGCJGG7LTCAGGTT GCTLJCJAGGLG7-GC 'GCAGAACTGGCACGTCGGGGTIJLJA..GCGT1AAAA' ’GAGC' T A G AAGCGLJTTATA LJLJTTTAGCLJ-J-.A1ACC7" GCATTIJLJJTT..-ACAGG'LJ' GCGGG' ’TCTGGAA‘ GGAT TLJLJTTATATCAATCCGAGCGGTIJLJTTATACAAJ'1AAACGAGAA AGAT7AAAGLJA ACCC 'GACCACCGATAAA/-GC.AGL1JAGCACCGCJGT.7\TA' GCAGLJTGAGCAGCJCTGAGCTCTG .IJGI'G-AT'AGCGC"’"111A11/1"1"TGCACGC‘"T'1""I.'1‘G.A"’1/11CIC1A1Z‘JGC'1'1‘CG/111J1WI‘.r1 1 "GAG 1G‘ "AJCfJfAG’TCTGACCGT '1AGCAG7C"’-"1"-‘G'1‘GGr SGTAG’1‘G-‘T 1GCGG'1GG'T1CA 1G1CGG'1‘21th CA.GA'T '1IGT'1C'CA TCCAGAGTGCGGCAA1"‘A1CAGCGCA/1G "GCGG _r~(j<.1r ‘\ "r‘ r1' ‘\rV1‘\rV1 V" 7 rI1IT r~ 'V 7" "‘ I.J 12‘1L3LJ1IA1A1C3AA113L31A1L1: GCAGAAJMJ('x u» \* , > . I-J ﬁ "1/‘1’ r-x er V."" ... ~r1IJ’I»Jr~»nITIrI1ITlnI ‘ Nr‘I - L, -LJLJALJL.A.GI’JLJ’JGAAALJ J: 1 - -.I»I.J . 1.1}. 5AALLJ'AG n ITK rI1 ITK rI1 "I K" .r‘."- J"J' J. J. A" "'LJ TC!73‘CL,'J71"":[\CrugariAD '1'GG""V‘TC'CA’TCAAAC 'GGZ-AATTAZ‘TC1'1‘TAG (S 71": 11) NO: 299)- Antibody C22 V511I32Wy chain 1111111110 AC1D SF. "ENCE 117L211KQSC113131.170? JCTISTJS1Z1T'JL G C31JTI111117:1f11/1LLJS J.KG1..EW1JG1J711AJS‘JI'3G111'."DYI1T '-F'1SRLGTKKDNSKSQFFK1V11\£S1..LJI'G;IDTCARA-JYYFYTFAYNGI’JG7T1J7AAS'."' ’G1J1J’I W Wnﬁﬂ m m mWﬂW. W C 'CJA'CJVAAGCJJTCJGT GJAC'CICTJTICJ'CJC'CJ' A 'CJCAJGCJAGCC.TT C) hﬂhm / Jk'CJV'CJACJALJCTACJA TCJT'CJLJA/J 'CJIJ':WﬂWnI nmm.Wﬁ7 Wm TGAAA ACAJAGCCAGCAAC/JCCJAACJIJGLJACJAACJAAACTTGAGLCCCAJJATCTTCTJAC’A AA’CJTCAC CATCJCJCCJAC'CJCJTCJCCCACCAJCCTGAACT'CJC TCGCGLCCLJTCJAGTCTTCCTC TTCCCCCCAAJAACJCCJAACJCJACACCCTCATGATCTCCJ ICA'CCCCTGJGGTCJJACJJA"CJCCJ' CG !CCCTGA -‘IG'I‘CZ JIIJCT'TCAAC'ICGIAC31"GGACCI5CGTGGA VAJC\JTCJCJTCA er‘ﬁrﬂ ~\ AAIGTLJLJ. IIIAA. IJAJCTJCJAA ~r~r A" 7 1T.) CJJJrTJJCJL‘wCJJ 'ITACT' CIbLJ »: » A:m A C} L) IC7E[JCT I {3:1IC7V) 5A1VJ } (A, _) C .1 In] C .l LJ‘ C .1 CI1 _JG1 Gf} L) (.l (N(1 {>1TI: J‘ ('\ CNA:WWI,NA:V:L AGAACCZT?G"mﬁm"crcIgccc:IAECGCGGGAGGAGATGAC7ATGAAGCAGGTCAGG c- ‘"C""CT"A/AG"11*1xmCCAGGGACATCJCGG" JA7TJJGAGAGCAATG ijgccmuGTga CAGCJICCTCLCCG"CCACAZGACCAGJTJGCAGCAGG C‘TCTTCTCA‘CC CCGGZTG 7ATGAGGCTCTGCP£ ‘CCTCTCCCTC‘C‘CCGC ‘AAA" JGQSGQCTSPN’JJ'TI’DGP‘YIMYS'CGCC GGCJCJG‘I J'IJS CJI‘CSDNHGSSGTC CJTNIHWYI’JTTI‘IGSPRT JTKTASESTC'GIPS'RFJGCJGJJCOC'J'DFTLST NS‘I/"E'CELTADYYC‘ICWNNII‘ITTC’CAJ’CJ.1.ICIJEIJKRTVAAPS‘JE.EPPSDEQ JVCJIJL NNEYPREAI«CVQWIxVD:IAIJ-CJSCJAS'QE'J-mCi;ﬂ.JJQI)JKI‘\~TY§IJSJ-TIJ'ALSKADYEKPK‘WACJ JI‘fT HQCIJCJSI‘ZTKSENRGEC* (SEQ TLJ TIC): 3"!" NUCLEOTIDE ENCE CJAJA'J'CJLJ 'CJt—‘GJI Ci.,«W’\7\‘/'Wr W’A m m WTWI Wr‘V. W’A m :IC‘J JCJACJTCJCJJL‘T 'C CCTCJG :IJJL"Wm. Ilr- CJLACI‘JGTCUPT; JACCJ'CJ' 'LJA'.I/‘Wr' ,W7'\ ’Im’I Wr‘ r‘I7 .WI.'CjT ALJ'CJ‘J'JI'CJ7 WhW. W Wﬁ 'CJ JJTTJGCJLJGT LJ'CJLJCJm ﬁ7ﬂWWﬂW WthWmWﬁmﬁ'CJ‘J'JI'CJI' JJLLJGAJL 'CJLJ'CJLJTT‘J'J'ACJCJ JJCJCC LJJL T LJ'CJLJATAAmHWﬁWﬂ h n ﬁmmﬁWﬂ7hv1 TLJAT'CJIJ'JICJACJ TTACJ,I/‘V‘r. WrW'W Irv-I "C GGT ACJLJ'CJA'CJATC-Ti GC' LJLACOCA JAC’JLJ'CJMV'CJ‘J'J'TGTTTm W/‘VW W7\ .r‘ .m mC ‘7‘ vIW‘n;W ,IW/‘VWr‘Wrﬁr‘i TAT-"T mﬁmﬁ/‘V ’W : Wr‘ - TLJT"TLJLJ'CJT'CJ/J 'CJI-V CCCGCCCAAJCGTCTCACJCJ TTAGC' CCCJJJCCJCJCJA’}CJC JCJ/JCCATTGCJ'CAJCCJAAACATTCATT L! mm TC CACCAL-CG GCA‘JCCC .JLJLJCJLmarmmﬁr‘TJL LJC GATTAAATATCC .JACJCJ'CJAAA'CJCAT JC TTCCGAGCCCCTTTAC JJ 1m C'LJJL"AJCGL’JCAJJLJLJLJLJA\-‘Lj-PJAAiiiiACLJCJLJ-ALJ JATTr1" f‘"/"‘" n mr‘imrﬂ vlrv7rwrx ..

-. .J. AJAJA.

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LJ LJ‘JAH'LJiLJGLJLJGL." -LJLJLJ CTC GFJG GGCTCA‘3LJ J LJ J LJAGCJ T‘JLJ '-H'LJALJGJAJi CAGL’JCJLJLJ-ALJ‘J LJA "\7\ GGT CACCT‘C(r; LCit'FI.C .r‘..._,11CA2»CTCGLA/~1* \._'."'\J jTG'A'L-J-\ 'TC TCTAAACC""‘17"7GCTT2JT1»CCTT'"11C 7C -7TT191'T‘-291C. TCCAT" GGGTTAAACACCC TC-CCG A. ."-‘\ ~r1.~JJ~Jm~anLJLALJLJbiL-Ji H1L36A17 1J .n 'L3 ' 1.1CTT1 -T11TC2917 ACTCACCACC" "CCCACT1‘TATT2;C"1 CTCCA 7GCTA TCL"ACJLJ1LCAJL.w..." -J.CTCA4I22‘JCGTTA 7CAGCG LEA-"‘1CTTCT -72"1CCC2»C2»CCCCCCA ATTATGA 7CGCA-2JCTCCCCCTCC AC C.ATG {"73£1 CC1'"1’1C7CC2"17C1GC1»CCCTT CCT‘AT ATGA ‘LTTGCTATCACC 7.1TTTATCA1AC 2»AACTGGCAAGCCCTCT r 1r~r~1 \LJKJ \{Wr771 \Cl‘ 1 \JLV \;._1& M111115pec111c activatabie antibody 34954CZZSVS13111H15,12111011 001111311535 Eight 91121111 204425 plasmid a11dheavy chain C225V5OKT3~1111~E—N piasmid.

Antibody (7225175 Tightcl1ai1’1 AUCTTO ACTD SE JUC\1C:LL L11 D--LL1QST'V1T SVSBG ETL1.7S':'SC1:ASQSTCTNJHUY J‘L51:TI‘JCST‘RLLTEXASES SC BSA FSGSGSGTDFTTJSTLTSXWS"CDT-T CYY CJ‘QLJINNWBT : : CL-CTI LET K. TVA-AAGV': -3: EBSL Z CELKSCTASV’CTHAITI3PEAKVQTKVDJIALQCG:TSCZSVTEQDSLDST1 SLSST LSK AD EKITVIA-CCUTT‘QLJ SPVTKSET-TR CJ'EC* (SEQ :L- CEO: .311) TIDE SEQUENCE L'J'_"LJJ'"\_TVTL LTJC' L'JL.ACCAG LJLJ‘LJV'LJIV'I'TGL;L1 L"V "\7\ F‘ m mCV 'ncxr"I "V/"J"VrV"Vn 23mm" r"Iﬁ'7\r"V "V "VrVﬁrV 'JALJLJLJTLJ'LJV‘LJV GCG ALCC' L'LJmf‘ Ir'7r'"VI'LJA'LJ‘LJT TTAGC' 'TCLJVJCGVTEATEVVG'GCATTGCJ'CACCAAACA " CAT' GGT TCA ACTTTA’TCAA CGGCAGC CJCGCJCTGC 'GATTAAATATGCGAGCGAA' GGATTALJC CATIC'C‘AGC'G'GC TTTAGCGVJCJCAJ'CGGCAGITGGIT’CCGAJT1TACCC1GAGI’TATTAACAGCGTCLJAAAGGGAAIwJLJ VLJ JG. mm'nmm "V . .mI»"Vr"I"rr~"V—\ "I r"Ir"I 1 LAL LAT 'LJ J'LJV IJ‘LJA‘L'JAI;CI;ALJAALJJLI I'."I;"VKI"V/"V"V7\ "VrVA ’J'LJ J'LJ'LJ‘LI"\_'LJ\VACLJL L LGL’JCJLJ'LJLJ'LJLJ'LJA‘V ﬁrIHﬁ/"Vﬁﬂﬁ/"V'r (L1 GAAAC' GGAACTGAAAGGTJAIVVGTGGCTGLV'ACC‘A1CTTTTTTCAT7\/"I"‘ .TTLV'CC ATTTGJ LJ'-‘LJIV-I."V "VrV'rrjI JITILL: A;.AL.JJLL'J'L21"‘;A_Amﬂl’u‘lﬁf‘7 I»"V. 'LJ '"VrV"V. ‘LJ LL31 1 LJJLLJJLLJLJLJTLJLJTIV'IAA1AA;C1 L C1A1"LJLJ'LJA‘I";LJ_"Vm "Imrﬂﬁmﬁmﬁ ("II"V "V/"V r"Ir"I IITI‘7‘lTI/‘V f‘V7r" J I:I 'JIV-‘LJAAALjiALLJA7m—Lﬂ—V-‘v ~JJ—J-J mmﬁmﬁ -, n—LI‘ A . AATLJLJ‘IJLJ‘man—L A; LJ'LLJ'C-rJ'AGLJALAJLJ' GT"ITI 'JJLL'J'LJVI"; '.L‘TI'ILJNTAALCLJLJLJJLL"Inf"Jmmc ("xJA‘LJA'L'JA'L'JCA;GL3ACAGLJAALJ«1 «1A A J m 7mg JA‘LJA'LJI-VA CLJ' LEAL.A «7‘ an J—J-J mmﬁ Jan—JIM II—V—J-J «7‘ 'JLJL.JJL LJAI'I‘LJJ"\LJCLJLLJ CL'JLJL'LJA'LJ AA. A(—JIrJ7rJIm v—J GIT AGACTA T'GA’TAAA’TLL7217»AG1VT '1AC GCLV"I'GCGAA L3'1'L.ALV‘VA1 C1";I') erx I.—~=r~Ir-.'\r.1 xn ‘G VI'1rurV'LJ1L31'V'L3IV1IVL3LJI'Vf'n’lwarVrN -AG".T'I'C7"/\"‘/\VI"IV:IV‘I_AGAC'1 GTGGAGG'TGGA'l'VT TGGE»GG‘I‘G’TC’TG‘1—; H .IF1>1;? ("\(.ll C'1I'VT '"GGC-ITA J W." LIL. mm. JJA L GIV: IV IVIV- 'V-IVI 'I-I'V-AI'V- -JA_LV AIG- VGIG‘I LIIJJJIJC7 MI." J7J C‘CTTTACCCG '1‘ 'I'A'I'A’TCATGC’T1 ITIVTC '1‘ TAALI TGGTT1‘Z‘TCAA ACCC'CAC"1C ' ‘ 'JI ~"IGCACCGCCTZTATGCA AGIVPJAGCGCV"111111A"1"IVCAC"CT’1TATF'GAichiATAGC"TFTA11HF 'VIIV1VAVJLJCAVIVAV.-V1IA-V'V'I-I1"1LGC("x r~.. LI," w J "J7IJJVJJJJLWJILN 7 G‘ mumLJGLJLLJJL'QLJ'LLH'IL'LL‘LGGIr1"IGL:GLZJ«JJJH. WGJJIJ 7 J JJLJ7,VL.»J.»7I..~.»W'V-JI311 A GAT'1'3'1'TC" -JA "C’TAV’TAV’TTCI’VCI‘JVAA1"""'1" -JA JE'V‘LJT'V‘AAG' 'CCGG («urn 7VJLLVVJ'L‘VVAALJ11ALV"V\ 7 JJ ~mm7 'VWJJTJJ 7» mm. ~ , f‘;"‘. ~. A "Ia «Am J ‘53. Jr 'LVI‘C' J1"‘L3‘Vit71aL-AL3IV 1'L‘VL3LV'1'Z‘V1'A1'CI11A1IIVV'L31A1‘ ~r~ .v .v JJ .v 'n 7 -qw {15‘3" L3 .J'IVIIVAIV LV'L;1'1_rXIV'I'V A »7 ~~J~~7 «A» Anman L3IVAIV'IAL‘VVAAAn» p» "'\" JJ "VA‘V .L'V'L3IV'LJIV'VL‘1AL‘1'V"I1 1m wJJJJnJVAJTJnJJTJm.»JJ~7» :1» r "V\ »7 H JJ ,J ' «JJJGI» A»ULAAIVLVIV'V17 JVVJ‘AH ' C ' '1'11 1 11110111.»ka VH1}LUCAAT'M 'L31(«Hr"rump-.VVVV TZV CTLAC"ATTACTCG' 'IIGVAAITICAGAAGA'I'GC;»_GCAACCTA"1"2"i‘1'"1"-‘T ‘f ' ' I . ‘. C". "I"1"1‘ZC‘CT‘l G' 7L3C2"C‘:Z§;2'XZ§L:"GGFI.AZ\\T1-7\u\rV1LJL_';'I {131913415} Table 11 shows exainpies of pairing of heavy chain (EC) and light chain (LC) sequences to make a muiiispeciﬁc dy 01‘ muiiispeeiﬁc activaiable aniibedy of the diseiosure. AS used herein when referring to Eight chains, dy light chains cmnprising CZZSVS are also referred to as Eight chains comprising C225.

Tahie ii. Muhispeeiﬁe antibodies and mui‘rispeciiic activa‘rable antibodies of {he disclosure Protein name Piasmidinucieatide} hairs fhr transfection €225v5nOKT3mvi-int23 HG nOKTSmIHc E C C???- 3‘3154—‘1294—6225V5OKT3m—HN HC C225V5~OKT3m—Hni\i LC (3225—3553544 264 C225v5~b§29?QnOKT3m~H—N HC C225v5~N237Q~UKT3mvH~N LC (3225 CE25V5AN29?Q*OKT3FY3"L*C Hi) ~NZSﬁQ LC C225-Oii'i'ﬁmuL-C 3954-12(34-(3225v5w253?Q~OKT3m—H~C HC C225v5~N29?Q~OKT3m—H~C LC 6322539544264 VS refers its version 5 of C225 EXAMPLE 6: Binding 0f muitispeeiﬁc amibodies m C7038 rm durkat T Ceiis {999M373 This Exarhpie deim‘msirs’tes the ability ofmuitispeciﬁc ahiihodies ofthe discldsure to bind to 'i‘ ceiis.

EGMMEE} Three formats ofmuiiispeciﬁc aiitibddies were tested: 4: i) C225vS~OKT3rn— H~N, a multispeciﬁc ari‘ribody in which an OKT3m scFV ( which binds CD38 (also referred to herein as CD3e and CD3) on T ceiis) was attached t0 the rminus of the heavy chain {ifahti—EGFR antibody 7225x157; (2) C22Sv5—OKT3m—H—C, a muitispeeiﬁc hddy iii which an m scFv was attached to the C~termious of the heavy ch aim of anii~EGFR antibody C225v5; and (3) C225v5-OKT3m-L-C, a multispecific antibody in which an OKT3m scFv was attached to the C-terminus of the light chain of anti-EGFR antibody C225v5. The amino acid sequences of these multispecific antibodies are provided herein, as are the amino acid ces of the C225v5 and OKT3 antibodies. Also tested was an isotype control, namely human IgG1 isotype control, Enzo, Catalog ALX133-C100.

To determine if the three multispecific antibody formats could bind to CD3- positive Jurkat T cells (also referred to herein as Jurkat cells and Jurkats), a flow cytometrybased binding assay was performed. Jurkat T cells (Clone E6-1, ATCC, TIB-152) were cultured in 640 with GlutaMAX™ (Life Technologies, Catalog 72400-120), 10% Heat Inactivated-Fetal Bovine Serum (HI-FBS, Life Technologies, Catalog 10438-026), 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Catalog 15140-122) (also referred to herein as te media) according to ATCC guidelines. Cells were harvested by centrifugation (200xg, 4°C, 5 min) and re-suspended in PBS with 2% HI-FBS (FACS Buffer). About 0 Jurkat cells per well were transferred to a 96-well U-bottom plate, harvested, and re-suspended in 50 microliters (also referred to herein as µL or ul) of the antibodies being tested. The ng concentration of the antibodies was 100 nM for the multispecific dies and 166.7 nM for the isotype control, followed by 5-fold serial dilutions for a total of 8 trations for each dy.

The cells and antibodies were incubated at 4°C with shaking for about 1 hour, harvested, and washed 3 times with 200 µL FACS Buffer. The resultant Jurkat cells were resuspended in 50 µl of AlexaFluor® 647 conjugated anti-human IgG (H+L) (Jackson ImmunoResearch, Catalog 709149) and incubated at 4°C with shaking for about 30 min. The resultant Jurkat cells were harvested, washed 3 times with 200 µL FACS Buffer, and resuspended in a final volume of 150 µL of FACS Buffer with 2.5 µg/ml 7-AAD (BD Pharmingen, Catalog 559925). Samples were analyzed on a BD Accuri C6 flow cytometer (BD Biosciences), and the median scence intensity (MFI) of viable cells was ated using FlowJo V10 (Treestar). 7-AAD staining showed that a forward side scatter gate was sufficient to identify viable cells. EC50 values were calculated in GraphPad Prism 6 by curve fitting the data to onist) vs. response (three parameters).

Figure 16A demonstrates that all three multispecific antibody formats bound Jurkat T cells with EC50 values g from single-digit nM to sub-nM.

EXAMPLE 7: Multispeeific antibodies bind to CBSa—ennressing 1‘ cells and recombinant human EGFR EtltltleZ} This Example demonstrates the ability ol‘inultispeeilic antibodies of the disclosure to bind to both (also referred to herein as cabind) T cells and target antigen containing cells. t-23} To determine if innltisbecitic antibody CZZSVS--Ol Also tested was an isotype control. namely hunian ’1',ng isotype control. Fnzo Catalog [XLX-htldulﬁ-Cllltl.

Etltltldld} lurkat cells were cultured and harvested as described herein. About 500,000 Jurkat cells per well were transfen‘ed to a U-bottoni plate, harvested, and res ed in 50 lull of r-lll nM ntultispeciﬁc antibody or 67 nh'l e antibody. Cells were incubated at dot: with shaking for about l hour, harvested, and washed 3 times with 2th) uL FAQS Buffer. The resultant 'at cells (binding a niultispecilic dy of the sure) were resuspended in St) ul biotinylated recombinant human FGFR protein (Abcani, Catalog abl68702) starting at about 290 anl ed by 5-fold serial dilutions for a total of 8 concentrations. Cells were ted at 4°C with shaking for about l hour; harvested, and washed 3 times with 200 ill, FAQ‘S Buffer. The resultant .lurlrat cells were resuspended in fat) ul l0 ug/rnl Streptavidin, R—l’hycoerythrin Conjugate (Life Technologies, 8866) and ted at dot: with shaking for about l hour. Cells were harvested, washed once with 200 uL FACE; Buffer, and pended in lStl uL FACE Buffer. Samples were analyzed on a Bl) Accuri C6 and MEI calculated as described herein. ‘8ng vali es were calculated in Graphllad Prism 6 as described herein.

EtltltleS} Figure loB demonstrates that all three inultispecilic antibody formats exhibited concentration—dependent binding of EGFR that required concomitant binding to CD32 on T cells. ECSQ values ranged from sub—nM to single digit nMi EXAMPLE 8: ’l‘argetndependent activation of T cells by multispeclfic antibodies {tidbit-26} This Example demonstrates the ability of niultispecitic antibodies of the disclosure to activate T cells in a —dependent manner.

Elltllldllﬂ To ine ifco—engagement of lurkat T cells and BEER—positive target cells by multispecilic antibody CZ25vS—OKT3rn—ltl—N, C225V5—0KT3Ul—H-C, or CZZSVS— OK'T‘Sni—LC could induce tion of .l'urlrat cells, a flow cytometry~based assay was employed. lurkat cells and CFSE-labeled target cells were eta-cultured for about 15 h and T cell activation assessed by staining for surface sion of the early T cell activation marker, C1369. ldlllldld} EGFR-positive SW480 cells, also referred to as SW480 cells, (A’l’CC, Catalog Oils—228) and EGER—negative {1266 cells, also referred to as U266 cells, (ATCC, Catalog TlB—l96) were each cultured in RPMl-l 640 with GlutaMAXTM, l0% S, l00 U/ml penicillin, and l00 pg/ml streptomycin (referred to as complete media) according to A’l'CC guidelines. These target cells were harvested, washed once with PBS, and re-- suspended in PBS at 2Xl06 cells/ml. A 5 lel CFSE stock (Life Technologies, CellTraceTM CESF Cell Proliferation Kit, Catalog C34554) was prepared in DMSO and then diluted to nM in PBS. The FSE working stock was used immediately. Equal volumes of the target cells and CFSE stock were combined for a ﬁnal CFSE concentration of l5 nlvl and ﬁnal cell density of lxll)6 cells/ml. Cells were incubated for l5 min at 37°C. ng was quenched by adding an equal volume. of lll—FBS, 'l'he incuba ed target cells were harvested, washed once with complete media, and rel-suspended in complete media at 5le5 cells/ml. 50 uL ofcell suspension per well was added to a l flat bottom plate for a total of 25,000 target cells per well. dlh} Jurkat T cells were cultured as bed herein. The lurhat cells were harvested, re--suspended in complete media at tall}6 cells/ml, and 50 iii otcell suspension per well were added to the flat bottom plate containing target cells for a total of about 50,000 .lurlrat cells per well, The ratio of effector T cells to target cells was 21: l, {Mild-ﬁll Three times --concentrated antibody stocks were prepared in complete media.

The highest concentrations used were l2 nM for each of the multispecitic antibody formats and l.5 nM for OKT3 (Bioltegend, Catalog H7304), CZZSVS (the arnino acid sequence of which is ed herein), and isotype control dies. Five-fold serial dilutions were prepared in complete media for a total of 8 concentrations for each antibody. Fifty pL of antibody per well were added to the flat bottom plate containing the T cells and target cells, diluting all stocl Elllllléllil} Alter about l5 h, cells were erred to a ll—bottorn plate and harvested.

To maximize recovery, the cell culture plate was washed with lSl) uL FACES , and the wash was transferred to the Uvbottom plate. Cells were harvested, ire—suspended in 50 till anti-CD69 PE conjugated antibody (BD Biosciences, Catalog 555531, used at the rnanulacturer’s recommended concentration), and stained for l h at 4°C with shaking. Cells were washed once with 290 uL FACS Buffer and then pended in a final volume of l50 uL. Single color controls were used to set compensation on the BD Accuri C6. lll cells were collected in a forward side scatter, CFSE—negative gate and l‘vlFl calculated using Flowlo. Elisa values were calculated in GraphPad Prism 6 as described herein. {$88432} Figure l7 denujnstrates that the most potent {1369 activation, evident beginning at sub-prl concentrations depended on each inultispecitic antibody format being co—cultured in the presence of both lurltat T cells and EGFR—positive S‘Wdélﬂ cells, in contrast, '1" cell tion induced by co~culturing with EGFR—negative U266 cells was signiﬁcantly less efﬁcient, exhibiting sub-hid ECSU values and a 75% reduction in maximal CD969 induction relative to tion induced by ECE'ER—positive cells. The EGFR— independent activation of the niultispecitic antibodies, as seen when ll266 cells were used, was similar to that of OKTS antibody, and negligible C1369 ng was observed with C225v5 and isotype control antibodies, EXAMPLE 9: Targetudependent tion of primary (338+ T cells by multispecific antibodies Elllll3433} This e demonstrates the ability ltispecilic antibodies of the sure to activate prirnary Cl)?» positive (CD8 l) T cells. {b88434} To determine if iniiltispecitic antibody CZESVS—OK'l‘Srndl—N, C225v5~ OK'l‘Bm-ll-C, or C225v5-0lé’l‘31n-L-C could mediate EGFR-dependent activation of primary human (338* T cells, a llow cytometry based assay was performed. CDlll' T cells d from human peripheral blood rnononuclear cells (PBMCs) and DDAO—SE labeled target cells were co-cultured overnight, and activation was assessed by ng for the early activation inarlter, €369. llllllldilﬁl Elfililiapositive S‘Wzlllll cells were cultured and labeled as described herein with the following exceptions: (l) SWdSQ target cells were labeled with Cell'l'raceTM FarRed DEAD-SE (Life Technologies, Catalog (734553) and (2,) complete media was supplemented with 25 U/rnl lL—Z (R&D Systems, Catalog 202~lL—ii5ll/CF). Fifty uL of target cell suspension (at Sal ()5 cells/nil) per well were added to a 96—well, tlat bottom plate for a total of25,llllll targets cells per well, {$88436} Fresh normal peripheral blood C138+ Cytotoxic T cells (AllCella Catalog PBOOQ-S) were obtained from AllCells (Alameda, CA), harvested by centrifugation (ZOﬂxg, RT, l5 min), and re—suspended in complete media mented with l l ~2 at l .SX l06 cells/ml. Fifty all of cell suspension per well were added to the flat bottom plate containing target cells for a total of about 753% CBS+ cytotoxic T cells per well and a 3:1 ratio ol‘T cells to target cells. lllllll'n’lil’fl Three times—concentrated antibody stocks were prepared in complete media supplemented with 25U/l’lll ill—2., The highest concentration was 6:90 plvl followed by Svfold serial dilutions for a total of eight concentrations for each antibody. Fifty pl; of antibody per well were added to the llat bottom plate containing CD8+ cells and target cells, diluting all stocks 3—fold for a starting concentration of 2th) pill/l. ltlllll438} After an overnight incubation, the flat bottom plate was centrifuged and 100 uL of supernatant was removed for the scent eytotoxicity assay described herein.

The remaining atant was transferred to a U—hottom plate, and the cells in the flat bottom plate were detached with 0.25% n (Life Technologies, Catalog 95 6): ’l‘rypsin ty was quenched hy adding 3 volumes of FACS , and the cell suspension was trarisl‘erred to the U—hottorn plate. After harvesting, the cells were resuspended in 50 "all of either an anti—CD69 PE/Anti—CDS FlTC cocktail (anti—CD8 FlTC, Bl) Biosciences, g Sol 948), Fl’l‘C isotype control (Bl) BioSciencesﬂ Catalog ), or PE isotype l (BID BioSciences, Catalog 34tl76l). All antihodies were used at the manufacturer’s irnended ct‘ineentrations. Cells were stained for l h at 4%? with shaking, harvested and re~snspended in a final volume of lSO pl. FACS Buffer with 2.5 l 7--AAD. Single color controls were used to set compensation on a Bl) Accuri on, and a ﬁxed volurne ol‘cell suspension was collected. Gating on CDltl' cells was sufﬁcient to distinguish between target cells and viable T cells. Activation was quantified as the percentage of T cells with expression of C369 above the PE isotype control.

Etltltldfilll Figure lllA demonstrates that all three multispecific antibody formats demonstrated concennation—dependent activation of primary CD? T cells with single digit prl £ng values. Treatment with OKT3, C225VS, or human lgGl isotype control (Enzo) antibodies resulted in negligible CD69 induction.

EXAMPLE l9: Targetwdependent killing of target cells by inultispecillc antibodies {999449} This Example demonstrates the ability tispecitic antibodies of the disclosure to induce "l‘ cell—directed target—dependent cell lysis {999441} To ine if inultispecitic antibody CZZSVS--0Kfl‘3in--ll-N, C225v5-— OKTBin—l-l—C, or (IZZSVS—OK'HnnL—C was capable of inducing T cell—directed, target— dependent cell lysis, SW489 cells were co—cultured with CD8+ 'l"~cells at a 3 :l effector—to— target cell ratio as described herein, After an overnight incubation, llll) bill; of the supernatant was assayed in white wall 96—well plates (Greiner Bio One Catalog ) for distinct protease activity ated with cytotoxicity ing manufacturer’s protocol (Cyto'l‘oX-Glol'M Cytotoxicity Assay," Catalog {39292, Promega), which uses a luminogenic peptide substrate to measure activity of proteases released by cells that have lost membrane integrity and have subsequently undergone cytolysis. lvlultispecitic antibosly—dependent xicity ol‘target cells was expressed in luminescence after background subtraction of untreated values and plotted in Prism with curve ﬁtting analysis log(agonist) vs. response (three parameters). {999442} Figure lSB demonstrates that all three iniiltispecitic antibody formats induced "l‘wcellnmediated killing of SW489 cells. As expected, Oli’l‘Zl antibody alone exhibited no detectible killing of Ell/V499 cells, suggesting a requirement for co—engagement of both CD3 and tumor target for effective cytotoxicitv by the rnultispecitic dies.

EXAMPLE ll: —dependent Twcell tion and killing of target cells by inultispeciilc antibodies 3} This Example demonstrates the ability of multispecific antibodies of the disclosure to induce ’l‘ cell-directed, -dependent activation of (IDS; T cells and killing ot‘target cells {999444} To determine if the observed "l‘—cell activation was dependent on EGFR expression by the target cell: abeled ositive SW489 cells or EGFR-negative U266 cells were co~cultured with human CD9+ T cells in the presence of multispecilic antibody CZESVS—OKTSrn—ll—N or GRIT}, each in a 5—fold dilution series starting at 299 pl‘v’l as described above CFSE labeling of the target cells (39 anl) was used to distinguish the target cells from the T cells using a method described herein. Frozen normal eral blood {7109* Cytotoxic T cells (AllCellsg Catalog P899931?) were thawed as speciﬁed by the manufacturen After an overnight incubation, the ls (CFSE negative cells) were assayed for surface expression of the early activation marker, CD69 as described herein.

Brieﬂy, cells were d from the assay plate, adherent cells were lifted using trypsin (Life Technologies} and cells were washed once with FACS Buffer. Cells were stained for l hour with artti--CD69--PE (Bl) Bioscience). Cells were washed and analyzed on the Bl) Accuri C6 for cell surface CD69 expression. MFl values were calculated as bed herein. Results were expressed as the percentage of T cells with expression of CD69 ahove isotype control using FCS Express analysis software and plotted in Prism with curve ﬁtting analysis logtagonist) vs. se (three parameters).

Elllllla’la’lS} As shown in Figure lSC, n'iultispecilic antibody CZZSVS—OKThndl—N trated potent 'l"~cell activation at suh—pl‘vl concentrations in the presence of SW48!) cells and only minimal Tncell activation by U266 cells, even at concentrations that were greater than 3 logs higher than concentrations used with SWL‘lSll cells. The Glilfl antibody exhibited minimal T—cell activation. These results demonstrated that ive T—cell tion is dependent on target cell expression of EGFR.

Elllllléléltl} To ine the dependence oi‘EGFR expression tor target cell ltilling, lilt) ul of supernatant of the T cell co~culture with S‘Wéltll} or U266 cells was measured for protease ty associated with cytotoxicity (Cyto'l'ox-Glo, Promega). Results were expressed in luminescence without background subtraction to show the U266 response and plotted in Prism with curve litting analysis log,'r--{LiloTM Cytotoxicity Assay (Proinega) was added directly to the plates (lSO ill of supernatant) to measure released protease activity, with the exception of the U266 samples, where lilt) ul of the ll266 supernatant was used to assay for protease activity, Results were sed in luminescence after background subtraction of untreated values and plotted in l’risni with curve fitting analysis logtagonist) vs, response (three parameters), ltllllltlétll Figure l9 demonstrates that all EGFR—expressing cell lines treated with rntrltispeci’ric antibody C225vS—Ol4'l‘3rnllvN in the presence of CDX‘" T cells exhibited dose-dependent cytotoxicity. in contrast, EGE’R-negative U266 cells remained unaffected by inultispeciﬁc dy CZQSvS—OKTZlmd-l—N. None of the cell lines treated with anti— EGFR antibody CEZSVS at 200 plvl exhibited any evidence ot‘cytotoxicity, regardless of their EGFR expression EXAMPLE l3: Attenuation of EGFR binding hy rnultispecific aetivatahle antibodies {$99451} This Example demonstrates that EGFR binding by a ispecitic activatable antibody of the disclosure is ated compared to EGFR binding by a niultisnecific antibody of the disclosure. This Example also demonstrates that EGER binding of the niultispecilic table antibody, which includes a protease cleavable moiety, is restored upon cleavage of the rnultispecit‘ic aetivatable antibody by such protease {999452} Multispecitic activatable antibody 3954--l2(l4--C.225vﬁ"OlQlBin-ll-N, activated multispecilic activatable antibody 395442tl4—C22SvS—OKT3rn—l-l—N, and multis eci’ric antibody CZZSvS—Olé’ldrnllvN were tested for their abilities to bind to EGFR-expressing Silt/48% cells.

Etltltldﬁill Activation of the inultispecil'ic activatable dy was conducted as follows: 8.25 ug ol'rnultispecitic activatable antibody 204-CZ25vS—OKT3rn—ltl—N in PBS was cleaved by the addition of active site~titrated niatriptase (also ed to herein as M'l’-—SPl and M’l‘SPl; ble from R8510 Systems, Catalog 3946K-SE-Qltl) to a final concentration of llltl nM. The digest was incubated at 37°C overnight, and cleavage was confirmed by removing an aliquot for capillary ophoresis analysis (GK—ll Capillary Electrophoresis, Perkin . se and the d mashing moiety were removed by Protein A puriﬁcation. Briefly, the digested sample was diluted to 2 ml with PBS and loaded onto equilibrated MahSelect SuReTM beads (GE Healthcare Lille Sciences, Product l l~0026—0l AD). Beads were washed with 5 column volumes (CV ) of lxl’BS, ed by CV of SXPBS mented with 5% isopropyl alcohol (lPA‘), and finally with 5 CV of leBS. Antibody was eluted with ltl CV ol‘0.l M Glycine, pH 3.0, and tractions were neutralized with l M Tris, pH 8.0, pooled, concentrated and buffer exchanged into PBS.

Ellllllzlﬁél} SW480 cells, which s EGFR, were lifted with cell dissociation buffer (Sigma, Catalog ), washed, and incubated for l hour with a 5—fold dilution of l000 nh’l of niultispecilie actiyatable antibody 3954—l204—C225VS—OKT3rn-ll—N, activated niultispecitic activatable antibody 204—C225VS—0K’l‘3m~H—N, multispecific antibody CZZSV’S-{lK’l‘Llni-HuN, activatable antibody 3954(3225V5, or antibody CZZSVS in FACES Butler on ice. Cells were washed 3 times with FACES Butler and incubated with l :400 of a secondary antibody, namely anti—human FeGarnnia specitic AFth (Jackson linrnunoResearch Catalog 109098) in FACS Buffer on ice. "ells were washed 3 times with FACE. Butler, and the Ml:l ol‘AF488 was read on the Bl") Accuri (BD Biosciences) tlow cytometer. The thl of a sarnple comprising only the secondary antibody control was subtracted from the experimental MFI and plotted in Prism with curve fitting analysis logtagonist} vs. se (_three ters). {06894553 Figure 20A demonstrates that EGFR binding by multispecil‘ic activatable antibody 3954— l 204~l3225V’5 —OKT3rn—l>l—N was attenuated compared EGFR binding by multispeeific dy C225VS—0Kl‘3indlvN, but EGFR binding of the multispecitic table antibody was fully restored upon protease cleavage of the multispeciiic activatable antibody by rnatriptase. Figure 203 shows that EGFR binding by antibody C225y5 and by aetivatable antibody 3954l~l20ril~C225VS were comparable to EGFR binding by innltispecitic antibody C7225VS-Oléfl‘3in-ll-N and multispecilic activatable antibody 3954p-l204-C225V5-0K’l‘3mil-N, respectively, demonstrating that the anti-CD38 moiety present in the specitic format did not alter the EGFR binding proﬁle of either the rnultispecitic antibody or multispecilic activatable antibody.

EXAb'lPLE l4: Ability of multispeeifie aetiyatable dies to bind to (733+ '33 cells {000456} This Example trates that a rnultispecitic activatable antibody; an activated rnultispeeilic activatable antibody, and a rnultispecitic antibody, all of the embodiments, are each able to bind to CD}? T cells. '7} To determine if CD35 binding had been affected by masking of the EGFR binding site of an anti~EGFR multispecifie aetivatahle antibody, a Jurkat T—eeil binding assay was performed as bed herein with multispeciiie aetivatabie antibody 3954— 225vS—0KT3rn-l>l~N, activated niultispeeilie activatable antibody 39544204— (ﬁ225v5—OK’I‘3m—H—N, and inultispeeitie antibody C225vS—0K’l‘3inllvN. lnrlrat cells were incubated with a 5-fold dilution of 1000 nM of inuitispeeitic aetivatable antibody 3954-- l2(l4—CZ25V5~OKT3m—H~N, inatriptase—activated multispeeilie activatahle antibody 3954— i204—(2225vii-OKTSm—H—N, or multispeeitie antibody C225v5—0KT3ni—H—N in FAQ'S Buffer for l hour at 4°C" Cells were washed 3 times and incubated with a secondary antibody, namely 1:400 of anti-human FeGamma specific AF488 (Jackson lnumtnoReseareh). Cells were washed 3 times, and the MFl of AF488 was read on the BI) Aceuri (Bl?) Bioseienees) flow eytorneter. The MR of a sample comprising only the secondary antibody l was subtracted from the experimental MEI and plotted in Prism with curve g analysis log£agonist) vs. response (three parameters). {999458} Figure 2 l A demonstrates that multispeeilie activatable antibody 04— C225V5vOKT3m~H~N and inultispeeitie antibody C225vS—Oi4'l‘3m—llvN exhibit equivalent binding to t T cells. Figure 2 18 demonstrates that innltispeeifie activatable antibody 39544204—(3225v5—Ol{T3ni~l-l—N and activated niultispeeilie table dy 3954— i204—022SvS—OKThn-PLN exhibit equivalent binding to Jurkat T cells. These results indicate that masking of the EGER binding moiety of the niuitispeeific aetivatable antibody does not affect the ability of the inultispeeiiic activatahie antibody to engage T eells.

EXAMPLE ES: 'l‘argetmdependent Tneell activation by multispeciﬁe atttivatable antibodies EillllldSQ} This Example demonstrates that target—dependent T—eell activation by a multispeeiiie table antibody of the disclosure is attenuated compared to activation exhibited by a multispeciiie antibody of the disclosure. This Example also demonstrates that —dependent T—ceil activation by the speeitie activatable antibody, which includes a protease cleavable moiety, is restored upon cleavage of the multispeeitie aetiyatable antibody by such protease {ﬁlllldtitl} To determine if masking of the EGFR binding site of an GFR nruitispeeitie activatable antibody attenuates target~dependent T~celi activation and to determine if protease activation of the niultispeeitie activatable dy restores aetivationt a inrkat activation assay was performed, as described herein, g m'nltispecilic activatable dy 3954—l204—C225v5—0KT3m—H-N, activated pecitic activatable antibody 2tl4—C225vS—OKT3m~ll—N, mnltispecitic dy C225v5~OKl'3ni—H—N, anti-EGE'R antibody CZZSVS, and a Synagis (Medimrnune) isotype control as described herein. 1} Figure 22 demonstrates that EGFR~dependent activation, as determined by (7969 induction ol‘lnrlrat 'l‘ cells co-—cnltured with EGFR—expressing Silt/48% cells, by maltispecilic table antibody 2G4~C£25v5—OKT3ni~l-l—N was attenuated compared to ependent activation exhibited by n'niltispecitic antibody CZZSvS— OKTSm—H~N, The figure also tes that EGF‘ere aendent tion by the rnnltispecific activatable antibody was fully restored upon protease cleavage of the rnnltispecitic activatable antibody by matriptase. Neither anti—EGER C225v5 nor the isotype control antibody exhibited activation ofT—cells.

EXAMPLE id: Target-dependent T—cell activatinn and killing ot’target cells by multispeeific activatable antibodies {$88462} This Example demonstrates that target~dependent T—eell activation and killing of target cells by a nialtispeciiic activatable antibody of the disclosure is attenuated compared to activation exhibited by a innltispecil'ic antibody of the disclosure. This Example also demonstrates that target~dependent T—cell activation and killing of target cells by the speciﬁc activatable antibody, which includes a protease cleavable moiety, is restored upon cleavage of the mnltispecitic activatable antibody by such se. lilillla’iﬁii} To determine if masking of the EGFR binding site of an anti—EGFR mnltispecitic activatable antibody attenuates target—dependent activation and to determine if se activation of the ninltispeciﬁc table antibody restores activation, at Jorlrat activation assay was performed, as bed herein, testing intiltispecitic activatable dy 3954420442225,vS—OKT3irnll—N, activated maltispecilic activatabie antibody 39544 2G4—C225v50K'l‘3m—H~N, mnltispecitic antibody C225v5~OKl'3ni—H—N, anti—CD38 antibody OKT3, antiaEGFR antibody CZZSvS, and an isotype control as described herein, 4} To determine the impact of masking the EGFR binding site of an antinGF‘R. mnltispecific activatable antibody on cytotoxicity and the ability of protease activation to restore cytotoxic activity of the activated innltispecitic activatable antibody, EGFR— expressing SW480 cells were ctr—cultured with 'l"~cells at a 5:1 effector—to—target cell ratio with a 5—fold dilution series, starting at 200 pM, ot‘rnultispecitic activatabie antibody 3954— l204—022SVS—0KT3tn-llSN, activated niultispeeilic actiyatable antibody 3954-1204— C225v5—OKT3ni—H—N, or ntultispeeifie antibody C225v5~0Kfl'3ni—ll—N} 209 ply/fl of OKTB, CZZSVS, and isotype control Synagis (Medinnnune) lgGl antibodies were used as controls.

After an ght incubation, lOO ttL of the supernatant was assayed for in white wall 9t» well plates distinct protease activity associated with cytotoxicity (Cytol'oX-Glo, Prornega).

Results were expressed in scence at‘ter background subtraction of untreated values and plotted in Prisrn with curve ﬁtting analysis logtagonist) vs response (three parameters). {$630465} Figure 23A demonstrates that EGFRedepen dent activation as determined by (7969 induction of primary T cells tured with EGFR-expressing SW480 cells, by niultispecilic activatable antibody 3954—l204~C£25v5—OKT3ni~l-l—N was attenuated cornpared to ependent activation exhibited by ispecitic dy CZZSVS— OK'i‘Bin-H-N. The figure also indicates that EGFR-dependent activation by the rnultispecitic activatabie antibody was fully restored upon protease cleavage of the rnultispecitic activatable antibody by niatriptase. OKT3, GFR 02256, and s lgGl e control antibodies exhibited negligible activation of 'l'~ceils. ltltltldtiti} Figure 233 demonstrates that EGE’R-dependent lysis of SW’dStl cells by niultispecilic table antibody 3954—1204~t3225v5—OKT3ni~l-l—N was attenuated cornpared to EGFR-dependent cytotoxicity exhibited by ispecitic antibody CZZSVS— OK'i‘Bin-H-N. The figure also indicates that EGFR-dependent cytotoxicity by the rnultispecitic activatabie antibody was fully restored upon protease cleavage of the rnultispecitic activatable antibody by niatriptase. CZZSVS and OKT3 and Synagis lgGl e control antibodies exhibited negligible eytotoxicity.

Qtlier Embodiments {$630467} While the invention has been described in conjunction with the detailed description thereof, the foregoing description is ed to illustrate and not limit the scope of the invention, which is deﬁned by the scope of the appended claims Other aspects, advantages, and modiﬁcations are within the scope of the following claims.

Definitions of the specific ments of the invention as claimed herein follow.

According to a first embodiment of the invention, there is provided a multispecific activatable antibody that in an activated state binds two or more targets or two or more epitopes or a ation thereof, the multispecific activatable antibody comprising: at least an antibody (AB1) that specifically binds a first target or epitope, wherein AB1 comprises an IgG antibody comprising a heavy chain and a light chain; at least an scFv (AB2) that ically binds a second target or epitope, wherein an AB2 is fused to the amino terminus of each heavy chain of AB1; at least a first masking moiety (MM1) coupled to the AB1 that inhibits the g of the AB1 to its target when the multispecific activatable antibody is in an uncleaved state; at least a first cleavable moiety (CM1) coupled to the AB1, wherein the CM1 is a polypeptide that functions as a substrate for a first protease, and wherein at least a portion of the multispecific activatable antibody in an uncleaved state has a structural arrangement from N-terminus to C-terminus as follows: MM1-LP1-CM1-LP2-AB1, wherein LP1 is an optionally present first linking peptide and LP2 is an optionally present second linking peptide; at least a second masking moiety (MM2) coupled to the AB2 that inhibits the g of the AB2 to its target when the multispecific activatable antibody is in an uncleaved state; at least a second ble moiety (CM2) coupled to the AB2, wherein the CM2 is a polypeptide that ons as a substrate for a second protease, and wherein at least a portion of the multispecific activatable antibody in an uncleaved state has a structural arrangement from N-terminus to C-terminus as follows: MM2-CM2-AB2, wherein the structural arrangement optionally comprises a linkage peptide between MM2 and CM2 and optionally comprises a linkage peptide between CM2 and AB2.

According to a second embodiment of the invention, there is ed a pharmaceutical composition comprising the multispecific table antibody of the first embodiment and a carrier.

According to a third embodiment of the ion, there is provided an ed nucleic acid molecule encoding the multispecific activatable dy of the first ment. ing to a fourth embodiment of the invention, there is provided a vector comprising the isolated nucleic acid molecule of the third embodiment.

According to a fifth embodiment of the invention, there is ed a method of manufacturing the multispecific activatable antibody of the first embodiment, the method comprising culturing a cell comprising a nucleic acid construct that encodes the multispecific table antibody of the first embodiment, under conditions that lead to expression of the multispecific activatable dy of the first embodiment.

According to a sixth embodiment of the invention, there is provided use of the multispecific activatable antibody of the first embodiment or the pharmaceutical composition of the second embodiment, in the preparation of a medicament for alleviating a symptom of a clinical indication associated with a disorder in a subject, wherein the ment is to be administered to a subject in need thereof in an amount sufficient to alleviate the symptom of the clinical tion associated with the disorder, wherein the disorder is cancer, inflammatory disease, or autoimmune disease.

Claims

What is claimed is:

1. A multispecific activatable antibody that in an activated state binds two or more targets or two or more epitopes or a combination thereof, the multispecific table antibody comprising: at least an antibody (AB1) that specifically binds a first target or epitope, n AB1 comprises an IgG antibody comprising a heavy chain and a light chain; at least an scFv (AB2) that specifically binds a second target or epitope, wherein an AB2 is fused to the amino terminus of each heavy chain of AB1; at least a first masking moiety (MM1) coupled to the AB1 that inhibits the binding of the AB1 to its target when the multispecific activatable antibody is in an uncleaved state; at least a first cleavable moiety (CM1) coupled to the AB1, wherein the CM1 is a ptide that functions as a substrate for a first protease, and wherein at least a portion of the multispecific activatable antibody in an uncleaved state has a ural arrangement from N-terminus to C-terminus as follows: MM1-LP1-CM1-LP2-AB1, wherein LP1 is an optionally t first linking peptide and LP2 is an ally present second linking peptide; at least a second g moiety (MM2) coupled to the AB2 that inhibits the binding of the AB2 to its target when the multispecific activatable antibody is in an uncleaved state; at least a second cleavable moiety (CM2) coupled to the AB2, wherein the CM2 is a polypeptide that functions as a substrate for a second protease, and wherein at least a portion of the multispecific activatable antibody in an uncleaved state has a structural arrangement from inus to C-terminus as follows: MM2-CM2-AB2, wherein the ural arrangement optionally ses a linkage peptide between MM2 and CM2 and optionally comprises a linkage peptide between CM2 and AB2.

2. The multispecific activatable antibody of claim 1, wherein the MM1 has an equilibrium dissociation constant for binding to the AB1 which is greater than the equilibrium dissociation constant of the AB1 to its target or epitope, wherein the CM1 is positioned in the activatable antibody such that, in an uncleaved state, the MM1 interferes with specific binding of the AB1 to the first target or first e, wherein the MM1 does not interfere or compete with the AB1 for binding to the first target or first epitope when the multispecific activatable antibody is in a cleaved state, wherein the MM1 polypeptide sequence is different from that of the first target or first epitope, wherein the MM1 polypeptide sequence is no more than 50% cal to any natural binding partner of the AB1, and/or wherein the MM1 is a polypeptide of no more than 40 amino acids in length.

3. The multispecific activatable antibody of any one of claims 1-2, wherein the MM2 has an equilibrium dissociation constant for binding to the AB2 which is greater than the equilibrium dissociation constant of the AB2 to its target or e, wherein the CM2 is positioned in the activatable antibody such that, in the uncleaved state, the MM2 interferes with specific g of the AB2 to the second target or second epitope, wherein the MM2 does not interfere or compete with the AB2 for binding to the second target or second epitope when the multispecific table antibody is in a cleaved state, wherein the MM2 polypeptide sequence is different from that of the second target or second epitope, wherein the MM2 polypeptide sequence is no more than 50% identical to any natural g partner of the AB2, and/or wherein the MM2 is a polypeptide of no more than 40 amino acids in length.

4. The multispecific activatable antibody of any one of claims 1-3, wherein the protease that s CM1 is co-localized with the first target or e in a tissue, and wherein the protease cleaves the CM1 in the multispecific activatable dy when the multispecific activatable antibody is d to the protease.

5. The multispecific activatable antibody of any one of claims 1-4, n the multispecific activatable antibody comprises the structural arrangement from N-terminus to C-terminus as s: MM1-CM1-AB1; MM1-LP1-CM1-AB1; or MM1-CM1-LP2-AB1.

6. The multispecific activatable antibody of any one of claims 1-5, wherein LP1 and LP2 are not cal to each other.

7. The multispecific activatable antibody of any one of claims 1-6, wherein each of LP1 and LP2 is a peptide of about 1 to 20 amino acids in length.

8. The multispecific activatable antibody of any one of claims 1-7, wherein the CM1 is a substrate for a protease selected from the group consisting of ADAMS, ADAMTS, ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, Aspartate ses, BACE, Renin, Aspartic cathepsins, Cathepsin D, sin E, Caspases, Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, e 7, e 8, e 9, Caspase 10, Caspase 14, Cysteine cathepsins, Cathepsin B, Cathepsin C, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V/L2, Cathepsin X/Z/P, Cysteine proteinases, Cruzipain, Legumain, Otubain-2, KLKs, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, Metallo proteinases, Meprin, Neprilysin, PSMA, BMP-1, MMPs, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27, Serine proteases, activated protein C , Cathepsin A, Cathepsin G, Chymase, coagulation factor ses, FVIIa, FIXa, FXa, FXIa, FXIIa, Elastase, Granzyme B, Guanidinobenzoatase, HtrA1, Human Neutrophil Elastase, Lactoferrin, Marapsin, NS