EA043382B1 - ACTIVATE ANTIBODIES AGAINST CTLA-4 AND THEIR APPLICATION - Google Patents

Description

Cross reference to related applications

This application claims priority to US Provisional Application No. 62/417,212, filed November 2016, which is incorporated herein by reference in its entirety.

Link to sequence list

Presented in electronic form via EFS-WEB.

The contents of the electronically submitted sequence listing (name: 3338_059PC02_SeqListing.txt; size: 527968 bytes; and creation date: October 27, 2017) are incorporated herein by reference in their entirety.

State of the art

The immune system is able to control tumor development and mediate tumor regression. This requires the generation and activation of tumor antigen-specific T cells. Multiple T cell co-stimulatory receptors and T cell negative regulators or co-inhibitory receptors act together to control T cell activation, proliferation, and gain or loss of effector function. Some of the earliest and most characteristic co-stimulatory and co-inhibitory molecules for T cells are CD28 and CTLA-4. Rudd et al. (2009), Immunol. Rev., 229:12. CD28 provides co-stimulatory signals for T cell receptor engagement by binding to B7-1 and B7-2 ligands on antigen presenting cells, whereas CTLA-4 provides a negative signal that suppresses T cell proliferation and function. CTLA-4, which also binds B7-1 (CD80) and B7-2 (CD86) ligands but with higher affinity than CD28, acts as a negative regulator of T cell function through both cell autonomous (or intrinsic) and and cellular non-autonomous (or extrinsic) pathways. Intrinsic control of CD8 and CD4 T effector ( _Teff ) function is mediated by inducible surface expression of CTLA-4 through T cell activation and inhibition of T cell proliferation and cytokine proliferation through multivalent recruitment of B7 ligands to opposing cells. Peggs et al. (2008), Immunol. Rev. 224:141.

Antibodies against CTLA-4, when cross-linked, suppress T cell function in vitro. Krummel & Allison (1995), J. Exp. Med., 182:459; Walunas et al. (1994), Immunity, 1:405. Regulatory T cells (T _reg ), which constitutively express CTLA-4, control T cell effector functions (T _eff ) in a non-cell autonomous manner. T _regs deficient in CTLA-4 have impaired suppressive capacity (Wing et al. (2008), Science, 322:271) and antibodies that block the interaction of CTLA-4 with B7 can inhibit T _reg function (Read et al. (2000), J. Exp. Med., 192:295; Quezada et al. (2006), J. Clin. Invest., 116:1935). More recently, T _effs have been shown to control T cell function through extrinsic pathways (Corse & Allison (2012), J. Immunol., 189:1123; Wang et al. (2012), J. Immunol., 189:1118) . Extrinsic control of T cell function by T _reg and T _eff occurs through the ability of CTLA-4-positive cells to remove B7 ligands on antigen-presenting cells, thereby limiting their co-stimulatory potential. Qureshi et al. (2011), Science, 332:600; Onishi et al. (2008), Proc. Nat'l Acad. Sci. (USA), 105:10113. Antibody blockade of CTLA-4/B7 interactions is thought to promote _Teff activation by interfering with the negative signals transmitted by CTLA-4 interactions; this intrinsic control of T cell activation and proliferation may promote both _Teff and _Treg proliferation (Krummel & Allison (1995), J. Exp. Med., 182:459; Quezada et al. (2006), J. Clin Invest., 116:1935). Early studies in animal models showed that antibody blockade of CTLA-4 enhanced autoimmunity. Perrin et al. (1996), J. Immunol., 157:1333; Hurwitz et al. (1997), J. Neuroimmunol., 73:57. The expansion of tumor immunity, the ability of anti-CTLA-4 to induce regression of established tumors, is a prime example of the therapeutic potential of CTLA-4 blockade. Leach et al. (1996), Science, 271:1734.

Human antibodies to human CTLA-4, ipilimumab and tremelimumab, were selected to inhibit CTLA-4-B7 interactions (Keler et al. (2003), J. Immunol., 171:6251; Ribas et al. (2007), Oncologist, 12:873) and tested in various clinical trials on multiple malignancies. Hoos et al. (2010), Semin. Oncol 37:533; Ascierto et al. (2011), J. Transl. Med., 9:196. Tumor regression and disease stabilization were frequently observed, and treatment with these antibodies was associated with side effects with inflammatory infiltrates that could affect various organ systems. In 2011, the IgG1 constant region ipilimumab was approved in the US and EU for the treatment of unresectable or metastatic melanoma based on improved overall survival in a phase III trial of previously treated patients with advanced melanoma. Hodi et al. (2010), N. Engl. J Med 363:711.

However, treatment with ipilimumab is hampered by dose-limiting toxicities such as colitis. Di Giacomo et al. (2010), Seminars in Oncology, 37:499. Accordingly, there is a need for improved anti-CTLA-4 antibodies, such as modified forms of ipilimumab, with reduced toxicity but comparable antitumor efficacy. Such improved anti-CTLA-4 antibodies may be more effective antitumor agents than the developed antibodies.

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Brief description of the invention

Provided herein are activated anti-human CTLA-4 antibodies containing a heavy chain including a VH domain and a light chain including a masking fragment (MM), a cleavage fragment (CM) and a VL domain. Such activated anti-human CTLA-4 antibodies have CTLA-4 binding activity in the tumor microenvironment, where the masking fragment is removed by proteolytic cleavage of the cleaved fragment by tumor-specific proteases, but exhibit significantly reduced binding to CTLA-4 outside the tumor. Thus, the activated anti-human CTLA-4 antibodies of the present invention retain antitumor activity while reducing the side effects associated with off-tumor anti-CTLA-4 activity.

Provided herein are improved anti-CTLA-4 antibodies, such as improved ipilimumab, in particular an activated antibody that upon activation binds to cytotoxic T-lymphocyte antigen 4 (CTLA-4). In some embodiments, the activated anti-human CTLA-4 antibody comprises (i) a heavy chain comprising a heavy chain variable domain (VH) comprising

CDRH1: SYTMH (SEQ ID NO: 557);

CDRH2: FISYDGNNKYYADSVKG (SEQ ID NO: 558); And

CDRH3: TGWLGPFDY (SEQ ID NO: 559); and (ii) a light chain comprising (a) a light chain variable domain (VL) containing

CDRL1: RASQSVGSSYLA (SEQ ID NO: 560);

CDRL2: GAFSRAT (SEQ ID NO: 561); And

CDRL3: QQYGSSPWT (SEQ ID NO: 562);

(b) cleavable fragment (CM); and (c) a masking fragment (MM), wherein the MM is selected from the group consisting of YV01 (SEQ ID NO: 1), YV02 (SEQ ID NO: 2), YV03 (SEQ ID NO: 3), YV04 (SEQ ID NO: 4), YV09 (SEQ ID NO: 9), YV23 (SEQ ID NO: 23), YV24 (SEQ ID NO: 24), YV35 (SEQ ID NO: 35), YV39 (SEQ ID NO: 39), YV51 (SEQ ID NO: 51), YV61 (SEQ ID NO: 60), YV62 (SEQ ID NO: 61), YV63 (SEQ ID NO: 62), YV64 (SEQ ID NO: 63), YV65 (SEQ ID NO : 64) and YV66 (SEQ ID NO: 65), where the light chain has a structural arrangement from N-terminus to C-terminus as follows: MM-CM-VL.

In some embodiments, the CM is a protease substrate selected from the group consisting of MMP1, MMP2, MMP3, MMP8, MMP9, MMP11, MMP13, MMP14, MMP17, legumain, matriptase, and uPA.

In some embodiments, the CM is selected from the group consisting of 2001 (SEQ ID NO: 297), 2003 (SEQ ID NO: 298), 2005 (SEQ ID NO: 299), 2006 (SEQ ID NO: 300), 2007 ( SEQ ID NO: 301), 2008 (SEQ ID NO: 302), 2009 (SEQ ID NO: 303), 2011 (SEQ ID NO: 304), 2012 (SEQ ID NO: 305), 3001 (SEQ ID NO: 306 ), 3006 (SEQ ID NO: 307), 3007 (SEQ ID NO: 308), 3008 (SEQ ID NO: 309), 3009 (SEQ ID NO: 310), 3011 (SEQ ID NO: 311) and 3012 (SEQ ID NO:312).

In some embodiments, the MM is YV04 (SEQ ID NO: 4), YV23 (SEQ ID NO: 23), YV24 (SEQ ID NO: 24), YV39 (SEQ ID NO: 39), YV61 (SEQ ID NO: 60), YV62 (SEQ ID NO: 61), YV63 (SEQ ID NO: 62) or YV64 (SEQ ID NO: 63).

In some embodiments, the MM is YV39 (SEQ ID NO: 39).

In some embodiments, the CM is 2001 (SEQ ID NO: 297), 2011 (SEQ ID NO: 304), or 2012 (SEQ ID NO: 305).

In some embodiments, the CM is 2011 (SEQ ID NO: 304).

In some embodiments, the activated anti-human CTLA-4 antibody comprises (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 345; and (ii) a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 563, 564 and 565.

The activated anti-human CTLA-4 antibody (see above) contains (i) a heavy chain containing the amino acid sequence of SEQ ID NO: 345; and (ii) a light chain containing the amino acid sequence of SEQ ID NO: 564.

The activated anti-human CTLA-4 antibody (see above) contains (i) a heavy chain containing the sequence SEQ ID NO: 350 of the human IgG1 constant domain; and (ii) a light chain comprising the sequence SEQ ID NO: 346 of the human light chain constant kappa domain.

In some embodiments, the activated anti-human CTLA-4 antibody further comprises a first linker peptide (LP1) between MM and SM or between SM and VL.

In some embodiments, the activated anti-human CTLA-4 antibody

- 2043382 further contains a second linker peptide (LP2), wherein the activated anti-human CTLA-4 antibody has an N-terminal to C-terminal structural arrangement MM-LP1-CM-LP2-VL or MM-LP2CM-LP1-VL.

In some embodiments of the invention, LP1 and LP2 are not identical to each other.

In some embodiments, the activated anti-human CTLA-4 antibody further comprises a spacer and has an N-terminal to C-terminal structural arrangement, spacerMM-CM-VL.

In some embodiments, the activated anti-human CTLA-4 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 353; and light chain, contains the amino acid sequence of SEQ ID NO: 448.

In some embodiments, the activated anti-human CTLA-4 antibody is conjugated to a toxic agent, wherein the activated anti-human CTLA-4 antibody comprises (i) a heavy chain comprising a heavy chain variable domain (VH) containing

CDRH1: SYTMH (SEQ ID NO: 557);

CDRH2: FISYDGNNKYYADSVKG (SEQ ID NO: 558); And

CDRH3: TGWLGPFDY (SEQ ID NO: 559); and (ii) a light chain comprising (a) a light chain variable domain (VL) containing

CDRL1: RASQSVGSSYLA (SEQ ID NO: 560);

CDRL2: GAFSRAT (SEQ ID NO: 561); And

CDRL3: QQYGSSPWT (SEQ ID NO: 562);

(b) cleavable fragment (CM); and (c) a masking fragment (MM), wherein the MM is selected from the group consisting of YV01 (SEQ ID NO: 1), YV02 (SEQ ID NO: 2), YV03 (SEQ ID NO: 3), YV04 (SEQ ID NO: 4), YV09 (SEQ ID NO: 9), YV23 (SEQ ID NO: 23), YV24 (SEQ ID NO: 24), YV35 (SEQ ID NO: 35), YV39 (SEQ ID NO: 39), YV51 (SEQ ID NO: 51), YV61 (SEQ ID NO: 60), YV62 (SEQ ID NO: 61), YV63 (SEQ ID NO: 62), YV64 (SEQ ID NO: 63), YV65 (SEQ ID NO : 64) and YV66 (SEQ ID NO: 65), and where the light chain has a structural arrangement from N-terminus to C-terminus as follows: MM-CM-VL.

In some embodiments, the toxic agent is conjugated to the activated antibody via a cleavable linker.

In some embodiments, the activated anti-human CTLA-4 antibody further comprises a detectable fragment.

In some embodiments of the invention, the detectable fragment is a diagnostic agent.

The claimed invention relates to pharmaceutical compositions containing an activated anti-human CTLA-4 antibody and a carrier.

In some embodiments of the invention, the pharmaceutical composition contains an additional therapeutic agent.

The invention relates to an isolated nucleic acid molecule encoding the heavy chain and light chain of an activated anti-human CTLA-4 antibody.

The invention relates to a vector containing an isolated nucleic acid molecule.

The invention relates to a method for producing an activated anti-human CTLA-4 antibody, comprising (i) culturing a cell containing (a) a vector (see above) or (b) a vector containing an isolated nucleic acid encoding the heavy chain of an activated anti-CTLA-4 antibody 4 (see above), and a separate vector containing an isolated nucleic acid encoding the heavy chain of an activated anti-CTLA-4 antibody (see above), under conditions that lead to expression of the activated antibody; and (ii) isolating the activated antibody.

The invention relates to a method of reducing CTLA-4 activity in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition.

The invention relates to a method of treating, ameliorating a symptom, or delaying the progression of a solid tumor in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition.

In some embodiments, the solid tumor is bladder cancer, bone cancer, breast cancer, carcinoid, cervical cancer, colon cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, genitourinary cancer or urothelial cancer.

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In some embodiments of the invention, the solid tumor is a melanoma.

The activated anti-CTLA-4 antibodies of the invention are activated when the cleavable fragment is cleaved by a protease. In some embodiments, the protease is produced by a tumor that is in proximity to T cells that express CTLA-4. In some embodiments, the protease is produced by a tumor that colocalizes with T cells that express CTLA-4. In some embodiments of the invention, the protease is selected from the group of proteases presented in table. 1 shown below. In some embodiments, the protease is selected from the group consisting of matrix metalloprotease (MMP), thrombin, neutrophil elastase, cysteine protease, legumain, and a serine protease such as matriptase or urokinase (uPA). In some embodiments, the protease is selected from the group consisting of MMP1, MMP2, MMP3, MMP8, MMP9, MMP11, MMP13, MMP14, MMP17, legumain, matriptase and uPA, or a combination of one or more such proteases. In some embodiments, SM is cleaved by matrix metalloprotease (MMP) and serine protease. In some embodiments, SM is cleaved by matrix metalloprotease (MMP), serine protease, and legumain.

Table 1

Typical proteases and/or enzymes

ADAMS, AD AMTS, e.g. Cysteine proteinases Serine proteases, e.g. eg ADAM8 Kruzipain activated protein C ADAM9 Legumain Cathepsin A ADAM10 Otubain-2 Cathepsin G ADAM12 Himaza ADAMI 5 KLK, for example.. protease factors coagulability ADAMI 7/T ACE KLK4 (e.g. FVIIa, FIXa, FXa, FXIa, FXIIa) ADAMDEC1 KLK5 AD AMTS 1 KLK6 Elastase ADAMTS4 KLK7 Granzyme B ADAMTS5 KLK8 G y anidinobenzoatase KLK10 HtrAl

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Aspartate proteases, KLK11 Neutrophil Elastase eg Human VASE KLK13 Lactoferrin Renin KLK14 Marapsin NS3/4A Aspartic Metalloproteinases, RACE4 cathepsins, for example... eg Cathepsin D Meprin Plasmin Cathepsin E Neprilysin P.S.A. PSMA tPA Caspases, e.g. VMR-1 Thrombin Caspase 1 Tryptase Caspase 2 MMR, for example.. IRA Caspase 3 MMP1 Caspase 4 MMP2 Transmembrane Serine Protease Type II (TTSPs), Caspase 5 MMRZ eg Caspase 6 MMP7 DESC1 Caspase 7 MMP8 DPP-4 Caspase 8 MMP9 FAP Caspase 9 MMP10 Gepsin Caspase 10 MMP11 Matriptase-2 Caspase 14 MMP12 MT-SP1/Matriptase MMP13 TMPRSS2 Cysteine Cathepsins, MMP14 TMPRSS3 eg Cathepsin B MMP15 TMPRSS4 Cathepsin C MMP16 Cathepsin K MMP17 Cathepsin L MMP19 Cathepsin S MMP20 Cathepsin V/L2 MMP23 Cathepsin X/Z/P MMP24 MMP26 MMP27

The claimed invention relates to activated antibodies against CTLA-4, which additionally contain one or more linker peptides. In some embodiments of the invention, the linker peptide is located between MM and SM. In some embodiments, the linker peptide is located between CM and VL. In some embodiments, the activated antibody comprises a first linker peptide (LP1) and a second linker peptide (LP2). In some embodiments, the activated antibody comprises a heavy chain and a light chain such that the light chain has a structural arrangement from N-terminus to C-terminus of the light chain MM-LP1-CMLP2-VL. In some embodiments of the invention, LP1 and LP2 are not identical to each other. In some embodiments of the invention, LP1 and LP2 are identical to each other. In some embodiments, the prodomain comprises MM-LP1-CM-LP2.

In some embodiments, LP1 and/or LP2 comprise a glycine-serine polymer. In some embodiments, LP1 and/or LP2 comprise an amino acid sequence selected from the group consisting of (GS)n (SEQ ID NO: 532), (GGS)n (SEQ ID NO: 533), (GSGGS)n (SEQ ID NO: 534) and (GGGS)n (SEQ ID NO: 535), where n is an integer of at least one. In some embodiments, LP1 contains the amino acid sequence GGGSSGGS (SEQ ID NO: 542). In some embodiments, LP2 contains the amino acid sequence GGGS (SEQ ID NO: 543).

The claimed invention relates to activated antibodies against CTLA-4, which also contain a spacer. In some embodiments, the spacer is connected directly to the MM and has a structural arrangement from N-terminus to C-terminus as follows: spacer-MM-CM-VL. In some embodiments, the spacer comprises an amino acid sequence selected from the group consisting of QGQSGQG (SEQ ID NO: 544), GQSGQG (SEQ ID NO: 545), QGQSGS (SEQ ID NO: 546), QGQSGQ (SEQ ID NO: 547 ), QSGQG (SEQ ID NO: 548), GQSGS (SEQ ID NO: 549), QGQSG (SEQ ID NO: 550), SGQG (SEQ ID NO: 551), QSGS (SEQ ID NO: 552), QGQS (SEQ ID NO: 553), GQG, SGS, QGQ, QG, GS, G, S and Q. In some embodiments, the spacer and MM contain the amino acid sequence QGQSGSCRTQLYGYNLCPY (SEQ ID NO: 556).

The claimed invention relates to activated antibodies that contain toxic

- 5 043382 agent such as dolastatin, auristatin, auristatin E, monomethyl auristatin E (MMAE), maytansinoid, duocarmycin, calicheamicin, pyrrolobenzodiazepine or a derivative thereof. In some embodiments, the toxic agent is conjugated to the activated antibody via a linker. In some embodiments of the invention, the linker is a cleavable linker. In some embodiments of the invention, the linker is a non-cleavable linker.

The claimed invention relates to activated antibodies against CTLA-4, which contain a detectable fragment. In some embodiments of the invention, the detectable fragment is a diagnostic agent.

The claimed invention relates to pharmaceutical compositions containing an activated anti-CTLA-4 antibody described herein. In some embodiments of the invention, the pharmaceutical composition contains an additional therapeutic agent.

The claimed invention relates to isolated nucleic acid molecules encoding the heavy and/or light chains of the activated anti-CTLA-4 antibodies described herein, vectors that contain one or more isolated nucleic acid molecules, and methods for producing an activated antibody by culturing a cell containing vector or vectors, under conditions that result in expression of the antibody being activated.

The claimed invention relates to methods for producing an activatable antibody, comprising (a) culturing a cell containing a nucleic acid construct that encodes an activatable antibody described herein under conditions that result in expression of the activatable antibody; and (b) reconstituting the activated antibody.

The claimed invention relates to methods of reducing the activity of CTLA-4, comprising administering an effective amount of an activated antibody described herein, or pharmaceutical compositions containing an activated anti-CTLA-4 antibody described herein, to a subject in need thereof.

The claimed invention relates to methods of treating, alleviating a symptom, or delaying the progression of a disorder associated with CTLA-4, comprising administering a therapeutically effective amount of an activated antibody described herein, or pharmaceutical compositions containing an activated anti-CTLA-4 antibody described herein, subject who needs it. In some embodiments, the CTLA-4 related disorder is cancer. In some embodiments, the cancer is melanoma, such as unresectable or metastatic melanoma, breast cancer, colorectal cancer, gastric cancer, glioblastoma, head and neck cancer, lung cancer, ovarian cancer, endometrial cancer, pancreatic cancer, prostate cancer, kidney cancer, sarcoma or skin cancer. In some embodiments, the CTLA-4-related disorder is a disorder known to be treatable with ipilimumab.

Where aspects or embodiments of the invention are described in terms of the Markush group or other group of alternatives, the present invention covers not only the entire group listed as a whole, but also each member of the group individually and all possible subgroups of the main group, and when in the main group is missing one or more group members. The present invention also provides for the express exclusion of one or more group members in the claimed invention.

Brief description of drawings/figures

In fig. 1A-1C show tumor volumes as a function of days after tumor implantation in mice (n=10) treated with (i) unrelated mouse IgG2a antibody (FIG. 1A), (ii) mouse anti-CTLA-4 IgG2a antibody (9D9) (FIG. 2B) or (iii) activated antibody 9D9 (Fig. 1C). All antibodies and activated antibodies were dosed at 25 μg/mouse. The 9D9 activation antibody contains MY11 (SEQ ID NO: 294) as a masking fragment and 2001 (SEQ ID NO: 297) as a cleavable fragment. TF denotes the number of tumor-free mice at the end of each experiment. An unrelated mouse IgG2a antibody and a mouse anti-CTLA-4 IgG2a antibody (9D9) were used as controls.

In fig. 2A-2C show the frequency of regulatory T cells in the tumor (Fig. 2A) and the proliferation and activation of regulatory T cells in the spleen (Figs. 2B and 2C) of mice treated with different activated anti-CTLA-4 mouse IgG2a antibodies (9D9). Various activated 9D9 antibodies contain (i) either MY03 (SEQ ID NO: 293) or MY11 (SEQ ID NO: 294) as a masking fragment and (2) 0003 (SEQ ID NO: 320), 1004 (SEQ ID NO: 323) or 2001 (SEQ ID NO: 297) as a cleavable fragment. An unrelated mouse IgG2a antibody (DT 1D12 mg2a) and a mouse anti-CTLA-4 IgG2a antibody (9D9) (9D9 mg2a) were used as controls. In fig. Figure 2A shows the frequency of regulatory T cells as a percentage of total CD4+ T cells that are Foxp3+ in the tumor. In fig. 2B and 2C show the frequency of proliferating (Ki-67+) and activated (ICOS+) regulatory T cells as a percentage of Foxp3+ T cells in the spleen, respectively.

In fig. 3A-3E show the ability of various activated anti-CTLA-4 antibodies (isotype

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Human IgG1) bind to human CTLA-4 as measured in vitro by ELISA binding assay. Ipilimumab (YV1) was used as a control in all experiments. In fig. 3A, the activated anti-CTLA-4 antibodies contain YV04 (SeQ ID NO: 4), Yv06 (SEQ ID NO: 6), YV09 (SEQ ID NO: 9) or YV23 (SEQ ID NO: 23) as a masking moiety. In fig. 3B activated anti-CTLA-4 antibodies contain YV27 (SEQ ID NO: 27), YV29 (SEQ ID NO: 29), YV32 (SEQ ID NO: 32) or YV33 (SEQ ID NO: 33) as a masking fragment. In fig. 3C activated anti-CTLA-4 antibodies contain YV35 (SEQ ID NO: 35) or YV41 (SEQ ID NO: 41) as a masking fragment. In fig. 3D activated anti-CTLA-4 antibodies contain YV24 (SEQ ID NO: 24), YV39 (SEQ ID NO: 39), YV51 (SEQ ID NO: 51), YV52 (SEQ ID NO: 52) or YV53 (SEQ ID NO: 53) as a masking fragment. In fig. 3E activated anti-CTLA-4 antibodies contain YV54 (SEQ ID NO: 54), YV55 (SEQ ID NO: 55), YV56 (SEQ ID NO: 56), YV57 (SEQ ID NO: 57) or YV58 (SEQ ID NO: 58) as a masking fragment. In fig. 3A-3E all activated anti-CTLA-4 antibodies contain 2001 (SEQ ID NO: 297) as a cleavable fragment.

In fig. 4A-4D show the ability of additional activated antibodies against CTLA-4 (human IgG1 isotype) to bind to human CTLA-4, as measured in vitro using an ELISA binding assay. Ipilimumab (YV1) was used as a control in all experiments. In fig. 4A activated anti-CTLA-4 antibodies contain YV04, YV06, YV09, YV23, YV27 or YV29 as a masking moiety. In fig. 4B activated anti-CTLA-4 antibodies contain YV32, YV33, YV35 or YV41 as a masking moiety. In fig. 4C activated anti-CTLA-4 antibodies contain YV24, YV39, YV51, YV52 or YV53 as a masking moiety. In fig. 4D activated anti-CTLA-4 antibodies contain YV54, YV55, YV56, YV57 or YV58 as a masking moiety. In fig. 4A-4D, all activated anti-CTLA-4 antibodies contain 3001 as a cleavable fragment.

In fig. 5A-5F show the ability of several activated anti-CTLA-4 antibodies (mouse IgG2a isotype) to bind human CTLA-4 as measured in vitro by ELISA binding assay. Ipilimumab (YV1) was used as a control. In fig. 5A activated anti-CTLA-4 antibodies contain YV04 as a masking fragment and 2001 (SEQ ID NO: 297), 2006 (SEQ ID NO: 300), 2007 (SEQ ID NO: 301), 2008 (SEQ ID NO: 302) or 2009 (SEQ ID NO: 303) as a cleavable fragment. In fig. 5B activated anti-CTLA-4 antibodies contain YV04 or YV23 as a masking fragment and 2001, 2006, 2007, 2008 or 2009 as a cleavable fragment. In fig. 5C activated anti-CTLA-4 antibodies contain YV39 as a masking fragment and 2001, 2006, 2008 or 2009 as a cleavable fragment. In fig. 5D activated anti-CTLA-4 antibodies contain YV61 (SEQ ID NO: 60), YV62 (SEQ ID NO: 61), YV63 (SEQ ID NO: 62), YV64 (SEQ ID NO: 63) or YV39 (SEQ ID NO: 39) as a mask fragment and 2001 or 2012 as a split fragment. In fig. 5E activated anti-CTLA-4 antibodies contain YV65 (SEQ ID NO: 64), YV66 (SEQ ID NO: 65), YV01 (SEQ ID NO: 1), YV02 (SEQ ID NO: 2) or YV39 (SEQ ID NO: 39) as a mask fragment and 2001 or 2012 as a split fragment. In fig. 5F activated anti-CTLA-4 antibodies contain YV39 or YV03 (SEQ ID NO: 3) as a masking fragment and 2001 or 2012 as a cleavable fragment.

In fig. 6A and 6B compare the ability of activated anti-CTLA-4 antibodies having either the mouse IgG2a isotype (Fig. 6A) or the human IgG1 isotype (Fig. 6B) to bind to human CTLA-4, as measured in vitro by ELISA binding assay. Ipilimumab (YV1) was used as a control. In both figs. 6A and 6B, the activated anti-CTLA-4 antibodies contain YV39 as a masking fragment and 2001, 2008, 2011 or 2012 as a cleavable fragment. In the modified antibody of the invention (YV39-NSUB), the cleavage fragment was replaced by a protease-resistant linker (NSUB) containing the amino acid sequence GGSGGSGGGSGGGS (SEQ ID NO: 570).

In fig. 7A-7D show the ability of various activated anti-CTLA-4 antibodies to bind 58 α'β' cells overexpressing human CTLA-4 as measured by flow cytometry. Binding is reported as arbitrary fluorescence units (mean fluorescence intensity, MFI, or geometric mean fluorescence intensity, gMFI) as a function of the concentration of anti-CTLA-4 antibody added. In fig. 7A activated anti-CTLA-4 antibodies contain YV04, YV23, YV24 or YV39 as a masking fragment and 2001 as a cleavable fragment. In fig. 7B activated anti-CTLA-4 antibodies contain YV61, YV62, YV64 or YV39 as a masking fragment and 2001 or 2011 as a cleavable fragment. In fig. 7C activated anti-CTLA-4 antibodies contain YV39 as a masking fragment and for the cleavable fragment, 2011 (Ipi YV39 2011) or three variants of Ipi YV39 2011: (i) monolinked (Ipi YV39 MMP monolinked''), (ii) fully linked MMP (Ipi YV39 MMP) or (iii) fully bonded uPA (Ipi YV39 2011 uPA). In fig. 7D shows EC ₅₀ values for the various activated antibodies shown in FIG. 7C. Ipilimumab was used as a control for Fig. 7A-7D.

In fig. 8 shows the activity of an activated anti-CTLA-4 antibody containing YV39 as a masking fragment and 2011 as a cleavage fragment (Ipi YV39 2011) (square) at various concentrations, as measured in vitro using the SEB (staphylococcal enterotoxin B) assay ).

Antibody activity is demonstrated through IL-2 production by human PBMCs following SEB stimulation. Controls included unrelated human IgG1 isotype (triangle), ipilimumab (circle), and SEB stimulation alone (x).

In fig. 9A-9F show tumor volume as a function of days after tumor implantation in human CTLA-4 mice (n=10) treated with different activated anti-human CTLA-4 antibodies (mouse IgG2a isotype) dosed once at 10 mg/kg . An unrelated mouse IgG2a antibody (Fig. 9A) and mouse IgG2a isotype ipilimumab (Fig. 9B) were used as controls. In fig. 9C-9F activated antibodies contain YV04, YV23, YV24 and YV39, respectively, as a masking fragment and 2001 as a cleavable fragment.

In fig. 10A-10F show tumor volume as a function of days after tumor implantation in human CTLA-4 mice (n=10) injected with different activated anti-human CTLA-4 antibodies (human IgG1 isotype). Antibodies were administered once at a dose of 200 μg/mouse on day 7 after implantation. An unrelated human IgG1 antibody (Fig. 10A) and human IgG1 isotype ipilimumab (Fig. 10B) were used as controls. In fig. 10C-10F, the activated antibodies contain YV39 as a masking fragment and 2001, 2012, 2011 or 2008 as a cleavable fragment. Cleavable fragments 2012, 2011 and 2008 were modified to overcome the deamidation site in 2001.

In fig. 11A-11G show tumor volume as a function of days after tumor implantation in CTLA-4 mice (n=16) that were administered different doses of an activated anti-CTLA antibody containing YV39 as a mask fragment and 2011 as a cleavage fragment (Ipi YV39 2011) (Figs. 11E-11G). The antibody was administered once at a dose of 10 mg/kg (Fig. 11E), 3 mg/kg (Fig. 11F) or 1 mg/kg (Fig. 11G) on day 7 after tumor implantation. Control animals were treated with ipilimumab (10, 3 or 1 mg/kg; Fig. 11B-11D, respectively) or an unrelated human IgG1 antibody (Fig. 11A).

In fig. 12A-12D show the frequency of regulatory T cells in the tumor (FIGS. 12A and 12B) or spleen (FIGS. 12C and 12D) of human CTLA-4 mice (n=10) treated with different activated anti-human CTLA-4 antibodies mouse IgG2a isotype. All antibodies were dosed once at 10 mg/kg. The activated antibodies contain YV04, YV23, YV24 or YV39 as a masking fragment and 2001 as a cleavable fragment. Labels on the abscissas of Figs. 12C and 12D also refer to FIGS. 12A and 12B respectively. An unrelated human IgG1 antibody and ipilimumab with the mouse IgG2a isotype were used as controls. In fig. 12A and 12C, the frequency of regulatory T cells is shown as the percentage of total CD4+ T cells that are Foxp3+. In fig. 12B and 12D, the frequency of regulatory T cells is shown as the percentage of total CD45+ T cells that are Foxp3+. In fig. 12E and 12F show the frequency of activated (ICOS+) cells and proliferating (Ki-67+) cells as a percentage of regulatory T cells in the spleen.

In fig. 13A-13C show the frequency of regulatory T cells in the tumor (FIGS. 13A and 13B) or spleen (FIG. 13C) of human CTLA-4 mice treated with an activated anti-CTLA-4 antibody. The activated antibody used contained YV39 as a masking moiety and was either the mouse IgG2a isotype or the human IgG1 isotype. An unrelated human IgG1 antibody and human IgG1 isotype ipilimumab were used as controls. In fig. 13A and 13C, the frequency of regulatory T cells is shown as the percentage of total CD4+ T cells that are Foxp3+. In fig. 13B, the frequency of regulatory T cells is shown as the percentage of total CD45+ T cells that are Foxp3+. In fig. 13D and 13E show the frequency of proliferating (Ki-67+) and activated (ICOS+) cells as a percentage of regulatory T cells in the spleen.

In fig. 14A-14C show the frequency of regulatory T cells (FIGS. 14A and 14B) or CD4+ effector T cells (FIG. 14C) in tumors from mice treated with various activated anti-CTLA-4 antibodies. In fig. 14D and 14E show regulatory T cells in the spleen. Activated anti-CTLA-4 antibodies contain YV39 as a masking fragment and 2012, 2011, 2008 or 2001 as a cleavable fragment. An unrelated human IgG1 antibody and human IgG1 isotype ipilimumab were used as controls. In fig. 14A and 14D, the frequency of regulatory T cells is shown as the percentage of total CD4+ T cells that are Foxp3+. In fig. 14B and 14E the frequency of regulatory T cells is shown as the percentage of total CD45+ T cells that are Foxp3+. In fig. 14C shows the frequency of CD4+ effector T cells as a percentage of the total number of CD45+ T cells in the tumor. In fig. 14F and 14G show the percentage of proliferating (Ki67+) and activated (ICOS+) regulatory T cells in the spleen.

In fig. 15 shows the frequency of regulatory T cells in tumors from human CTLA-4 mice (n=8) that were treated with varying doses of either ipilimumab or an activated anti-CTLA-4 antibody containing YV39 as a masking fragment and 2011 as a cleavable fragment (Ipi YV39 2011). Antibodies were dosed once at 10, 3, or 1 mg/kg on day 7 after tumor implantation. An unrelated human IgG1 antibody was used as a control.

In fig. 16A and 16B show the percentages of activated (ICOS+) and proliferating (Ki-67+) regulatory T cells in the spleen of human CTLA-4 mice (n=8) treated with varying doses of either ipilimumab or activated anti- CTLA-4 containing YV39 as a masking fragment and 2011 as a cleavage fragment (Ipi YV39 2011). Antibodies were dosed once at 10, 3, or 1 mg/kg on day 7 after tumor implantation. An unrelated human IgG1 antibody was used as a control.

In fig. 17A-17D show tumor volume as a function of days after tumor implantation in human CTLA-4 mice (n=10) treated with varying doses of ipilimumab (Ipi) (FIG. 17B), a non-fucosylated version of ipilimumab. (Ipi NF) (Fig. 17C) or a non-fucosylated version of the activated anti-CTLA-4 antibody containing YV39 as a masking fragment and 2011 as a cleavable fragment (Ipi YV39 2011 NF) (Fig. 17D). Antibodies were dosed once at 10, 3, or 1 mg/kg (left panel, middle panel, and right panel, respectively, in Figures 17B-17D). Control animals received an unrelated human IgG1 antibody (FIG. 17A).

In fig. 18 shows the frequency of regulatory T cells in tumors from human CTLA-4 mice (n=5) that were treated with either a non-fucosylated version of ipilimumab (Ipi NF) or a non-fucosylated version of an activated anti-CTLA-4 antibody containing YV39 as a masking moiety and 2011 as a cleavable fragment (NF Ipi YV39 2011). Antibodies were dosed once at 200 μg/mouse on day 7 after tumor implantation. An unrelated human IgG1 antibody was used as a control.

In fig. 19 shows the binding affinities (Kd) for both ipilimumab (Ipi) and the non-fucosylated version of ipilimumab (Ipi NF) to various human, macaque and mouse Fc receptors.

In fig. 20 shows the average percentage of Ki67+ CD4+ T cells in the blood of cynomolgus macaques following administration of an activated anti-CTLA-4 antibody. The activated anti-CTLA-4 antibody contains YV39 as a masking fragment and 2001 as a cleavable fragment. Vehicle and ipilimumab were used as controls.

Detailed Description of the Invention

In order that the present disclosure may be more easily understood, specific terms are first clarified. Additional definitions are set forth throughout the detailed description of the invention.

It should be noted that the term refers to one or more objects; for example, by nucleotide sequence is meant one or more nucleotide sequences. As such, the terms one or more and at least one may be used interchangeably herein.

In addition, and/or when used herein should be understood as specifically disclosing each of the two specified features or components, with or without the other. Thus, the term and/or, when used in a phrase such as A and/or B herein, is intended to include A and B, A or B, A (alone) and B (alone). Likewise, the term and/or, when used in a phrase such as A, B and/or C, is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (separately); B (separately); and C (separately).

It is understood that wherever aspects are described herein in terms of containing, similar aspects described in terms of consisting of and/or consisting essentially of are also provided.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide one skilled in the art with a common vocabulary for many of the terms used herein.

Units, prefixes and symbols are designated in the accepted form of the Systeme International de Unites (SI). Numeric ranges include numbers that define a range. Unless otherwise noted, nucleotide sequences are written from left to right in a 5' to 3' orientation. Amino acid sequences are written from left to right in amino to carboxy orientation. The headings set forth herein are not intended to limit various aspects of the disclosure that may appear with reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

As used herein, the term cytotoxic T lymphocyte antigen 4 or CTLA-4 refers to a receptor that is a member of the immunoglobulin superfamily that is expressed by activated T cells and conveys an inhibitory signal to T cells. CTLA-4 is homologous to the T cell co-stimulatory protein, CD28, and both molecules bind to CD80 and CD86, also called B7-1 and B7-2, respectively, on antigen-presenting cells. CTLA4 is also found in regulatory T cells and contributes to its inhibitory function. CTLA-4 is also called cytotoxic T-lymphocyte-associated protein 4, CD152, insulin-dependent diabetes mellitus 12 (IDDM12), celiac disease 3 (CELIAC3), GRD4 and GSE. The term CTLA-4 includes any variants or isoforms of CTLA-4 that are naturally expressed by cells.

As used herein, the term T cell is defined as a thymus-derived lymphocyte that participates in various cellular immune responses. As used herein, the term regulatory T cell refers to a CD4+CD25+Foxp3+ T cell with suppressive properties. T _reg is the abbreviation used herein for regulatory T cell.

As used herein, the term helper T cell refers to a CD4+ T cell; helper T cells recognize antigen bound to MHC class II molecules. There are at least two types of helper T cells, Th1 and Th2, which produce different cytokines. Helper T cells become CD25+ when activated, but only transiently become Foxp3+.

As used herein, the term cytotoxic T cell refers to a CD8+ T cell; cytotoxic T cells recognize antigen bound to MHC class I molecules.

The term antibody refers to immunoglobulin molecules and the immunologically active parts of immunoglobulin (Ig) molecules, i.e. molecules that contain an antigen binding site that specifically binds (immunoreacts to) an antigen. By specific binding or immunoreaction with or immunospecific binding 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 to them with much lower affinity (Kd>10 ^-6 ). Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, domain antibodies, single chain, Fab and F(ab') ₂ fragments, scFv and Fab expression library.

It is known that the basic structural unit of an antibody contains a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, with each pair having one light (about 25 kDa) and one heavy (about 50-70 kDa) chain. The amino-terminal portion of each chain includes a variable region of about 100 to about 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines the constant region primarily responsible for effector function. In general, antibody molecules obtained from humans fall into any of the classes IgG, IgM, IgA, IgE and IgD, which differ from each other by the nature of the heavy chain present in the molecule. Some classes also have subclasses such as IgG1, IgG2 and others. Additionally, in humans, the light chain may be a kappa chain or a lambda chain.

As used herein, the term activated antibody refers to an antibody that also contains a masking fragment (MM) and a cleavable fragment (CM), where the MM is attached to the VL of the antibody via a CM that is cleaved by a protease. As used herein, the term prodomain includes the N-terminal fragment that is attached to the VL domain of activated anti-human CTLA-4 antibodies and, as such, includes MM and SM. In some embodiments, the light chain of the activated antibody has a structural arrangement from N-terminus to C-terminus as follows: MM-CM-VL. In some embodiments, the prodomain is located in the VH domain of an anti-human CTLA-4 antibody. The activated antibody is designed to cleave the activated proteolytic activity present in most, if not all cancers. Such proteolytic cleavage or activation removes the prodomain and releases the active antibody, that is, the activated, activated antibody. Activation of protease-activated antibodies in normal tissue is significantly reduced due to the tight control of proteolytic activity in normal tissues. As such, activated antibodies remain largely inert in the bloodstream and normal tissues.

An activated antibody, because its prodomain masks the antigen binding domain, thereby inhibiting the ability of the antigen binding domain to bind its target, has a lower affinity for binding to the target than an activated activated antibody in which the MM has been removed by proteolytic cleavage of the SM, thereby releasing active antibody. Such released antibody exhibits a higher binding affinity to its target. In some embodiments, MM specifically interacts with the antigen binding domain of ipilimumab to reduce the ability of the antibody to bind its target. When MM is removed by proteolytic cleavage of the activated antibody, the released antibody binds to its target with an affinity similar to the parent ipilimumab.

Schematic representations of the activated antibodies of the present invention, for example MM-CM-VL, are not intended to be limiting. Other sequence elements, such as linkers, spacers, and signal sequences, may be present before, after, or between the listed sequence elements in such diagrammatic representations. It should also be understood that the prodomain containing MM and CM may be attached to the VH of the antibody rather than to the VL of the antibody, such that the heavy chain has a structural arrangement from N-terminus to C-terminus as MM-CM-VH.

As used herein, the term monoclonal antibody (mAb) or monoclonal antibody composition refers to a population of antibody molecules that contain only one

- 10 043382 molecular species of an antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of a monoclonal antibody are identical in all molecules of the population. MAbs contain an antigen-binding site or domain that is capable of immunoreacting with a specific epitope of an antigen characterized by a unique binding affinity for it. Monoclonal antibody molecules typically contain two heavy chains and two light chains.

The term antigen-binding domain refers to that portion of the immunoglobulin molecule that is involved 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 regions in the V regions of the heavy and light chains, called hypervariable regions, are located between more conserved flanking regions known as framework regions or FRs. Thus, the term FR refers to amino acid sequences that naturally occur between and adjacent to hypervariable regions in immunoglobulins. In an antibody molecule, three hypervariable regions of the light chain and three hypervariable regions of the heavy chain are located relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each of the heavy and light chains are called complementarity determining regions or CDRs. The assignment of amino acids to each domain follows the definitions of the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)); or Chothia & Lesk J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:878-883 (1989).

As used herein, the term epitope includes any protein determinant capable of specifically binding to an immunoglobulin, scFv, or T-cell receptor. The term epitope includes any protein determinant capable of specifically binding to an immunoglobulin or T-cell receptor. Epitope determinants typically consist of reactive surface moieties of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics and specific charge characteristics. For example, antibodies can be raised against the N-terminal or C-terminal peptides of a polypeptide. An antibody is considered to specifically bind to an antigen when the dissociation constant is =1 μM; preferably =100 nM and most preferably =10 nM.

As used herein, the terms specific binding, immunological binding and immunological binding properties refer to non-covalent interactions of the type that occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (Kd) of the interaction, where a lower Kd indicates greater affinity. The immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method involves measuring the rates of formation and dissociation of an antigen-binding site/antigen complex, which rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters, which influence the rate equally in both directions. Thus, both the association rate constant (k _on ) and the dissociation rate constant (k _off ) can be determined by calculating the concentrations and actual rates of association and dissociation. (See Nature, 361:186-87 (1993)). The koff/kon ratio cancels out all non-affinity parameters and is equal to the dissociation constant Kd. (See generally Davies et al. (1990), Annual. Rev. Biochem., 59:439-473). The antibody of the present invention is believed to specifically bind to CTLA-4 when the binding equilibrium constant (Kd) is: 11 μM, preferably = 100 nM, more preferably = 10 nM, and most preferably = 100 pM to about 1 pM, as measured with using assays such as radioligand binding assays or similar assays known to those skilled in the art.

As used herein, the term isolated polynucleotide refers to a polynucleotide of genomic, cDNA or synthetic origin, or some combination thereof, which, by virtue of its isolated polynucleotide origin, (1) is not associated with all or part of the polynucleotide in which the isolated polynucleotide is found in nature, (2) functionally linked to a polynucleotide with which it is not naturally associated, or (3) not found in nature as part of a larger sequence.

The polynucleotides of the invention include nucleic acid molecules encoding heavy chain immunoglobulin molecules shown herein and nucleic acid molecules encoding light chain immunoglobulin molecules shown herein.

The term isolated protein as used herein means a protein of cDNA, recombinant RNA or synthetic origin, or some combination thereof, which by virtue of its origin or the source of the isolated protein (1) is not related to naturally occurring proteins (1). 2) does not contain other proteins from the same source, for example does not contain mouse proteins, (3) is expressed by a cell of another species, or (4) does not occur in nature.

The term polypeptide is used herein as a general term to refer to native protein, fragments or analogues of a polypeptide sequence. Therefore, native protein fragments and analogues are species of the polypeptide genus. Polypeptides in accordance with the invention include heavy chain immunoglobulin molecules shown herein and light chain immunoglobulin molecules shown herein, as well as antibody molecules formed by combinations containing heavy chain immunoglobulin molecules with light chain immunoglobulin molecules, such as immunoglobulin molecules kappa light chain, and vice versa, as well as their fragments and analogues.

The term naturally occurring is used herein to refer to an object, referring to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a natural source and that has not been intentionally modified by humans in a laboratory is naturally occurring.

As used herein, the term operably coupled refers to provisions of components that are described in such a way as to enable them to function as intended. A control sequence operably linked to the coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the control sequences.

As used herein, the term control sequence refers to polynucleotide sequences that are necessary to effect expression and processing of the coding sequences to which they are ligated. The nature of such control sequences varies depending on the host organism in prokaryotes, such control sequences typically include a promoter, a ribosome binding site and a transcription termination sequence in eukaryotes, typically such control sequences include promoters and a transcription termination sequence. The term control sequences is intended to include, at a minimum, all components whose presence is essential for expression and processing, and may also include additional components whose presence is beneficial, for example, leader sequences and fusion partner sequences. The term polynucleotide as used herein means nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides, or a modified form of any type of nucleotide. The term includes single-stranded and double-stranded forms of DNA.

As used herein, the term oligonucleotide includes naturally occurring and modified nucleotides linked together by naturally occurring and non-naturally occurring oligonucleotide linkages. Oligonucleotides are a subset of polynucleotides, typically 200 bases or less in length. Preferably, the oligonucleotides are from 10 to 60 bases in length, and most preferably are from 12, 13, 14, 15, 16, 17, 18, 19 or 20 to 40 bases in length. Oligonucleotides are usually single-stranded, for example for probes, although oligonucleotides can be double-stranded, for example for use in constructing a gene mutant. The oligonucleotides of the invention are sense or antisense oligonucleotides.

As used herein, the term naturally occurring nucleotides includes deoxyribonucleotides and ribonucleotides. As used herein, the term modified nucleotides includes nucleotides with modified or substituted sugar groups and the like. As used herein, the term oligonucleotide linkages includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphodiselenoate, phosphoroanilotioate, fosoraniladate, phosphoronmidate, and the like. See, for example, LaPlanche et al., Nucl. Acids Res. 14:9081 (1986); Stec et al., J. Am. Chem. Soc., 106:6077 (1984); Stein et al., Nucl. Acids Res. 16:3209 (1988); Zon et al., Anti-Cancer Drug Design, 6:539 (1991); Zon et al., Oligonucleotides and Analogues: A Practical Approach, p. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec et al., US Patent No. 5151510; Uhlmann, Peyman, Chemical Reviews, 90:543 (1990). If desired, the oligonucleotide may include a detection tag.

As used herein, the twenty common amino acids and their abbreviations are as used in traditional use. See Immunology - A Synthesis (2nd edition, E.S. Golub, D.R. Gren, eds., Sinauer Associates, Sunderland Mass. (1991)). Stereoisomers (e.g., D-amino acids) of twenty traditional amino acids, unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid and other non-traditional amino acids may also be

- 12 043382 suitable components for the polypeptides of the present invention. Examples of non-traditional amino acids include 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyl-lysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ- N-methylarginine and other similar amino acids and imino acids (for example, 4-hydroxyproline). In the polypeptide designation used herein, the left direction is the amino-terminal direction and the right direction is the carboxy-terminal direction according to standard usage and convention.

As applied to polypeptides, the term substantial identity means that two peptide sequences, when optimally aligned, for example using the GAP or BESTFIT programs using default gap weights, share at least 80% sequence identity, preferably at least 90 % sequence identity, more preferably at least 95% sequence identity, and most preferably at least 99% sequence identity.

As discussed herein, small variations in the amino acid sequences of antibodies or immunoglobulin molecules are considered to be covered by the present invention provided that the variations in the amino acid sequence remain at least 75%, more preferably at least 80, 90, 95%, and most preferably 99 % sequence identity. In particular, conservative amino acid substitutions are provided. Conservative substitutions are those that occur within a family of amino acids in their side chains. Genetically encoded amino acids are generally divided into the following families:

(1) acidic amino acids are aspartate, glutamate;

(2) the basic amino acids are lysine, arginine, histidine;

(3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine.

Hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine. Other families of amino acids include (i) serine and threonine, which are the aliphatic hydroxy family;

(ii) asparagine and glutamine, which are the amide-containing family;

(iii) alanine, valine, leucine and isoleucine, which are the aliphatic family; and (iv) phenylalanine, tryptophan and tyrosine, which are the aromatic family.

In the case of antibodies, it is reasonable to expect that an isolated substitution of isoleucine or valine for leucine, glutamate for aspartate, serine for threonine, or a similar amino acid substitution for a structurally related amino acid will not have a significant effect on the binding or properties of the resulting molecule, especially if the substitution does not involve an amino acid in the CDR or framework region. Whether an amino acid substitution results in a functional peptide can be readily determined by analyzing the specific activity of the derived polypeptide. The assays are described in detail herein. Fragments or analogues of antibodies or immunoglobulin molecules can be readily prepared by those skilled in the art. Preferred amino and carboxy termini of fragments or analogues occur near the boundaries of functional domains. Structural and functional domains can be identified by comparing nucleotide and/or amino acid sequence data with publicly available or proprietary sequence databases. Preferably, computational comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function. Methods for identifying protein sequences that fold into a known three-dimensional structure are known. Bowie et al., Science, 253:164 (1991). Thus, the above examples demonstrate that those skilled in the art can recognize sequence motifs and structural conformations that can be used to define structural and functional domains in accordance with the invention.

Preferred amino acid substitutions are those that (1) reduce susceptibility to proteolysis in regions of the activated antibody other than the cleavable linker containing the CM;

(2) reduce sensitivity to oxidation;

(3) alter binding affinity to form protein complexes;

(4) change binding affinity; and (5) impart or alter other physicochemical or functional properties of such analogues.

Analogues may include various muteins of sequence different from the naturally occurring peptide sequence. For example, single or multiple amino acids

- 13 043382 substitutions (preferably conservative amino acid substitutions) can be made in a naturally occurring sequence (preferably in a portion of the polypeptide outside the intermolecular junction domain(s). A conservative amino acid substitution must not significantly alter the structural characteristics of the parent sequence (eg, the substituting amino acid must not disrupt a helix that occurs in the parent sequence or disrupt other types of secondary structure that characterize the parent sequence). Examples of prior art secondary and tertiary structures of polypeptides are described in Proteins, Structures and Molecular Principles (Creighton, ed., W.H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden, J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al., Nature, 354:105 (1991).

As used herein, the term polypeptide fragment refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion and/or one or more internal deletions, but where the remaining amino acid sequence is identical to corresponding positions in a naturally occurring sequence predicted, for example, from a complete cDNA sequence . The fragments are typically at least 5, 6, 8 or 10 amino acids in length, preferably at least 14 amino acids, more preferably at least 20 amino acids, typically at least 50 amino acids, and even more preferably at least 70 amino acids. As used herein, the term analog refers to polypeptides that contain a segment of at least 25 amino acids that has substantial identity to a portion of the predicted amino acid sequence and that specifically binds to CTLA-4 under suitable binding conditions. Typically, polypeptide analogues contain a conservative amino acid substitution (or addition or deletion) with respect to the naturally occurring sequence. The analogues are typically at least 20 amino acids in length, preferably at least 50 amino acids or more, and can often be as long as a full-length naturally occurring polypeptide.

As used herein, the term agent means a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.

As used herein, the term labeled or labeled refers to the incorporation of a detectable marker, for example, by the inclusion of a radiolabeled amino acid or the attachment of biotinyl moieties to the polypeptide that can be detected by labeled avidin (eg, streptavidin containing a fluorescent marker, or enzymatic activity that can be detected by optical or calorimetric methods). In certain situations, a label or marker may also be therapeutic. Various methods for labeling polypeptides and glycoproteins are known and can be used in the art. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., ³ H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹ⁿ In, ¹²⁵ I, ¹³¹ I) fluorescent labels (e.g. FITC, rhodamine, lanthanide phosphorescents), enzyme tags (e.g., horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups, predefined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, the tags are attached with spacer groups of varying lengths to reduce potential steric hindrance.

Other chemical terms herein are used in accordance with common usage in the art, as shown in The McGraw-Hill Dictionary of Chemical Terms (Parker, S., ed., McGraw-Hill, San Francisco (1985)).

As used herein, the term substantially pure means that the species of the item is the presence of a predominant species (i.e., on a molar basis, it is more abundant than any other single species in the composition), and preferably, the substantially purified fraction is a composition that is in which the species of the object constitutes at least about 50% (on a molar basis) of the presence of all macromolecular species. Typically, a substantially pure composition will contain greater than about 80% presence of all macromolecular species in the composition, more preferably greater than about 85, 90, 95 and 99%. Most preferably, the species of the object is purified to substantial homogeneity (the contaminant species cannot be detected in the composition by conventional detection methods), where the composition consists of essentially one macromolecular species.

As used herein, the term treatment is an approach to obtain useful or desired clinical results. Beneficial or desired clinical outcomes may include, but are not limited to, any one or more of relief of one or more symptoms, reduction in the extent of disease, stabilized (ie, not worsening) disease state, prevention or delay of spread (eg, metastasis) of disease, prevention or delaying the onset or recurrence of the disease, delaying or slowing the progression of the disease, improvement of the disease and remission (partial or complete).

The term treatment also includes reducing the pathological effects of a proliferative disease such as cancer. The methods presented herein provide for any one or more of these aspects of treatment.

As used herein, the term effective amount refers to an amount of a compound or composition, when used alone or in combination with a second therapy, sufficient to treat a particular disorder, condition or disease, such as ameliorating, alleviating, reducing and/or delaying one or more of its symptoms. With respect to cancer or other unwanted cell proliferation, an effective amount includes an amount sufficient to cause a tumor to shrink and/or reduce the rate of tumor growth (such as to inhibit tumor growth) or to prevent or delay other unwanted cell proliferation. An effective amount may be administered in one or more administrations.

As used herein, the term combination therapy means that the first agent is administered in combination with another agent. In combination with refers to the use of one treatment modality in addition to another treatment modality. As such, in combination with refers to the use of one treatment before, during, or after delivery of another treatment to an individual.

As used herein, the term pharmaceutical agent or drug refers to a chemical compound or composition capable of producing a desired therapeutic effect when properly administered to a subject.

As used herein, the term pharmaceutically acceptable or pharmacologically compatible means a material that is not biologically or otherwise objectionable, e.g., the material can be included in a pharmaceutical composition administered to an individual or subject without causing any significant adverse biological effects or in a harmful manner. interacting with any other component of the composition in which it is contained. Pharmaceutically acceptable carriers or excipients, for example, meet required toxicological and manufacturing testing standards and/or are included in the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

The terms cancer, cancerous or malignant refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, for example, melanoma, such as unresectable or metastatic melanoma, leukemia, lymphoma, blastoma, carcinoma and sarcoma. More specific examples of such cancers include chronic myeloid leukemia, acute lymphoblastic leukemia, Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL), squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, glioma, gastrointestinal cancer, renal cancer, cancer ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma colon, as well as head and neck cancer, gastric cancer, germ cell tumor, childhood sarcoma, sinonasal natural killer, multiple myeloma, acute myelogenous leukemia (AML) and chronic lymphocytic leukemia (CML).

Leukemia refers to progressive malignant diseases of the hematopoietic organs and is usually characterized by distorted distribution and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is usually clinically classified based on (1) the duration and nature of the disease - acute or chronic;

(2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous) or monocytic; and (3) an increase or absence of an increase in the number of abnormal cells in the blood - leukemic or aleukemic (subleukemic).

Leukemia includes, for example, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, leukocythaemic leukemia, basophilic leukemia, undifferentiated leukemia, leukemia cattle, chronic myelocytic leukemia, hematodermatosis, embryonal leukemia, eosinophilic leukemia, Gross leukemia, hairy cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia , leukemia with lymphosarcoma cells, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Nigeli leukemia, plasma leukemia tic cells, plasmacytic leukemia, promyelocytic leukemia, Reader cell leukemia, leukemia Schilling, stem cell leukemia, subleukemic leukemia and undifferentiated cell leukemia. In certain aspects, the present invention relates to the treatment of chronic myeloid leukemia, acute lymphoblastic leukemia, and/or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL).

I. Activated antibodies against CTLA-4.

The present invention relates to improved anti-CTLA-4 antibodies that are as effective as traditional anti-CTLA-4 antibodies (eg, ipilimumab), but with higher, i.e. improved security profile. Specifically, the improved anti-CTLA-4 antibodies are activated monoclonal antibodies (mAbs) that specifically bind to human CTLA-4 upon activation. These improved anti-CTLA-4 antibodies, also referred to herein as activated anti-CTLA-4 antibodies or CTLA-4-activated antibodies, are used in methods of treating, preventing, slowing the progression, attenuating and/or alleviating a symptom of a disease or disorder, including but not limited to without limitation, a disease or disorder associated with the expression and/or activity of aberrant CTLA-4. For example, activated anti-CTLA-4 antibodies are used in methods of treating, preventing, slowing the progression, attenuating and/or alleviating symptoms of cancer or other neoplastic condition. Activated antibodies are described, for example, in US Patent No. 8513390; 8518404; 9120853; 9127053 and international publication No. WO 2016/149201.

In some embodiments, the activated anti-CTLA-4 antibodies provided herein include (i) ipilimumab or an antigen binding domain (AB) thereof, such as ipilimumab variable light chain (VL);

(ii) cleavable fragment (CM); and (iii) masking fragment (MM).

In some embodiments, VL is associated with MM such that binding to MM reduces the ability of ipilimumab to bind CTLA-4. In some embodiments, the MM is linked to the VL through a cleavable fragment (CM) (also known as a substrate linker) that includes a substrate for a protease, for example, a protease that is overexpressed in the tumor microenvironment.

Antibody or antigen-binding fragment.

In some embodiments, the antibody or antigen binding domain (AB) thereof comprises anti-CTLA-4 ipilimumab complementarity determining regions (CDRs), designated 10D1 in US Pat. Nos. 6,984,720 and 7,605,238, which are incorporated herein by reference in their entirety. Ipilimumab (also known as MDX-010 and BMS-734016) is marketed under the brand name YERVOY® and has been approved for the treatment of metastatic melanoma and is in clinical trials in other forms of cancer. See Hoos et al. (2010), Semin. Oncol 37:533; Hodi et al. (2010), N. Engl. J Med 363:711; Pardoll (2012), Nat. Immunol., 13(12):1129.

Ipilimumab has the human IgG1 isotype, which binds best to most human Fc receptors (Bruhns et al. (2009), Blood, 113:3716) and is considered equivalent to mouse IgG2a with respect to the types of activating Fc receptors it binds. Because IgG1 binds to the activating receptor CD16 (FcyRIIIa) expressed by human NK cells and monocytes, ipilimumab may mediate ADCC. The IgG1 isotype ipilimumab was initially isolated directly from a hybridoma but was subsequently cloned and expressed in Chinese hamster ovary (CHO) cells. Although the isotype that mediates ADCC and/or CDC may be undesirable in an antibody targeting a receptor on T cells that seeks to enhance the immune response, the IgG1 antibody isotype was retained in part because it enhanced vaccine response in Javanese macaque and was considered functional. Ipilimumab has been shown to increase the number of activated T cells in the blood, as evidenced, for example, by a significant increase in the expression of HLA-DR on the surface of CD4+ and CD8+ cells after treatment, as well as an increase in the absolute number of lymphocytes (Ku et al. (2010), Cancer, 116:1767; Attia et al. (2005), J. Clin. Oncol., 23:6043; Maker et al. (2005), J. Immunol., 175:7746; Berman et al. (2009), J. Clin. Oncol., 27(suppl):15s.3020; Hamid et al. (2009), J. Clin. Oncol., 27(suppl):15s.9008), indicating that T-depletion cells does not occur in the periphery in humans. Ipilimumab demonstrated only moderate levels of ADCC in activated T cells using IL-2-activated PBMCs as effector cells; however, the use of T _regs as targets has not been tested. There have been minor changes in the frequency of peripheral _Treg in the blood of patients treated with ipilimumab (Maker et al. (2005), J. Immunol., 175:7746), but little information is available on the effect of ipilimumab on intratumoral _Treg . However, a positive correlation has been described between a high CD8+ to T _reg ratio and tumor necrosis in biopsies from metastatic melanoma lesions in patients treated with ipilimumab. Hodi et al. (2008), Proc. Nat'l Acad. Sci. (USA), 105:3005. In addition, tumor tissue from bladder cancer patients treated with ipilimumab had a lower percentage of CD4+ Foxp3+ T cells than tumors from untreated bladder cancer patients. Liakou et al. (2008), Proc. Nat'l Acad. Sci.

- 16 043382 (USA), 105:14987.

In some embodiments, the activated anti-CTLA-4 antibody comprises a combination of variable heavy chain CDR1 (VH CDR1, also referred to herein as CDRH1), CDR2 (VH CDR2, also referred to herein as CDRH2), and CDR3 (VH CDR3, also referred to herein as herein CDRH3) and variable light chain CDR1 (VL CDR1, also referred to herein as CDRL1), CDR2 (VL CDR2, also referred to herein as CDRL2), and CDR3 (VL CDR3, also referred to herein as CDRL3). The sequences of these CDRs are presented in table. 2.

table 2

Heavy and light chain CDR sequences for ipilimumab

CHAIN CDR1 CDR2 CDR3 EASY RASQSVGSSYLA (SEQIDNO: 560) GAFSRAT (SEQIDNO: 561) QQYGSSPWT (SEQ ID NO: 562) HEAVY SYTMH (SEQ Ш NO: 557) FISYDGNNKYYADSV KG (SEQ ID NO: 558) TGWLGPFDY (SEQ ID NO: 559)

Ipilimumab-VL chain

EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFS RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 344)

Ipilimumab-VH chain

QVQLVESGGGVVQPGRSLRLSCAASGFFSSYTMHWVRQAPGKGLEWVTFIS YDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDY WGQGTLVTVSS (SEQ ID NO: 345) Various other sequences, as indicated, are presented below. Human LC kappa constant

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 346)

Kappa constant mouse light chain

RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVL NSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSSTSPIVKSFNRNEC (SEQ ID NO: 347)

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Ipilimumab-Kappa LC Human

EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFS RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 348)

Ipilimumab-Kappa LC Mice

EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFS RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRADA APTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSK DSTYSMSSTLTLTKDEYERHNSYTC EATHKTSSTSPIVKSFNRNEC (SEQ ID NO: 349)

Constant human IgGl NS

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 350)

Mouse IgGl constant NS

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYASTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 351)

Mouse IgG2a constant NS

AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTF PAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCP APNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQT QTHREDYNSTLRVVSALPIQHQD WMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAP QVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDG SYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO: 352)

Ipilimumab-VH-Constant NS Human IgGl

QVQLVESGGGVVQPGRSLRLSCAASGFFSSYTMHWVRQAPGKGLEWVTFIS YDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL

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TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<ALPAPIEI<T IS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 353)

Ipilimumab-VH-Constant HC IgGl mouse

QVQLVESGGGVVQPGRSLRLSCAASGFFSSYTMHWVRQAPGKGLEWVTFIS YDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ Ш NO: 354)

Ipilimumab-VH-Constant HC Mouse IgG2a

QVQLVESGGGVVQPGRSLRLSCAASGFFSSYTMHWVRQAPGKGLEWVTFIS YDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDY WGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSL SSGVHTFPAVLQSDLYTLSSSVTVTSSTW PSQSITCNVAHPASSTKVDKKIEPRGPTIKP CPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNV EVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKP KGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEW TNNGKTELNYKNTE PVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO: 355)

In some embodiments, the antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one CDR sequence contains 1, 2, 3, 4 or more differences in amino acid sequences compared to the CDR sequences shown in table. 2, including conserved amino acid differences.

In some embodiments, the activated anti-CTLA-4 antibody comprises a heavy chain variable domain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identical to the group consisting of SEQ ID NO: 345. In some embodiments, the activated anti-CTLA-4 antibody comprises a light chain variable domain, not including any MM, CM, linker, spacer, or other sequence added in the creation of the activated form of the antibody, that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the group consisting of SEQ ID NO: 563-56.

In some embodiments, the antibody or antigen binding fragment thereof that binds CTLA-4 in activated antibodies may include modifications, particularly in the Fc region of the antibody or antigen binding fragment thereof. For example, the interaction of antibodies with FcyR can be enhanced by modifying the glycan moiety attached to each Fc fragment at residue N297. Specifically, the absence of core fucose residues significantly enhances ADCC through improved IgG binding to activating FcyRIIIA without altering antigen or CDC binding. Natsume et al. (2009), Drug Des. Devel. Ther., 3:7. There is compelling evidence that afucosylated tumor-specific antibodies confer enhanced therapeutic activity in an in vivo mouse model. Nimmerjahn & Ravetch (2005), Science, 310:1510; Mossner et al. (2010), Blood, 115:4393.

Modification of antibody glycosylation can be accomplished, for example, by expressing the antibody in a host cell with a modified glycosylation mechanism. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which the recombinant antibodies of the present invention are expressed to thereby produce an altered glycosylation antibody. For example, the Ms704, Ms705 and Ms709 cell lines lack the glycosyltransferase gene, FUT8 (a-(1,6)-fucosyltransferase) (see US Patent Application Publication No. 20040110704; Yamane-Ohnuki et al. (2004), Biotechnol. Bioeng., 87:614), so that antibodies expressed in these cell lines lack fucose on their carbohydrates. As another example, EP 1176195 also describes a cell line with a functionally disrupted FUT8 gene, and cell lines that have little or no activity in adding fucose to N-acetylglucosamine, which binds to the Fc region of the antibody, on

- 19 043382 example, rat myeloma cell line YB2/0 (ATCC CRL 1662). PCT Publication WO 03/035835 describes a variant of the CHO cell line, Lec13, with a reduced ability to attach fucose to Asn(297)-linked carbohydrates, which also results in hypofucosylation of antibodies expressed in that host cell. See also Shields et al. (2002), J. Biol. Chem., 277:26733. Antibodies with a modified glycosylation profile can also be produced in chicken eggs, as described in PCT Publication No. WO 2006/089231. Alternatively, antibodies with a modified glycosylation profile can be produced in plant cells such as Lemna. See, for example, US Publication No. 2012/0276086. PCT Publication No. WO 99/54342 describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (eg, beta(1,4)-Acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased structural separation GlcNac, which leads to increased activity of ADCC antibodies. See also Umana et al. (1999), Nat. Biotech., 17:176. Alternatively, the fucose residues of the antibody can be cleaved using the enzyme fucosidase. For example, fucosyl residues from antibodies are removed by the enzyme alpha-L-fucosidase. Tarentino et al. (1975), Biochem., 14:5516. Core fucosylation can be reduced by culturing antibody-producing cells in the presence of low molecular weight fucose analogues, such as those described in EP 2282773B1, or in the presence of castanospermine, as described in WO 08/052030.

Cleavable fragment.

In some embodiments, the CM is specific for a protease that is useful for enhancing dysregulated protease activity in tumor cells for targeted activation of activated antibodies at the site of treatment and/or diagnosis. Numerous studies have demonstrated correlations in the levels of aberrant proteases, e.g., uPA, legumain, MT-SP1, and matrix metalloproteases (MMPs), in solid tumors (see, e.g., Murthy R.V. et al., Legumain expression in relation to clinicopathologic and biological variables in colorectal cancer, Clin. Cancer Res., 11(2005):2293-2299; Nielsen B. S. et al., Urokinase plasminogen activator is localized in stromal cells in ductal breast cancer, Lab. Invest., 81(2001):1485 -1501; Look O.R. et al., In situ localization of gelatinolytic activity in the extracellular matrix of metastases of colon cancer in rat liver using quenched fluorogenic DQ-gelatin, J. Histochem. Cytochem., 51(2003):821-829) .

A general overview of this process is discussed in patents No. 7666817, 8513390 and 9120853 and international publications No. WO 2016/118629 and WO 2016/149201, which are incorporated herein by reference in their entirety. The cleavable fragment selection process is used to identify cleavable fragments that have a number of desirable characteristics. For example, the selected cleavage fragments are systemically stable (i.e., stable in the subject's systemic circulation), generally not susceptible to cleavage by circulating proteases such as plasmin, thrombin, tissue plasminogen activator (tPA), or kallikrein (KLK), such as KLK -5 and/or KLK-7, non-toxic, generally not subject to degradation at potential sites of toxicity such as skin by proteases such as ADAM 9, ADAM 10, ADAM 17 and/or kallikreins such as KLK-5 and KLK -7, and are active at the intended site of treatment and/or diagnosis. In some embodiments, identified cleavage fragments are selected for proteases that are overexpressed at the intended therapeutic and/or diagnostic site but are not typically expressed on or in normal, healthy, or otherwise uninfected or undamaged tissue, and the selected substrates are then subjected to a challenge screen against proteases. , expressed in normal, for example, unaffected tissue. Typical proteases and/or enzymes are presented in table. 1, as stated earlier.

In some embodiments of the invention, the cleavable fragment is selected from the group consisting of 2001 and 3001, and derivatives thereof. In some embodiments, the cleavable fragment is selected from the group consisting of 2001 (SEQ ID NO: 297), 2006 (SEQ ID NO:

(SEQ (SEQ (SEQ (SEQ

ID

ID

ID

NO

NO

NO

ID NO:

301), 2008 (SEQ ID NO: 302), 2009 (SEQ ID NO: 303), 2012 (SEQ IDNO

304), 2003 (SEQ ID NO: 298), 3001 (SEQ ID NO: 306), 3006 (SEQ IDNO

308), 3008 (SEQ ID NO: 309), 3009 (SEQ ID NO: 310), 3012 (SEQ IDNO

300), 2007

305), 2011

313), 3007

312), 3011

311) and 2005 (SEQ ID NO: 299). In table 3 provides additional cleavable fragments that can be used with the activated anti-CTLA-4 antibodies disclosed herein.

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Table 3

Cleavable fragments of activated anti-CTLA-4

SEQUENCE ID SM sequence 313 LSGRSDNH 314 LSGRSANPRG 315 TGRGPSWV 316 PLTGRSGG 317 TARGPSFK 318 NTLSGRSENHSG 319 NTLSGRSGNHGS 320 TSTSGRSANPRG 321 TSGRSANP 322 VHMPLGFLGP 306 AVGLAPGGLSGRDSDNH 307 AVGLLAPPGGLSGRSDDH 308 AVGLLAPPGGLSGRSDIH 309 AVGLLAPPGGLSGRSDQH 310 AVGLLAPPGGLSGRSDTH 338 AVGLLAPPGGLSGRSDYH 339 AVGLAPGGLSGRSANI 340 AVGLAPGGLSGRSDNI 312 AVGLLAPPGGLSGRSANP 311 AVGLLAPPGGLSGRSDNP 299 AVGLLAPPSGRSANPRG 323 AVGLAP 324 AQNLLGMV 325 QNQALRMA 326 LAAPLGLL 327 STFPFGMF 328 ISSGLLSS 329 PAGLWLDP 330 VAGRSMRP 331 VVPEGRS 332 ILPRSPAF 333 MVLGRSLL 334 VAGRSMRP 335 QGRAITFI 336 SPRSIMLA 337 SMLRSMPL 297 ISSGLLSGRSDNH 300 ISSGLLSGRSDDH 301 ISSGLLSGRSDIH 302 ISSGLLSGRSDQH 303 ISSGLLSGRSDTH 341 ISSGLLSGRSDYH 342 ISSGLLSGRSANI 343 ISSGLLSGRSDNI 305 ISSGLLSGRSANP 304 ISSGLLSGRSDNP 298 ISSGLLSGRSANPRG

Masking fragment.

The activated anti-CTLA-4 antibodies provided herein contain a masking fragment (MM). In some embodiments, the MM is an amino acid sequence that is linked or otherwise attached to an anti-CTLA-4 antibody and is located in an activated anti-CTLA-4 antibody construct such that the MM reduces the ability of the anti-CTLA-4 antibody to specifically bind CTLA-4. In some embodiments, the MM specifically binds to the antigen binding domain. Suitable MMs are identified using any of a variety of known techniques. For example, peptide MMs are identified using methods described in Patent Applications

US Application No. 2009/0062142, Daugherty et al.; and 2012/0244154, Daugherty et al., the contents of which are incorporated herein by reference in their entirety.

In some embodiments of the invention, the MM is selected from the group consisting of YV01-YV66,

- 21 043382 and contains an amino acid sequence selected from the table below. 4.

Table 4

Anti-CTLA-4 masking fragments (MM)

RESEARCH IDENTIFICATION POSLEDOVA BODY MM RESEARCH IDENTIFICATION POSLEDOVA BODY MM 1 DFSCLHSMYNVCLDP 147 EHCDVWMFGFNLCPY 2 QPCAQMYGYSMCPHT 148 EPCDYWMFGVNLCPY 3 LHCRTQMYGYNLCPY 149 EQCTMWMYGFNLCPY 4 LHCRTQLYGYNLCPY 150 ES AC SLRMYE VCLQP 5 CTYSFFNVC 151 ESCASMYGYSMCPRT 6 CAQMYGYSMC 152 ESC S YWMFGYNLCP Y 7 CPNHPMC 153 FSNTCPHHPMCYDYR 8 GTACTYSFFNVCLDP 154 FWNTCPHHPMCHDYK 9 FGTACPNHPMCHDWQ 155 FYQNCYPPTWCSMFS 10 SACAYWMFGVNLCPY 156 GECSYWMFGYNLCPY AND CRTQLYGYNLC 157 GGSCMYSFFNICLDP 12 CRTQIYGYNLC 158 GGSCVYVMYNVCLDP 13 LHCRTQIYGYNLCPY 159 GHCLMHMYGYNLCPK 14 CPNHPMCHDWQ 160 GHCRMSMYEMTLCPR 15 GTACPNHPMCHDWQ 161 GISCVHIMFNFCLDP 16 CAYWMFGVNLCPY 162 GLCVMYMFGVNLCPY 17 QECHLYMYGVNLCPY 163 GSCDYWMFGYNLCPY 18 CHLYMYGVNLCPY 164 GSYCMYVMYNVCLDP 19 GQCQFYMFGYNLCPY 165 GTKCIYSFYNVCLDP 20 LSTCMYSFFNVCLDP 166 GTSTCPYHPMCHDYR 21 CLHSMYNVCLDP 167 GTTCTYSFFNVCLDP 22 CLHSMYNVC 168 GVCHFFMYGVSMCPA 23 CLHSLYNVCLDP 169 GVPCWYSMYNVCLDP 24 CLHSAYNVCLDP 170 GVSCMYSMFNICLDP 25 CMYSFFNVCLDP 171 HAKCVYSFFNVCLDP 26 CMYSFFNVC 172 HDSCMYSMYNFCLDP 27 QPCAQMYGYSMC 173 HGNTCPNHPMCHDYQ 28 CAQLYGYSMCPHT 174 HKGCLYSFYNICLDP 29 CAQMYGYSMCAHT 175 HKGCLYSFYNVCLDP thirty CAQMYGYSMCPAT 176 HLSCMYIMYNVCLDP

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31 CAQMYGYSMCPHT 177 HSSCIYSMFNVCLDP 32 CPNHPLCHDWQ 178 HTNMCPYHPMCYDYK 33 CPNHPMCADWQ 179 HTPCTYSFFNVCLDP 34 CPNHPMCHAWQ 180 IMNTCPYHPMCHDYQ 35 CPNHPMCHDAQ 181 IVPCTYMMFGVCLQP 36 CPNHPMCHDWA 182 KKCDYWFYGVNLCPY 37 GTACPNHPMC 183 KNTCVYSFFNVCLDP 38 LHCRTQLYGYNLC 184 KPCAQMYGYSMCPHP 39 CRTQLYGYNLCPY 185 KPSCMYSFFNVCLDP 40 CRTQLYGYNLCAY 186 KRPCMYSFYNVCLDP 41 CRTQLYGYNLCPA 187 KTSCMYSFYNICLDP 42 FGTACPNHPLCHDWQ 188 KTTCTYSFFNVCLDP 43 CPNHPLCHDFQ 189 LDCQMYWWFGACGDM 44 CPNHPLCHDYQ 190 LHCAIYMYGYNLCPF 45 CPNHPLCPY 191 LHCPFQMYGYNLCPH 46 CPNHPLCPA 192 LHC SMYM YGFNLCPN 47 CMYSFFNVCYP 193 RECMAYMYGYNLCPY 48 CMYSFFNVCYA 194 RHCQMHMFGYDLCPY 49 CLYSFFNVCYP 195 LIHCRYVMYGMCLEP 50 CLYSFFNVCYA 196 LLPCEVMGPSRCKHD 51 FGAACPNHPICHDWQ 197 LPCHAYMYGYSLCPY 52 FGAACPNHPLCHDWQ 198 LPCLAYMYGVNLCPN 53 FGAACPNHPMCHDAQ 199 LPCMAYMFGFNLCPH 54 CLHSAYNACLDP 200 LPCNFHMFGFNLCPY 55 CAHSAYNVCLDP 201 LQCAMYMYGYNLCPY 56 CLHSAYNVCADP 202 LSSCTYSFFNVCLDP 57 CLHSAYNVCLAP 203 LTCPFQMYGYNLCPY 58 CLHSAYNVCLDA 204 LTSQCSPWYWCQIYD 59 KNTCTYVMYNVCLDP 205 LYCPYMMYGYNLCPY 60 YISDCPYHPMCHDYQ 206 LYHCTYSFYNVCLDP 61 FRNTCPYHPMCHDYR 207 LYRCIYSFYNVCLDP 62 RECHMWMFGVNLCPY 208 MGC SMRMWGMELCPE 63 AVCHMYMYGYNLCPF 209 MKCDYWLYGYNLCPY 64 RSCPQMYGYSMCPHT 210 MNHCTLHMYNICMDP 65 QPCAQMFGYSMCPHT 211 MNPECPHHPMCHNSN 66 TAKCTYSFFNVCLDP 212 MPACTYSFFNICLDP 67 DFSCLYSMYNVCLDP 213 MPQCHVIMYNLCLDP 68 DVSCMYMMYNFCLDP 214 MSTCTYSFFNVCLDP 69 CPNHPMC 215 MTCNYWFYGVNLCPY 70 CMYSFFNVCPY 216 MYCHQSMFGFRMCPD 71 CMYSFFNVCPA 217 NACAQMYGYSMCPHT 72 CTYSFFNVCPY 218 NDCDISMFDQSLCPY 73 CTYSFFNVCPA 219 NF SC VYVMFNVCLDP 74 GFPCMYSMFNVCLDP 220 NFTCALTMYEVCLDP

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75 GLSCMYSMYGYCLDP 221 NLCHAFMFGFNLCPY 76 IPCDYWMFGVNLCPY 222 NLNNCPHHPMCHDYQ 77 QVCHAYMYGYNLCPY 223 NPPCMYSFFNICLDP 78 RMYCTYSFYNVCLDP 224 NSACTYSFFNVCLDP 79 ALSCMYIMYNVCLDP 225 NVCTVSMFGVMLCPS 80 DF SCMYVMFNVCLDP 226 PACATLMYSVPLCPA 81 DFSCVYSMFNVCLDP 227 PAPCMYSFYNVCLDP 82 DMNTCPNHPMCYDYR 228 PLCAEMYGYSMCPHN 83 DMNTCPRHPMCHDYH 229 PQCHLYMYGYNLCPY 84 DSRCMYVMYNVCLDP 230 PRPCMYSFYNVCLDP 85 EHLCTYSFYNVCLDP 231 QHCPFQMYGYNLCPY 86 ELSCVYSMFGFCLDP 232 QHCQMHMFGYNLCPY 87 FTNNCPYHPMCHDYL 233 QHSCMYSFFNVCLDP 88 GF SCT YIM YD VCLDP 234 QKCHSYLYGVNLCPY 89 GSS CM YSMYN VCLDP 235 QKCNMFMFGYNLCPY 90 HF S CM YIM YN VCLDP 236 QMNDCPNHPMCHDYH 91 LHCGMWMFGVNLCPK 237 QPCAQMYGYSMCPAT 92 LPCQMWMFGHNLCPH 238 QPCAQMYGYSMCPRT 93 LPCTMYMYGYNLCPY 239 RECHFFFFYGVNLCPY 94 LTCHHWMFGVNLCPY 240 LNCGMFMYGYNLCPY 95 NFSCMYSMFNVCLDP 241 RLCTSYMFGYNLCPQ 96 NNHCMYSFFNICLDP 242 RL S CM YSMFN VCLDP 97 NRSCMYIMYNVCLDP 243 RNCPFVMFGVNLCPY 98 NSCTMFMFGVNLCPY 244 RNGCMYSFFN VCLDP 99 NTCELYMFGVNLCPY 245 RNGCVYSFFNVCLDP 100 QHCDMWMFGYNLCPY 246 RPCHLYMFGYNLCPD 101 QHCPMYMFGYNLCPF 247 RPCHSYMYGINLCPY 102 QVCHIQMYGFDLCPH 248 RSCDMIMFGFNLCPY 103 RACDYWMYGVNLCPY 249 RSCPMWFYGVNLCPY 104 RQCHMQMFGYDLCPF 250 RSTVCFYDFCGPWER 105 S GS CL YSFYN VCLDP 251 RTCHFYMYGVNLCPY 106 SNGCTYSFFNVCLDP 252 RTC SMVMFGVNLCP Y 107 STCAQMYGYSMCPH 253 SGKCTYSFFNVCLDP 108 SYKCLYSFYNVCLDP 254 SIVCDLYWEATCLRP 109 VLYCTYVMYN VCLDP 255 SLSCTYSFFNICLDP 110 VNCGMWMFGYNLCPK 256 SMNTCPYHPMCFDYK 111 YGSCLYSFYNICLDP 257 SQCWMWMYGYNLCPK 112 YPCAQMYGYSMCPHT 258 SSSCMYSFFNVCLDP FROM AACDLWMFGVNLCPY 259 STACTYSFYNVCLDP 114 AFCTLAPYNQACIAN 260 STCAQMYGYSMCPHT 115 AGS CL YSMYN VCLDP 261 STRCVYSFYNVCLDP 116 ALCENTMYGYHLCPW 262 TACGAWMFGVNLCPY 117 ALSCMYIMYGVCLDP 263 TGACMYSFYNVCLDP 118 APVCDVLMFGFCMQP 264 TLS CM YSMYN VCLDP

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119 AQVCSIMMYGTCLMP 265 TSCTTVMYQISMCPY 120 ASTCMYSFYNVCLDP 266 VGGCRHSFYNVCLDP 121 AVCEFWMFGFNLCPY 267 VHCQMYMYGYNLCPY 122 DANTCPNHPMCYDYH 268 VHNCMYSFFNVCLDP 123 DFSCIYIMFDVCLDP 269 VMCKLHMYGIPVCPK 124 DF SCMYVMYGFCLDP 270 VNFCNYSMYGICLLP 125 DFTCMYSMYNVCLDP 271 VNFCYACYCMSCVFS 126 DFTCTYSMYNVCLDP 272 VNQCTYSFFNVCLDP 127 DHYCTYIMYSICLDP 273 VPCPFHMFGYNLCPY 128 DICTNFMFGVNLCPY 274 VRCQMWMYGFNLCPH 129 DINTCPYHPMCHDYH 275 VRPCTYSFFNVCLDP 130 DKNTCPLHPMCHDYR 276 VSGCTYSFFNICLDP 131 DMNMCPNHPMCHDWH 277 YCSSWDTMTIPACNN 132 DMNSCPNHPMCHDYH 278 YDCDLSMFGIEMCPQ 133 DMNSCPNHPMCYDYR 279 YGNTCPFHPMCHDYK 134 DMNTCPNHPMCFDYR 280 YGYCMYSFFNVCLDP 135 DMNTCPNHPMCHDFQ 281 YHCTMHMFGYNLCPF 136 DMNTCPNHPMCHDYR 282 YMNTCPNHPMCFDYQ 137 DMNTCPNHPMCYDYH 283 YMNTCPYHPMCHDYL 138 DMNTCPNHPMCYDYK 284 YMNTCPYHPMCHDYR 139 DMSTCPNHPMCHDYM 285 YNNCTYSFFNVCLDP 140 DRNMCPYHPMCYDYR 286 YPGCQYSFFNVCLDP 141 DSCAFMFGVNLCPY 287 YRSCTHIMYNVCLDP 142 DSCRSVFDMVWNCWN 288 YSFCDMLMYDVCLVP 143 DTPNCPHHPMCHNHM 289 YSIDCGLSWWCGGMT 144 DVSCLYVMYSVCLDP 290 YSTTCPYHPMCHDYH 145 DWCASMMFGYNLCPY 291 YVNTCPHHPMCHDYH 146 EFSCMYSMFNVCLDP 292 YVNTCPYHPMCHDYN

In some embodiments, the Kd of an activated anti-CTLA-4 antibody comprising a MM disclosed herein on target is at least 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, 1000 times greater or 5-10, 10-100, 10-200, 10-500, 10-1000 times greater than Kd AB, unmodified MM or parent AB relative to the target.

In some embodiments, MM is not a natural binding partner of the antibody being activated. In some embodiments, the MM contains no or substantially no homology to any natural binding partner of the antibody being activated. In some embodiments, the MW is no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80% identical to any natural binding partner of the antibody being activated. In some embodiments, the MW is no more than 50%, 25%, 20%, or 10% identical to any natural binding partner of the antibody being activated. In some embodiments, the MM is no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80% identical to human CTLA-4. In some embodiments, the MM is no more than 50, 25, 20, or 10% identical to human CTLA-4.

Typical activated antibodies against CTLA-4.

The specific antibodies described herein are activated anti-CTLA-4 antibodies containing any combination of masking fragments, cleavable fragments, light chain variable domains (VL) (or corresponding CDRs) and heavy chain variable domains (VH) (or corresponding CDRs) ), described in table. 2-6. In some embodiments, the activated anti-CTLA-4 antibody comprises a light chain comprising YV01 (SEQ ID NO: 1) as a masking fragment, LSGRSDNH (SEQ ID NO: 313) as a cleavable fragment, and an ipilimumab light chain variable domain (VL) (SEQ ID NO: 344). In some embodiments, the activated anti-CTLA-4 antibody comprises a light chain comprising YV01 (SEQ ID NO: 1) as a mask fragment, ISSGLLSGRSDNH (2001) (SEQ ID NO: 297) as a cleavable fragment, and a light chain variable domain CDR (VL) ipilimumab (SEQ ID NO: 560, 561 and 562, respectively). In some embodiments, the activated anti-CTLA-4 antibody comprises the heavy chain variable domain (VH) of ipilimumab (SEQ ID NO: 345) or only the corresponding CDRs (SEQ ID NOs: 557, 558 and 559).

In some embodiments, the activated anti-CTLA-4 contains YV39 (SEQ ID NO: 39) as a masking fragment and ISSGLLSGRSDNP (2011) (SEQ ID NO: 304) as a cleavable fragment, as well as ipilimumab heavy and light chain variable domains ((SEQ ID NO: 345 and 344, respectively) where MM and CM are associated with VL in the arrangement MM-CM-VL.

In some embodiments, the activated anti-CTLA-4 antibody includes a signal peptide. The signal peptide can be linked to the activated anti-CTLA-4 antibody using a spacer. In some embodiments, the spacer is conjugated to an activated antibody in the absence of a signal peptide. In some embodiments of the invention, spe

- 25 043382 ser is connected directly to the MM of the activated antibody. In some embodiments, the spacer has the amino acid sequence QGQSGS (SEQ ID NO: 546). In some embodiments, the activated antibody comprises a spacer sequence QGQSGS (SEQ ID NO: 546) linked directly to the MM sequence CRTQLYGYNLCPY (YV39) (SEQ ID NO: 39) in the structural arrangement N-terminal to C-terminal spacer-MM- CM-VL or spacerMM-CM-AV.

In some embodiments, the activated anti-CTLA-4 antibody comprises a linker peptide (LP) between MM and SM. In some embodiments, the activated anti-CTLA-4 antibody comprises a linker peptide between the CM and the antibody or antigen binding domain (AB) thereof. In some embodiments, the activated anti-CTLA-4 antibody comprises a first linker peptide (LP1) and a second linker peptide (LP2), wherein the activated anti-CTLA-4 antibody has a structural arrangement from N-terminus to C-terminus as follows: MM-LP1 -CM-LP2-AB. In some embodiments, the light chain of the activated anti-CTLA-4 antibody has a structural arrangement from N-terminus to C-terminus as follows: MM-LP1-CM-LP2-VL. In some embodiments, the two linker peptides need not be identical to each other. Examples of linker peptides that can be used with activated anti-CTLA-4 antibodies as described herein are provided in US Patent Publication No. 2016/0193332 and International Publication WO 2016/149201, ibid.

The invention also provides a modified anti-CTLA-4 antibody that contains an MM that is attached to the light chain of the antibody via a non-protease-cleavable linker. In some embodiments, the non-protease-cleavable linker comprises the amino acid sequence set forth in SEQ ID NO: 570. In some embodiments, such a modified anti-CTLA-4 antibody has a light chain containing YV39 and a non-protease-cleavable linker. In some embodiments, the light chain of the modified anti-CTLA-4 antibody comprises the amino acid sequence

QGQSGSCRTQLYGYNLCPYGGGSSGGSGGSGGSGGGSGGGSGGSEIVLTQSPGTLSL SPGERATLSCRASQS VGS S YLAWYQQKPGQ APRLLIYGAF SRATGIPDRF SGSGSGTD FTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ GO NO: 530) or

CRTQLYGYNLCPYGGGSSGGSGGSGGSGGGSGGGSGGSEIVLTQSPGTLSLSPGERAT LSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRL EPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ GO NO: 531).

Linkers suitable for use in the compositions described herein are generally linkers that provide flexibility to the activated anti-CTLA-4 antibody to facilitate inhibition of binding of the activated antibody to the target. Such linkers are commonly referred to as flexible linkers (also referred to herein as linker peptides). Suitable linkers can be easily selected and can be of any suitable varying length, such as 1 amino acid (eg Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 to 10 amino acids , 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length.

Exemplary flexible linkers include glycine polymers (G) _n , glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (GSGGS is SEQ ID NO: 534) and (GGGS)n (GGGS is SEQ ID NO: 535), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and therefore may be able to serve as a neutral bond between components. Glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem., 11173-142 (1992)). Exemplary flexible linkers include, but are not limited to, Gly-Gly-Ser-Gly (SEQ ID NO: 536), Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 537), Gly-Ser-Gly-Ser-Gly ( SEQ ID NO: 538), Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 539), Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 540), Gly-Ser-Ser-Ser-Gly (SEQ ID NO: 541), etc. One of ordinary skill in the art will appreciate that the design of the activated antibodies may include linkers that are fully or partially flexible, such that the linker may include a flexible linker as well as one or more portions that impart a less flexible structure to provide the structure of the desired activated antibodies.

In some embodiments, the activated anti-CTLA-4 antibodies include the VL and VH (or corresponding CDRs) of ipilimumab and the combination of MM and SM presented in Table below. 5, so any MM in column 2 can be combined with any MM in column 4.

Table 5

Combinations of activated anti-CTLA-4 antibodies

SEQ ID NO. Masking Fragment (MM) SEQ ID NO. Cleavable Fragment (CM) 1 (YV01) DFSCLHSMYNVCLDP 313 LSGRSDNH 2 (YV02)QPCAQMYGYSMCPHT 314 LSGRSANPRG 3 (YV03) LHCRTQMYGYNLCPY 315 TGRGPSWV 4 (YV04) LHCRTQLYGYNLCPY 316 PLTGRSGG 5 (YV05) CTYSFFNVC 317 TARGPSFK 6 (YV06) CAQMYGYSMC 318 NTLSGRSENHSG 7 (YV07) CPNHPMC 319 NTLSGRSGNHGS 8 (YV08) GTACTYSFFNVCLDP 320 TSTSGRSANPRG 9 (YV09)FGTACPNHPMCHDWQ 321 TSGRSANP 10 (YV10) SACAYWMFGVNLCPY 322 VHMPLGFLGP AND (YV11)CRTQLYGYNLC 323 AVGLAP 12 (YV12)CRTQIYGYNLC 324 AQNLLGMV 13 (YV13) LHCRTQIYGYNLCPY 325 QNQALRMA 14 (YV14) CPNHPMCHDWQ 326 LAAPLGLL 15 (YV15) GTACPNHPMCHDWQ 327 STFPFGMF 16 (YV16) CAYWMFGVNLCPY 328 ISSGLLSS 17 (YV17)QECHLYMYGVNLCPY 329 PAGLWLDP 18 (YV18)CHLYMYGVNLCPY 330 VAGRSMRP 19 (YV19)GQCQFYMFGYNLCPY 331 VVPEGRS 20 (YV20)LSTCMYSFFNVCLDP 332 ILPRSPAF 21 (YV21)CLHSMYNVCLDP 333 MVLGRSLL 22 (YV22)CLHSMYNVC 334 VAGRSMRP 23 (YV23)CLHSLYNVCLDP 335 QGRAITFI 24 (YV24)CLHSAYNVCLDP 336 SPRSIMLA 25 (YV25) CMYSFFNVCLDP 337 SMLRSMPL 26 (YV26) CMYSFFNVC 297 ISSGLLSGRSDNH 27 (YV27)QPCAQMYGYSMC 300 ISSGLLSGRSDDH 28 (YV28)CAQLYGYSMCPHT 301 ISSGLLSGRSDIH 29 (YV29)CAQMYGYSMCAHT 302 ISSGLLSGRSDQH thirty (YV30) CAQMYGYSMCPAT 303 ISSGLLSGRSDTH 31 (YV31) CAQMYGYSMCPHT 341 ISSGLLSGRSDYH 32 (YV32) CPNHPLCHDWQ 342 ISSGLLSGRSANI 33 (YV33) CPNHPMCADWQ 343 ISSGLLSGRSDNI 35 (YV34) CPNHPMCHAWQ 305 ISSGLLSGRSANP 35 (YV35) CPNHPMCHDAQ 304 ISSGLLSGRSDNP 36 (YV36) CPNHPMCHDWA 298 ISSGLLSGRSANPRG 37 (YV37) GTACPNHPMC 306 AVGLAPGGLSGRDSDNH 38 (YV38) LHCRTQLYGYNLC 307 AVGLLAPPGGLSGRSDDH 39 (YV39)CRTQLYGYNLCPY 308 AVGLLAPPGGLSGRSDIH 40 (YV40) CRTQLYGYNLCAY 309 AVGLLAPPGGLSGRSDQH 41 (YV41) CRTQLYGYNLCPA 310 AVGLLAPPGGLSGRSDTH 42 (YV42)FGTACPNHPLCHDWQ 338 AVGLLAPPGGLSGRSDYH

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43 (YV43) CPNHPLCHDFQ 339 AVGLAPGGLSGRSANI 44 (YV44) CPNHPLCHDYQ 340 AVGLAPGGLSGRSDNI 45 (YV45) CPNHPLCPY 312 AVGLLAPPGGLSGRSANP 46 (YV46) CPNHPLCPA 311 AVGLLAPPGGLSGRSDNP 47 (YV47) CMYSFFNVCYP 299 AVGLLAPPSGRSANPRG 48 (YV48) CMYSFFNVCYA 49 (YV49) CLYSFFNVCYP 50 (YV50) CLYSFFNVCYA 51 (YV51)FGAACPNHPICHDWQ 52 (YV52)FGAACPNHPLCHDWQ 53 (YV53)FGAACPNHPMCHDAQ 54 (YV54)CLHSAYNACLDP 55 (YV55) CAHSAYNVCLDP 56 (YV56)CLHSAYNVCADP 57 (YV57)CLHSAYNVCLAP 58 (YV58)CLHSAYNVCLDA 59 (YV60) KNTCTYVMYNVCLDP 60 (YV61)YISDCPYHPMCHDYQ 61 (YV62)FRNTCPYHPMCHDYR 62 (YV63)RECHMWMFGVNLCPY 63 (YV64) AVCHMYMYGYNLCPF 64 (YV65)RSCPQMYGYSMCPHT 65 (YV66)QPCAQMFGYSMCPHT

In some embodiments, the activated anti-CTLA-4 antibodies include the specific combination of MM and SM presented in table. 6 below.

Table 6

Typical Combination of Activated Anti-CTLA-4 Antibody

Comb. No. Masking Fragment (MM) Cleavable Fragment (CM) 1 CRTQLYGYNLCPY (SEQIDNO: 39) ISSGLLSGRSDNH (SEQ ID NO: 297) 2 CRTQLYGYNLCPY (SEQ Ш NO: 39) ISSGLLSGRSDNP (SEQ ID NO: 304) 3 CRTQLYGYNLCPY (SEQ ID NO: 39) ISSGLLSGRSANP (SEQIDNO: 305) 4 CRTQLYGYNLCPY (SEQIDNO: 39) ISSGLLSGRSDQH (SEQ ID NO: 302) 5 CRTQLYGYNLCPY (SEQIDNO: 39) ISSGLLSGRSDDH (SEQ ID NO: 300) 6 CRTQLYGYNLCPY (SEQ ID NO: 39) ISSGLLSGRSDTH (SEQIDNO: 303) 7 LHCRTQMYGYNLCPY ISSGLLSGRSDNH

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(SEQ Ш NO: 3) (SEQ Ш NO: 297) 8 LHCRTQMYGYNLCPY (SEQ ID NO: 3) AVGLLAPPGGLSGRSDNH (SEQ Ш NO: 306) 9 LHCRTQMYGYNLCPY (SEQ Ш NO: 3) ISSGLLSGRSDDH (SEQ Ш NO: 300) 10 LHCRTQMYGYNLCPY (SEQ Ш NO: 3) ISSGLLSGRSDIH (SEQ Ш NO: 301) AND LHCRTQMYGYNLCPY (SEQ Ш NO: 3) ISSGLLSGRSDQH (SEQ Ш NO: 302) 12 LHCRTQMYGYNLCPY (SEQ Ш NO: 3) ISSGLLSGRSDTH (SEQ Ш NO: 303) 13 CAQMYGYSMC (SEQ ID NO: 06) ISSGLLSGRSDNH (SEQ Ш NO: 297) 14 CAQMYGYSMC (SEQ ID NO: 06) AVGLLAPPGGLSGRSDNH (SEQ Ш NO: 306) 15 FGTACPNHPMCHDWQ (SEQ ID NO: 09) ISSGLLSGRSDNH (SEQ Ш NO: 297) 16 FGTACPNHPMCHDWQ (SEQ ID NO: 09) AVGLLAPPGGLSGRSDNH (SEQ Ш NO: 306) 17 CLHSLYNVCLDP (SEQ ID NO: 23) ISSGLLSGRSDNH (SEQ Ш NO: 297) 18 CLHSLYNVCLDP (SEQ ID NO: 23) ISSGLLSGRSDDH (SEQ Ш NO: 300) 19 CLHSLYNVCLDP (SEQ ID NO: 23) ISSGLLSGRSDIH (SEQ Ш NO: 301) 20 CLHSLYNVCLDP (SEQ ID NO: 23) ISSGLLSGRSDQH (SEQ ID NO: 302) 21 CLHSLYNVCLDP (SEQ ID NO: 23) ISSGLLSGRSDTH (SEQ Ш NO: 303) 22 CLHSLYNVCLDP (SEQ ID NO: 23) AVGLLAPPGGLSGRSDNH (SEQ Ш NO: 306) 23 CLHSAYNVCLDP (SEQ ID NO: 24) ISSGLLSGRSDNH (SEQ Ш NO: 297) 24 CLHSAYNVCLDP (SEQ ID NO: 24) AVGLLAPPGGLSGRSDNH (SEQ Ш NO: 306) 25 QPCAQMYGYSMC (SEQ ID NO: 27) ISSGLLSGRSDNH (SEQ Ш NO: 297) 26 QPCAQMYGYSMC (SEQ ID NO: 27) AVGLLAPPGGLSGRSDNH (SEQ Ш NO: 306) 27 CAQMYGYSMCAHT (SEQ ID NO: 29) ISSGLLSGRSDNH (SEQ Ш NO: 297) 28 CAQMYGYSMCAHT (SEQ ID NO: 29) AVGLLAPPGGLSGRSDNH (SEQ Ш NO: 306) 29 CPNHPLCHDWQ ISSGLLSGRSDNH

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(SEQ Ш NO: 32) (SEQ ID NO: 297) thirty CPNHPLCHDWQ (SEQ ID NO: 32) AVGLLAPPGGLSGRSDNH (SEQ ID NO: 306) 31 CPNHPMCADWQ (SEQ Ш NO: 33) ISSGLLSGRSDNH (SEQ ID NO: 297) 32 CPNHPMCADWQ (SEQ ID NO: 33) AVGLLAPPGGLSGRSDNH (SEQ Ш NO: 306) 33 CPNHPMCHDAQ (SEQ Ш NO: 35) ISSGLLSGRSDNH (SEQ ID NO: 297) 34 CPNHPMCHDAQ (SEQ Ш NO: 35) AVGLLAPPGGLSGRSDNH (SEQ ID NO: 306) 35 CRTQLYGYNLCPY (SEQ Ш NO: 39) AVGLLAPPGGLSGRSDNH (SEQ ID NO: 306) 36 CRTQLYGYNLCPA (SEQ Ш NO: 41) ISSGLLSGRSDNH (SEQ ID NO: 297) 37 CRTQLYGYNLCPA (SEQ Ш NO: 41) AVGLLAPPGGLSGRSDNH (SEQ ID NO: 306) 38 FGAACPNHPICHDWQ (SEQ Ш NO: 51) ISSGLLSGRSDNH (SEQ ID NO: 297) 39 FGAACPNHPICHDWQ (SEQ Ш NO: 51) AVGLLAPPGGLSGRSDNH (SEQ ID NO: 306) 40 FGAACPNHPLCHDWQ (SEQ ID NO: 52) ISSGLLSGRSDNH (SEQ ID NO: 297) 41 FGAACPNHPLCHDWQ (SEQ ID NO: 52) AVGLLAPPGGLSGRSDNH (SEQ Ш NO: 306) 42 FGAACPNHPMCHDAQ (SEQ ID NO: 53) ISSGLLSGRSDNH (SEQ ID NO: 297) 43 FGAACPNHPMCHDAQ (SEQ ID NO: 53) AVGLLAPPGGLSGRSDNH (SEQ ID NO: 306) 44 CLHSAYNACLDP (SEQ ID NO: 54) ISSGLLSGRSDNH (SEQ ID NO: 297) 45 CLHSAYNACLDP (SEQ ID NO: 54) AVGLLAPPGGLSGRSDNH (SEQ ID NO: 306) 46 CAHSAYNVCLDP (SEQ ID NO: 55) ISSGLLSGRSDNH (SEQ ID NO: 297) 47 CAHSAYNVCLDP (SEQ ID NO: 55) AVGLLAPPGGLSGRSDNH (SEQ ID NO: 306) 48 CLHSAYNVCADP (SEQ ID NO: 56) ISSGLLSGRSDNH (SEQ ID NO: 297) 49 CLHSAYNVCADP (SEQ ID NO: 56) AVGLLAPPGGLSGRSDNH (SEQ Ш NO: 306) 50 CLHSAYNVCLAP (SEQ ID NO: 57) ISSGLLSGRSDNH (SEQ ID NO: 297) 51 CLHSAYNVCLAP AVGLAPGGLSGRDSDNH (SEQ ID NO: 57) (SEQ Ш NO: 306) 52 CLHSAYNVCLDA (SEQ ID NO: 58) ISSGLLSGRSDNH (SEQ Ш NO: 297) 53 CLHSAYNVCLDA (SEQ ID NO: 58) AVGLLAPPGGLSGRSDNH (SEQ Ш NO: 306) 54 YISDCPYHPMCHDYQ (SEQ ID NO: 60) ISSGLLSGRSDNH (SEQ Ш NO: 297) 55 FRNTCPYHPMCHDYR (SEQ Ш NO: 61) ISSGLLSGRSDNH (SEQ Ш NO: 297) 56 AVCHMYMYGYNLCPF (SEQ ID NO: 63) ISSGLLSGRSDNH (SEQ Ш NO: 297) 57 RSCPQMYGYSMCPHT (SEQ ID NO: 64) ISSGLLSGRSANP (SEQ Ш NO: 305) 58 QPCAQMFGYSMCPHT (SEQ ID NO: 65) ISSGLLSGRSANP (SEQ Ш NO: 305)

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In some embodiments, the activated anti-CTLA-4 antibodies described herein also include an agent conjugated to the activated antibody. In some embodiments, the conjugate agent is a therapeutic agent, such as an antitumor agent. In some embodiments, the agent is conjugated to a carbohydrate moiety of the activated antibody, preferably when the carbohydrate moiety is located outside the antigen binding region of the antibody or antigen binding moiety in the activated antibody. In some embodiments, the agent is conjugated to a sulfhydryl group of an antibody or antigen-binding moiety in an activated antibody. In some embodiments, the agent is conjugated to the amino group of an antibody or antigen-binding fragment of an activated antibody. In some embodiments, the agent is conjugated to a carboxylic acid group of an antibody or an antigen binding moiety of an activated antibody.

In some embodiments, the agent is a cytotoxic agent, such as a toxin (eg, an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (ie, a radioconjugate).

In some embodiments, the conjugated activation antibody may be modified for site-specific conjugation through modified amino acid sequences inserted or otherwise included in the sequence of the activation antibody. These modified amino acid sequences are intended to provide controlled placement and/or dosage of the conjugated agent in the conjugated, activated anti-CTLA-4 antibody. For example, an activated antibody can be designed to include cysteine substitutions at positions on the light and heavy chains that provide reactive thiol groups and do not adversely affect protein folding and assembly and alter antigen binding. In some embodiments, the activation antibody may be designed to include or otherwise introduce one or more non-naturally occurring amino acid residues in the activation antibody to provide suitable sites for conjugation. In some embodiments, the activated antibody may be designed to include or otherwise introduce enzymatically activated peptide sequences into the sequence of the activated antibody.

In some embodiments, the agent is a detectable moiety, such as, for example, a tag or other marker. For example, the agent is or includes a radiolabeled amino acid, one or more biotinyl groups that can be detected using labeled avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods), one or several radioisotopes or radionuclides, one or more fluorescent tags, one or more enzyme tags and/or one or more chemiluminescent agents. In some embodiments of the invention, the detectable fragments are joined using linker molecules.

Antibody-cytotoxic agent conjugates can be prepared using a variety of bifunctional protein binding agents such as N-succinimidyl-3-(2-pyridyldithiol)npropionate (SPDP), iminothiolane (IT), bifunctional imidoester derivatives (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (such as bis(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene-2,6diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is a typical chelating agent for radionucleotide-antibody conjugation (see WO 94/11026).

Those skilled in the art will appreciate that a large number of possible fragments may be associated with the resulting antibodies of the invention (see, e.g., Conjugate Vaccines, Contributions to Microbiology and Immunology, J.M. Cruse, R.E. Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

II. Applications of activated antibodies against CTLA-4.

The therapeutic compositions of the invention, which include an activated anti-CTLA-4 antibody, are used to prevent, treat, or otherwise alleviate a disease or disorder, including, but not limited to, a disease or disorder associated with aberrant expression and/or activity of CTLA-4. For example, therapeutic compositions of the invention that include an activated anti-CTLA-4 antibody are used as cancer immunotherapy, for example, to enhance the endogenous immune response in a subject suffering from cancer, thereby treating the subject, where the method includes administering to the subject a therapeutically effective amount of any of the activated anti-CTLA-4 antibodies described herein.

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Examples of cancers that can be treated using the immunotherapeutic methods of the present invention include bone cancer, pancreatic cancer, skin cancer, head or neck cancer, breast cancer, lung cancer, cutaneous or intraocular malignant melanoma, unresectable or metastatic melanoma, cancer kidney, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, carcinoma esophagus, small bowel cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, hematological malignancy, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney cancer or ureter, renal pelvis cancer, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal cord tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell carcinoma, environmentally induced cancer, including asbestos-related cancers, metastatic cancers, and any combination of these cancers. In some embodiments, the cancer is selected from MEL, RCC, squamous NSCLC, non-squamous NSCLC, CRC, CRPC, squamous cell carcinoma of the head and neck, and carcinomas of the esophagus, ovary, gastrointestinal tract, and breast. The present methods are also useful for the treatment of metastatic cancers.

Other cancers include hematologic malignancies including, for example, multiple myeloma, B-cell lymphoma, Hodgkin's lymphoma/primary B-cell mediastinal lymphoma, non-Hodgkin's lymphomas, acute myeloid lymphoma, chronic myelogenous leukemia, chronic lymphoid leukemia, follicular leukemia, diffuse B -large cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, B-lymphoblastic leukemia from progenitor cells, mantle cell lymphoma, acute lymphoblastic leukemia, fungoid mycosis, anaplastic large cell lymphoma, T-cell lymphoma, T-lymphoblastic leukemia from progenitor cells and any combinations of these types of cancer.

It is known that increased proteolysis is a hallmark of cancer (see, for example, Affara N.I. et al., Delineating protease functions during cancer development, Methods Mol. Biol., 539(2009):1-32). Tumor progression, invasion and metastasis are the result of several interdependent processes in which proteases are involved. This process is generally described in US Publication No. 2016/0193332 A1, which is incorporated in its entirety.

In some embodiments of these methods of treating a subject suffering from cancer, the activated antibodies of the present invention, for example, activated ipilimumab, are administered to the subject as monotherapy. In some embodiments, stimulation or blockade of immunomodulatory targets can be effectively combined with standard cancer treatment modalities, including chemotherapy regimens, radiation, surgery, hormonal deprivation, and angiogenesis inhibitors. The activated anti-CTLA-4 antibody may be associated with an antitumor agent (as an immunoconjugate) or may be administered separately from the agent. In the latter case (separate administration), the antibody may be administered before, after, or simultaneously with the agent, or may be administered in conjunction with other known therapeutic agents. Chemotherapy drugs include, but are not limited to, doxorubicin (ADRIAMYCIN®), cisplatin, carboplatin, bleomycin sulfate, carmustine, chlorambucil (LEUKERAN®), cyclophosphamide (CYTOXAN®; NEOSAR®), lenalidomide (REVLIMID®), bortezomib (VELCADE®), dexamethasone, mitoxantrone, etoposide, cytarabine, bendamustine (TREANDA®), rituximab (RITUXAN®), ifosfamide, vincristine (ONCOVIN®), fludarabine (FLUDARA®), thalidomide (THALOMID®), alemtuzumab (CAMRATH®, ofatumumab (ARZERRA®) everolimus (AFINITOR®, ZORTRESS®) and carfilzomib (KYPROLISTM) Coadministration of anticancer agents that act through different mechanisms may help overcome the development of drug resistance or changes in tumor cell antigenicity.

The activated anti-CTLA-4 antibodies of the present invention, such as activated ipilimumab, can also be used in combination with other immunomodulatory agents, such as antibodies against other immunomodulatory receptors or their ligands. Several other co-stimulatory and inhibitory receptors and ligands have been identified that regulate T cell responses. Examples of stimulatory receptors include inducible T-cell costimulator (ICOS), CD137 (4-1BB), CD134 (OX40), CD27, glucocorticoid-induced TNFR-related protein (GITR) and herpes virus entry mediator (HVEM), while examples of inhibitory receptors include programmed death-1 (PD-1), programmed death ligand-1 (PD-L1), B and T lymphocyte attenuator (BTLA), T cell immunoglobulin and mucin domain-3 (TIM-3), gene-3 lymphocyte activation receptor (LAG-3), adenosine receptor A2a (A2aR), killer cell lectin-like receptor G1 (KLRG-1), natural killer cell receptor 2B4 (CD244), CD160, T cell immunoreceptor with Ig and ITIM domains (TIGIT ) and receptor for Ig suppressor V activation domain

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T cells (VISTA). Mellman et al. (2011), Nature, 480:480; Pardoll (2012), Nat. Rev. Cancer, 12:252; Baitsch et al. (2012), PloS One, 7:e30852.

The anti-PD-1 antibodies OPDIVO® (nivolumab) and KEYTRUDA® (pembrolizumab), as well as the anti-PD-L1 antibody TECENTRIQ® (atezolizumab), have been approved for use in the treatment of cancer and can be combined with the activated anti-CLTA-4 antibody of the present invention. for example, activated by ipilimumab. These receptors and their ligands provide targets for therapeutic agents designed to stimulate, or prevent suppression, of the immune response, thereby attacking tumor cells. Weber (2010), Semin. Oncol., 37:430; Flies et al. (2011), Yale J. Biol. Med., 84:409; Mellman et al. (2011), Nature, 480:480; Pardoll (2012), Nat. Rev. Cancer, 12:252. Stimulatory receptors or receptor ligands are targets for agonists, whereas inhibitory receptors or receptor ligands are targets for blocking agents. One of the most promising approaches to enhance immunotherapeutic antitumor activity is blockade of so-called immune checkpoints, which refer to a variety of inhibitory signaling pathways that regulate the immune system and are critical for maintaining self-tolerance and modulating the duration and amplitude of physiological immune responses in peripheral tissues for in order to minimize damage to collateral tissues. See, for example, Weber (2010), Semin. Oncol., 37:430; Pardoll (2012), Nat. Rev. Cancer, 12:252. Because many of the immune checkpoints are initiated by ligand-receptor interactions, they can be easily blocked by antibodies or modulated by recombinant forms of the ligands or receptors.

Antibodies against PD-1 suitable for the invention.

Any anti-PD-1 antibody that is known in the art can be used in the methods described herein. In particular, various human monoclonal antibodies that specifically bind to PD-1 with high affinity have been disclosed in US Pat. No. 8,008,449. Each of the humanized anti-PD-1 antibodies disclosed in US Pat. No. 8,008,449 has been demonstrated to have one or several of the following characteristics:

(a) binds to human PD-1 with a K _D of 1x10 ^-7 M or less, as determined by surface plasmon resonance using the Biacore biosensor system;

(b) practically does not bind to human C28, CTLA-4 or ICOS;

(c) increases T cell proliferation in a mixed lymphocyte response (MLR) assay;

(d) increases interferon-γ production in the MLR assay;

(e) increases IL-2 secretion in the MLR assay;

(f) binds to human PD-1 and cynomolgus PD-1;

(g) inhibits the binding of PD-L1 and/or PD-L2 to PD-1;

(h) stimulates antigen-specific memory responses;

(i) stimulates antibody responses; and (j) inhibits tumor cell growth in vivo.

Anti-PD-1 antibodies used in the present invention include monoclonal antibodies that specifically bind to human PD-1 and exhibit at least one, in some embodiments at least five, of the preceding characteristics.

Other anti-PD-1 monoclonal antibodies have been described, for example, in US Patent Nos. 6808710, 7488802, 8168757 and 8354509, US Publication No. 2016/0272708 and PCT Publications No. WO 2012/145493, WO 2008/156712, WO 2015/1129 00, WO 2012/145493, WO 2015/112800, WO 2014/206107, WO 2015/35606, WO 2015/085847, WO 2014/179664, WO 2017/020291, WO 2017/020858, WO 20 16/197367, WO 2017/024515, WO 2017/025051, WO 2017/123557, WO 2016/106159, WO 2014/194302, WO 2017/040790, WO 2017/133540, WO 2017/132827, WO 2017/024465, WO 2 017/025016, WO 2017/106061, each of which is incorporated by reference in its entirety.

In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab (also known as OPDIVO®; previously designated 5C4, BMS-936558, MDX-1106, or ONO-4538), pembrolizumab (Merck, also known as KEYTRUDA® , lambrolizumab and MK-3475; see WO 2008156712 A1), PDR001 (Novartis; see WO 2015/112900), MEDI-0680 (AstraZeneca; AMP-514; see WO 2012/145493), REGN-2810 (Regeneron; see WO 2015/112800), JS001 (TAIZHOU JUNSHI PHARMA; see Si-Yang Liu et al., J. Hematol. Oncol., 10:136 (2017)), BGB-A317 (Beigene; see WO 2015/ 35606 and US 2015/0079109), INCSHR1210 (SHR-1210; Jiangsu Hengrui Medicine; see WO 2015/085847; Si-Yang Liu et al., J. Hematol. Oncol., 10:136 (2017)), TSR- 042 (ANB011; Tesaro Biopharmaceutical; see WO 2014/179664), GLS-010 (WBP3055; Wuxi/Harbin Gloria Pharmaceuticals; see Si-Yang Liu et al., J. Hematol. Oncol., 10:136 (2017) ), AM-0001 (Armo), STI-1110 (Sorrento Therapeutics; see WO 2014/194302), AGEN2034 (Agenus; see WO 2017/040790) and MGD013 (Macrogenics).

In one embodiment of the invention, the anti-PD-1 antibody is nivolumab. Nivolumab is a fully human IgG4 antibody (S228P), an immune checkpoint inhibitor of PD-1 that selectively prevents interaction with PD-1 ligands (PD-L1 and

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PD-L2), thereby blocking the suppression of antitumor function of T cells (US Patent No. 8008449;

Wang et al., 2014, Cancer Immunol. Res., 2(9):846-56).

In another embodiment, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab is a humanized IgG4 monoclonal antibody directed against the human cell surface receptor PD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab is described, for example, in US patent No. 8354509 and 8900587; see also www.cancer.gov/drugdictionary?cdrid=695789 (last accessed December 14, 2014). Pembrolizumab has been approved by the FDA for the treatment of relapsed or refractory melanoma.

Anti-PD-1 antibodies used in the disclosed methods also include isolated antibodies that specifically cross-bind to human PD-1 and compete for human PD-1 binding with any anti-PD-1 antibody disclosed herein, e.g., nivolumab (See for example US Patent Nos. 8008449 and 8779105; WO 2013/173223). In some embodiments, the anti-PD-1 antibody binds to the same epitope as any of the anti-PD-1 antibodies described herein, such as nivolumab. The ability of antibodies to cross-compete for binding to an antigen indicates that these monoclonal antibodies bind to the same epitope region of the antigen and sterically interfere with the binding of other cross-competing antibodies to that particular epitope region. These cross-competing antibodies are expected to have functional properties very similar to those of a reference antibody, such as nivolumab, due to their binding to the same PD-1 epitope region. Cross-competing antibodies can be readily identified based on their ability to cross-compete with nivolumab in standard PD-1 binding assays such as the Biacore assay, ELISA assays or flow cytometry (see, for example, WO 2013/173223).

In specific embodiments, antibodies that cross-compete for binding to human PD-1 with or bind to the same epitope region of the anti-human PD-1 antibody, nivolumab, are monoclonal antibodies. For administration to human subjects, these cross-competing antibodies are chimeric antibodies, engineered antibodies, or humanized antibodies, or human antibodies. Such chimeric, engineered, humanized or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

Anti-PD-1 antibodies used in the methods of the disclosed invention also include antigen binding portions of the above antibodies. It has been shown that the antigen-binding function of an antibody can be carried out by fragments of a full-length antibody.

Anti-PD-1 antibodies suitable for use in the disclosed methods or compositions are antibodies that bind to PD-1 with high specificity and affinity, block the binding of PD-L1 and/or PD-L2, and inhibit the immunosuppressive effect of the PD-1 signaling pathway. 1. In any of the compositions or methods disclosed herein, the anti-PD-1 antibody includes an antigen binding moiety or fragment that binds to the PD-1 receptor and exhibits functional properties similar to those of whole antibodies in inhibiting ligand binding and activating the immune system. In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof competes with nivolumab for binding to human PD-1.

Antibodies against PD-L1 suitable for the invention.

Any anti-PD-L1 antibody can be used in the methods of the present invention. Examples of anti-PD-L1 antibodies useful in the methods of the present invention include those disclosed in US Pat. No. 9,580,507. Each of the human anti-PD-L1 monoclonal antibodies disclosed in US Pat. No. 9,580,507 has been demonstrated to have one or more of the following characteristics:

(a) binds to human PD-L1 with a KD of 1x10' ⁷ M or less, as determined by surface plasmon resonance using the Biacore biosensor system;

(b) increases T cell proliferation in a mixed lymphocyte response (MLR) assay;

(c) increases interferon-γ production in the MLR assay;

(d) increases IL-2 secretion in the MLR assay;

(e) stimulates antibody responses; and (f) reverses the effect of T regulatory cells on effector and/or dendritic cells of T cells.

Anti-PD-L1 antibodies useful in the present invention include monoclonal antibodies that specifically bind to human PD-L1 and exhibit at least one, in some embodiments at least five, of the preceding characteristics.

In specific embodiments, the anti-PD-L1 antibody is selected from the group consisting of BMS-936559 (formerly 12A4 or MDX-1105; see, for example, US Pat. No. 7,943,743 and WO 2013/173223), MPDL3280A (also known as RG7446, atezolizumab and TECENTRIQ®; US 8217149; see also Herbst et al. (2013), J. Clin. Oncol. 31(suppl):3000), durvalumab (IMFINZI™; MEDI-4736; Astra- 34 043382

Zeneca; see WO 2011/066389), avelumab (Pfizer; MSB-0010718C; BAVENCIO®; see WO 2013/079174),

STI-1014 (Sorrento; see WO 2013/181634), CX-072 (CytomX; see WO 2016/149201), KN035 (3D

Med/Alphamab; see Zhang et al., Cell Discov., 7:3 (March 2017), LY3300054 (Eli Lilly Co.; see e.g.

WO 2017/034916) and CK-301 (Checkpoint Therapeutics; see Gorelik et al., AACR: abstract 4606 (Apr 2016)).

In specific embodiments, the anti-PD-L1 antibody is atezolizumab (TECENTRIQ®). Atezolizumab is a fully humanized anti-PD-L1 IgG1 monoclonal antibody.

In specific embodiments, the anti-PD-L1 antibody is durvalumab (IMFINZI™). Durvalumab is a human IgG1 kappa monoclonal antibody against PD-L1.

In specific embodiments, the anti-PD-L1 antibody is avelumab (BAVENCIO®). Avelumab is a human IgG1 lambda monoclonal antibody against PD-L1.

In other embodiments, the anti-PD-L1 monoclonal antibody is selected from the group consisting of 28-8, 28-1, 28-12, 29-8, 5H1, and any combination thereof.

Anti-PD-L1 antibodies used in the disclosed methods also include isolated antibodies that specifically bind to human PD-L1 and compete for binding to human PD-L1 with any anti-PD-L1 antibody disclosed herein, e.g. atezolizumab and/or avelumab. In some embodiments, the anti-PD-L1 antibody binds to the same epitope as any of the anti-PD-L1 antibodies described herein, for example, atezolizumab and/or avelumab. The ability of antibodies to cross-compete for binding to an antigen indicates that these antibodies bind to the same epitope region of the antigen and sterically interfere with the binding of other cross-competing antibodies to that particular epitope region. These cross-competing antibodies are expected to have functional properties very similar to those of the reference antibody, such as atezolizumab and/or avelumab, due to their binding to the same PD-L1 epitope region. Cross-competing antibodies can be readily identified based on their ability to cross-compete with atezolizumab and/or avelumab in standard PD-L1 binding assays such as the Biacore assay, ELISA assays or flow cytometry (see, for example, WO 2013/173223) .

In specific embodiments, antibodies that compete for binding to human PD-L1 or bind to the same epitope region of a human anti-PD-L1 antibody as atezolizumab and/or avelumab are monoclonal antibodies. For administration to human subjects, these cross-competing antibodies are chimeric antibodies, engineered antibodies, or humanized antibodies or human antibodies. Such chimeric, engineered, humanized or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

Antibodies against PD-L1 used in the methods of the disclosed invention also include antigen binding portions of the above antibodies. It has been shown that the antigen-binding function of an antibody can be carried out by fragments of a full-length antibody.

Anti-PD-L1 antibodies suitable for use in the disclosed methods or compositions are antibodies that bind to PD-L1 with high specificity and affinity, block PD-1 binding, and inhibit the immunosuppressive effect of the PD-1 signaling pathway. In any of the compositions or in any of the methods disclosed herein, the anti-PD-L1 antibody includes an antigen binding moiety or fragment that binds to PD-L1 and exhibits functional properties similar to those of whole antibodies in inhibiting receptor binding and upregulating the immune system . In certain embodiments, the anti-PD-L1 antibody or antigen-binding portion thereof cross-competes with atezolizumab and/or avelumab for binding to human PD-L1.

The effectiveness of preventing, ameliorating or treating is determined in combination with any known method of diagnosing or treating a disease or disorder, including, but not limited to, a disease or disorder associated with aberrant expression and/or activity of CTLA-4. Increasing the lifespan of a subject or otherwise delaying the progression of a disease or disorder, including, but not limited to, a disease or disorder associated with aberrant expression and/or activity of CTLA-4 in the subject, indicates that the activated antibody provides a clinical benefit.

It is understood that therapeutic entities in accordance with the invention will be administered with suitable carriers, excipients and other agents that are formulated to provide improved transport, delivery, tolerability and the like. Many suitable formulations can be found in a reference book known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, Pa. (1975)), particularly in Chapter 87 by Blaug, Seymour. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (such as Lipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in and water-in emulsions. -oil, carbowax emulsions (polyethylene glycols of various molecular weights), semi-solid gels and semi-solid mixtures containing carbowax. Any of the above mixtures may be suitable for treatment and therapy in accordance with the present invention, provided that the active ingredient in the composition is not inactivated by the composition and the composition is physiologically compatible and tolerated by the route of administration. See also Baldrick P., Pharmaceutical excipient development: the need for preclinical guidance, Regul. Toxicol. Pharmacol., 32(2):210-8 (2000); Wang W., Lyophilization and development of solid protein pharmaceuticals, Int. J. Pharm., 203(1-2):1-60 (2000); Charman W.N., Lipids, lipophilic drugs, and oral drug delivery—some emerging concepts, J. Pharm. Sci., 89(8):967-78 (2000); Powell et al., Compendium of excipients for parenteral formulations. PDA J. Pharm. Sci Technol., 52:238-311 (1998) and references therein for additional information relating to formulations, excipients and carriers well known to pharmaceutical chemists.

Activated anti-CTLA-4 antibodies can be administered in the form of pharmaceutical compositions. The principles and considerations associated with the preparation of such compositions and recommendations for the selection of components are given, for example, in Remington: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro et al., editors), Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, vol. 4), 1991, M. Dekker, New York.

The composition may also contain more than one active compound that is needed for the particular indication being treated, preferably one that has additional activities that do not adversely affect each other. Alternatively or in addition, the composition may contain an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent or growth inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the intended purpose.

Active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation or interfacial polymerization techniques, e.g. hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g. liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions.

Formulations used for in vivo administration must be sterile. This is easily achieved by filtration through sterile filter membranes.

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, these matrices being in the form of shaped articles such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactides (US Pat. No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-P-glutamate, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres formulated from lactic acid-glycolic acid copolymer leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene vinyl acetate and lactic acid-glycolic acid allow molecules to be released for more than 100 days, some hydrogels release proteins over shorter periods of time.

In some embodiments of the invention, the activated antibody contains a detectable label. An intact antibody or fragment thereof (eg Fab, scFv or F(ab) ₂ ) may be used. The term labeled with respect to a probe or antibody is intended to cover the direct labeling of a probe or antibody by binding (i.e., physical binding) of a detectable substance to the probe or antibody, as well as the indirect labeling of a probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin so that it can be detected using fluorescently labeled streptavidin. The term biological specimen is intended to include tissues, cells and body fluids isolated from a subject and tissues, cells and fluids present in the subject. Therefore, the term biological sample includes blood and its fraction or component, including blood serum, blood plasma or lymph. For example, the antibody may be labeled with a radioactive marker, the presence and location of which in a subject can be detected by standard imaging techniques.

III. Pharmaceutical compositions.

The activated anti-CTLA-4 antibodies of the invention (also referred to herein as active compounds) and their derivatives, fragments, analogs and homologues can be included in pharmaceutical compositions suitable for administration. Such compositions typically contain an activated antibody and a pharmaceutically acceptable carrier. As used herein, the term pharmaceutically acceptable carrier is intended to include any and all solutions

- 36 043382 agents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, the standard reference text in the field, which is incorporated herein by reference. Preferred 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 can also be used. The use of such media and agents for pharmaceutically active substances is known in the art. Unless the conventional media or agent is incompatible with the active compound, their use in pharmaceutical compositions is possible. Additional active compounds may also be included in the compositions.

The pharmaceutical composition of the invention is formulated so that it is compatible with the intended route of administration. Examples of routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (ie, topical), transmucosal, and rectal. Solutions or suspensions used for parenteral, intradermal or subcutaneous use may include the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; and agents to adjust tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. The parenteral drug may be contained in ampoules, single-use syringes, or multiple-dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable vehicles include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be liquid to such an extent that it can be easily used in a syringe. It must be stable under the conditions of production and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg glycerin, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Proper flow can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved using various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases it will be preferable to include isotonic agents in the composition, for example, sugars, polyhydric alcohols such as mannitol, sorbitol, or sodium chloride. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injection solutions can be prepared by incorporating the active compound in the required amount in a suitable diluent with one or a combination of the ingredients listed above, as required, followed by filter sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains the basic dispersion medium and the necessary other ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying (lyophilization), which yield a powder of the active ingredient plus any additional required ingredient from a pre-sterile filtered solution.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser that contains a suitable propellant, for example a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be done via the transmucosal or transdermal route. For transmucosal or transdermal administration, the formulation uses penetrants suitable to penetrate the barrier they are intended to penetrate. 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 achieved through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated as ointments, rubs, gels or creams, generally

- 37 043382 known in this field of technology.

The activated antibodies of the present invention can also be administered subcutaneously in combination with agents to facilitate the administration of large volumes at a single site (agents for dispersing drugs in the intercellular space), such as soluble neutral active hyaluronidase glycoproteins (sHASEGP), for example, water-soluble glycoproteins PH-20 hyaluronidases such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Some exemplary sHASEGPs and their uses, including rHuPH20, are described in US Patent Publications No. 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

The compounds may also be formulated as suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In some embodiments, the active compounds are formulated with carriers that will protect the compound from rapid elimination from the body, such as controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. Methods for preparing such compositions will be apparent to those skilled in the art. Materials may also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (including liposomes targeting infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. They can be prepared in accordance with methods known to those skilled in the art, for example, as described in US Pat. No. 4,522,811.

It is particularly preferred to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Unit dosage form, as used herein, refers to physically discrete units suitable as unitary dosages for mammalian subjects to be treated; each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect, in combination with the required pharmaceutical carrier. The specification of the dosage forms of the invention is dictated by and directly depends on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and on the limitations inherent in the nature of the preparation of such active compound for the treatment of individuals.

The pharmaceutical compositions may be included in a container, package or dispenser along with instructions for administration.

Embodiments of the present disclosure may be further defined by reference to the following non-limiting examples, which describe in detail the preparation of specific antibodies of the present invention and methods of using the antibodies of the present invention. Those skilled in the art will appreciate that many modifications, both materials and methods, can be made without departing from the scope of the present disclosure.

The invention will be further described in the following examples, which do not limit the scope of the invention as described in the claims.

Examples

Example 1: Identification of Masking Fragments for Activable Anti-CTLA-4 Antibody.

To identify masking fragments (MMs) that reduce the binding of anti-CTLA-4 antibodies to their target protein, anti-CTLA-4 antibody (i.e., ipilimumab) was used to screen peptide libraries using methods similar to those described in PCT International Publication No. WO 2009/025846, WO 2010/081173 and WO 2016/149201, the contents of which are hereby incorporated by reference in their entirety. Screening consisted of two rounds of magnetically activated cell sorting (MACS) purification followed by three rounds of fluorescence-activated cell sorting (FACS).

Initial MACS purification was performed using Dynabeads® protein-A (Invitrogen) and anti-CTLA-4 antibody at a concentration of 100 nM. Approximately 10 ¹¹ cells were screened for binding and 6x106 cells were collected. A second MACS purification was performed using DYNABEADS® streptavidin (Thermo Fisher Scientific) and biotinylated anti-CTLA-4 antibody at a concentration of 100 nM. The eluate from the initial MACS purification was expanded, approximately 10 ¹¹ cells were tested for binding, and approximately 10 ⁷ cells were collected. The yield from the previously described MACS purification was subjected to successive rounds of FACS sorting with decreasing concentrations of antiCTLA-4 labeled with Alexa Fluor® 488 (Thermo Fisher Scientific). Labeled anti-CTLA4 antibodies were used at concentrations of 10, 1 nM, and 200 pM for the first, second, and third sorts, respectively. Individual peptide clones from the third sort were identified using

- 38043382 sequences and subsequently tested for their ability to bind anti-CTLA4 antibody.

Two peptide consensus sequences were selected for affinity maturation:

XXCXXXMYGYNLCPY (SEQ ID NO: 554) and XXXCXHSMYNVCLDP (SEQ ID NO: 555).

Affinity maturation libraries were constructed from these consensus sequences as described in Table 1 . 7. Lines 1 and 3 represent the consensus sequence, and lines 2 and 4 represent the nucleotide sequences encoding the peptide libraries that were inserted into the display system using a method similar to that described in PCT International Publication No. WO 2010/081173, ibid.

Table 7

Maturation libraries

1 X X WITH X X X m Y G Y N L WITH R Y 2 NN NN TG NN NN NN NT TW GG KW AA st TG SS TA TO TO WITH TO TO TO T T G T T G WITH G T 3 X X X WITH X n S m Y N V WITH L D R 4 NN NN NN TG NN NW A.G. NT TW AA NT TG st GA SS TO TO TO WITH TO T T T T T T WITH T T T

Maturation libraries were screened in a manner similar to that described for the naïve libraries described above. Screening consisted of one round of MACS and subsequent rounds of FACS triage. MACS was performed using DYNABEADS® protein-A (Thermo Fisher Scientific) and anti-CTLA-4 antibody at 100 nM. For MACS 10 ¹¹ cells were tested for binding and approximately 10 ⁸ cells were selected. The MACS eluate was expanded and approximately 10 ¹¹ cells were subjected to successive rounds of FACS sorting with decreasing concentrations of Alexa Fluor® 488 labeled anti-CTLA4 antibody. Labeled anti-CTLA4 antibodies were used at concentrations of 100, 20, 5, 1, and 1 nM for the first, second, third, fourth, and fifth sorts, respectively. Individual peptide clones of the fourth and fifth sorts were identified by sequence analysis and subsequently tested for their ability to bind anti-CTLA4 antibody. The sequences of the anti-CTLA-4 masking fragments identified using the methods described above are presented in table. 4 and 5. Four consensus sequences can be obtained from the mask sequences listed in Table. 4 and 5.

Consensus 1. C(L/M/V/T)Y(S/V/I)(F/L/M/A)(Y/F)N(V/I)CLDP (SEQ ID NO: 566)

Consensus 2. CAQMYGYSMC(P/A)(H/R/A)T (SEQ ID NO: 567)

Consensus 3. CX(M/VY/L/N/F)(Y/W/F/Q/T)(M/Y)YG(Y/V/F)(N/D)LCP(Y/F) (SEQ ID NO: 568)

Consensus 4. (N/T)(S/T/M/A)CP(N/Y)HP(M/L)C(H/F/Y)D(Y/FAV) (SEQ ID NO: 569)

Example 2: Construction and characterization of activated anti-muCTLA-4 antibodies.

To demonstrate proof of concept that activated anti-CTLA-4 antibodies can be used to treat tumors, six activated mouse anti-CTLA-4 antibodies (based on clone 9D9) were constructed using techniques similar to those described in Examples 1 and 3 of this document. These antibodies contain either MY11 or MY03 as a masking fragment and a cleavable fragment 0003 having the amino acid sequence TSTSGRSANPRG (SEQ ID NO: 320), 1004 having the amino acid sequence AVGLLAPP (SEQ ID NO: 323) or 2001 having the amino acid sequence ISSGLLSGRSDNH ( SEQ ID NO: 297). All antibodies were of the mouse IgG2a isotype. Mouse monoclonal antibody against CTLA-4 (9D9) (9D9 mg2a) and human anti-diphtheria toxin antibody with mIgG2a isotype (mg2a) were used as controls.

On day 0, BALB/c mice were injected subcutaneously with ^1x106 CT26 tumor cells. The administration of various antibodies began on day 7 after tumor implantation. Before administration, tumor size was measured and mice were randomized to different treatment groups to have comparable mean tumor volumes (eg, 39-44 mm ³ ). Tumors were measured with calipers in two dimensions, and tumor volume was calculated as

Lx(W2/2),

L=length (longest of 2 dimensions),

W=width.

Mice were then injected intraperitoneally (i.p.) with the designated antibody (eg, 25 μg/dose). Tumor volume was measured twice a week. On day 12 after tumor implantation, some mice from each group were sacrificed, and tumors and spleens were collected for immunomonitoring to examine the effect of antibodies on T-cell populations. Some or all of the remaining mice from the different groups were used for subsequent pharmacokinetics (PK) and/or pharmacodynamics (PD) analyses.

As shown in FIG. 1A, mice that received an unrelated mouse IgG2a antibody (ie, human anti-diphtheria toxin antibody) lacked tumor control. In contrast, as shown in FIG. 1C, mice that received an activated mouse anti-CTLA-4 antibody (containing MY11 as a masking fragment and 2001 as a cleavable fragment) had tumor size controlled almost identically to mice that received an anti-CTLA-4 mAb (9D9) mice (Fig. 1B). These data demonstrate that a tumor-specific protease can cleave the cleaved fragment, resulting in removal of the masking fragment and binding of the released antibody to its target protein.

To determine whether activated anti-mouse CTLA-4 antibodies are active in the periphery, the proliferation and activity of Foxp3+ regulatory T cells was determined in the spleen, and the number of regulatory T cells was determined in tumor samples for comparison, as described in Example 5, infra. In accordance with the data of Fig. 1B and 1C, all activated anti-CTLA-4 antibodies behave similarly to the mouse anti-CTLA-4 mAb (9D9) in the tumor (Fig. 2A). In contrast, the activated antibodies resembled the unrelated mouse IgG2a antibody in the spleen (Fig. 2B and 2C). These data suggest that the masking fragment-containing prodomain of the activated mouse anti-CTLA-4 antibody remains intact and binds to the antibody in the spleen, blocking antibody activity, while the prodomain is cleaved by tumor-specific proteases to produce fully active anti-CTLA-4 antibody in the tumor .

Example 3: Construction of activated antibodies against human CTLA-4.

Activated anti-CTLA4 antibodies containing an anti-CTLA4 masking fragment, a cleavable fragment, and an anti-CTLA4 antibody (eg, ipilimumab) of the invention were prepared according to methods similar to those described in PCT publication No. WO 2009/025846, ibid.; WO 2010/081173, ibid; and WO 2016/118629, ibid. Activable anti-CTLA4 antibodies were expressed in EXPI293™ cells (Thermo Fisher Scientific) and purified by protein A chromatography (MabSelect SuRe, GE Healthcare) according to the manufacturers' protocols. Quality control of the resulting activated antibodies showed that most contained at least 95% monomer.

To evaluate the utility of the activated anti-CTLA-4 antibodies described herein in human settings, the antibodies were generated as human IgG1 heavy chain (Hc) (hIgG1) and human kappa light chain (Lc) (hK). All activated antibodies contain the antibody or its antigen binding domain of ipilimumab. The cleavable fragment was selected from the group consisting of the cleavable fragment designated herein as 2001 and containing the sequence ISSGLLSGRSDNH (SEQ ID NO: 297), and its derivatives and the cleavable fragment designated herein as 3001 and comprising the sequence AVGLLAPPGGLSGRSDNH (SEQ ID NO: 306), and its derivatives. In some embodiments, the cleavable fragment was selected from the group consisting of ISSGLLSGRSDNH (SEQ ID NO: 297), also referred to herein as 2001; ISSGLLSGRSDDH (SEQ ID NO: 300), also referred to herein as 2006; ISSGLLSGRSDIH (SEQ ID NO: 301), also referred to herein as 2007; ISSGLLSGRSDQH (SEQ ID NO: 302), also referred to herein as 2008; ISSGLLSGRSDTH (SEQ ID NO: 303), also referred to herein as 2009; ISSGLLSGRSANP (SEQ ID NO: 305), also referred to herein as 2012; ISSGLLSGRSDNP (SEQ ID NO: 304), also referred to herein as 2011; ISSGLLSGRSANPRG (SEQ ID NO: 298), also referred to herein as 2003; AVGLLAPPGGLSGRSDNH (SEQ ID NO: 306), also referred to herein as 3001; AVGLLAPPGGLSGRSDDH (SEQ ID NO: 307), also referred to herein as 3006; AVGLLAPPGGLSGRSDIH (SEQ ID NO: 308), also referred to herein as 3007; AVGLLAPPGGLSGRSDQH (SEQ ID NO: 309), also referred to herein as 3008; AVGLLAPPGGLSGRSDTH (SEQ ID NO: 310), also referred to herein as 3009; AVGLLAPPGGLSGRSANP (SEQ ID NO: 312), also referred to herein as 3012; AVGLLAPPGGLSGRSDNP (SEQ ID NO: 311), also referred to herein as 3011; and AVGLLAPPSGRSANPRG (SEQ ID NO: 299), also referred to herein as 2005. The masking fragment was selected from the group of masking fragments presented in table. 4 and 5. In some embodiments, the masking fragment was CRTQLYGYNLCPY (SEQ ID NO: 39), referred to herein as YV39. Some of the activated anti-CTLA-4 antibodies also included spacer sequences and/or linker peptides.

Example 4: In vitro characterization of activated anti-human CTLA-4 antibodies.

To evaluate the ability of activated antibodies to bind CTLA-4 in the absence of protease activity, an enzyme-linked immunosorbent assay (ELISA) was used to determine binding affinity. Briefly, Nunc MaxiSorp® plates were coated overnight at 40°C with 100 μl/well of a solution of 1 μg/ml human CTLA-4 protein (Sino Biological) in PBS, pH 7.4. The plates were then washed three times with PBST (PBS, pH 7.4, 0.05% Tween-20) and the wells were blocked with 200 μl/well of 10 mg/ml bovine serum albumin (BSA) in PBST for 2 h at room temperature. After this, the plates were washed three more times with PBST. The activated antibodies were then serially diluted as shown in the table below. 8.

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Table 8

Serial dilution of activated anti-CTLA-4 antibodies for binding assay

[Antibody] = nM Columns 1-3 [activated antibody 1] = nM Columns 4-6 [activated antibody 2] = nM Columns 7-9 [activated antibody 3] = nM Columns 10-12 A 10 1000 1000 1000 IN 3.33 333 333 333 WITH 1D1 111 111 111 D 0.37 37 37 37 E 0.123 12.3 12.3 12.3 F 0.041 4.1 4.1 4.1 G 0.0137 1.34 1.34 1.34 N .0046 0.45 0.45 Empty

In this example, the highest concentration used for the parent antibody and the activated antibodies was 10 and 100 nM, respectively. However, concentrations can be increased or decreased to obtain fully saturated binding curves for activated antibodies with stronger or weaker masking.

Diluted antibodies were added to the plates and incubated for 1 h at room temperature. The plates were then washed three times with PBST. 100 μl of goat anti-human IgG (Fab-specific, Sigma cat #A0293; diluted 1:4000 in 10 mg/ml BSA in PBST) was then added to each well and the plate was incubated for an additional 1 h at room temperature. The plates were then treated with tetramethylbenzidine (TMB) and 1N. HCl. Absorbance at 450 nm was then measured and reported as optical density (OD 450 nm).

As shown in FIG. 3A-3E, activated anti-CTLA-4 antibodies generally had reduced binding to CTLA-4 compared to ipilimumab (YV1). See also FIG. 4A-4D, 5A-5F and 6A, 6B. Such data demonstrate that masking moieties effectively hide the antigen binding domain on activated anti-CTLA-4 antibodies.

To further evaluate binding capacity, human activated anti-CTLA-4 antibodies were serially diluted (eg, 60 to 0.0003 μg/ml) and added to 58 a-e-CTLA-4/CD3Z cells that stably express human CTLA-4. After 30 min of incubation at 4°C, allophycocyanin (APC)-labeled anti-human antibody was added and activated anti-human CTLA-4 antibodies were coupled to human CTLA-4 using a Canto flow cytometer. Geometric mean fluorescence intensity (GMFI) was determined using FlowJo® analysis software. Ipilimumab was used as a control. As shown in FIG. 7A and 7B, the activated anti-human CTLA-4 antibodies did not bind to human CTLA-4 as efficiently as ipilimumab. These data further demonstrate that, in the absence of specific proteases, the masking fragment of activated antibodies inhibits the binding of such activated antibodies to human CTLA-4.

To confirm that the reduced binding observed with the activated anti-CTLA-4 antibodies was due to the masking moiety, studies were performed on mono-truncated, fully truncated MMP, and fully truncated uPA forms of the activated antibody containing YV39 as the masking fragment and 2011 as a fissile fragment. A mono-truncated form of the antibody was generated by expressing a construct producing one intact light chain (including the masking fragment) and a second light chain truncated at the same position as if it had been cleaved by MMP14. Fully truncated forms of MMP or uPA were expressed from constructs with both light chains truncated as if they had been cleaved by MMP or pPA, respectively. As shown in FIG. 7C and 7D, the activated monotruncated antibody had intermediate binding (EC ₅₀ =2.8 nM) compared to the activated non-truncated antibody (EC ₅₀ =22 nM) and ipilimumab (EC ₅₀ =0.54 nM). In contrast, fully truncated MMP or uPA activated antibodies behaved similarly to ipilimumab (truncated MMP: _EC50 =0.65 nM; truncated pPA: EC50 =0.76). Such data confirm that the reduced binding observed with the activated anti-CTLA-4 antibody is due to the masking moiety.

Next, to determine whether the observed reduced binding to CTLA-4 correlated with reduced activity, the activity of an activated human anti-CTLA-4 antibody containing YV39 as a masking fragment and 2011 as a cleavable fragment (Ipi YV39 2011) was characterized in in vitro functional assay using staphylococcal enterotoxin B (SEB). SEB is a superantigen that potently activates T cells and stimulates cytokine secretion. Whole fresh peripheral blood mononuclear cells (PBMCs) were isolated from healthy human donors using a standard Ficoll-Paque separation method.

- 41 043382

Antibodies were serially diluted (eg, 40 to 0.01 μg/mL) and plated in triplicate in a 96-well flat-bottom tissue culture plate. Antibodies used included (i) Ipi YV39 2011;

(ii) ipilimumab; and (iii) an unrelated isotype control.

Isolated PBMCs were then resuspended in T cell assay medium (RPMI medium + 10% heat-inactivated fetal bovine serum (HI-FBS) + 1% HEPES buffer + 1% nonessential amino acid MEM + 1% Na-pyruvate) and added to the plate at quantity 1x105 cells/well. Cells were stimulated with a suboptimal concentration (eg, 85 ng/ml, determined by SEB titration and observed stimulation of T cell proliferation) of SEB. Cells were incubated at 37°C for 3 days. The concentration of IL-2 in the supernatants was then measured using homogeneous time-resolved fluorescence (HTRF). HTRF data were analyzed using Softmax Pro and presented graphically using GraphPad Prism.

As shown in FIG. 8, ipilimumab enhanced SEB-mediated IL-2 production by PBMCs in a dose-dependent manner. In contrast, the activated antibody Ipi YV39 2011 had activity similar to that of the isotype control, suggesting that the masking fragment (YV39) is effective in blocking the functional activity of ipilimumab. These data are consistent with the binding data described above and demonstrate that in the absence of specific proteases, activated anti-human CTLA-4 antibodies exhibit reduced activity.

Example 5 In Vivo Characterization of Activated Anti-Human CTLA-4 Antibodies.

In order to characterize the antibodies disclosed herein in vivo, four activated anti-human CTLA-4 antibodies (ipilimumab based) were generated using mouse IgG2a. Antibodies include YV04, YV23, YV24 or YV39 as a masking fragment and 2001 as a cleavable fragment (Ipi YV04 2001, Ipi YV23 2001, Ipi YV24 2001 and Ipi YV39 2001, respectively). Ipilimumab (Ipi mg2a) and an unrelated human anti-diphtheria toxin (control mg2a) were used as controls. The activity of these activated anti-CTLA-4 antibodies was assessed using the MC38 tumor model as described below.

Briefly, on day 0, human CTLA-4 C57BL/6 mice were injected subcutaneously with 2x106 MC38 colon adenocarcinoma cells into their left lower abdomen. Tumors were measured with calipers in two dimensions, and tumor volume was calculated as

Lx(W ² /2),

L=length (longest of 2 dimensions),

W=width.

Mice were then randomized into different groups to have the same average tumor volumes (eg, 37 mm ³ ). Antibody administration began on day 7 after tumor implantation in mice receiving a single dose (eg, 200 μg/mouse) of the appropriate antibody via intraperitoneal (ip) injection. On day 12 after tumor implantation, several mice from each group were sacrificed, and tumors and spleens were collected for immunomonitoring to examine the effect of antibodies on T-cell populations. Some or all of the remaining mice from the different groups were used for subsequent pharmacokinetics (PK) and/or pharmacodynamics (PD) analyses.

Immunomonitoring of T-cell populations.

The harvested tumor and spleen were processed using a gentleMACS Octo Dissociator™ (Miltenyi, San Diego, CA). Single cell suspensions were stained with the following T cell markers: CD4, CD8, CD19, ICOS, CD45, FoxP3, CTLA-4, CD3, Ki-67, PD-1, Granzyme B and LIVE/DEAD®.

PK/PD analysis.

Mice were checked daily for postural, grooming, and respiratory changes, as well as lethargy. Group tumors and body weights were recorded twice weekly until death, euthanasia, or the end of the study period. Treatment response was measured as a function of tumor growth inhibition (TGI), which was calculated as follows:

% TGI= {1 -[(Tt-To)/(Ct-Co)]} x 100,

Tt=tumor volume of the treatment group on a given day,

Then = initial tumor volume,

Ct=tumor volume of the control group on a given day,

Co=initial tumor volume of the control group.

Animals were sacrificed if the tumor reached a volume greater than approximately 2500 mm ³ or appeared ulcerated.

Statistical analysis.

Microsoft Excel was used to calculate the mean standard deviation (SD) and median values of tumor volumes and body weights. Mean and median values were calculated when 100% and at least 60% of study animals remained in each treatment group, respectively. GraphPad Prism® v software was used to construct the graph. 4.

- 42 043382

As expected, mice that received the unrelated control antibody did not have tumor growth controlled (Fig. 9A), whereas all mice that received ipilimumab had effective tumor growth control (Fig. 9B). Mice that received various activated anti-human CTLA-4 antibodies had tumor growth control comparable to ipilimumab (Figures 9C-9F). Of the activated antibodies, Ipi YV39 2001 most closely resembled the efficacy of ipilimumab in controlling tumor growth (Fig. 9F).

Regarding the frequency of regulatory T cells in the tumor and spleen of treated mice, as previously observed with activated anti-mouse CTLA-4 antibodies (see Example 2), activated anti-human CTLA-4 antibodies (mouse IgG2a isotype) behave similarly to ipilimumab in tumors (Figs. 12A and 12B), but in the spleen the activated antibodies were more comparable to the unrelated control antibodies (Figs. 12C-12F).

The data presented herein collectively demonstrate that the activateable anti-human CTLA-4 antibodies disclosed herein can effectively control tumors similar to conventional ipilimumab while exhibiting a reduced risk of unwanted side effects.

Example 6: In vivo characterization of activated anti-human CTLA-4 antibodies containing modified cleavable fragments.

To address a possible deamidation site at specific cleavage sequences (see Example 10), activated anti-human CTLA4 antibodies were generated using human IgG1 and various CM sequences. Activated antibodies contain YV39 as a masking fragment and one of several variants of the 2001 cleavage fragment: WT (2001), ANP (2012), DNP (2011) or Q (2008) (Ipi YV39 2001, Ipi YV39 2012, Ipi YV39 2011 and Ipi YV39 2008 respectively). Ipilimumab and an unrelated human anti-diphtheria toxin were again used as controls.

To measure the activity of activated antibodies against CTLA-4, the MC38 tumor model was used as described above in Example 5. For the dose titration study (Fig. 11A-11F), mice were treated with ipilimumab or an activated antibody containing YV39 as a masking fragment and 2011 as a cleavable fragment. fragment (Ipi YV39 2011), at doses of 200, 60 and 20 μg/dose.

As shown in FIG. 10A and 10B, mice treated with control antibody were tumor free, whereas 6 of 10 mice treated with ipilimumab were tumor free at the end of the experiment. In mice treated with various activated antibodies, tumors were able to be controlled as was observed with traditional ipilimumab (Figures 10C-10F). See also FIG. 11В-1У.

Regarding the frequency of regulatory T cells in the tumor and spleen of treated mice, as noted previously, a tumor-specific protease was required to cleave cleavable fragment variants 2001. In tumors, these activated antibodies behaved similarly to ipilimumab in reducing the frequency of Foxp3+ regulatory T cells (Fig. 13A , 13B, 14A and 14B). See also FIG. 15. In the spleen, the antibodies more closely mirrored the unrelated control antibody (FIGS. 13C-13E, 14D-14G and 16A-16B), demonstrating that the masking fragment remains associated with the activated antibody in the absence of specific tumor-associated proteases.

Example 7: In vivo characterization of a non-fucosylated version of activated anti-human CTLA-4 antibodies.

As described above, the absence of fucose residues in the core can strongly enhance ADCC through improved IgG binding to activating FcyRIIIA without altering antigen or CDC binding. Natsume et al. (2009), Drug Des. Devel. Ther., 3:1'. Non-fucosylated forms of ipilimumab (Ipi NF) and ipi YV39 2011 (Ipi YV39 2011 NF) were prepared. Ipi and Ipi NF binding were determined for various mouse, human, and cynomolgus Fc receptors. The results are presented in Fig. 19. As expected, Ipi NF showed dramatically increased affinity (i.e., lower Kd) for the activating receptors human CD16a (FcyRIIIa), macaque CD16 (FcyRIII), and mouse FcyRIV.

Ipi YV39 2011 NF and Ipi-NF were tested at various doses in the MC38 tumor model described in Example 5. Ipilimumab and unrelated hIgG1 were used as controls. The results are presented in Fig. 17A-17D. Ipi NF was slightly more effective at limiting or preventing tumor growth than ipilimumab (compare Figs. 17B and 17C), and Ipi YV39 2011 NF was equivalent to Ipi NF (compare Figs. 17C and 17D). In addition, Foxp3+ regulatory T cells were also depleted in tumors from mice treated with Ipi NF and Ipi YV39 2011 antibodies (see FIG. 18). Both experiments showed that Ipi YV39 2011 NF is fully activated in the tumor.

These results confirm that the methods of the present invention are equally applicable to non-fucosylated forms of ipilimumab, including non-fucosylated activated anti-CTLA-4 antibodies, such as YV39 2011 NF.

Example 8 In vivo characterization of activated anti-human CTLA-4 antibodies in cynomolgus monkeys.

To evaluate anti-CTLA-4 antibodies in primates, cynomolgus monkeys were administered active

- 43 043382 a variable antibody containing YV39 as a masking fragment and 2001 as a cleavable fragment. Vehicle and ipilimumab were used as controls. Each monkey received 10 mg of antibody or activated antibody against CTLA-4, and blood was collected on days 0, 4, 8, 15, 22, 36, and 43 after antibody administration. As shown in FIG. 20, ipilimumab-treated monkeys exhibited a surge in CD4+ T cell proliferation, as measured by Ki67 staining, approximately 8–15 days after antibody administration. In contrast, the activated anti-CTLA-4 antibody behaved similarly to vehicle controls and did not induce CD4+ T cell proliferation in monkeys. These data demonstrate that even in primates, activated anti-CTLA-4 antibody exhibits little, if any, activation, indicating the absence of specific proteases.

Taken together, the data presented in FIG. 1-20 demonstrate that the activated anti-CTLA-4 antibodies described herein offer an improvement over ipilimumab. The activated antibodies control tumor growth as effectively as ipilimumab, while reducing the risk of serious adverse events often observed with ipilimumab treatment.

Example 9 Kapp and ME values for CTLA-4 activated antibodies.

In table 9 presents Kapp values and masking efficiency (ME) for the activated antibodies disclosed herein, which include various masking fragments and cleavable fragments in human IgG1 format. The values presented in this table were calculated based on the data depicted in the figures. K _app illustrates the binding affinity of the activated antibody under measurement conditions, in this example binding by ELISA; however, it should be understood that binding affinity can also be measured by binding to CTLA-4 expressed on primary or transfected cells or by other physical methods such as, but not limited to, surface plasmon resonance or equilibrium dialysis. Masking efficiency (ME) is calculated by dividing the Kapp of the activated antibody by the KD of ipilimumab measured under the same conditions.

Table 9

K _app and ME values

SM 2001 SM 3001 SM 2008 SM 2011 SM 2012 NSUB Carr nM M.E. Carr nM M.E. Carr nM M.E. Carr nM m E Carr nM M.E. Carr nM M.E. YV04- YV1 17.8 57 YV06- YV1 0.6 2 YV09- YV1 33.6 112 44.4 126 YV23- YV1 I,4 38 13.8 39 YV24- YV1 9.0 29 YV27- 0.7 2.3 0.8 2.3

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YV1 YV29- YV1 0.7 2.3 0.8 2.3 YV32- YV1 0.9 3.0 1.2 3.4 YV33- YV1 1.3 4.3 1.9 5 YV35- YV1 3.7 12, 3 5.3 15 YV39- YV1 16.9 56 14.3 41 31.4 135 13.2 57 14.9 64 31.8 137 YV41- YV1 14.4 48 22.6 65 YV51- YV1 4.4 15 4.9 14 YV52- YV1 0.8 2.7 0.9 2.6 YV53- YV1 4.1 14 5.3 15 YV54- YV1 0.6 2 1.0 2.8 YV55- YV1 4.8 16 6.0 18 YV56- YV1 0.4 1.3 0.4 1 YV57- YV1 0.4 1.3 1.6 4.6 YV58- YV1 o, s 1 0.4 1

In table 10 shows the Kapp and ME values for the activated antibodies disclosed herein, including various masking fragments and cleavable fragments in the mouse YV1 Ig2a format. The values presented were calculated based on the data depicted in the figures.

Table 10

K _app and ME values

SM 2001 SM 2006 SM 2007 SM 2008 SM 2009 Carr nM M.E. Carr nM M.E. Carr nM M.E. Carr nM M.E. Carr nM M.E. YV04-YV1 5.7 16.2 26.4 75 19.3 55 19.1 54 16.4 47 YV23-YV1 12.5 36 7.8 22 2.7 8 9.4 27 YV39-YV1 18.0 51 23.9 68 17.6 50 18.0 51

In table 11 shows the Kapp and ME values for activated antibodies including masking fragments having higher ME values and the 2012 cleavage fragment in mouse IgG2a YY1 format. The values presented were calculated based on the data depicted in the figures.

Table 11

K _app and ME values

SM 2001 SM 2011 SM 2012 NSUB Carr nM M.E. Carr nM M.E. Carr nM M.E. Carr nM M.E. YV39-YV1 18.0 51 18.0 51 12.9 144 29.8 85 YV61-YV1 17.9 200 YV62-YV1 15.5 173 YV63-YV1 104 1170 YV64-YV1 56.5 631 YV65-YV1 12.3 156 YV66-YV1 18.9 242 YV01-YV1 38.6 493 YV02-YV1 14.8 189

Example 10. Evaluation of deamidation, isomerization and stabilization for CTLA-4 activated antibodies.

As suggested in Example 6, to address a possible deamidation site at certain

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Claims (16)

cleavage fragment (CM) sequences in certain human anti-CTLA-4 activated antibodies, such activated antibodies were generated using different CM sequences (ie, 2001, 2011, 2012, and 2008). In cleavages 2011, 2012, and 2008, the DNH sequence found in cleavage 2001 was replaced by DNP, ANP, and DQH, respectively. These activated CTLA-4 antibodies were generated by transiently transfecting the corresponding constructs in HEK 293 cells and subjected to peptide mapping liquid chromatography-mass spectroscopy (LC-MS) to detect potential degradation products. Cleavable fragment 2001 (DNH), which was originally selected for use in the activated anti-CTLA-4 antibodies of the present invention, showed deamidation of the asparagine (N) residue (6.4%) after 7 days in PBS at 4°C. Stability studies by accelerated aging showed an increase in deamidation from 18.5 to 32.8% when stored at 25°C for 4 weeks and to 36.5 and 66.6% when stored at 40°C for one week and four weeks respectively. The 2008, 2011 and 2012 cleavable fragments were chosen to try to overcome the problem of deamidation from 2001 in these CTLA-4 activated antibodies. All had 0.1% or less deamidation when stored at 40°C for one week in PBS, compared to 6.4% deamidation in 2001. However, further stability analysis (also using LC-MS) showed that although These activated anti-CTLA-4 antibodies containing the cleavable fragment 2008 (DQH) showed minimal deamidation, it showed significant isomerization of the aspartate moiety under various conditions (see Table 12). In contrast, 2011 (DNP) showed minimal aspartate isomerization. Aspartate isomerization was not relevant for 2012 (ANP), in which the aspartate residue is replaced by alanine. Table 12 Isomerization values Temperature Time Cleavable Fragment - Isomerization Values 2011 (DNP) 2012 (ANP) 2008 (DQH) -80°С 0 days (To) 0.1% N/A 1.8% 4°C 0 days (To) 0.1% N/A 2.4% 25°C 3 months 0.2% N/A 8.2% 40°C 3 months 0.2% N/A 34.5% However, in vitro stability studies in sera from mice, rats and cynomolgus monkeys showed significant cleavage between asparagine and proline residues in 2012 (ANP) (see Table 13) in these activated anti-CTLA-4 antibodies. 2011 (DNP) remained a cleavable fragment with acceptably low levels of deamidation, aspartate isomerization, and light chain truncation. Table 13 Degree of truncation between asparagine and proline residues Serum Cleavable Fragment - Truncation Between Asparagine and Proline Residues 2011 (DNP) 2012 (ANP) Mouse - ++ macaque +/- +++ All publications, patents, patent applications, Internet sites and accession numbers/sequence databases (including both polynucleotide and polypeptide sequences) cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as that each individual publication, patent, patent application, Internet site or accession number/sequence database be specifically and individually designated for inclusion by reference. CLAIM 1. An activated anti-human CTLA-4 antibody comprising (i) a heavy chain comprising a heavy chain variable domain (VH) containing CDRH1: SYTMH (SEQ ID NO: 557); CDRH2: FISYDGNNKYYADSVKG (SEQ ID NO: 558); And CDRH3: TGWLGPFDY (SEQ ID NO: 559); and (ii) a light chain comprising (a) a light chain variable domain (VL) containing CDRL1: RASQSVGSSYLA (SEQ ID NO: 560); CDRL2: GAFSRAT (SEQ ID NO: 561); And CDRL3: QQYGSSPWT (SEQ ID NO: 562); (b) cleavable fragment (CM); And - 46 043382 (c) a masking fragment (MM), in which the MM is selected from the group consisting of YV01 (SEQ ID NO: 1), YV02 (SEQ ID NO: 2), YV03 (SEQ ID NO: 3), YV04 ( SEQ ID NO: 4), YV09 (SEQ ID NO: 9), YV23 (SEQ ID NO: 23), YV24 (SEQ ID NO: 24), YV35 (SEQ ID NO: 35), YV39 (SEQ ID NO: 39), YV51 (SEQ ID NO: 51), YV61 (SEQ ID NO : 60), YV62 (SEQ ID NO: 61), YV63 (SEQ ID NO: 62), YV64 (SEQ ID NO: 63), YV65 (SEQ ID NO: 64) and YV66 (SEQ ID NO: 65), where The light chain has a structural arrangement from N-terminus to C-terminus as follows: MM-CM-VL. 2. The activated anti-human CTLA-4 antibody according to claim 1, wherein the SM is a protease substrate selected from the group consisting of MMP1, MMP2, MMP3, MMP8, MMP9, MMP11, MMP13, MMP14, MMP17, legumain, matriptase and uPA. 3. The activated anti-human CTLA-4 antibody according to claim 2, wherein the CM is selected from the group consisting of 2001 (SEQ ID NO: 297), 2003 (SEQ ID NO: 298), 2005 (SEQ ID NO: 299), 2006 (SEQ ID NO: 300), 2007 (SEQ ID NO: 301), 2008 (SEQ ID NO: 302), 2009 (SEQ ID NO: 303), 2011 (SEQ ID NO: 304), 2012 (SEQ ID NO : 305), 3001 (SEQ ID NO: 306), 3006 (SEQ ID NO: 307), 3007 (SEQ ID NO: 308), 3008 (SEQ ID NO: 309), 3009 (SEQ ID NO: 310), 3011 (SEQ ID NO: 311) and 3012 (SEQ ID NO: 312). 4. The activated anti-human CTLA-4 antibody according to claim 3, wherein the MM is YV04 (SEQ ID NO: 4), YV23 (SEQ ID NO: 23), YV24 (SEQ ID NO: 24), YV39 (SEQ ID NO: 39), YV61 (SEQ ID NO: 60), YV62 (SEQ ID NO: 61), YV63 (SEQ ID NO: 62) or YV64 (SEQ ID NO: 63). 5. The activated anti-human CTLA-4 antibody of claim 4, wherein the MM is YV39 (SEQ ID NO: 39). 6. The activated anti-human CTLA-4 antibody of claim 5, wherein the CM is 2001 (SEQ ID NO: 297), 2011 (SEQ ID NO: 304) or 2012 (SEQ ID NO: 305). 7. The activated anti-human CTLA-4 antibody of claim 6, wherein the CM is 2011 (SEQ ID NO: 304). 8. The activated anti-human CTLA-4 antibody according to claim 1, comprising (i) a heavy chain containing the amino acid sequence of SEQ ID NO: 345; and (ii) a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 563, 564 and 565. 9. The activated anti-human CTLA-4 antibody according to claim 8, comprising (i) a heavy chain containing the amino acid sequence of SEQ ID NO: 345; and (ii) a light chain comprising the amino acid sequence of SEQ ID NO: 564. 10. The activated anti-human CTLA-4 antibody of claim 9, wherein (i) the heavy chain comprises the sequence SEQ ID NO: 350 of a human IgG1 constant domain; and (ii) the light chain comprises the sequence SEQ ID NO: 346 of the human light chain constant kappa domain. 11. The activated anti-human CTLA-4 antibody according to claim 1, further comprising a first linker peptide (LP1) between MM and SM or between SM and VL. 12. The activated anti-human CTLA-4 antibody of claim 11, further comprising a second linker peptide (LP2), wherein the activated anti-human CTLA-4 antibody has a structural arrangement from N-terminus to C-terminus MM-LP1-CM-LP2- VL or MM-LP2-CM-LP1-VL. 13. The activated anti-human CTLA-4 antibody according to claim 12, wherein LP1 and LP2 are not identical to each other. 14. The activated anti-human CTLA-4 antibody according to any one of claims 1 to 13, further comprising a spacer and having a structural arrangement from N-terminus to C-terminus, spacer-MM-CM-VL. 15. An activated anti-human CTLA-4 antibody comprising a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO: 353; and the light chain contains the amino acid sequence of SEQ ID NO: 448. 16. An activated anti-human CTLA-4 antibody conjugated to a toxic agent, wherein the activated anti-human CTLA-4 antibody comprises (i) a heavy chain comprising a heavy chain variable domain (VH) containing CDRH1: SYTMH (SEQ ID NO: 557); CDRH2: FISYDGNNKYYADSVKG (SEQ ID NO: 558); And CDRH3: TGWLGPFDY (SEQ ID NO: 559); and (ii) a light chain comprising (a) a light chain variable domain (VL) containing CDRL1: RASQSVGSSYLA (SEQ ID NO: 560); CDRL2: GAFSRAT (SEQ ID NO: 561); And CDRL3: QQYGSSPWT (SEQ ID NO: 562); (b) cleavable fragment (CM); and (c) a masking fragment (MM), in which the MM is selected from the group consisting of YV01 -