Activatable masked anti-CTLA4 binding protein

Abstract

The present invention provides activatable masked anti-CTLA4 binding proteins (e.g., antibodies, bispecific antibodies, and chimeric receptors) and their uses in the treatment and prevention of cancer, as well as compositions and kits comprising the activatable masked anti-CTLA4 binding proteins.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Application No. 62 / 785,111, filed December 26, 2018, the contents of which are incorporated by reference in their entirety.

[0002] Submission of sequence listing as an ASCII text file The contents of the following submission, an ASCII text file, are incorporated herein by reference in their entirety: Computer Readable Form (CRF) of the Sequence Listing (Filename: 737762001640.txt, Created: December 23, 2019, Size: 518KB).

[0003] FIELD OF THEINVENTION The present invention relates to activatable masked anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) binding proteins (eg, anti-CTLA4 antibodies) and methods relating to their use. [Background technology]

[0004] 2. Background of the Invention Cancer is the second leading cause of death in the United States, accounting for more deaths than the next five leading causes (chronic respiratory disease, stroke, accidents, Alzheimer's disease, and diabetes). Although significant progress has been made, especially in targeted therapy, much research remains in this field. Immunotherapy and the field's branch, immuno-oncology, have produced viable and exciting treatment options for treating malignancies. In particular, it is now recognized that one hallmark of cancer is immune evasion, and remarkable efforts have been made to identify targets and develop therapies against these targets to reactivate the immune system to recognize and treat cancer. In fact, ipilimumab, an anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) antibody, has resulted in long-term survival for patients with stage III / IV malignant melanoma. Ipilimumab is an immune checkpoint antagonist that interrupts T-cell inhibition by blocking CTLA4, which can result in depletion of T regulatory cells (Tregs). [Korman, A., et al., 2005. Tumor immunotherapy: preclinical and clinical activity of anti-CTLA4 antibodies. Current Opinion in Investigational Drugs 6: 582-591 (Non-Patent Document 1), Quezada et al., J. Exp. Med., 206 (8): 1717-1725, 2009 (Non-Patent Document 2), Selby et al. Cancer Immunol Res., 1 (1); 32-42, 2013 (Non-Patent Document 3)] Unfortunately, ipilimumab causes systemic (not tumor-specific) activation of T cell-dependent immune responses that result in potentially life-threatening immune-related adverse effects, often limiting dose and duration of treatment (Weber, JS, et al., 2008. Phase I / II study of ipilimumab for patients with metastatic melanoma. Journal of Clinical Oncology 26: 5950-5956 (Non-Patent Document 4)). These include enteritis, dermatitis, hypophysitis, uveitis, hepatitis, nephritis, and death.Enterocolitis is the most common primary toxicity (affecting about 20% of patients). Due to the severe safety risks associated with immune-mediated adverse reactions, the FDA approved ipilimumab with a risk evaluation and mitigation strategy (REMS). Recently, it has been shown that the combination of ipilimumab with a second immune checkpoint regulator targeting PD1 (e.g., nivolumab) significantly increases the efficacy of melanoma immunotherapy compared to ipilimumab alone. However, this increase was associated with an increased frequency of grade 3 / 4 adverse effects affecting more than 50% of patients receiving the combination therapy (Wolchok, JD, et al. 2013. Nivolumab plus Ipilimumab in Advanced Melanoma. N Engl J Med (Non-Patent Document 5)).

[0005] These findings illustrate the need for the development of anti-CTLA4 protein therapeutics that effectively target tumors without the side effects associated with systemic immune activation. Provided herein are anti-CTLA binding proteins, compositions thereof, and methods of use thereof to address this need.

[0006] All references cited herein, including patent applications, patent publications, and scientific literature, are incorporated by reference in their entirety as if each individual reference was specifically and individually indicated to be incorporated by reference. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Korman, A., et al., 2005. Tumor immunotherapy: preclinical and clinical activity of anti-CTLA4 antibodies. Current Opinion in Investigational Drugs 6:582-591 [Non-Patent Document 2] Quezada et al.,J.Exp.Med.,206(8):1717-1725,2009 [Non-Patent Document 3] Selby et al.Cancer Immunol Res.,1(1);32-42,2013 [Non-Patent Document 4] Weber,JS,et al.,2008.Phase I / II study of ipilimumab for patients with metastatic melanoma.Journal of Clinical Oncology 26:5950-5956 [Non-Patent Document 5] Wolchok,JD,et al.2013.Nivolumab plus Ipilimumab in Advanced Melanoma.N Engl J Med Summary of the Invention

[0008] Provided herein are activatable masked anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) binding proteins, compositions comprising same, and methods of using same.

[0009] Provided herein is a masked antibody comprising an antibody or antigen-binding fragment thereof that binds to CTLA4, the antibody or antigen-binding fragment thereof comprising a first chain and a second chain, the masking peptide comprising an amino acid sequence selected from SEQ ID NOs: 1-46, the masking peptide being linked to the amino or carboxy terminus of the first or second chain of the antibody or antigen-binding fragment thereof via a linker comprising a cleavable peptide. In some embodiments, the first chain is a light chain and the second chain is a heavy chain.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof comprises two first chains and two second chains. In some embodiments, the first chain is or comprises a light chain variable domain and the second chain is or comprises a heavy chain variable domain. In some of any such embodiments, the antigen-binding fragment is a dAb, Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragment. In some of any such embodiments, the amino- or carboxy-terminus of the masking peptide is linked to a linker comprising a cleavable peptide. In some of any such embodiments, the linker comprising a cleavable peptide comprises a spacer linker and a cleavable peptide. In some of any such embodiments, the cleavable peptide comprises an amino acid sequence selected from SEQ ID NOs: 47-88, 464-469, and 479-508. In some of any such embodiments, the spacer linker is linked directly to the N-terminus and / or C-terminus of the cleavable peptide. In some of any such embodiments, the spacer linker comprising an amino acid sequence is selected from SEQ ID NOs: 89-112 and 415-420. In some of any such embodiments, at least one amino acid but not more than 20 amino acids is directly linked to the N-terminus of the masking peptide. In some of any such embodiments, at least one amino acid is alanine (A) or glycine-alanine (GA).

[0011] In some of any such embodiments, the masked antibody comprises, from N-to-C-terminal or C-to-N-terminal direction, a) a masking peptide, b) a cleavable peptide, and c) an antibody or antigen-binding fragment thereof that binds CTLA4. In some of any such embodiments, the masked antibody comprises a spacer between the masking peptide and the cleavable peptide, and the masked antibody comprises a spacer linker between the cleavable peptide and the antibody or antigen-binding fragment thereof that binds CTLA4.

[0012] In some of any such embodiments, the antibody is a mouse antibody. In some of any such embodiments, the antibody is a humanized antibody, a chimeric antibody, or a human antibody. In some of any such embodiments, the antibody has an IgG1, IgG2, IgG3, or IgG4 isotype. In some of any such embodiments, the IgG1 has the amino acid substitutions S298A, E333A, and K334A; S239D and I332E; S239D, A330L, and I332E; P247I and A339D or A339Q; D280H, K290S with or without S298D, or S298V; F243L, R292P, and Y300L; F243L, R292P, Y3 00L, and P396L; F243L, R292P, Y300L, V305I, and P396L; G236A, S239D, and I332E; K326A and E333A; K326W and E333S; or K290E or K290N, S298G, T299A, and / or K326E, wherein the amino acid residues are numbered according to the EU index as in Kabat.

[0013] In some of any such embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 402 or 408, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 403 or 409, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 404 or 410, and / or the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 405 or 411, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 406 or 412, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 407 or 413. In some of any such embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 232, and / or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 233.

[0014] In some of any such embodiments, the antibody or antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 402, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 403, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 404, and / or the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 405, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 406, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 407. In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 402, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 403, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 404, and the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 405, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 406, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 407.

[0015] In some of any such embodiments, the antibody or antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 432, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 433, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 444, and / or the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 435, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 436, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 437. In some of any such embodiments, the antibody or antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 432, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 433, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 434, and the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 435, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 436, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 437.

[0016] In some of any such embodiments, the antibody or antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 408, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 409, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 410, and / or the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 411, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 412, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 413. In some of any such embodiments, the antibody or antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 408, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 409, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 410, and the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 411, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 412, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 413.

[0017] In some of any such embodiments, the antibody or antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 438, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 439, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 440, and / or the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 441, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 442, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 443. In some of any such embodiments, the antibody or antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 438, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 439, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 440, and the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 441, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 442, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 443.

[0018] In some of any such embodiments, the antibody comprises a light chain comprising an amino acid sequence selected from SEQ ID NOs: 237-318, and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 319 or 320. In some of any such embodiments, the antibody or antigen-binding fragment comprises a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 321 or 322, and / or a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 323 or 324. In some of any such embodiments, the antibody or antigen-binding fragment comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 321, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 323. In some of any such embodiments, the antibody or antigen-binding fragment comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 322, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 324.

[0019] In some of any such embodiments, the antibody comprises a light chain comprising an amino acid sequence selected from SEQ ID NOs: 327-341, and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 366-380, 421, and 478. In some of any such embodiments, the antibody comprises a light chain comprising an amino acid sequence selected from SEQ ID NOs: 327, 334, or 342-365, and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 366 or 380-397. In some of any such embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising an amino acid sequence of SEQ ID NO: 327, and a heavy chain comprising an amino acid sequence of SEQ ID NO: 36 ...478. In some of any such embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising an amino acid sequence of SEQ ID NO: 334, and a heavy chain comprising an amino acid sequence of SEQ ID NO: 380. In some of any such embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO:334, and a heavy chain comprising the amino acid sequence of SEQ ID NO:421.

[0020] In some of any such embodiments, the cleavable peptide is a substrate for a protease that is co-localized in a region that includes cells or tissues expressing CTLA4. In some of any such embodiments, the cleavable peptide is a substrate for a protease that is co-localized in a region that includes cells or tissues expressing CTLA4. In some of any such embodiments, the cleavable peptide is a substrate for a protease that is co-localized in a region that includes cells or tissues expressing CTLA4. , ADAMTS20, ADAMTS3, ADAMTS4, ABHD17B, ADAMTS5, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADAMTSL1, ADAMTSL2, ADAMTSL3, ABHD17C, ADAMTS L5, ASTL, BMP1, CELA1, CELA2A, CELA2B, CELA3A, CELA3B, ADAM10, ADAM15, ADAM17, ADAM9, ADAMTS4, CTSE, CTSF, ADAMTSL4, CMA1, CTRB1, CTRC , CTSO, CTRl, CTSA, CTSW, CTSB, CTSC, CTSD, ESP1, CTSG, CTSH, GZMA, GZMB, GZMH, CTSK, GZMM, CTSL, CTSS, CTSV, CTSZ, HTRA4, KLK10, KLK11, K LK13, KLK14, KLK2, KLK4, DPP4, KLK6, KLK7, KLKB1, ECE1, ECE2, ECEL1, MASP2, MEP1A, MEP1B, ELANE, FAP, GZMA, MMP11, GZMK, HGFAC, HPN, HTRA 1, MMP11, MMP16, MMP17, MMP19, HTRA2, MMP20, MMP21, HTRA3, HTRA4, KEL, MMP23B, MMP24, MMP25, MMP26, MMP27, MMP28, KLK5, MMP3, MMP7, MMP 8, MMP9, LGMN, LNPEP, MASP1, PAPPA, PAPPA2, PCSK1, NAPSA, PCSK5, PCSK6, MME, MMP1, MMP10, PLAT, PLAU, PLG, PRSS1, PRSS12, PRSS2, PRSS21,It is cleaved by one or more enzymes selected from the group consisting of PRSS3, PRSS33, PRSS4, PRSS55, PRSS57, MMP12, PRSS8, PRSS9, PRTN3, MMP13, MMP14, ST14, TMPRSS10, TMPRSS11A, TMPRSS11D, TMPRSS11E, TMPRSS11F, TMPRSS12, TMPRSS13, MMP15, TMPRSS15, MMP2, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS5, TMPRSS6, TMPRSS7, TMPRSS9, NRDC, OVCH1, PAMR1, PCSK3, PHEX, TINAG, TPSAB1, TPSD1, and TPSG1.

[0021] In some of any such embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of ADAM17, HTRA1, PRSS1, FAP, GZMK, NAPSA, MMP1, MMP2, MMP9, MMP10, MMP7, MMP12, MMP28, ADAMTS9, HGFAC, and HTRA3. In some of any such embodiments, the antibody or antigen-binding fragment thereof is conjugated to an agent. In some of any such embodiments, the agent is an inhibitor of tubulin polymerization, a DNA damaging agent, or a DNA synthesis inhibitor. In some of any such embodiments, the agent is a maytansinoid, an auristatin, a pyrrolobenzodiazepine (PBD) dimer, a calicheamicin, a duocarmycin, an indolinobenzodiazepine dimer, or an exatecan derivative Dxd.

[0022] In some of any such embodiments, the masked antibodies provided herein exhibit an optimal occlusion ratio of about 20 to about 10,000. In further embodiments, the optimal occlusion ratio is about 20 to about 1,000. In further embodiments, the optimal occlusion ratio is about 80 to about 100.

[0023] In some of any such embodiments, the masked antibody comprises the amino acid sequence of SEQ ID NO: 421 and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 358 and 422-431.

[0024] Also provided herein is a masked bispecific antibody comprising a first pair of light and heavy chains that specifically bind to CTLA4, a second pair of light and heavy chains that specifically bind to an antigen, and a masking peptide comprising an amino acid sequence selected from SEQ ID NOs: 1-46, wherein the masking peptide is linked to the amino or carboxy terminus of the first pair of light or heavy chains via a linker that comprises a cleavable peptide. In some embodiments, the amino or carboxy terminus of the masking peptide is linked to a linker that comprises a cleavable peptide. In some of any such embodiments, the linker that comprises a cleavable peptide comprises a spacer linker and a cleavable peptide.

[0025] In some of any such embodiments, the cleavable peptide comprises an amino acid sequence selected from SEQ ID NOs: 47-88, 464-469, and 479-508. In some of any such embodiments, the spacer linker is directly linked to the N-terminus or C-terminus of the cleavable peptide. In some of any such embodiments, the spacer linker comprising an amino acid sequence is selected from SEQ ID NOs: 89-112 and 415-420. In some of any such embodiments, at least one amino acid, but not more than 20 amino acids, is directly linked to the N-terminus of the masking peptide. In some of any such embodiments, at least one amino acid is alanine (A) or glycine-alanine (GA).

[0026] In some of any such embodiments, the light or heavy chain of the first pair comprises, in an N to C-terminal or C to N-terminal direction, a) a masking peptide, b) a cleavable peptide, and c) a light or heavy chain. In some of any such embodiments, the first pair comprises a spacer linker between the masking peptide and the cleavable peptide, and the first pair comprises a spacer linker between the cleavable peptide and the light or heavy chain.

[0027] In some of any such embodiments, the bispecific antibody is a murine antibody. In some of any such embodiments, the bispecific antibody is a humanized antibody, a chimeric antibody, or a human antibody. In some of any such embodiments, the bispecific antibody has an IgG1, IgG2, IgG3, or IgG4 isotype. In some of any such embodiments, the IgG1 has the following amino acids: S298A, E333A, and K334A; S239D and I332E; S239D, A330L, and I332E; P247I and A339D or A339Q; D280H, K290S with or without S298D, or S298V; F243L, R292P, and Y300L; F243L, R292P, Y300L, and P 396L; F243L, R292P, Y300L, V305I, and P396L; G236A, S239D, and I332E; K326A and E333A; K326W and E333S; or K290E or K290N, S298G, T299A, and / or K326E, where amino acid residues are numbered according to the EU index, as in Kabat.

[0028] In some of any such embodiments, the first pair comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 402 or 408, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 403 or 409, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 404 or 410, and / or the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 405 or 411, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 406 or 412, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 407 or 413.

[0029] In some of any such embodiments, the first pair comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 402, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 403, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 404, and / or the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 405, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 406, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 407. In some embodiments, the first pair comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 402, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 403, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 404, and the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 405, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 406, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 407.

[0030] In some of any such embodiments, the first pair comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 432, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 433, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 444, and / or the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 435, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 436, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 437. In some of any such embodiments, the first pair comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 432, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 433, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 434, and the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 435, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 436, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 437.

[0031] In some of any such embodiments, the first pair comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 408, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 409, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 410, and / or the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 411, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 412, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 413. In some of any such embodiments, the first pair comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 408, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 409, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 410, and the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 411, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 412, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 413.

[0032] In some of any such embodiments, the first pair comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 438, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 439, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 440, and / or the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 441, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 442, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 443. In some of any such embodiments, the first pair comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 438, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 439, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 440, and the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 441, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 442, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 443.

[0033] In some of any such embodiments, the first pair comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 232 and / or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 233. In some of any such embodiments, the first pair comprises a light chain comprising an amino acid sequence selected from SEQ ID NOs: 237-318 and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 319 or 320. In some of any such embodiments, the first pair comprises a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 321 or 322 and / or a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 323 or 324. In some of any such embodiments, the first pair comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 321 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 323. In some of any such embodiments, the first pair comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 322 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 324.

[0034] In some of any such embodiments, the first pair comprises a light chain comprising an amino acid sequence selected from SEQ ID NOs: 327-341, and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 366-380, 421, and 478. In some of any such embodiments, the first pair comprises a light chain comprising an amino acid sequence selected from SEQ ID NOs: 327, 334, or 342-365, and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 366 or 380-397. In some of any such embodiments, the first pair comprises a light chain comprising an amino acid sequence of SEQ ID NO: 327, and a heavy chain comprising an amino acid sequence of SEQ ID NO: 36 ...478. In some of any such embodiments, the first pair comprises a light chain comprising an amino acid sequence of SEQ ID NO: 334, and a heavy chain comprising an amino acid sequence of SEQ ID NO: 380. In some of any such embodiments, the first pair comprises a light chain comprising the amino acid sequence of SEQ ID NO:334, and a heavy chain comprising the amino acid sequence of SEQ ID NO:421.

[0035] In some of any such embodiments, the cleavable peptide is a substrate for a protease that is co-localized in a region that includes cells or tissues expressing CTLA4. In some of any such embodiments, the cleavable peptide is a substrate for a protease that is co-localized in a region that includes cells or tissues expressing CTLA4. In some of any such embodiments, the cleavable peptide is a substrate for a protease that is co-localized in a region that includes cells or tissues expressing CTLA4. , ADAMTS20, ADAMTS3, ADAMTS4, ABHD17B, ADAMTS5, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADAMTSL1, ADAMTSL2, ADAMTSL3, ABHD17C, ADAMTS L5, ASTL, BMP1, CELA1, CELA2A, CELA2B, CELA3A, CELA3B, ADAM10, ADAM15, ADAM17, ADAM9, ADAMTS4, CTSE, CTSF, ADAMTSL4, CMA1, CTRB1, CTRC , CTSO, CTRl, CTSA, CTSW, CTSB, CTSC, CTSD, ESP1, CTSG, CTSH, GZMA, GZMB, GZMH, CTSK, GZMM, CTSL, CTSS, CTSV, CTSZ, HTRA4, KLK10, KLK11, K LK13, KLK14, KLK2, KLK4, DPP4, KLK6, KLK7, KLKB1, ECE1, ECE2, ECEL1, MASP2, MEP1A, MEP1B, ELANE, FAP, GZMA, MMP11, GZMK, HGFAC, HPN, HTRA 1, MMP11, MMP16, MMP17, MMP19, HTRA2, MMP20, MMP21, HTRA3, HTRA4, KEL, MMP23B, MMP24, MMP25, MMP26, MMP27, MMP28, KLK5, MMP3, MMP7, MMP 8, MMP9, LGMN, LNPEP, MASP1, PAPPA, PAPPA2, PCSK1, NAPSA, PCSK5, PCSK6, MME, MMP1, MMP10, PLAT, PLAU, PLG, PRSS1, PRSS12, PRSS2, PRSS21,and is cleaved by one or more enzymes selected from the group consisting of PRSS3, PRSS33, PRSS4, PRSS55, PRSS57, MMP12, PRSS8, PRSS9, PRTN3, MMP13, MMP14, ST14, TMPRSS10, TMPRSS11A, TMPRSS11D, TMPRSS11E, TMPRSS11F, TMPRSS12, TMPRSS13, MMP15, TMPRSS15, MMP2, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS5, TMPRSS6, TMPRSS7, TMPRSS9, NRDC, OVCH1, PAMR1, PCSK3, PHEX, TINAG, TPSAB1, TPSD1, and TPSG1. In some of any such embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of ADAM17, HTRA1, PRSS1, FAP, GZMK, NAPSA, MMP1, MMP2, MMP9, MMP10, MMP7, MMP12, MMP28, ADAMTS9, HGFAC, and HTRA3. In some of any such embodiments, the bispecific antibody is conjugated to an agent. In some of any such embodiments, the agent is an inhibitor of tubulin polymerization, a DNA damaging agent, or a DNA synthesis inhibitor. In some of any such embodiments, the agent is a maytansinoid, an auristatin, a pyrrolobenzodiazepine (PBD) dimer, a calicheamicin, a duocarmycin, an indolinobenzodiazepine dimer, or an exatecan derivative Dxd.

[0036] In some of any such embodiments, the first pair and the second pair of masked bispecific antibodies provided herein each exhibit an optimal occlusion ratio that may be the same or different from each other. In some embodiments, the optimal occlusion ratio is about 20 to about 10,000. In further embodiments, the optimal occlusion ratio is about 20 to about 1,000. In further embodiments, the optimal occlusion ratio is about 80 to about 100.

[0037] Also provided herein is a masked chimeric receptor comprising a ligand binding domain comprising a first chain and a second chain that binds to CTLA4, a masking peptide comprising an amino acid sequence selected from SEQ ID NOs: 1-46, a transmembrane domain, and an intracellular signaling domain comprising a signaling domain, wherein the masking peptide is linked to the amino terminus or carboxy terminus of the first chain or the second chain of the ligand binding domain via a linker comprising a cleavable peptide.

[0038] In some embodiments, the first chain is a light chain variable domain and the second chain is a heavy chain variable domain. In some embodiments, the amino or carboxy terminus of the masking peptide is linked to a linker comprising a cleavable peptide. In some of any such embodiments, the linker comprising a cleavable peptide comprises a spacer linker and a cleavable peptide. In some of any such embodiments, the cleavable peptide comprises an amino acid sequence selected from SEQ ID NOs: 47-88, 464-469, and 479-508. In some of any such embodiments, the spacer linker is directly linked to the N-terminus or C-terminus of the cleavable peptide. In some of any such embodiments, the spacer linker comprising an amino acid sequence is selected from SEQ ID NOs: 89-112 and 415-420. In some of any such embodiments, at least one amino acid, but not more than 20 amino acids, is directly linked to the N-terminus of the masking peptide. In some of any such embodiments, at least one amino acid is alanine (A) or glycine-alanine (GA). In some of any such embodiments, the first or second chain of the ligand binding domain comprises, in an N-to-C-terminal or C-to-N-terminal direction, a) a masking peptide, b) a cleavable peptide, and c) the first or second chain. In some of any such embodiments, the ligand binding domain comprises a spacer linker between the masking peptide and the cleavable peptide, and the ligand binding domain comprises a spacer linker between the cleavable peptide and the first or second chain.

[0039] In some of any such embodiments, the ligand binding domain comprises a first chain and a second chain, wherein the first chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 402 or 408, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 403 or 409, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 404 or 410, and / or the second chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 405 or 411, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 406 or 412, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 407 or 413.

[0040] In some of any such embodiments, the first chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 402, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 403, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 404, and / or the second chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 405, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 406, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 407. In some embodiments, the first chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 402, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 403, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 404, and the second chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 405, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 406, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 407.

[0041] In some of any such embodiments, the first chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 432, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 433, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 444, and / or the second chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 435, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 436, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 437. In some of any such embodiments, the first chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 432, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 433, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 434, and the second chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 435, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 436, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 437.

[0042] In some of any such embodiments, the first chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 408, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 409, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 410, and / or the second chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 411, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 412, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 413. In some of any such embodiments, the first chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 408, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 409, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 410, and the second chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 411, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 412, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 413.

[0043] In some of any such embodiments, the first chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 438, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 439, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 440, and / or the second chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 441, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 442, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 443. In some of any such embodiments, the first chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 438, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 439, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 440, and the second chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 441, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 442, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 443.

[0044] In some of any such embodiments, the first chain comprises the amino acid sequence of SEQ ID NO: 232 and / or the second chain comprises the amino acid sequence of SEQ ID NO: 233. In some of any such embodiments, the first chain comprises the amino acid sequence selected from SEQ ID NO: 321 or 322 and / or the second chain comprises the amino acid sequence selected from SEQ ID NO: 323 or 324. In some of any such embodiments, the first chain comprises the amino acid sequence of SEQ ID NO: 321 and the second chain comprises the amino acid sequence of SEQ ID NO: 323. In some of any such embodiments, the first chain comprises the amino acid sequence of SEQ ID NO: 322 and the second chain comprises the amino acid sequence of SEQ ID NO: 324. In some of any such embodiments, the cleavable peptide is a substrate for a protease that is co-localized in a region that includes a cell or tissue that expresses CTLA4.

[0045] In some of any such embodiments, the cleavable peptides are ABHD12, ADAM12, ABHD12B, ABHD13, ABHD17A, ADAM19, ADAM20, ADAM21, ADAM28, ADAM30, ADAM33, ADAM8, ABHD17A, ADAMDEC1, ADAMTS1, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS2, ADAMTS20, ADAMTS3, ADAMTS4, ABHD17B, ADAMTS5, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADAMTSL1, ADAMTSL2, ADAMTSL3, ABHD17C, ADAMTSL5, ASTL, BMP1, CELA1, CELA2A, CELA2B, CELA3A, CELA3B, ADAM10, ADAM15, ADAM17, ADAM9, ADAMTS4, CTSE, CTSF, ADAMTSL4, CMA1, CTRB1, CTRC, CTSO, CTRl, CTSA, CTSW, CTSB, CTSC, CTSD, ESP1, CTSG, CTSH, GZMA, GZMB, GZMH, CTSK, GZMM, CTSL, CTSS, CTSV, CTSZ, HTRA4, KLK10, KLK11, KLK13, KLK14, KLK2, KLK4, DPP4, KLK6, KLK7, KLKB1, ECE1, ECE2, ECEL1, MASP2, MEP1A, MEP1B, ELANE, FAP, GZMA, MMP11, GZMK, HGFAC, HPN, HTRA1, MMP11, MMP16, MMP17, MMP19, HTRA2, MMP20, MMP21, HTRA3, HTRA4, KEL, MMP23B, MMP24, MMP25, MMP26, MMP27, MMP28, KLK5, MMP3, MMP7, MMP8, MMP9, LGMN, LNPEP, MASP1, PAPPA, PAPPA2, PCSK1, NAPSA, PCSK5, PCSK6, MME, MMP1, MMP10, PLAT, PLAU, PLG, PRSS1, PRSS12, PRSS2, PRSS21, PRSS3, PRSS33, PRSS4, PRSS55, PRSS57, MMP12, PRSS8, PRSS9, PRTN3, MMP13, MMP14, ST14,TMPRSS10, TMPRSS11A, TMPRSS11D, TMPRSS11E, TMPRSS11F, TMPRSS12, TMPRSS13, MMP15, TMPRSS15, MMP2, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS5, TMPRSS6, TMPRSS7, TMPRSS9, NRDC, OVCH1, PAMR1, PCSK3, PHEX, TINAG, TPSAB1, TPSD1, and TPSG1. In some of any such embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of ADAM17, HTRA1, PRSS1, FAP, GZMK, NAPSA, MMP1, MMP2, MMP9, MMP10, MMP7, MMP12, MMP28, ADAMTS9, HGFAC, and HTRA3.

[0046] In some of any such embodiments, the masked chimeric receptors provided herein exhibit an optimal occlusion ratio of about 20 to about 10,000. In further embodiments, the optimal occlusion ratio is about 20 to about 1,000. In further embodiments, the optimal occlusion ratio is about 80 to about 100.

[0047] Nucleic acids are also provided that encode the masked antibody, masked bispecific antibody, or masked chimeric receptor of any one of the preceding embodiments.Vectors are also provided that include the nucleic acids of the preceding embodiments.In some embodiments, the vector is an expression vector.Host cells are also provided that include the preceding nucleic acid embodiments.

[0048] Methods of producing a masked antibody, masked bispecific antibody, or masked chimeric receptor are also provided, comprising culturing the aforementioned host cell under conditions to produce the masked antibody, masked bispecific antibody, or masked chimeric receptor. In some embodiments, the host cell has an alpha 1,6-fucosyltransferase (Fut8) knockout. In some embodiments, the host cell overexpresses β1,4-N-acetylglucosaminyltransferase III (GnT-III). In some embodiments, the host cell further overexpresses Golgi μ-mannosidase II (ManII). Some of any such embodiments further comprise recovering the masked antibody, masked bispecific antibody, or masked chimeric receptor produced by the host cell. In some embodiments, the masked bispecific antibody or masked chimeric receptor produced by the aforementioned method.

[0049] Also provided is a composition comprising the masked antibody, masked bispecific antibody, or masked chimeric receptor of any one of the preceding embodiments. Some embodiments include a composition comprising the masked antibody, masked bispecific antibody, or masked chimeric receptor of the preceding embodiments. In some embodiments, the composition is a pharmaceutical composition.

[0050] Also provided is a kit comprising the masked antibody, masked bispecific antibody, masked chimeric receptor, or composition of any one of the preceding embodiments.

[0051] Also provided is a method for treating or preventing a neoplastic disease in a subject, comprising administering to the subject an effective amount of a masked antibody, a masked bispecific antibody, a masked chimeric receptor, or a composition of any one of the above-mentioned embodiments.In one embodiment, the neoplastic disease is cancer.In some embodiments, the cancer is leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, splenic cancer, melanoma, breast cancer, neuroblastoma, lung cancer, ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer, or testicular cancer.

[0052] It should be understood that one, some, or all of the characteristics of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the present invention will be apparent to those skilled in the art. These and other embodiments of the present invention are further described in the following detailed description. [Brief description of the drawings]

[0053] [Figure 1] Figures 1A and 1B are a series of graphs showing in vitro analysis of the blockage of anti-CTLA4 antibodies by masking peptides. Surface plasmon resonance (SPR) was performed to examine A) the reaction kinetics and B) the affinity between the 9D9 antibody (ligand) and the masking peptide CNLIVEGHC (analyte). Analytes were run at increasing concentrations of 2-fold serial dilutions from 0.1 to 50 µM at 37 °C. Reference subtracted responses were plotted. SPR was performed in triplicate and a representative trace from one experiment is shown. [Diagram 2]Figures 2A-2C are a series of graphs showing protease activation of masked mouse anti-CTLA4 antibodies. Figure 2A) The activatable masked anti-CTLA4 antibody exhibited 90-fold lower binding to mouse CTLA4-Fc (9D9, black squares) compared to the parental anti-CTLA4 antibody (9D9, black circles). Protease activation of the activatable masked anti-CTLA4 antibody fully restored binding to mouse CTLA4-Fc (protease activated, asterisks) at levels comparable to the parental CTLA4 antibody. The x-axis indicates the amount of antibody tested. Figure 2B) The activatable masked anti-CTLA4 antibody (masked 9D9, black inverted triangles) and the non-cleavable masked anti-CTLA4 antibody (NC masked 9D9, white squares) exhibited approximately 156-fold and approximately 218-fold lower binding to mouse CTLA4, respectively, compared to the parental anti-CTLA4 antibody (9D9, black circles). The X-axis indicates the amount of antibody tested. Figure 2C) EC50 was determined by ELISA for activatable masked anti-CTLA4 antibody (masked 9D9), non-cleavable masked anti-CTLA4 antibody (NC masked 9D9), and parental anti-CTLA4 antibody (9D9) in the absence or presence of recombinant MMP2. Each group was performed at least three times and the average EC50 + / - SE is reported. [Diagram 3] Figures 3A-3D are a series of graphs showing that the efficacy of a protease-activated masked anti-CTLA4 antibody (masked 9D9) in reducing tumor volume was comparable to that of a parental anti-CTLA4 antibody (9D9 IgG2a). MC38 syngeneic tumors were implanted into C57BI / 6 mice. A single 200 μg dose of Figure 3A) muIgG2a control antibody, Figure 3B) parental anti-CTLA4 antibody with IgG2a isotype (9D9.IgG2a), Figure 3C) activatable masked anti-CTLA4 antibody (masked 9D9) was administered intraperitoneally to mice when tumors reached 60-120 mm3 in size, and Figure 3D) humanized IgG1 anti-CTLA4 antibody 1 (Antibody 1) demonstrated similar in vivo efficacy in reducing tumor volume in the MC38 mouse tumor model compared to mice treated with 3B) parental anti-CTLA4 antibody (9D9 IgG2a) or 3A) muIgG2a control antibody. [Figure 4]Graph showing comparable depletion of regulatory T lymphocytes (Tregs) in tumor infiltrates by protease-activated masked anti-CTLA4 antibody (masked 9D9) and parental anti-CTLA4 antibody (9D9 IgG2a). Control shows treatment with muIgG2a control antibody. [Diagram 5] 5A and 5B are a series of graphs showing that a protease-activated masked anti-CTLA4 antibody (mask 9D9) significantly reduced proliferation of A) CD4+ T cells and B) CD8+ T cells in the spleens of treated mice. Arrows point to the average values ​​between treatment groups with parental anti-CTLA4 antibody (9D9 IgG2a) and protease-activated masked anti-CTLA4 antibody (mask 9D9). [Figure 6] Figures 6A-6D are a series of graphs showing efficacy and safety studies of masked anti-CTLA4 antibodies. The tumor suppressive activity of Figure 6A) isotype control antibody (IgG2a control), Figure 6B) non-cleavable masked anti-CTLA4 antibody (NC mask 9D9), Figure 6C) parental anti-CTLA4 antibody (9D9), and Figure 6D) activatable masked anti-CTLA4 antibody (mask 9D9) was examined in MC38 tumor-bearing C57BL / 6 mice (n=10). [Figure 7] Figures 7A and 7B are a series of graphs showing A) regulatory T lymphocyte (Treg) depletion in tumors and B) Treg expansion in the spleens of mice (n=6) treated with an isotype control antibody (IgG2a control), a parental anti-CTLA4 antibody (9D9), an activatable masked anti-CTLA4 antibody (masked 9D9), and a non-cleaving masked anti-CTLA4 antibody (NC masked 9D9). [Figure 8]Graph showing protease activation of masked humanized anti-CTLA4 antibody 1. Masked humanized anti-CTLA4 antibody 1 exhibited lower binding to human CTLA4-Fc (masked, black squares) compared to parental humanized anti-CTLA4 antibody 1 (parent, white circles). Protease activation of masked humanized anti-CTLA4 antibody completely restored binding to human CTLA4-Fc (protease activated, asterisk) at a level comparable to parental humanized anti-CTLA4 antibody 1. The x-axis indicates the amount of antibody tested. RLU of parental molecules / RLU on the y-axis indicates the percentage of relative light units compared to parental humanized anti-CTLA4 antibody 1. [Figure 9] Figures 9A and 9B are graphs showing binding of unmasked anti-CTLA4 antibodies to human CTLA4-Fc over a range of antibody concentrations. Figure 9A shows binding of forms of Antibody 1 (Antibody 1-1 and Antibody 1-2) to human CTLA4-Fc over a range of antibody concentrations, showing similar binding between the antibodies shown. Figure 9B shows binding of forms of Antibody 2 (Antibody 2-1, Antibody 2-2, Antibody 2-3, Antibody 2-4, and Antibody 2-5) to human CTLA4-Fc over a range of antibody concentrations, showing similar binding between the antibodies shown. [Figure 10A] Figures 10A and 10B are graphs showing binding of masked anti-CTLA4 antibodies to human CTLA4-Fc over a range of antibody concentrations. Figure 10A shows binding by the masked antibodies in the absence of protease activation. Figure 10B shows binding by the masked antibodies after protease activation. Antibody 2-6 is the unmasked parent antibody of Antibody 2-7, Antibody 2-8, Antibody 2-9, Antibody 2-10, Antibody 2-11, Antibody 2-12, and Antibody 2-13. Figures 10A and 10B show that after protease activation, the masked antibodies exhibit similar binding properties to the unmasked parent antibody, Antibody 2-6. [Figure 10B] See legend to Figure 10A. [Figure 11-1]Figures 11A-11H show radar plots of masked forms of Antibody 2 containing various cleavable peptide sequences. Antibody 2-15 contains a cleavable peptide sequence that is not cleavable. Activation of each antibody shown in Figures 11A-11H by each protease shown on the radar plot is shown using radar plots where activation ranges from 0 (no activation) to 1.0 (full activation). [Figure 11-2] See description of Figure 11-1. [Figure 11-3] Figures 11I-11M show the results of SDSP-PAGE Western blot analysis of protease cleavage using selected antibodies in the plasma of healthy mice (Figures 11I and 11J) and in the plasma of MC38 tumor-bearing mice (Figures 11K-11M). [Figure 11-4] See description of Figure 11-3. [Figure 11-5] See description of Figure 11-3. [Figure 12] Figures 12A and 12B are graphs showing binding of masked anti-CTLA4 antibodies to human CTLA4-Fc in the presence or absence of protease activation. In the presence of a protease, the masked antibody is "activated" by protease cleavage of the cleavable peptide. Figure 12A includes as controls an isotype control that is not activatable, and a masked antibody (Antibody 2-15) that is not activatable because its cleavable peptide sequence is non-cleavable. Figure 12B includes as controls an isotype control, and a masked antibody (Antibody 2-10) that is not activatable because its cleavable peptide sequence is non-cleavable. Activation of Antibody 2-14 fully restored its binding to human CTLA4-Fc at levels similar to the unmasked parent antibody, Antibody 2-6 (Figure 12B). [Figure 13A]Figures 13A-13D are graphs showing IL-2 levels (pg / mL) (Figures 13A and 13C), or fold change in IL-2 levels (Figures 13B and 13D), as determined using a Staphylococcal Enterotoxin B (SEB) assay, for unmasked forms of Antibody 1 and Antibody 2. Unmasked forms of Antibody 1 (Antibody 1-1 and 1-2) (Figures 13A and 13B), as well as unmasked forms of Antibody 2 (Antibody 2-1, Antibody 2-2, Antibody 2-3, Antibody 2-4, and Antibody 2-5) (Figures 13C and 13D), were tested for their ability to promote IL-2 production from peripheral mononuclear cells using the SEB assay. All tested forms of Antibody 1 and Antibody 2 demonstrated the ability to increase IL-2 levels compared to non-antibody controls (Figures 13A and 13C). [Figure 13B] See legend to Figure 13A. [Figure 13C] See legend to Figure 13A. [Figure 13D] See legend to Figure 13A. [Figure 14A]Figures 14A-14D are graphs showing IL-2 levels (pg / mL) (Figures 14A and 14C) or fold change in IL-2 levels (Figures 14B and 14D) for a masked form of Antibody 2, as determined using a Staphylococcal Enterotoxin B (SEB) assay. The unmasked form of Antibody 2 (Antibody 2-6) was tested along with masked forms of Antibody 2-6 (Antibody 2-7, Antibody 2-8, Antibody 2-9, Antibody 2-10, Antibody 2-11, Antibody 2-12, and Antibody 2-13). Figures 14A and 14B show the effect of antibodies on IL-2 levels (Figure 14A) and fold change in IL-2 levels (Figure 14B) in a non-activated state (i.e., without exposure to protease). The unmasked parent antibody, Antibody 2-6, shows the ability to significantly increase IL-2 levels, while only one of the masked antibodies (Antibody 2-12) shows a significant increase in IL-2 levels, but at a much lower level than Antibody 2-6 (Figures 14A and 14B). Figures 14C and 14D show the effect of the antibodies on IL-2 levels (Figure 14C) and the fold change in IL-2 levels (Figure 14D) in the activated state (i.e., after exposure to protease). Figures 14C and 14D show that exposure to protease rescues the ability of Antibody 2-7, Antibody 2-8, and Antibody 2-9 to promote IL-2 production at levels similar to that of the unmasked parent antibody, Antibody 2-6. [Figure 14B] See legend to Figure 14A. [Figure 14C] See legend to Figure 14A. [Figure 14D] See legend to Figure 14A. [Figure 15A]Figures 15A-15D show the extent of in vivo cleavage of masked anti-CTLA4 antibodies in plasma of healthy mice as shown by Western blot analysis. In vivo cleavage of the masked forms of antibody 2 (antibody 2-14, antibody 2-19, and antibody 2-20) in healthy mice is shown at days 2, 4, and 7 after intraperitoneal administration of the antibodies. Standard samples treated with masked antibodies in the presence or absence of proteases in vitro were used as cleavage controls. Both antibody 2-14 (Figure 15A) and antibody 2-20 (Figure 15C) were activated in vivo. Figure 15B shows the lack of activation of antibody 2-19. The increase in the percentage of activation from day 2 to day 7 is shown in Figure 15D for antibody 2-14 and antibody 2-20. [Figure 15B] See legend to Figure 15A. [Figure 15C] See legend to Figure 15A. [Figure 15D] See legend to Figure 15A. [Figure 16A] Figures 16A-16D show the extent of in vivo cleavage of masked anti-CTLA4 antibodies in the plasma of tumor-bearing mice as shown by Western blot analysis. In vivo cleavage of the masked forms of Antibody 2 (Antibody 2-14, Antibody 2-19, and Antibody 2-20) in MC38 tumor-bearing mice is shown at days 2, 4, and 7 after intraperitoneal administration of the antibodies. Standard samples treated with masked antibodies in the presence or absence of proteases in vitro were used as cleavage controls. Both Antibody 2-14 (Figure 16A) and Antibody 2-20 (Figure 16C) were activated in vivo. Figure 16B shows the lack of activation of Antibody 2-19. The increase in the percentage of activation from day 2 to day 7 is shown in Figure 16D for Antibody 2-14 and Antibody 2-20. The activation rate of antibody 2-14 was significantly greater in MC38 tumor-bearing mice compared to healthy non-tumor-bearing mice as determined by two-way ANOVA (adjusted P value=0.0113) (FIG. 16D). [Figure 16B] See legend to Figure 16A. [Figure 16C] See legend to Figure 16A. [Figure 16D] See legend to Figure 16A. [Figure 17] 17 is a graph showing the ability of masked anti-CTLA4 antibodies to promote ADCC activity as determined using an ADCC reporter bioassay in a masked and "activated" state as a result of prior exposure to proteases. FIG. 17 shows the degree of reporter activation by the unmasked parent antibody (Antibody 2-6) and masked versions of Antibody 2-6 (Antibody 2-14 and Antibody 2-15). Antibody 2-15 contains a cleavable peptide sequence that is non-cleavable to serve as a negative control. An isotype control was also tested. Antibodies with prior exposure to proteases are shown as "activated". When tested without having prior exposure to proteases, the masked antibodies Antibody 2-14 and Antibody 2-15 showed reduced reporter activation compared to the unmasked parent antibody (Antibody 2-6) (FIG. 17). The reduced reporter activation of Antibody 2-15, which is not protease-activatable, was not rescued by prior exposure to proteases (FIG. 17). Protease-activatable antibody 2-14 rescued reporter activation to levels similar to that induced by the unmasked parent antibody, antibody 2-6 (FIG. 17). [Figure 18A]Figures 18A and 18B are graphs showing the ability of anti-CTLA4 antibodies to block the interaction of CTLA4 with its ligands CD80 and CD86, as determined using a CTLA4 blocking bioassay. Figure 18A shows the ability of the unmasked form of antibody 2 (antibody 2-2) and the masked form of antibody 2 (antibody 2-14) to block CTLA4 binding to its ligands. Antibody 2-14 was tested in a masked state (i.e., without previous exposure to proteases) and in an "activated" state due to previous exposure to proteases. For comparison, the assay was also run using an isotype control and without antibody (i.e., as a no antibody control). Figure 18A shows that when in a masked (non-activated) form due to the absence of proteases, antibody 2-14 did not show the ability to effectively block CTLA4 binding to its ligands. Analysis of fold changes over no antibody control showed no significant difference between the ability of antibody 2-14 when in masked (non-activated) form and the ability of the isotype control to block CTLA4 binding to its ligand (Figure 18B). However, Figure 18A shows that when antibody 2-14 is in an "activated" form due to previous exposure to proteases, it showed the ability to effectively block CTLA4 binding to its ligand over a range of concentrations. The ability of "activated" antibody 2-14 to block CTLA4 binding to its ligand was similar to that of unmasked antibody 2-2 (Figures 18A and 18B). Analysis of fold changes over no antibody control showed no significant difference ("ns") between the ability of "activated" antibody 2-14 and unmasked antibody 2-2 to block CTLA4 binding to its ligand (Figure 18B). [Figure 18B] See legend to Figure 18A. [Figure 19A]Figures 19A-19D show the results of immunophenotyping studies evaluating the percentage of CD45+ cells expressing markers including: CD3+ / ICOS+, CD3+ T cells, CD4+ / Ki67+, CD3+ / Ki67+, CD4+ / ICOS+, CD4+ T cells, CD8+ / ICOS+, CD8+ T cells, Tregs+ / ICOS+, CD8+ / Ki67+, Treg, Treg+ / Ki67+. [Figure 19B] See legend to Figure 19A. [Figure 19C] See legend to Figure 19A. [Figure 19D] See legend to Figure 19A. [Figure 19E] Figures 19E and 19F provide statistics comparing the antibodies tested (Group 1: IgG, Group 2: Antibody 2-1, Group 3: Antibody 2-10, Group 4: Ipilimumab, Group 5: Ipilimumab-aFuc). [Figure 19F] See legend to Figure 19E. [Figure 20A] Figures 20A and 20B show graphs depicting tumor volume over time following administration of a single injection of 20 μg, 7 μg, or 2 μg of test antibody. [Figure 20B] See legend to Figure 20A. [Figure 21] Graphs showing the results of two sets of experiments (Experiment 1 and Experiment 2) in which pharmacodynamic effects in cynomolgus monkeys were evaluated by assessing the percentage of Ki67+ cells among total CD4+ cells following administration of Antibody 2-6, Antibody 2-10, Antibody 2-14, Antibody 2-16, or an isotype control. [Figure 22] Figure 22 shows the results of an experiment evaluating the pharmacokinetics in cynomolgus monkeys following administration of one of the following test antibodies: RSV-m control, ipilimumab, antibody 2-6, antibody 2-10, antibody 2-14, antibody 2-16, or ipilimumab-m-mask. Figure 22 shows a graph showing the levels of each administered antibody in μg / mL over a 14 day period. [Figure 23A]Graphs showing the results of efficacy studies on the percentage of CD4+Ki67+ cells (left) and CD4+ICOS+ cells (right) in peripheral blood 5 days after administration, which represents the level of T cell activation. [Figure 23B] Graphs showing tumor weight (left) and CD8 / Treg ratio (right) assessed on day 7 in mice treated with 10 mg / kg (RSV-m control) or 3 mg / kg (ipilimumab, antibody 2-6, antibody 2-14, antibody 2-15). [Figure 23C] Graphs showing regulatory T cells in the tumor microenvironment (left) and CD8+ T cells in the tumor microenvironment (right) following administration of RSV-m control, ipilimumab, antibody 2-6, antibody 2-14, or antibody 2-15 in mice. [Figure 23D] 1 shows a graph showing tumor volume (mm3) over time in mice following administration of 0.3 mg / kg, 1 mg / kg, 3 mg / kg, or 10 mg / kg of antibodies, including RSV-m control, ipilimumab, antibody 2-6, antibody 2-14, and antibody 2-15. [Figure 24A] 1 shows the results of SDS-PAGE Western blot analysis using MCA205 fibrosarcoma cells assessing cleavage of Antibody 2-6, Antibody 2-14, Antibody 2-16, and Antibody 2-10 in plasma, kidney, liver, and tumor tissue. [Figure 24B] 1 shows the results of SDS-PAGE Western blot analysis using MB49 cells assessing cleavage of Antibody 2-6, Antibody 2-14, Antibody 2-16, and Antibody 2-10 in plasma, kidney, and liver tissue. [Figure 24C] 1 shows the results of SDS-PAGE Western blot analysis using MC38 cells assessing cleavage of Antibody 2-6, Antibody 2-14, Antibody 2-16, and Antibody 2-10 in plasma, kidney, liver, and tumor tissue. [Figure 24D] The results of SDS-PAGE Western blot analysis using MC38 cells assessed for cleavage are shown, providing the percentage of cleavage for selected samples. The percentage of cleavage was calculated based on densitometric intensity (% cleavage = lower band / (lower band + upper band)). [Figure 24E] Graphs showing the binding between each antibody and CTLA4 are shown, with each antibody isolated from plasma, kidney, liver, or spleen following administration to MB49-bearing B-hCTLA4 transgenic mice. [Figure 24F] 1 shows heat maps of in vitro cleavage of cleavable peptide substrates by a panel of exemplary proteases. [Figure 25A] Figures 25A-25F show radar plots showing the frequency of cleavage in conditioned media using identified mouse models (C57 / Bl6-wt, MC38-wt, MC38-hCTLA4, MB49-wt, MCA205-wt, B16-wt). Media was conditioned with tissues as indicated, such as spleen, liver, kidney, plasma, or tumor. [Figure 25B] See legend to Figure 25A. [Figure 25C] See legend to Figure 25A. [Figure 25D] See legend to Figure 25A. [Figure 25E] See legend to Figure 25A. [Figure 25F] See legend to Figure 25A. [Figure 26A] Figures 26A and 26B show the results of an in vivo cleavage study in healthy cynomolgus monkey plasma using antibody 2-14. As shown in Figure 26A, capillary electrophoresis (CE) and mass spectrometry (MS) were used to calculate the cleavage of antibody 2-14 as indicated. Figure 26B shows a graph showing the percentage of intact cleavage products of antibody 2-14 at 1 hour, 24 hours, and 72 hours. [Figure 26B] See legend to Figure 26A. [Figure 26C] As indicated, results of an in vivo cleavage study in plasma of healthy cynomolgus monkeys using antibody 2-16 are shown, including assessment of calculated cleavage using capillary electrophoresis (CE) and mass spectrometry (MS). [Figure 27A]Figures 27A and 27B show the results of ex vivo cleavage studies using medium conditioned from human lung or colon tumor samples. [Figure 27B] See legend to Figure 27A. [Figure 28-1] Figures 28A-28D show an analysis of the percentage of cleaved molecules (Antibody 2-14, Figure 28A; Antibody 2-15, Figure 28B; Antibody 2-16, Figure 28C; Antibody 2-10, Figure 28D) using mass spectrometry with colon tumor supernatants. [Figure 28-2] See description of Figure 28-1. [Figure 28-3] FIG. 28E shows the results of an ELISA assay performed using Antibody 2-6, Antibody 2-14, Antibody 2-15, Antibody 2-10, Antibody 2-16, and masked ipilimumab-m, where each tested antibody was cultured in either colon tumor conditioned media (Colon 2 4sept colon tumor samples) or RPMI as a control. [Figure 29] Cellular cytotoxicity measured using an LDH release assay completed in serum-free or fetal calf serum (FCS) supplemented medium prepared from the indicated cell lines or tumor / NAT tissues is shown. [Diagram 30] Substrate cleavage in MC38 and H2228 cell lines is shown as iceLogo graphics. [Figure 31A] 31A and 31B show substrate cleavage of NAT and tumor (TUM) tissue as iceLogo. [Figure 31B] See legend to Figure 31A. [Figure 31C] The Z-score difference at the P4-P4' positions is shown as a heatmap, highlighting residues favored for NAT or tumor-specific cleavage. Norleucine (n) is a proxy for Met in the library. [Figure 31D] The Z-score difference at the P4-P4' position is shown as a heatmap, highlighting residues favored for H2228-specific cleavage compared to either NAT or tumor samples. Norleucine (n) is a proxy for Met in the library. [Figure 32A]Figures 32A and 32B show product formation curves of selected substrates with MC38 conditioned medium; AK10-01 (Figure 32A, left), AK10-02 (Figure 32A, right), AK10-04 (Figure 32B, left), and AK10-05 (Figure 32B, right). [Figure 32B] See legend to Figure 32A. [Diagram 33] 1 shows the relative efficiencies of the major cleavage products from MC38 AMSP-MS reactions with AK10 library peptides. [Figure 34A] Figures 34A and 34B show product formation curves of selected substrates with H2228 conditioned medium; AK10-01 (Figure 34A, top left), AK10-02 (Figure 34A, top right), AK10-04 (Figure 34A, bottom), AK10-09 (Figure 34B, left), and AK10-10 (Figure 34B, right). [Figure 34B] See legend to Figure 34A. [Diagram 35] The top substrates cleaved by H2228 conditioned medium via AMSP-MS along with their relative efficiencies are shown. [Figure 36A] Figures 36A-36C show product formation curves of selected substrates with tumor conditioned media; AK10-01 (Figure 36A, top left), AK10-02 (Figure 36A, top center), AK10-03 (Figure 36A, bottom), AK10-04 (Figure 36B, top left), and AK10-05 (Figure 36B, top right), AK10-09 (Figure 36B, bottom), AK10-09 oxidized peptide (Figure 36C, left), and AK10-10 (Figure 36C, right). [Figure 36B] See legend to Figure 36A. [Figure 36C] See legend to Figure 36A. [Figure 36D] 1 shows the top substrates cleaved by tumor conditioned media via AMSP-MS along with their relative efficiencies. [Figure 37A]Figures 37A-37D show product formation curves for selected substrates with NAT conditioned medium; AK10-01 (Figure 37A, top), AK10-02 (Figure 37A, bottom), AK10-04 (Figure 37B, top), and AK10-05 (Figure 37B, bottom), AK10-09 (Figure 37C, top), AK10-09 oxidized peptide (Figure 37C, bottom), and AK10-10 (Figure 37D). [Figure 37B] See legend to Figure 37A. [Figure 37C] See legend to Figure 37A. [Figure 37D] See legend to Figure 37A. [Figure 37E] 1 shows the top truncations produced by NAT conditioned medium with the AK10 library of peptides along with their relative efficiencies. [Figure 38] Differentially expressed proteins are shown in tumor vs. NAT tissue samples as a volcano plot. Statistically significant proteins have a threshold fold difference of > 2-fold change. Proteins significantly enriched in tumor (top right, section 2) or NAT (top left, section 1) are present in these sections. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] Detailed Description Treatments such as checkpoint inhibitors show unprecedented responses in cancer, but their use is limited by immune-related adverse events (irAEs) and other toxicities (e.g., hypophysitis). For example, protein therapeutics are provided herein to specifically bind CTLA4 after activation by proteases at target sites in tumor microenvironment, achieving sustained increased response rates and significantly improved safety profiles. Protein therapeutics provided herein are engineered to precisely target pharmacological activity to tumor microenvironment by taking advantage of high local concentrations of active proteases, one of the hallmarks of cancer. This feature of tumor microenvironment is used to transform systemically inactive molecules into locally active drugs. Activation of drugs in tumor microenvironment significantly reduces systemic toxicity that may be associated with drugs administered to subjects in active form.

[0055] I. Definition Before describing the invention in detail, it should be understood that the invention is not limited to a particular composition or biological system, which can of course vary. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only, and are not intended to be limiting. As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to an antibody optionally includes a combination of two or more such antibodies, and the like.

[0056] As used herein, the term "about" refers to the normal range of error for the respective value, readily known to one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments that are inherently directed to that value or parameter.

[0057] It is understood that aspects and embodiments of the invention described herein include "comprising," "consisting," and "consisting essentially of" aspects and embodiments.

[0058] The term "antibody" includes polyclonal antibodies, monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single chain molecules, as well as antibody fragments (e.g., Fab, F(ab')2, and Fv). The term "immunoglobulin (Ig)" is used interchangeably with "antibody" herein.

[0059] The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five basic heterotetrameric units with an additional polypeptide called the J chain and contain ten antigen-binding sites, whereas IgA antibodies consist of two to five basic four-chain units that can polymerize to form multivalent aggregates in combination with the J chain. For IgG, the four-chain unit is generally about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, whereas the two H chains are linked to each other by one or more disulfide bonds, depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each α and γ chain, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus followed by a constant domain at its other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain of the heavy chain (CH1). Certain amino acid residues are thought to form an interface between the light and heavy chain variable domains. The pairing of VH and VL together forms a single antigen-binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0060] L chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domain (CH) of their heavy chains, immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses based on relatively minor differences in CH sequence and function, for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. IgG1 antibodies may exist in multiple polymorphic variants called allotypes (Jefferis and Lefranc 2009.mAbs Vol 1 Issue 4 1-7), any of which are suitable for use in the present invention. Common allotypic variants in the human population are designated by the letters a, f, n, and z.

[0061] An "isolated" antibody is an antibody that has been identified, separated, and / or recovered from a component of its production environment (e.g., natural or recombinant). In some embodiments, an isolated polypeptide is free of association with all other components from its production environment. Contaminating components of its production environment, such as those resulting from recombinant transfected cells, are materials that typically interfere with research, diagnostic, or therapeutic uses of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the polypeptide is purified to (1) greater than 95% by weight, and in some embodiments, greater than 99% by weight, of the antibody as determined, for example, by the Lowry method, (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a rolling cup sequencer, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions with Coomassie blue or silver staining. Since at least one component of the antibody's natural environment is absent, an isolated antibody includes the antibody in situ, within recombinant cells. Ordinarily, however, an isolated polypeptide or antibody will be prepared by at least one purification step.

[0062] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. In some embodiments, the monoclonal antibody has a C-terminal truncation in the heavy and / or light chain. For example, 1, 2, 3, 4, or 5 amino acid residues are truncated at the C-terminus of the heavy and / or light chain. In some embodiments, the C-terminal truncation removes the C-terminal lysine from the heavy chain. In some embodiments, the monoclonal antibody has an N-terminal truncation in the heavy and / or light chain. For example, 1, 2, 3, 4, or 5 amino acid residues are truncated at the N-terminus of the heavy and / or light chain. In some embodiments, truncated forms of the monoclonal antibody may be produced by recombinant techniques. In some embodiments, the monoclonal antibody is highly specific and directed to a single antigenic site. In some embodiments, monoclonal antibodies are highly specific and directed against multiple antigenic sites (e.g., bispecific or multispecific antibodies, etc.). The modifier "monoclonal" indicates the character of the antibody as obtained from a population of substantially homogeneous antibodies, but is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be made by a variety of techniques, including, for example, hybridoma methods, recombinant DNA methods, phage display techniques, and techniques for producing human or human-like antibodies in animals that have some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences.

[0063] The term "naked antibody" refers to an antibody that is not conjugated to a cytotoxic moiety or radiolabel.

[0064] The term "parent antibody" refers to an antibody prior to modification, such as masking of the antibody with a masking peptide.

[0065] The term "masked antibody" refers to an antibody that has been modified to include a masking peptide and, in some embodiments, other components that allow for activation or removal of the masking peptide in favorable circumstances.

[0066] "Antibody-drug conjugate" or "ADC" refers to an antibody conjugated to one or more heterologous molecule(s), including, but not limited to, a cytotoxic agent.

[0067] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically, whole antibodies include those having heavy and light chains, including the Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, an intact antibody may have one or more effector functions.

[0068] "Antibody fragments" include portions of intact antibodies, antigen binding, and / or variable regions of intact antibodies. Examples of antigen-binding antibody fragments include domain antibodies (dAbs), Fab, Fab', F(ab')2, and Fv fragments; antibodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062

[1995] ); single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Single heavy chain antibodies or single light chain antibodies can be engineered or, in the case of heavy chains, isolated from camels, sharks, libraries, or mice engineered to generate single heavy chain molecules.

[0069] Papain digestion of antibodies produced two identical antigen-binding fragments called "Fab" fragments and a residual "Fc" fragment, a designation reflecting their ability to crystallize readily. The Fab fragment consists of the entire L chain together with the variable region domain of the H chain (VH) and the first constant domain of one heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of antibodies yields a single large F(ab')2 fragment that roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activities, and are still capable of cross-linking antigen. The Fab' fragment differs from the Fab fragment by having a few additional residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domain bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0070] The Fc fragment contains the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of the antibody are determined by sequences and glycans within the Fc region, the region also recognized by Fc receptors (FcRs) found on certain cell types.

[0071] "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of one heavy chain variable region domain and one light chain variable region domain in tight non-covalent association. From the folding of these two domains, six hypervariable loops (three loops from each H chain and L chain) are generated, which contribute amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv that contains only three HVRs specific for an antigen) has the ability to recognize and bind antigen, but with lower affinity than the entire binding site.

[0072] A "single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising the VH and VL antibody domains linked in a single polypeptide chain. In some embodiments, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains, thereby enabling the sFv to form the desired structure for antigen binding. For a general review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0073] "Functional fragments" of the antibodies of the present invention include portions of an intact antibody, generally including the antigen-binding or variable regions of the intact antibody, or the Fv regions of the antibody that retain or have modified FcR binding ability. Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0074] Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequences of antibodies from a particular species or belonging to a particular antibody class or subclass, while the remaining chain(s) are identical or homologous to the corresponding sequences of antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include PRIMATIZED® antibodies, in which the antigen-binding region of the antibody is derived from an antibody generated, for example, by immunizing macaque monkeys with the antigen of interest. As used herein, "humanized antibodies" are used as a subset of "chimeric antibodies."

[0075] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's HVR are replaced by residues from an HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and / or capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications may be made to further refine antibody performance, such as binding affinity. Generally, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a non-human immunoglobulin, but the FR regions may contain one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. In some embodiments, the number of these amino acid substitutions in the FR is six or less in the H chain and three or less in the L chain. The humanized antibody also optionally comprises at least a portion of an immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986), Riechmann et al., Nature 332:323-329 (1988), and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.In some embodiments, the humanized antibody is directed against a single antigenic site. In some embodiments, the humanized antibody is directed against multiple antigenic sites. Alternative humanization methods are described in U.S. Patent No. 7,981,843 and U.S. Patent Application Publication No. 2006 / 0134098.

[0076] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. Thus, as used herein, "variable region" and "variable domain" may be used interchangeably. The variable domains of the heavy and light chains may be referred to as "VH" and "VL", respectively. These domains are generally the most variable parts of an antibody (relative to other antibodies of the same class) and contain the antigen-binding site. The variable domains of the heavy and light chains may be determined using any available method or numbering scheme, and may include, for example, the variable domains described in WO2018 / 207701, the contents of which are incorporated herein by reference. In some embodiments, the variable domains of the heavy and / or light chains may lack one or more amino acid residues on the carboxyl terminus of the variable domain (i.e., at the carboxyl terminus of the fourth framework domain) or may otherwise be included in the description of the variable domain based on a particular numbering scheme. In some embodiments, the heavy and / or light chain variable domains may comprise one or more amino acid residues on the carboxyl terminus of the variable domain (i.e. at the carboxyl terminus of the fourth framework domain) that may not otherwise be included in the description of the variable domain based on a particular numbering scheme.

[0077] As used herein, "hypervariable region", "HVR" or "HV" refers to the region of an antibody variable domain that is hypervariable in sequence and / or forms a structurally defined loop. Generally, an antibody contains six HVRs, three in VH (H1, H2, H3) and three in VL (L1, L2, L3). In natural antibodies, H3 and L3 show the most diversity of the six HVRs, and H3 in particular is thought to play a unique role in conferring fine specificity to antibodies. See, for example, Xu et al. Immunity 13:37-45 (2000); Johnson and Wu in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003)). In fact, naturally occurring camelid antibodies consisting of only heavy chains are functional and stable in the absence of light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993) and Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0078] Several HVR descriptions are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs), HVRs, are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda, MD (1991)). Chothia HVRs instead refer to the location of structural loops (Chothia and Lesk J. Mol. Biol 196:901-917 (1987)). The "contact" HVRs are based on analysis of available complex crystal structures. Residues from each of these HVRs are listed below. TIFF0007680954000001.tif52128

[0079] Unless otherwise indicated, variable domain residues (HVR and framework region residues) are numbered according to Kabat et al., supra.

[0080] "Framework" or "FR" residues are those variable domain residues other than HVR residues as herein defined.

[0081] The phrase "variable domain residue numbering according to Kabat" or "amino acid position numbering according to Kabat", or variations thereof, refers to the numbering system used for the heavy or light chain variable domains of the above compilation of antibodies of Kabat et al. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or an insertion into, the FR or HVR of the variable domain. For example, the heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat numbering of residues may be determined for a given antibody by alignment of the antibody's sequence with the "standard" Kabat numbered sequence at the region of homology.

[0082] For the purposes of this specification, an "acceptor human framework" is a framework that comprises the amino acid sequence of a VL or VH framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework that is "derived" from a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence or may comprise pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.

[0083] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and does not take into account any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be achieved using various methods within the skill of the art, such as publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, etc. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the full length of the sequences to be compared. For example, the amino acid sequence identity % of a given amino acid sequence A to a given amino acid sequence B (which may alternatively be referred to as a given amino acid sequence A having or containing a certain amino acid sequence identity % to a given amino acid sequence B, to a given amino acid sequence B, or to a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y where X is the number of amino acid residues scored as identical matches by the sequences in the alignment of A and B in the program, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A.

[0084] An antibody that "binds," "specifically binds," or is "specific for" a particular polypeptide or epitope on a particular polypeptide is an antibody that binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptides or polypeptide epitopes. In some embodiments, the binding of an activatable masked anti-CTLA4 binding protein described herein (e.g., an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof) to an unrelated anti-CTLA4 polypeptide is less than about 10% of the antibody binding to CTLA4 as measured by methods known in the art (e.g., enzyme-linked immunosorbent assay (ELISA)). In some embodiments, a binding protein (e.g., an antibody) that binds to CTLA4 (e.g., mouse CTLA4 and / or human CTLA4) has an affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦2 nM, ≦1 nM, ≦0.7 nM, ≦0.6 nM, ≦0.5 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 Equilibrium dissociation constant (K D ).

[0085] The term "CTLA4" or "CTLA4 protein" as provided herein includes any recombinant or naturally occurring form of cytotoxic T-lymphocyte-associated protein 4 (CTLA4) or a variant or homolog thereof that maintains CTLA4 protein activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to CTLA4). In some aspects, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to a naturally occurring CTLA4 polypeptide. In some embodiments, the CTLA4 is a protein identified by the NCBI sequence reference GI:83700231, a homolog, or a functional fragment thereof. In some embodiments, the CTLA4 is human CTLA4. In some embodiments, the CTLA4 is mouse CTLA4.

[0086] Antibody "effector functions" refer to the biological activities attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence variant Fc region) and vary with antibody isotype. Examples of antibody effector functions include C1q binding and complement dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0087] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound on Fc receptors (FcR) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) allows these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. Antibodies are required to "arm" the cytotoxic cells and kill the target cells by this mechanism. The primary cells for mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Fc expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). In some embodiments, the activatable masked anti-CTLA4 binding protein (e.g., activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof) described herein is engineered or expressed in cells that lack the ability to fucosylate Fc glycans to enhance ADCC. To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as the assay described in U.S. Pat. No. 5,500,362 or 5,821,337, can be performed. Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or in addition, the ADCC activity of a molecule of interest can be assessed in vivo in an animal model, such as, for example, that disclosed in Clynes et al., PNAS USA 95:652-656 (1998). Other Fc variants that alter ADCC activity and other antibody properties include those described by Ghetie et al., Nat Biotech. 15:637-40, 1997; Duncan et al., Nature 332:563-564, 1988; Lund et al., J.Immunol 147:2657-2662,1991, Lund et al,Mol Immunol 29:53-59,1992, Alegre et al,Transplantation 57:1537-1543,1994, Hutchins et al.,Proc Natl.Acad Sci USA Jefferis et al, J Immunol 157:4963-4969,1996, Armor et al., Eur J Immunol 29:2613-2624,1999, Idusogie et al,J Immunol 164:4178-4184,200, Reddy et al,J Immunol 164:1925-1933,2000, Xu et al.,Cell Immunol 200:16-26,2000, Idusogie et al,J Immunol 166:2571-2575,2001, Shields et al.,J Biol Chem 276:6591-6604,2001, Jefferis et al,Immunol Lett 82:57-65.2002, Presta et al.,Biochem Soc Trans 30:487-490,2002, Lazar et al. al.,Proc.Natl.Acad.Sci.USA 103:4005-4010,2006, and those disclosed by U.S. Patent Nos. 5,624,821, 5,885,573, 5,677,425, 6,165,745, 6,277,375, 5,869,046, 6,121,022, 5,624,821, 5,648,260, 6,194,551, 6,737,056, 6,821,505, 6,277,375, 7,335,742, and 7,317,091.

[0088] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of an immunoglobulin heavy chain Fc region can vary, the Fc region of a human IgG heavy chain is usually defined to extend from an amino acid residue at position Cys226, or from an amino acid residue at position Pro230, to its carboxyl-terminus. Suitable native sequence Fc regions for use in the antibodies of the invention include human IgG1, IgG2, IgG3, and IgG4.

[0089] As used herein, "binding affinity" refers to the strength of a non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). In some embodiments, the affinity of a binding protein (e.g., an antibody) for CTLA4 is generally determined by the equilibrium dissociation constant (K D Affinity can be measured by common methods known in the art, including those described herein.

[0090] As used herein, "binding affinity" refers to the strength of binding between multiple binding sites of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen).

[0091] An "isolated" nucleic acid molecule encoding an antibody herein is a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule that is normally associated with the environment in which it is produced.In some embodiments, an isolated nucleic acid is free from all components associated with the environment in which it is produced.An isolated nucleic acid molecule encoding a polypeptide and an antibody herein is in a form other than the form or environment in which it is found in nature.Therefore, an isolated nucleic acid molecule is distinguished from the nucleic acid encoding a polypeptide and an antibody herein that naturally exists in a cell.

[0092] The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered. Such formulations are sterile.

[0093] As used herein, "carrier" includes pharma- ceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations used. Physiologically acceptable carriers are often aqueous pH buffered solutions. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0094] As used herein, the term "treatment" refers to a clinical intervention designed to change the natural history of the individual or cell being treated during the course of clinical pathology. The desired effects of treatment include slowing the rate of disease progression, improving or mitigating the disease state, and remission or improving prognosis. An individual is successfully "treated" if, for example, one or more symptoms associated with a disorder (e.g., a neoplastic disease) are alleviated or eliminated. For example, an individual is successfully "treated" if treatment results in an improvement in the quality of life of the individual suffering from the disease, a reduction in the dose of other drugs required to treat the disease, a reduction in the frequency of disease recurrence, a reduction in the severity of the disease, a delay in the onset or progression of the disease, and / or an extension of the individual's survival.

[0095] As used herein, "in conjunction with" or "in combination with" refers to the administration of one therapeutic modality in addition to another therapeutic modality. Thus, "in conjunction with" or "in combination with" refers to the administration of one therapeutic modality before, during, or after the administration of another therapeutic modality to an individual.

[0096] As used herein, the term "prevention" includes providing prophylaxis with respect to the occurrence or recurrence of a disease in an individual. The individual may be predisposed to, susceptible to, or at risk of developing a disorder, but has not been diagnosed with the disorder. In some embodiments, the activatable masked anti-CTLA4 binding proteins (e.g., activatable masked anti-CTLA4 antibodies) described herein are used to delay the onset of a disorder.

[0097] As used herein, an individual who is "at risk" of developing a disorder may or may not have detectable disease or symptoms of a disease, and may or may not show detectable disease or symptoms of a disease prior to the treatment methods described herein. "At risk" refers to an individual having one or more risk factors, which are measurable parameters that correlate with the development of a disease, as known in the art. Individuals who have one or more of these risk factors have a higher probability of developing a disorder than individuals who do not have one or more of these risk factors.

[0098] "Effective amount" refers to an amount effective at least at the dosage and for the period of time necessary to achieve a desired or indicated effect, including a therapeutic or preventative result. An effective amount may be provided in one or more administrations. A "therapeutically effective amount" is at least the minimum concentration necessary to affect a measurable improvement of a particular disorder. A therapeutically effective amount herein may vary depending on factors such as the disease state, age, sex, and weight of the patient, as well as the ability of the antibody to elicit a desired response in an individual. A therapeutically effective amount may also be an amount in which the therapeutically beneficial effects outweigh any toxic or harmful effects of the antibody. A "prophylactically effective amount" refers to an amount effective at the dosage and for the period of time necessary to achieve a desired prophylactic result. Typically, but not necessarily, a prophylactic dose is used in subjects at a pre- or early stage of disease, so that the prophylactically effective amount may be less than the therapeutically effective amount.

[0099] "Chronic" administration refers to the administration of a pharmaceutical agent(s) continuously, as opposed to an acute mode, so as to predominate the initial therapeutic effect (activity) over an extended period of time. "Intermittent" administration is treatment that is not given continuously without interruption, but rather is cyclic in nature.

[0100] As used herein, an "individual" or "subject" is a mammal. For purposes of treatment, "mammals" include humans, domestic and farm animals, as well as zoo, sports, or pet animals, such as dogs, horses, rabbits, cows, pigs, hamsters, gerbils, mice, ferrets, rats, cats, etc. In some embodiments, the individual or subject is a human.

[0101] II. Activatable Masked Anti-CTLA4 Binding Proteins In one aspect, an activatable masked cytotoxic T-lymphocyte-associated protein 4 (CTLA4) binding protein is provided, comprising: (i) a CTLA4 binding domain; (ii) a CTLA4 binding domain masking peptide (also referred to herein as a "masking peptide"); and (iii) a linker comprising a cleavable peptide that connects the masking peptide to the CTLA4 binding domain. In some embodiments, the activatable masked CTLA4 binding protein is a masked anti-CTLA4 antibody or an antigen-binding fragment thereof. In some embodiments, the activatable masked CTLA4 binding protein is a masked bispecific antibody that binds to CTLA4. In some embodiments, the activatable masked CTLA4 binding protein is a masked chimeric receptor that binds to CTLA4.

[0102] The activatable masked CTLA4 binding proteins provided herein can bind to CTLA4 from various species, for example, some bind to human CTLA4 and / or mouse CTLA4, or cynomolgus monkey CTLA4. In some embodiments, the activatable masked anti-CTLA4 binding proteins described herein have one or more of the following characteristics: (1) bind CTLA4 (e.g., human CTLA4), (2) bind CTLA4 with higher affinity after protease cleavage of the peptide linker that connects the masking peptide to the binding protein (e.g., activation), and (3) bind CTLA4 in vivo at the tumor site.

[0103] In one aspect, provided herein is an activatable masked CTLA4 binding protein that is particularly useful for treating neoplastic diseases in which CTLA4 plays a role. Provided herein is an activatable masked CTLA4 binding protein that comprises a binding domain that can interact with (e.g., bind to) CTLA4 protein expressed on the surface of a cell (e.g., cancer cell or T cell). In some embodiments, the binding domain is linked to a masking peptide by a linker that comprises a cleavable peptide, such that the masking peptide prevents the CTLA4 binding domain from binding to the CTLA4 protein. Upon cleavage of the cleavable peptide, the masking peptide is released, thereby allowing the binding domain to interact with the CTLA4 protein.

[0104] Also provided herein in some embodiments is a masked CTLA4 binding protein (e.g., a masked anti-CTLA4 antibody or antigen-binding fragment thereof) comprising (a) a CTLA4 binding protein (e.g., an anti-CTLA4 antibody or antigen-binding fragment thereof comprising a first chain and a second chain), and (b) a masking peptide. In some embodiments, the CTLA4 binding protein is an anti-CTLA4 antibody or antigen-binding fragment thereof comprising a first chain and a second chain, and the masking peptide is linked to the amino or carboxy terminus of the first or second chain of the antibody or antigen-binding fragment thereof via a linker comprising a cleavable peptide. In some embodiments, the first chain is or comprises a heavy chain, and the second chain is or comprises a light chain, or the first chain is or comprises a light chain, and the second chain is or comprises a heavy chain. In some embodiments, the first chain is or comprises a heavy chain variable region, and the second chain is or comprises a light chain variable region, or the first chain is or comprises a light chain variable region, and the second chain is or comprises a heavy chain variable region. In some embodiments, the linker comprising a cleavable peptide comprises, from the amino terminus to the carboxy terminus, a spacer linker, a cleavable peptide, and a spacer linker. In some embodiments, the C-terminus of the masking peptide is linked to the N-terminus of the linker comprising a cleavable peptide, and the C-terminus of the linker comprising a cleavable peptide is linked to the N-terminus of the first chain, e.g., the light chain or the light chain variable region.

[0105] CTLA4-binding protein The term "CTLA4 binding protein" as provided herein refers to a polypeptide comprising a CTLA4 binding domain capable of binding to or otherwise exhibiting affinity for a CTLA4 protein. In some embodiments, the CTLA4 binding protein is an anti-CTLA4 antibody or antigen-binding fragment thereof, a bispecific antibody, an antigen-binding fragment thereof, a single-chain antibody, or the like. In some embodiments, the CTLA4 binding protein is an antibody or antigen-binding fragment thereof that binds to CTLA4. In some embodiments, the antibody or antigen-binding fragment thereof that binds to CTLA4 is an anti-CTLA4 antibody or antigen-binding fragment thereof. Thus, in some embodiments, the CTLA4 binding protein is an anti-CTLA4 antibody or antigen-binding fragment thereof. In some embodiments, the CTLA4 binding protein is a component of a chimeric antigen receptor that binds to CTLA4.

[0106] The term "CTLA4 binding domain" refers to a recombinantly expressed polypeptide domain capable of binding to or otherwise exhibiting affinity for a CTLA4 protein found in or on a cell. Methods for determining the degree of binding of a CTLA4 binding domain to CTLA4 are well known in the art.

[0107] In some embodiments, the antibody is a humanized antibody, a chimeric antibody, or a human antibody. In some embodiments, the anti-CTLA4 antibody or antigen-binding fragment thereof described herein is a monoclonal antibody. In some embodiments, the anti-CTLA4 antibody or antigen-binding fragment thereof described herein is an antibody fragment (including an antigen-binding fragment), such as a dAb, Fab'-SH, Fv, scFv, or (Fab')2 fragment. In some embodiments, the antibody or antigen-binding fragment thereof is a dimer. In some embodiments, the antibody or antigen-binding fragment thereof is a homodimer. In some embodiments, the antibody or antigen-binding fragment thereof is a heterodimer. In some embodiments, the antibody or antigen-binding fragment thereof is a heterodimer comprising a first chain and a second chain, such as a heterodimer comprising a heavy chain and a light chain. In some embodiments, the antibody or antigen-binding fragment thereof comprises a first chain and a second chain. In some embodiments, the first chain is or comprises a heavy chain, and the second chain is or comprises a light chain, or the first chain is or comprises a light chain, and the second chain is or comprises a heavy chain. In some embodiments, the first chain is or comprises a heavy chain variable region, and the second chain is or comprises a light chain variable region, or the first chain is or comprises a light chain variable region, and the second chain is or comprises a heavy chain variable region. In some embodiments, the antibody or antigen-binding fragment thereof comprises a first chain and a second chain (e.g., a light chain and a heavy chain). In some embodiments, the antibody or antigen-binding fragment thereof comprises two first chains and two second chains (e.g., two light chains and two heavy chains).In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 402 or 408, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 403 or 409, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 404 or 410, and / or the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 405 or 411, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 406 or 412, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 407 or 413.

[0108] In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 402, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 403, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 404, and / or the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 405, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 406, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 407. In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 402, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 403, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 404, and the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 405, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 406, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 407.

[0109] In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 432, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 433, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 434, and / or the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 435, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 436, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 437. In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 432, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 433, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 434, and the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 435, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 436, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 437.

[0110] In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 408, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 409, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 410, and / or the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 411, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 412, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 413. In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 408, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 409, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 410, and the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 411, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 412, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 413.

[0111] In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 438, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 439, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 440, and / or the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 441, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 442, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 443. In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 438, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 439, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 440, and the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 441, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 442, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 443.

[0112] In some embodiments, the antibody or antigen-binding fragment has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO:232 or comprises an amino acid sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology. and / or a heavy chain variable region comprising an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO:233 or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:233. In some embodiments, the antibody or antigen-binding fragment has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO:232 or an amino acid sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:232. and a heavy chain variable region comprising an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 233. In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 232 and / or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:233.In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:232 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:233.

[0113] In some embodiments, the antibody or antigen-binding fragment thereof comprises CDR-L1, CDR-L2, and CDR-L3 contained within a VL domain comprising the amino acid sequence of SEQ ID NO: 321, and CDR-H1, CDR-H2, and CDR-H3 contained within a VH domain comprising the amino acid sequence of SEQ ID NO: 323. In some embodiments, the antibody or antigen-binding fragment thereof comprises CDR-L1, CDR-L2, and CDR-L3 contained within a VL domain comprising the amino acid sequence of SEQ ID NO: 322, and CDR-H1, CDR-H2, and CDR-H3 contained within a VH domain comprising the amino acid sequence of SEQ ID NO: 324.

[0114] In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 321 or 322, and / or a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 323 or 324. In some embodiments, the antibody or antigen-binding fragment comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 321 or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology. and / or a heavy chain variable region comprising an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO:323 or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:323. In some embodiments, the antibody or antigen-binding fragment has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 321 or an amino acid sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO: 321. and a heavy chain variable region comprising an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO:323 or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:323.In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 322 or an amino acid sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology, and / or a heavy chain variable region having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 324 or an amino acid sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology. In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 322 or an amino acid sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology, and a heavy chain variable region having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 324 or an amino acid sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology. In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 322, and / or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 324.In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:322 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:324.

[0115] In some embodiments, the antibody or antigen-binding fragment thereof has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 334 or has about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO: 334. and / or a heavy chain comprising an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 90%, about 91%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:421. In some embodiments, the antibody or antigen-binding fragment thereof has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:334. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO:334 and / or a heavy chain comprising the amino acid sequence of SEQ ID NO:421. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO:334 and / or a heavy chain comprising the amino acid sequence of SEQ ID NO:421. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO:334 and / or a heavy chain comprising the amino acid sequence of SEQ ID NO:421.

[0116] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising an amino acid sequence selected from SEQ ID NO: 237-318, and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 319 or 320. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising an amino acid sequence selected from SEQ ID NO: 327-341, and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 366-380, 421, and 478. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising an amino acid sequence selected from SEQ ID NO: 327, 334, or 342-365, and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 366 or 380-397. In some embodiments, the antibody or antigen-binding fragment thereof has an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the antibody or antigen-binding fragment thereof has an IgG1 isotype comprising an amino acid substitution that enhances effector function as described herein.

[0117] In some embodiments, the CTLA4 binding domain comprises the light and heavy chains of the antigen-binding arms of a bispecific antibody. In some embodiments of the bispecific antibody, the light chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 402 or 408, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 403 or 409, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 404 or 410, and / or the heavy chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 405 or 411, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 406 or 412, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 407 or 413. In some embodiments of the bispecific antibody, the light chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 402, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 403, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 404, and the heavy chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 405, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 406, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 407. In some embodiments of the bispecific antibody, the light chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 432, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 433, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 434, and the heavy chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 435, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 436, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 437. In some embodiments of the bispecific antibody, the light chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 408, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 409, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 410, and the heavy chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 411, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 412, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 413.In some embodiments of the bispecific antibody, the light chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 438, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 439, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 440, and the heavy chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 441, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 442, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 443.

[0118] In some embodiments of the bispecific antibody, the light chain comprises CDR-L1, CDR-L2, and CDR-L3 contained within a VL domain comprising the amino acid sequence of SEQ ID NO: 321, and the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3 contained within a VH domain comprising the amino acid sequence of SEQ ID NO: 323. In some embodiments of the bispecific antibody, the light chain comprises CDR-L1, CDR-L2, and CDR-L3 contained within a VL domain comprising the amino acid sequence of SEQ ID NO: 322, and the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3 contained within a VH domain comprising the amino acid sequence of SEQ ID NO: 324.

[0119] In some embodiments of the bispecific antibody, the light chain has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:232. and / or the heavy chain comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:233. In some embodiments of the bispecific antibody, the light chain has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:321. and / or the heavy chain comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:323.In some embodiments of the bispecific antibody, the light chain has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:322. and / or the heavy chain comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:324.

[0120] In some embodiments of the bispecific antibody, the light chain comprises the amino acid sequence of SEQ ID NO: 232 and / or the heavy chain comprises the amino acid sequence of SEQ ID NO: 233. In some embodiments of the bispecific antibody, the light chain comprises an amino acid sequence selected from SEQ ID NO: 321 or 322 and / or the heavy chain comprises an amino acid sequence selected from SEQ ID NO: 323 or 324. In some embodiments of the bispecific antibody, the light chain comprises an amino acid sequence selected from SEQ ID NO: 237-318 and / or the heavy chain comprises an amino acid sequence selected from SEQ ID NO: 319 or 320.

[0121] In some embodiments of the bispecific antibody, the light chain comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 327-341. and / or the heavy chain comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 366-380, 421, and 478. In some embodiments of the bispecific antibody, the light chain comprises an amino acid sequence selected from SEQ ID NOs: 327-341 and / or the heavy chain comprises an amino acid sequence selected from SEQ ID NOs: 366-380, 421, and 478. In some embodiments of the bispecific antibody, the light chain comprises an amino acid sequence selected from SEQ ID NOs: 327, 334, or 342-365, and / or the heavy chain comprises an amino acid sequence selected from SEQ ID NOs: 366, 380-397, 421, and 478.In some embodiments of the bispecific antibody, the light chain has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:334. and / or the heavy chain comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 421 or about 70%, about 75%, about 80%, about 85%, about 90%, about 90%, about 91%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO: 421. In some embodiments of the bispecific antibody, the light chain comprises the amino acid sequence of SEQ ID NO: 334 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 421. In some embodiments of the bispecific antibody, the CTLA4 is human CTLA4. In some embodiments of the bispecific antibody, the CTLA4 is mouse CTLA4. In some embodiments, the bispecific antibody is a murine antibody. In some embodiments, the bispecific antibody is a humanized antibody, a chimeric antibody, or a human antibody. In some embodiments, the bispecific antibody has an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the bispecific antibody has an IgG1 isotype that includes amino acid substitutions that enhance effector function as described herein.

[0122] In some embodiments, the CTLA4 binding domain comprises a first chain and a second chain that bind to CTLA4, such as a portion of a ligand binding domain for use in a chimeric receptor. In some embodiments of the chimeric receptor, the first chain is a light chain variable domain. In some embodiments, the second chain is a heavy chain variable domain. In some embodiments of the chimeric receptor, the first chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 402 or 408, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 403 or 409, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 404 or 410, and / or the second chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 405 or 411, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 406 or 412, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 407 or 413. In some embodiments of the chimeric receptor, the first chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 402, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 403, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 404, and the second chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 405, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 406, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 407. In some embodiments of the chimeric receptor, the first chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 432, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 433, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 434, and the second chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 435, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 436, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 437.In some embodiments of the chimeric receptor, the first chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 408, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 409, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 410, and the second chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 411, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 412, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 413. In some embodiments of the chimeric receptor, the first chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 438, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 439, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 440, and the second chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 441, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 442, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 443.

[0123] In some embodiments of the chimeric receptor, the first chain comprises CDR-L1, CDR-L2, and CDR-L3 contained within a VL domain comprising the amino acid sequence of SEQ ID NO: 321, and the second chain comprises CDR-H1, CDR-H2, and CDR-H3 contained within a VH domain comprising the amino acid sequence of SEQ ID NO: 323. In some embodiments of the chimeric receptor, the first chain comprises CDR-L1, CDR-L2, and CDR-L3 contained within a VL domain comprising the amino acid sequence of SEQ ID NO: 322, and the second chain comprises CDR-H1, CDR-H2, and CDR-H3 contained within a VH domain comprising the amino acid sequence of SEQ ID NO: 324.

[0124] In some embodiments of the chimeric receptor, the first chain has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:232. and / or the second strand comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO:233 or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology. In some embodiments of the chimeric receptor, the first chain has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:321. and / or the second strand comprises an amino acid sequence that has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 323 or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology.In some embodiments of the chimeric receptor, the first chain has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:322. In some embodiments of the chimeric receptor, the first chain comprises an amino acid sequence of SEQ ID NO:232 and / or the second chain comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 324. In some embodiments of the chimeric receptor, the first chain comprises an amino acid sequence of SEQ ID NO:232 and / or the second chain comprises an amino acid sequence of SEQ ID NO:233. In some embodiments of the chimeric receptor, the first chain comprises an amino acid sequence selected from SEQ ID NO: 321 or 322 and / or the second chain comprises an amino acid sequence selected from SEQ ID NO: 323 or 324. In some embodiments of the chimeric receptor, the first chain comprises the amino acid sequence of SEQ ID NO: 322 and the second chain comprises the amino acid sequence of SEQ ID NO: 324.

[0125] Masking Peptides The CTLA4 binding domain masking peptide (also referred to as "masking peptide") provided herein refers to a peptide that can bind to or otherwise exhibit affinity for the CTLA4 binding domain. When bound to the CTLA4 binding domain, the masking peptide blocks, occludes, inhibits (e.g., reduces) or otherwise prevents (e.g., masks) the activity or binding of the CTLA4 binding domain to its cognate receptor or protein (i.e., CTLA4). Methods for determining the degree of binding of the CTLA4 binding domain to the CTLA4 protein are well known in the art.

[0126] In embodiments, the masking peptide has a length of at least 4 amino acids. In some embodiments, the masking peptide is a linear peptide. In some embodiments, the linear peptide is a 4-mer to 24-mer. In embodiments, the masking peptide is a cyclic peptide. In embodiments, the cyclic peptide is a 3-mer to 12-mer, as defined by the number of amino acids between the two cysteines. In embodiments, the cyclic peptide is a 3-mer to 20-mer. When the masking peptide is a cyclized peptide, the cyclized peptide is formed by a di-sulfide bond linking two cysteine ​​amino acid residues. In some embodiments, the cysteine ​​amino acid residues are terminal cysteines (i.e., located at or near the N-terminus and / or C-terminus of the masking peptide). In embodiments, a di-sulfide bond links the N-terminal cysteine ​​to the C-terminal cysteine.

[0127] In some embodiments, the masking peptide is linked to the N-terminus of the light or heavy chain of the anti-CTLA4 antibody or antigen-binding fragment thereof. In some embodiments, the masking peptide is linked to the N-terminus of the light or heavy chain variable region of the anti-CTLA4 antibody or antigen-binding fragment thereof. In some embodiments, the masking peptide is linked to the C-terminus of the light or heavy chain of the anti-CTLA4 antibody or antigen-binding fragment thereof. In some embodiments, the masking peptide is linked to the C-terminus of the light or heavy chain variable region of the anti-CTLA4 antibody or antigen-binding fragment thereof.

[0128] In some embodiments, the masking peptide is linked to the N-terminus of the light or heavy chain of the anti-CTLA4 antibody or antigen-binding fragment thereof via a linker comprising a cleavable peptide. In some embodiments, the masking peptide is linked to the N-terminus of the light chain of the anti-CTLA4 antibody or antigen-binding fragment thereof via a linker comprising a cleavable peptide. In some embodiments, the masking peptide is linked to the N-terminus of the light or heavy chain variable region of the anti-CTLA4 antibody or antigen-binding fragment thereof via a linker comprising a cleavable peptide. In some embodiments, the masking peptide is linked to the N-terminus of the light or heavy chain variable region of the anti-CTLA4 antibody or antigen-binding fragment thereof via a linker comprising a cleavable peptide. In some embodiments, the masking peptide is linked to the C-terminus of the light or heavy chain variable region of the anti-CTLA4 antibody or antigen-binding fragment thereof via a linker comprising a cleavable peptide. In some embodiments, the masking peptide is linked to the C-terminus of the light or heavy chain variable region of the anti-CTLA4 antibody or antigen-binding fragment thereof via a linker comprising a cleavable peptide.

[0129] In some embodiments, the masking peptide comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to an amino acid sequence selected from SEQ ID NOs: 1-46. Thus, in embodiments, the masking peptide comprises The amino acid sequence of TIFF0007680954000002.tif181147 has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to the amino acid sequence of TIFF0007680954000002.tif181147.

[0130] In some embodiments, the masking peptide comprises an amino acid sequence having about 90% homology to an amino acid sequence selected from SEQ ID NOs: 1 to 46. For example, the masking peptide comprises an amino acid sequence having about 90% homology to the amino acid sequence of SEQ ID NO: 1.

[0131] In some embodiments, the masking peptide comprises an amino acid sequence having about 80% homology to an amino acid sequence selected from SEQ ID NOs: 1 to 46. For example, the masking peptide comprises an amino acid sequence having about 80% homology to the amino acid sequence of SEQ ID NO: 1.

[0132] In some embodiments, the masking peptide comprises an amino acid sequence having about 70% homology to an amino acid sequence selected from SEQ ID NOs: 1 to 46. For example, the masking peptide comprises an amino acid sequence having about 70% homology to the amino acid sequence of SEQ ID NO: 1.

[0133] In some embodiments, the masking peptide amino acid sequence is an amino acid sequence selected from SEQ ID NOs: 1 to 46. For example, the masking peptide amino acid sequence is the amino acid sequence of SEQ ID NO: 1.

[0134] In some embodiments, the masking peptide comprises an amino acid sequence that has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or has about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the masking peptide comprises the amino acid sequence of SEQ ID NO:5.

[0135] In some embodiments, the masking peptide comprises an amino acid sequence that has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or has about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO: 19. In some embodiments, the masking peptide comprises the amino acid sequence of SEQ ID NO:19.

[0136] In some embodiments, at least one amino acid but not more than 20 amino acids are directly linked to the N-terminus of the masking peptide. In some embodiments, at least one amino acid but not more than 30 amino acids are directly linked to the N-terminus of the masking peptide. In some embodiments, at least one amino acid but not more than 40 amino acids are directly linked to the N-terminus of the masking peptide. In some embodiments, at least one amino acid but not more than 50 amino acids are directly linked to the N-terminus of the masking peptide. In some embodiments, at least one amino acid directly linked to the N-terminus of the masking peptide is alanine (A) or glycine-alanine (GA). In some embodiments, at least one amino acid directly linked to the N-terminus of the masking peptide is a detectable tag. In some embodiments, at least one amino acid directly linked to the N-terminus of the masking peptide is TIFF0007680954000003.tif21128. In some embodiments, at least one amino acid but not more than 50 amino acids is linked directly to the N-terminus of a masking peptide comprising an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-46. In some embodiments, at least one amino acid, but not more than 50 amino acids, is directly linked to the N-terminus of a masking peptide selected from the group consisting of SEQ ID NOs: 1-46. In some embodiments, at least one amino acid, but not more than 50 amino acids, is directly linked to the N-terminus of a masking peptide selected from the group consisting of SEQ ID NOs: 1-46, and at least one amino acid is alanine (A) or glycine-alanine (GA). In some embodiments, at least one amino acid, but not more than 50 amino acids, is directly linked to the N-terminus of a masking peptide selected from the group consisting of SEQ ID NOs: 1-46, and at least one amino acid is alanine (A).

[0137] Linker In some embodiments, the activatable masked anti-CTLA4 binding protein comprises a linker, e.g., a spacer linker. In some embodiments, the activatable masked anti-CTLA4 binding protein comprises a linker, e.g., a first spacer linker and a second spacer linker. In some embodiments, the activatable masked anti-CTLA4 binding protein comprises a linker comprising a cleavable peptide. As used herein, a "linker comprising a cleavable peptide" refers to an enzymatically cleavable linker that is covalently attached to the CTLA4 binding domain and covalently attached to the masking peptide. In some embodiments, the linker comprising a cleavable peptide is recombinantly expressed. In some embodiments, the linker comprising a cleavable peptide is a linker formed by reacting a functional (reactive) group attached to the linker with the masking peptide, e.g., using conjugation chemistry. In some embodiments, the linker comprising a cleavable peptide is a linker formed by reacting a functional (reactive) group attached to the linker with the CTLA4 binding domain, e.g., using conjugation chemistry. In some embodiments, a linker comprising a cleavable peptide joins the masking peptide to the N-terminus of the CTLA4 binding domain (e.g., the N-terminus of the light chain). In some embodiments, a linker comprising a cleavable peptide joins the masking peptide to the C-terminus of the CTLA4 binding domain (e.g., the C-terminus of the light chain).

[0138] In some embodiments, the linker comprising a cleavable peptide is fused to a masking peptide, such as when a nucleic acid encodes the linker and the masking peptide, and expressed from the cell as an amino acid sequence encoding the linker and the masking peptide. In some embodiments, the linker comprising a cleavable peptide is fused to a CTLA4 binding domain, such as when a nucleic acid encodes the linker and the CTLA4 binding domain, and expressed from the cell as an amino acid sequence encoding the linker and the CTLA4 binding domain. In some embodiments, the linker comprising a cleavable peptide links the masking peptide to the N-terminus of the CTLA4 binding domain (e.g., the N-terminus of the light chain). In some embodiments, the linker comprising a cleavable peptide links the masking peptide to the C-terminus of the CTLA4 binding domain (e.g., the C-terminus of the light chain).

[0139] In some embodiments, the linker comprising a cleavable peptide is a flexible linker comprising one or more glycine, serine, alanine, histidine, and / or proline residues. In some embodiments, the linker comprising a cleavable peptide comprises a spacer linker directly linked to the N-terminus and / or C-terminus of the cleavable peptide. In some embodiments, the spacer linker comprises one or more glycine, serine, alanine, histidine, and / or proline residues. In some embodiments, the linker comprising a cleavable peptide comprises a spacer linker and a cleavable peptide. In some embodiments, the linker comprising a cleavable peptide comprises a first spacer linker, a cleavable peptide, and a second spacer linker. Thus, in some embodiments, the masked CTLA4 binding protein (e.g., a masked anti-CTLA4 antibody or antigen-binding fragment thereof) comprises a linker comprising a cleavable peptide comprising a spacer linker (e.g., a first spacer linker, or spacer linker 1) linked to the N-terminus of the cleavable peptide and a spacer linker (e.g., a second spacer linker, or spacer linker 2) linked to the C-terminus of the cleavable peptide, each spacer linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 89-112 and 415-420. In some embodiments, the C-terminus of the cleavable peptide-comprising linker is linked to a light chain or light chain variable domain of the anti-CTLA4 antibody or antigen-binding fragment thereof, and the N-terminus of the cleavable peptide-comprising linker is linked to a masking peptide. In some embodiments, the C-terminus of the cleavable peptide-comprising linker is linked to a heavy chain or heavy chain variable domain of the anti-CTLA4 antibody or antigen-binding fragment thereof, and the N-terminus of the cleavable peptide-comprising linker is linked to a masking peptide. In some embodiments, the N-terminus of the linker comprising the cleavable peptide is linked to the light chain or light chain variable domain of the anti-CTLA4 antibody or antigen-binding fragment thereof, and the C-terminus of the linker comprising the cleavable peptide is linked to a masking peptide.In some embodiments, the N-terminus of the linker comprising the cleavable peptide is linked to a heavy chain or heavy chain variable domain of an anti-CTLA4 antibody or antigen-binding fragment thereof, and the C-terminus of the linker comprising the cleavable peptide is linked to a masking peptide.

[0140] In some embodiments, the spacer linker comprises an amino acid sequence selected from SEQ ID NOs: 89-112 and 415-420. In some embodiments, the spacer linker is directly linked to the N-terminus of the cleavable peptide and comprises an amino acid sequence selected from SEQ ID NOs: 89-100. In some embodiments, the spacer linker is directly linked to the C-terminus of the cleavable peptide and comprises an amino acid sequence selected from SEQ ID NOs: 101-112 and 415-420. In some embodiments, a masking peptide described herein is directly linked to the N-terminus of the spacer linker. Thus, in some embodiments, a linker comprising a cleavable peptide comprises, from the N-terminus to the C-terminus, 1) a spacer linker (e.g., a spacer linker comprising an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 89-112 and 415-420), 2) a cleavable peptide such as those described herein (e.g., a cleavable peptide comprising an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 47-88, 464-469, and 479-508), and 3) a spacer linker (e.g., a spacer linker comprising an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 89-112 and 415-420).

[0141] In some embodiments, the linker comprising a cleavable peptide comprises, from the N-terminus to the C-terminus, 1) a spacer linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 89-112 and 415-420, 2) a cleavable peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-88, 464-469, and 479-508, and 3) a spacer linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 89-112 and 415-420.

[0142] In some embodiments, the linker comprising a cleavable peptide comprises, from N-terminal to C-terminal direction, 1) a spacer linker comprising the amino acid sequence of SEQ ID NO:420, 2) a cleavable peptide comprising the amino acid sequence of SEQ ID NO:50, and 3) a spacer linker comprising the amino acid sequence of SEQ ID NO:102.

[0143] In some embodiments, the linker comprising a cleavable peptide comprises, from N-terminal to C-terminal direction, 1) a spacer linker comprising the amino acid sequence of SEQ ID NO:96, 2) a cleavable peptide comprising the amino acid sequence of SEQ ID NO:86, and 3) a spacer linker comprising the amino acid sequence of SEQ ID NO:102.

[0144] In some embodiments, the linker comprising a cleavable peptide comprises, from N-terminal to C-terminal direction, 1) a spacer linker comprising the amino acid sequence of SEQ ID NO:415, 2) a cleavable peptide comprising the amino acid sequence of SEQ ID NO:86, and 3) a spacer linker comprising the amino acid sequence of SEQ ID NO:102.

[0145] In some embodiments, the linker comprising a cleavable peptide comprises, from N-terminal to C-terminal direction, 1) a spacer linker comprising the amino acid sequence of SEQ ID NO:416, 2) a cleavable peptide comprising the amino acid sequence of SEQ ID NO:47, and 3) a spacer linker comprising the amino acid sequence of SEQ ID NO:102.

[0146] In some embodiments, the linker comprising a cleavable peptide comprises, from N-terminal to C-terminal direction, 1) a spacer linker comprising the amino acid sequence of SEQ ID NO:417, 2) a cleavable peptide comprising the amino acid sequence of SEQ ID NO:57, and 3) a spacer linker comprising the amino acid sequence of SEQ ID NO:102.

[0147] In some embodiments, the linker comprising a cleavable peptide comprises, from N-terminal to C-terminal direction, 1) a spacer linker comprising the amino acid sequence of SEQ ID NO:418, 2) a cleavable peptide comprising the amino acid sequence of SEQ ID NO:48, and 3) a spacer linker comprising the amino acid sequence of SEQ ID NO:102.

[0148] In some embodiments, the linker comprising a cleavable peptide comprises, from N-terminal to C-terminal direction, 1) a spacer linker comprising the amino acid sequence of SEQ ID NO:417, 2) a cleavable peptide comprising the amino acid sequence of SEQ ID NO:72, and 3) a spacer linker comprising the amino acid sequence of SEQ ID NO:102.

[0149] In some embodiments, the linker comprising a cleavable peptide comprises, from N-terminal to C-terminal direction, 1) a spacer linker comprising the amino acid sequence of SEQ ID NO:418, 2) a cleavable peptide comprising the amino acid sequence of SEQ ID NO:51, and 3) a spacer linker comprising the amino acid sequence of SEQ ID NO:102.

[0150] In some embodiments, the linker comprising a cleavable peptide comprises, from N-terminal to C-terminal direction, 1) a spacer linker comprising the amino acid sequence of SEQ ID NO:419, 2) a cleavable peptide comprising the amino acid sequence of SEQ ID NO:54, and 3) a spacer linker comprising the amino acid sequence of SEQ ID NO:102.

[0151] In some embodiments, the linker comprising a cleavable peptide comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 454-462. In some embodiments, the linker comprising a cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 454-462. In some embodiments, the linker comprising a cleavable peptide comprises the amino acid sequence of SEQ ID NO: 454. In some embodiments, the linker comprising a cleavable peptide comprises the amino acid sequence of SEQ ID NO:455.

[0152] The linker may be conjugated to the masking peptide and / or the CTLA4 binding protein by a variety of methods well known in the art. The terms "conjugate" and "conjugation chemistry" refer to reactions with known reactive groups that proceed under relatively mild conditions. These include, but are not limited to, nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions), and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reactions, Diels-Alder additions). These and other useful reactions are discussed, for example, in March, Advanced Organic Chemistry, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson, Bioconjugate Techniques, Academic Press, San Diego, 1996; and Feeney et al., Modification of Proteins; Advances in Chemistry Series, Vol. 198, American Chemical Society, Washington, DC, 1982.

[0153] Useful reactive functional groups for use in the conjugation chemistry herein include, for example, (a) carboxyl groups and various derivatives thereof, including, but not limited to, N-hydroxysuccinimide esters, N-hydroxybenztriazole esters, acid halides, acylimidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl, and aromatic esters; (b) hydroxyl groups, which can be converted to esters, ethers, aldehydes, and the like; (c) haloalkyl groups, where the halide can be subsequently displaced with a nucleophilic group, such as an amine, a carboxylate anion, a thiol anion, a carbanion, or an alkoxide ion, thereby resulting in the covalent attachment of a new group at the site of the halogen atom; (d) dienophile groups, such as, for example, a maleimide group, which can participate in Diels-Alder reactions; (e) functional groups, such as, for example, a Grignard addition or an alkyllithium addition, and the like. (f) sulfonyl halogen groups for subsequent reaction with amines to form, for example, sulfonamides; (g) thiol groups, which can be converted to disulfides, reacted with acyl halides, or attached to metals such as gold; (h) amine or sulfhydryl groups, which can be, for example, acylated, alkylated, or oxidized; (i) alkenes, which can undergo, for example, cycloaddition, acylation, Michael addition, etc.; (j) epoxides, which can react, for example, with amines and hydroxyl compounds; (k) phosphoramidites and other standard functional groups useful in nucleic acid synthesis; (i) metal-silicon oxide linkages; and (l) metal linkages to reactive phosphorus groups (e.g., phosphines) to form, for example, a phosphodiester linkage.

[0154] The reactive functional groups can be selected so as not to participate in or interfere with the chemical stability of the compositions described herein, or alternatively, the reactive functional groups can be protected from participating in the crosslinking reaction by the presence of a protecting group.

[0155] In some embodiments, the linker can be engineered to be fused to the masking peptide and / or CTLA4 binding protein by various methods well known in the art. For example, a nucleic acid can be engineered to encode a linker that includes a masking peptide and / or a CTLA4 binding protein to generate a fusion protein when recombinantly expressed from a host cell.

[0156] Cleavable peptides The masked CTLA4 binding proteins provided herein (e.g., masked anti-CTLA4 antibodies or antigen-binding fragments thereof) in some embodiments comprise a cleavable peptide. In some embodiments, the cleavable peptide is included within a linker that comprises a cleavable peptide. The linkers provided herein that comprise a cleavable peptide may comprise a protease cleavage site within the cleavable peptide. As used herein, "cleavage site" refers to a recognizable site for cleavage of a portion of a linker (e.g., a linker that comprises a cleavable peptide as described above) found in the CTLA4 binding proteins described herein. Thus, the cleavage site may be found in the sequence of the cleavable peptide described herein, including embodiments thereof. In some embodiments, the cleavage site is an amino acid sequence that is recognized and cleaved by a cleavage agent. Exemplary cleavage agents include proteins, enzymes, DNAzymes, RNAzymes, metals, acids, and bases. The cleavable peptide may be any peptide that comprises a protease cleavage site. Exemplary cleavable peptides are shown in Table 1.

[0157] Table 1. Representative cleavable peptides TIFF0007680954000004.tif216160

[0158] Thus, in some embodiments, the cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-88, 464-469, and 479-508. In some embodiments, the cleavable peptide comprises an amino acid sequence of SEQ ID NO: 50. In some embodiments, the cleavable peptide comprises an amino acid sequence of SEQ ID NO: 86. In some embodiments, the cleavable peptide comprises an amino acid sequence of SEQ ID NO: 47. In some embodiments, the cleavable peptide comprises an amino acid sequence of SEQ ID NO: 57. In some embodiments, the cleavable peptide comprises an amino acid sequence of SEQ ID NO: 48. In some embodiments, the cleavable peptide comprises an amino acid sequence of SEQ ID NO: 72. In some embodiments, the cleavable peptide comprises an amino acid sequence of SEQ ID NO: 51. In some embodiments, the cleavable peptide comprises an amino acid sequence of SEQ ID NO: 54.

[0159] In some embodiments, the protease cleavage site is a tumor-associated protease cleavage site. As provided herein, a "tumor-associated protease cleavage site" is an amino acid sequence recognized by a protease, the expression of which is specific to a tumor cell or its tumor cell environment. In some embodiments, the protease cleavage site is selected from the group consisting of ABHD12, ADAM12, ABHD12B, ABHD13, ABHD17A, ADAM19, ADAM20, ADAM21, ADAM28, ADAM30, ADAM33, ADAM8, ABHD17A, ADAMDECl, ADAMTS1, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19 ... AMTS2, ADAMTS20, ADAMTS3, ADAMTS4, ABHD17B, ADAMTS5, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADAMTSL1, ADAMTSL2, ADAMTSL3, AB HD17C, ADAMTSL5, ASTL, BMP1, CELA1, CELA2A, CELA2B, CELA3A, CELA3B, ADAM10, ADAM15, ADAM17, ADAM9, ADAMTS4, CTSE, CTSF, ADAMT SL4, CMA1, CTRB1, CTRC, CTSO, CTRl, CTSA, CTSW, CTSB, CTSC, CTSD, ESP1, CTSG, CTSH, GZMA, GZMB, GZMH, CTSK, GZMM, CTSL, CTSS, CTS V, CTSZ, HTRA4, KLK10, KLK11, KLK13, KLK14, KLK2, KLK4, DPP4, KLK6, KLK7, KLKB1, ECE1, ECE2, ECEL1, MASP2, MEP1A, MEP1B, ELANE, F AP, GZMA, MMP11, GZMK, HGFAC, HPN, HTRA1, MMP11, MMP16, MMP17, MMP19, HTRA2, MMP20, MMP21, HTRA3, HTRA4, KEL, MMP23B, MMP24, MMP 25, MMP26, MMP27, MMP28, KLK5, MMP3, MMP7, MMP8, MMP9, LGMN, LNPEP, MASP1, PAPPA, PAPPA2, PCSK1, NAPSA, PCSK5, PCSK6, MME, MMP1,The cleavage sites are recognized by one or more enzymes selected from the group consisting of MMP10, PLAT, PLAU, PLG, PRSS1, PRSS12, PRSS2, PRSS21, PRSS3, PRSS33, PRSS4, PRSS55, PRSS57, MMP12, PRSS8, PRSS9, PRTN3, MMP13, MMP14, ST14, TMPRSS10, TMPRSS11A, TMPRSS11D, TMPRSS11E, TMPRSS11F, TMPRSS12, TMPRSS13, MMP15, TMPRSS15, MMP2, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS5, TMPRSS6, TMPRSS7, TMPRSS9, NRDC, OVCH1, PAMR1, PCSK3, PHEX, TINAG, TPSAB1, TPSD1, and TPSG1. In some embodiments, the protease cleavage site is a cleavage site recognized by one or more enzymes selected from the group consisting of ADAM17, HTRA1, PRSS1, FAP, GZMK, NAPSA, MMP1, MMP2, MMP9, MMP10, MMP7, MMP12, MMP28, ADAMTS9, HGFAC, and HTRA3.

[0160] In embodiments, the protease cleavage site is a matrix metalloprotease (MMP) cleavage site, a diintegrin and metalloprotease domain-containing (ADAM) metalloprotease cleavage site, a prostate-specific antigen (PSA) protease cleavage site, a urokinase-type plasminogen activator (uPA) protease cleavage site, a membrane-type serine protease 1 (MT-SP1) protease cleavage site, a matriptase protease cleavage site (ST14), or a legumain protease cleavage site. In embodiments, the matrix metalloprotease (MMP) cleavage site is an MMP9 cleavage site, an MMP13 cleavage site, or an MMP2 cleavage site. In embodiments, the biintegrin and metalloprotease domain-containing (ADAM) metalloprotease cleavage site is an ADAM9 metalloprotease cleavage site, an ADAM10 metalloprotease cleavage site, or an ADAM17 metalloprotease cleavage site. The protease cleavage site may be specified by a specific amino acid sequence.

[0161] In some embodiments, the cleavable peptides are ABHD12, ADAM12, ABHD12B, ABHD13, ABHD17A, ADAM19, ADAM20, ADAM21, ADAM28, ADAM30, ADAM33, ADAM8, ABHD17A, ADAMDEC1, ADAMTS1, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS2, ADAMTS20, ADAMTS3, ADAMTS4, ABHD17B, ADAMTS5, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADAMTSL1, ADAMTSL2, ADAMTSL3, ABHD17C, ADAMTSL5, ASTL, BMP1, CELA1, CELA2A, CELA2B, CELA3A, CELA3B, ADAM10, ADAM15, ADAM17, ADAM9, ADAMTS4, CTSE, CTSF, ADAMTSL4, CMA1, CTRB1, CTRC, CTSO, CTRl, CTSA, CTSW, CTSB, CTSC, CTSD, ESP1, CTSG, CTSH, GZMA, GZMB, GZMH, CTSK, GZMM, CTSL, CTSS, CTSV, CTSZ, HTRA4, KLK10, KLK11, KLK13, KLK14, KLK2, KLK4, DPP4, KLK6, KLK7, KLKB1, ECE1, ECE2, ECEL1, MASP2, MEP1A, MEP1B, ELANE, FAP, GZMA, MMP11, GZMK, HGFAC, HPN, HTRA1, MMP11, MMP16, MMP17, MMP19, HTRA2, MMP20, MMP21, HTRA3, HTRA4, KEL, MMP23B, MMP24, MMP25, MMP26, MMP27, MMP28, KLK5, MMP3, MMP7, MMP8, MMP9, LGMN, LNPEP, MASP1, PAPPA, PAPPA2, PCSK1, NAPSA, PCSK5, PCSK6, MME, MMP1, MMP10, PLAT, PLAU, PLG, PRSS1, PRSS12, PRSS2, PRSS21, PRSS3, PRSS33, PRSS4, PRSS55, PRSS57, MMP12, PRSS8, PRSS9, PRTN3, MMP13, MMP14, ST14, TMPRSS10,The peptide is cleaved by one or more enzymes selected from the group consisting of TMPRSS11A, TMPRSS11D, TMPRSS11E, TMPRSS11F, TMPRSS12, TMPRSS13, MMP15, TMPRSS15, MMP2, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS5, TMPRSS6, TMPRSS7, TMPRSS9, NRDC, OVCH1, PAMR1, PCSK3, PHEX, TINAG, TPSAB1, TPSD1, and TPSG1. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of ADAM17, HTRA1, PRSS1, FAP, GZMK, NAPSA, MMP1, MMP2, MMP9, MMP10, MMP7, MMP12, MMP28, ADAMTS9, HGFAC, and HTRA3.

[0162] In embodiments, the cleavable peptide is a 5mer (i.e., a peptide that is 5 amino acids long), a 6mer (i.e., a peptide that is 6 amino acids long), a 7mer (i.e., a peptide that is 7 amino acids long), an 8mer (i.e., a peptide that is 8 amino acids long), a 9mer (i.e., a peptide that is 9 amino acids long), a 10mer (i.e., a peptide that is 10 amino acids long), an 11mer (i.e., a peptide that is 11 amino acids long), a 12mer (i.e., a peptide that is 12 amino acids long), or a 13mer (i.e., a peptide that is 13 amino acids long).

[0163] Thus, in some embodiments, a linker comprising a masking peptide and a cleavable peptide comprises, in the N-terminal to C-terminal direction, 1) a masking peptide (e.g., a masking peptide comprising an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1-46), 2) a spacer linker (e.g., a spacer linker comprising an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 89-112 and 415-420), 3) a cleavable peptide such as those described herein (e.g., a cleavable peptide comprising an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 47-88, 464-469, and 479-508), and 4) a spacer linker (e.g., a spacer linker comprising an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 89-112 and 415-420). In some embodiments, at least one amino acid, but not more than 20 amino acids, is directly linked to the N-terminus of the masking peptide. In some embodiments, at least one amino acid, but not more than 30 amino acids, is directly linked to the N-terminus of the masking peptide. In some embodiments, at least one amino acid, but not more than 40 amino acids, is directly linked to the N-terminus of the masking peptide. In some embodiments, at least one amino acid, but not more than 50 amino acids, is directly linked to the N-terminus of the masking peptide. In some embodiments, the at least one amino acid directly linked to the N-terminus of the masking peptide is alanine (A) or glycine-alanine (GA). In some embodiments, the at least one amino acid directly linked to the N-terminus of the masking peptide is a detectable tag. In some embodiments, the at least one amino acid directly linked to the N-terminus of the masking peptide is The file is TIFF0007680954000005.tif13135.

[0164] In some embodiments, the activatable masked anti-CTLA4 binding proteins described herein include a peptide comprising a masking peptide, a spacer linker, and a cleavable peptide, wherein the peptide comprises an amino acid sequence selected from SEQ ID NOs: 113-231. In some embodiments, the activatable masked anti-CTLA4 binding proteins described herein include a peptide comprising a masking peptide, a spacer linker, and a cleavable peptide, wherein the peptide comprises an amino acid sequence selected from SEQ ID NOs: 113-193. In some embodiments, the activatable masked anti-CTLA4 binding proteins described herein include a peptide comprising a masking peptide, a spacer linker, and a cleavable peptide, wherein the peptide comprises an amino acid sequence selected from SEQ ID NOs: 194-206. In some embodiments, the activatable masked anti-CTLA4 binding proteins described herein include a peptide comprising a masking peptide, a spacer linker, and a cleavable peptide, wherein the peptide comprises an amino acid sequence selected from SEQ ID NOs: 207-231.

[0165] In some embodiments, the activatable masked anti-CTLA4 binding proteins described herein include a peptide comprising a masking peptide, a first spacer linker, a cleavable peptide, and a second spacer linker, wherein the peptide comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 113-231, 444, 446-448, and 450-453, or having about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology. In some embodiments, the activatable masked anti-CTLA4 binding proteins described herein comprise a peptide comprising a masking peptide, a first spacer linker, a cleavable peptide, and a second spacer linker, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 113-231, 444, 446-448, and 450-453. In some embodiments, the activatable masked anti-CTLA4 binding proteins described herein include a peptide comprising a masking peptide, a first spacer linker, a cleavable peptide, and a second spacer linker, wherein the peptide comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 444, 446-448, and 450-453, or having about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology.In some embodiments, the activatable masked anti-CTLA4 binding proteins described herein comprise a peptide comprising a masking peptide, a first spacer linker, a cleavable peptide, and a second spacer linker, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 444, 446-448, and 450-453.

[0166] Exemplary Masked CTLA4 Binding Proteins Certain exemplary embodiments of masked CTLA4 binding proteins that incorporate certain features described above are described below, which are merely exemplary and should not be construed as limiting.

[0167] In some embodiments, provided herein is a masked antibody comprising: a) an antibody or antigen-binding fragment thereof that binds to CTLA4 (e.g., human CTLA4), where the antibody or antigen-binding fragment thereof comprises a first chain and a second chain; and b) a masking peptide comprising an amino acid sequence selected from SEQ ID NOs: 1-46, where the masking peptide is linked to the amino or carboxy terminus of the first or second chain of the antibody or antigen-binding fragment thereof via a linker comprising a cleavable peptide. In some embodiments, the antibody or antigen-binding fragment thereof that binds to CTLA4 is any anti-CTLA4 antibody or antigen-binding fragment thereof described herein. In some embodiments, the antibody or antigen-binding fragment thereof comprises two first chains and two second chains. In some embodiments, the first chain is a light chain and the second chain is a heavy chain. In some embodiments, the first chain is a light chain variable domain and the second chain is a heavy chain variable domain. In some embodiments, the cleavable peptide comprises an amino acid sequence selected from SEQ ID NOs: 47-88, 464-469, and 479-508. In some embodiments, the spacer linker is directly linked to the N-terminus and / or C-terminus of the cleavable peptide. In some embodiments, the spacer linker comprises an amino acid sequence selected from SEQ ID NOs: 89-112 and 415-420. In some embodiments, at least one amino acid, but not more than 20, 30, 40, or 50 amino acids, is directly linked to the N-terminus of the masking peptide. In some embodiments, the at least one amino acid is alanine (A) or glycine-alanine (GA). In some embodiments, the at least one amino acid directly linked to the N-terminus of the masking peptide is a detectable tag. In some embodiments, the at least one amino acid directly linked to the N-terminus of the masking peptide is The file is TIFF0007680954000006.tif21128.

[0168] In some embodiments, a masked antibody is also provided herein, comprising: a) an antibody or antigen-binding fragment thereof that binds to CTLA4 (e.g., human CTLA4), wherein the antibody or antigen-binding fragment thereof comprises a first chain and a second chain; and b) a masking peptide comprising an amino acid sequence selected from SEQ ID NOs: 1-46, wherein the masking peptide is linked to the amino termini of the first chain and the second chain of the antibody or antigen-binding fragment thereof via a linker comprising a cleavable peptide. In some embodiments, the antibody or antigen-binding fragment thereof that binds to CTLA4 is any anti-CTLA4 antibody or antigen-binding fragment thereof described herein. In some embodiments, the antibody or antigen-binding fragment thereof comprises two first chains and two second chains. In some embodiments, the first chain is a light chain and the second chain is a heavy chain. In some embodiments, the first chain is a light chain variable domain and the second chain is a heavy chain variable domain. In some embodiments, the cleavable peptide comprises an amino acid sequence selected from SEQ ID NOs: 47-88, 464-469, and 479-508. In some embodiments, the spacer linker is directly linked to the N-terminus and / or C-terminus of the cleavable peptide. In some embodiments, the spacer linker comprises an amino acid sequence selected from SEQ ID NOs: 89-112 and 415-420. In some embodiments, at least one amino acid, but not more than 20, 30, 40, or 50 amino acids, is directly linked to the N-terminus of the masking peptide. In some embodiments, the at least one amino acid is alanine (A) or glycine-alanine (GA). In some embodiments, the at least one amino acid directly linked to the N-terminus of the masking peptide is a detectable tag. In some embodiments, the at least one amino acid directly linked to the N-terminus of the masking peptide is The file is TIFF0007680954000007.tif21128.

[0169] Further provided herein in some embodiments is a masked antibody comprising: a) an antibody or antigen-binding fragment thereof that binds to CTLA4 (e.g., human CTLA4), wherein the antibody or antigen-binding fragment thereof comprises a first chain and a second chain; and b) a masking peptide comprising an amino acid sequence selected from SEQ ID NOs: 1-46, wherein the masking peptide is linked to the carboxy termini of the first chain and the second chain of the antibody or antigen-binding fragment thereof via a linker comprising a cleavable peptide. In some embodiments, the antibody or antigen-binding fragment thereof that binds to CTLA4 is any anti-CTLA4 antibody or antigen-binding fragment thereof described herein. In some embodiments, the antibody or antigen-binding fragment thereof comprises two first chains and two second chains. In some embodiments, the first chain is a light chain and the second chain is a heavy chain. In some embodiments, the first chain is a light chain variable domain and the second chain is a heavy chain variable domain. In some embodiments, the cleavable peptide comprises an amino acid sequence selected from SEQ ID NOs: 47-88, 464-469, and 479-508. In some embodiments, the spacer linker is directly linked to the N-terminus and / or C-terminus of the cleavable peptide. In some embodiments, the spacer linker comprises an amino acid sequence selected from SEQ ID NOs: 89-112 and 415-420. In some embodiments, at least one amino acid, but not more than 20, 30, 40, or 50 amino acids, is directly linked to the N-terminus of the masking peptide. In some embodiments, the at least one amino acid is alanine (A) or glycine-alanine (GA). In some embodiments, the at least one amino acid directly linked to the N-terminus of the masking peptide is a detectable tag. In some embodiments, the at least one amino acid directly linked to the N-terminus of the masking peptide is The file is TIFF0007680954000008.tif21128.

[0170] Further provided herein, in some embodiments, is a) an antibody or antigen-binding fragment thereof that binds to CTLA4 (e.g., human CTLA4), and b) a masking peptide comprising an amino acid sequence selected from SEQ ID NOs: 1-46, wherein the masking peptide is linked to the C-terminus or N-terminus of a first chain of the antibody via a linker comprising a cleavable peptide, and the masking peptide is linked to the C-terminus or N-terminus of a second chain of the antibody via a linker comprising a cleavable peptide. In some embodiments, the antibody or antigen-binding fragment thereof that binds to CTLA4 is any anti-CTLA4 antibody or antigen-binding fragment thereof described herein. In some embodiments, a) the first chain of the antibody is a light chain and the second chain of the antibody is a light chain, b) the first chain of the antibody is a heavy chain and the second chain of the antibody is a heavy chain, or c) the first chain of the antibody is a light chain and the second chain of the antibody is a heavy chain. Thus, in some embodiments, the isolated antibody comprises a masking peptide on the C-terminus and / or N-terminus of each of the two light chains and the C-terminus and / or N-terminus of each of the two heavy chains. In some embodiments, the cleavable peptide comprises an amino acid sequence selected from SEQ ID NOs: 47-88, 464-469, and 479-508. In some embodiments, a spacer linker is directly linked to the N-terminus and / or C-terminus of the cleavable peptide. In some embodiments, the spacer linker comprises an amino acid sequence selected from SEQ ID NOs: 89-112 and 415-420. In some embodiments, at least one amino acid, but not more than 20, 30, 40, or 50 amino acids, is directly linked to the N-terminus of the masking peptide. In some embodiments, the at least one amino acid is alanine (A) or glycine-alanine (GA). In some embodiments, at least one amino acid directly linked to the N-terminus of the masking peptide is a detectable tag. In some embodiments, at least one amino acid directly linked to the N-terminus of the masking peptide is a The file is TIFF0007680954000009.tif13130.

[0171] In some embodiments, also provided herein is a masked anti-CTLA4 antibody or antigen-binding fragment thereof comprising: a) a light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 438, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 439, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 440, and a heavy chain variable region comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 441, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 442, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 443; b) a masking peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-46; and c) a cleavable peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-88, 464-469, and 479-508. In some embodiments, the cleavable peptide comprises the amino acid sequence of SEQ ID NO: 50. In some embodiments, the cleavable peptide comprises the amino acid sequence of SEQ ID NO: 86. In some embodiments, at least one amino acid, but not more than 20, 30, 40, or 50 amino acids, is linked directly to the N-terminus of the masking peptide. In some embodiments, the at least one amino acid is alanine (A) or glycine-alanine (GA). In some embodiments, the at least one amino acid linked directly to the N-terminus of the masking peptide is a detectable tag. In some embodiments, the at least one amino acid linked directly to the N-terminus of the masking peptide is TIFF0007680954000010.tif13131. In some embodiments, the masking peptide is linked to a cleavable peptide, and the cleavable peptide is linked to a light chain variable region or a heavy chain variable region. In some embodiments, the masked anti-CTLA4 antibody or antigen-binding fragment thereof further comprises a spacer linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 89-112 and 415-420 that links the masking peptide to the cleavable peptide, and further comprises a spacer linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 89-112 and 415-420 that links the cleavable peptide to the light chain variable region or the heavy chain variable region. In some embodiments, the spacer linker that links the masking peptide to the cleavable peptide comprises the amino acid sequence of SEQ ID NO: 420, and the spacer linker that links the cleavable peptide to the light chain variable region or the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 102. In some embodiments, the spacer linker linking the masking peptide to the cleavable peptide comprises the amino acid sequence of SEQ ID NO:96, and the spacer linker linking the cleavable peptide to the light chain variable region or the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:102. In some embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO:322, and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:324. In some embodiments, the masked anti-CTLA4 antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:421, a light chain comprising the amino acid sequence of SEQ ID NO:334, and a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:113-231 and 444-453. In some embodiments, the masked anti-CTLA4 antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:421 and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:358 and 422-431.

[0172] In one aspect, provided herein is an activatable masked anti-CTLA4 antibody, or antigen-binding fragment thereof, comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 232, and / or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 233. In a further aspect, provided herein is an activatable masked anti-CTLA4 antibody, or antigen-binding fragment thereof, comprising a light chain comprising an amino acid sequence selected from SEQ ID NOs: 237-318, and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 319 or 320.

[0173] In one aspect, provided herein is an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 321 or 322, and / or a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 323 or 324. In some embodiments, provided herein is a masked anti-CTLA4 antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an amino acid sequence of SEQ ID NO: 322, and / or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 324. In a further aspect, provided herein is an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprising a light chain comprising an amino acid sequence selected from SEQ ID NO: 327-341, and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 366-380, 421, and 478. In yet another further aspect, provided herein is an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprising a light chain comprising an amino acid sequence selected from SEQ ID NO: 327, 334, or 342-365, and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 366 or 380-397. In some embodiments, provided herein is a masked anti-CTLA4 antibody or antigen-binding fragment thereof comprising a light chain comprising an amino acid sequence of SEQ ID NO: 334, and a heavy chain comprising an amino acid sequence of SEQ ID NO: 421. In some embodiments, provided herein is a masked anti-CTLA4 antibody or antigen-binding fragment thereof comprising a light chain comprising an amino acid sequence of SEQ ID NO: 327, and a heavy chain comprising an amino acid sequence of SEQ ID NO: 478.

[0174] In some embodiments, provided herein is an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof, comprising a heavy chain variable domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 233. In some embodiments, provided herein is an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof, comprising a heavy chain variable domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from SEQ ID NO: 323 or 324. In some embodiments, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity contains substitutions, insertions, or deletions relative to the reference sequence, but an antibody containing the amino acid sequence retains the ability to bind to CTLA4 (e.g., human CTLA4). In some embodiments, the substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, or 5 amino acids) occur in a region outside the HVR (i.e., within the FR). In some embodiments, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:233. In some embodiments, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising an amino acid sequence selected from SEQ ID NO:323 or 324.

[0175] In some embodiments, provided herein is an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof, comprising a light chain variable domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 232. In some embodiments, provided herein is an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof, comprising a light chain variable domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from SEQ ID NO: 321 or 322. In some embodiments, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity contains substitutions, insertions, or deletions relative to the reference sequence, but an antibody containing the amino acid sequence retains the ability to bind to CTLA4 (e.g., human CTLA4). In some embodiments, the substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, or 5 amino acids) occur in a region outside the HVR (i.e., within the FR). In some embodiments, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 232. In some embodiments, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprises a light chain variable domain comprising an amino acid sequence selected from SEQ ID NO: 321 or 322.

[0176] In some embodiments, provided herein is an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof, comprising: a) an amino acid sequence comprising a masking peptide, a linker comprising a cleavable peptide, and a light chain; and b) an amino acid sequence comprising a heavy chain. In some embodiments, the amino acid sequence comprising the masking peptide, the linker comprising a cleavable peptide, and the light chain is selected from the group consisting of SEQ ID NOs: 358, 422, 424-426, and 428-431. In some embodiments, the amino acid sequence comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 421. In some embodiments, the amino acid sequence comprising the masking peptide, the linker comprising a cleavable peptide, and the light chain is selected from the group consisting of SEQ ID NOs: 358, 422, 424-426, and 428-431, and the amino acid sequence comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 421.

[0177] In some embodiments, the amino acid sequence has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 358 and 422-431, or comprises an amino acid sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 358 and 422-431, and / or a sequence Provided herein is an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprising an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of No. 421 or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology. In some embodiments, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 358 and 422-431 or has about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% , about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:421, including an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO:421 or an amino acid sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:421.In some embodiments, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 358 and 422-431, and / or comprises the amino acid sequence of SEQ ID NO: 421. In some embodiments, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 358 and 422-431, and comprises the amino acid sequence of SEQ ID NO: 421.

[0178] In some embodiments, the amino acid sequence of SEQ ID NO:422 has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity or is about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:422, and comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:421. Provided herein is an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprising an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to, or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to, in some embodiments, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:422, and the amino acid sequence of SEQ ID NO:421.

[0179] In some embodiments, the amino acid sequence of SEQ ID NO: 358 has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity or is about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to the amino acid sequence of SEQ ID NO: 358, and the amino acid sequence of SEQ ID NO: 421. Provided herein is an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprising an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to, or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to, in some embodiments, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:358 and the amino acid sequence of SEQ ID NO:421.

[0180] In some embodiments, the present invention comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO:423 or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:423 and comprising an amino acid sequence having 70%, 75%, 80%, 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:421. Provided herein is a masked anti-CTLA4 antibody or antigen-binding fragment thereof comprising an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 423 or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO: 423. In some embodiments, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 423 and the amino acid sequence of SEQ ID NO: 421.

[0181] In some embodiments, the amino acid sequence of SEQ ID NO:424 has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:424, or has about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to the amino acid sequence of SEQ ID NO:421. Provided herein is an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprising an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to, or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to, In some embodiments, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:424, and the amino acid sequence of SEQ ID NO:421.

[0182] In some embodiments, the amino acid sequence of SEQ ID NO:425 has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity or is about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:425, and comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:421. Provided herein is an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprising an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to, or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to, in some embodiments, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:425, and the amino acid sequence of SEQ ID NO:421.

[0183] In some embodiments, the amino acid sequence of SEQ ID NO:426 has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity or is about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:426, and comprises an amino acid sequence of SEQ ID NO:421. Provided herein is an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprising an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to, or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to, In some embodiments, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:426, and the amino acid sequence of SEQ ID NO:421.

[0184] In some embodiments, the present invention comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO:427 or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:427 and comprising an amino acid sequence having 70%, 75%, 80%, 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:421. Provided herein is a masked anti-CTLA4 antibody or antigen-binding fragment thereof comprising an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 427, or about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% homology to the amino acid sequence of SEQ ID NO: 427. In some embodiments, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 427, and the amino acid sequence of SEQ ID NO: 421.

[0185] In some embodiments, the amino acid sequence of SEQ ID NO:428 has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity or is about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:428, and comprises an amino acid sequence of SEQ ID NO:421. Provided herein is an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprising an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to SEQ ID NO: 428, and SEQ ID NO: 421. In some embodiments, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 428, and the amino acid sequence of SEQ ID NO: 421.

[0186] In some embodiments, an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO:429 or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:421, Provided herein is an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprising an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to, or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to, in some embodiments, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:429, and the amino acid sequence of SEQ ID NO:421.

[0187] In some embodiments, the amino acid sequence of SEQ ID NO:430 includes an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:430, and the amino acid sequence of SEQ ID NO:421. Provided herein is an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprising an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to, or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to, in some embodiments, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:430, and the amino acid sequence of SEQ ID NO:421.

[0188] In some embodiments, the amino acid sequence of SEQ ID NO:431 has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity or is about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:431, and comprises the amino acid sequence of SEQ ID NO:421. Provided herein is an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprising an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to, or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to, in some embodiments, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:431, and the amino acid sequence of SEQ ID NO:421.

[0189] There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses, for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. IgG1 antibodies may exist in multiple polymorphic variants called allotypes (Jefferis and Lefranc 2009.mAbs Vol 1 Issue 4 1-7), any of which are suitable for use in some of the embodiments herein. Common allotypic variants in the human population are those designated by the letters a, f, n, z, or combinations thereof. In some of the embodiments herein, the antibodies have an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof has an IgG1 isotype (e.g., a human IgG1 isotype). In some embodiments, the antibodies provided herein comprise a heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 235 or 236. In some embodiments, the antibodies provided herein comprise a heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 326. In some embodiments, the antibodies provided herein comprise a heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 463.

[0190] In some embodiments, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof binds CTLA4 upon cleavage by a protease, such as the proteases described herein, in some embodiments, the cleavable peptide is a substrate for a protease that is co-localized in a region that contains cells or tissues that express CTLA4.

[0191] In one aspect of the present invention, a polynucleotide is provided that encodes an activatable masked anti-CTLA4 antibody or an antigen-binding fragment thereof. In certain embodiments, a vector is provided that comprises a polynucleotide that encodes an activatable masked anti-CTLA4 antibody or an antigen-binding fragment thereof. In certain embodiments, a host cell is provided that comprises such a vector. In another aspect of the present invention, a composition is provided that comprises an activatable masked anti-CTLA4 antibody as described herein or a polynucleotide that encodes an activatable masked anti-CTLA4 antibody as described herein. In certain embodiments, the composition of the present invention is a pharmaceutical preparation for the treatment of neoplastic diseases in which CTLA4 plays a role, such as those listed herein.

[0192] In some embodiments, the CTLA4 binding protein provided herein is a bispecific antibody that can bind to CTLA4.A bispecific antibody is a monoclonal antibody that has binding specificity for at least two different antigens.In some embodiments, one of the binding specificities is for CTLA4, and the other is for any other antigen.In certain embodiments, a bispecific antibody can bind to two different epitopes of CTLA4.

[0193] In some aspects, provided herein is a masked bispecific antibody comprising a) a first pair of light and heavy chains that specifically bind to CTLA4, b) a second pair of light and heavy chains that specifically bind to an antigen, and c) a masking peptide comprising an amino acid sequence selected from SEQ ID NOs: 1-46, wherein the masking peptide is linked to the amino terminus of the first pair of light and / or heavy chains via a linker comprising a cleavable peptide. In some aspects, provided herein is a masked bispecific antibody comprising a) a first pair of light and heavy chains that specifically bind to CTLA4, b) a second pair of light and heavy chains that specifically bind to an antigen, and c) a masking peptide comprising an amino acid sequence selected from SEQ ID NOs: 1-46, wherein the masking peptide is linked to the carboxy terminus of the first pair of light and / or heavy chains via a linker comprising a cleavable peptide. In some embodiments, the antigen is an antigen different from CTLA4. In some embodiments, the first pair or the second pair of light chains is any light chain described herein. In some embodiments, the heavy chain of the first pair or the second pair is any light chain described herein. In some embodiments, the light chain of the first pair comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 438, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 439, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 440, and the heavy chain of the first pair comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 441, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 442, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 443. In some embodiments, the light chain of the second pair comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 438, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 439, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 440, and the heavy chain of the second pair comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 441, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 442, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 443. In some embodiments, the antigens are directed to different epitopes of CTLA4. In some embodiments, the cleavable peptide comprises an amino acid sequence selected from SEQ ID NOs: 47-88, 464-469, and 479-508.In some embodiments, the spacer linker is directly linked to the N-terminus and / or C-terminus of the cleavable peptide. In some embodiments, the spacer linker comprises an amino acid sequence selected from SEQ ID NOs: 89-112 and 415-420. In some embodiments, at least one amino acid, but not more than 20, 30, 40, or 50 amino acids, is directly linked to the N-terminus of the masking peptide. In some embodiments, the at least one amino acid is alanine (A) or glycine-alanine (GA). In some embodiments, the at least one amino acid directly linked to the N-terminus of the masking peptide is a detectable tag. In some embodiments, the at least one amino acid directly linked to the N-terminus of the masking peptide is. The file is TIFF0007680954000011.tif21128.

[0194] Bispecific antibodies contemplated herein for use in a masked bispecific antibody include murine bispecific antibodies, humanized bispecific antibodies, chimeric bispecific antibodies, and human bispecific antibodies. In some of the embodiments herein, the bispecific antibody has an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the bispecific antibody provided herein has an IgG1 isotype (e.g., a human IgG1 isotype). In some embodiments, the antibody is expressed by a cell that has an IgG1 isotype that includes an amino acid substitution or does not have a reduced ability to fucosylate Fc glycans that enhance the effector functions described herein. In some embodiments, the masked bispecific antibody provided herein includes a heavy chain constant domain that includes the amino acid sequence of SEQ ID NO: 235 or 236. In some embodiments, the masked bispecific antibody provided herein includes a heavy chain constant domain that includes the amino acid sequence of SEQ ID NO: 326. In some embodiments, the masked bispecific antibody provided herein includes a heavy chain constant domain that includes the amino acid sequence of SEQ ID NO: 463.

[0195] In one aspect, provided herein is an activatable masked anti-CTLA4 bispecific antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 232 and / or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 233. In a further aspect, provided herein is an activatable masked anti-CTLA4 bispecific antibody comprising a light chain comprising an amino acid sequence selected from SEQ ID NOs: 237-318 and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 319 or 320.

[0196] In one aspect, provided herein is an activatable masked anti-CTLA4 bispecific antibody comprising a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 321 or 322, and / or a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 323 or 324. In a further aspect, provided herein is an activatable masked anti-CTLA4 bispecific antibody comprising a light chain comprising an amino acid sequence selected from SEQ ID NO: 327-341, and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 366-380, 421, and 478. In yet another further aspect, provided herein is an activatable masked anti-CTLA4 bispecific antibody comprising a light chain comprising an amino acid sequence selected from SEQ ID NO: 327, 334, or 342-365, and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 366 or 380-397.

[0197] In some embodiments, provided herein is an activatable masked anti-CTLA4 bispecific antibody comprising a heavy chain variable domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 233. In some embodiments, provided herein is an activatable masked anti-CTLA4 bispecific antibody comprising a heavy chain variable domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from SEQ ID NO: 323 or 324. In some embodiments, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity contains substitutions, insertions, or deletions relative to the reference sequence, but an antibody containing the amino acid sequence retains the ability to bind to CTLA4 (e.g., human CTLA4). In some embodiments, the substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, or 5 amino acids) occur in a region outside the HVR (i.e., within the FR). In some embodiments, an activatable masked anti-CTLA4 bispecific antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 233. In some embodiments, an activatable masked anti-CTLA4 bispecific antibody comprises a heavy chain variable domain comprising an amino acid sequence selected from SEQ ID NO: 323 or 324.

[0198] In some embodiments, provided herein is an activatable masked anti-CTLA4 bispecific antibody comprising a light chain variable domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 232. In some embodiments, provided herein is an activatable masked anti-CTLA4 bispecific antibody comprising a light chain variable domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from SEQ ID NO: 321 or 322. In some embodiments, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity contains substitutions, insertions, or deletions relative to the reference sequence, but an antibody containing the amino acid sequence retains the ability to bind to CTLA4 (e.g., human CTLA4). In some embodiments, the substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, or 5 amino acids) occur in a region outside the HVR (i.e., within the FR). In some embodiments, an activatable masked anti-CTLA4 bispecific antibody comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 232. In some embodiments, an activatable masked anti-CTLA4 bispecific antibody comprises a light chain variable domain comprising an amino acid sequence selected from SEQ ID NO: 321 or 322.

[0199] In some embodiments, the CTLA4 binding proteins provided herein are chimeric receptors (e.g., chimeric antigen receptors (CARs)) capable of binding to CTLA4. CARs are molecules that combine antibody-based specificity for a desired antigen (e.g., CTLA4) with a T cell receptor activation intracellular domain to generate a chimeric protein that exhibits specific anti-tumor cell activity. In one embodiment, chimeric receptors engineered to include an extracellular domain having a CTLA4 binding domain described herein fused to an intracellular signaling domain of a T cell antigen receptor complex zeta chain (e.g., CD3 zeta). The CTLA4 binding domain is engineered to be linked to a masking peptide, such as those described herein, via a linker that includes a cleavable peptide. The activatable masked chimeric receptors provided herein, when expressed in T cells, can redirect antigen recognition based on antigen binding specificity upon cleavage by a protease that recognizes the cleavable peptide. In some embodiments, the CTLA4 binding domain is preferably fused to an intracellular domain from one or more of a costimulatory molecule and a zeta chain. In some embodiments, the CTLA4 binding domain is fused to one or more intracellular domains selected from the group of a CD137 (4-1BB) signaling domain, a CD28 signaling domain, a CD3 zeta signaling domain, and any combination thereof.

[0200] In some aspects, provided herein is a masked chimeric receptor comprising: a) a ligand binding domain comprising a first chain and a second chain that binds to CTLA4; b) a masking peptide comprising an amino acid sequence selected from SEQ ID NOs: 1-46; c) a transmembrane domain; and d) an intracellular signaling domain comprising a signaling domain, wherein the masking peptide is linked to the amino terminus of the first chain and / or the second chain of the ligand binding domain via a linker comprising a cleavable peptide. In some embodiments, the first chain is a light chain variable domain and the second chain is a heavy chain variable domain. In some of the embodiments of the activatable masked chimeric receptor described herein, the first chain comprises an amino acid sequence of SEQ ID NO: 232 and / or the second chain comprises an amino acid sequence of SEQ ID NO: 233. In some embodiments, the first chain comprises an amino acid sequence selected from SEQ ID NOs: 321 or 322 and / or the second chain comprises an amino acid sequence selected from SEQ ID NOs: 323 or 324.

[0201] In some aspects, provided herein is a masked chimeric receptor comprising: a) a ligand binding domain comprising a first chain and a second chain that binds to CTLA4; b) a masking peptide comprising an amino acid sequence selected from SEQ ID NOs: 1-46; c) a transmembrane domain; and d) an intracellular signaling domain comprising a signaling domain, wherein the masking peptide is linked to the carboxy terminus of the first chain and / or the second chain of the ligand binding domain via a linker comprising a cleavable peptide. In some embodiments, the first chain is a light chain variable domain and the second chain is a heavy chain variable domain. In some of the embodiments of the activatable masked chimeric receptor described herein, the first chain comprises an amino acid sequence of SEQ ID NO: 232 and / or the second chain comprises an amino acid sequence of SEQ ID NO: 233. In some embodiments, the first chain comprises an amino acid sequence selected from SEQ ID NOs: 321 or 322 and / or the second chain comprises an amino acid sequence selected from SEQ ID NOs: 323 or 324.

[0202] In some of the embodiments of the activatable masked chimeric receptor described herein, the cleavable peptide comprises an amino acid sequence selected from SEQ ID NOs: 47-88, 464-469, and 479-508. In some embodiments, a spacer linker is directly linked to the N-terminus and / or C-terminus of the cleavable peptide. In some embodiments, the spacer linker comprises an amino acid sequence selected from SEQ ID NOs: 89-112 and 415-420. In some embodiments, at least one amino acid, but not more than 20, 30, 40, or 50 amino acids, is directly linked to the N-terminus of the masking peptide. In some embodiments, the at least one amino acid is alanine (A) or glycine-alanine (GA). In some embodiments, the at least one amino acid directly linked to the N-terminus of the masking peptide is a detectable tag. In some embodiments, the at least one amino acid directly linked to the N-terminus of the masking peptide is The file is TIFF0007680954000012.tif21128.

[0203] 1.Binding affinity The strength, or affinity, of an immunological binding interaction, such as that between an antibody and the antigen for which the antibody is specific, is determined by the equilibrium dissociation constant (K D ), and K D A smaller K represents a higher affinity. The immunological binding properties of a protein can be quantified using methods well known in the art. For example, one method involves measuring the rate of antigen-binding protein (e.g., antibody) / antigen complex formation and dissociation, which depends on the concentration of the complex partners, the affinity of the interaction, and geometric parameters that affect the rate in both directions equally. Both the "on-rate constant" (Kon) and the "off-rate constant" (Koff) can be determined by calculation of the concentration and the actual rates of association and dissociation. The rate of Koff / Kon allows the removal of all parameters that are not related to affinity, and is the equilibrium dissociation constant K DSee Davies et al., Annual Rev Biochem. 59:439-473, (1990).

[0204] In some aspects, the activatable masked anti-CTLA4 binding proteins described herein (e.g., activatable masked anti-CTLA4 antibodies or antigen-binding fragments thereof) bind to CTLA4 with about the same or higher affinity upon cleavage with a protease compared to a parent anti-CTLA4 binding protein that does not include a cleavable peptide. In certain embodiments, the anti-CTLA4 binding proteins provided herein bind to CTLA4 with an affinity of ≦1 μM, ≦150 nM, ≦100 nM, ≦50 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 Equilibrium dissociation constant (K D In some embodiments, the anti-CTLA4 binding proteins provided herein (e.g., anti-CTLA4 antibodies or antigen-binding fragments thereof) have an equilibrium dissociation constant (K D ) to a target protein (e.g., CTLA4 protein). Assays for assessing binding affinity are well known in the art, such as the assays described in Examples 3A and 3B herein.

[0205] In some embodiments, an activatable masked anti-CTLA4 binding protein (e.g., an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof) is provided that exhibits a desired blockage ratio. As used herein, the term "blockage ratio" refers to the ratio of (a) the maximum detectable level of a parameter under a first set of conditions to (b) the minimum detectable value of that parameter under a second set of conditions. For example, in the context of an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof, the blockage ratio refers to the ratio of (a) the maximum detectable level of a target protein (e.g., CTLA4 protein) that binds to the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof in the presence of at least one protease capable of cleaving the cleavable peptide of the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof, to (b) the minimum detectable level of a target protein (e.g., CTLA4 protein) that binds to the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof in the absence of the protease. The blocking ratio of an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof can be calculated as the ratio of the dissociation constant of the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof before cleavage by a protease to the dissociation constant of the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof after cleavage by a protease. In some embodiments, a larger blocking ratio of an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof indicates that the target protein (e.g., CTLA4 protein) bound by the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof occurs to a greater extent (e.g., occurs primarily) in the presence of a protease capable of cleaving the cleavable peptide of the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof than in the absence of the protease. In some embodiments, an activatable masked anti-CTLA4 binding protein having an optimal blocking ratio is provided herein.In some embodiments, the optimal blocking ratio of an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof indicates that the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof has desirable properties useful in the methods or compositions contemplated herein. In some embodiments, the activatable masked anti-CTLA4 binding proteins provided herein exhibit an optimal blocking ratio of about 20 to about 10,000, e.g., about 80 to about 100. In further embodiments, the occlusion ratio is about 20 to about 7,500, about 20 to about 5,000, about 20 to about 2,500, about 20 to about 2,000, about 20 to about 1,000, about 20 to about 900, about 20 to about 800, about 20 to about 700, about 20 to about 600, about 20 to about 500, about 20 to about 400, about 20 to about 300, about 20 to about 200, about 20 to about 100, about 20 to about 50, about 30 to about 100, about 40 to about 100, about 50 to about 100, about 60 to about 100, about 70 to about 100, about 80 to about 100, or about 100 to about 1,000. In some embodiments, the activatable masked anti-CTLA4 binding proteins provided herein exhibit an optimal occlusion ratio of about 80 to about 100. In some embodiments, the activatable masked anti-CTLA4 binding proteins provided herein exhibit an optimal occlusion ratio of about 20 to about 1,000. Binding of the activatable masked anti-CTLA4 binding proteins to a target protein (e.g., CTLA4 protein) prior to cleavage and / or following cleavage by a protease can be determined using techniques well known in the art, such as ELISA.

[0206] In some aspects, the masking peptides described herein bind to an anti-CTLA4 binding protein (e.g., an anti-CTLA4 antibody or antigen-binding fragment thereof) with an affinity lower than the affinity between the anti-CTLA4 binding protein and a target protein (e.g., a CTLA4 protein). In certain embodiments, the masking peptides provided ... -5 M or less, e.g., 10-5 M~10 -13 M, for example, 10 -5 M~10 -7 Equilibrium dissociation constant (K D ) to an anti-CTLA4 binding protein (e.g., an anti-CTLA4 antibody or antigen-binding fragment thereof). In some embodiments, the masking peptides provided herein bind to an anti-CTLA4 binding protein (e.g., an anti-CTLA4 antibody or antigen-binding fragment thereof) with an equilibrium dissociation constant (K D ) to an anti-CTLA4 binding protein (e.g., an anti-CTLA4 antibody or antigen-binding fragment thereof). An exemplary assay for assessing the binding affinity between a masking peptide and an anti-CTLA4 binding protein (e.g., an anti-CTLA4 antibody or antigen-binding fragment thereof) can be found in Example 2 herein.

[0207] 2. Biological Activity Assay In some embodiments, the activatable masked anti-CTLA4 binding proteins described herein reduce tumor volume in an in vivo mouse tumor model. Assays for assessing tumor volume reduction are well known in the art, such as the assays described in Examples 4A and 4B herein.

[0208] III. Preparation of Masked Anti-CTLA4 Binding Protein The masked anti-CTLA4 binding proteins described herein are prepared using techniques available in the art, exemplary methods of which are described in more detail in the following sections.

[0209] 1. Masked anti-CTLA4 binding proteins: antibody fragments The present invention encompasses antibody fragments as masked anti-CTLA4 binding proteins.Masked antibody fragments can be produced by conventional means such as enzyme digestion or by recombinant technology.In certain circumstances, there are advantages to using masked antibody fragments rather than whole antibodies.For a review of certain antibody fragments, see Hudson et al.(2003)Nat.Med.9:129-134.

[0210] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992), and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv, and ScFv antibody fragments can all be expressed in and secreted from E. coli and other cell types, thus allowing the facile production of large amounts of these masked fragments. Alternatively, masked Fab'-SH fragments can be directly recovered from the culture medium and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). According to another approach, masked F(ab')2 fragments can be directly isolated from recombinant host cell culture. Masked Fab and F(ab')2 fragments with increased in vivo half-life containing FcRN / salvage receptor binding epitope residues are described in U.S. Pat. No. 5,869,046. Other techniques for the production of masked antibody fragments will be apparent to those skilled in the art. In certain embodiments, the masked antibody is a single chain Fv fragment (scFv). See WO93 / 16185, U.S. Pat. Nos. 5,571,894, and 5,587,458. Fv and scFv are the only species with intact binding sites that lack constant regions, and therefore, they may be suitable for reduced non-specific binding during in vivo use. scFv fusion proteins can be constructed to provide fusion of effector proteins at either the amino or carboxy terminus of the scFv. See Antibody Engineering, ed. Borrebaeck, supra. Also, a dual scFv comprising two scFvs linked via a polypeptide linker can be used as a bispecific antibody, or a multi-scFv comprising three or more scFvs can be used as a multispecific antibody.

[0211] The present invention includes linear antibodies (e.g., as described in U.S. Pat. No. 5,641,870) or single-chain immunoglobulins comprising antibody heavy and light chain sequences linked via a suitable linker. Such linear antibodies or immunoglobulins may be monospecific or bispecific. Such single-chain immunoglobulins can be dimerized, thereby maintaining a structure and activity similar to that of an originally tetrameric antibody. The antibody of the present invention may also be an antibody having a single heavy chain variable region and no light chain sequence. Such antibodies are called single domain antibodies (sdAbs) or nanobodies. These antibodies are also encompassed within the meaning of functional fragments of antibodies according to the present invention.

[0212] 2. Masked anti-CTLA4 binding protein: humanized antibody The present invention encompasses masked humanized antibodies. Humanized antibodies are masked according to the guidance provided herein. Various methods for humanizing non-human antibodies are known in the art. For example, humanized antibodies can have one or more amino acid residues introduced from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter (Jones et al. (1986) Nature 321:522-525, Riechmann et al. (1988) Nature 332:323-327, Verhoeyen et al. (1988) Science 239:1534-1536) by replacing the sequences of hypervariable regions with the corresponding sequences of human antibodies. Such "humanized" antibodies are therefore chimeric antibodies (U.S. Patent No. 4,816,567) in which substantially less of an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0213] 3. Masked anti-CTLA4 binding protein: human antibody The human anti-CTLA4 antibodies of the present invention can be constructed by combining the Fv clone variable domain sequence(s) selected from a human-derived phage display library with a known human constant domain sequence(s). Alternatively, the human monoclonal anti-CTLA4 antibodies of the present invention can be produced by hybridoma technology. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies are described, for example, by Kozbor J.Immunol., 133:3001 (1984), Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987), and Boerner et al., J.Immunol., 147:86 (1991). Human antibodies are masked according to the guidance provided herein.

[0214] 4. Masked Anti-CTLA4 Binding Proteins: Bispecific Antibodies A bispecific antibody is a monoclonal antibody that has binding specificities for at least two different antigens. In certain embodiments, the bispecific antibody is a human or humanized antibody. In certain embodiments, one of the binding specificities is for CTLA4 and the other is for any other antigen. In certain embodiments, the bispecific antibody may bind to two different epitopes of CTLA4. The bispecific antibody may be used to localize cytotoxic agents to cells expressing CTLA4. The bispecific antibody may be prepared as a full-length antibody or an antibody fragment (e.g., F(ab')2 bispecific antibody). The bispecific antibody is masked according to the guidance provided herein.

[0215] Methods for making bispecific antibodies are known in the art. See Milstein and Cuello, Nature, 305:537 (1983), WO93 / 08829 published May 13, 1993, Traunecker et al., EMBO J., 10:3655 (1991), Kontermann and Brinkmann, Drug Discovery Today, 20(7):838-847. For further details on the production of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986). Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be bound to avidin and the other to biotin. Heteroconjugate antibodies can be made using any convenient cross-linking method. Suitable cross-linking agents, along with a number of cross-linking techniques, are well known in the art and are disclosed in US Pat. No. 4,676,980.

[0216] 5. Masked anti-CTLA4 binding proteins: single domain antibodies In some embodiments, single domain antibodies are masked according to the guidance provided herein. Single domain antibodies are single polypeptide chains that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, single domain antibodies are human single domain antibodies (Domantis, Inc., Waltham, Mass., see, e.g., U.S. Patent No. 6,248,516 B1). In one embodiment, single domain antibodies consist of all or part of the heavy chain variable domain of an antibody.

[0217] 6. Masked Anti-CTLA4 Binding Protein: Antibody Variants In some embodiments, amino acid sequence modification(s) of the masked antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the masked antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate changes into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions into and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics. Amino acid changes may be introduced into the subject antibody amino acid sequence at the time the sequence is made.

[0218] A useful method for identifying certain residues or regions of an antibody that are preferred positions for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. Here, a residue or group of target residues is identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to affect the interaction of the amino acid with the antigen. Those amino acid positions that show functional sensitivity to the substitution are then refined by introducing additional or other variants at or for the substitution site. Thus, the site for introducing amino acid sequence mutations is predetermined, but the nature of the mutation itself need not be predetermined. For example, to analyze the performance of a mutation at a given site, Ala scanning or random mutagenesis is performed at the target codon or region, and the expressed immunoglobulin is screened for the desired activity.

[0219] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing hundreds or more residues, as well as intersequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody to an enzyme or a polypeptide to extend the serum half-life of the antibody.

[0220] In some embodiments, FcRn mutations that improve pharmacokinetics include, but are not limited to, M428L, T250Q / M428L, M252Y / S254T / T256E, P257I / N434H, D376V / N434H, P257I / Q3111, N434A, N434W, M428L / N434S, V259I / V308F, M252Y / S254T / T256E, V259I / V308F / M428L, T307Q / N434A, T307Q / N434S, T307Q / E380A / N434A, V308P / N434A, N434H, V308P. In some embodiments, such mutations enhance antibody binding to FcRn at low pH but do not alter antibody affinity at neutral pH.

[0221] In certain embodiments, the antibody of the present invention is modified to increase or decrease the extent to which the antibody is glycosylated. Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid other than proline, are recognition sequences for the enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0222] Addition or deletion of glycosylation sites to an antibody is conveniently accomplished by altering the amino acid sequence to create or remove one or more of the above-mentioned tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by adding, deleting, or substituting one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0223] If the antibody comprises an Fc region, the carbohydrate attached thereto can be modified. For example, an antibody having a mature carbohydrate structure lacking fucose attached to the Fc region of the antibody is described in US Patent Application No. US2003 / 0157108 (Presta, L.). See also US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). An antibody having a bisecting N-acetylglucosamine (GlcNAc) in the carbohydrate attached to the Fc region of the antibody is referenced in WO2003 / 011878, Jean-Mairet et al., and U.S. Patent No. 6,602,684, Umana et al. An antibody having at least one galactose residue in the oligosaccharide attached to the Fc region of the antibody is reported in WO1997 / 30087, Patel et al. See also WO1998 / 58964 (Raju, S.) and WO1999 / 22764 (Raju, S.) concerning antibodies with altered carbohydrate attached to the Fc region thereof. See also US2005 / 0123546 (Umana et al.) concerning antigen-binding molecules with modified glycosylation.

[0224] In certain embodiments, the glycosylation variant comprises an Fc region, and the carbohydrate structure attached to the Fc region lacks fucose or has reduced fucose. Such variants have improved ADCC function. Optionally, the Fc region further comprises one or more amino acid substitutions therein, such as substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region, which further improve ADCC. Examples of publications related to "afucosylated", "defucosylated" or "fucose-deficient" antibodies include US2003 / 0157108, WO2000 / 61739, WO2001 / 29246, US2003 / 0115614, US2002 / 0164328, US2004 / 00 93621, US2004 / 0132140, US2004 / 0110704, US2004 / 0110282, US2004 / 0109865, WO2003 / 085119, WO2003 / 084570, WO2005 / 035586, WO2005 / 035778, WO2005 / 053742, Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004), Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines that produce defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US2003 / 0157108A1; Presta, L; and WO2004 / 056312A1; Adams et al., especially Example 11), and knockout cell lines such as the alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004)), and cells overexpressing β1,4-N-acetylglucosaminyltransferase III (GnT-III) and Golgi μ-mannosidase II (ManII).

[0225] In any of the embodiments herein, the masked anti-CTLA4 binding protein may be engineered to improve antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In some embodiments, the masked anti-CTLA4 binding protein may be produced in a cell line with an alpha 1,6-fucosyltransferase (Fut8) knockout. In some further embodiments, the masked anti-CTLA4 binding protein may be produced in a cell line that overexpresses β1,4-N-acetylglucosaminyltransferase III (GnT-III). In further embodiments, the cell line further overexpresses Golgi μ-mannosidase II (ManII). In some of the embodiments herein, the masked anti-CTLA4 binding protein may include at least one amino acid substitution in the Fc region that improves ADCC activity.

[0226] In one embodiment, the masked antibody is modified to improve its serum half-life. To increase the serum half-life of an antibody, one can incorporate an FcRN / salvage receptor binding epitope into the antibody (especially an antibody fragment), for example, as described in US Patent No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule (US2003 / 0190311, US Patent No. 6,821,505, US Patent No. 6,165,745, US Patent No. 5,624,821, US Patent No. 5,648,260, US Patent No. 6,165,745, US Patent No. 5,834,597).

[0227] Another type of variant is amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by a different residue. The target site for substitution mutagenesis includes hypervariable regions, but FR modification is also contemplated. Conservative substitutions are shown in Table 2 under the heading of "preferred substitutions". If such substitutions cause desirable changes in biological activity, larger substitution changes can be introduced and the products screened, as shown in Table 2 under "exemplary substitutions" or as detailed below for classes of amino acids.

[0228] (Table 2) TIFF0007680954000013.tif189138

[0229] Substitutional modifications in the biological properties of antibodies are achieved by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the region of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the target site molecule, or (c) the bulk of the side chain. Amino acids can be grouped according to the similarity of the properties of their side chains (ALLehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)). (1) Non-polar: Ala(A), Val(V), Leu(L), Ile(I), Pro(P), Phe(F), Trp(W), Met(M) (2) Uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q) (3) Acidic: Asp(D), Glu(E) (4) Basic: Lys(K), Arg(R), His(H)

[0230] Alternatively, the naturally occurring residues can be divided into groups based on the following common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that influence chain directionality: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0231] Non-conservative substitutions entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or into the remaining (non-conserved) sites.

[0232] One type of substitution variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further development have modified (e.g., improved) biological properties relative to the parent antibody from which they were generated. A convenient method for generating such substitution variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibodies thus generated are displayed from filamentous phage particles as fusions to at least a portion of a phage coat protein (e.g., the gene III product of M13) packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity). To identify candidate hypervariable region sites for modification, scanning mutagenesis (e.g., alanine scanning) can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively, or in addition, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues are candidates for substitution by techniques known in the art, including those described herein. Once such variants are generated, a panel of variants can be screened using techniques known in the art, including those described herein, and antibodies with superior properties in one or more relevant assays can be selected for further development.

[0233] Nucleic acid molecules encoding masked antibody amino acid sequence variants are prepared by a variety of methods known in the art, including, but not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of variant or non-variant versions of previously prepared antibodies.

[0234] It may be desirable to introduce one or more amino acid modifications into the Fc region of an antibody of the invention, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., a substitution) at one or more amino acid positions, including the hinge cysteine ​​amino acid positions.

[0235] In some embodiments, the activatable masked anti-CTLA4 binding proteins provided herein (e.g., activatable masked anti-CTLA4 antibodies or antigen-binding fragments thereof, or activatable masked anti-CTLA4 bispecific antibodies) have an IgG1 isotype with enhanced effector function. In some embodiments, the activatable masked anti-CTLA4 antibodies or antigen-binding fragments thereof are afucosylated. In some embodiments, the activatable masked anti-CTLA4 bispecific antibodies are afucosylated. In some embodiments, the activatable masked anti-CTLA4 antibodies or antigen-binding fragments thereof have increased levels of mannose moieties. In some embodiments, the activatable masked anti-CTLA4 antibodies or antigen-binding fragments thereof have increased levels of bisecting glycan moieties. In some embodiments, the activatable masked anti-CTLA4 bispecific antibodies have increased levels of mannose moieties. In some embodiments, the IgG1 comprises an amino acid mutation.

[0236] In some embodiments, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof, or activatable masked anti-CTLA4 bispecific antibody provided herein has an IgG1 isotype (e.g., a human IgG1 isotype). In one embodiment, the IgG1 comprises amino acid substitutions S298A, E333A, and K334A, where the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises amino acid substitutions S239D and I332E, where the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises amino acid substitutions S239D, A330L, and I332E, where the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises amino acid substitutions P247I and A339D or A339Q, where the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgG1 comprises amino acid substitutions D280H, K290S with or without S298D, or S298V, where the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgG1 comprises amino acid substitutions F243L, R292P, and Y300L, where the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgG1 comprises amino acid substitutions F243L, R292P, Y300L, and P396L, where the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgG1 comprises amino acid substitutions F243L, R292P, Y300L, V305I, and P396L, where the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgG1 comprises amino acid substitutions G236A, S239D, and I332E, where the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions K326A and E333A, where the amino acid residues are numbered according to the EU index as in Kabat.In one embodiment, the IgG1 comprises the amino acid substitutions K326W and E333S, where the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions K290E or K290N, S298G, T299A, and / or K326E, where the amino acid residues are numbered according to the EU index as in Kabat.

[0237] According to this description and the teachings of the art, it is contemplated that in some embodiments, the antibody of the present invention may contain one or more modifications, for example, in the Fc region, compared to the corresponding wild-type antibody. However, these antibodies will retain substantially the same characteristics required for therapeutic utility compared to their wild-type counterparts. For example, certain modifications may be made in the Fc region that will result in modified (i.e., either improved or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC), as described, for example, in WO99 / 51642. See also Duncan & Winter Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO94 / 29351 for other examples of Fc region variants. WO00 / 42072 (Presta) and WO2004 / 056312 (Lowman) describe antibody variants with improved or reduced binding to FcR. The contents of these patent publications are specifically incorporated herein by reference. See also Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001). Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) are described in US2005 / 0014934A1 (Hinton et al.). These antibodies include an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Polypeptide variants with altered Fc region amino acid sequences and increased or decreased C1q binding ability are described in U.S. Patent No. 6,194,551B1, WO99 / 51642. The contents of these patent publications are specifically incorporated herein by reference. See also Idusogie et al. J Immunol.164:4178-4184(2000).

[0238] 7. Masked antibody-drug conjugates The present invention also provides masked antibody-drug conjugates (ADCs) comprising an activatable masked anti-CTLA4 binding protein provided herein conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioactive isotope.

[0239] In one embodiment, the one or more drugs conjugated to the antibody-drug conjugate include, but are not limited to, maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064, and European Patent EP 0425235 B1); auristatins such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatins; calicheamicin or a derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993), and Lode et al., Cancer Res. 58:2925-2928 (1998); anthracyclines such as daunomycin or doxorubicin (Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 16:358-362 (2006); 12:1529-1532 (2002), King et al., J. Med. Chem. 45:4336-4343 (2002), and U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

[0240] In another embodiment, the one or more drugs conjugated to the antibody-drug conjugate include, but are not limited to, inhibitors of tubulin polymerization (e.g., maytansinoids and auristatins), DNA damaging agents (e.g., pyrrolobenzodiazepine (PBD) dimers, calicheamicins, duocarmycins, and indolinobenzodiazepine dimers), and DNA synthesis inhibitors (e.g., exatecan derivatives Dxd).

[0241] In another embodiment, the antibody-drug conjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0242] In another embodiment, the antibody-drug conjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for generating radioconjugates. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 When a radioactive complex is used for detection, the radioactive complex may be, for example, tc 99m Or I 123or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0243] Conjugates of an activatable masked anti-CTLA4 binding protein (e.g., an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof) and a cytotoxic agent can be prepared using a variety of compounds, including N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipidine diamine HCl), active esters (succinimidyl esters), and cytotoxic agents. Various bifunctional protein coupling agents can be used to generate such as disuccinimidyl verate, aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO94 / 11026. The linker may be a "cleavable linker" that facilitates the release of the cytotoxic drug in cells. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992), U.S. Patent No. 5,208,020) may be used.

[0244] The ADCs herein expressly contemplate such conjugates prepared with cross-linking reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology Inc., Rockford, Ill., USA).

[0245] 8. Vectors, host cells, and recombinant methods For recombinant production of the activatable masked anti-CTLA4 binding protein of the present invention, the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (amplification of DNA) or for expression. The DNA encoding the antibody is easily isolated and sequenced by using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the antibody). Many vectors are available. The choice of vector depends in part on the host cell used. Generally, the host cell is either of prokaryotic or eukaryotic (generally mammalian) origin. It will be understood that any isotype of constant region can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions can be obtained from any human or animal species.

[0246] 9. Production of Binding Proteins Using Prokaryotic Host Cells a) Vector construction Polynucleotide sequences encoding the polypeptide components of the activatable masked anti-CTLA4 binding proteins of the present invention can be obtained using standard recombinant techniques. The desired polynucleotide sequences may be isolated and sequenced from antibody producing cells, such as hybridoma cells. Alternatively, polynucleotides can be synthesized using a nucleotide synthesizer or PCR techniques. Once obtained, the sequences encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a prokaryotic host. Many vectors available and known in the art can be used for the purposes of the present invention. The selection of an appropriate vector depends primarily on the size of the nucleic acid to be inserted into the vector and the specific host cell to be transformed with the vector. Each vector contains various components depending on its function (amplification or expression of heterologous polynucleotides, or both) and its compatibility with the specific host cell in which it resides. Vector components generally include, but are not limited to, an origin of replication, a selectable marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, a heterologous nucleic acid insert, and a transcription termination sequence.

[0247] Generally, plasmid vectors containing replicons and control sequences derived from species compatible with the host cell are used in connection with these hosts. The vectors usually carry a replication site, as well as a marking sequence that can provide phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from E. coli species. pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance, thus providing an easy means for identifying transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophages may also contain, or be modified to contain, promoters that can be used by the microorganism for the expression of endogenous proteins. An example of a pBR322 derivative used to express a particular antibody is described in Carter et al., U.S. Pat. No. 5,648,237.

[0248] Additionally, phage vectors containing replicons and control sequences compatible with host microorganisms can be used as transformation vectors in conjunction with these hosts. For example, bacteriophages such as λGEM.TM.-11 can be utilized in making recombinant vectors that can be used to transform susceptible host cells such as E. coli LE392.

[0249] The expression vector of the present invention may contain two or more promoter-cistron pairs, each encoding a polypeptide component. A promoter is a non-translated regulatory sequence located upstream (5') of a cistron that regulates its expression. Prokaryotic promoters are typically divided into two classes: inducible and homeostatic. An inducible promoter is a promoter that initiates an increase in the level of transcription of the cistron under its control in response to a change in culture condition, such as the presence or absence of a nutrient or a change in temperature.

[0250] Numerous promoters that are recognized by various potential host cells are well known. The selected promoter can be operably linked to the cistron DNA encoding the light or heavy chain by removing the promoter from the source DNA via restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the present invention. Both the native promoter sequence and many heterologous promoters can be used to direct the amplification and / or expression of the target gene. In some embodiments, heterologous promoters are utilized because they generally allow a higher transcription amount and a higher yield of the expressed target gene compared to the native target polypeptide promoter.

[0251] Suitable promoters for use with prokaryotic hosts include PhoA promoter, β-galactamase and lactose promoter system, tryptophan (Trp) promoter system, and hybrid promoters such as tac or trc promoter.However, other promoters that function in bacteria (such as other known bacterial or phage promoters) are also suitable.Their nucleotide sequences are published, allowing those skilled in the art to operably ligate them to the cistrons encoding target light and heavy chains, using linkers or adapters to provide any necessary restriction sites (Siebenlist et al. (1980) Cell 20:269).

[0252] In one aspect of the invention, each cistron in the recombinant vector contains a secretion signal sequence component that directs the translocation of the expressed polypeptide across the membrane. In general, the signal sequence may be a component of the vector or may be part of the target polypeptide DNA inserted into the vector. The signal sequence selected for the purposes of the present invention should be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native signal sequence for the heterologous polypeptide, the signal sequence is replaced by a prokaryotic signal sequence selected from the group consisting of, for example, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leader, LamB, PhoE, PelB, OmpA, and MBP. In one embodiment of the present invention, the signal sequences used in both cistrons of the expression system are STII signal sequences or variants thereof.

[0253] In another embodiment, the production of immunoglobulins according to the present invention does not require the presence of a secretion signal sequence in each cistron, since it can occur in the cytoplasm of the host cell. In this regard, immunoglobulin light and heavy chains are expressed, folded and assembled with or without sequences such as masking peptides, linker sequences, etc. to form functional immunoglobulins in the cytoplasm. Certain host strains (e.g., E. coli trxB strains) provide cytoplasmic conditions favorable for disulfide bond formation, thereby allowing proper folding and assembly of the expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

[0254] The activatable masked anti-CTLA4 binding proteins of the invention can also be produced by using an expression system in which the quantitative ratio of the expressed polypeptide components can be adjusted to maximize the yield of secreted and properly assembled antibodies of the invention, such adjustment being achieved at least in part by simultaneously adjusting the translational strength of the polypeptide components.

[0255] Suitable prokaryotic host cells for expressing the activatable masked anti-CTLA4 binding protein of the present invention include, for example, archaea and eubacteria, such as gram-negative or gram-positive organisms. Examples of useful bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B. subtilis), Enterobacteria, species of Pseudomonas (e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, gram-negative cells are used. In one embodiment, E. coli cells are used as hosts in the present invention. Examples of E. coli strains include W3110 strains (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No. 27,325), including 33D3 strains with the genotype W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8 ΔompT Δ (nmpc-fepE) degP41 kanR (U.S. Patent No. 5,639,635), and derivatives thereof. Other strains such as E. coli 294 (ATCC 31,446), E. coli B, E. coli λ 1776 (ATCC 31,537), and E. coli RV308 (ATCC 31,608) and their derivatives are also suitable. These examples are illustrative rather than limiting. Methods for constructing derivatives of any of the above-mentioned bacteria with defined genotypes are known in the art and are described, for example, in Bass et al., Proteins, 8:309-314 (1990).It is generally necessary to select an appropriate bacterium, taking into consideration the replicability of the replicon in the bacterial cell. For example, when using a well-known plasmid such as pBR322, pBR325, pACYC177, or pKN410 to supply the replicon, species of Escherichia coli, Serratia, or Salmonella can be suitably used as the host. Typically, the host cell should secrete a minimal amount of proteolytic enzymes, and additional protease inhibitors can desirably be incorporated into the cell culture.

[0256] b) Binding protein production Host cells are transformed with the above-described expression vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0257] Transformation means introducing DNA into a prokaryotic host so that the DNA is replicable either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is performed using standard techniques appropriate for such cells. Calcium treatment using calcium chloride is commonly used for bacterial cells that contain substantial cell wall barriers. Another method for transformation uses polyethylene glycol / DMSO. Yet another technique used is electroporation.

[0258] The prokaryotic cells used to produce the activatable masked anti-CTLA4 binding protein of the present invention are grown in media known in the art and suitable for the culture of the selected host cells. Examples of suitable media include Luria Broth (LB) plus necessary nutritional supplements. In some embodiments, the media also contains a selection agent selected based on the construction of the expression vector, selectively allowing the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the media for the growth of cells expressing an ampicillin resistance gene.

[0259] Any necessary supplements other than carbon, nitrogen, and inorganic phosphate sources may also be introduced at appropriate concentrations, either alone or in admixture with the medium, such as another supplement or a complex nitrogen source. Optionally, the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, dithioerythritol, and dithiothreitol.

[0260] The prokaryotic host cells are cultured at a suitable temperature. In certain embodiments, for growth of E. coli, the growth temperature ranges from about 20° C. to about 39° C., from about 25° C. to about 37° C., or about 30° C. The pH of the medium may be any pH ranging from about 5 to about 9, depending primarily on the host organism. In certain embodiments, for E. coli, the pH is about 6.8 to about 7.4, or about 7.0.

[0261] When an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for the activation of the promoter.In one aspect of the present invention, the PhoA promoter is used to control the transcription of the polypeptide.Therefore, the transformed host cell is cultured in a phosphate-limited medium for induction.In a particular embodiment, the phosphate-limited medium is CRAP medium (see, for example, Simmons et al., J.Immunol.Methods (2002), 263:133-147).As is known in the art, various other inducers may be used according to the vector construct used.

[0262] In one embodiment, the expressed activatable masked anti-CTLA4 binding protein of the present invention is secreted into the periplasm of host cells and recovered from the periplasm of host cells. Protein recovery typically involves disrupting the microorganism, generally by such means as osmotic shock, sonication, or lysis. Once the cells are disrupted, cell debris or whole cells can be removed by centrifugation or filtration. The protein can be further purified, for example, by affinity resin chromatography. Alternatively, the protein can be transported to and isolated in the culture medium. The cells can be removed from the culture, and the culture supernatant is filtered and concentrated for further purification of the produced protein. The expressed polypeptide can be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assay.

[0263] In one aspect of the present invention, activatable masked anti-CTLA4 binding protein production is carried out in large quantities by fermentation processes. A variety of large-scale fed-batch fermentation procedures are available for recombinant protein production. Large-scale fermentation has a capacity of at least 1000 liters, and in certain embodiments, a capacity of about 1,000 to 100,000 liters. These fermenters use agitator impellers to distribute oxygen and nutrients, especially glucose. Small-scale fermentation generally refers to fermentation in fermentors with a volumetric capacity of about 100 liters or less, and which may range from about 1 liter to about 100 liters.

[0264] In fermentation processes, induction of protein expression is typically initiated after the cells have grown under suitable conditions to a desired density, an OD550 of about 180-220, at which stage the cells are in early stationary phase. Various inducers are known in the art and may be used according to the vector construct used, as described above. The cells may be grown for a shorter period of time before induction. The cells are usually induced for about 12-50 hours, although longer or shorter induction times may be used.

[0265] Various fermentation conditions can be modified to improve the production yield and quality of the polypeptide of the present invention. For example, to improve the proper assembly and folding of secreted antibody polypeptides, the host prokaryotic cells can be co-transformed with additional vectors overexpressing chaperone proteins such as Dsb proteins (DsbA, DsbB, DsbC, DsbD, and / or DsbG) or FkpA (peptidyl prolyl cis, trans-isomerase with chaperone activity). Chaperone proteins have been demonstrated to promote proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. (1999) J. Biol. Chem. 274:19601-19605; Georgiou et al., U.S. Patent No. 6,083,715; Georgiou et al., U.S. Patent No. 6,027,888; Bothmann and Pluckthun(2000) J.Biol.Chem.275:17100-17105;Ramm and Pluckthun(2000)J.Biol.Chem.275:17106-17113;Arie et al.(2001)Mol.Microbiol.39:199-210.

[0266] In order to minimize the proteolysis of expressed heterologous proteins (especially proteins that are proteolytically sensitive), certain host strains that are deficient in proteolytic enzymes can be used in the present invention.For example, host cell strains can be modified to affect genetic mutation(s) in genes encoding known bacterial proteases, such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI, and combinations thereof.Some E. coli protease-deficient strains are described, for example, in Joly et al. (1998) above; Georgiou et al., U.S. Patent No. 5,264,365; Georgiou et al., U.S. Patent No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996).

[0267] In one embodiment, an E. coli strain that is deficient in a proteolytic enzyme and transformed with a plasmid overexpressing one or more chaperone proteins is used as a host cell in the expression system of the present invention.

[0268] c) Binding protein purification In one embodiment, the masked antibody protein produced herein is further purified to obtain a substantially homogeneous preparation for further assays and uses.Standard protein purification methods known in the art can be used.The following procedures are examples of suitable purification procedures: fractionation on an immunoaffinity column or an ion exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cation exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, for example, Sephadex G-75.

[0269] In one embodiment, Protein A immobilized on a solid phase is used for immunoaffinity purification of the antibody products of the invention. Protein A is a 41 kD cell wall protein from Staphylococcus aureus that binds with high affinity to the Fc region of antibodies. Lindmark et al (1983) J. Immunol. Meth. 62:1-13. The solid phase on which Protein A is immobilized can be a glass or silica surface, or a column, including controlled pore glass columns or silicic acid columns. In some applications, the column may be coated with a reagent, such as glycerol, to prevent nonspecific adhesion of contaminants.

[0270] As a first step of purification, the preparation from the above-mentioned cell culture can be applied to a Protein A-immobilized solid phase to allow specific binding of the antibody of interest to Protein A. The solid phase is then washed to remove contaminants non-specifically bound to the solid phase. Finally, the antibody of interest is recovered from the solid phase by elution.

[0271] 10. Production of Binding Proteins Using Eukaryotic Host Cells Vectors for use in eukaryotic host cells generally include one or more of the following, but are not limited to, a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0272] a) Signal Sequence Component Vectors for use in eukaryotic host cells may also contain a signal sequence or other polypeptide with a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. The heterologous signal sequence selected may be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, such as the herpes simplex gD signal, are available. The DNA of such precursor region is ligated in reading frame to the DNA encoding the antibody.

[0273] b) Origin of Replication Generally, the origin of replication component is not needed for mammalian expression vectors. For example, the SV40 origin may typically be used only because it contains the early promoter.

[0274] c) Selection Gene Components Expression and cloning vectors may contain a selection gene, also referred to as a selection marker. Typical selection genes encode (a) a protein that confers resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, (b) a protein that complements an auxotrophic deficiency, if relevant, or (c) a protein that supplies a vital nutrient that is not available from complex media.

[0275] One example of a selection scheme utilizes drugs to stop the growth of host cells. Those cells that are successfully transformed with the heterologous gene produce a protein that confers drug resistance and therefore survive the selection regime. Examples of such dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin.

[0276] Other examples of suitable selectable markers for mammalian cells are those that allow the identification of cells capable of extracting an activatable masked anti-CTLA4 binding protein encoding nucleic acid such as DHFR, thymidine kinase, metallothionein-I and -II, primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, and the like.

[0277] For example, in some embodiments, cells transformed with the DHFR selection gene are first identified by culturing all transformants in a culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR. In some embodiments, when wild-type DHFR is used, a suitable host cell is a Chinese hamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCC CRL-9096).

[0278] Alternatively, host cells transformed or co-transformed with DNA sequences encoding an activatable masked anti-CTLA4 binding protein, wild-type DHFR protein, and another selectable marker, such as aminoglycoside 3'-phosphotransferase (APH), particularly wild-type hosts containing endogenous DHFR, can be selected by growing the cells in a medium containing a selection agent for the selectable marker, such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199. Host cells may include NS0, including cell lines deficient in glutamine synthetase (GS). Methods for using GS as a selectable marker for mammalian cells are described in U.S. Pat. Nos. 5,122,464 and 5,891,693.

[0279] d) Promoter components Expression and cloning vectors usually contain a promoter that is recognized by the host organism and operably linked to the nucleic acid encoding the activatable masked anti-CTLA4 binding protein of interest. Promoter sequences are known for eukaryotes. For example, nearly all eukaryotic genes have an AT-rich region located approximately 25-30 bases upstream from the site where transcription is initiated. Another sequence found 70-80 bases upstream from the start of transcription of many genes is the CNCAAT region, where N can be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence that may be a signal for the addition of a polyA tail to the 3' end of the coding sequence. In certain embodiments, any or all of these sequences may be suitably inserted into a eukaryotic expression vector.

[0280] Transcription from the vector in a mammalian host cell is controlled by a promoter derived, for example, from the genome of a virus such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and Simian Virus 40 (SV40), from a heterologous mammalian promoter, for example, the actin promoter or an immunoglobulin promoter, from a heat shock promoter, provided that such promoter is compatible with the host cell system.

[0281] The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. A system for expressing DNA in a mammalian host using bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification of this system is described in U.S. Pat. No. 4,601,978. See also Reyes et al., Nature 297:598-601 (1982), who describe the expression of human β-interferon cDNA in mouse cells under the control of the thymidine kinase promoter from herpes simplex virus. Alternatively, the Rous sarcoma virus long terminal repeat can be used as a promoter.

[0282] e) Enhancer element component Transcription of DNA encoding the antibody of the present invention by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, one uses an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the human cytomegalovirus early promoter enhancer, the mouse cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982), which describes enhancer elements for the activation of eukaryotic promoters. The enhancer may be spliced ​​into the vector at a 5' or 3' position to the antibody polypeptide coding sequence, but is generally located at a site 5' from the promoter.

[0283] f) Transcription termination component Expression vectors used in eukaryotic host cells may also contain sequences necessary for the termination of transcription and stabilization of mRNA. Such sequences are commonly available from the 5' and sometimes 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94 / 11026 and the expression vector disclosed therein.

[0284] g) Selection and transformation of host cells Suitable host cells for cloning or expressing the DNA in the vectors herein include the higher eukaryotic cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines include monkey kidney CV1 transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NYAcad.Sci.383:44-68(1982)); MRC5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[0285] Host cells are transformed with the above-described expression or cloning vectors for production of an activatable masked anti-CTLA4 binding protein and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0286] h) Cultivation of host cells The host cells used to produce the activatable masked anti-CTLA4 binding protein of the present invention may be cultured in various media. Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO90 / 03430, WO87 / 00195, or U.S. Patent Reissue 30,985 may be used as the culture medium for the host cells. Any of these media may be supplemented, if necessary, with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphates), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the GENTAMYCIN™ drug), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other supplements may also be included at appropriate concentrations that would be known to one of ordinary skill in the art. Culture conditions such as temperature, pH, etc. will be those previously used with the host cell selected for expression and will be apparent to one of ordinary skill in the art.

[0287] i) Purification of binding proteins When using recombinant techniques, the activatable masked anti-CTLA4 binding protein can be produced intracellularly or directly secreted into the medium. If the antibody is produced intracellularly as a first step, particulate debris, either host cells or lysed fragments, can be removed, for example, by centrifugation or ultrafiltration. If the activatable masked anti-CTLA4 binding protein is secreted into the medium, the supernatant from such an expression system can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors, such as PMSF, can be included in any of the aforementioned steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of adventitious contaminants.

[0288] The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a convenient technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human gamma 1, gamma 2, or gamma 4 heavy chains (Lindmark et al., J. Immunol. Methods 62:1-13 (1983)). Protein G is recommended for all mouse isotypes, and human gamma 3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached can be agarose, although other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation on ion exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available depending on the antibody to be recovered.

[0289] Following any preliminary purification step(s), the mixture containing the masked binding protein of interest and contaminants may be subjected to further purification, for example, by low pH hydrophobic interaction chromatography performed at a low salt concentration (e.g., about 0-0.25 M salt) and using an elution buffer at a pH of about 2.5-4.5.

[0290] Generally, a variety of methodologies for preparing antibodies for use in research, testing, and clinical use are established in the art, consistent with the methodologies described above and / or as deemed appropriate by the skilled artisan for a particular antibody of interest.

[0291] IV. Composition Also provided herein, in some embodiments, are compositions (eg, pharmaceutical compositions) comprising any of the activatable masked anti-CTLA4 binding proteins described herein.

[0292] Therapeutic formulations are prepared for storage by mixing the active ingredient having the desired purity with any pharma- ceutically acceptable carrier, excipient, or stabilizer (Remington: The Science and Practice of Pharmacy, 20th Ed., Lippincott Williams & Wiklins, Pub., Gennaro Ed., Philadelphia, Pa. 2000). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include buffers; antioxidants, including ascorbic acid, methionine, vitamin E, sodium pyrosulfite; preservatives, isotonicity agents, stabilizers, metal complexes (e.g., Zn-protein complexes); chelating agents, such as EDTA and / or non-ionic surfactants.

[0293] Buffers can be used to adjust the pH within a range that optimizes therapeutic efficacy, especially when stability is pH dependent. Buffers can be present at concentrations ranging from about 20 mM to about 250 mM. Suitable buffers for use with the present invention include both organi...

Claims

1. a) an anti-CTLA4 antibody or antigen-binding fragment thereof comprising a light chain variable (VL) domain and a heavy chain variable (VH) domain; b) a masking peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 19; A masked antibody comprising: the masking peptide is linked to the amino- or carboxy-terminus of the VL domain or the VH domain via a linker comprising a cleavable peptide; and a) the VL domain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 402, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 403, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 404, and the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 405, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 406, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 407, or b) the VL domain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 408, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 409, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 410, and the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 411, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 412, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 413; or c) the VL domain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 432, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 433, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 434, and the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 435, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 436, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 437; or d) the VL domain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 438, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 439, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 440, and the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 441, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 442, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 443; The masked antibody.

2. The masked antibody of claim 1, wherein the linker comprising a cleavable peptide comprises a cleavable peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-88, 464-469, and 479-508.

3. 3. The masked antibody of claim 1 or 2, wherein the cleavable peptide comprises an amino terminus and a carboxy terminus, and the linker comprising the cleavable peptide comprises a first spacer linker and a second spacer linker, the first spacer linker is linked to the amino terminus of the cleavable peptide and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 89-112 and 415-420, and the second spacer linker is linked to the carboxy terminus of the cleavable peptide and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 89-112 and 415-420.

4. The masked antibody of any one of claims 1 to 3, wherein the linker comprising a cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 454-462.

5. The masked antibody of any one of claims 1 to 4, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 113-231 and 444-453.

6. The masked antibody of any one of claims 1 to 5, wherein the anti-CTLA4 antibody or antigen-binding fragment thereof, or ligand-binding fragment that binds to CTLA4, is a humanized or chimeric antibody.

7. a) the VL domain comprises the amino acid sequence of SEQ ID NO: 321 and the VH domain comprises the amino acid sequence of SEQ ID NO: 323, or b) the VL domain comprises the amino acid sequence of SEQ ID NO: 322 and the VH domain comprises the amino acid sequence of SEQ ID NO: 324; A masked antibody according to any one of claims 1 to 6.

8. The masked antibody of any one of claims 1 to 7, wherein the VL domain is comprised within a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 327-341, and the VH domain is comprised within a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 366-380 and 421.

9. The masked antibody of any one of claims 1 to 8, comprising the amino acid sequence of SEQ ID NO: 421 and comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 358 and 422 to 431.

10. The masked antibody of any one of claims 1 to 9, wherein the anti-CTLA4 antibody or antigen-binding fragment thereof is conjugated to an agent.

11. The masked antibody of claim 10, wherein the drug is an inhibitor of tubulin polymerization, a DNA damaging agent, or a DNA synthesis inhibitor, a maytansinoid, an auristatin, a pyrrolobenzodiazepine (PBD) dimer, a calicheamicin, a duocarmycin, an indolinobenzodiazepine dimer, or an exatecan derivative Dxd.

12. A masked bispecific antibody comprising the masked antibody of any one of claims 1 to 9.

13. A nucleic acid encoding the masked antibody of any one of claims 1 to 9 or the masked bispecific antibody of claim 12.

14. A vector or host cell comprising the nucleic acid of claim 13.

15. 15. A method for producing a masked antibody or a masked bispecific antibody, comprising culturing a host cell according to claim 14 under conditions to produce the masked antibody.

16. A pharmaceutical composition comprising the masked antibody of any one of claims 1 to 9 or the masked bispecific antibody of claim 12 and a pharma- ceutically acceptable carrier.

17. A therapeutic kit comprising a masked antibody according to any one of claims 1 to 9 or a masked bispecific antibody according to claim 12.

18. Use of a composition comprising a masked antibody according to any one of claims 1 to 9 or a masked bispecific antibody according to claim 12 for the manufacture of a medicament for treating or preventing a neoplastic disease in a subject.

19. a) an anti-CTLA4 antibody or antigen-binding fragment thereof comprising a light chain variable (VL) domain and a heavy chain variable (VH) domain; b) a masking peptide comprising the amino acid sequence of SEQ ID NO:5; A masked antibody comprising: the masking peptide is linked to the amino terminus of the VL domain via a linker comprising a cleavable peptide; the linker comprising a cleavable peptide comprises a cleavable peptide comprising the amino acid sequence of SEQ ID NO:86; (a) the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 408, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 409, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 410, and the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 411, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 412, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 413; or (b) the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 438, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 439, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 440, and the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 441, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 442, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 443; Masked antibodies.

20. a) an anti-CTLA4 antibody or antigen-binding fragment thereof comprising a light chain variable (VL) domain and a heavy chain variable (VH) domain; b) a masking peptide comprising the amino acid sequence of SEQ ID NO: 19; A masked antibody comprising: the masking peptide is linked to the amino terminus of the VL domain via a linker comprising a cleavable peptide; the linker comprising a cleavable peptide comprises a cleavable peptide comprising the amino acid sequence of SEQ ID NO:50; (a) the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 408, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 409, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 410, and the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 411, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 412, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 413; or (b) the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 438, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 439, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 440, and the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 441, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 442, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 443; Masked antibodies.

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