TW202126686A - Activatable masked anti-ctla4 binding proteins - Google Patents

Abstract

The invention provides activatable masked anti-CTLA4 binding proteins (e.g., antibodies, bispecific antibodies, and chimeric receptors) and their use in treating and preventing cancer, as well as compositions and kits comprising the activatable masked anti-CTLA4 binding proteins.

Description

Activated masked anti-CTLA4 binding protein

The present invention relates to activatable and masked anti-cytotoxic T lymphocyte-associated protein 4 (CTLA4) binding proteins (such as anti-CTLA4 antibodies) and related methods of use.

Cancer is the second leading cause of death in the United States. It causes more deaths than the last five major causes (chronic respiratory disease, stroke, accident, Alzheimer's disease and diabetes). Although great progress has been made (especially with targeted therapies), a lot of work is still needed in this field. Immunotherapy and its branch, immuno-oncology, are producing viable and exciting treatment options for the treatment of malignant diseases. In particular, it has been discovered that one of the characteristics of cancer is to avoid immunity and a lot of work has been done to identify targets and develop therapies for these targets, so as to reactivate the immune system to recognize and treat cancer. The fact is that the anti-cytotoxic T lymphocyte-associated protein 4 (CTLA4) antibody, ipilimumab, achieves long-term survival of patients suffering from stage III/IV malignant melanoma. Ipelizumab is an immune checkpoint antagonist, which interrupts the suppression of T cells by blocking CTLA4 and can cause the depletion of T regulatory cells (Treg). [Korman, A. et al., 2005. Tumor immunotherapy: preclinical and clinical activity of anti-CTLA4 antibodies. Current Opinion in Investigational Drugs 6:582-591; Quezada et al., J. Exp. Med., 206(8): 1717-1725, 2009; Selby et al. Cancer Immunol Res., 1(1); 32-42, 2013]. Unfortunately, Ipelizumab causes systemic (non-tumor-specific) activation of T cell-dependent immune responses, which cause immune-related side effects that can be life-threatening and usually limit the 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). These side effects include enterocolitis, dermatitis, hypophysitis, uveitis, hepatitis, nephritis and death. Enterocolitis is the most common serious toxicity (affecting approximately 20% of patients). The major safety risks associated with immune-mediated adverse reactions prompted the FDA to approve the use of Ipelizumab for Risk Evaluation and Mitigation Strategy (REMS). Recently, it has been proved that the co-administration of Ipelizumab and a second immune checkpoint modulator that targets PD1 (such as nivolumab) can significantly increase melanin compared with Ipelizumab alone. The efficacy of tumor immunotherapy. However, this gain is associated with an increased incidence of grade 3/4 side effects, which affects more than 50% of patients receiving combination therapy (Wolchok, J.D. et al., 2013. Nivolumab plus Ipilimumab in Advanced Melanoma. N Engl J Med).

These results indicate the need to develop anti-CTLA4 protein therapeutics that can effectively target tumors without the side effects associated with systemic immune activation. Provided herein are anti-CTLA binding proteins, their compositions, and methods of use to address this need.

All references cited in this article (including patent applications, patent publications, and scientific documents) are incorporated herein by reference in their entirety, as if each reference was specifically and individually indicated to be incorporated by reference .

Provided herein are activatable and masked anti-cytotoxic T lymphocyte-associated protein 4 (CTLA4) binding proteins, compositions containing them, and methods of use.

Provided herein is a masked antibody, which contains an antibody or antigen-binding fragment thereof that binds to CTLA4, wherein the antibody or antigen-binding fragment thereof contains a first chain and a second chain, and comprises an antibody selected from SEQ ID NO: 1-46 A masking peptide of an amino acid sequence, wherein the masking peptide is connected to the amino or carboxyl end of the first chain or the second chain of an antibody or an antigen-binding fragment thereof via a linker containing a cleavable peptide. In some embodiments, the first chain is a light chain; and the second chain is a heavy chain.

In some embodiments, the antibody or antigen-binding fragment thereof contains two first chains and two second chains. In some embodiments, the first chain is or includes a light chain variable domain; and the second chain is or includes 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 amine or carboxy terminus of the masking peptide is attached to a linker comprising a cleavable peptide. In some of any such embodiments, the linker comprising the cleavable peptide contains a spacer linker and a cleavable peptide. In some of any such embodiments, the cleavable peptide contains an amino acid sequence selected from SEQ ID NO: 47-88, 464-469, and 479-508. In some of any such embodiments, the spacer linker is directly connected to the N-terminus and/or C-terminus of the cleavable peptide. In some of any such embodiments, the spacer linker contains an amino acid sequence selected from SEQ ID NO: 89-112 and 415-420. In some of any such embodiments, at least one amino acid but no more than 20 amino acids are directly attached 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 masked antibody contains in the N-to-C-terminal or C-to-N-terminal orientation: a) a masking peptide; b) a cleavable peptide; and c) an antibody or antigen thereof that binds to CTLA4 Combine fragments. In some of any such embodiments, the masked antibody contains a spacer linker between the masking peptide and the cleavable peptide; and the masked antibody is between the cleavable peptide and the antibody or antigen-binding fragment thereof that binds to CTLA4 Contains spacer linkers between.

In some of any such embodiments, the antibody is a murine antibody. In some of any such embodiments, the antibody is a humanized antibody, chimeric antibody, or human antibody. In some of any such embodiments, the antibody has an IgG1, IgG2, IgG3, or IgG4 isotype. In some of any such embodiments, IgG1 contains 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, Y300L and P396L; F243L, R292P, Y300L, V305I and P396L; G236A, S239D and I332E; K326A and E333A; K326W and E333S; or K290E or K290N, S/298G, T299A and Or K326E; wherein the amino acid residues are numbered according to the EU index as in Kabat.

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

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

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) CDR-L1, which comprises SEQ ID NO: 432 (Ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 433, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 444; and/or The heavy chain variable region includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 435, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 436, and (iii) ) CDR-H3, which includes 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) CDR-L1, which comprises SEQ ID NO: 432 (Ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 433, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 434; and heavy chain The variable region includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 435, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 436, and (iii) CDR- H3, which includes 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) CDR-L1, which comprises SEQ ID NO: 408 (Ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 409, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 410; and/or The heavy chain variable region includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 411, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 412, and (iii) ) CDR-H3, which includes 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) CDR-L1, which comprises SEQ ID NO: 408 (Ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 409, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 410; and heavy chain The variable region includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 411, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 412, and (iii) CDR-H2. H3, which includes 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) CDR-L1, which comprises SEQ ID NO: 438 (Ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 439, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 440; and/or The heavy chain variable region includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 441, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 442, and (iii) ) CDR-H3, which includes 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) CDR-L1, which comprises SEQ ID NO: 438 (Ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 439, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 440; and heavy chain The variable region includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 441, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 442, and (iii) CDR- H3, which includes the amino acid sequence of SEQ ID NO:443.

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

In some of any such embodiments, the antibody contains a light chain comprising an amino acid sequence selected from SEQ ID NO: 327-341; and/or a heavy chain comprising a light chain selected from SEQ ID NO: 366-380 , 421 and 478 amino acid sequences. In some of any such embodiments, the antibody contains a light chain, which comprises an amino acid sequence selected from SEQ ID NO: 327, 334, or 342-365; and/or a heavy chain, which comprises a light chain selected from SEQ ID NO : 366 or 380-397 amino acid sequence. 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: 327, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 366. 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: 327, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 478. 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: 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.

In some of any such embodiments, the cleavable peptide is a substrate for a protease that is co-located in a region with cells or tissues expressing CTLA4. In some of any such embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of 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, ADAMTS5ADA, ADAMTSADA, ADAMTS6 ADAMTSL3, ABHD17C, ADAMTSL5, ASTL, BMP1, CELA1, CELA2A, CELA2B, CELA3A, CELA3B, ADAM10, ADAM15, ADAM17, ADAM9, ADAMTS4, CTSE, CTSF, ADAMTSL4, CMA1, CTRB1, CTRC, CTSO, CTRSW, CTSA, CTSA, CTSB, CTSC, CTSD, ESP1, CTSG, CTSH, GZMA, GZMB, GZMH, CTSK, GZMM, CTSL, CTSS, CTSV, CTSZ, HTRA4, KLK10, KLK11, KLK13, KLK14, KLK2, KLK4, DPP4, KLK, 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, ST1 4. TMPRSS10, TMPRSS11A, TMPRSS11D, TMPRSS11E, TMPRSS11F, TMPRSS12, TMPRSS13, MMP15, TMPRSS15, MMP2, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS5, TMPRSS6, TMPRSS7, TMPRSS9, TIN, OVCH1, PAMR1, PCTIN 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 antibody or antigen-binding fragment thereof binds to the agent. In some of any such embodiments, the agent is a tubulin polymerization inhibitor, a DNA damaging agent, or a DNA synthesis inhibitor. In some of any such embodiments, the agent is maytansinoid, auristatin, pyrrolobenzodiazepine (PBD) dimer, calicheamicin, Duocarmycin, indo-linobenzodiazepine dimer or exatecan derivative Dxd.

In some of any such embodiments, the masked antibodies provided herein exhibit an optimal occlusion rate of about 20 to about 10,000. In another embodiment, the optimal occlusion rate is about 20 to about 1,000. In another embodiment, the optimal occlusion rate is about 80 to about 100.

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 NO: 358 and 422-431.

Also provided herein is a masked bispecific antibody, which contains 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 contains selected from SEQ ID NO : A masking peptide of the amino acid sequence of 1-46, wherein the masking peptide is connected to the amine or carboxyl end of the first pair of light or heavy chains via a linker containing a cleavable peptide. In some embodiments, the amine or carboxy terminus of the masking peptide is connected to a linker comprising a cleavable peptide. In some of any such embodiments, the linker comprising the cleavable peptide contains a spacer linker and a cleavable peptide.

In some of any such embodiments, the cleavable peptide contains an amino acid sequence selected from SEQ ID NO: 47-88, 464-469, and 479-508. In some of any such embodiments, the spacer linker is directly connected to the N-terminus or C-terminus of the cleavable peptide. In some of any such embodiments, the spacer linker contains an amino acid sequence selected from SEQ ID NO: 89-112 and 415-420. In some of any such embodiments, at least one amino acid but no more than 20 amino acids are directly attached 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 pair of light or heavy chains in N-to-C-terminal or C-to-N-terminal orientation contains: a) masking peptide; b) cleavable peptide; and c) light chain or heavy chain chain. In some of any such embodiments, the first pair contains a spacer linker between the masking peptide and the cleavable peptide; and the first pair contains a spacer linker between the cleavable peptide and the light or heavy chain.

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, chimeric antibody, or 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, IgG1 contains amino acid substitutions, such as 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 P396L; F243L, R292P, Y300L, V305I and P396L; G236A, S239D and I332E; K326A and E333A; K326W and E333S; or K290E or KT299N, S298G, S298G And/or K326E, wherein the amino acid residues are numbered according to the EU index as in Kabat.

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

In some of any such embodiments, the first pair includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region includes (i) CDR-L1, which includes the amine of SEQ ID NO: 402 Base acid sequence, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 403, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 404; and/or its weight The chain variable region includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 405, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 406, and (iii) CDR -H3, which includes the amino acid sequence of SEQ ID NO:407. In some embodiments, the first pair includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 402, ( ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 403, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 404; and the heavy chain variable region includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 405, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 406, and (iii) CDR-H3, which includes SEQ ID NO : 407 amino acid sequence.

In some of any such embodiments, the first pair includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region includes (i) CDR-L1, which includes the amine of SEQ ID NO: 432 Base acid sequence, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 433, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 444; and/or its weight The chain variable region includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 435, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 436, and (iii) CDR -H3, which includes the amino acid sequence of SEQ ID NO:437. In some of any such embodiments, the first pair includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region includes (i) CDR-L1, which includes the amine of SEQ ID NO: 432 Base acid sequence, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 433, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 434; and heavy chain variable The region includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 435, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 436, and (iii) CDR-H3, It includes the amino acid sequence of SEQ ID NO:437.

In some of any such embodiments, the first pair includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region includes (i) CDR-L1, which includes the amine of SEQ ID NO: 408 Base acid sequence, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 409, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 410; and/or its weight The chain variable region includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 411, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 412, and (iii) CDR -H3, which includes the amino acid sequence of SEQ ID NO: 413. In some of any such embodiments, the first pair includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region includes (i) CDR-L1, which includes the amine of SEQ ID NO: 408 Base acid sequence, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 409, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 410; and heavy chain variable The region includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 411, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 412, and (iii) CDR-H3, It includes the amino acid sequence of SEQ ID NO: 413.

In some of any such embodiments, the first pair includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region includes (i) CDR-L1, which includes the amine of SEQ ID NO: 438 Base acid sequence, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 439, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 440; and/or its weight The chain variable region includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 441, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 442, and (iii) CDR -H3, which includes the amino acid sequence of SEQ ID NO:443. In some of any such embodiments, the first pair includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region includes (i) CDR-L1, which includes the amine of SEQ ID NO: 438 Base acid sequence, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 439, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 440; and heavy chain variable The region includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 441, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 442, and (iii) CDR-H3, It includes the amino acid sequence of SEQ ID NO:443.

In some of any such embodiments, the first pair contains a light chain variable region comprising the amino acid sequence of SEQ ID NO: 232; and/or a heavy chain comprising the amino acid sequence of SEQ ID NO: 233 Variable region. In some of any such embodiments, the first pair contains a light chain comprising an amino acid sequence selected from SEQ ID NO: 237-318; and/or includes an amino group selected from SEQ ID NO: 319 or 320 The heavy chain of the acid sequence. In some of any such embodiments, the first pair contains a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 321 or 322; and/or comprises a light chain variable region selected from SEQ ID NO: 323 or 324 The variable region of the heavy chain of the amino acid sequence. In some of any such embodiments, the first pair includes a light chain variable region comprising the amino acid sequence of SEQ ID NO: 321, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 323 can be Variable area. In some of any such embodiments, the first pair includes a light chain variable region comprising the amino acid sequence of SEQ ID NO: 322, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 324 may Variable area.

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

In some of any such embodiments, the cleavable peptide is a substrate for a protease that is co-located in a region with cells or tissues expressing CTLA4. In some of any such embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of 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, ADAMTS5ADA, ADAMTSADA, ADAMTS6 ADAMTSL3, ABHD17C, ADAMTSL5, ASTL, BMP1, CELA1, CELA2A, CELA2B, CELA3A, CELA3B, ADAM10, ADAM15, ADAM17, ADAM9, ADAMTS4, CTSE, CTSF, ADAMTSL4, CMA1, CTRB1, CTRC, CTSO, CTRSW, CTSA, CTSA, CTSB, CTSC, CTSD, ESP1, CTSG, CTSH, GZMA, GZMB, GZMH, CTSK, GZMM, CTSL, CTSS, CTSV, CTSZ, HTRA4, KLK10, KLK11, KLK13, KLK14, KLK2, KLK4, DPP4, KLK, 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, ST1 4. TMPRSS10, TMPRSS11A, TMPRSS11D, TMPRSS11E, TMPRSS11F, TMPRSS12, TMPRSS13, MMP15, TMPRSS15, MMP2, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS5, TMPRSS6, TMPRSS7, TMPRSS9, TIN, OVCH1, PAMR1, PCTIN 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 binds to the agent. In some of any such embodiments, the agent is a tubulin polymerization inhibitor, a DNA damaging agent, or a DNA synthesis inhibitor. In some of any such embodiments, the agent is a maytansinoid, auristatin, pyrrolobenzodiazepine (PBD) dimer, calicheamicin, becarcinomycin, indoline benzene Diazadiazepine dimer or ixinotecan derivative Dxd.

In some of any such embodiments, the first pair and the second pair of masked bispecific antibodies provided herein each exhibit an optimal occlusion rate, which may be the same or different from each other. In some embodiments, the optimal occlusion rate is about 20 to about 10,000. In another embodiment, the optimal occlusion rate is about 20 to about 1,000. In another embodiment, the optimal occlusion rate is about 80 to about 100.

Also provided herein is a masked chimeric receptor, which contains a ligand binding domain, which includes a first chain and a second chain that bind to CTLA4; a masked peptide, which includes an amine selected from SEQ ID NO: 1-46 Base acid sequence, that is, transmembrane domain; and intracellular signal transduction domain comprising a signal transduction domain, wherein the masking peptide is connected to the amine group of the first chain or the second chain of the ligand binding domain via a linker comprising a cleavable peptide End or carboxyl end.

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 amine or carboxy terminus of the masking peptide is connected to a linker comprising a cleavable peptide. In some of any such embodiments, the linker comprising the cleavable peptide contains a spacer linker and a cleavable peptide. In some of any such embodiments, the cleavable peptide contains an amino acid sequence selected from SEQ ID NO: 47-88, 464-469, and 479-508. In some of any such embodiments, the spacer linker is directly connected to the N-terminus or C-terminus of the cleavable peptide. In some of any such embodiments, the spacer linker contains an amino acid sequence selected from SEQ ID NO: 89-112 and 415-420. In some of any such embodiments, at least one amino acid but no more than 20 amino acids are directly attached 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 strand of the ligand binding domain in the N-to-C-terminal or C-to-N-terminal direction contains: a) a masking peptide; b) a cleavable peptide; and c ) The first chain or the second chain. In some of any such embodiments, the ligand binding domain contains a spacer linker between the masking peptide and the cleavable peptide; and the ligand binding domain is between the cleavable peptide and the first or second chain Contains spacer linkers.

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

In some of any such embodiments, the first chain comprises (i) CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 402, and (ii) CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 403 Amino acid sequence, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 404; and/or the second chain includes (i) CDR-H1, which includes the amine group of SEQ ID NO: 405 The acid sequence, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 406, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 407. In some embodiments, the first chain includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 402, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 403, And (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 404; and/or the second chain includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 405, (ii ) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 406, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 407.

In some of any such embodiments, the first chain comprises (i) CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 432, and (ii) CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 433 Amino acid sequence, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 444; and/or the second chain includes (i) CDR-H1, which includes the amine group of SEQ ID NO: 435 The acid sequence, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 436, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 437. In some of any such embodiments, the first chain comprises (i) CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 432, and (ii) CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 433 Amino acid sequence, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 434; and the second chain includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 435 , (Ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 436, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 437.

In some of any such embodiments, the first chain comprises (i) CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 408, and (ii) CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 409 Amino acid sequence, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 410; and/or the second chain includes (i) CDR-H1, which includes the amino group of SEQ ID NO: 411 The acid sequence, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 412, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 413. In some of any such embodiments, the first chain comprises (i) CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 408, and (ii) CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 409 Amino acid sequence, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 410; and the second chain includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 411 , (Ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 412, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 413.

In some of any such embodiments, the first chain comprises (i) CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 438, (ii) CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 439 Amino acid sequence, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 440; and/or the second chain includes (i) CDR-H1, which includes the amine group of SEQ ID NO: 441 The acid sequence, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 442, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 443. In some of any such embodiments, the first chain comprises (i) CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 438, (ii) CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 439 Amino acid sequence, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 440; and the second chain includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 441 , (Ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 442, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 443.

In some of any such embodiments, the first strand contains the amino acid sequence of SEQ ID NO: 232; and/or the second strand contains the amino acid sequence of SEQ ID NO: 233. In some of any such embodiments, the first strand contains an amino acid sequence selected from SEQ ID NO: 321 or 322; and/or the second strand contains an amino acid selected from SEQ ID NO: 323 or 324 sequence. In some of any such embodiments, the first strand includes the amino acid sequence of SEQ ID NO: 321, and the second strand includes the amino acid sequence of SEQ ID NO: 323. In some of any such embodiments, the first strand includes the amino acid sequence of SEQ ID NO: 322, and the second strand includes 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-located in a region with cells or tissues expressing CTLA4.

In some of any such embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of 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, ADAMTS5ADA, ADAMTSADA, ADAMTS6 ADAMTSL3, ABHD17C, ADAMTSL5, ASTL, BMP1, CELA1, CELA2A, CELA2B, CELA3A, CELA3B, ADAM10, ADAM15, ADAM17, ADAM9, ADAMTS4, CTSE, CTSF, ADAMTSL4, CMA1, CTRB1, CTRC, CTSO, CTRSW, CTSA, CTSA, CTSB, CTSC, CTSD, ESP1, CTSG, CTSH, GZMA, GZMB, GZMH, CTSK, GZMM, CTSL, CTSS, CTSV, CTSZ, HTRA4, KLK10, KLK11, KLK13, KLK14, KLK2, KLK4, DPP4, KLK, 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, ST1 4. TMPRSS10, TMPRSS11A, TMPRSS11D, TMPRSS11E, TMPRSS11F, TMPRSS12, TMPRSS13, MMP15, TMPRSS15, MMP2, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS5, TMPRSS6, TMPRSS7, TMPRSS9, TIN, OVCH1, PAMR1, PCTIN 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 masked chimeric receptors provided herein exhibit an optimal occlusion rate of about 20 to about 10,000. In another embodiment, the optimal occlusion rate is about 20 to about 1,000. In another embodiment, the optimal occlusion rate is about 80 to about 100.

A nucleic acid is also provided, which encodes a masked antibody, a masked bispecific antibody or a masked chimeric receptor as in any one of the preceding embodiments. A vector is also provided, which comprises the nucleic acid as in the foregoing embodiment. In some embodiments, the vector is a performance vector. A host cell is also provided, which comprises the aforementioned nucleic acid examples.

It also provides a method for preparing a masked antibody, a masked bispecific antibody or a masked chimeric receptor, which involves the production of a masked antibody, a masked bispecific antibody or a masked chimera The aforementioned host cells are cultured under the conditions of the recipient. In some embodiments, the host cell has an α1,6-trehalosyltransferase (Fut8) gene knockout. In some embodiments, wherein the host cell overexpresses β1,4-N-acetylglycosamine polyglycosyltransferase III (GnT-III). In some embodiments, the host cell also overexpresses Golgi μ-mannosidase II (ManII). Some of any such embodiments further include recovering masked antibodies, masked bispecific antibodies, or masked chimeric receptors produced by the host cell. In some embodiments, masked bispecific antibodies or masked chimeric receptors are produced by the aforementioned methods.

A composition is also provided, which contains the masked antibody, the masked bispecific antibody or the masked chimeric receptor as in any one of the preceding embodiments. Some embodiments encompass a composition comprising a masked antibody, a masked bispecific antibody, or a masked chimeric receptor as in the previous embodiment. In some embodiments, the composition is a pharmaceutical composition.

A kit is also provided, which contains a masked antibody, a masked bispecific antibody, a masked chimeric receptor or a composition as in any one of the foregoing embodiments.

A method for treating or preventing neoplastic diseases in an individual is also provided, the method comprising administering to the individual an effective amount of a masked antibody, a masked bispecific antibody, Masked chimeric receptor or composition. In one embodiment, the neoplastic disease is cancer. In some embodiments, the cancer is leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma, lung cancer, ovarian cancer, osteosarcoma, bladder cancer, children Cervical cancer, liver cancer, kidney cancer, skin cancer or testicular cancer.

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

Submit the sequence list as an ASCII text file

The following content submitted in the form of an ASCII text file is incorporated into this article by reference in its entirety: Sequence table in computer readable form (CRF) (file name: 737762001641.txt, creation date: December 27, 2019, size: 519) KB).

Therapeutic agents such as checkpoint inhibitors show unprecedented responses in cancer, but their use is limited by immune-related adverse events (irAE) and other toxicities such as hypophysitis. Provided herein are protein therapeutics that specifically bind to CTLA4 after being activated by a protease at a target site, such as in the tumor microenvironment, to achieve an increased sustained response rate and a significantly improved safety profile. The protein therapeutics provided herein are engineered to precisely target the pharmacological activity of the tumor microenvironment by using a marker of cancer, namely the high local concentration of active protease. Use this feature of the tumor microenvironment to transform systemically inert molecules into locally active drugs. The activation of the drug in the tumor microenvironment can significantly reduce the systemic toxicity that can be associated with the administration of the drug to the individual in an active form. I. Definition

Before describing the present invention in detail, it should be understood that the present invention is not limited to a specific composition or biological system, which can of course be changed. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to be limiting. Unless the content clearly indicates otherwise, the singular form "一 (a/an)" and "the (the)" used in this specification and the accompanying patent application include plural indicators. Thus, for example, a reference to "antibody" optionally includes a combination of two or more such antibodies and their analogs.

As used herein, the term "about" refers to the common error range of individual values that are easily known to those skilled in the art. Herein, reference to "about" a value or parameter includes (and describes) an embodiment for the value or parameter itself.

It should be understood that the aspects and embodiments of the present invention described herein include “including” aspects and embodiments, “consisting of aspects and embodiments”, and “basically consisting of aspects and embodiments”.

The term "antibody" includes multiple antibodies, monoclonal antibodies (including full-length antibodies with immunoglobulin Fc regions), antibody compositions with multiple epitope specificities, and multispecific antibodies (e.g., bispecific antibodies, bifunctional antibodies). Antibodies and single chain molecules) and antibody fragments (e.g. Fab, F(ab')2 and Fv). In this article, the term "immunoglobulin" (Ig) is used interchangeably with "antibody".

The basic 4-chain antibody unit is a heterotetraglycan protein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibody is composed of 5 basic heterotetramer units and other polypeptides called J chain and contains 10 antigen binding sites, while IgA antibody contains 2-5 basic 4 chain units, which can be polymerized to combine with J chain Form multivalent aggregates. In the case of IgG, the 4-chain unit is usually about 150,000 Daltons. Each L chain is connected to the H chain by a covalent disulfide bond, and the two H chains are connected to each other by one or more disulfide bonds depending on the same type of the H chain. Each H chain and L chain also has regularly spaced intra-chain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) of each α and γ chain, and four CH domains of the same type of μ and ε. Each L chain has a variable domain (VL) at the N-terminus, and then a constant domain at the other end. VL is aligned with VH and CL is aligned with the first constant domain (CH1) of the heavy chain. It is believed that specific amino acid residues form an interface between the light chain and heavy chain variable domains. VH and VL pair together to form 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, No. Page 71 and Chapter 6.

The L chain from any vertebrate species can be classified into one of two distinct types (called kappa and lambda) based on the amino acid sequence of its constant domain. Depending on the amino acid sequence of the constant domain of the immunoglobulin heavy chain (CH), it can be classified into different classes or homotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, which have heavy chains called α, δ, ε, γ, and μ, respectively. Based on the relatively small differences in CH sequence and function, the gamma and alpha classes are further divided into subclasses. For example, humans exhibit the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. IgG1 antibodies can exist in the form of multiple polymorphic variants called allotypes (reviewed in Jefferis and Lefranc 2009. mAbs Volume 1, Issue 4 1-7), any of which is suitable for use in the present invention. Common heteromorphic variants in the human population are the variants designated by the letters a, f, n, and z.

"Isolated" antibodies are antibodies that have been identified, separated, and/or recovered (for example, in natural or recombinant manner) from components of the environment in which they were produced. In some embodiments, the isolated polypeptide is unrelated to all other components from the environment in which it was produced. The pollutant components of the preparation environment (such as those produced by recombinant transfected cells) are substances that usually interfere with the research, diagnostic or therapeutic uses of antibodies, and may include enzymes, hormones, and other protein or non-protein solutes. In some embodiments, the polypeptide is purified to (1) greater than 95% by weight of the antibody, as determined by, for example, the Lowry method, and in some embodiments, purified to greater than 99% by weight; (1) It is sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using, for example, a rotating cup sequencer; or (3) homogenization, which uses Coomassie blue or silver dye in Africa It can be achieved by SDS-PAGE under reducing or reducing conditions. Isolated antibodies include antibodies that are present in recombinant cells in situ, because at least one component of the antibody's natural environment will not be present. However, generally, the isolated polypeptide or antibody system is prepared by at least one purification step.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies that make up the population except for mutations that may occur naturally and/or translations that may exist in small amounts Except for the modification (for example, isomerization, amination), the rest is the same. In some embodiments, the monoclonal antibody has C-terminal cleavage at the heavy chain and/or light chain. For example, 1, 2, 3, 4, or 5 amino acid residues are cleaved at the C-terminus of the heavy chain and/or light chain. In some embodiments, C-terminal cleavage removes the C-terminal lysine from the heavy chain. In some embodiments, the monoclonal antibody has N-terminal cleavage at the heavy chain and/or light chain. For example, 1, 2, 3, 4, or 5 amino acid residues are cleaved at the N-terminus of the heavy chain and/or light chain. In some embodiments, truncated forms of monoclonal antibodies can be produced by recombinant technology. In some embodiments, monoclonal antibodies are highly specific for a single antigenic site. In some embodiments, monoclonal antibodies are highly specific for multiple antigenic sites (such as bispecific antibodies or multispecific antibodies). The modifier "monoclonal" indicates that the characteristic of the antibody is obtained from a substantially homogeneous antibody population, and should not be understood as requiring the production of the antibody by any specific method. For example, the monoclonal antibodies to be used in accordance with the present invention can be produced by a variety of techniques, including, for example, fusion tumor methods, recombinant DNA methods, phage display techniques, and use in encoding human immunoglobulins that have part or all of them. The technology of producing human antibodies or human-like antibodies in animals with human immunoglobulin loci or genes of protein sequences.

The term "naked antibody" refers to an antibody that has not been conjugated with a cytotoxic moiety or radiolabel.

The term "parent antibody" refers to the antibody before modification (such as masking the antibody with a masking peptide).

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 activation or removal of the masking peptide in a better environment.

"Antibody-drug conjugate" or "ADC" refers to an antibody that binds to one or more heterogeneous molecules (including but not limited to cytotoxic agents).

The terms "full-length antibody," "complete antibody," or "complete antibody" are used interchangeably to refer to an antibody that is in a substantially complete form compared to antibody fragments. Specifically, a complete antibody includes an antibody having a heavy chain and a light chain including an Fc region. The constant domain may be a native sequence constant domain (for example, a human native sequence constant domain) or an amino acid sequence variant thereof. In some cases, intact antibodies may have one or more effector functions.

"Antibody fragments" include a part of a complete antibody, the antigen binding region and/or variable region of the complete antibody. Examples of antigen-binding antibody fragments include domain antibodies (dAb), Fab, Fab', F(ab')2, and Fv fragments; bifunctional antibodies; linear antibodies (see U.S. Patent 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, can be isolated from camels, sharks, libraries, or mice that have been engineered to produce single heavy chain molecules.

The papain digestion of the antibody produces two identical antigen-binding fragments, called the "Fab" fragment and the residual "Fc" fragment, which reflects the ability to be easily crystallized. The Fab fragment is composed of a complete L chain and the variable region domain of the H chain (VH) and the first constant domain (CH1) of a heavy chain. Each Fab fragment is monovalent in terms of antigen binding, that is, it has a single antigen binding site. Pepsin treatment of the antibody produces a single large F(ab')2 fragment, which roughly corresponds to two disulfide-bonded Fab fragments with different antigen-binding activities and still capable of cross-linking with the antigen. The difference between the Fab' fragment and the Fab fragment is that the Fab' fragment has several other residues at the carboxyl end of the CH1 domain, including one or more cysteine from the hinge region of the antibody. Fab'-SH is the name of Fab' in which the cysteine residue of the constant domain has a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines in between. Other chemical couplings of antibody fragments are also known.

The Fc fragment contains the carboxy terminal portions of two H chains held together by disulfide bonds. The effector function of an antibody is determined by the sequence and glycans in the Fc region, which is also recognized by the Fc receptor (FcR) found on certain types of cells.

"Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a dimer of a heavy chain and a light chain variable domain in a tight non-covalent association. Thus, the folding of the two domains produces six hypervariable loops (3 loops each from the H chain and the L chain), which provide 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 containing only three HVRs specific to the antigen) still has the ability to recognize and bind to the antigen, but the affinity is lower than the complete binding site.

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

The "functional fragment" of the antibody of the present invention includes a part of a complete antibody, and these fragments usually include the antigen binding region or variable region of the complete antibody or the Fv region of an antibody that retains or has modified FcR binding ability. Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

In this context, monoclonal antibodies specifically include "chimeric" antibodies (immunoglobulins), in which a part of the heavy chain and/or light chain is identical or identical to the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass. Homologous, and the rest of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, as long as they exhibit the required biological activity (US Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Related chimeric antibodies herein include PRIMATIZED® antibodies, where the antigen binding region of the antibody is derived from an antibody produced by, for example, immunizing a rhesus monkey with a related antigen. As used herein, "humanized antibodies" are used as a subset of "chimeric antibodies".

The "humanized" form of non-human (e.g., murine) antibodies is a chimeric antibody that contains the smallest sequence derived from non-human immunoglobulin. In one embodiment, the humanized antibody is a human immunoglobulin (acceptor antibody) in which the residues of the HVR from the recipient are replaced by the residues of the HVR from a non-human species (donor antibody), the non-human Species such as mice, rats, rabbits or non-human primates with the required specificity, affinity and/or ability. In some cases, FR residues of human immunoglobulins are replaced with corresponding non-human residues. In addition, humanized antibodies may contain residues that are not found in the recipient antibody or the donor antibody. These modifications can be made to further optimize antibody performance, such as binding affinity. Generally, a humanized antibody will contain substantially at least one and typically all of two variable domains, wherein all or substantially all hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all All FR regions are FR regions of human immunoglobulin sequences, but FR regions may include one or more individual FR residue substitutions that improve antibody performance (such as binding affinity, isomerization, immunogenicity, etc.). In some embodiments, the number of such amino acid substitutions in the FR does not exceed 6 in the H chain, and does not exceed 3 in the L chain. The humanized antibody will optionally also contain at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. For other details, see, for example, 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 also, for example, 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. Patent Nos. 6,982,321 and 7,087,409. In some embodiments, the humanized antibody system is directed to a single antigenic site. In some embodiments, the humanized antibody system targets 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.

The "variable region" or "variable domain" of an antibody refers to the amino terminal domain of the heavy or light chain of the antibody. Therefore, the terms "variable region" and "variable domain" as used herein can be used interchangeably. The variable domains of the heavy chain and light chain can be referred to as "VH" and "VL", respectively. These domains are usually the most variable part of the antibody (relative to other antibodies of the same class) and contain antigen binding sites. The variable domains of the heavy chain and the light chain can be determined using any available method or numbering scheme and may include, for example, the variable domains described in WO 2018/207701, the contents of which are incorporated herein by reference. In some embodiments, the variable domain of the heavy chain and/or light chain may not have one or more amino acid residues on the carboxy terminus of the variable domain (that is, the carboxy terminus of the fourth framework domain), The one or more amino acid residues can be included in the description of variable domains based on certain numbering schemes in other ways. In some embodiments, the variable domain of the heavy chain and/or the light chain may include one or more amino acid residues on the carboxy terminus of the variable domain (that is, the carboxy terminus of the fourth framework domain), The one or more amino acid residues can be included in the description of variable domains based on certain numbering schemes in other ways.

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

A variety of HVRs have been described and covered in this article. The HVR as the Kabat complementarity determining region (CDR) is based on sequence variability and is the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institute of Health, Bethesda, MD (1991) ). Chothia mentions the position of the structural loop instead (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). "Contact" HVR is based on the analysis of available composite crystal structures. The residues from each of these HVRs are labeled below. Ring Kabat Chothia Contact L1 L24-L34 L26-L34 L30-L36 L2 L50-L56 L50-L56 L46-L55 L3 L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H32 H30-H35B (Kabat number) H1 H31 -H35 H26-H32 H30-H35 (Chothia number) H2 H50-H65 H53-H56 H47-H58 H3 H95-H102 H95-H102 H93-H101

Unless otherwise indicated, variable domain residues (HVR residues and framework region residues) are numbered according to Kabat et al., see above.

"Framework" or "FR" residues are variable domain residues other than HVR residues as defined herein.

The expressions "such as the number of variable domain residues in Kabat" or "such as the number of amino acid positions in Kabat" and their variants refer to Kabat et al., see above for the heavy chain variable domains used in antibody compilation or The numbering system of light chain variable domains. Using this numbering system, the actual linear amino acid sequence may contain fewer or other amino acids, which correspond to the shortening or insertion of the FR or HVR of the variable domain. For example, the heavy chain variable domain may include a single amino acid insert after residue 52 of H2 (residue 52a, according to Kabat), and an inserted residue after heavy chain FR residue 82 (e.g., residue 82a). , 82b and 82c, etc., according to Kabat). For a given antibody, the Kabat numbering of residues can be determined by aligning the homology regions of the antibody sequence with the "standard" Kabat numbering sequence.

For the purposes herein, "acceptor human framework" is a framework comprising amino acid sequences derived from the human immunoglobulin framework or the VL or VH framework of the human common framework. The acceptor human framework "derived from" the human immunoglobulin framework or the human common framework may comprise the same amino acid sequence of the human immunoglobulin framework or the human common framework, or it may contain 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 more. Fewer, 4 or less, 3 or less, or 2 or less.

The "% of amino acid sequence identity (%)" relative to the reference polypeptide sequence is defined as after aligning the sequences and introducing gaps as necessary to achieve the maximum sequence identity, and without considering any conservative substitutions as sequence identity In the case of part of sex, the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence. The alignment for the purpose of determining the percent identity of amino acid sequences can be achieved in various ways within the skill of this technology, such as using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software . Those skilled in the art can determine the parameters suitable for the alignment of sequences, including any algorithms required to achieve the maximum alignment over the full length of the sequence being compared. For example, a given amino acid sequence A is relative to, with or with respect to the amino acid sequence of a given amino acid sequence B. The amino acid sequence identity% (or, it can be expressed as having relative to, and or with respect to, the given amino acid sequence B Or a predetermined amino acid sequence A) containing a certain amino acid sequence identity% is calculated as follows: 100 times the fraction X/Y Where X is the number of amino acid residues evaluated as a consistent match by sequence in the comparison program of A and B, and where Y is the total number of amino acid residues in B. It should be understood that when the length of the amino acid sequence A is not equal to the length of the amino acid sequence B, the amino acid sequence identity of A relative to B is the same as the amino acid sequence identity of B relative to A. not equal.

"Binds to", "specifically binds to" a specific polypeptide or an epitope on a specific polypeptide or an antibody "specific for a specific polypeptide or an epitope on a specific polypeptide" is one that binds to a specific polypeptide or a specific polypeptide The epitope does not substantially bind to any other polypeptide or polypeptide epitope antibody. In some embodiments, the activatable masked anti-CTLA4 binding protein described herein (e.g., the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof) binds to irrelevant, non-CTLA4 polypeptides less than that of antibodies and About 10% of the binding of CTLA4, as measured by methods known in the art (for example, enzyme-linked immunosorbent assay (ELISA)). _{In some embodiments, the equilibrium dissociation constant (K D} ) of a binding protein (such as an antibody) that binds to CTLA4 (such as murine CTLA4 and/or human CTLA4) is ≤ 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 to 10 ^-13 M, e.g. 10 ^{- 9} M to 10 ^-13 M).

The term "CTLA4" or "CTLA4 protein" as provided herein includes any of the recombinant or naturally-occurring forms of cytotoxic T lymphocyte-associated protein 4 (CTLA4) or it retains the activity of CTLA4 protein (e.g., when compared with CTLA4) Than at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity) variants or homologs. In some aspects, compared with the naturally occurring CTLA4 polypeptide, the variant or homologue spans the entire sequence or part of the sequence (for example, 50, 100, 150, or 200 consecutive amino acid moieties) with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity. In some embodiments, CTLA4 is a protein, homologue or functional fragment thereof identified by NCBI sequence reference GI: 83700231. In some embodiments, CTLA4 is human CTLA4. In some embodiments, CTLA4 is murine CTLA4.

The "effector function" of an antibody refers to the biological activity attributable to the Fc region of the antibody (for example, the native sequence Fc region or the amino acid sequence variant Fc region) and varies with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors (such as B cell receptors) Down regulation; and B cell activation.

"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to some form of cytotoxicity in which the secreted Ig binds to certain cytotoxic cells (such as natural killer (NK) cells, neutrophils). The Fc receptors (FcR) on leukocytes and macrophages allow these cytotoxic effector cells to specifically bind to target cells carrying antigens and then use cytotoxins to kill the target cells. Antibodies "arm" cytotoxic cells and need to use this mechanism to kill target cells. The native cells (NK cells) used to mediate ADCC only express FcyRIII, while monocytes express FcyRI, FcyRII and FcyRIII. The expression of Fc 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 described herein (for example, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof) does not have the ability to trehalose Fc glycan Engineered or expressed in cells with enhanced ADCC. In order to evaluate the ADCC activity of related molecules, an in vitro ADCC analysis method can be performed, such as described in U.S. Patent No. 5,500,362 or U.S. Patent No. 5,821,337. Effector cells suitable for this type of analysis include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of related molecules can be assessed in vivo, for example in animal models such as those disclosed in Clynes et al., PNAS USA 95:652-656 (1998). Other Fc variants that alter ADCC activity and other antibody properties include those disclosed by: Ghetie et al., Nat Biotech. 15:637-40, 1997; Duncan et al., Nature 332:563-564, 1988; Lund et al. Human, 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 92:11980-11984, 1995; Jefferis et al., Immunol Lett. 44:111-117, 1995; Lund et al., FASEB J9:115-119, 1995; Jefferis et al., Immunol Lett 54:101-104, 1996; Lund et al., J Immunol 157:4963-4969, 1996; Armour 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., Proc. Natl. Acad. Sci. USA 103:4005-4010, 2006; US Patent No. 5,624,821; No. 5,885,573; No. 5,677,425; No. 6,165,745; No. 6,277,375; No. 5,869,046; No. 6,121,022; No. 5,624,821; No. 5,648,260; No. 6,194,551; No. 6,737,056; ; No. 6,277,375; No. 7,335,742; and No. 7, No. 317,091.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc region and variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the Fc region of a human IgG heavy chain is generally defined as an amino acid residue at position Cys226 or extending from Pro230 to its carboxy terminus. The native sequence Fc region of the antibody suitable for the present invention includes human IgG1, IgG2, IgG3 and IgG4.

As used herein, "binding affinity" refers to the strength of the non-covalent interaction between a single binding site of a molecule (such as an antibody) and its binding partner (such as an antigen). In some embodiments, the affinity of the binding protein (eg, antibody) to CTLA4 can generally be represented by the equilibrium dissociation constant (K _D ). Affinity can be measured by common methods known in the art (including the methods described herein).

As used herein, "binding affinity" refers to the binding strength of multiple binding sites of a molecule (such as an antibody) and its binding partner (such as an antigen).

As used herein, an "isolated" nucleic acid molecule encoding an antibody is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule to which the at least one contaminating nucleic acid molecule is usually associated in its production environment. In some embodiments, the isolated nucleic acid is not associated with all components associated with the production environment. In this context, isolated nucleic acid molecules encoding polypeptides and antibodies are in a form different from the form or environment found in nature. Therefore, the isolated nucleic acid molecule is different from the nucleic acid encoding the polypeptide and antibody naturally occurring in the cell herein.

The term "pharmaceutical formulation" refers to a preparation in a form that allows the biological activity of the active ingredient to be effective, and does not contain other components that have unacceptable toxicity to the individual to which the formulation will be administered. Such formulations are sterile.

As used herein, "carrier" includes a pharmaceutically acceptable carrier, excipient or stabilizer, which is non-toxic to cells or mammals exposed to it at the dose and concentration used. Generally, the physiologically acceptable carrier is an aqueous pH buffered solution. 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 white Protein, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, aspartame, 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 relative ions, such as sodium; and/or nonionic surfactants , Such as TWEEN™, polyethylene glycol (PEG) and PLURONICS™.

As used herein, the term "treatment" refers to a clinical intervention designed to change the natural course of the individual or cell being treated during the course of clinical pathology. The desired therapeutic effects include reducing the rate of disease progression, ameliorating or alleviating the condition of the disease, and relieving or improving the prognosis. For example, if one or more of the symptoms associated with the disorder (such as neoplastic disease) is relieved or eliminated, the individual is successfully "treated". For example, if the treatment results in improving the quality of life of the individual suffering from the disease, reducing the dose of other drugs needed to treat the disease, reducing the recurrence rate of the disease, reducing the severity of the disease, delaying the development or progression of the disease, and/or prolonging the individual During the survival period, the individual is successfully "treated".

As used herein, "in combination with" or "in combination with" means that in addition to one form of treatment, another form of treatment is also administered. Therefore, "in combination with" or "in combination with" refers to the administration of one form of treatment before, during, or after the administration of another form of treatment to the individual.

As used herein, the term "prevention" includes providing prevention and treatment regarding the occurrence or recurrence of a disease in an individual. The individual may be susceptible to, susceptible to, or at risk of developing the disease, but has not yet been diagnosed with the disease. In some embodiments, the use of the activatable masked anti-CTLA4 binding protein described herein (e.g., the activatable masked anti-CTLA4 antibody) delays the progression of the disorder.

As used herein, an individual "at risk of developing a disease" may or may not have detectable disease or disease symptoms, and may or may not have shown detectable disease before the treatment methods described herein Or symptoms of disease. "At risk" means that an individual has one or more risk factors, which are measurable parameters related to the development of the disease, as known in this technology. Individuals who have one or more of these risk factors have a higher probability of developing symptoms than individuals who do not have one or more of these risk factors.

"Effective amount" refers to an amount that is at least effective to achieve the desired or indicated effect (including therapeutic or preventive results) at the required dose and time period. The effective amount can be provided in one or more administrations. The "therapeutically effective amount" is at least the minimum concentration required to achieve a measurable improvement in a particular condition. In this context, the therapeutically effective amount may vary according to factors such as the patient's disease state, age, sex, and weight, and the ability of the antibody to cause the desired response in the individual. A therapeutically effective amount can also be an amount in which the therapeutically beneficial effects of the antibody outweigh any toxic or deleterious effects. "Prophylactically effective amount" refers to the amount that is effective to achieve the desired preventive result at the necessary dose and time period. Usually, but not necessarily, the prophylactically effective amount may be less than the therapeutically effective amount because the individual uses the prophylactic dose before or in the early stages of the disease.

"Chronic" administration means that, contrary to the acute mode, the drug is administered in a continuous mode in order to maintain the initial therapeutic effect (activity) for a long time. "Intermittent" administration is a treatment that is not performed continuously without interruption, but is actually performed cyclically.

As used herein, an "individual" is a mammal. For therapeutic purposes, "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, The cat waits. In some embodiments, the individual is a human. II. Activated masked anti- CTLA4 binding protein

In one aspect, an activatable masked cytotoxic T lymphocyte-associated protein 4 (CTLA4) binding protein is provided, which comprises (i) a CTLA4 binding domain; (ii) a CTLA4 binding domain masking peptide (also referred to herein as Is a "masking peptide"); and (iii) a linker, which includes 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 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.

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

In one aspect, provided herein is an activatable masked CTLA4 binding protein that is particularly suitable for treating neoplastic diseases in which CTLA4 plays a role. The activatable masked CTLA4 binding protein as provided herein includes a binding domain that is capable of interacting (eg, binding) with CTLA4 protein expressed on the surface of cells (eg, cancer cells or T cells). In some embodiments, the binding domain is connected to the masking peptide via a linker comprising a cleavable peptide such that the masking peptide prevents the binding of the CTLA4 binding domain to the CTLA4 protein. After cleavage of the cleavable peptide, the masking peptide is released, thereby allowing the binding domain to interact with the CTLA4 protein.

In some embodiments, a masked CTLA4 binding protein (e.g., a masked anti-CTLA4 antibody or antigen-binding fragment thereof) is also provided herein, which comprises (a) a CTLA4 binding protein (e.g., an anti-CTLA4 antibody or an antigen-binding fragment thereof, It includes 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 an antigen-binding fragment thereof, which comprises a first chain and a second chain, and the masking peptide is connected to the first chain of the antibody or its antigen-binding fragment via a linker comprising a cleavable peptide. The amine or carboxyl end of the first or second chain. In some embodiments, the first chain is or includes a heavy chain and the second chain is or includes a light chain; or the first chain is or includes a light chain and the second chain is or includes a heavy chain. In some embodiments, the first chain is or includes the variable region of the heavy chain, and the second chain is or includes the variable region of the light chain; or the first chain is or includes the variable region of the light chain, and the second chain is or Contains the variable region of the heavy chain. In some embodiments, the linker including the cleavable peptide includes a spacer linker, a cleavable peptide, and a spacer linker in an amino-terminal to carboxy-terminal direction. In some embodiments, the C-terminus of the masking peptide is connected to the N-terminus of the linker comprising the cleavable peptide, and the C-terminus of the linker comprising the cleavable peptide is connected to the first chain (e.g., light chain or light chain variable region). ) Of the N terminal. CTLA4 binding protein

As provided herein, the term "CTLA4 binding protein" refers to a polypeptide comprising a CTLA4 binding domain, which can bind to the CTLA4 protein or otherwise exhibit affinity for it. In some embodiments, the CTLA4 binding protein is an anti-CTLA4 antibody or an antigen-binding fragment thereof, a bispecific antibody, an antigen-binding fragment, 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. Therefore, 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 the chimeric antigen receptor that binds to CTLA4.

The term "CTLA4 binding domain" refers to a polypeptide domain expressed in a recombinant manner that can bind to or otherwise exhibit affinity for CTLA4 proteins found in or on cells. The method for determining the degree of binding of CTLA4 binding domain to CTLA4 is well known in the art.

In some embodiments, the antibody is a humanized antibody, chimeric antibody, or 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 antibodies or antigen-binding fragments thereof described herein are antibody fragments (including antigen-binding fragments), such as dAb, Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragments. 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 includes a first chain and a second chain. In some embodiments, the first chain is or includes a heavy chain and the second chain is or includes a light chain; or the first chain is or includes a light chain and the second chain is or includes a heavy chain. In some embodiments, the first chain is or includes the variable region of the heavy chain, and the second chain is or includes the variable region of the light chain; or the first chain is or includes the variable region of the light chain, and the second chain is or Contains the variable region of the heavy chain. In some embodiments, the antibody or antigen-binding fragment thereof comprises a first chain and a second chain (e.g., light chain and 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) CDR-L1, which comprises the amine of SEQ ID NO: 402 or 408 Base acid sequence, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 403 or 409, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 404 or 410; and / Or wherein the heavy chain variable region comprises (i) CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 405 or 411, (ii) CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 406 or 412 Acid sequence, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 407 or 413.

In some embodiments, the antibody or antigen-binding fragment includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 402 , (Ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 403, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 404; and/or wherein the heavy chain is variable The region includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 405, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 406, and (iii) CDR-H3, It includes the amino acid sequence of SEQ ID NO:407. In some embodiments, the antibody or antigen-binding fragment includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 402 , (Ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 403, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 404; and the heavy chain variable region includes ( i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 405, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 406, and (iii) CDR-H3, which includes SEQ ID NO: 406 ID NO: 407 amino acid sequence.

In some embodiments, the antibody or antigen-binding fragment includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 432 , (Ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 433, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 434; and/or wherein the heavy chain is variable The region includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 435, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 436, and (iii) CDR-H3, It includes the amino acid sequence of SEQ ID NO:437. In some embodiments, the antibody or antigen-binding fragment includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 432 , (Ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 433, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 434; and the heavy chain variable region includes ( i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 435, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 436, and (iii) CDR-H3, which includes SEQ ID NO: 436 ID NO: 437 amino acid sequence.

In some embodiments, the antibody or antigen-binding fragment includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 408 , (Ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 409, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 410; and/or wherein the heavy chain is variable The region includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 411, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 412, and (iii) CDR-H3, It includes the amino acid sequence of SEQ ID NO: 413. In some embodiments, the antibody or antigen-binding fragment includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 408 , (Ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 409, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 410; and the heavy chain variable region includes ( i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 411, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 412, and (iii) CDR-H3, which includes SEQ ID NO: 412 ID NO: 413 amino acid sequence.

In some embodiments, the antibody or antigen-binding fragment includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 438 , (Ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 439, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 440; and/or wherein the heavy chain is variable The region includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 441, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 442, and (iii) CDR-H3, It includes the amino acid sequence of SEQ ID NO:443. In some embodiments, the antibody or antigen-binding fragment includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 438 , (Ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 439, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 440; and the heavy chain variable region includes ( i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 441, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 442, and (iii) CDR-H3, which includes SEQ ID NO: 442 ID NO: 443 amino acid sequence.

In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region, which comprises the amino acid sequence of SEQ ID NO: 232 or about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of amino acid sequences; and/or include heavy chain variable regions, which include Have or have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% with the amino acid sequence of SEQ ID NO: 233 , 98%, 99% or 100% homology of amino acid sequences. In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region, which comprises the amino acid sequence of SEQ ID NO: 232 or about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of amino acid sequences; and comprising the heavy chain variable region, which comprises the same as SEQ The amino acid sequence of ID NO: 233 has or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% or 100% homology of amino acid sequences. In some embodiments, the antibody or antigen-binding fragment includes a light chain variable region, which includes the amino acid sequence of SEQ ID NO: 232; and/or a heavy chain variable region, which includes the amino acid sequence of SEQ ID NO: 233 Acid sequence. In some embodiments, the antibody or antigen-binding fragment includes a light chain variable region, which includes the amino acid sequence of SEQ ID NO: 232; and a heavy chain variable region, which includes the amino acid sequence of SEQ ID NO: 233 .

In some embodiments, the antibody or antigen-binding fragment thereof includes CDR-L1, CDR-L2, and CDR-L3 contained in a VL domain that includes the amino acid sequence of SEQ ID NO: 321, and includes The CDR-H1, CDR-H2, and CDR-H3 contained in the VH domain include the amino acid sequence of SEQ ID NO: 323. In some embodiments, the antibody or antigen-binding fragment thereof includes CDR-L1, CDR-L2, and CDR-L3 contained in a VL domain that includes the amino acid sequence of SEQ ID NO: 322, and includes The CDR-H1, CDR-H2, and CDR-H3 contained in the VH domain include 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 an amino acid sequence selected from SEQ ID NO: 321 or 322; and/or a heavy chain variable region comprising a variable region selected from SEQ ID NO: 323 or 324 amino acid sequence. In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region, which comprises the amino acid sequence of SEQ ID NO: 321 or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of amino acid sequences; and/or include heavy chain variable regions, which include Have or have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% with the amino acid sequence of SEQ ID NO: 323 , 98%, 99% or 100% homology of amino acid sequences. In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region, which comprises the amino acid sequence of SEQ ID NO: 321 or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of amino acid sequences; and comprising the heavy chain variable region, which comprises the same as SEQ The amino acid sequence of ID NO: 323 has or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% or 100% homology of amino acid sequences. In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region, which comprises the amino acid sequence of SEQ ID NO: 322 or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of amino acid sequences; and/or include heavy chain variable regions, which include The amino acid sequence of SEQ ID NO: 324 has or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% or 100% homology of amino acid sequences. In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region, which comprises the amino acid sequence of SEQ ID NO: 322 or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of amino acid sequences; and comprising the heavy chain variable region, which comprises the same as SEQ The amino acid sequence of ID NO: 324 has or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% or 100% homology of amino acid sequences. In some embodiments, the antibody or antigen-binding fragment includes a light chain variable region, which includes the amino acid sequence of SEQ ID NO: 322; and/or a heavy chain variable region, which includes the amine of SEQ ID NO: 324 Base acid sequence. In some embodiments, the antibody or antigen-binding fragment includes a light chain variable region, which includes the amino acid sequence of SEQ ID NO: 322; and includes a heavy chain variable region, which includes the amino acid of SEQ ID NO: 324 sequence.

In some embodiments, the antibody or antigen-binding fragment comprises a light chain, which comprises the amino acid sequence of SEQ ID NO: 334 or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology of amino acid sequences; and/or include a heavy chain, which includes the same as SEQ ID NO: 421 The amino acid sequence has or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% Or 100% homologous amino acid sequence. In some embodiments, the antibody or antigen-binding fragment comprises a light chain, which comprises the amino acid sequence of SEQ ID NO: 334 or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of the amino acid sequence; and comprising the heavy chain, which comprises the amine with SEQ ID NO: 421 The base acid sequence has or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. % Homology of amino acid sequence. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain, which comprises the amino acid sequence of SEQ ID NO: 334; and/or comprises a heavy chain, which comprises the amino acid sequence of SEQ ID NO: 421. In some embodiments, the antibody or antigen-binding fragment thereof includes a light chain, which includes the amino acid sequence of SEQ ID NO: 334; and a heavy chain, which includes the amino acid sequence of SEQ ID NO: 421.

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 a light chain selected from SEQ ID NO: 319 or 320 The amino acid sequence. 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 a light chain selected from SEQ ID NO: 366-380 , 421 and 478 amino acid sequences. 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 a light chain selected from SEQ ID NO : 366 or 380-397 amino acid sequence. 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 the IgG1 isotype, which includes amino acid substitutions that enhance the effector function as described herein.

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

In some embodiments of the bispecific antibody, the light chain includes CDR-L1, CDR-L2, and CDR-L3 contained in a VL domain that includes the amino acid sequence of SEQ ID NO: 321, and a heavy The chain includes CDR-H1, CDR-H2, and CDR-H3 contained in the VH domain, which includes the amino acid sequence of SEQ ID NO: 323. In some embodiments of the bispecific antibody, the light chain includes CDR-L1, CDR-L2, and CDR-L3 contained in a VL domain that includes the amino acid sequence of SEQ ID NO: 322, and a heavy The chain includes CDR-H1, CDR-H2, and CDR-H3 contained in the VH domain, which includes the amino acid sequence of SEQ ID NO:324.

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

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

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

In some embodiments, the CTLA4 binding domain comprises a first chain and a second chain that bind to CTLA4, such as as part of a ligand binding domain for a chimeric receptor. In some embodiments of chimeric receptors, 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 includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 402 or 408, and (ii) CDR-L2, which includes SEQ ID NO: 403 or 409 amino acid sequence, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 404 or 410; and/or wherein the second chain includes (i) CDR-H1, which includes SEQ The amino acid sequence of ID NO: 405 or 411, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 406 or 412, and (iii) CDR-H3, which includes SEQ ID NO: 407 or The amino acid sequence of 413. In some embodiments of the chimeric receptor, the first chain includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 402, and (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 403. Amino acid sequence, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 404; and the second chain, which includes (i) CDR-H1, which includes the amino group of SEQ ID NO: 405 The acid sequence, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 406, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 407. In some embodiments of the chimeric receptor, the first chain includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 432, and (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 433. Amino acid sequence, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 434; and the second chain includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 435 , (Ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 436, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 437. In some embodiments of the chimeric receptor, the first chain includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 408, and (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 409 Amino acid sequence, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 410; and the second chain includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 411 , (Ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 412, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 413. In some embodiments of the chimeric receptor, the first chain includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 438, and (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 439. Amino acid sequence, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 440; and the second chain includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 441 , (Ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 442, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 443.

In some embodiments of the chimeric receptor, the first chain includes CDR-L1, CDR-L2, and CDR-L3 contained in a VL domain that includes the amino acid sequence of SEQ ID NO: 321, and The second chain includes CDR-H1, CDR-H2, and CDR-H3 contained in the VH domain, which includes the amino acid sequence of SEQ ID NO: 323. In some embodiments of the chimeric receptor, the first chain includes CDR-L1, CDR-L2, and CDR-L3 contained in a VL domain that includes the amino acid sequence of SEQ ID NO: 322, and The second chain includes CDR-H1, CDR-H2, and CDR-H3 contained in the VH domain, which includes the amino acid sequence of SEQ ID NO:324.

In some embodiments of the chimeric receptor, the first chain comprises the amino acid sequence of SEQ ID NO: 232 or about 70%, 75%, 80%, 85%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of the amino acid sequence; and/or the second chain contains the amino acid sequence of SEQ ID NO: 233 The sequence has or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical Source amino acid sequence. In some embodiments of the chimeric receptor, the first chain contains or has about 70%, 75%, 80%, 85%, 90%, 91%, 92% of the amino acid sequence of SEQ ID NO: 321 , 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of the amino acid sequence; and/or the second strand contains the amino acid sequence of SEQ ID NO: 323 The sequence has or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical Source amino acid sequence. In some embodiments of the chimeric receptor, the first chain includes the amino acid sequence of SEQ ID NO: 322 or about 70%, 75%, 80%, 85%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of the amino acid sequence; and/or the second strand contains the amino acid sequence of SEQ ID NO: 324 The sequence has or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical Source amino acid sequence. In some embodiments of the chimeric receptor, the first strand includes the amino acid sequence of SEQ ID NO: 232; and/or the second strand includes the amino acid sequence of SEQ ID NO: 233. In some embodiments of the chimeric receptor, the first strand includes an amino acid sequence selected from SEQ ID NO: 321 or 322; and/or the second strand includes an amino acid selected from SEQ ID NO: 323 or 324 sequence. In some embodiments of the chimeric receptor, the first strand includes the amino acid sequence of SEQ ID NO: 322, and the second strand includes the amino acid sequence of SEQ ID NO: 324. Masking peptide

The CTLA4 binding domain masking peptide (also referred to as "masking peptide") as provided herein refers to a peptide capable of binding to the CTLA4 binding domain or exhibiting affinity for it in other ways. When binding to the CTLA4 binding domain, the masking peptide blocks, inhibits, inhibits (eg reduces) or otherwise prevents (eg masks) the activity or binding of the CTLA4 binding domain to its cognate receptor or protein (ie, CTLA4) . The method for determining the degree of binding of the CTLA4 binding domain to the CTLA4 protein is well known in the art.

In an embodiment, the length of the masking peptide is at least 4 amino acids. In some embodiments, the masking peptide is a linear peptide. In some embodiments, linear peptides are 4-mer to 24-mer. In the examples, the masking peptide is a cyclic peptide. In the embodiment, the cyclic peptide is a 3-mer to a 12-mer, as defined by the number of amino acids between two cysteines. In the examples, the cyclic peptides are 3-mer to 20-mer. When the masking peptide is a cyclized peptide, the cyclized peptide is formed by a disulfide bond connecting two cysteine amino acid residues. In some embodiments, the cysteine amino acid residue is a terminal cysteine (ie, at or near the N-terminus and/or C-terminus of the masking peptide). In the embodiment, the disulfide bond connects the N-terminal cysteine and the C-terminal cysteine.

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

In some embodiments, the masking peptide is connected to the N-terminus of the light chain or the 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 connected 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 connected to the N-terminus of the light chain variable region or the 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 connected to the N-terminus of the light 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 connected to the C-terminus of the light chain 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 connected to the C-terminus of the light chain variable region or the 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 comprises an amino acid sequence selected from SEQ ID NO: 1-46 having or having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% %, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of amino acid sequences. Therefore, in an embodiment, the masking peptide comprises about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of amino acid sequences: CNLIVEGHC (SEQ ID NO: 1), MQTRCKEYPRWCEHWL (SEQ ID NO: 2), CKHAPYALC (SEQ ID NO: 3), CPFPAKILC (SEQ ID NO: 4), CPGKGLPSC (SEQ ID NO: 5), NWLGEWLPPGKV (SEQ ID NO: 6), QFIECPNFPRQCPGKN (SEQ ID NO: 7), VRQQCSLNPGRCPYLV (SEQ ID NO: 8), VWQECHTAPQLCPGKI (SEQ ID NO :9), DSYTCRGPTWMCAGNM (SEQ ID NO: 10), FNHDCSGHWMRCLDQQ (SEQ ID NO: 11), NKSPCRPKMVACYGIL (SEQ ID NO: 12), PTPQCWNQYYECWIPS (SEQ ID NO: 13), SQKCPWTKETCMHYM (SEQ ID NO: 14), WHLSMYPK (SEQ ID NO: 15), WHTDGFYTRLPA (SEQ ID NO: 16), CIHAPYAKC (SEQ ID NO: 17), CPAKIGQEC (SEQ ID NO: 18), CPFPALELC (SEQ ID NO: 19), CTKPAKALC (SEQ ID NO: 20), DTATCYTTTGWCEGMV (SEQ ID NO: 21), NSDNCGPAKSTCMYND (SEQ ID NO: 22), PPGKCTQPHRCPPLN (SEQ ID NO: 23), DDPVCWDSNPTCQTIA (SEQ ID NO: 24), ISDQCSVLFLSCNTRV (SEQ ID NO: 25), ACHFPHPEGC ( SEQ ID NO: 26), CLPPFPTKC (SEQ ID NO: 27), CPDHVFPKC (SEQ ID NO: 28), CWLPKPDMC (SEQ ID NO: 29), CWSWPSKAC (SEQ ID NO: 30), CYPFGKYEC (SEQ ID NO: 31 ), ALTPAKWLPADD (SEQ ID NO: 32), DD KECDWMHFACTGPQ (SEQ ID NO: 33), DEMKCAWSLEMCVRTS (SEQ ID NO: 34), DPILCPNTRMSCDNQT (SEQ ID NO: 35), GNALYDSPGTML (SEQ ID NO: 36), KNYECREVMPPCEPNT (SEQ ID NO: 37), NSYTSPYWLPDS (SEQ ID NO :38), SLTPPYWIPREW (SEQ ID NO: 39), SPLTPHDRPSFL (SEQ ID NO: 40), TADVFSSSRYTR (SEQ ID NO: 41), TDLQCPPSSPICQIEH (SEQ ID NO: 42), TKCHCDGNCVMCYQMQ (SEQ ID NO: 43), TLAYETPLLWLP (SEQ ID NO: 44), TNWHCNNDGSSCNVRA (SEQ ID NO: 45) or CNLIVQGHC (SEQ ID NO: 46).

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

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

In some embodiments, the masking peptide comprises an amino acid sequence having about 70% homology with an amino acid sequence selected from SEQ ID NO: 1-46. For example, the masking peptide includes an amino acid sequence having about 70% homology with the amino acid sequence of SEQ ID NO:1.

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

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

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

In some embodiments, at least one amino acid but no more than 20 amino acids are directly connected to the N-terminus of the masking peptide. In some embodiments, at least one amino acid but no more than 30 amino acids are directly connected to the N-terminus of the masking peptide. In some embodiments, at least one amino acid but no more than 40 amino acids are directly connected to the N-terminus of the masking peptide. In some embodiments, at least one amino acid but no more than 50 amino acids are directly connected to the N-terminus of the masking peptide. In some embodiments, the at least one amino acid directly connected 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 connected to the N-terminus of the masking peptide is a detectable label. In some embodiments, the at least one amino acid directly linked to the N-terminus of the masking peptide is YPYDVPDYA (SEQ ID NO: 398), DYKDDDDK (SEQ ID NO: 399), EQKLISEEDL (SEQ ID NO: 400) or GLNDIFEAQKIEWHE (SEQ ID NO: 401). In some embodiments, at least one amino acid but no more than 50 amino acids is directly connected to the N-terminus of the masking peptide, the masking peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-46 Have or have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology The amino acid sequence of sex. In some embodiments, at least one amino acid but no more than 50 amino acids is directly connected to the N-terminus of a masking peptide selected from the group consisting of SEQ ID NO: 1-46. In some embodiments, at least one amino acid but no more than 50 amino acids is directly connected to the N-terminus of the masking peptide selected from the group consisting of SEQ ID NO: 1-46, wherein the at least one amino acid is propylamine Acid (A) or Glycine-Alanine (GA). In some embodiments, at least one amino acid but no more than 50 amino acids is directly connected to the N-terminus of the masking peptide selected from the group consisting of SEQ ID NO: 1-46, wherein the at least one amino acid is propylamine Acid (A). Linker

In some embodiments, the activatable masked anti-CTLA4 binding protein comprises a linker, such as a spacer linker. In some embodiments, the activatable masked anti-CTLA4 binding protein comprises more than one linker, such as 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, "linker comprising a cleavable peptide" refers to a linker that can be cleaved enzymatically, which is covalently bonded to the CTLA4 binding domain and covalently bonded to the masking peptide. In some embodiments, the linker comprising the cleavable peptide is expressed recombinantly. In some embodiments, the linker comprising the cleavable peptide is a linker formed by reacting a functional (reactive) group connected to the linker with a masking peptide using, for example, a conjugation chemical reaction. In some embodiments, the linker comprising the cleavable peptide is a linker formed by reacting a functional (reactive) group attached to the linker with the CTLA4 binding domain using, for example, a binding chemical reaction. In some embodiments, a linker comprising a cleavable peptide connects 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 connects the masking peptide to the C-terminus of the CTLA4 binding domain (e.g., the C-terminus of the light chain).

In some embodiments, the linker comprising the cleavable peptide is fused with the masking peptide, such as when the nucleic acid encodes the linker and the masking peptide and is expressed by the cell as an amino acid sequence encoding the linker and the masking peptide. In some embodiments, the linker comprising the cleavable peptide is fused to the CTLA4 binding domain, such as when the nucleic acid encodes the linker and the CTLA4 binding domain and is expressed by the cell in the form of amino acid sequences encoding the linker and the CTLA4 binding domain. In some embodiments, a linker comprising a cleavable peptide connects 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 connects the masking peptide to the C-terminus of the CTLA4 binding domain (e.g., the C-terminus of the light chain).

In some embodiments, the linker comprising the cleavable peptide is a flexible linker, which includes one or more glycine residues, serine residues, alanine residues, histidine residues, and/ Or proline residues. In some embodiments, the linker comprising the cleavable peptide contains a spacer linker directly connected to the N-terminus and/or C-terminus of the cleavable peptide. In some embodiments, the spacer linker includes one or more glycine residues, serine residues, alanine residues, histidine residues, and/or proline residues. In some embodiments, the linker including the cleavable peptide includes a spacer linker and a cleavable peptide. In some embodiments, the linker comprising the cleavable peptide includes a first spacer linker, a cleavable peptide, and a second spacer linker. Therefore, in some embodiments, a masked CTLA4 binding protein (eg, a masked anti-CTLA4 antibody or antigen-binding fragment thereof) comprises a linker comprising a cleavable peptide, which comprises a spacer connected to the N-terminus of the cleavable peptide A linker (for example, the first spacer linker, or spacer linker 1), and a spacer linker (for example, the second spacer linker, or spacer linker 2) connected to the C-terminus of the cleavable peptide, wherein each spacer linker Contains an amino acid sequence selected from the group consisting of SEQ ID NO: 89-112 and 415-420. In some embodiments, the C-terminus of the linker containing the cleavable peptide is connected to the light chain or light chain variable domain of the anti-CTLA4 antibody or antigen-binding fragment thereof, and the N-terminus of the linker containing the cleavable peptide is connected to the masking Peptide. In some embodiments, the C-terminus of the linker containing the cleavable peptide is connected to the heavy chain or heavy chain variable domain of the anti-CTLA4 antibody or antigen-binding fragment thereof, and the N-terminus of the linker containing the cleavable peptide is connected to the masking Peptide. In some embodiments, the N-terminus of the linker comprising the cleavable peptide is connected 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 connected to the masking Peptide. In some embodiments, the N-terminus of the linker containing the cleavable peptide is connected to the heavy chain or heavy chain variable domain of the anti-CTLA4 antibody or antigen-binding fragment thereof, and the C-terminus of the linker containing the cleavable peptide is connected to the masking Peptide.

In some embodiments, the spacer linker comprises an amino acid sequence selected from SEQ ID NO: 89-112 and 415-420. In some embodiments, the spacer linker is directly connected to the N-terminus of the cleavable peptide and includes an amino acid sequence selected from SEQ ID NO: 89-100. In some embodiments, the spacer linker is directly connected to the C-terminus of the cleavable peptide and includes an amino acid sequence selected from SEQ ID NO: 101-112 and 415-420. In some embodiments, the masking peptide described herein is directly linked to the N-terminus of the spacer linker. Therefore, in some embodiments, the linker comprising the cleavable peptide in the N-terminal to C-terminal direction comprises: 1) a spacer linker (for example, comprising an amino acid sequence selected from SEQ ID NO: 89-112 and 415-420 The spacer linker of the amino acid sequence), 2) a cleavable peptide, such as the cleavable peptide described herein (for example, comprising an amino acid selected from SEQ ID NO: 47-88, 464-469 and 479-508) The cleavable peptide of the amino acid sequence of the sequence), and 3) the spacer linker (for example, the spacer linker including the amino acid sequence selected from the amino acid sequence of SEQ ID NO: 89-112 and 415-420).

In some embodiments, the linker comprising the cleavable peptide in the N-terminal to C-terminal direction comprises: 1) a spacer linker, which comprises an amino acid selected from the group consisting of SEQ ID NO: 89-112 and 415-420 Sequence; 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 a group selected from SEQ ID NO : The amino acid sequence of the group consisting of 89-112 and 415-420.

In some embodiments, the linker comprising the cleavable peptide comprises in the N-terminal to C-terminal direction: 1) a spacer linker, which comprises the amino acid sequence of SEQ ID NO: 420; 2) a cleavable peptide, which comprises SEQ The amino acid sequence of ID NO: 50; and 3) a spacer linker, which includes the amino acid sequence of SEQ ID NO: 102.

In some embodiments, the linker comprising the cleavable peptide comprises in the N-terminal to C-terminal direction: 1) a spacer linker, which comprises the amino acid sequence of SEQ ID NO: 96; 2) a cleavable peptide, which comprises SEQ The amino acid sequence of ID NO: 86; and 3) a spacer linker, which includes the amino acid sequence of SEQ ID NO: 102.

In some embodiments, the linker comprising the cleavable peptide comprises in the N-terminal to C-terminal direction: 1) a spacer linker, which comprises the amino acid sequence of SEQ ID NO: 415; 2) a cleavable peptide, which comprises SEQ The amino acid sequence of ID NO: 86; and 3) a spacer linker, which includes the amino acid sequence of SEQ ID NO: 102.

In some embodiments, the linker comprising the cleavable peptide comprises in the N-terminal to C-terminal direction: 1) a spacer linker, which comprises the amino acid sequence of SEQ ID NO: 416; 2) a cleavable peptide, which comprises SEQ The amino acid sequence of ID NO: 47; and 3) a spacer linker, which includes the amino acid sequence of SEQ ID NO: 102.

In some embodiments, the linker comprising the cleavable peptide comprises in the N-terminal to C-terminal direction: 1) a spacer linker, which comprises the amino acid sequence of SEQ ID NO: 417; 2) a cleavable peptide, which comprises SEQ The amino acid sequence of ID NO: 57; and 3) a spacer linker, which includes the amino acid sequence of SEQ ID NO: 102.

In some embodiments, the linker comprising the cleavable peptide comprises in the N-terminal to C-terminal direction: 1) a spacer linker, which comprises the amino acid sequence of SEQ ID NO: 418; 2) a cleavable peptide, which comprises SEQ The amino acid sequence of ID NO: 48; and 3) a spacer linker, which includes the amino acid sequence of SEQ ID NO: 102.

In some embodiments, the linker comprising the cleavable peptide comprises in the N-terminal to C-terminal direction: 1) a spacer linker, which comprises the amino acid sequence of SEQ ID NO: 417; 2) a cleavable peptide, which comprises SEQ The amino acid sequence of ID NO: 72; and 3) a spacer linker, which includes the amino acid sequence of SEQ ID NO: 102.

In some embodiments, the linker comprising the cleavable peptide comprises in the N-terminal to C-terminal direction: 1) a spacer linker, which comprises the amino acid sequence of SEQ ID NO: 418; 2) a cleavable peptide, which comprises SEQ The amino acid sequence of ID NO: 51; and 3) a spacer linker, which includes the amino acid sequence of SEQ ID NO: 102.

In some embodiments, the linker comprising the cleavable peptide comprises in the N-terminal to C-terminal direction: 1) a spacer linker, which comprises the amino acid sequence of SEQ ID NO: 419; 2) a cleavable peptide, which comprises SEQ The amino acid sequence of ID NO: 54; and 3) a spacer linker, which includes the amino acid sequence of SEQ ID NO: 102.

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

The linker can be combined with the masking peptide and/or CTLA4 binding protein by various methods well known in the art. The terms "binding" and "binding chemical reaction" refer to reactions with known reactive groups that are carried out under relatively mild conditions. These reactions include, but are not limited to, nucleophilic substitution (such as the reaction of amines and alcohols with acyl halides, active esters), electrophilic substitution (such as enamine reactions), and multiple bonds with carbon-carbon and carbon-heteroatoms Addition (for example, Michael reaction, Diels-Alder addition). These and other applicable reactions are discussed in, for example, March, Advanced Organic Chemistry, 3rd Edition, 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, Volume 198, American Chemical Society, Washington, DC, 1982.

Reactive functional groups suitable for the binding chemical reaction herein include, for example: (a) carboxyl and various derivatives thereof, including (but not limited to) N-hydroxysuccinimide and N-hydroxybenzotriazole ester , Acid halides, amidazoles, thioesters, p-nitrophenyl esters, alkyl esters, alkenyl esters, alkynyl esters and aromatic esters; (b) hydroxyl groups that can be converted into esters, ethers, aldehydes, etc.; (c) Haloalkyl, in which the halide ion can be subsequently replaced by a nucleophilic group (such as an amine, carboxylate anion, thiol anion, carbanion or alkoxide ion), thereby causing a new position at the halogen atom Covalent linkage of groups; (d) dienophilic groups, which can participate in Diels-Alder reactions, such as maleimide groups; (e) aldehyde or ketone groups, making it possible to pass through Formation of carbonyl derivatives (such as imines, hydrazones, hemicarbhydrazones or oximes) or subsequent derivatization via mechanisms such as Grignard addition or alkyl lithium addition; (f) for subsequent derivatization with amines The reacted sulfonyl halide groups, for example, to form sulfonamides; (g) thiol groups, which can be converted into disulfides, react with sulfonyl halides, or bond to metals, such as gold; (h) Amine or sulfhydryl group, which can be, for example, acylated, alkylated or oxidized; (i) olefin, which can undergo, for example, cycloaddition, acylation, Michael addition, etc.; (j) epoxide, which can be combined with For example, the reaction of amines and hydroxyl compounds; (k) amino phosphate and other standard functional groups suitable for nucleic acid synthesis; (i) metal silica bonding; and (l) metals, which are bonded to reactive phosphorus groups ( For example, phosphine) to form, for example, a phosphodiester bond.

The reactive functional group can be selected so that it does not participate in or interfere with the chemical stability of the composition described herein. Alternatively, the presence of a protecting group can protect the reactive functional group from participating in the cross-linking reaction.

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, when expressed recombinantly by a host cell, the nucleic acid can be engineered to encode a linker with a masking peptide and/or CTLA4 binding protein to produce a fusion protein. Cleavable peptide

In some embodiments, the masked CTLA4 binding protein provided herein (eg, a masked anti-CTLA4 antibody or antigen binding fragment thereof) comprises a cleavable peptide. In some embodiments, the cleavable peptide is contained in a linker comprising the cleavable peptide. The linker comprising a cleavable peptide provided herein may include a protease cleavage site within the cleavable peptide. As used herein, "cleavage site" refers to the cleavage of a part of the linker (for example, the linker comprising a cleavable peptide as described above) found in the CTLA4 binding protein described herein Recognizable site. Therefore, the cleavage site can be found in the sequence of the cleavable peptide as described herein (including the examples thereof). In some embodiments, the cleavage site is an amino acid sequence that is recognized and cleaved by the cleavage agent. Exemplary lysing agents include proteins, enzymes, DNases, RNases, metals, acids, and bases. The cleavable peptide can be any peptide that includes a protease cleavage site. Exemplary cleavable peptides are shown in Table 1. Table 1. Representative cleavable peptides Exemplary cleavable peptide MPYDLYHP (SEQ ID NO:47) RAAAVKSP (SEQ ID NO: 72) IYDQKT (SEQ ID NO: 481) GGIGQLTA (SEQ ID NO: 48) DLLAVVAAS (SEQ ID NO: 73) AHNYKT (SEQ ID NO:482) DLGRFQTF (SEQ ID NO: 49) VQTVTWPD (SEQ ID NO: 74) MMDQAN (SEQ ID NO:483) DSGGFMLT (SEQ ID NO: 50) AIPMSIPP (SEQ ID NO: 75) MLGEFVSE (SEQ ID NO: 484) TSVLMAAP (SEQ ID NO: 51) GYEVHHQK (SEQ ID NO: 76) GLVALRGA (SEQ ID NO:485) TSEFVFAPDQ (SEQ ID NO: 52) VHHQKLVF (SEQ ID NO: 77) KEHKYKAE (SEQ ID NO:486) KLVLPVLP (SEQ ID NO: 53) IRRVSYSF (SEQ ID NO: 78) LAQAVRSS (SEQ ID NO:487) KPILFFRL (SEQ ID NO: 54) MPYDLYHPILFFRL (SEQ ID NO:79) LGGSGRSNAQVRLE (SEQ ID NO: 488) ANQLKG (SEQ ID NO: 55) GGIGQLTSVLMAAP (SEQ ID NO: 80) LGGSGRKASLSLE (SEQ ID NO: 489) QSQLKE (SEQ ID NO: 56) DSGGFMLTLVLPVLP (SEQ ID NO: 81) SGRIGFLRTA (SEQ ID NO: 490) HEQLTV (SEQ ID NO: 57) TSEFVFAPDLGRFQTF (SEQ ID NO: 82) SGAIGFLRTA (SEQ ID NO:491) PANLVAPDP (SEQ ID NO: 58) TSTSGRSANPR (SEQ ID NO: 83) RPARSGRSAGGSVA (SEQ ID NO:492) PAPGVYPGP (SEQ ID NO: 59) TSTSGRSANPG (SEQ ID NO: 84) VTGRGDSPASS (SEQ ID NO:493) APAGLIVPYN (SEQ ID NO: 60) TSTSGRSANPH (SEQ ID NO: 85) PRFKIIGG (SEQ ID NO:494) PQALVA (SEQ ID NO: 61) VPLSLY (SEQ ID NO: 86) LSGRIGFLRTA (SEQ ID NO:495) VGNLNF (SEQ ID NO: 62) TSASGASASAA (SEQ ID NO: 87) LSGRSNAGGIGQLTA (SEQ ID NO: 496) VANLLYE (SEQ ID NO: 63) PSSPGGGSSP (SEQ ID NO: 88) LSGRSNAVPLSLY (SEQ ID NO: 497) VYNLMD (SEQ ID NO: 64) ISSGLLSGRSDNH (SEQ ID NO:464) LSGRSNADSGGFMLT (SEQ ID NO: 498) TFNIKQ (SEQ ID NO: 65) AVGLLAPPGGLSGRSDNH (SEQ ID NO:465) LSGRSNAHEQLTA (SEQ ID NO: 499) DLWKLLP (SEQ ID NO: 66) VPLSLYSG (SEQ ID NO: 466) LSGRSNARAAAVKSP (SEQ ID NO: 500) PGSTKRA (SEQ ID NO: 67) RQARVVG (SEQ ID NO:467) LSGRSNATSVLMAAP (SEQ ID NO:501) QQYRALKS (SEQ ID NO: 68) LSGRSNAMPYDLYHP (SEQ ID NO: 468) VPLSLYLSGRSNA (SEQ ID NO:502) YVPRAVL (SEQ ID NO: 69) MPYDLYHPRQARVVG (SEQ ID NO: 469) DSGGFMLTLSGRSNA (SEQ ID NO:503) GVNKWPT (SEQ ID NO: 70) IPESLRAG (SEQ ID NO:479) GGIGQLTALSGRSNA (SEQ ID NO:504) LAQAVRSS (SEQ ID NO: 71) IPVSLRSG (SEQ ID NO:480) MPYDLYHPLSGRSNA (SEQ ID NO:505) HEQLTVLSGRSNA (SEQ ID NO:506) RAAAVKSPLSGRSNA (SEQ ID NO:507) TSVLMAAPLSGRSNA (SEQ ID NO:508)

Therefore, 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 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, the cleavable peptide comprises the amino acid sequence of SEQ ID NO:47. In some embodiments, the cleavable peptide comprises the amino acid sequence of SEQ ID NO:57. In some embodiments, the cleavable peptide comprises the amino acid sequence of SEQ ID NO:48. In some embodiments, the cleavable peptide comprises the amino acid sequence of SEQ ID NO:72. In some embodiments, the cleavable peptide comprises the amino acid sequence of SEQ ID NO:51. In some embodiments, the cleavable peptide comprises the amino acid sequence of SEQ ID NO:54.

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, and its performance is specific to tumor cells or their tumor cell environment. In some embodiments, the protease cleavage site is a cleavage site recognized by one or more enzymes selected from the group consisting of 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, ADAMTSADA, SLADA8, ADAMTSADAMTS1, ADAMTS5, ADASL , ADAMTSL3, ABHD17C, ADAMTSL5, ASTL, BMP1, CELA1, CELA2A, CELA2B, CELA3A, CELA3B, ADAM10, ADAM15, ADAM17, ADAM9, ADAMTS4, CTSE, CTSF, ADAMTSL4, CMA1, CTRB1, CTRC, CTSO, CTSWl, CTSA , CTSB, CTSC, CTSD, ESP1, CTSG, CTSH, GZMA, GZMB, GZMH, CTSK, GZMM, CTSL, CTSS, CTSV, CTSZ, HTRA4, KLK10, KLK11, KLK13, KLK14, KLK2, KLK4, DPP4, KL7K6, KLK6 , 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, PABEX1, TINPR1, TIN 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.

In the embodiment, the protease cleavage site is a matrix metalloprotease (MMP) cleavage site, a metalloprotease cleavage site containing disintegrin and a metalloprotease domain (ADAM), and a prostate specific antigen (PSA) protease cleavage site , Urokinase-type plasminogen activator (uPA) protease cleavage site, membrane serine protease 1 (MT-SP1) protease cleavage site, interstitial protease protease cleavage site (ST14) or podrin protease Cleavage site. In an embodiment, the matrix metalloprotease (MMP) cleavage site is the MMP9 cleavage site, the MMP13 cleavage site, or the MMP2 cleavage site. In an embodiment, the metalloprotease cleavage site containing disintegrin and metalloprotease domain (ADAM) is an ADAM9 metalloprotease cleavage site, an ADAM10 metalloprotease cleavage site or an ADAM17 metalloprotease cleavage site. The protease cleavage site can be specified by a specific amino acid sequence.

In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of 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, ADAMTS1, ADAMTSSL, ADAMTSSL, ADAMTSSL, ADAMTS1, ADAMTSSL5 , 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, KECEKB1, ECEKB1 , 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, TMPR The 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.

In the embodiment, the cleavable peptide is a 5-mer (that is, a peptide with a length of 5 amino acids), a 6-mer (that is, a peptide with a length of 6 amino acids), and a 7-mer (also That is, a peptide with a length of 7 amino acids), an 8-mer (that is, a peptide with a length of 8 amino acids), a 9-mer (that is, a peptide with a length of 9 amino acids), 10 Polymers (that is, peptides of 10 amino acids in length), 11-mers (that is, peptides of 11 amino acids in length), 12-mers (that is, peptides of 12 amino acids in length) Peptide) or 13-mer (that is, a peptide with a length of 13 amino acids).

Therefore, in some embodiments, the masking peptide and the linker comprising the cleavable peptide comprise in the N-terminal to C-terminal direction: 1) the masking peptide (for example, an amine comprising an amino acid sequence selected from SEQ ID NO: 1-46 Base acid sequence masking peptide), 2) spacer linker (for example, spacer linker comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 89-112 and 415-420), 3) cleavable peptide , Such as the cleavable peptide described herein (for example, a cleavable peptide comprising an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 47-88, 464-469 and 479-508), and 4) spacer Linker (for example, a spacer linker including an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 89-112 and 415-420). In some embodiments, at least one amino acid but no more than 20 amino acids are directly connected to the N-terminus of the masking peptide. In some embodiments, at least one amino acid but no more than 30 amino acids are directly connected to the N-terminus of the masking peptide. In some embodiments, at least one amino acid but no more than 40 amino acids are directly connected to the N-terminus of the masking peptide. In some embodiments, at least one amino acid but no more than 50 amino acids are directly connected to the N-terminus of the masking peptide. In some embodiments, the at least one amino acid directly connected 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 connected to the N-terminus of the masking peptide is a detectable label. In some embodiments, the at least one amino acid directly connected to the N-terminus of the masking peptide is YPYDVPDYA (SEQ ID NO: 398), DYKDDDDK (SEQ ID NO: 399), EQKLISEEDL (SEQ ID NO: 400) or GLNDIFEAQKIEWHE (SEQ ID NO: 401).

In some embodiments, the activatable masked anti-CTLA4 binding protein described herein comprises a peptide comprising a masking peptide, a spacer linker and a cleavable peptide, wherein the peptide comprises a peptide selected from SEQ ID NO: 113-231 The amino acid sequence. In some embodiments, the activatable masked anti-CTLA4 binding protein described herein comprises a peptide comprising a masking peptide, a spacer linker and a cleavable peptide, wherein the peptide comprises a peptide selected from SEQ ID NO: 113-193 The amino acid sequence. In some embodiments, the activatable masked anti-CTLA4 binding protein described herein comprises a peptide comprising a masking peptide, a spacer linker, and a cleavable peptide, wherein the peptide comprises a peptide selected from SEQ ID NO: 194-206 The amino acid sequence. In some embodiments, the activatable masked anti-CTLA4 binding protein described herein comprises a peptide comprising a masking peptide, a spacer linker and a cleavable peptide, wherein the peptide comprises a peptide selected from SEQ ID NO: 207-231 The amino acid sequence.

In some embodiments, the activatable masked anti-CTLA4 binding protein described herein comprises a peptide comprising a masking peptide, a first spacer linker, a cleavable peptide, and a second spacer linker, wherein the peptide comprises and The amino acid sequence selected from the group consisting of or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% or 100% homology of amino acid sequences: SEQ ID NO: 113-231, 444, 446-448 and 450-453. In some embodiments, the activatable masked anti-CTLA4 binding protein described herein comprises a peptide comprising a masking peptide, a first spacer linker, a cleavable peptide, and a second spacer linker, wherein the peptide comprises a selection Amino acid sequence from the group consisting of: SEQ ID NO: 113-231, 444, 446-448, and 450-453. In some embodiments, the activatable masked anti-CTLA4 binding protein described herein comprises a peptide comprising a masking peptide, a first spacer linker, a cleavable peptide, and a second spacer linker, wherein the peptide comprises and The amino acid sequence selected from the group consisting of or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% or 100% homology of amino acid sequences: SEQ ID NO: 444, 446-448 and 450-453. In some embodiments, the activatable masked anti-CTLA4 binding protein described herein comprises a peptide comprising a masking peptide, a first spacer linker, a cleavable peptide, and a second spacer linker, wherein the peptide comprises a selection Amino acid sequence from the group consisting of: SEQ ID NO: 444, 446-448, and 450-453. Exemplary masked CTLA4 binding protein

The following describes certain illustrative examples of masked CTLA4 binding proteins containing certain characteristics as described above. These examples are merely illustrative and should not be construed as restrictive.

In some embodiments, a masked antibody is provided herein, which comprises 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 NO: 1-46, wherein the masking peptide is connected to the first or second chain of the antibody or antigen-binding fragment thereof via a linker comprising a cleavable peptide The amino or carboxyl end. 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 NO: 47-88, 464-469, and 479-508. In some embodiments, the spacer linker is directly connected 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 NO: 89-112 and 415-420. In some embodiments, at least one amino acid but no more than 20, 30, 40, or 50 amino acids are directly connected 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 connected to the N-terminus of the masking peptide is a detectable label. In some embodiments, the at least one amino acid directly connected to the N-terminus of the masking peptide is YPYDVPDYA (SEQ ID NO: 398), DYKDDDDK (SEQ ID NO: 399), EQKLISEEDL (SEQ ID NO: 400) or GLNDIFEAQKIEWHE (SEQ ID NO: 401).

In some embodiments, a masked antibody is also provided herein, which comprises a) an antibody or antigen-binding fragment thereof that binds to CTLA4 (for example, 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 NO: 1-46, wherein the masking peptide is connected to the first chain and the second chain of the antibody or its antigen-binding fragment via a linker comprising a cleavable peptide The amino end of the chain. 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 NO: 47-88, 464-469, and 479-508. In some embodiments, the spacer linker is directly connected 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 NO: 89-112 and 415-420. In some embodiments, at least one amino acid but no more than 20, 30, 40, or 50 amino acids are directly connected 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 connected to the N-terminus of the masking peptide is a detectable label. In some embodiments, the at least one amino acid directly connected to the N-terminus of the masking peptide is YPYDVPDYA (SEQ ID NO: 398), DYKDDDDK (SEQ ID NO: 399), EQKLISEEDL (SEQ ID NO: 400) or GLNDIFEAQKIEWHE (SEQ ID NO: 401).

In some embodiments, a masked antibody is also provided herein, which comprises a) an antibody or antigen-binding fragment thereof that binds to CTLA4 (for example, 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 NO: 1-46, wherein the masking peptide is connected to the first chain and the first chain of the antibody or its antigen-binding fragment via a linker comprising a cleavable peptide The carboxyl end of the two chain. 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 NO: 47-88, 464-469, and 479-508. In some embodiments, the spacer linker is directly connected 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 NO: 89-112 and 415-420. In some embodiments, at least one amino acid but no more than 20, 30, 40, or 50 amino acids are directly connected 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 connected to the N-terminus of the masking peptide is a detectable label. In some embodiments, the at least one amino acid directly connected to the N-terminus of the masking peptide is YPYDVPDYA (SEQ ID NO: 398), DYKDDDDK (SEQ ID NO: 399), EQKLISEEDL (SEQ ID NO: 400) or GLNDIFEAQKIEWHE (SEQ ID NO: 401).

In some embodiments, a masking antibody is also provided herein, which comprises a) an antibody or antigen-binding fragment thereof that binds to CTLA4 (for example, human CTLA4), and b) a masking peptide, which comprises a peptide selected from SEQ ID NO: 1-46 The amino acid sequence of the antibody in which the masking peptide is connected to the C-terminus or N-terminus of the first chain of the antibody via a linker containing a cleavable peptide and the masking peptide is connected to the C-terminus of the second chain of the antibody via a linker containing the cleavable peptide End or N end. 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 the light chain and the second chain of the antibody is the light chain; b) the first chain of the antibody is the heavy chain and the second chain of the antibody is the heavy chain; or c) the first chain of the antibody One chain is the light chain and the second chain of the antibody is the heavy chain. Therefore, in some embodiments, the isolated antibody comprises 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 Masking peptide. In some embodiments, the cleavable peptide comprises an amino acid sequence selected from SEQ ID NO: 47-88, 464-469, and 479-508. In some embodiments, the spacer linker is directly connected 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 NO: 89-112 and 415-420. In some embodiments, at least one amino acid but no more than 20, 30, 40, or 50 amino acids are directly connected 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 connected to the N-terminus of the masking peptide is a detectable label. In some embodiments, the at least one amino acid directly connected to the N-terminus of the masking peptide is YPYDVPDYA (SEQ ID NO: 398), DYKDDDDK (SEQ ID NO: 399), EQKLISEEDL (SEQ ID NO: 400) or GLNDIFEAQKIEWHE (SEQ ID NO: 401).

In some embodiments, a masked anti-CTLA4 antibody or antigen-binding fragment thereof is also provided herein, which comprises: a) an anti-CTLA4 antibody or antigen-binding fragment thereof, which comprises a light chain variable region, and the light chain variable region 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 comprising SEQ ID CDR-H3 of the amino acid sequence of NO: 443; b) a masking peptide, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-46; and c) a cleavable peptide, which comprises a peptide selected from the group consisting of SEQ ID NO: 1-46 ID NO: 47-88, 464-469 and 479-508 amino acid sequence. 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 no more than 20, 30, 40, or 50 amino acids are directly connected 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 connected to the N-terminus of the masking peptide is a detectable label. In some embodiments, the at least one amino acid directly connected to the N-terminus of the masking peptide is YPYDVPDYA (SEQ ID NO: 398), DYKDDDDK (SEQ ID NO: 399), EQKLISEEDL (SEQ ID NO: 400) or GLNDIFEAQKIEWHE (SEQ ID NO: 401). 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 an amino acid sequence selected from the group consisting of SEQ ID NO: 89-112 and 415-420, which links the masking peptide to the cleavable peptide The spacer linker, and further comprises a spacer comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 89-112 and 415-420 to connect the cleavable peptide to the light chain variable region or the heavy chain variable region Linker. In some embodiments, the spacer linker connecting the masking peptide to the cleavable peptide comprises the amino acid sequence of SEQ ID NO: 420, and the spacer linking the cleavable peptide to the light chain variable region or the heavy chain variable region The linker includes the amino acid sequence of SEQ ID NO:102. In some embodiments, the spacer linker connecting the masking peptide to the cleavable peptide comprises the amino acid sequence of SEQ ID NO: 96, and the spacer linking the cleavable peptide to the light chain variable region or the heavy chain variable region The linker includes the amino acid sequence of SEQ ID NO:102. In some embodiments, the light chain variable region includes the amino acid sequence of SEQ ID NO: 322, and the heavy chain variable region includes 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 and 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 NO: 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 NO: 358 and 422-431 .

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

In one aspect, provided herein is an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof, which comprises a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 321 or 322 and/ Or comprising a heavy chain variable region selected from the amino acid sequence of SEQ ID NO: 323 or 324. In some embodiments, provided herein is a masked anti-CTLA4 antibody or antigen-binding fragment thereof, which comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 322, and a light chain variable region comprising SEQ ID NO: 324 The variable region of the heavy chain of the amino acid sequence. In another aspect, provided herein is an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof, which comprises a light chain comprising an amino acid sequence selected from SEQ ID NO: 327-341 and/or There is a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 366-380, 421, and 478. In another aspect, provided herein is an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof, which comprises a light chain comprising an amino acid sequence selected from SEQ ID NO: 327, 334, or 342-365 And/or comprise 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, which comprises a light chain comprising the amino acid sequence of SEQ ID NO: 334 and an amino acid comprising SEQ ID NO: 421 The heavy chain of the sequence. In some embodiments, provided herein is a masked anti-CTLA4 antibody or antigen-binding fragment thereof, which comprises a light chain comprising the amino acid sequence of SEQ ID NO: 327, and an amino acid comprising SEQ ID NO: 478 The heavy chain of the sequence.

In some embodiments, provided herein is an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 233 An amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, provided herein is an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable domain comprising a heavy chain variable domain selected from SEQ ID NO: 323 or 324 The amino acid sequence has an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity is compared with the reference sequence Antibodies containing substitutions, insertions or deletions but containing the amino acid sequence retain the ability to bind to CTLA4 (such as human CTLA4). In some embodiments, substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, or 5 amino acids) are present in regions outside the HVR (ie, in 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.

In some embodiments, provided herein is an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof, which comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 232 An amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, provided herein is an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof, which comprises a light chain variable domain comprising a light chain variable domain selected from SEQ ID NO: 321 or 322 The amino acid sequence has an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity is compared with the reference sequence Antibodies containing substitutions, insertions or deletions but containing the amino acid sequence retain the ability to bind to CTLA4 (such as human CTLA4). In some embodiments, substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, or 5 amino acids) are present in regions outside the HVR (ie, in 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.

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

In some embodiments, provided herein is an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 358 and 422-431 having or having approximately 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of amine groups Acid sequence; and/or include the amino acid sequence of SEQ ID NO: 421 or have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99% or 100% homology of amino acid sequences. In some embodiments, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof contains about 70%, 75% of an amino acid sequence selected from the group consisting of SEQ ID NO: 358 and 422-431 , 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of amino acid sequences; and Have or have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% with the amino acid sequence of SEQ ID NO: 421 , 98%, 99% or 100% homology of amino acid sequences. 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 NO:358 and 422-431; and/or comprises SEQ ID NO:421 The amino acid sequence. 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 NO: 358 and 422-431; and an amine comprising SEQ ID NO: 421 Base acid sequence.

In some embodiments, an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof is provided herein, which contains or has about 70%, 75%, 80%, or about 70%, 75%, 80%, and the amino acid sequence of SEQ ID NO: 422. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of amino acid sequences; and include amino acid sequences with SEQ ID NO The amino acid sequence of 421 has or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of amino acid sequence. 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.

In some embodiments, an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof is provided herein, which contains or has about 70%, 75%, 80%, or about 70%, 75%, 80%, and the amino acid sequence of SEQ ID NO: 358. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of amino acid sequences; and include amino acid sequences with SEQ ID NO The amino acid sequence of 421 has or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of amino acid sequence. 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.

In some embodiments, provided herein is a masked anti-CTLA4 antibody or antigen-binding fragment thereof, which comprises the amino acid sequence of SEQ ID NO: 423 having or having about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology of amino acid sequences; and include those with SEQ ID NO: 421 The amino acid sequence has or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or Amino acid sequence with 100% homology. 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.

In some embodiments, an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof is provided herein, which contains or has about 70%, 75%, 80%, or about 70%, 75%, 80%, and the amino acid sequence of SEQ ID NO: 424. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of amino acid sequences; and include amino acid sequences with SEQ ID NO The amino acid sequence of 421 has or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of amino acid sequence. 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.

In some embodiments, provided herein is an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof, which contains or has about 70%, 75%, 80%, or about 70%, 75%, 80%, and the amino acid sequence of SEQ ID NO: 425. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of amino acid sequences; and include amino acid sequences with SEQ ID NO The amino acid sequence of 421 has or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of amino acid sequence. 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.

In some embodiments, an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof is provided herein, which contains or has about 70%, 75%, 80%, or about 70%, 75%, 80%, and the amino acid sequence of SEQ ID NO: 426. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of amino acid sequences; and include amino acid sequences with SEQ ID NO The amino acid sequence of 421 has or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of amino acid sequence. 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.

In some embodiments, provided herein is a masked anti-CTLA4 antibody or antigen-binding fragment thereof, which comprises the amino acid sequence of SEQ ID NO: 427 or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology of amino acid sequences; and include those with SEQ ID NO: 421 The amino acid sequence has or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or Amino acid sequence with 100% homology. 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.

In some embodiments, an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof is provided herein, which comprises or has about 70%, 75%, 80%, and the amino acid sequence of SEQ ID NO: 428. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of amino acid sequences; and include amino acid sequences with SEQ ID NO The amino acid sequence of 421 has or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of amino acid sequence. 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.

In some embodiments, an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof is provided herein, which contains or has about 70%, 75%, 80%, and the amino acid sequence of SEQ ID NO: 429. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of amino acid sequences; and SEQ ID NO: The amino acid sequence of 421 has or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % Or 100% homology of amino acid sequence. 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.

In some embodiments, provided herein is an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof, which comprises or has about 70%, 75%, 80%, or about 70%, 75%, 80%, and the amino acid sequence of SEQ ID NO: 430. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of amino acid sequences; and include amino acid sequences with SEQ ID NO The amino acid sequence of 421 has or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of amino acid sequence. 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. In some embodiments, an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof is provided herein, which comprises or has about 70%, 75%, 80%, or about 70%, 75%, 80%, and the amino acid sequence of SEQ ID NO: 431. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of amino acid sequences; and include amino acid sequences with SEQ ID NO The amino acid sequence of 421 has or has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of amino acid sequence. 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.

There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, which have heavy chains called α, δ, ε, γ, and μ, respectively. The gamma and alpha classes are further divided into subclasses. For example, humans exhibit the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. IgG1 antibodies can exist in a variety of polymorphic variants called allotypes (reviewed in Jefferis and Lefranc 2009. mAbs Vol. 1, No. 4, 1-7), any of which is applicable to some examples herein . The common heteromorphic variants in the human population are the variants designated by the letters a, f, n, z or a combination thereof. In some embodiments herein, the antibody has an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof provided herein has an IgG1 isotype (e.g., a human IgG1 isotype). In some embodiments, the antibodies provided herein comprise a heavy chain constant domain, which comprises the amino acid sequence of SEQ ID NO: 235 or 236. In some embodiments, the antibodies provided herein comprise a heavy chain constant domain, which comprises the amino acid sequence of SEQ ID NO:326. In some embodiments, the antibodies provided herein comprise a heavy chain constant domain, which comprises the amino acid sequence of SEQ ID NO:463.

In some embodiments, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof binds CTLA4 after cleavage by a protease (such as the protease described herein). In some embodiments, the cleavable peptide is a substrate for a protease that is co-located in a region with cells or tissues expressing CTLA4.

In one aspect of the present invention, a polynucleotide encoding an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof is provided. In certain embodiments, a vector is provided comprising polynucleotides that encode activatable masked anti-CTLA4 antibodies or antigen-binding fragments thereof. In certain embodiments, host cells containing such vectors are provided. In another aspect of the present invention, there is provided a composition comprising the activatable masked anti-CTLA4 antibody described herein or a polynucleus encoding the activatable masked anti-CTLA4 antibody described herein Glycidic acid. In certain embodiments, the composition of the present invention is a pharmaceutical formulation for the treatment of neoplastic diseases in which CTLA4 functions, such as the diseases listed herein.

In some embodiments, the CTLA4 binding protein provided herein is a bispecific antibody capable of binding to CTLA4. Bispecific antibodies are monoclonal antibodies that have binding specificities for at least two different antigens. In some embodiments, one binding specificity is for CTLA4 and the other is for any other antigen. In certain embodiments, bispecific antibodies can bind to two different epitopes of CTLA4.

In some aspects, a masked bispecific antibody is provided herein, which comprises a) a first pair of light and heavy chains, which specifically bind to CTLA4; b) a second pair of light and heavy chains, which are specific Sexually binds to the antigen; and c) a masking peptide comprising an amino acid sequence selected from SEQ ID NO: 1-46, wherein the masking peptide is connected to the light chain in the first pair via a linker comprising a cleavable peptide and/ Or the amino end of the heavy chain. In some aspects, a masked bispecific antibody is provided herein, which comprises a) a first pair of light and heavy chains, which specifically bind to CTLA4; b) a second pair of light and heavy chains, which are specific Sexually binds to the antigen; and c) a masking peptide comprising an amino acid sequence selected from SEQ ID NO: 1-46, wherein the masking peptide is connected to the light chain in the first pair via a linker comprising a cleavable peptide and/ Or the carboxyl end of the heavy chain. In some embodiments, the antigen is a different antigen from CTLA4. In some embodiments, the light chain in the first pair or the second pair is any light chain described herein. In some embodiments, the heavy chain in the first pair or the second pair is any light chain described herein. In some embodiments, the light chain in the first pair includes CDR-L1 including the amino acid sequence of SEQ ID NO: 438, CDR-L2 including the amino acid sequence of SEQ ID NO: 439, and includes SEQ ID CDR-L3 of the amino acid sequence of NO: 440; and the heavy chain in the first pair includes CDR-H1 including the amino acid sequence of SEQ ID NO: 441, including the amino acid sequence of SEQ ID NO: 442 CDR-H2 and CDR-H3 including the amino acid sequence of SEQ ID NO:443. In some embodiments, the light chain in the second pair includes CDR-L1 including the amino acid sequence of SEQ ID NO: 438, CDR-L2 including the amino acid sequence of SEQ ID NO: 439, and includes SEQ ID CDR-L3 of the amino acid sequence of NO: 440; and the heavy chain of the second pair includes CDR-H1 including the amino acid sequence of SEQ ID NO: 441, including the amino acid sequence of SEQ ID NO: 442 CDR-H2 and CDR-H3 including the amino acid sequence of SEQ ID NO:443. In some embodiments, the antigen lines are directed to different epitopes of CTLA4. In some embodiments, the cleavable peptide comprises an amino acid sequence selected from SEQ ID NO: 47-88, 464-469, and 479-508. In some embodiments, the spacer linker is directly connected 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 NO: 89-112 and 415-420. In some embodiments, at least one amino acid but no more than 20, 30, 40, or 50 amino acids are directly connected 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 connected to the N-terminus of the masking peptide is a detectable label. In some embodiments, the at least one amino acid directly connected to the N-terminus of the masking peptide is YPYDVPDYA (SEQ ID NO: 398), DYKDDDDK (SEQ ID NO: 399), EQKLISEEDL (SEQ ID NO: 400) or GLNDIFEAQKIEWHE (SEQ ID NO: 401).

Bispecific antibodies for masked bispecific antibodies encompassed herein include murine bispecific antibodies, humanized bispecific antibodies, chimeric bispecific antibodies, and human bispecific antibodies. In some embodiments herein, the bispecific antibody has an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the bispecific antibodies provided herein have an IgG1 isotype (e.g., a human IgG1 isotype). In some embodiments, the antibody has the IgG1 isotype, which contains amino acid substitutions or is expressed by cells that do not have the ability to trehalose Fc glycans to have reduced ability to trehalose Fc glycans, the Fc poly Sugar enhances the effector function as described herein. In some embodiments, the masked bispecific antibody provided herein comprises a heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 235 or 236. In some embodiments, the masked bispecific antibody provided herein comprises a heavy chain constant domain comprising the amino acid sequence of SEQ ID NO:326. In some embodiments, the masked bispecific antibody provided herein comprises a heavy chain constant domain, which comprises the amino acid sequence of SEQ ID NO:463.

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

In one aspect, an activatable masked anti-CTLA4 bispecific antibody is provided herein, which 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 selected from the amino acid sequence of SEQ ID NO: 323 or 324. In another aspect, an activatable masked anti-CTLA4 bispecific antibody is provided herein, which comprises a light chain comprising an amino acid sequence selected from SEQ ID NO: 327-341 and/or comprises A heavy chain selected from the amino acid sequence of SEQ ID NO: 366-380, 421 and 478. In another 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 it contains a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 366 or 380-397.

In some embodiments, provided herein is an activatable masked anti-CTLA4 bispecific antibody comprising a heavy chain variable region comprising at least the amino acid sequence of SEQ ID NO: 233 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences. In some embodiments, provided herein is an activatable masked anti-CTLA4 bispecific antibody comprising a heavy chain variable domain comprising an amine group selected from SEQ ID NO: 323 or 324 The acid sequence has an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity is compared with the reference sequence Antibodies containing substitutions, insertions or deletions but containing the amino acid sequence retain the ability to bind to CTLA4 (such as human CTLA4). In some embodiments, substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, or 5 amino acids) are present in regions outside the HVR (ie, in the FR). In some embodiments, the 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, the 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.

In some embodiments, provided herein is an activatable masked anti-CTLA4 bispecific antibody comprising a light chain variable domain comprising at least the amino acid sequence of SEQ ID NO: 232 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences. In some embodiments, provided herein is an activatable masked anti-CTLA4 bispecific antibody comprising a light chain variable domain comprising an amine group selected from SEQ ID NO: 321 or 322 The acid sequence has an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity is compared with the reference sequence Antibodies containing substitutions, insertions or deletions but containing the amino acid sequence retain the ability to bind to CTLA4 (such as human CTLA4). In some embodiments, substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, or 5 amino acids) are present in regions outside the HVR (ie, in the FR). In some embodiments, the 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, the 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.

In some embodiments, the CTLA4 binding protein provided herein is a chimeric receptor capable of binding to CTLA4 (for example, a chimeric antigen receptor (CAR)). CAR is a molecule that combines antibody-based specificity against a desired antigen (such as CTLA4) and T cell receptor activating intracellular domain to produce a chimeric protein that exhibits specific anti-tumor cell activity. In one embodiment, a chimeric receptor is provided herein, which is engineered to include a CTLA4 binding as described herein fused to the intracellular signaling domain of the T cell antigen receptor complex zeta chain (eg CD3ζ) The extracellular domain of the domain. The CTLA4 binding domain is engineered so that it is connected to a masking peptide, such as the masking peptide described herein, via a linker comprising a cleavable peptide. When expressed in T cells, the activatable masked chimeric receptor provided herein can redirect antigen recognition based on antigen binding specificity after cleavage by a protease that recognizes a cleavable peptide. In some embodiments, the CTLA4 binding domain is preferably fused with an intracellular domain derived from one or more of the costimulatory molecule and the zeta chain. In some embodiments, the CTLA4 binding domain is fused with one or more intracellular domains selected from the group consisting of: CD137(4-1BB) signaling domain, CD28 signaling domain, CD3ζ signaling domain, and any combination thereof.

In some aspects, a masked chimeric receptor is provided herein, which comprises a) a ligand binding domain comprising a first chain and a second chain, which binds to CTLA4; b) a masking peptide, which comprises selected from The amino acid sequence of SEQ ID NO: 1-46, c) a transmembrane domain; and d) an intracellular signaling domain comprising a signaling domain, wherein the masking peptide is connected to the ligand via a linker comprising a cleavable peptide The amino end of the first chain and/or the second chain of the binding domain. 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 of the activatable masked chimeric receptor described herein, the first strand includes the amino acid sequence of SEQ ID NO: 232; and/or the second strand includes the amino acid sequence of SEQ ID NO: 233 Amino acid sequence. In some embodiments, the first strand includes an amino acid sequence selected from SEQ ID NO: 321 or 322; and/or the second strand includes an amino acid sequence selected from SEQ ID NO: 323 or 324.

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

In some embodiments of the activatable masked chimeric receptor described herein, the cleavable peptide comprises an amino acid sequence selected from SEQ ID NO: 47-88, 464-469, and 479-508. In some embodiments, the spacer linker is directly connected 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 NO: 89-112 and 415-420. In some embodiments, at least one amino acid but no more than 20, 30, 40, or 50 amino acids are directly connected 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 connected to the N-terminus of the masking peptide is a detectable label. In some embodiments, the at least one amino acid directly connected to the N-terminus of the masking peptide is YPYDVPDYA (SEQ ID NO: 398), DYKDDDDK (SEQ ID NO: 399), EQKLISEEDL (SEQ ID NO: 400) or GLNDIFEAQKIEWHE (SEQ ID NO: 401). 1. Binding affinity

The strength or affinity of an immunological binding interaction (such as between an antibody and an antigen specific to the antibody) can be expressed by the equilibrium dissociation constant (K _D ) used with each other, where a smaller K _D indicates a larger affinity. The immunological binding properties of proteins can be quantified using methods well known in the art. For example, one method involves measuring the rate of formation and dissociation of an antigen-binding protein (such as an antibody)/antigen complex, where these rates depend on the concentration of the complex partner, the affinity of the interaction, and the rate in both directions. Depends on the geometric structure parameters. The "Association Rate Constant" (Kon) and "Dissociation Rate Constant" (Koff) can be determined by calculating the concentration and the actual rate of association and dissociation. The ratio of Koff/Kon makes it possible to eliminate all parameters that have nothing to do with affinity, and is equal to the equilibrium dissociation constant K _D. See Davies et al., Annual Rev Biochem. 59:439-473, (1990).

In some aspects, the activatable masked anti-CTLA4 binding protein described herein (e.g., can activate the masked anti-CTLA4 antibody or its antigen The binding fragment) binds to CTLA4 with approximately the same or higher affinity after protease cleavage. In certain embodiments, the equilibrium dissociation constant (K _D ) of the anti-CTLA4 binding protein provided herein is ≤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 lower, such as 10 ^-8 M to 10 ^-13 M, such as 10 ^-9 M to 10 ^-13 M). In some embodiments, the anti-CTLA4 binding protein provided herein (such as an anti-CTLA4 antibody or antigen-binding fragment thereof) binds to a target protein (such as a CTLA4 protein) with an _{equilibrium dissociation constant (K D) of about 50 pM to about 5 nM} . The analytical methods used to assess binding affinity are well known in the art, such as the analytical methods described in Example 3A and Example 3B herein.

In some aspects, an activatable masked anti-CTLA4 binding protein (such as an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof) that exhibits a desired occlusion rate is provided. As used herein, the term "occlusion rate" refers to the ratio of (a) the maximum detection value of the parameter under the first set of conditions to (b) the minimum detection value of the parameter under the second set of conditions. For example, in the case of an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof, the occlusion rate refers to (a) in the presence of at least one activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof In the case of the protease that can cleave the peptide, the maximum detectable amount of the target protein (such as CTLA4 protein) bound to the activatable masked anti-CTLA4 antibody or its antigen-binding fragment and (b) in the absence of protease , The ratio of the minimum detectable amount of target protein (such as CTLA4 protein) bound to the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof. The occlusion rate of the activatable masked anti-CTLA4 antibody or its antigen-binding fragment can be calculated as the dissociation constant of the activatable masked anti-CTLA4 antibody or its antigen-binding fragment before protease cleavage and the activatability after protease cleavage The ratio of the dissociation constants of the masked anti-CTLA4 antibody or antigen-binding fragment thereof. In some embodiments, the greater the entrapment rate of the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof, indicates that the masked anti-CTLA4 antibody capable of cleaving the activatable masked anti-CTLA4 antibody is present compared to the absence of protease. In the case of the protease of the cleavable peptide of the antibody or its antigen-binding fragment, the activatable masked anti-CTLA4 antibody or its antigen-binding fragment binds to the target protein (such as CTLA4 protein) to a greater extent (e.g., significant progress) ). In some embodiments, provided herein is an activatable masked anti-CTLA4 binding protein with the best occlusion rate. In some embodiments, the optimal occlusion rate of the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof indicates that the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof has a method or combination suitable for use in this context The ideal characteristics of things. In some embodiments, the activatable masked anti-CTLA4 binding protein provided herein exhibits an optimal occlusion rate of about 20 to about 10,000, for example, about 80 to about 100. In another embodiment, the occlusion rate 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 protein provided herein exhibits an optimal occlusion rate of about 80 to about 100. In some embodiments, the activatable masked anti-CTLA4 binding protein provided herein exhibits an optimal occlusion rate of about 20 to about 1,000. Techniques well known in the art (such as ELISA) can be used to determine the binding of the activatable masked anti-CTLA4 binding protein to the target protein (eg CTLA4 protein) before protease cleavage and/or after protease cleavage.

In some aspects, the masking peptide described herein binds to an anti-CTLA4 binding protein (such as an anti-CTLA4 antibody or its Antigen-binding fragment). In certain embodiments, the masking peptides provided herein are ≤1 mM, ≤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 ^-5 M or less, e.g. 10 ^-5 M to 10 ^-13 M, e.g. 10 ^-5 M to 10 ^-7 M) with equilibrium dissociation constant (K _D ) bound to the anti-CTLA4 binding protein ( For example, anti-CTLA4 antibody or antigen-binding fragment thereof). In some embodiments, the masking peptide provided herein binds to an anti-CTLA4 binding protein (eg, an anti-CTLA4 antibody or antigen-binding fragment thereof) with an _{equilibrium dissociation constant (K D) of about 50 nM to about 50 µM.} An exemplary analytical method for evaluating the binding affinity between a masking peptide and an anti-CTLA4 binding protein (eg, an anti-CTLA4 antibody or an antigen-binding fragment thereof) can be found in Example 2 herein. 2. Biological activity analysis method

In some aspects, the activatable masked anti-CTLA4 binding protein described herein reduces tumor volume in an in vivo murine tumor model. The analytical methods used to assess the reduction in tumor volume are well known in the art, such as the analytical methods described in Example 4A and Example 4B herein. III. Preparation of masked anti- CTLA4 binding protein

The masked anti-CTLA4 binding protein described herein was prepared using the techniques available in this technology, an exemplary method of which is described in more detail in the following section. 1. Masked anti-CTLA4 binding protein: antibody fragment

The present invention encompasses antibody fragments that are masked anti-CTLA4 binding proteins. The masked antibody fragments can be produced by traditional means (such as enzymatic digestion) or by recombinant technology. In certain situations, the use of masked antibody fragments instead of full antibodies may have advantages. For a review of certain antibody fragments, see Hudson et al. (2003) Nat. Med. 9:129-134.

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments are obtained by proteolytic digestion of whole antibodies (see, for example, 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 and secreted in E. coli and other cell types, thus making it easy to produce a large number of these masked fragments. Alternatively, the masked Fab'-SH fragments can be recovered directly 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 method, the masked F(ab')2 fragment can be isolated directly from recombinant host cell culture. Masked Fab and F(ab')2 fragments with extended half-life in vivo and containing FcRN/rescue receptor binding epitope residues are described in U.S. Patent No. 5,869,046. Other preparation techniques for masked antibody fragments will be obvious to those familiar with this technique. In certain embodiments, the masked antibody is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Patent No. 5,571,894; and No. 5,587,458. Fv and scFv are the only types that have complete combination sites without constant regions; therefore, they are suitable for non-specific binding that decreases during in vivo use. The scFv fusion protein can be constructed to produce the effector protein fusion at the amine or carboxy terminus of the scFv. See Antibody Engineering, Borrebaeck, ed., supra. In addition, a dual scFv comprising two scFvs linked via a polypeptide linker can be used as a bispecific antibody. Alternatively, multiple scFvs comprising three or more scFvs can be used as multispecific antibodies.

The present invention includes linear antibodies (such as described in U.S. Patent No. 5,641,870) or single chain immunoglobulins, which comprise the heavy and light chain sequences of the antibody connected via a suitable linker. Such linear antibodies or immunoglobulins can be monospecific or bispecific. Such single-chain immunoglobulins can dimerize to thereby maintain structure and activity similar to antibodies (which were originally tetramers). In addition, the antibody of the present invention may be an antibody having a single heavy chain variable region and no light chain sequence. Such antibodies are called single domain antibodies (sdAb) or nanoantibodies. These antibodies are also encompassed in the meaning of functional fragments of the antibodies of the present invention. 2. Masked anti-CTLA4 binding protein: humanized antibody

The present invention encompasses masked humanized antibodies. Mask the humanized antibodies according to the guidelines provided herein. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from the "import" variable domain. Humanization can basically follow 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 substituting the hypervariable region sequence for the corresponding sequence of the human antibody. Therefore, such "humanized" antibodies are chimeric antibodies (US Patent No. 4,816,567) in which substantially smaller than complete human variable domains have been substituted with corresponding sequences from non-human species. In fact, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues have been replaced with residues from similar sites in rodent antibodies. 3. Masked anti-CTLA4 binding protein: human antibody

The human anti-CTLA4 antibody of the present invention can be constructed by combining Fv cloned variable domain sequences selected from a human-derived phage display library with known human constant domain sequences. Alternatively, the human monoclonal anti-CTLA4 antibody of the present invention can be produced by the fusion tumor method. Human myeloma and murine-human fusion myeloma cell lines used to prepare human monoclonal antibodies have been described, for example, by Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, 51 -63 pages (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991). Mask human antibodies according to the guidelines provided herein. 4. Masked anti-CTLA4 binding protein: bispecific antibody

Bispecific antibodies are monoclonal antibodies that have binding specificities for at least two different antigens. In certain embodiments, the bispecific antibody is a human or humanized antibody. In certain embodiments, one binding specificity is for CTLA4 and the other is for any other antigen. In certain embodiments, bispecific antibodies can bind to two different epitopes of CTLA4. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing CTLA4. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). Mask bispecific antibodies according to the guidelines provided herein.

Methods for preparing bispecific antibodies are known in the art. See Milstein and Cuello, Nature, 305: 537 (1983); WO 93/08829 published on May 13, 1993; Traunecker et al., EMBO J., 10: 3655 (1991); Kontermann and Brinkmann, Drug Discovery Today, 20(7): 838-847. For other 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 "heterobinding" antibodies. For example, one antibody in the heteroconjugate can be coupled to an antibiotic protein, and the other antibody can be coupled to biotin. Heterobinding antibodies can be prepared using any suitable cross-linking method. Suitable crosslinking agents and many crosslinking techniques are well known in the art and are disclosed in US Patent No. 4,676,980. 5. Masked anti-CTLA4 binding protein: single domain antibody

In some embodiments, single domain antibodies are masked according to the guidelines provided herein. A single domain antibody is a single polypeptide chain that contains 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, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, Mass.; see, for example, U.S. Patent No. 6,248,516 B1). In one embodiment, a single domain antibody is composed of all or part of the heavy chain variable domain of the antibody. 6. Masked anti-CTLA4 binding protein: antibody variants

In some embodiments, the amino acid sequence modifications of the masked antibodies described herein are encompassed. For example, it may be necessary to improve the binding affinity and/or other biological properties of the masked antibody. The amino acid sequence variants of the antibody can be prepared by introducing suitable changes into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be performed to obtain the final structure, and the limitation is that the final structure has the required characteristics. When the sequence is prepared, amino acid changes can be introduced into the target antibody amino acid sequence.

A method suitable for identifying certain residues or regions in antibodies that are preferred locations for mutagenesis is called "alanine scanning mutagenesis", as described by Cunningham and Wells (1989) Science, 244:1081-1085 . Herein, a residue or a set of target residues (for example, charged residues, such as Arg, Asp, His, Lys, and Glu) are identified and consists of neutral or negatively charged amino acids (for example, alanine or polyalanine). Replacement to affect the interaction between amino acid and antigen. Next, by introducing additional or other variants at the substitution site or for the substitution site, the positions of amino acids that exhibit functional sensitivity to substitution are optimized. Therefore, although the site for introducing amino acid sequence changes is predetermined, the nature of the mutation itself need not be predetermined. For example, in order to analyze the mutation efficiency at the established site, Ala scanning or random mutagenesis is performed at the target codon or region, and the expressed immunoglobulin is screened according to the desired activity.

Amino acid sequence insertions include amino-terminal and/or carboxy-terminal fusions ranging from one residue to a polypeptide containing one hundred or more residues, and within the sequence of single or multiple amino acid residues insert. Examples of terminal insertions include antibodies with N-terminal methionine residues. Other insertion variants of antibody molecules include fusions of the N-terminus or C-terminus of the antibody with enzymes or polypeptides that extend the serum half-life of the antibody.

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 the binding of the antibody to FcRn at low pH, but do not change the affinity of the antibody at neutral pH.

In certain embodiments, the antibodies of the invention are modified to increase or decrease the degree of glycosylation of the antibody. Polypeptide glycosylation is usually N-linked or O-linked. N linkage refers to the linkage of the carbohydrate moiety to the side chain of the asparagine residue. The tripeptide sequence asparagine-X-serine and asparagine-X-threonine, where X is any amino acid other than proline, used for carbohydrate moiety and asparagine Recognition sequence for enzymatic ligation of side chains. Therefore, the presence of any of these tripeptide sequences in a polypeptide can create potential glycosylation sites. O-linked glycosylation refers to the connection of one of the sugars N-acetylgalactosamine, galactose or xylose with hydroxyl amino acids, most commonly serine or threonine, but it can also be used 5-hydroxyproline or 5-hydroxylysine.

It is advisable to change the amino acid sequence so that one or more of the above-mentioned tripeptide sequences are generated or removed to achieve the addition or deletion of glycosylation sites for antibodies (for N-linked glycosylation sites) . Changes can also be achieved by adding, deleting or substituting one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

In the case where the antibody contains an Fc region, the carbohydrate linked to it can be changed. For example, an antibody with a mature carbohydrate structure (which does not have trehalose linked to the Fc region of the antibody) is described in US Patent Application No. US 2003/0157108 (Presta, L.). See also US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibodies with equal parts of N-acetylglucosamine (GlcNAc) in the carbohydrates linked to the Fc region of the antibody are described in WO 2003/011878 by Jean-Mairet et al. and U.S. Patent No. 6,602,684 by Umana et al. middle. Antibodies having at least one galactose residue in the oligosaccharide linked to the Fc region of the antibody are reported in Patel et al. WO 1997/30087. For antibodies with altered carbohydrates linked to the Fc region, see also WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.). For antigen binding molecules with modified glycosylation, see also US 2005/0123546 (Umana et al.).

In certain embodiments, the glycosylation variant comprises an Fc region, wherein the carbohydrate structure linked to the Fc region does not have trehalose or has reduced trehalose. Such variants have improved ADCC function. Optionally, the Fc region further includes one or more amino acid substitutions that further improve ADCC, such as substitutions at positions 298, 333, and/or 334 (Eu numbering of residues) in the Fc region. Examples of publications concerning "no trehalose", "de-trehalose" or "trehalose deficient" antibodies include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614 US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778 ; 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 anti-trehaloseylated antibodies include protein trehalosylation-deficient Lec13 CHO cells (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US 2003/ 0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al., especially in Example 11), and gene knock-out cell lines, such as α-1,6-trehalosyltransferase gene (FUT8) knock-out CHO Cells (Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)), and overexpress β1,4-N-acetylglycosamine polysaccharyltransferase III (GnT-III) and Golgi μ (Golgi μ )-Mannosidase II (ManII) cells.

In any of the embodiments herein, the masked anti-CTLA4 binding protein can be engineered to improve antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In some embodiments, the masked anti-CTLA4 binding protein can be produced in a cell line with α1,6-trehalosyltransferase (Fut8) gene knockout. In some other embodiments, the masked anti-CTLA4 binding protein can be produced in cell lines that have overexpressed β1,4-N-acetylglycosamine polysaccharyltransferase III (GnT-III). In other embodiments, the cell line also overexpresses Golgi mu-mannosidase II (ManII). In some embodiments herein, the masked anti-CTLA4 binding protein may include at least one amino acid substitution in the Fc region that improves ADCC activity.

In one embodiment, the masked antibody is modified to improve its serum half-life. In order to extend the serum half-life of antibodies, FcRN/rescue receptor binding epitopes can be incorporated into antibodies (especially antibody fragments), as described in, for example, US Patent No. 5,739,277. As used herein, the term "rescue receptor binding epitope" refers to the epitope of the Fc region of an IgG molecule (such as IgG1, IgG2, IgG3 or IgG4), which is responsible for prolonging the in vivo serum half-life of IgG molecules (US 2003/0190311, U.S. Patent No. 6,821,505; U.S. Patent No. 6,165,745; U.S. Patent No. 5,624,821; U.S. Patent No. 5,648,260; U.S. Patent No. 6,165,745; U.S. Patent No. 5,834,597).

Another variant is the amino acid substitution variant. These variants have at least one amino acid residue replaced by a different residue in the antibody molecule. The relevant sites induced by substitution mutations include hypervariable regions, but also FR changes. Conservative substitutions are shown in Table 2 under the heading of "preferred substitutions". If such substitutions result in a desired change in biological activity, more substantial changes can be introduced, which are referred to as "exemplary substitutions" in Table 2 or described further below with reference to amino acid categories, and the products are screened. Table 2. Original residue Exemplary substitution Better replace Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Leucine Leu Leu (L) Leucine; Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Leucine Leu

The substantial modification of the biological properties of the antibody is achieved by selective substitutions, which are significantly different in their effects on maintaining the following: (a) the structure of the polypeptide backbone in the substituted region, such as sheet or helical configuration, (b) The charge or hydrophobicity of the molecule at the target site, or c) the volume of the side chain. Amino acids can be grouped according to the similarity of the characteristics of their side chains (A. L. Lehninger, Biochemistry, Second Edition, pp. 73-75, Worth Publishers, New York (1975)): (1) Non-polarity: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M) (2) Without electrical polarity: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q) (3) Acidity: Asp (D), Glu (E) (4) Basicity: Lys (K), Arg (R), His (H)

Alternatively, naturally occurring residues can be grouped based on common side chain properties: (1) Hydrophobicity: Leucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln; (3) Acidity: Asp, Glu; (4) Alkaline: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatics: Trp, Tyr, Phe.

A non-conservative substitution will necessarily accompany the replacement of a member of one of these categories with another category. Such substituted residues can also introduce conservative substitution sites or introduce other (non-conservative) sites.

A 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 selected for further development has modified (e.g., improved) biological properties relative to the parent antibody from which it was produced. A suitable way for generating such substitution variants involves the use of affinity maturation presented by phage. In short, mutate several hypervariable regions (e.g., 6-7 sites) to generate all possible amino acid substitutions at each site. The resulting antibody is presented from filamentous phage particles in the form of a fusion with at least a part of the phage sheath protein (for example, the gene III product of M13) encapsulated in each particle. Then, the phage is screened for the biological activity (such as binding affinity) of the variant. To identify candidate hypervariable region sites for modification, scanning mutagenesis (such as alanine scan) can be performed to identify hypervariable region residues that significantly contribute to antigen binding. Alternatively or additionally, it may be useful to analyze the crystal structure of the antigen-antibody complex to identify contact points between the antibody and the antigen. Such contact residues and adjacent residues are candidates for substitution according to techniques known in the art, including those described in detail herein. After such variants are produced, use known techniques in the art (including those described in this article) to screen the set of variants, and select antibodies with excellent characteristics in one or more related analysis methods For further development.

Nucleic acid molecules encoding amino acid sequence variants of masked antibodies are prepared by various methods known in the art. These methods include (but are not limited to) isolation from natural sources (in the case of naturally-occurring amino acid sequence variants), or by oligonucleotide-mediated early preparation of variants or non-variant versions of antibodies. Guided (or site-directed) mutagenesis, PCR mutagenesis and cassette mutagenesis are prepared.

It may be necessary to introduce one or more amino acid modifications into the Fc region of the antibody of the present invention, thereby generating Fc region variants. 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 amine at one or more amino acid positions (including the amino acid positions of hinge cysteine) Base acid modification (e.g. substitution).

In some embodiments, the activatable masked anti-CTLA4 binding protein provided herein (for example, an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof, or an activatable masked anti-CTLA4 bispecific antibody ) Has the IgG1 isotype, which has enhanced effector functions. In some embodiments, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof is non-trehalosylated. In some embodiments, the activatable masked anti-CTLA4 bispecific antibody is non-trehalosylated. In some embodiments, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof has an increased content of mannose moieties. In some embodiments, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof has an increased content of aliquots of glycan moieties. In some embodiments, the activatable masked anti-CTLA4 bispecific antibody has an increased content of mannose moieties. In some embodiments, IgG1 contains amino acid mutations.

In some embodiments, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof or the activatable masked anti-CTLA4 bispecific antibody provided herein has an IgG1 isotype (e.g., a human IgG1 isotype). In one embodiment, IgG1 includes 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, IgG1 contains amino acid substitutions S239D and I332E, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgG1 contains amino acid substitutions S239D, A330L and I332E, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgG1 contains amino acid substitutions P247I and A339D or A339Q, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgG1 contains 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 includes 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 contains 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 contains 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 includes 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, IgG1 includes amino acid substitutions K326A and E333A, where the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgG1 contains amino acid substitutions K326W and E333S, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgG1 contains amino acid substitutions K290E or K290N, S298G, T299A and/or K326E, wherein the amino acid residues are numbered according to the EU index as in Kabat.

According to the present specification and the teachings of the technology, it is expected that in some embodiments, the antibody of the present invention may contain one or more changes, such as in the Fc region, compared to the wild-type corresponding antibody. These antibodies will still retain substantially the same characteristics required for therapeutic utility compared to their wild-type counterparts. For example, it is believed that certain changes can be made in the Fc region, which will cause changes (ie, improvement or reduction) of C1q binding and/or complement dependent cytotoxicity (CDC), such as described in WO99/51642. For other examples of Fc region variants, see also Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO94/29351. WO00/42072 (Presta) and WO 2004/056312 (Lowman) describe antibody variants with improved or reduced binding to FcRs. 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). The half-life is prolonged and is associated with the neonatal Fc receptor (FcRn) 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)) ) Binding improved antibodies are described in US 2005/0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions that improve the binding of the Fc region to FcRn. Polypeptide variants with altered Fc region amino acid sequence 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). 7. Masked antibody-drug conjugate

The present invention also provides a masked antibody-drug conjugate (ADC), which contains one or more cytotoxic agents (such as chemotherapeutics or drugs), growth inhibitors, toxins (such as protein toxins, bacteria, fungi, plants or Enzymatically active toxins of animal origin, or fragments thereof) or radioisotopes conjugated with the activatable masked anti-CTLA4 binding protein provided herein.

In one embodiment, one or more drugs that bind to the antibody-drug conjugate include, but are not limited to, maytansinoid (see U.S. Patent No. 5,208,020, No. 5,416,064, and European Patent Case EP 0 425 235 B1); auristatin, such as the monomethyl auristatin drug part DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483 and 5,780,588 and 7,498,298); Apnea toxin ( dolastatin); calicheamicin or its derivatives (see U.S. Patent 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)); anthracycline, such as daunorubicin or small Cranberries (see 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 12:1529-1532 (2002 ); King et al., J. Med. Chem. 45:4336-4343 (2002); U.S. Patent No. 6,630,579); Methotrexate; Vindesine; Taxane, such as Docetaxel, paclitaxel, larotaxel, tesetaxel and ortataxel; trichothecene; and CC1065.

In another embodiment, one or more drugs combined with the antibody-drug conjugate include (but are not limited to) tubulin polymerization inhibitors (such as maytansinoids and auristatin), DNA damaging agents (such as pyrrolo Benzodiazepine (PBD) dimers, calicheamicin, becarcinomycin, and indoline benzodiazepine dimers) and DNA synthesis inhibitors (for example, ixinotecan derivative Dxd).

In another embodiment, the antibody-drug conjugate comprises an antibody as described herein that binds to an enzymatically active toxin or fragment thereof, the enzymatically active toxin or fragment thereof includes, but is not limited to, the diphtheria A chain (diphtheria A chain). ), non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, Modisin Modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S) ), momordica charantia inhibitor, curcin, crotonin (crotin), sapaonaria officinalis inhibitor, gelonin, mitogen (mitogellin), limited kojimycin ( restrictocin), phenomycin (phenomycin), inomycin (enomycin) and crescent toxin (tricothecenes).

In another embodiment, the antibody-drug conjugate comprises an antibody as described herein that binds to a radioactive atom to form a radioactive conjugate. A variety of radioisotopes can be used to produce radioactive conjugates. Examples include At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and radioisotopes of Lu. When the radioactive conjugate is used for detection, it can contain radioactive atoms used in scintigraphic research, such as tc ^99m or I ¹²³ , or used for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI) rotation Labels, such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gamma, manganese or iron.

A variety of bifunctional protein coupling agents can be used to prepare conjugates of activatable and masked anti-CTLA4 binding proteins (for example, activatable and masked anti-CTLA4 antibodies or antigen-binding fragments thereof) and cytotoxic agents. These bifunctional proteins Coupling agents such as N-succinimidyl-3-(2-pyridyldisulfide) propionate (SPDP), succinimidyl-4-(N-maleimidinyl (Methyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidate (such as dimethyl adipimidate hydrochloride) , Active esters (such as dibutyldiimide suberate), aldehydes (such as glutaraldehyde), diazide compounds (such as bis(p-azidobenzyl) hexamethylene diamine), double nitrogen Derivatives (such as bis(p-diazobenzyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and dual-reactive fluorine compounds (such as 1,5-difluoro-2,4- Dinitrobenzene). For example, the ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the binding of radionucleotides to antibodies. See WO94/11026. The linker can be a "cleavable linker" that promotes the release of cytotoxic drugs in the cell. For example, acid labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or linkers containing disulfide bonds can be used (Chari et al., Cancer Res. 52:127- 131 (1992); U.S. Patent No. 5,208,020).

The ADC herein clearly covers (but is not limited to) such conjugates prepared with cross-linking agents, 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, and SVSB (butyl Diamido-(4-vinylsulfonate) benzoate), and these cross-linking agents are commercially available (for example, from Pierce Biotechnology, Inc., Rockford, IL., USA). 8. Vectors, host cells and recombination methods

For the recombinant production of the activatable and masked anti-CTLA4 binding protein of the present invention, the nucleic acid encoding it is isolated and inserted into a replicable vector for further selection (DNA amplification) or for expression. The DNA encoding the antibody is easily isolated and sequenced using conventional procedures (for example, using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody). Many carriers are available. The choice of vector depends in part on the host cell used. Generally, host cells are of prokaryotic or eukaryotic (usually mammalian) origin. It should be understood that constant regions of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and such constant regions can be obtained from any human or animal species. 9. Use prokaryotic host cells to produce binding proteins a) Carrier construction

Standard recombinant techniques can be used to obtain polynucleotide sequences encoding the polypeptide components of the activatable masked anti-CTLA4 binding protein of the present invention. The desired polynucleotide sequence can be isolated and sequenced from antibody-producing cells (such as fusion tumor cells). Alternatively, polynucleotides can be synthesized using a nucleotide synthesizer or PCR technology. After obtaining, the sequence encoding the polypeptide is inserted into a recombinant vector capable of replicating in a prokaryotic host and expressing the heterologous polynucleotide. Many vectors available and known in the art can be used for the purpose of the present invention. The choice of a suitable vector will mainly depend on the size of the nucleic acid inserted into the vector and the specific host cell transformed with the vector. Each vector contains various components, depending on its function (amplification or performance of heterologous polynucleotide, or both) and compatibility with the specific host cell in which it resides. The vector components usually 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.

Generally, plastid vectors containing replicons and control sequences derived from species compatible with the host cell are used in combination with these hosts. The vector usually carries a replication site and a marker sequence capable of providing phenotypic selection in the transformed cell. For example, E. coli is typically transformed using pBR322 (a plastid derived from the E. coli species). pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance and therefore provides a way to easily identify transformed cells. pBR322, its derivatives, or other microbial plastids or bacteriophages can also contain or be modified to contain a promoter that can be used by microorganisms to express endogenous proteins. Examples of pBR322 derivatives for the expression of specific antibodies are described in detail in Carter et al., US Patent No. 5,648,237.

In addition, phage vectors containing replicons and control sequences compatible with the host microorganism can be used as transformation vectors in conjunction with these hosts. For example, bacteriophages such as λGEM.TM.-11 can be used to prepare recombinant vectors, which can be used to transform sensitive host cells such as E. coli LE392.

The expression vector of the present invention may include two or more promoter-cistronic pairs encoding each of the polypeptide components. A promoter is an untranslated regulatory sequence located upstream (5') of the cistron that regulates its performance. Prokaryotic promoters are typically divided into two categories: inducible and constitutive. An inducible promoter is a promoter that initiates an increase in cistronic transcription in response to changes in culture conditions (for example, the presence or absence of nutrients or temperature changes) under its control.

Many promoters recognized by a variety of potential host cells are well known. The selected promoter is operably linked to the cistronic DNA encoding the light chain or the heavy chain by removing the promoter from the source DNA by digestion with restriction enzymes and inserting the isolated promoter sequence into the present invention The carrier to achieve. Both the native promoter sequence and many heterologous promoters can be used to guide the amplification and/or expression of the target gene. In some embodiments, as compared to native target polypeptide promoters, heterologous promoters generally achieve more transcription and higher yield of the expressed target gene, so heterologous promoters are used.

Promoters suitable for use with prokaryotic hosts include the PhoA promoter, β-galactase and lactose promoter systems, tryptophan (Trp) promoter systems, and hybrid promoters, such as tac or trc promoters. However, other promoters that function in bacteria (such as other known bacterial or phage promoters) are also suitable. Its nucleotide sequence has been published, thereby enabling skilled workers to operably use linkers or adaptors to join the nucleotide sequence to the cistron encoding the target light chain and heavy chain (Siebenlist et al. (1980) Cell 20: 269) to provide any required restriction sites.

In one aspect of the invention, each cistron in the recombinant vector contains a secretory signal sequence component that directs the translocation of the expressed polypeptide across the membrane. Generally, the signal sequence can be a component of the vector, or it can be a part of the target polypeptide DNA inserted into the vector. The signal sequence selected for the purpose of the present invention should be a signal sequence that is recognized and processed (ie, cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native signal sequence of a heterologous polypeptide, the signal sequence is replaced with a prokaryotic signal sequence selected from, for example, alkaline phosphatase, penicillinase, Ipp, or thermostable enterotoxin II (STII) leader, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of the present invention, the signal sequence used in the two cistrons of the expression system is the STII signal sequence or a variant thereof.

In another aspect, the production of the immunoglobulin according to the present invention can be carried out in the cytoplasm of the host cell, and therefore does not require the presence of a secretion signal sequence within each cistron. In this regard, immunoglobulin light chains and heavy chains are expressed, folded and assembled in the presence or absence of sequences of masking peptides, linking sequences, etc. to form functional immunoglobulins in the cytoplasm. Certain host strains (such as the E. coli trxB-strain) provide cytoplasmic conditions that are conducive to the formation of disulfide bonds, thereby achieving the correct folding and assembly of the expressed protein subunits. Proba and Pluckthun, Gene, 159:203 (1995).

The activatable and masked anti-CTLA4 binding protein of the present invention can also be produced using an expression system, in which the quantitative rate of expressed polypeptide components can be adjusted to maximize the secretion and production of appropriately assembled antibodies of the present invention. Such regulation is achieved, at least in part, by simultaneously regulating the translation intensity of the polypeptide components.

Prokaryotic host cells suitable for expressing the activatable and masked anti-CTLA4 binding protein of the present invention include Archaebacteria and Eubacteria, such as Gram-negative or Gram-positive. -positive) organisms. Examples of suitable bacteria include Escherichia (e.g. Escherichia), bacilli (e.g. B. subtilis), intestinal bacteria, pseudomonas species (e.g. Pseudomonas aeruginosa ( P. aeruginosa)), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobium Genus (Rhizobia), Vitreoscilla (Vitreoscilla) or Paracoccus (Paracoccus). In one embodiment, Gram-negative cells are used. In one embodiment, E. coli cells are used as the host of the present invention. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, Volume 2 (Washington, DC: American Society for Microbiology, 1987), pages 1190-1219; ATCC deposit number 27,325) and derivatives thereof, including Genotype W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8 ΔompTΔ(nmpc-fepE) degP41 kanR strain 33D3 (US Patent No. 5,639,635). Other strains and their derivatives, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli lambda 1776 (ATCC 31,537) and E. coli RV308 (ATCC 31,608) are also suitable. These examples are illustrative and not restrictive. Methods for constructing derivatives of any of the above-mentioned bacteria having a defined genotype are known in the art and described in, for example, Bass et al., Proteins, 8:309-314 (1990). Considering the replicability of the replicon in bacterial cells, it is usually necessary to select suitable bacteria. For example, when a well-known plastid (such as pBR322, pBR325, pACYC177 or pKN410) is used to supply the replicon, it is preferable to use Escherichia coli, Serratia or Salmonella species as the host. Generally, host cells should secrete the least amount of proteolytic enzymes, and other protease inhibitors should be incorporated into the cell culture. b) Binding protein production

The host cell is transformed with the above-mentioned expression vector and cultured in a conventional nutrient medium modified as appropriate to induce a promoter, select a transformant, or amplify a gene encoding a desired sequence.

Transformation means the introduction of DNA into a prokaryotic host so that the DNA can be replicated in the form of extrachromosomal elements or by chromosomal integrants. Depending on the host cell used, transformation is performed using standard techniques suitable for such cells. Calcium treatment with calcium chloride is usually used for bacterial cells with substantial cell wall barriers. Another transformation method uses polyethylene glycol/DMSO. Another technique used is electroporation.

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

In addition to carbon, nitrogen, and inorganic phosphate sources, any necessary supplements at appropriate concentrations may also be included, which are introduced alone or as a mixture with other supplements or culture media such as complex nitrogen sources. Optionally, the medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, dithioerythritol, and dithiothreitol.

Prokaryotic host cells are cultured at a suitable temperature. In certain embodiments, for E. coli growth, the growth temperature is in the range of about 20°C to about 39°C; in the range of about 25°C to about 37°C; or about 30°C. The pH of the culture medium can be any pH in the range of about 5 to about 9, depending on the host organism. In certain embodiments, for E. coli, the pH is about 6.8 to about 7.4, or about 7.0.

If an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for activating the promoter. In one aspect of the invention, the PhoA promoter is used to control the transcription of the polypeptide. Therefore, the transformed host cells are cultured in a phosphate-restricted medium for induction. In certain embodiments, the phosphate-restricted medium is C.R.A.P. medium (see, for example, Simmons et al., J. Immunol. Methods (2002), 263:133-147). Depending on the vector construct used, a variety of other inducers can be used, as are known in the art.

In one embodiment, the expressed activatable masked anti-CTLA4 binding protein of the present invention is secreted into the periplasm of the host cell and recovered from the periplasm of the host cell. Protein recovery typically involves the destruction of microorganisms, usually by means such as osmotic shock, sonication or lysis. After cell destruction, cell debris or complete 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 into the medium and separated from it. The cells can be removed from the culture, and the culture supernatant can be filtered and concentrated to further purify the protein produced. The expressed polypeptides can be further separated and identified using commonly known methods (such as polyacrylamide gel electrophoresis (PAGE) and Western blot analysis).

In one aspect of the present invention, a large amount of activatable masked anti-CTLA4 binding protein is produced by a fermentation process. Various large-scale batch feed fermentation procedures can be used to produce recombinant protein. Large-scale fermenters have a capacity of at least 1,000 liters and, in certain embodiments, have a capacity of about 1,000 to 100,000 liters. These fermenters use agitator impellers to distribute oxygen and nutrients, especially glucose. Small-scale fermenting usually refers to fermenting in a fermenter whose volume capacity does not exceed about 100 liters and can range from about 1 liter to about 100 liters.

In the fermentation process, protein expression is typically induced after the cells have grown to a desired density (for example, an OD550 of about 180-220) under suitable conditions. At this stage, the cells are in an early stationary phase. Depending on the vector construct used, a variety of inducers can be used, as is known in the art and described above. Cells can grow for a shorter period of time before induction. Cells are usually induced for about 12-50 hours, but longer or shorter induction times can be used.

In order to improve the yield and quality of the polypeptide of the present invention, various fermentation conditions can be modified. For example, in order to improve the correct assembly and folding of the secreted antibody polypeptide, other carriers that have expressed chaperone proteins (such as Dsb protein (DsbA, DsbB, DsbC, DsbD and/or DsbG) or FkpA (with chaperone activity) can be used. The peptidyl proline cis and trans-isomerase)) co-transform host prokaryotic cells. It has been proven that companion proteins can promote the correct 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.

In order to minimize the proteolysis of expressed heterologous proteins (especially heterologous proteins that are sensitive to proteolysis), the present invention can use certain proteolytic enzyme-deficient host strains. For example, the host cell strain can be modified to achieve genetic mutations 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 protease-deficient E. coli strains are available and described in, for example, Joly et al. (1998), see 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).

In one embodiment, an E. coli strain lacking proteolytic enzymes and transformed into a plastid expressing one or more accompanying proteins is used as the host cell in the expression system of the present invention. c) Binding protein purification

In one embodiment, the masked antibody protein produced herein is further purified to obtain a substantially homogeneous preparation for other analytical methods and uses. Standard protein purification methods known in the art can be used. The following procedures illustrate suitable purification procedures: immunoaffinity or fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or cation exchange resins (such as DEAE), chromatographic focusing, SDS- PAGE, ammonium sulfate precipitation, and gel filtration using, for example, Sephadex G-75.

In one aspect, the antibody product of the present invention is immunoaffinity purified using protein A immobilized on a solid phase. Protein A is a 41 kD cell wall protein from Staphylococcus aureas, which binds to the Fc region of antibodies with high affinity. Lindmark et al. (1983) J. Immunol. Meth. 62:1-13. The solid phase for immobilizing protein A can be a column containing glass or silica surface, or a controlled microporous glass column or silicic acid column. In some applications, the column is coated with a reagent such as glycerol to possibly prevent non-specific adhesion of contaminants.

As the first step of purification, the cell culture-derived preparation as described above can be applied to the solid phase of protein A immobilization to achieve specific binding of related antibodies to protein A. The solid phase is then washed to remove contaminants that are non-specifically bound to the solid phase. Finally, the relevant antibodies are recovered from the solid phase by dissociation. 10. Use eukaryotic host cells to produce binding proteins

Vectors used in eukaryotic host cells usually include one or more of the following non-limiting components: a signal sequence, an origin of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences. a) Signal sequence components

Vectors used in eukaryotic host cells may also contain signal sequences or other polypeptides with specific cleavage sites at the N-terminus of the relevant mature protein or polypeptide. The selected heterologous signal sequence can be a signal sequence that is recognized and processed (ie, cleaved by a signal peptidase) by the host cell. In mammalian cell performance, mammalian signal sequences and viral secretory leaders can be used, such as herpes simplex gD signal. The DNA of this precursor region is joined to the DNA encoding the antibody in reading frame. b) Origin of replication

Generally, mammalian expression vectors do not require an origin of replication component. For example, the SV40 origin can usually be used simply because it contains an early promoter. c) Select gene components

Expression and selection vectors can contain selection genes, also known as selectable markers. Typical selection genes encode the following proteins: (a) confer resistance to antibiotics or other toxins (such as ampicillin, neomycin, methotrexate or tetracycline), (b) insufficient complement auxotrophy (if relevant), Or (c) Provide important nutrients that cannot be obtained from the complex medium.

An example of the selection process uses drugs to retard the growth of host cells. These cells successfully transformed by the heterologous gene produce proteins that confer drug resistance and therefore survive selective therapies. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.

Another example of a selectable marker suitable for mammalian cells is a marker that enables the identification of cells capable of absorbing the activatable masked anti-CTLA4 binding protein-encoding nucleic acid, such as DHFR, thymidine kinase, metallothionein-I And metallothionein-II, primate metallothionein gene, adenosine deaminase, ornithine decarboxylase, etc.

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

Alternatively, the coded activatable can be selected by cell growth in a medium containing a selection agent for a selectable marker (such as an aminoglycoside antibiotic, such as kanamycin, neomycin, or G418) The masked anti-CTLA4 binding protein, wild-type DHFR protein and another selectable marker (such as aminoglycoside 3'-phosphotransferase (APH)) transformed or co-transformed host cells (especially those containing endogenous DHFR) Wild-type host). See U.S. Patent No. 4,965,199. Host cells may include NS0, including glutamine synthase (GS)-deficient cell lines. The method of using GS as a selectable marker for mammalian cells is described in U.S. Patent No. 5,122,464 and U.S. Patent No. 5,891,693. d) Promoter components

Expression and selection vectors usually contain a promoter that is recognized by the host organism and operably linked to the nucleic acid encoding the relevant activatable masked anti-CTLA4 binding protein. The promoter sequence of eukaryotes is known. For example, almost all eukaryotic genes have AT-rich regions, which are located about 25 to 30 bases upstream of the transcription start site. Another sequence found 70 to 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 the AATAAA sequence, which can be a signal for adding the poly A tail region to the 3'end of the coding sequence. In certain embodiments, any or all of these sequences can be appropriately inserted into the eukaryotic expression vector.

It can control the transcription from the vector in mammalian host cells, for example, by the promoter obtained from the following: the genome of viruses, such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine nipple Oncovirus, avian sarcoma virus, cell megavirus, retrovirus, hepatitis B virus and simian virus 40 (SV40); heterologous mammalian promoters, such as actin promoter or immunoglobulin promoter; heat shock promoter The restriction is that such promoters are compatible with the host cell system.

The early and late promoters of the SV40 virus are preferably obtained in the form of SV40 restriction fragments that also contain the origin of replication of the SV40 virus. The immediate early promoter of human cell megavirus is preferably obtained as a HindIII E restriction fragment. A system for expressing DNA using bovine papilloma virus as a vector in a mammalian host is disclosed in US Patent No. 4,419,446. The modification of this system is described in US Patent No. 4,601,978. See also Reyes et al., Nature 297:598-601 (1982), which describes the expression of human beta-interferon cDNA in murine cells under the control of the thymidine kinase promoter from herpes simplex virus. Alternatively, Rous Sarcoma Virus (Rous Sarcoma Virus) long terminal repeat can be used as a promoter. e) Strengthen the sub-element components

The transcription of the DNA encoding the antibody of the present invention by higher eukaryotes is usually increased by inserting enhancer sequences into the vector. Many enhancer sequences from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin) are now known. However, usually enhancers from eukaryotic cell viruses will be used. Examples include the SV40 enhancer on the late side of the replication source (bp 100-270), the human cell megavirus early promoter enhancer, the murine cell megavirus early promoter enhancer, and the polyoma virus on the late side of the replication source Enhancer and adenovirus enhancer. See also Yaniv, Nature 297:17-18 (1982), which describes enhancer elements for the activation of eukaryotic promoters. The enhancer can be spliced into the vector at the 5'or 3'position of the antibody polypeptide coding sequence, but is usually located at the 5'position relative to the promoter. f) Transcription termination component

Expression vectors used in eukaryotic host cells may also contain sequences required for transcription termination and mRNA stabilization. Such sequences are usually available from the 5'and occasionally 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. A suitable transcription termination component is the polyadenylation region of bovine growth hormone. See WO94/11026 and the expression vectors disclosed therein. g) Selection and transformation of host cells

Host cells suitable for the selection or expression of DNA in vectors herein include the higher eukaryotic cells described herein, including vertebrate host cells. The propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of suitable mammalian host cell lines are monkey kidney CV1 cell lines transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell lines (293 or 293 sub-selected to grow in suspension culture) Cells, 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)); murine sertoli cell (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cell (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (buffalo rat liver cell) (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); murine breast tumors (MMT 060562, ATCC CCL51); TRI cells (Mather Et al., Annals N. Y Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human liver tumor cell line (Hep G2).

The host cell is transformed with the above-mentioned expression or selection vector used for the production of activatable masked anti-CTLA4 binding protein and cultured in a modified conventional nutrient medium as appropriate to induce promoter, selective transformant or amplification The gene encoding the desired sequence. h) Cultivation of host cells

The host cells used to produce the activatable and masked anti-CTLA4 binding protein of the present invention can be cultured in a variety of 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 cell. In addition, any of the media described in the following documents can be used as the host cell culture medium: Ham et al., Meth. Enz. 58:44 (1979); Barnes et al., Anal. Biochem. 102:255 (1980) ; US Patent No. 4,767,704, No. 4,657,866, No. 4,927,762, No. 4,560,655 or No. 5,122,469; WO 90/03430; WO 87/00195; or US Patent Reexamination Case 30,985. Any of these media can be supplemented with hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ drugs), trace elements (defined as inorganic compounds usually present in a final concentration in the micromolar range), and glucose or equivalent energy sources . It is also possible to include any other supplements at suitable concentrations known to those skilled in the art. The culture conditions (such as temperature, pH, and the like) are previously used for the host cells selected for expression, and will be obvious to those skilled in the art. i) Purification of binding protein

When using recombinant technology, the activatable masked anti-CTLA4 binding protein can be produced intracellularly or directly secreted into the culture medium. If the antibody is produced in the cell, as the first step, the particulate debris (host cell or lysed fragment) can be removed, for example, by centrifugation or ultrafiltration. In the case where the activatable masked anti-CTLA4 binding protein is secreted into the culture medium, a commercially available protein concentration filter (such as an Amicon or Millipore Pellicon ultrafiltration unit) can be used first to concentrate the supernatant from such a performance system. Protease inhibitors such as PMSF can be included in any previous steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants.

The antibody composition prepared from the cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, among which affinity chromatography is a convenient technique. The suitability of protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Methods 62:1-13 (1983)). It is recommended that protein G be used for all murine isotypes and human γ3 (Guss et al., EMBO J. 5: 15671575 (1986)). The matrix to which the affinity ligand is attached can be agarose, but other matrices can be used. Compared to what can be achieved with agarose, mechanically stable matrices such as controlled microporous glass or poly(styrene divinyl)benzene achieve faster flow rates and shorter processing times. Where the antibody contains a CH3 domain, Bakerbond ABX™ resin (J. T. Baker, Phillipsburg, N.J.) is suitable for purification. Depending on the antibody to be recovered, other protein purification techniques can also be used, such as fractionation on an ion exchange column, ethanol precipitation, reverse phase HPLC, silica chromatography, heparin SEPHAROSE™ chromatography, anion or cation exchange resin (Such as polyaspartic acid column) chromatography, chromatographic focusing, SDS-PAGE and ammonium sulfate precipitation.

After any preliminary purification steps, the mixture containing the relevant masked binding proteins and contaminants can undergo further purification, for example by performing at low salt concentrations (e.g., about 0-0.25 M salt) using a pH of about 2.5- Low pH hydrophobic interaction chromatography with dissociation buffer between 4.5.

Generally, various methods for preparing antibodies for research, testing, and clinical use are recognized in the art, conform to the above-mentioned methods, and/or are deemed suitable for specific related antibodies by those familiar with the technology. IV. Composition

In some aspects, also provided herein are compositions (eg, pharmaceutical compositions) that include any of the activatable masked anti-CTLA4 binding proteins described herein.

The therapeutic formulation for storage is prepared by mixing the active ingredient with the required purity and optionally pharmaceutically acceptable carriers, excipients or stabilizers (Remington: The Science and Practice of Pharmacy, 20th edition, published by Lippincott Williams & Wiklins, edited by Gennaro, Philadelphia, Pa. 2000). Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the dose and concentration used, and include buffers, antioxidants (including ascorbic acid, methionine, vitamin E, and sodium metabisulfite); Preservatives, isotonic agents (such as sodium chloride), stabilizers, metal complexes (such as Zn-protein complexes); chelating agents, such as EDTA and/or nonionic surfactants.

The buffer can be used to control the pH within the range of optimal therapeutic effectiveness, especially when the stability is pH-dependent. The buffer may be present in a concentration in the range of about 20 mM to about 250 mM. Suitable buffers for the present invention include organic acids and inorganic acids and their salts. For example, citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. In addition, the buffer may contain histidine and trimethylamine salts, such as Tris.

Preservatives can be added to prevent the growth of microorganisms, and are usually present in the range of about 0.2%-1.0% (w/v). Preservatives suitable for use in the present invention include octadecyl dimethyl benzyl ammonium chloride; hexahydroxy quaternary ammonium chloride; benzalkonium halides (such as chloride, bromide, iodide), benzethon chloride Ammonium; thimerosal, phenol, butanol or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol, 3- Amyl alcohol and m-cresol.

Tonicity agents (sometimes called "stabilizers") may be present to adjust or maintain the tonicity of the liquid in the composition. When used with large charged biomolecules (such as proteins and antibodies), they are often referred to as "stabilizers" because they can interact with the charged groups of the amino acid side chains, thereby reducing intermolecular and intramolecular The possibility of interaction. Considering the relative amounts of other ingredients, the tonicity agent may be present in any amount between about 0.1% to about 25% by weight or between about 1 to about 5% by weight. In some embodiments, tonicity agents include polyhydric sugar alcohols, triols, or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol, and mannitol.

Other excipients include agents that can act as one or more of the following: (1) bulking agent, (2) solubility enhancer, (3) stabilizer, and (4) agent that prevents denaturation or adhesion to the container wall . Such excipients include: polyhydroxy sugar alcohols (listed above); amino acids, such as alanine, glycine, glutamic acid, asparagine, histidine, arginine, lysine Acid, ornithine, leucine, 2-phenylalanine, glutamine, threonine, etc.; organic sugars or sugar alcohols, such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose , Xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclic alcohol (such as inositol), polyethylene glycol; sulfur-containing reducing agent, Such as urea, glutathione, lipoic acid, sodium thioacetate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin or others Immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g. xylose, mannose, fructose, glucose; disaccharides (e.g. lactose, maltose, sucrose); trisaccharides such as raffinose; and Polysaccharides, such as dextrin or polydextrose.

Non-ionic surfactants or detergents (also known as "humectants") may be present to help dissolve the therapeutic agent and protect the therapeutic protein from aggregation induced by stirring. It also allows the formulation to be exposed to shear surface stress without causing Denaturation of active therapeutic proteins or antibodies. The nonionic surfactant is present in the range of about 0.05 mg/ml to about 1.0 mg/ml or about 0.07 mg/ml to about 0.2 mg/ml. In some embodiments, the nonionic surfactant is present in the range of about 0.001% to about 0.1% w/v or about 0.01% to about 0.1% w/v or about 0.01% to about 0.025% w/v.

Suitable nonionic surfactants include polysorbate (20, 40, 60, 65, 80, etc.), polyoxamer (184, 188, etc.), PLURONIC® polyol, TRITON®, polyoxyethylene Sorbitan monoether (TWEEN®-20, TWEEN®-80, etc.), lauryl alcohol 400, polyethylene glycol 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate Acid esters, sucrose fatty acid esters, methyl cellulose and carboxymethyl cellulose. Usable anionic detergents include sodium lauryl sulfate, sodium dioctyl sulfosuccinate and sodium dioctyl sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

In order for the formulation to be used for in vivo administration, it must be sterile. The formulation can be sterilized by filtering through a sterile filter membrane. The therapeutic composition herein is usually placed in a container with a sterile access port, such as an intravenous solution bag or bottle with a stopper pierceable by a hypodermic injection needle.

The route of administration is based on known and acceptable methods, such as by single or multiple rapid injections or infusions in a suitable manner over a long period of time, for example, by subcutaneous, intravenous, intraperitoneal, intramuscular, and intraarterial , Injection or infusion via intralesional or intraarticular route, local administration, inhalation or by sustained release or sustained release methods.

The activatable masked anti-CTLA4 binding proteins described herein (eg, activatable masked anti-CTLA4 antibodies or antigen-binding fragments thereof) can be used alone or in combination with other therapeutic agents, such as the methods described herein middle. The term "combination" encompasses two or more therapeutic agents (e.g., activatable masked anti-CTLA4 binding protein and therapeutic agents) included in the same or separate formulations. In some embodiments, "combination" refers to "simultaneous" administration, in which case, the administration of the activatable masked anti-CTLA4 binding protein of the present invention is carried out simultaneously with the administration of one or more other therapeutic agents (for example, in The interval between the administration of the activatable masked anti-CTLA4 binding protein and the administration of one or more other therapeutic agents at the same time or within one hour). In some embodiments, "combination" refers to sequential administration, in which case, the administration of the activatable masked anti-CTLA4 binding protein of the present invention is performed before and/or after the administration of one or more other therapeutic agents (For example, the interval between the administration of the activatable masked anti-CTLA4 binding protein and the administration of one or more other therapeutic agents is greater than one hour). The agents covered herein include (but are not limited to) cytotoxic agents, cytokines, agents targeting immune checkpoint molecules, agents targeting immunostimulatory molecules, or growth inhibitors.

The formulation herein may also contain more than one active compound necessary for the specific indication being treated, preferably active compounds with complementary activities that do not adversely affect each other. Alternatively or in addition, the composition may include a cytotoxic agent, a cytokine, an agent targeting an immune checkpoint molecule or a stimulating molecule, or a growth inhibitory agent. Such molecules are preferably present in a combination of amounts effective to achieve the desired purpose. V. Treatment methods

Provided herein is a method for treating or preventing a disease in an individual, which comprises administering to the individual an effective amount of an activatable masked anti-CTLA4 binding protein described herein (e.g., an activatable masked anti-CTLA4 antibody Or an antigen-binding fragment thereof) or a composition thereof. In some embodiments, the individual (e.g., human patient) has been diagnosed with a neoplastic disorder (e.g. cancer) or is at risk of developing such a disorder.

For the prevention or treatment of diseases, as defined above, the appropriate dosage of the active agent will depend on the type of disease to be treated, the severity and course of the disease, the administration of the agent for prophylactic or therapeutic purposes, previous therapies, and the individual It depends on the clinical history and response to the medication and the judgment of the attending physician. It is advisable to administer the agent to the individual at one time or over a series of treatments. In some embodiments of the methods described herein, the interval between administrations of the described activatable masked anti-CTLA4 binding protein (e.g., activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof) is About a month or more. In some embodiments, the interval between administrations is about two months, about three months, about four months, about five months, about six months, or longer. As used herein, the interval between administrations refers to the period of time between one antibody administration and the next antibody administration. As used herein, an interval of about one month includes four weeks. In some embodiments, the interval between administrations is about two weeks, about three weeks, about four weeks, about eight weeks, about twelve weeks, about sixteen weeks, about twenty weeks, about twenty four weeks, or longer time. In some embodiments, treatment includes multiple administrations of the antibody, wherein the interval between administrations can be different. For example, the interval between the first administration and the second administration is about one month, and the interval between subsequent administrations is about three months. In some embodiments, the interval between the first administration and the second administration is about one month, the interval between the second administration and the third administration is about two months, and the interval between subsequent administrations For about three months. In some embodiments, the activatable masked anti-CTLA4 binding protein described herein (e.g., the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof) is administered at a uniform dose. Depending on the type and severity of the disease, about 1 µg/kg to 15 mg/kg (for example, 0.1 mg/kg-10 mg/kg) of antibodies can be the initial candidate dose for administration to patients, whether for example by One or more independent administrations or by continuous infusion. Depending on the factors mentioned above, a typical daily dose may range from about 1 μg/kg to 100 mg/kg or more. For repeated administrations over several days or longer, depending on the condition, the treatment will usually continue until the necessary suppression of disease symptoms occurs. An exemplary dose of antibody would be in the range of about 0.05 mg/kg to about 10 mg/kg. Therefore, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or any combination thereof) can be administered to the patient. In some embodiments, the activatable masked anti-CTLA4 binding protein described herein (e.g., activatable masked anti-CTLA4 antibody) is administered to the individual in a dose of about 25 mg to about 500 mg per administration. Or its antigen-binding fragment). In some embodiments, the activatable masked anti-CTLA4 binding described herein is administered to the individual at a dose of about 0.1 mg/kg to about 10 mg/kg or about 1.0 mg/kg to about 10 mg/kg Protein (e.g., activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof). At about 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 3.0 mg/kg, 3.5 mg/kg, 4.0 mg/kg, 4.5 mg/kg kg, 5.0 mg/kg, 5.5 mg/kg, 6.0 mg/kg, 6.5 mg/kg, 7.0 mg/kg, 7.5 mg/kg, 8.0 mg/kg, 8.5 mg/kg, 9.0 mg/kg, 9.5 mg/ A dose of either kg or 10.0 mg/kg administers the activatable masked anti-CTLA4 binding protein described herein (e.g., activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof) as described herein. In some embodiments, between 0.1 mg/kg and 10 mg/kg or between about 0.1 mg/kg and 10 mg/kg, between 10 mg/kg and 20 mg/kg, or between about 10 mg/kg and Between 20 mg/kg, between 20 mg/kg and 30 mg/kg, or between about 20 mg/kg and 30 mg/kg, between 30 mg/kg and 40 mg/kg, or between about 30 mg/kg and Between 40 mg/kg, between 40 mg/kg and 50 mg/kg, or between about 40 mg/kg and 50 mg/kg, between 50 mg/kg and 60 mg/kg, or between about 50 mg/kg and Between 60 mg/kg, between 60 mg/kg and 70 mg/kg, or between about 60 mg/kg and 70 mg/kg, between 70 mg/kg and 80 mg/kg, or between about 70 mg/kg and The activatable masked anti-CTLA4 binding protein described herein (e.g., the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof) is administered to an individual at a dose of between 80 mg/kg. Any of the dosing frequencies described above can be used.

The method of treatment encompassed herein is the use of the activatable masked anti-CTLA4 binding protein described herein (e.g., an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof) to treat a condition or disease. Conditions or diseases that can be treated with the formulation of the present invention include leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma, lung cancer, ovarian cancer, osteosarcoma, Bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer (e.g. Merkel cell carcinoma) or testicular cancer.

In some embodiments, provided herein is a treatment or prevention by administering the activatable masked anti-CTLA4 binding protein described herein (for example, an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof) Methods of cancer. As used herein, the term "cancer" refers to all types of cancers, neoplasms or malignant tumors found in mammals, including leukemias, lymphomas, melanomas, neuroendocrine tumors, carcinomas and sarcomas. Exemplary cancers that can be treated with the activatable masked anti-CTLA4 binding protein (e.g., activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof), pharmaceutical compositions or methods provided herein include lymphoma, sarcoma, bladder Cancer, bone cancer, brain tumor, cervical cancer, colon cancer, esophageal cancer, stomach cancer, head and neck cancer, kidney cancer, myeloma, thyroid cancer, leukemia, prostate cancer, breast cancer (e.g. triple negative, ER positive, ER negative, chemical Therapy resistance, Herceptin resistance, HER2 positive, doxorubicin resistance, tamoxifen resistance, ductal cancer, lobular cancer, primary, metastatic), ovarian Cancer, pancreatic cancer, liver cancer (e.g. hepatocellular carcinoma), lung cancer (e.g. non-small cell lung cancer, squamous cell lung cancer, adenocarcinoma, large cell lung cancer, small cell lung cancer, carcinoid, sarcoma), pleomorphic nerve Glioblastoma, glioma, melanoma, prostate cancer, castration-resistant prostate cancer, breast cancer, triple-negative breast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cell carcinoma (e.g., head, neck Or esophagus), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B-cell lymphoma, or multiple myeloma. Other examples include thyroid, endocrine system, brain, breast, cervix, colon, head and neck, esophagus, liver, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach , Cancer of the uterus, or Medulloblastoma, Hodgkin's Disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, glioma, pleomorphism Glioblastoma, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumor, cancer, malignant insulinoma, malignant carcinoid, bladder cancer, premalignant skin lesions , Testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, endocrine or exocrine pancreatic neoplasm, medullary thyroid Cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, Paget's Disease of the nipple, phyllodes tumor, lobular carcinoma, ductal carcinoma, pancreatic stellate Form cell carcinoma, hepatic stellate cell carcinoma, or prostate cancer.

In some embodiments, provided herein is a treatment or prevention by administering the activatable masked anti-CTLA4 binding protein described herein (for example, an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof) The method of leukemia. The term "leukemia" broadly refers to a progressive and malignant disease of the hematopoietic organs, and is usually characterized by the abnormal proliferation and development of white blood cells and their precursors in the blood and bone marrow. Leukemia is usually classified clinically based on the following: (1) the duration and characteristics of the disease-acute or chronic; (2) the cell type involved; bone marrow (myeloid), lymph (lymphogenesis) or monocytic; and ( 3) Increase or no increase in the number of abnormal cells in the blood-leukemia or leukemia (sub-leukemia). Exemplary leukemias that can be treated with the compounds, pharmaceutical compositions or methods provided herein include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, and acute promyelocytic leukemia , Adult T-cell leukemia, leukemia leukemia, leukocyte leukemia, basophilic leukemia, blastoblastic leukemia, bovine leukemia, chronic myelogenous leukemia, skin leukemia, embryonic leukemia, eosinophilic leukemia, Gross leukemia ( Gross' leukemia), hairy cell leukemia, hemoblastic leukemia, hematopoietic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenia leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia Leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocyte leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelogenous leukemia, myelogenous leukemia, Myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasma cell leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling leukemia ( Schilling's leukemia), stem cell leukemia, subleukemia or undifferentiated cell leukemia.

In some embodiments, provided herein is a treatment or prevention by administering the activatable masked anti-CTLA4 binding protein described herein (for example, an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof) The method of sarcoma. The term "sarcoma" generally refers to a tumor composed of materials such as embryonic connective tissue and usually composed of compact cells embedded in a fibrous or homogeneous material. Sarcomas that can be treated with the compounds, pharmaceutical compositions or methods provided herein include chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma (Abemethy's sarcoma), liposarcoma, lipid Sarcoma, soft tissue alveolar sarcoma, ameloblastic sarcoma, botryoid sarcoma, green tumor sarcoma, choriocarcinoma, embryonic sarcoma, Wilms' tumor sarcoma (Wilms' tumor sarcoma), endometrial sarcoma, stromal sarcoma, Ewing Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, B-cell immunoblastic sarcoma, lymph Tumors, T-cell immunoblastic sarcoma, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukemic sarcoma, malignant stromal tumor Sarcoma, extraperiosteal sarcoma, reticular cell sarcoma, Rous sarcoma, serous cystic sarcoma, synovial sarcoma, or capillary dilatation sarcoma.

In some embodiments, provided herein is a treatment or prevention by administering the activatable masked anti-CTLA4 binding protein described herein (for example, an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof) The method of melanoma. The term "melanoma" is used to mean tumors caused by the melanocyte system of the skin and other organs. Melanoma that can be treated with the compounds, pharmaceutical compositions or methods provided herein includes, for example, acro-freckle melanoma, amelanoma melanoma, benign juvenile melanoma, Cloudman's melanoma (Cloudman's melanoma) ), S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigines melanoma, malignant melanoma, nodular melanoma, scrotal melanoma or superficial Table diffuse melanoma.

In some embodiments, provided herein is a treatment or prevention by administering the activatable masked anti-CTLA4 binding protein described herein (for example, an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof) The method of cancer. The term "carcinoma" refers to a malignant new growth composed of epithelial cells that tend to infiltrate surrounding tissues and cause cancer metastasis. Exemplary carcinomas that can be treated with the compounds, pharmaceutical compositions or methods provided herein include, for example, medullary thyroid carcinoma, familial medullary thyroid carcinoma, acinar carcinoma, acinar carcinoma, adenosal carcinoma, adenoid Cystic cancer, carcinoma adenoma, carcinoma of the adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, basal cell carcinoma, basaloid carcinoma, basal squamous cell carcinoma, bronchioloalveolar carcinoma, Bronchiolar carcinoma, bronchial carcinoma, brain-like carcinoma, cholangiocarcinoma, choriocarcinoma, colloid carcinoma, acne carcinoma, uterine corpus carcinoma, ethmoid carcinoma, armored carcinoma, dermoid carcinoma , Cylindrical carcinoma, cylindrical cell carcinoma, ductal carcinoma, ductal carcinoma, sclerocarcinoma, embryonic carcinoma, brain-like carcinoma, epidermoid carcinoma, glandular epithelial carcinoma, exogenous carcinoma, ulcerative carcinoma, fibrous Carcinoma, glial carcinoma, colloid carcinoma, giant cell carcinoma, giant cell carcinoma, adenocarcinoma, granular cell carcinoma, hairy stromal carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma (Hurthle cell carcinoma) ), clear carcinoma, adrenoid carcinoma, infant embryonic carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, erosive ulcer (Krompecher's carcinoma), Kulchitzky-cell carcinoma (Kulchitzky-cell carcinoma, large cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatoid carcinoma, lobular carcinoma, lymphoepithelial carcinoma, medullary carcinoma, medullary carcinoma, melanoma , Medullary carcinoma, mucinous carcinoma, mucinous carcinoma, mucinous cell carcinoma, mucoepidermoid carcinoma, mucosal carcinoma, mucinous carcinoma, myxoma-like carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma, Osteoid carcinoma, papillary carcinoma, periportal carcinoma, carcinoma in situ, acanthocyte carcinoma, brain-like carcinoma, renal cell carcinoma of the kidney, reserve cell carcinoma, sarcomatoid carcinoma, Schneiderian carcinoma ), sclerocarcinoma, scrotal carcinoma, signet ring cell carcinoma, simplex carcinoma, small cell carcinoma, potato-like carcinoma, spheroid-like carcinoma, spindle cell carcinoma, medullary carcinoma, squamous carcinoma , Squamous Cell Carcinoma, Tethered Carcinoma, Capillary Dilatation Carcinoma, Vasodilatory Carcinoma, Transitional Cell Carcinoma, Massive Carcinoma, Tubular Carcinoma, Nodular Carcinoma, Verrucous Carcinoma or Villiform Carcinoma.

In some embodiments, provided herein is a treatment or prevention by administering the activatable masked anti-CTLA4 binding protein described herein (for example, an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof) Methods of metastatic cancer. As used herein, the terms "metastatic", "metastatic" and "metastatic cancer" are used interchangeably and refer to the spread of a neoplastic disease or condition (such as cancer) from one organ to another non-adjacent organ or Body part. Cancer occurs at an initial site (such as the breast), which is called a primary tumor, such as primary breast cancer. Some cancer cells in the primary tumor or initiation site acquire the ability to penetrate and infiltrate the surrounding normal tissues in the local area and/or the ability to penetrate the barriers of the lymphatic system or vascular system, and circulate through the system to other parts of the body Location and organization. Secondary clinically detectable tumors formed by cancer cells of the primary tumor are called metastatic or secondary tumors. When cancer cells metastasize, it is assumed that the metastatic tumor and its cells are similar to those of the original tumor. Therefore, if lung cancer metastasizes to the breast, the secondary tumor at the breast site is composed of abnormal lung cells and non-abnormal breast cells. The secondary tumor in the breast is called metastatic lung cancer. Therefore, the phrase metastatic cancer refers to a disease in which an individual has or has had a primary tumor and has one or more secondary tumors. The phrase non-metastatic cancer or a system with non-metastatic cancer refers to a disease in which an individual has a primary tumor but does not have one or more secondary tumors. For example, metastatic lung cancer refers to those in which the individual has a primary lung tumor or a history of a primary lung tumor and has one or more secondary tumors in a second location or multiple locations (e.g., in the breast) disease.

In some embodiments, diseases or disorders that can benefit from the masked CTLA4 binding protein described herein include diseases (completely or partially) caused by CTLA4 or CTLA4 activity or function (e.g., diabetes, cancer (e.g., prostate cancer) , Kidney cancer, metastatic cancer, melanoma, castration-resistant prostate cancer, breast cancer, triple-negative breast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cell carcinoma (e.g., head, neck, or esophagus), Colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B-cell lymphoma or multiple myeloma)) or symptoms of diseases (completely or partially) caused by CTLA4 or CTLA4 activity or function. VI. Products or sets

In another aspect, there is provided an article or kit comprising the activatable masked anti-CTLA4 binding protein described herein (for example, an activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof). The preparation or kit may further include instructions for using the binding protein in the method of the present invention. Therefore, in certain embodiments, the article or kit includes the use of an activatable masked anti-CTLA4 binding protein (e.g., an activatable masked The description of an anti-CTLA4 antibody or an antigen-binding fragment thereof includes administering to an individual an effective amount of an activatable masked anti-CTLA4 binding protein (for example, an activatable masked anti-CTLA4 antibody or an antigen-binding fragment thereof). In certain embodiments, the individual is a human. In some embodiments, the individual has a disease selected from the group consisting of leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma, lung cancer, Ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer or testicular cancer.

The article or kit may further comprise a container. Suitable containers include, for example, bottles, vials (such as dual-chamber vials), syringes (such as single-chamber syringes or dual-chamber syringes), and test tubes. The container can be formed of a variety of materials, such as glass or plastic. The container holds the formulation. In some embodiments, the formulation is a lyophilized formulation.

The article or kit may further include a label or package insert located on or associated with the container, which may indicate instructions for reconstitution and/or use of the formulation. The label or package insert may further indicate that the formulation is suitable or intended for subcutaneous, intravenous, or other modes of administration for the treatment or prevention of conditions (e.g., cancer) in the individual. The container for storing the formulation can be a single-use vial or a multiple-use vial, which allows repeated administration of the reconstituted formulation. The article or kit may further comprise a second container, which contains a suitable diluent. The product or kit may further include other materials required from a commercial, therapeutic, and user point of view, including other buffers, diluents, filters, needles, syringes, and drug instructions with instructions for use.

In a specific embodiment, the present invention provides a kit for a single-dose dosing unit. Such kits include containers for aqueous formulations of therapeutic antibodies, including single-chamber or multi-chamber pre-filled syringes. An exemplary pre-filled syringe is available from Vetter GmbH, Ravensburg, Germany.

Herein, the product or kit optionally further includes a container containing a second agent, wherein the activatable masked anti-CTLA4 binding protein (for example, the activatable masked anti-CTLA4 antibody or antigen-binding fragment thereof) is the first A medicament, and the product or kit further includes instructions on the label or package insert for treating the individual with an effective amount of the second medicament.

In another embodiment, provided herein is an article or kit comprising a formulation described herein for administration in an autoinjector device. An autoinjector can be described as an injection device that, after activation, will deliver its contents without other necessary operations from the patient or the administering person. It is especially suitable for self-administration of therapeutic formulations when the delivery rate must be constant and the delivery time needs to last for a period of time. Exemplary embodiment

The exemplary embodiments provided include: 1. A masked antibody comprising: a) an antibody or antigen-binding fragment thereof, which binds to 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 NO: 1-46, wherein the masking peptide is connected to the first chain of the antibody or antigen-binding fragment thereof via a linker comprising a cleavable peptide Or the amino or carboxyl end of the second chain. 2. The masked antibody of embodiment 1, wherein a) the first chain is a light chain; and b) the second chain is a heavy chain. 3. The masked antibody of embodiment 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises two first chains and two second chains. 4. The masked antibody of embodiment 1, wherein a) the first chain is a light chain variable domain; and b) the second chain is a heavy chain variable domain. 5. The masked antibody of any one of embodiments 1 to 4, wherein the antigen-binding fragment is a dAb, Fab, Fab'-SH, Fv, scFv or (Fab') ₂ fragment. 6. The masked antibody of any one of embodiments 1 to 5, wherein the amino or carboxyl end of the masked peptide is connected to a linker comprising a cleavable peptide. 7. The masked antibody of any one of embodiments 1 to 6, wherein the linker comprising the cleavable peptide comprises: a) a first spacer linker and a cleavable peptide; or b) a first spacer linker, Cleavable peptide and second spacer linker. 8. The masked antibody of any one of embodiments 1 to 7, wherein the cleavable peptide comprises an amino acid sequence selected from SEQ ID NO: 47-88, 464-469 and 479-508. 9. The masked antibody of any one of embodiments 1 to 8, wherein: a) the first spacer linker is directly connected to the N-terminus and/or C-terminus of the cleavable peptide; or b) the first The spacer linker is directly connected to the N-terminus of the cleavable peptide, and the second spacer linker is directly connected to the C-terminus of the cleavable peptide. 10. The masked antibody of any one of embodiments 7 to 9, wherein the first spacer linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 89-112 and 415-420; and/ Or the second spacer linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 89-112 and 415-420. 11. The masked antibody of any one of embodiments 1 to 10, wherein the linker comprising the cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 454-462. 12. The masked antibody of any one of embodiments 1 to 10, wherein at least one amino acid but no more than 20 amino acids is directly connected to the N-terminus of the masked peptide. 13. The masked antibody of embodiment 12, wherein the at least one amino acid is alanine (A) or glycine-alanine (GA). 14. The masked antibody of any one of embodiments 1 to 13, wherein the masked antibody in the N-to-C-terminal or C-to-N-terminal direction comprises: a) a masking peptide; b) a cleavable peptide; and c ) The first chain or the second chain of an antibody or antigen-binding fragment thereof that binds to CTLA4. 15. The masked antibody of embodiment 14, wherein a) the masked antibody comprises a spacer linker between the masking peptide and the cleavable peptide; and b) the masked antibody comprises the cleavable peptide and The spacer linker between the antibody or antigen-binding fragment thereof that binds to CTLA4. 16. The masked antibody of any one of embodiments 1 to 15, wherein the masked antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 113-231 and 444-453. 17. The masked antibody of any one of embodiments 1 to 16, wherein the antibody or antigen-binding fragment thereof is a murine antibody. 18. The masked antibody of any one of embodiments 1 to 16, wherein the antibody or antigen-binding fragment thereof is a humanized antibody, a chimeric antibody, or a human antibody. 19. The masked antibody of any one of embodiments 1 to 18, wherein the antibody or antigen-binding fragment thereof has an IgG1, IgG2, IgG3, or IgG4 isotype. 20. The masked antibody of embodiment 19, wherein the IgG1 comprises the following amino acid substitutions: a) S298A, E333A and K334A; b) S239D and I332E; c) S239D, A330L and I332E; d) P247I and A339D or A339Q; e) D280H, K290S (with or without S298D or S298V); f) F243L, R292P and Y300L; g) F243L, R292P, Y300L and P396L; h) F243L, R292P, Y300L, V305I and P396L; i) G236A , S239D and I332E; j) K326A and E333A; k) K326W and E333S; or 1) K290E or K290N, S298G, T299A and/or K326E; wherein the amino acid residues are numbered according to the EU index in Kabat. 21. The masked antibody of any one of embodiments 1 to 20, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein: a) the light chain variable region comprises (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 402, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 403, and (iii) CDR-L3, which includes The amino acid sequence of SEQ ID NO: 404; and/or the heavy chain variable region includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 405, (ii) CDR-H2, which includes The amino acid sequence of SEQ ID NO: 406, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 407; or b) the light chain variable region includes (i) CDR-L1, which Contains the amino acid sequence of SEQ ID NO: 408, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 409, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 410 Acid sequence; and/or the heavy chain variable region includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 411, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 412 Acid sequence, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 413; or c) the light chain variable region includes (i) CDR-L1, which includes the amine of SEQ ID NO: 432 Base acid sequence, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 433, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 434; and/or wherein The heavy chain variable region includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 435, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 436, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 437; or d) the light chain variable region includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 438, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 439, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 440; and/or wherein the heavy chain variable region includes ( i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 441, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 442, and (iii) CDR-H3, which includes SEQ ID NO: 442 ID NO: 443 amino acid sequence. 22. The masked antibody of any one of embodiments 1 to 21, wherein 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 comprises The heavy chain variable region of the amino acid sequence of SEQ ID NO: 233. 23. The masked antibody of embodiment 22, wherein 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 comprises a light chain selected from SEQ ID NO : The heavy chain of the amino acid sequence of 319 or 320. 24. The masked antibody of any one of embodiments 1 to 21, wherein the antibody or antigen-binding fragment thereof comprises: a) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 321; and/ Or the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 323; or b) the light chain variable region comprising the amino acid sequence of SEQ ID NO: 322; and/or the light chain variable region comprising the amino acid sequence of SEQ ID NO: 324 The variable region of the heavy chain of the amino acid sequence. 25. The masked antibody of embodiment 24, wherein the antibody comprises a light chain comprising an amino acid sequence selected from SEQ ID NO: 327-341; and/or comprises a light chain selected from SEQ ID NO: 366-380, The heavy chain of the amino acid sequence of 421 and 478. 26. The masked antibody of embodiment 24 or embodiment 25, wherein the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 334 and an amine comprising SEQ ID NO: 421 Or the antibody or the antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 327, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 478. 27. The masked antibody of any one of embodiments 1 to 26, wherein the cleavable peptide is a substrate for a protease, and the protease is co-located in a region with cells or tissues expressing CTLA4. 28. The masked antibody of any one of embodiments 1 to 27, wherein the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of 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, ADAMTS6, AB17ADAMT, ADAMTS6, AB17 ADAMTS7, ADAMTS8, ADAMTS9, ADAMTSL1, ADAMTSL2, ADAMTSL3, ABHD17C, ADAMTSL5, ASTL, BMP1, CELA1, CELA2A, CELA2B, CELA3A, CELA3B, ADAM10, ADAM15, ADAM17, ADAM9, ADAMTS4, CTSE, CTCSF, ADAMTSL 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, HTRA 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, The TINAG, TPSAB1, TPSD1 and TPSG1. 29. The masked antibody of embodiment 28, wherein 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. 30. The masked antibody of any one of embodiments 1 to 29, wherein the masked antibody comprises the amino acid sequence of SEQ ID NO: 421, and comprises a composition selected from SEQ ID NO: 358 and 422-431 The amino acid sequence of the group. 31. The masked antibody of any one of embodiments 1 to 30, wherein the antibody or antigen-binding fragment thereof binds to an agent. 32. The masked antibody of embodiment 31, wherein the agent is a tubulin polymerization inhibitor, a DNA damaging agent, or a DNA synthesis inhibitor. 33. The masked antibody of embodiment 32, wherein the agent is a maytansinoid, aristatin, pyrrolobenzodiazepine (PBD) dimer, calicheamicin, becarcinomycin, indole Doline benzodiazepine dimer or ixinotecan derivative Dxd. 34. 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, which comprises an amino acid sequence selected from SEQ ID NO: 1-46, wherein the masking peptide is connected to the amino terminal of the light chain or heavy chain of the first pair via a linker comprising a cleavable peptide Or the carboxyl end. 35. The masked bispecific antibody of embodiment 34, wherein the amino or carboxy terminus of the masking peptide is linked to a linker comprising a cleavable peptide. 36. The masked bispecific antibody of embodiment 34 or 35, wherein the linker comprising a cleavable peptide comprises: a) a first spacer linker and a cleavable peptide; or b) a first spacer linker, a Cleavage peptide and second spacer linker. 37. The masked bispecific antibody of any one of embodiments 34 to 36, wherein the cleavable peptide comprises an amino acid sequence selected from SEQ ID NO: 47-88, 464-469, and 479-508. 38. The masked bispecific antibody of embodiment 36 or embodiment 37, wherein: a) the first spacer linker is directly connected to the N-terminus or C-terminus of the cleavable peptide; or b) the first spacer The linker is directly connected to the N-terminus of the cleavable peptide, and the second spacer linker is directly connected to the C-terminus of the cleavable peptide. 39. The masked bispecific antibody of any one of embodiments 36 to 38, wherein the first spacer linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 89-112 and 415-420 And/or the second spacer linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 89-112 and 415-420. 40. The masked bispecific antibody of any one of embodiments 34 to 39, wherein the linker comprising a cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 454-462. 41. The masked bispecific antibody of any one of embodiments 34 to 40, wherein at least one amino acid but no more than 20 amino acids is directly connected to the N-terminus of the masked peptide. 42. The masked bispecific antibody of embodiment 41, wherein the at least one amino acid is alanine (A) or glycine-alanine (GA). 43. The masked bispecific antibody according to any one of embodiments 34 to 42, wherein the light chain or the heavy chain of the first pair in the N-to-C-terminal or C-to-N-terminal direction comprises: a) a masking peptide; b) cleavable peptide; and c) light chain or heavy chain. 44. The masked bispecific antibody of embodiment 43, wherein a) the first pair comprises a spacer linker between the masking peptide and the cleavable peptide; and b) the first pair comprises the cleavable peptide The spacer linker with the light chain or the heavy chain. 45. The masked bispecific antibody of any one of embodiments 34 to 44, wherein the masked bispecific antibody comprises an amine group selected from the group consisting of SEQ ID NO: 113-231 and 444-453 Acid sequence. 46. The masked bispecific antibody of any one of embodiments 34 to 45, wherein the bispecific antibody is a murine antibody. 47. The masked bispecific antibody of any one of embodiments 34 to 45, wherein the bispecific antibody is a humanized antibody, a chimeric antibody, or a human antibody. 48. The masked bispecific antibody of any one of embodiments 34 to 47, wherein the bispecific antibody has an IgG1, IgG2, IgG3, or IgG4 isotype. 49. The masked bispecific antibody of embodiment 48, wherein the IgG1 comprises the following amino acid substitutions: a) S298A, E333A and K334A; b) S239D and I332E; c) S239D, A330L and I332E; d) P247I And A339D or A339Q; e) D280H, K290S (with or without S298D or S298V); f) F243L, R292P and Y300L; g) F243L, R292P, Y300L and P396L; h) F243L, R292P, Y300L, V305I and P396L; i) G236A, S239D and I332E; j) K326A and E333A; k) K326W and E333S; or 1) K290E or K290N, S298G, T299A and/or K326E, where the amino acid residues are numbered according to the EU index as in Kabat . 50. The masked bispecific antibody of any one of embodiments 34 to 49, wherein the first pair comprises a light chain variable region and a heavy chain variable region, wherein: a) the light chain variable region comprises (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 402, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 403, and (iii) CDR-L3, which includes The amino acid sequence of SEQ ID NO: 404; and/or wherein the heavy chain variable region comprises (i) CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 405, (ii) CDR-H2, which Comprising the amino acid sequence of SEQ ID NO: 406, and (iii) CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 407; or b) the light chain variable region comprises (i) CDR-L1, It includes the amino acid sequence of SEQ ID NO: 408, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 409, and (iii) CDR-L3, which includes the amine of SEQ ID NO: 410 Base acid sequence; and/or wherein the heavy chain variable region comprises (i) CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 411, (ii) CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 412 Amino acid sequence, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 413; or c) the light chain variable region includes (i) CDR-L1, which includes SEQ ID NO: 432 (Ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 433, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 434; and/or Wherein the heavy chain variable region includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 435, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 436, and ( iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 437; or d) the light chain variable region includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 438, ( ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 439, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 440; and/or wherein the heavy chain variable region It includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 441, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 442, and (iii) CDR-H3, which Contains the amino acid sequence of SEQ ID NO:443. 51. The masked bispecific antibody of any one of embodiments 34 to 50, wherein the first pair comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 232; and/or comprises The heavy chain variable region of the amino acid sequence of SEQ ID NO: 233. 52. The masked bispecific antibody of embodiment 51, wherein the first pair comprises a light chain comprising an amino acid sequence selected from SEQ ID NO: 237-318; and/or comprises a light chain selected from SEQ ID NO : The heavy chain of the amino acid sequence of 319 or 320. 53. The masked bispecific antibody of any one of embodiments 34 to 50, wherein the first pair comprises: a) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 321; and/ Or the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 323; or b) the light chain variable region comprising the amino acid sequence of SEQ ID NO: 322; and/or the light chain variable region comprising the amino acid sequence of SEQ ID NO: 324 The variable region of the heavy chain of the amino acid sequence. 54. The masked bispecific antibody of embodiment 53, wherein the first pair comprises a light chain comprising an amino acid sequence selected from SEQ ID NO: 327-341; and/or comprises a light chain selected from SEQ ID NO : The heavy chain of the amino acid sequence of 366-380, 421 and 478. 55. The masked bispecific antibody of embodiment 53 or embodiment 54, wherein the first pair comprises a light chain comprising the amino acid sequence of SEQ ID NO: 334 and an amine comprising SEQ ID NO: 421 Or the first pair includes a light chain including the amino acid sequence of SEQ ID NO: 327, and a heavy chain including the amino acid sequence of SEQ ID NO: 478. 56. The masked bispecific antibody of any one of embodiments 34 to 55, wherein the cleavable peptide is a substrate for a protease that is co-located in a region with cells or tissues expressing CTLA4. 57. The masked antibody of any one of embodiments 34 to 56, wherein the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of 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, ADAMTS6, AB17ADAMT, ADAMTS6, AB17 ADAMTS7, ADAMTS8, ADAMTS9, ADAMTSL1, ADAMTSL2, ADAMTSL3, ABHD17C, ADAMTSL5, ASTL, BMP1, CELA1, CELA2A, CELA2B, CELA3A, CELA3B, ADAM10, ADAM15, ADAM17, ADAM9, ADAMTS4, CTSE, CTCSF, ADAMTSL 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, HTRA 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, TMPRSS7, PCPRSS0 , TINAG, TPSAB1, TPSD1 and TPSG1. 58. The masked bispecific antibody of embodiment 57, wherein 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. 59. The masked bispecific antibody of any one of embodiments 34 to 58, wherein the masked bispecific antibody comprises the amino acid sequence of SEQ ID NO: 421, and comprises the amino acid sequence selected from SEQ ID NO: The amino acid sequence of the group consisting of 358 and 422-431. 60. The masked bispecific antibody of any one of embodiments 34 to 59, wherein the masked bispecific antibody binds to an agent. 61. The masked antibody of embodiment 60, wherein the agent is a tubulin polymerization inhibitor, a DNA damaging agent, or a DNA synthesis inhibitor. 62. The masked antibody of embodiment 60, wherein the agent is maytansinoids, aristatin, pyrrolobenzodiazepine (PBD) dimer, calicheamicin, becarcinomycin, indole Doline benzodiazepine dimer or ixinotecan derivative Dxd. 63. A masked chimeric receptor, comprising: a) a ligand binding domain that binds to CTLA4, which comprises a first chain and a second chain; b) a masking peptide, which comprises a peptide selected from SEQ ID NO:1 -46 amino acid sequence, c) transmembrane domain; and d) intracellular signal transduction domain, which comprises a signal transduction domain, wherein the masking peptide is connected to the ligand binding domain via a linker comprising a cleavable peptide The amine or carboxyl end of the first or second chain. 64. The masked chimeric receptor of embodiment 63, wherein a) the first chain is a light chain variable domain; and b) the second chain is a heavy chain variable domain. 65. The masked chimeric receptor of embodiment 63 or 64, wherein the amino or carboxy terminus of the masked peptide is connected to a linker comprising a cleavable peptide. 66. The masked chimeric receptor of any one of embodiments 63 to 65, wherein the linker comprising a cleavable peptide comprises: a) a first spacer linker and a cleavable peptide; or b) a first spacer Linker, cleavable peptide and second spacer linker. 67. The masked chimeric receptor of any one of embodiments 63 to 66, wherein the cleavable peptide comprises an amino acid sequence selected from SEQ ID NO: 47-88, 464-469, and 479-508. 68. The masked chimeric receptor of embodiment 66 or embodiment 67, wherein: a) the first spacer linker is directly connected to the N-terminus or C-terminus of the cleavable peptide; or b) the first spacer The linker is directly connected to the N-terminus of the cleavable peptide, and the second spacer linker is directly connected to the C-terminus of the cleavable peptide. 69. The masked chimeric receptor of embodiment 68, wherein the first spacer linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 89-112 and 415-420; and/or the first spacer linker; The two-spacer linker includes an amino acid sequence selected from the group consisting of SEQ ID NO: 89-112 and 415-420. 70. The masked chimeric receptor of any one of embodiments 63 to 69, wherein the linker comprising a cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 454-462. 71. The masked chimeric receptor according to any one of embodiments 63 to 70, wherein at least one amino acid but no more than 20 amino acids is directly connected to the N-terminus of the masked peptide. 72. The masked chimeric receptor of embodiment 71, wherein the at least one amino acid is alanine (A) or glycine-alanine (GA). 73. The masked chimeric receptor of any one of embodiments 63 to 72, wherein the first chain or the second chain of the ligand binding domain in the N-to-C-terminal or C-to-N-terminal direction comprises: a ) Masking peptide; b) Cleavable peptide; and c) First or second chain. 74. The masked chimeric receptor of embodiment 73, wherein a) the ligand binding domain comprises a spacer linker between the masking peptide and the cleavable peptide; and b) the ligand binding domain comprises A spacer linker between the cleavable peptide and the first or second chain. 75. The masked chimeric receptor of any one of embodiments 63 to 74, wherein the masked chimeric receptor comprises an amine group selected from the group consisting of SEQ ID NO: 113-231 and 444-453 Acid sequence. 76. The masked chimeric receptor according to any one of embodiments 63 to 75, wherein: a) the first chain comprises (i) CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 402, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 403, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 404; and/or wherein the second chain includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 405, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 406, and (iii) CDR-H3, which includes The amino acid sequence of SEQ ID NO: 407; or b) the first chain includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 408, and (ii) CDR-L2, which includes SEQ ID The amino acid sequence of NO: 409, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 410; and/or wherein the second chain includes (i) CDR-H1, which includes SEQ ID The amino acid sequence of NO: 411, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 412, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 413; Or c) the first chain includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 432, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 433, and ( iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 434; and/or wherein the heavy chain variable region includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 435, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 436, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 437; or d) the first chain includes ( i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 438, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 439, and (iii) CDR-L3, which includes SEQ ID NO: 439 ID NO: 440 amino acid sequence; and/or wherein the heavy chain variable region comprises (i) CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 441, (ii) CDR-H2, which comprises The amino acid sequence of SEQ ID NO: 442, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 443. 77. The masked chimeric receptor of any one of embodiments 63 to 76, wherein the first chain comprises the amino acid sequence of SEQ ID NO: 232; and/or the second chain comprises SEQ ID NO: 233 amino acid sequence. 78. The masked chimeric receptor of any one of embodiments 63 to 76, wherein: a) the first chain comprises the amino acid sequence of SEQ ID NO: 321; and/or the second chain comprises SEQ The amino acid sequence of ID NO: 323; or b) the first chain includes the amino acid sequence of SEQ ID NO: 322; and/or the second chain includes the amino acid sequence of SEQ ID NO: 324. 79. The masked chimeric receptor of any one of embodiments 63 to 78, wherein the cleavable peptide is a substrate for a protease, and the protease is co-located in a region with cells or tissues expressing CTLA4. 80. The masked chimeric receptor of any one of embodiments 63 to 79, wherein the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of ABHD12, ADAM12, ABHD12B, ABHD13, ABHD17A , ADAM19, ADAM20, ADAM21, ADAM28, ADAM30, ADAM33, ADAM8, ABHD17A, ADAMDEC1, ADAMTS1, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS3, ADAMTSMTADA2, ADAMTS5, ADAMTSMT, ADAMTSMTS5 , ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADAMTSL1, ADAMTSL2, ADAMTSL3, ABHD17C, ADAMTSL5, ASTL, BMP1, CELA1, CELA2A, CELA2B, CELA3A, CELA3B, ADAM10, ADAM15, ADAM17, ADAM9, ADAMTSL, ADAMTSMA, 1 , 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, MMP17 , 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, PRT N3, MMP13, MMP14, ST14, TMPRSS10, TMPRSS11A, TMPRSS11D, TMPRSS11E, TMPRSS11F, TMPRSS12, TMPRSS13, MMP15, TMPRSS15, MMP2, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS5, TMPRSS6, TMPRSS5, TMPRSS6, TMPRSS1, TMPRSS7, PRSK PHEX, TINAG, TPSAB1, TPSD1 and TPSG1. 81. The masked chimeric receptor of embodiment 80, wherein 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. 82. A nucleic acid encoding the masked antibody of any one of embodiments 1 to 33, 100 to 112, and 139 to 148, such as the masked double of any one of embodiments 34 to 62 and 113 to 121 Specific antibodies or masked chimeric receptors as in any one of Examples 63 to 82 and 122 to 128. 83. A vector comprising the nucleic acid as in Example 82. 84. Like the vector of Example 83, it is a performance vector. 85. A host cell comprising the nucleic acid as in Example 82. 86. A method for preparing a masked antibody, a masked bispecific antibody or a masked chimeric receptor, which comprises producing the masked antibody, masked bispecific antibody or masked The host cells of Example 85 were cultured under the conditions of the chimeric receptor. 87. The method of embodiment 86, wherein the host cell has α1,6-trehalosyltransferase (Fut8) knockout. 88. The method of embodiment 86 or 87, wherein the host cell overexpresses β1,4-N-acetylglycosamine polyglycosyltransferase III (GnT-III). 89. The method of embodiment 88, wherein the host cell also overexpresses Golgi mu-mannosidase II (ManII). 90. The method of any one of embodiments 86 to 89, further comprising recovering the masked antibody, masked bispecific antibody or masked chimeric receptor produced by the host cell. 91. A masked antibody, masked bispecific antibody or masked chimeric receptor, which is produced by the method as in any one of Examples 86 to 90. 92. A composition comprising the masked antibody as in any one of embodiments 1 to 33, 100 to 112, and 139 to 148, and the masked antibody as in any one of embodiments 34 to 62 and 113 to 121 Bispecific antibodies, or masked chimeric receptors as in any one of Examples 63 to 82 and 122 to 128. 93. A composition comprising a masked antibody, a masked bispecific antibody or a masked chimeric receptor as in Example 91. 94. A pharmaceutical composition comprising the masked antibody as in any one of embodiments 1 to 33, 100 to 112, and 139 to 148, and the masked antibody as in any one of embodiments 34 to 62 and 113 to 121 The bispecific antibody or the masked chimeric receptor as in any one of Examples 63 to 82 and 122 to 128, and a pharmaceutically acceptable carrier. 95. A pharmaceutical composition comprising a masked antibody, a masked bispecific antibody or a masked chimeric receptor as in Example 91, and a pharmaceutically acceptable carrier. 96. A kit comprising the masked antibody as in any one of embodiments 1 to 33, 100 to 112 and 139 to 148, and the masked antibody as in any one of embodiments 34 to 62 and 113 to 121 Bispecific antibodies, masked chimeric receptors as in any of Examples 63 to 82 and 122 to 128, or compositions as in any of Examples 92 to 95. 97. A method for treating or preventing neoplastic diseases in an individual, the method comprising administering to the individual an effective amount of the masked antibody as in any one of Examples 1 to 33, 100 to 112, and 139 to 148 , As in the masked bispecific antibody of any one of embodiments 34 to 62 and 113 to 121, as in the masked chimeric receptor of any one of embodiments 63 to 82 and 122 to 128, or as implemented The composition of any one of Examples 92 to 95. 98. The method of embodiment 97, wherein the neoplastic disease is cancer. 99. The method of embodiment 98, wherein the cancer is leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma, lung cancer, ovarian cancer, osteosarcoma , Bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer or testicular cancer. 100. A masked antibody, comprising: a) an anti-CTLA4 antibody or an antigen-binding fragment thereof, which comprises a variable light (VL) domain and a variable heavy (VH) domain; and b) a masking peptide, which comprises An amino acid sequence selected from the group consisting of SEQ ID NO: 1-46; wherein the masking peptide is connected to the amino or carboxyl end of the VL domain or the VH domain via a linker containing a cleavable peptide. 101. The masked antibody of embodiment 100, wherein the linker comprising a cleavable peptide comprises a cleavable peptide comprising an amine selected from the group consisting of SEQ ID NO: 47-88, 464-469 and 479-508 Base acid sequence. 102. The masked antibody of embodiment 100 or 101, wherein the cleavable peptide comprises an amino terminal and a carboxy terminal, and the linker comprising the cleavable peptide comprises a first spacer linker and a second spacer linker, wherein The first spacer linker is connected to the amino terminal of the cleavable peptide and includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 89 to 112 and 415 to 420, and the second spacer linker is connected to the The carboxyl end of the cleavable peptide includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 89 to 112 and 415-420. 103. The masked antibody of any one of embodiments 100 to 102, wherein the linker comprising a cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 454-462. 104. The masked antibody of any one of embodiments 100 to 103, wherein the masked antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 113-231 and 444-453. 105. The masked antibody of any one of embodiments 100 to 104, wherein the anti-CTLA4 antibody or antigen-binding fragment thereof is a humanized antibody, a chimeric antibody, or a human antibody. 106. The masked antibody of any one of embodiments 100 to 105, wherein: a) the VL domain comprises (i) CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 402, (ii) CDR -L2, which includes the amino acid sequence of SEQ ID NO: 403, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 404; and/or the VH domain includes (i) CDR-H1 , Which includes the amino acid sequence of SEQ ID NO: 405, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 406, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 407 Amino acid sequence; or b) The VL domain includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 408, (ii) CDR-L2, which includes the amino acid of SEQ ID NO: 409 Sequence, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 410; and/or the VH domain includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 411, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 412, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 413; or c) the VL domain includes (i ) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 432, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 433, and (iii) CDR-L3, which includes SEQ ID The amino acid sequence of NO: 434; and/or the VH domain includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 435, and (ii) CDR-H2, which includes SEQ ID NO: 436 And (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 437; or d) the VL domain includes (i) CDR-L1, which includes the amine of SEQ ID NO: 438 Base acid sequence, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 439, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 440; and/or the VH The domain includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 441, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 442, and (iii) CDR-H3, It includes the amino acid sequence of SEQ ID NO:443. 107. The masked antibody of any one of embodiments 100 to 106, wherein: 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 Acid sequence; or b) the VL domain includes the amino acid sequence of SEQ ID NO: 322, and the VH domain includes the amino acid sequence of SEQ ID NO: 324. 108. The masked antibody of any one of embodiments 100 to 107, wherein the VL domain is contained within a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 327-341, And the VH domain is contained in the heavy chain, and the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 366-380, 421, and 478. 109. The masked antibody of any one of embodiments 100 to 108, wherein the masked antibody comprises the amino acid sequence of SEQ ID NO: 421, and comprises a composition selected from SEQ ID NO: 358 and 422-431 The amino acid sequence of the group. 110. The masked antibody of any one of embodiments 100 to 109, wherein the anti-CTLA4 antibody or antigen-binding fragment thereof binds to an agent. 111. The masked antibody of embodiment 110, wherein the agent is a tubulin polymerization inhibitor, a DNA damaging agent, or a DNA synthesis inhibitor. 112. The masked antibody of embodiment 110, wherein the agent is maytansinoid, aristatin, pyrrolobenzodiazepine (PBD) dimer, calicheamicin, becarcinomycin, indole Doline benzodiazepine dimer or ixinotecan derivative Dxd. 113. 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 the group consisting of SEQ ID NO: 1-46, wherein the masking peptide is connected to the light chain or heavy chain of the first pair via a linker comprising a cleavable peptide The amino or carboxyl end. 114. The masked bispecific antibody of embodiment 113, wherein the linker comprising a cleavable peptide comprises a cleavable peptide, which comprises a group selected from the group consisting of SEQ ID NO: 47-88, 464-469 and 479-508 The amino acid sequence of the group. 115. The masked bispecific antibody of embodiment 113 or 114, wherein the cleavable peptide comprises an amino terminal and a carboxy terminal, and the linker comprising the cleavable peptide comprises a first spacer linker and a second spacer linker Wherein the first spacer linker is connected to the amino end of the cleavable peptide and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 89-112 and 415-420, and the second spacer linker It is connected to the carboxyl terminal of the cleavable peptide and includes an amino acid sequence selected from the group consisting of SEQ ID NO: 89-112 and 415-420. 116. The masked bispecific antibody of any one of embodiments 113 to 115, wherein the linker comprising a cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 454-462. 117. The masked bispecific antibody of any one of embodiments 113 to 116, wherein the masked bispecific antibody comprises an amine group selected from the group consisting of SEQ ID NO: 113-231 and 444-453 Acid sequence. 118. The masked bispecific antibody of any one of embodiments 113 to 117, wherein the light chain of the first pair comprises a VL domain, and the heavy chain of the first pair comprises a VH domain, wherein: a) the The VL domain includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 402, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 403, and (iii) CDR-L3 , Which includes the amino acid sequence of SEQ ID NO: 404; and/or the VH domain includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 405, (ii) CDR-H2, which includes The amino acid sequence of SEQ ID NO: 406, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 407; or b) the VL domain includes (i) CDR-L1, which includes SEQ ID The amino acid sequence of NO: 408, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 409, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 410; And/or the VH domain includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 411, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 412, and (iii) ) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 413; or c) the VL domain includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 432, (ii) CDR- L2, which includes the amino acid sequence of SEQ ID NO: 433, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 434; and/or the VH domain includes (i) CDR-H1, It includes the amino acid sequence of SEQ ID NO: 435, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 436, and (iii) CDR-H3, which includes the amine of SEQ ID NO: 437 Base acid sequence; or d) the VL domain includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 438, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 439 , And (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 440; and/or the VH domain includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 441, ( ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 442, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 443. 119. The masked bispecific antibody of any one of embodiments 113 to 118, wherein: a) the VL domain comprises the amino acid sequence of SEQ ID NO: 321, and the VH domain comprises SEQ ID NO: 323 Or b) the VL domain includes the amino acid sequence of SEQ ID NO: 322, and the VH domain includes the amino acid sequence of SEQ ID NO: 324. 120. The masked bispecific antibody of any one of embodiments 113 to 119, wherein the light chain of the first pair comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 327-341, and the The heavy chain of the first pair comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 366-380, 421, and 478. 121. The masked bispecific antibody of any one of embodiments 113 to 120, wherein the masked bispecific antibody comprises the amino acid sequence of SEQ ID NO: 421, and comprises the amino acid sequence selected from SEQ ID NO: The amino acid sequence of the group consisting of 358 and 422-431. 122. A masked chimeric receptor, comprising: a) a ligand binding domain that binds to CTLA4, which comprises a VL domain and a VH domain; b) a masking peptide, which comprises a peptide selected from SEQ ID NO: 1-46 The amino acid sequence of the group consisting of; c) a transmembrane domain; and d) an intracellular signal transduction domain comprising a signal transduction domain, wherein the masking peptide is connected to the VL domain or the VH via a linker comprising a cleavable peptide The amino or carboxyl end of the domain. 123. The masked chimeric receptor of embodiment 122, wherein the linker comprising a cleavable peptide comprises a cleavable peptide, which comprises the group consisting of SEQ ID NO: 47-88, 464-469 and 479-508 The amino acid sequence of the group. 124. The masked chimeric receptor of embodiment 122 or 123, wherein the cleavable peptide comprises an amino terminal and a carboxy terminal, and the linker comprising the cleavable peptide comprises a first spacer linker and a second spacer linker Wherein the first spacer linker is connected to the amino end of the cleavable peptide and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 89-112 and 415-420, and the second spacer linker It is connected to the carboxyl terminal of the cleavable peptide and includes an amino acid sequence selected from the group consisting of SEQ ID NO: 89-112 and 415-420. 125. The masked chimeric receptor of any one of embodiments 122 to 124, wherein the linker comprising a cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 454-462. 126. The masked chimeric receptor of any one of embodiments 122 to 125, wherein the masked chimeric receptor comprises an amine group selected from the group consisting of SEQ ID NO: 113-231 and 444-453 Acid sequence. 127. The masked chimeric receptor of any one of embodiments 122 to 126, wherein: a) the VL domain comprises (i) CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 402, ( ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 403, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 404; and/or the VH domain includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 405, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 406, and (iii) CDR-H3, which includes SEQ ID NO : 407 amino acid sequence; or b) the VL domain includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 408, (ii) CDR-L2, which includes SEQ ID NO: 409 Amino acid sequence, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 410; and/or the VH domain includes (i) CDR-H1, which includes the amino group of SEQ ID NO: 411 Acid sequence, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 412, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 413; or c) the VL domain It includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 432, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 433, and (iii) CDR-L3, which Comprising the amino acid sequence of SEQ ID NO: 434; and/or the VH domain comprises (i) CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 435, (ii) CDR-H2, which comprises SEQ ID The amino acid sequence of NO: 436, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 437; or d) the VL domain includes (i) CDR-L1, which includes SEQ ID NO: The amino acid sequence of 438, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 439, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 440; and/ Or the VH domain includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 441, (ii) CDR-H2, which includes the amino acid sequence of SEQ ID NO: 442, and (iii) CDR -H3, which includes the amino acid sequence of SEQ ID NO:443. 128. The masked chimeric receptor of any one of embodiments 122 to 127, wherein: a) the VL domain comprises the amino acid sequence of SEQ ID NO: 321, and the VH domain comprises SEQ ID NO: 323 Or b) the VL domain includes the amino acid sequence of SEQ ID NO: 322, and the VH domain includes the amino acid sequence of SEQ ID NO: 324. 129. A nucleic acid encoding a masked antibody as in any one of embodiments 100 to 112, a masked bispecific antibody as in any one of embodiments 113 to 121 or as in any one of embodiments 122 to 128 A masked chimeric receptor. 130. A vector comprising the nucleic acid as in Example 129. 131. A host cell comprising the nucleic acid as in Example 129. 132. A method for preparing a masked antibody, a masked bispecific antibody, or a masked chimeric receptor, which comprises culturing the host cell of Example 131 under conditions that produce the masked antibody. 133. A masked antibody, masked bispecific antibody or masked chimeric receptor, which is produced by the method as in Example 132. 134. A composition comprising the masked antibody as in any one of embodiments 100 to 112, the masked bispecific antibody as in any one of embodiments 113 to 121 or as in embodiments 122 to 128 Any of the masked chimeric receptors. 135. A pharmaceutical composition comprising a masked antibody as in any one of embodiments 100 to 112, a masked bispecific antibody as in any one of embodiments 113 to 121 or as in embodiments 122 to 128 Any one of the masked chimeric receptors, and a pharmaceutically acceptable carrier. 136. A kit comprising a masked antibody as in any one of embodiments 100 to 112, a masked bispecific antibody as in any one of embodiments 113 to 121 or as in embodiments 122 to 128 Any of the masked chimeric receptors. 137. A method for treating or preventing neoplastic diseases in an individual, the method comprising administering to the individual an effective amount of the masked antibody as in any one of Examples 100 to 112, as in Examples 113 to 121 The masked bispecific antibody of any one of or the masked chimeric receptor of any one of Examples 122 to 128. 138. A method for treating or preventing neoplastic disease in an individual, the method comprising administering to the individual an effective amount of the pharmaceutical composition of Example 135. 139. A masked antibody comprising: a) an anti-CTLA4 antibody or an antigen-binding fragment thereof, comprising a variable light (VL) domain and a variable heavy (VH) domain; and b) a masking peptide comprising The amino acid sequence of SEQ ID NO: 5; wherein the masking peptide is connected to the amino terminal of the VL domain via a linker comprising a cleavable peptide; wherein the linker comprising a cleavable peptide comprises a cleavable peptide, which comprises SEQ The amino acid sequence of ID NO: 86; wherein (a) the VL domain includes CDR-L1 including the amino acid sequence of SEQ ID NO: 408, and CDR-L2 including the amino acid sequence of SEQ ID NO: 409 And CDR-L3 including the amino acid sequence of SEQ ID NO: 410; and the VH domain includes CDR-H1 including the amino acid sequence of SEQ ID NO: 411, including the amino acid sequence of SEQ ID NO: 412 CDR-H2 and CDR-H3 including the amino acid sequence of SEQ ID NO: 413; or (b) the VL domain includes CDR-L1 including the amino acid sequence of SEQ ID NO: 438, including SEQ ID NO CDR-L2 of the amino acid sequence of :439 and CDR-L3 of the amino acid sequence of SEQ ID NO:440; and the VH domain includes CDR-H1 of 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. 140. The masked antibody of embodiment 139, wherein the linker comprising the cleavable peptide comprises the amino acid sequence of SEQ ID NO:454. 141. The masked antibody of embodiment 139 or 140, wherein 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. 142. The masked antibody of any one of embodiments 139 to 141, wherein the VL domain is contained in a light chain comprising the amino acid sequence of SEQ ID NO: 334, and the VH domain is contained in a light chain comprising SEQ ID NO : 421 in the heavy chain of the amino acid sequence. 143. The masked antibody of any one of embodiments 139 to 142, wherein the masked antibody comprises the amino acid sequence of SEQ ID NO:358 and the amino acid sequence of SEQ ID NO:421. 144. A masked antibody, comprising: a) an anti-CTLA4 antibody or an antigen-binding fragment thereof, comprising a variable light (VL) domain and a variable heavy (VH) domain; and b) a masking peptide comprising The amino acid sequence of SEQ ID NO: 19; wherein the masking peptide is connected to the amino end of the VL domain via a linker comprising a cleavable peptide; wherein the linker comprising a cleavable peptide comprises a cleavable peptide, which comprises SEQ The amino acid sequence of ID NO: 50; wherein (a) the VL domain includes CDR-L1 including the amino acid sequence of SEQ ID NO: 408, and CDR-L2 including the amino acid sequence of SEQ ID NO: 409 And CDR-L3 including the amino acid sequence of SEQ ID NO: 410; and the VH domain includes CDR-H1 including the amino acid sequence of SEQ ID NO: 411, including the amino acid sequence of SEQ ID NO: 412 CDR-H2 and CDR-H3 including the amino acid sequence of SEQ ID NO: 413; or (b) the VL domain includes CDR-L1 including the amino acid sequence of SEQ ID NO: 438, including SEQ ID NO CDR-L2 of the amino acid sequence of :439 and CDR-L3 of the amino acid sequence of SEQ ID NO:440; and the VH domain includes CDR-H1 of 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. 145. The masked antibody of embodiment 144, wherein the linker comprising the cleavable peptide comprises the amino acid sequence of SEQ ID NO:455. 146. The masked antibody of embodiment 144 or 145, wherein 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. 147. The masked antibody of any one of embodiments 144 to 146, wherein the VL domain is contained in a light chain comprising the amino acid sequence of SEQ ID NO: 334, and the VH domain is contained in a light chain comprising SEQ ID NO : 421 in the heavy chain of the amino acid sequence. 148. The masked antibody of any one of embodiments 144 to 147, wherein the masked antibody comprises the amino acid sequence of SEQ ID NO:422 and the amino acid sequence of SEQ ID NO:421. Instance

The present invention will be more fully understood with reference to the following examples. However, it should not be construed as limiting the scope of the present invention. It should be understood that the examples and embodiments described in this article are for illustrative purposes only, and various modifications or changes will be made by those familiar with the technology and included in the spirit and scope of this application and the accompanying patent application. Within the scope of the scope. Example 1 : Preparation of activatable and masked anti- CTLA4 antibody

The anti-CTLA4 antibody 9D9 muIgG2b mAb (9D9 mAb) was purchased from BioXcell for biopanning experiments. For all other experiments, a parental anti-CTLA4 antibody (9D9) that is reactive against murine CTLA4, an activatable masked anti-CTLA4 antibody (masked 9D9), and an uncleavable masked antibody were produced and purified at ATUM Anti-CTLA4 antibody (non-cleavable masked 9D9).

Activable masked anti-CTLA4 antibody, parental anti-CTLA4 antibody, and non-cleavable masked anti-CTLA4 antibody system are prepared by expressing plastid pD2610-v5 using HEK293 transient expression preparation (ATUM, Newark CA) and using murine IgG2a Isotypes are produced by gene synthesis (ATUM, Newark CA). On the seventh day after transfection, collect the supernatant from the transfected cells and purify the masked anti-CTLA4 antibody and the parental anti-CTLA4 antibody with KanCapA resin (Kaneka), and change the buffer to phosphate buffered physiology Saline solution (PBS). Next, use Source S or Capto S ImpAct (GE) to further purify the antibody by ion exchange. The antibody concentration was determined by a spectrophotometer at the optical density of OD280.

The above-described masked 9D9 antibody and the masked peptide contained in the non-cleavable masked 9D antibody were obtained by biopanning the spatially occluded peptides from the phage display peptide library. First capture 9D9 muIgG2b mAb (BioXcell) on ProteinG Magnetic Beads (MB) (Thermo Fisher Scientific) at room temperature for 30 minutes. After blocking the MB fixed by 9D9 muIgG2b mAb with skim milk, Ph.D.TM-C7C phage peptide library (New England BioLabs) was added to screen for spatially occluded peptides. After incubating for 30 minutes at room temperature, and then thoroughly washing the unbound phage, the bound phage was eluted into a pH neutralization solution with 0.2 M glycine pH 2.2. The lysed phage were amplified by infection into E. coli K12 ER2738 (New England BioLabs) for use in the next round of biopanning. Three subsequent rounds of biopanning were performed, and an additional round of negative sorting was performed using the isotype control (BioXcell) to eliminate peptide binding outside the 9D9 mAb CDR. To achieve the identification and enrichment of peptides that specifically bind at the interface between 9D9 mAb and mouse CTLA4.

After the last round of biopanning, individual clones from the enriched population were evaluated for specific binding to 9D9 mAb by ELISA. In the presence or absence of isomolar muCTLA4-hsIgG (R&D Systems), 9D9 mAb was spread on a 96-well MaxiSorp dish (Thermo Fisher Scientific) at 4°C overnight. Individual phage presenting unique peptides were added to the skim milk-blocked dish in triplicate and incubated at room temperature for 1 hour. After washing the unbound phage thoroughly, HRP-conjugated anti-M13 antibody (GE Healthcare) and HRP substrate (Sigma-Aldrich) were used to detect the bound phage. The absorbance at 405 nm was measured in a microplate reader (Synergy HT, BioTek).

A pure line that produces a high 9D9 binding signal and inhibits the binding of murine CTLA4 to 9D9 mAb is selected, and DNA is extracted for sequencing. The sequence from the phage encoding the peptide CNLIVEGHC was selected for subsequent representative experiments.

Regarding the design of the activatable masked anti-CTLA4 antibody (masked 9D9), the structural characteristics of the complex of 9D9 Fab and the peptide CNLIVEGHC were determined at high resolution (1.8 Å, Table 3). At 37°C, 9D9 was lysed with papain resin (Sigma) overnight. Fab was purified by protein-A, cation exchange (HiTrap SP-FF, GE Healthcare) and size exclusion (Superdex S75, GE Healthcare) column chromatography. Using 0.2 M sodium formate and 23% (w/v) PEG 3350 as mother liquor, the purified Fab was crystallized in 10 mM Tris-HCl pH 8.0, 25 mM NaCl, 1 mM EDTA using the hanging drop vapor diffusion method. The crystals are collected in a cryoprotectant (1:1 mixture of 35% (w/v) mesoerythritol and stock solution) and collected with a Rigaku MicroMax007-HF rotating anode diffractometer with a Rigaku RAXISIV++ detector data. At 100 K, raw data was collected at 1.5418 Å and processed with XDS, resulting in a 98.5% complete data set of 1.8 Å. Use the Phaser in the CCP4 kit and the Trastuzumab Fab (PDB ID: 4IOI) as a search model to perform molecular replacement to generate the initial phase. See Donaldson JM et al., PNAS., 110:17456-61, (2013). Use several artificially constructed in Coot, and then use Phenix to optimize the loop to generate the final model. Data collection and optimization statistics are listed in Table 3. The software PDBePISA was used to characterize the antibody-peptide interface in Table 4. The CNLIVEGHC peptide was determined to bind to the CDR loop at the interface between the heavy and light chains of the 9D9 antibody. The peptide: 9D9 Fab interface is composed of 5 hydrogen bonds (Table 4), resulting in a mutual surface area of 375 Å2. The electrostatic surface of 9D9 Fab represents a large hydrophobic area at the interface between the antigen binding sites of the heavy chain and the light chain. The central Leu-Ile-Val motif of the CNLIVEGHC peptide is embedded at this interface. The model indicates that the C-terminus of the CNLIVEGHC peptide points to the N-terminus of the light chain. The distance between the carboxyl carbon of the peptide and the amide nitrogen of the light chain (residue Aspl) is 20.4 Å. The peptide containing the MMP2/7/9 cleavage site was selected to connect the 9D9 antibody to the peptide. See Turk BE et al., Nat Biotech, 19:661-667, (2001). Glycine and serine amino acids are used to bridge the gap between the N and C terminals. In addition, the MMP2/7/9 substrate sequence was mutated to generate an "uncleavable" version of the masked anti-CTLA4 antibody (uncleavable, masked 9D9). Table 3. X-ray diffraction data and optimization statistics Space group P22 ₁ 2 ₁ Unit cell size (Å) 37.0 × 88.5 × 128.7 PH value of crystallization conditions 8.5 Protein/peptide concentration (µM) 60/600 data set Wavelength, Å 1.5418 Resolution range 50-1.8 Unique reflection (total) 34911 (188519) Completion 98.5 (91.1) Redundancy 5.4% (4.9%) R _meas 0.073 (0.35) CC(1/2) 99.9 (93.0) I/σ 20.83 (5.14) NCS copy 1 Model optimization Resolution range, Å 33-1.88 Reflection number 34,855 Protein atom number 3,936 Number of water molecules 561 Residues in the model LC: 1-219, HC: 1-216, encapsulated peptide 1-10 Rwork/Rfree,% 16.31/20.6 Root mean square deviation Bond length, Å 0.009 Bond angle 1 Ramachandran plot The most favorable area,% 99.08 Other allowed areas,% 0.92 Non-permitted area 0 Table 4. Hydrogen bond between sterically occluded peptides and 9D9-Fab Space-occupied peptide 9D9-Fab Distance, Å ASN 3 [OD1] LC: TYR 101 [OH] 2.58 ASN 3 [ND2] LC: GLY 96 [O] 2.96 ASN 3 [ND2] LC: SER 97 [O] 3.16 LEU 4 [O] HC: TYR 33 [OH] 3.53 ILE 5 [N] LC: TYR 101 [OH] 3.78 Example 2 : Determination of the binding affinity between the masking peptide and anti- CTLA4 antibody

The binding affinity of synthetic CNLIVEGHC peptide against 9D9 antibody was determined. method

At 37°C, a GE Biacore T200 instrument was used for surface plasmon resonance. The 9D9 antibody and isotype control (reference channel) were coupled with a CM5 chip (GE Healthcare) at a fixed amount of 100 RU. The peptides were synthesized and diluted in HBS-EP+ operating buffer (GE Healthcare). Use Biacore evaluation software, version 3.0 to analyze the combined data. result

Using a stable model, the binding affinity of synthetic CNLIVEGHC peptide and 9D9 antibody as determined by SPR is 11 E-6 ± 2.3E-6 M (Figure 1). The negative control peptide does not bind to the 9D9 antibody and the CNLIVEGHC peptide does not bind to the isotype control antibody. These results confirm that the selected peptides from the phage library are specific to the 9D9 antibody. Example 3A : In vitro characterization of an activatable masked anti- CTLA4 antibody that binds to murine CTLA4 Method evaluation of masked antigen binding sites

The ELISA plate was coated with murine CTLA4-Fc (R&D Systems) in TBS overnight and then washed with TBS with 0.05% Tween 20 and blocked with PBS containing 1% BSA, followed by washing with TBS with Tween 20. The dilutions of the parental anti-CTLA4 antibody and the masked anti-CTLA4 antibody in PBS were bound to CTLA4 for 60 minutes, and then washed with TBS Tween 20. Use anti-muKappa-HRP (Abcam) and Super Signal Pico chemiluminescence to detect binding. The masking rate was determined from the EC50 of the pre-lysed masked anti-CTLA4 antibody and the EC50 of the parental anti-CTLA4 antibody. Protease cleavage test

The parental anti-CTLA4 antibody and the masked anti-CTLA4 antibody are incubated with 6 nM MMP2 (R&D Systems) in 150 mM NaCl, 50 mM Tris (pH 7.5), 10 mM CaCl2, 0.02% NP-40 at 37°C Overnight. Using "any precast polyacrylamide gel without Kd staining" (Bio-Rad), the binding of the antibody dilution to murine CTLA4-Fc was tested by the ELISA and reduced SDS-PAGE analysis described above. result

The activatable masked anti-CTLA4 antibody exhibited a 90-fold lower binding to murine CTLA4 (Figure 2A). The protease activation of the masked anti-CTLA4 antibody completely restored the binding of the antibody to murine CTLA4 at a level similar to that of the parental anti-CTLA4 antibody (Figure 2A). Example 3B : In vitro characterization of activatable masked anti- CTLA4 antibody and non-cleavable masked anti- CTLA4 antibody that bind to murine CTLA4 . Method Low-density antigen binding ELISA

All ELISAs are performed in a manner substantially similar to the following description and generally conform to methods known in the art. Polysterene 96-well microplate (Thermo Fisher Scientific) was coated with 3 ng muCTLA4hsIgG protein (Abcam)/well and stored at 4°C overnight. The plate was washed with TBS (TBS-T) containing 0.1% Tween, blocked with PBS containing 1% BSA (Fisher) at 4°C for 2 hours and washed with TBS-T. Add serial dilutions of the test antibody in PBS containing 0.05% Tween+1% BSA (PBS-TB) to the dish and incubate at room temperature for 1 hour. After washing with TBS-T, HRP-conjugated goat anti-mouse kappa light chain antibody (Abcam) diluted 1:4000 in PBS-TB was applied to each well and incubated at room temperature for 1 hour. The disc was washed with TBS-T, a chemiluminescent HRP substrate was applied to the disc, and the luminescence was recorded using a spectrophotometer (Synergy HT, BioTek). The data was analyzed with GraphPad Prism, version 7.02. Enzymatic activation of masked antibodies

The parental anti-CTLA4 antibody, the masked anti-CTLA4 antibody, and the non-lysable masked anti-CTLA4 antibody are each in a lysis buffer (150 mM NaCl; 50 mM Tris, pH 7.5; 10 mM CaCl2; 0.02% NP-40) Dilute to 1 μM solution. Recombinant MMP2 (R&D Systems) activated with 1 mM mercuric p-aminophenylacetate (AMPA) at 37°C for 1 hour was added to each antibody solution to a final concentration of 4 nM and incubated at 37°C overnight. The binding of the antibody dilutions to murine CTLA4 (muCTLA4hsIgG protein; Abcam) was tested by the low density antigen binding ELISA protocol as described above. result

Compared with the parental anti-CTLA4 antibody, the activatable masked anti-CTLA4 antibody and the non-cleavable masked anti-CTLA4 antibody showed approximately 156-fold and 218-fold lower binding to murine CTLA4, respectively (Figure 2B). The average EC50 of the parental anti-CTLA4 antibody is 0.31±0.08 nM, the average EC50 of the activatable masked anti-CTLA4 antibody is 48.4±7.2 nM and the average EC50 of the non-cleavable masked anti-CTLA4 antibody is 67.7±16.3 nM, As measured in three experiments (Figure 2C). When treated with recombinant MMP2, the activatable masked anti-CTLA4 antibody recovered to a binding similar to that of the parental anti-CTLA4 antibody (average EC50=0.29±0.03 nM, Figure 2C), while the uncleavable masked anti-CTLA4 antibody The binding was unchanged (average EC50=64.3±18.5 nM, Figure 2C). Example 4A : In vivo characterization of activatable and masked anti- CTLA4 antibodies. Method of preparing activatable and masked anti- CTLA4 antibodies for in vivo studies

By expressing pD2610-v5, HEK293 was used for transient expression to prepare masked anti-CTLA4 antibody (ATUM, Newark CA). On the seventh day after transfection, the supernatant from the transfected cells was collected and the masked anti-CTLA4 antibody was purified with KanCapA resin (Kaneka), and then purified by cation exchange using a Source S column (GE Healthcare) and Dissolve at pH 5 with a 0-1 M NaCl gradient. Next, the antibody buffer was changed to PBS. The antibody concentration was determined by a spectrophotometer at an optical density of OD280 and the endotoxin content was determined using Charles River Endotoxin kit. Tumor eradication with activatable, masked anti- CTLA4 antibody

A single dose of 200 μg IgG2a control (BioXcel), parental anti-CTLA4 antibody (9D9.IgG2a, light The chain includes SEQ ID NO: 237, the heavy chain includes SEQ ID NO: 319), an activatable masked anti-CTLA4 antibody reactive to murine CTLA4 (the light chain includes SEQ ID NO: 238, and the heavy chain includes SEQ ID NO: 319), masked anti-CTLA4 antibody 1005 (light chain includes SEQ ID NO: 240, heavy chain includes SEQ ID NO: 319) that is reactive to murine CTLA4 or has cross-reactivity with human CTLA4 and murine CTLA4 Anti-CTLA4 antibody 1 (light chain includes SEQ ID NO: 327, heavy chain includes SEQ ID NO: 366). The body weight and tumor size were measured every two weeks. T cell population analysis

On days 1, 4, and 8, three 200 μg doses of IgG2a control substance (BioXcel) and parental anti-CTLA4 antibody (9D9.IgG2a, light chain) were administered to C57BL/6 mice carrying 50-100 mm³ MC38 tumors. Containing SEQ ID NO: 237, heavy chain comprising SEQ ID NO: 319), masked anti-CTLA4 antibody 2 (light chain comprising SEQ ID NO: 238, heavy chain comprising SEQ ID NO: 319), masked anti-CTLA4 antibody 1005 (the light chain includes SEQ ID NO: 240, and the heavy chain includes SEQ ID NO: 319) or anti-CTLA4 antibody 1 that has cross-reactivity with human CTLA4 and murine CTLA4 (the light chain includes SEQ ID NO: 327, and the heavy chain includes SEQ ID NO: 366). On day 9, the mice were sacrificed and the tumors were dissociated using the gentleMACS™ protocol "Tumor Dissociation Kit" (Milteni), and filtered through a 70 micron cell filter and washed twice in PBS/2.5% FBS buffer. To remove the enzyme buffer. The spleen cells have tumor infiltrate and dissociate the spleen cells by gently grinding the tissue through the mesh. Red blood cells were lysed with ACK buffer (Thermo Fisher), followed by cell staining. Cells were stained with fluorescently labeled anti-CD45 (BioLegend), anti-CD3 (BioLegend), anti-CD4 (BioLegend), anti-CD8 (BD Biosciences), and anti-CD25 (BioLegend). The cells were infiltrated before staining with anti-FoxP3 (eBioscience) and anti-Ki67 (eBioscience). Regarding the percentage of live (purchased from Life’s Live/Dead Aqua) CD4+, CD8+ and CD25 Foxp3+ (Treg) cells of CD45 positive cells and the percentage of these three cell populations that are Ki67 positive (a sign of cell proliferation), by Fluorescence activated cell sorting (FACS) was used to analyze stained cells. result

Compared with the tumor volume in mice treated with IgG2a control antibody, the parental anti-CTLA4 antibody (9D9.IgG2a) reduced the tumor volume in mice (Figures 3A and 3B). Compared with the parental anti-CTLA4 antibody (Figure 3B), the activatable masked anti-CTLA4 antibody showed equivalent efficacy in reducing tumor volume (Figure 3C). Regulatory T lymphocytes (Treg) in TME are important for immunosuppression. Both the parental anti-CTLA4 antibody (9D9.IgG2a) and the activatable masked anti-CTLA4 antibody 2 (masked 9D9) preferentially deplete the regulatory T lymphocyte (Treg) population in the tumor through effector functions (Figure 4). The blockade of CTLA4 on T cells in vivo causes the proliferation of CD4+ and CD8+ T cells. After multiple doses of parental anti-CTLA4 antibody (9D9.IgG2a) or activatable masked anti-CTLA4 antibody 2 (masked 9D9), the proliferation of T cells in the spleen was evaluated by %Ki67+. Compared with the parent antibody (9D9.IgG2a), the masked anti-CTLA4 antibody showed reduced proliferation of CD4+ cells (Figure 5A) and CD8+ T cells (Figure 5B) in the surrounding area.

The humanized masked anti-CTLA4 antibody 1 has a higher affinity for human CTLA4 (7-fold and 38-fold, respectively) than Ipelizumab, and has cross-reactivity with cynomolgus CTLA4 and murine CTLA4. Compared with the parental anti-CTLA4 antibody (Figure 3B), the humanized masked anti-CTLA4 antibody 1 showed equivalent efficacy in reducing tumor volume (Figure 3D). Example 4B : In vivo characterization of activatable masked anti- CTLA4 antibodies

Using the MC38 mouse colorectal cancer model that was previously confirmed to be responsive to treatment with 9D9 antibody, the effect of activatable masked anti-CTLA4 antibody on tumor growth and immune parameters in the tumor and surrounding areas was studied. See Peggs KS et al., J Exp Med., 206:1717-1725, (2009) and Selby MJ et al., Cancer Immunol Res., 1:32-42, (2013). Methods Tumor eradication with activatable and masked anti- CTLA4 antibody

0.5×106 MC38 tumor cells were injected subcutaneously into female C57BL/6 aged between 8 and 12 weeks. When the average tumor volume reached 50-100 mm3, the mice were randomly divided into treatment groups with similar average tumor volume. To evaluate the anti-tumor efficacy, on the first day after randomization, the antibody formulated in PBS was administered intraperitoneally (ip) in a volume of <250 μL at a dose of 200 μg per dose. The tumor volume was measured twice a week. All mice were monitored individually and sacrificed when the tumor volume reached 1500 mm3 or on the 45th day after randomization. In order to evaluate the biological function of antibody on regulatory T cells (Treg) by FACS analysis as described below, on the first 1, 4, and 8 days after randomization, 200 μg per dose and a volume of <250 μL were carried Antibodies were administered intraperitoneally to mice with tumors. The control antibody was muIgG2a (BioXcell). All mice were sacrificed on day 9; tumors and spleens were collected for FACS analysis. T cell population analysis

Using the gentleMACS, mouse tumor samples were dissociated according to standard protocols and passed through a 70 μm cell filter. In a 70 μm cell strainer, use the back of the syringe to dissociate the spleen sample. Dissolve red blood cells with ACK buffer (Lonza). The final cell suspension was washed and resuspended in staining buffer (PBS pH 7.4, 2.5% FBS, 0.09% NaN3) at 2×107 cells/ml. Cells were treated with Mu Trustain fcX (BioLegend) to block Fc receptors, followed by treatments for CD45 (BioLegend), CD3 (BioLegend), CD4 (BioLegend), CD8 (BD Biosciences), CD25 (BioLegend) and Live/Dead Aqua ( ThermoFisher) antibody staining. For intracellular staining, cells are fixed, infiltrated and stained with antibodies against FoxP3 (eBioscience) and Ki-67 (eBioscience). Perform single-color dyeing and FMO to achieve the purpose of gate control. result

Administration of parental anti-CTLA4 antibody (Figure 6C) and activatable masked anti-CTLA4 antibody (Figure 6D) induced tumor regression of subcutaneously implanted MC38 tumors, while the tumors were treated with isotype control antibodies (Figure 6A) or were not lysable Fast growth in mice treated with the masked anti-CTLA4 antibody (Figure 6B). These results indicate that the activatable masked anti-CTLA4 antibody maintains anti-tumor activity consistent with the effective lysis and dissociation of the masking peptide in the tumor microenvironment. In addition, the full activity of the activatable masked anti-CTLA4 antibody depends on protease cleavage, because the non-cleavable, masked anti-CTLA4 antibody failed to inhibit tumor growth. These results are consistent with early observations indicating that local administration of anti-CTLA4 antibody can effectively induce tumor regression in mice. See Marabelle, A. et al., JCI, 123:2447-2463, (2013).

Analysis of intratumoral and systemic immune activation by administration of activatable masked anti-CTLA4 antibody. Previous work determined that muIgG2a anti-CTLA4 antibody depleted Treg cells intratumorally in an FcγR-dependent manner, but it caused the activation and expansion of Treg in the spleen of treated mice. See Selby M. J. et al., Cancer Immunol Res., 1:32-42, (2013). Therefore, in order to evaluate the local and systemic activities of the masked anti-CTLA4 antibodies, the abundance and proliferation status of Tregs in the tumor (Figure 7A) and spleen (Figure 7B) were evaluated, respectively. Tumor and spleen samples were collected 24 hours after the last three doses of antibody. Flow cytometry analysis showed that Tregs were significantly reduced in tumors in mice treated with the parental anti-CTLA4 antibody (Figure 7A). The activatable masked anti-CTLA4 antibody can reduce Treg as effectively as the parental anti-CTLA4 antibody (Figure 7A). In contrast, the non-cleavable, masked anti-CTLA4 antibody had no significant effect on intratumoral Treg abundance (Figure 7A). These results indicate that the activatable masked anti-CTLA4 antibody has similar activity to the parental anti-CTLA4 antibody in a manner that depends on the activity of the tumor-associated protease in the tumor environment.

In contrast to its effect on Treg cells in tumor tissue, the parental anti-CTLA4 antibody caused a significant increase in proliferation (Ki67+FOXP3+) Treg in the spleen of treated mice (Figure 7B). When compared with the parental anti-CTLA4 antibody, the activatable masked anti-CTLA4 antibody caused a marginal increase in proliferating splenic Treg, and when compared with the isotype control antibody, the non-cleavable, masked anti-CTLA4 antibody increased Treg proliferation No effect (Figure 7B). Taken together, these results support the conclusion that the tumor activation of the activatable masked anti-CTLA4 antibody is mainly limited by the tumor environment with reduced activity in normal tissues. To assess the binding of the human CTLA4 activatable vitro characterization of anti-CTLA4 antibody masked by masking of the antigen binding site: Example 5

The ELISA plate was coated with human CTLA4-Fc (R&D Systems) in TBS overnight and then washed with TBS with 0.05% Tween 20 and blocked with PBS containing 1% BSA, followed by washing with TBS with Tween 20. The dilutions of the parental anti-CTLA4 antibody and the masked anti-CTLA4 antibody in PBS were bound to CTLA4 for 60 minutes, and then washed with TBS Tween 20. Use anti-huKappa-HRP (Abcam) and Super Signal Pico chemiluminescence to detect binding. The masking rate was determined from the EC50 of the pre-lysed masked anti-CTLA4 antibody and the EC50 of the parental anti-CTLA4 antibody. Protease cleavage test

The parental anti-CTLA4 antibody and the masked anti-CTLA4 antibody were incubated with 6 nM 1) interstitial protease in 50 mM Tris (pH 9), 50 mM NaCl, and 0.01% Tween-20 at 37°C overnight. Using "any precast polyacrylamide gel without Kd staining" (Bio-Rad), the binding of the antibody dilution to human CTLA4 was tested by the ELISA and reduced SDS-PAGE analysis described above. result

The humanized masked anti-CTLA4 antibody 1 exhibited a 50-fold lower binding to human CTLA4 (Figure 8). The protease activation of the humanized masked anti-CTLA4 antibody 1 completely restored the binding of the antibody to human CTLA4 at a level similar to that of the parental anti-CTLA4 antibody (Figure 8). Example 6 : Enzymatic activation of masked antibodies

Recombinant protease was used to activate the masked antibody by the protocol described below.

The buffer of the masked antibody was replaced with MMP lysis buffer, followed by incubation with protease. For activation, proteases (Anaspec, ProsecBio, and R&D Systems) were diluted 100 ng/µL in MMP lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 10 mM CaCl2). Add APMA (10 mM in DMSO) to a final concentration of 1 mM. The enzyme was incubated at 37°C for 1 hour. Add the activated protease to the masked antibody at a ratio of 1:1000 antibody: protease (for example, 1 μg MMP: 1 mg antibody), or add the same amount of buffer to the negative control sample. The samples were incubated overnight at 37°C.

After incubation, the processed antibody was purified using protein A resin. Briefly, 150 µL of MabSure ProA slurry (GE Healthcare) was added to a 1.7 mL test tube. The slurry was spun in a mySpin 6 centrifuge for two minutes and washed twice with 1 mL of PBS. The activated antibody (500 µL) was added to the slurry and incubated at room temperature for thirty minutes or 4°C for three hours under shaking. The antibody-slurry mixture was spun in a mySpin 6 centrifuge for two minutes and the fluid was collected. The resin was washed twice with 1 mL of 1x PBS and the washing solution was collected. The antibody is eluted at least six times with 100 µL 25 mM sodium acetate pH 3.5 (into a new 1.7 mL test tube with 6 µL 1 M Tris pH 8.0). Measure the concentration of the leachate.

Make all the eluting solution and buffer replacement samples pooled into PBS. Add 500 µL of PBS to the pooled samples. The sample is added to the filter and rotated three times. Collect the pooled sample by adding 100 µL PBS to the top of the filter and mixing to remove the replaced protein. Measure the concentration of purified antibody. The sample was diluted to 1 mg/mL in PBS.

Use SDS-PAGE to analyze the activation of the sample. In short, 10 µL of sample (1 mg/mL) is mixed with 10 µL of loading buffer and boiled at 90°C for ten minutes. Load 10 µL of the sample on a 15-well Any-kDa non-staining gel (BioRad) and run at 200 V for 30 minutes. The protein is imaged using the BioRad imager to ensure complete activation by changing the migration size.

The complete activation of the masked antibody is demonstrated by the change in the migration size of the lysed antibody. Example 7 : In vitro characterization of anti- CTLA4 antibodies that bind to human CTLA4 : low-density antigen-binding ELISA method

A 96-well polystyrene microplate (Fisher #07-200-591) was coated with 1 µg/mL human CTLA4-Fc (R&D #7268-CT)/well and stored at 4°C overnight. The plate was washed with TBS (TBS-T) containing 0.05% Tween, blocked with 1% BSA (Sigma Aldrich #B4287-25G) and washed with TBS-T. Prepare serial dilutions of samples containing anti-CTLA4 antibody in assay buffer (PBS+0.05% Tween+1% BSA), add them to the dish and shake on the track at 100 RPM for one hour at room temperature. After washing with TBS-T, anti-human kappa light chain [pure line: SB81a]-HRP (Abcam #ab79115) diluted 1:8000 in assay buffer was applied to each well and shaken at room temperature for one hour. The disc was washed with TBS-T, HRP substrate (Super Signal Pico Chemiluminescent Substrate, Thermo #37069) was applied to the disc and the luminescence was recorded using a spectrophotometer (BioTek). Analyze the data with GraphPad Prism.

The antibodies tested include humanized anti-CTLA4 antibodies (referred to as Antibody 1 and Antibody 2), and their variants/forms/versions. According to a specific numbering scheme, Antibody 1 includes a light chain variable region with CDR-L1 including the amino acid sequence of SEQ ID NO: 402, and CDR including the amino acid sequence of SEQ ID NO: 403 -L2 and CDR-L3 including the amino acid sequence of SEQ ID NO: 404, and including the heavy chain variable region including CDR-H1 including the amino acid sequence of SEQ ID NO: 405 , CDR-H2 comprising the amino acid sequence of SEQ ID NO: 406 and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 407. Antibody 1 includes a VL domain including the amino acid sequence of SEQ ID NO: 321, and a VH domain including the amino acid sequence of SEQ ID NO: 323. According to the Kabat numbering scheme, Antibody 1 includes a light chain variable region having CDR-L1 including the amino acid sequence of SEQ ID NO: 432, and CDR including the amino acid sequence of SEQ ID NO: 433 -L2 and CDR-L3 including the amino acid sequence of SEQ ID NO: 434, and including the heavy chain variable region including CDR-H1 including the amino acid sequence of SEQ ID NO: 435 , CDR-H2 comprising the amino acid sequence of SEQ ID NO:436 and CDR-H3 comprising the amino acid sequence of SEQ ID NO:437. According to a specific numbering scheme, Antibody 2 includes a light chain variable region with CDR-L1 including the amino acid sequence of SEQ ID NO: 408, and CDR including the amino acid sequence of SEQ ID NO: 409 -L2 and CDR-L3 including the amino acid sequence of SEQ ID NO: 410, and including the heavy chain variable region including CDR-H1 including 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. According to the Kabat numbering scheme, Antibody 2 includes a light chain variable region with CDR-L1 including the amino acid sequence of SEQ ID NO: 438, CDR including the amino acid sequence of SEQ ID NO: 439 -L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 440, and comprising the 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. The various forms of antibody 1 are produced, each with the name "antibody 1-#", and the various forms of antibody 2 are produced, each with the name "antibody 2-#". result

Figures 9A and 9B show binding studies performed with humanized anti-CTLA4 antibodies called Antibody 1 and Antibody 2, or variants thereof. Figure 9A shows the binding between the version of Antibody 1 with the wild-type Fc region (Antibody 1-1) and the version of Antibody 1 with the S239D mutation and I332E mutation in the Fc region (Antibody 1-2) and human CTLA4-Fc The undetectable difference. Antibody 1-2 includes a VL domain including the amino acid sequence of SEQ ID NO: 327 and a VH domain including the amino acid sequence of SEQ ID NO: 478. Figure 9B compares the binding of CTLA4 in various forms of Antibody 2, including the version of Antibody 2 with wild-type Fc region (Antibody 2-1); the version of Antibody 2 with S239D mutation and I332E mutation in the Fc region (Antibody 2-2 ); Antibody 2 version with S239D mutation, I332E mutation in the Fc region and two hinge region mutations (antibody 2-3); Antibody 2 version with S239D, I332E and A330L mutations in the Fc region (antibody 2- 4); and antibody 2 version with S239D, I332E, A330L mutations in the Fc region and two hinge region mutations (antibody 2-5). As shown in Figure 9B, all forms of antibodies bind to human CTLA4-Fc in a similar manner. The average EC ₅₀ values of the tested antibodies are provided in Table 5 below. Table 5 Human antibody binding to CTLA4-Fc of Antibody EC ₅₀ (nM) R ² Antibody 1-1 0.32 ± 0.01 0.9984 Antibody 1-2 0.32 ± 0.01 0.998 Antibody 2-1 0.44 ± 0.02 0.9978 Antibody 2-2 0.39 ± 0.01 0.9979 Antibody 2-3 0.40 ± 0.02 0.9967 Antibody 2-4 0.40 ± 0.02 0.9962 Antibody 2-5 0.41 ± 0.02 0.9968 Example 8 : Method for the influence of protease activation on the binding of human CTLA4

The humanized anti-CTLA4 antibody was used in the low density antigen binding ELISA described in Example 7. To assess the activation of the masked activatable form of the humanized anti-CTLA4 antibody, recombinant proteases were used as outlined in Example 6.

Use the unmasked version of antibody 2 with the S239D mutation and I332E mutation in the Fc region (antibody 2-6) as an unmasked control for the same two mutations as the antibody, but also include The masking peptide (which includes the amine sequence of SEQ ID NO: 19 or 5) and the version of the spacer linker and the cleavable peptide were compared. The parental unmasked antibodies 2-6 include a variable heavy chain with the amino acid sequence of SEQ ID NO: 324, a variable light chain with the amino acid sequence of SEQ ID NO: 322, and a variable light chain with the amino acid sequence of SEQ ID NO: 322. The heavy chain of the amino acid sequence of 421 and the light chain of the amino acid sequence of SEQ ID NO:334. The masked antibodies from antibody 2-7 to antibody 2-21 include the variable heavy chain, variable light chain, heavy chain, and light chain sequences of the parental unmasked antibody 2-6, and further include masking peptides and spacers Linker 1, cleavable (or non-cleavable) peptide and spacer linker 2, as described in Table 6 and Table 9. Therefore, antibody 2-6, antibody 2-7, antibody 2-8, antibody 2-9, antibody 2-10, antibody 2-11, antibody 2-12, antibody 2-13, antibody 2-14, antibody 2- 15. Each of antibody 2-16, antibody 2-17, antibody 2-18, antibody 2-19, antibody 2-20, and antibody 2-21 includes an amino acid sequence comprising SEQ ID NO: 324 The VH domain, the VL domain including the amino acid sequence of SEQ ID NO: 322, the heavy chain including the amino acid sequence of SEQ ID NO: 421, and the light chain including the amino acid sequence of SEQ ID NO: 334. Unless otherwise indicated, the masked antibody system called Antibody 2-7 to Antibody 2-21 is made by connecting the following components to the parental unmasked antibody 2-6 in the N-terminal to C-terminal direction as follows: Chain to mask: 1) masking peptide; 2) spacer linker 1; 3) cleavable peptide; 4) spacer linker 2; the N-terminus of the light chain. Antibody 2-10 includes a non-cleavable cleavable peptide sequence for use as a negative control. Examples of the exemplary antibodies tested (including the sequences of spacer linkers and cleavable peptides in antibody 2-7 to antibody 2-13) are provided in Table 6. Table 6 Antibody Masking peptide Spacer linker 1 Cleavable peptide Spacer linker 2 Heavy chain Light chain + masking component Antibody 2-7 CPFPALELC (SEQ ID NO: 19) GGSGGS (SEQ ID NO: 415) VPLSLY (SEQ ID NO: 86) SGG (SEQ ID NO: 102) SEQ ID NO: 421 SEQ ID NO: 424 Antibody 2-8 CPFPALELC (SEQ ID NO: 19) GGPGSSP (SEQ ID NO: 416) MPYDLYHP (SEQ ID NO:47) SGG (SEQ ID NO: 102) SEQ ID NO: 421 SEQ ID NO: 425 Antibody 2-9 CPFPALELC (SEQ ID NO: 19) GGSSPP (SEQ ID NO:417) HEQLTV (SEQ ID NO: 57) SGG (SEQ ID NO: 102) SEQ ID NO: 421 SEQ ID NO: 426 Antibody 2-10 CPFPALELC (SEQ ID NO: 19) GGGSSGGSG (SEQ ID NO: 96) GSGGSG (SEQ ID NO: 414) SGG (SEQ ID NO: 102) SEQ ID NO: 421 SEQ ID NO: 427 Antibody 2-11 CPFPALELC (SEQ ID NO: 19) SSPSPSGG (SEQ ID NO: 418) GGIGQLTA (SEQ ID NO: 48) SGG (SEQ ID NO: 102) SEQ ID NO: 421 SEQ ID NO: 428 Antibody 2-12 CPFPALELC (SEQ ID NO: 19) GSPGSP (SEQ ID NO: 419) KPILFFRL (SEQ ID NO: 54) SGG (SEQ ID NO: 102) SEQ ID NO: 421 SEQ ID NO: 431 Antibody 2-13 CPFPALELC (SEQ ID NO: 19) GGSSPP (SEQ ID NO:417) RAAAVKSP (SEQ ID NO: 72) SGG (SEQ ID NO: 102) SEQ ID NO: 421 SEQ ID NO: 429

Serial dilutions of antibodies were prepared in assay buffer, added to 96-well microplates coated with human CTLA4-Fc, and shaken at room temperature for one hour. The dish was washed with TBS-T, blocked with BSA and washed again with TBS-T. Add antibody to the dish and shake. After washing, the anti-human kappa light chain (bound to HRP) in the assay buffer was applied to each well and shaken at room temperature. Wash the dishes again, and then add the HRP substrate. The luminescence was recorded using a spectrophotometer and the data was analyzed with GraphPad Prism. result

Figures 10A and 10B show binding studies with masked and unmasked versions of Antibody 2 in the absence (Figure 10A) or presence (Figure 10B) of protease. Figure 10A shows the binding of various versions of Antibody 2 to the IgG1 isotype in the absence of protease. Higher antibody concentrations are required to demonstrate binding, as evidenced by the right shift of the unmasked antibody 2-6 and IgG1 isotype (Figure 10A). It is believed that the masked antibody is concealed by the masking peptide. The occlusion of each masked antibody is shown in Table 7 below, and is calculated by dividing the EC50 of the masked antibody by the EC50 of the unmasked parent antibody (antibody 2-6). The variability of the occlusion amount confirms that the sequence of the spacer linker and/or the cleavable peptide affects the ability of the masking peptide to block antigen binding. The EC50 of the antibody and the calculated inclusion degree are shown in Table 7 below. Table 7 Table 2. Antibody binding to CTLA4 of the masked Antibody EC ₅₀ ± SE (nM) R ² Occlusion Antibody 2-6 0.69 ± 0.03 0.9967 Antibody 2-7 33.9 ± 0.7 0.9997 49 Antibody 2-8 54.8 ± 1.0 0.9999 79 Antibody 2-9 48.9 ± 1.0 0.9998 62 Antibody 2-10 29.4 ± 1.5 0.9985 43 Antibody 2-11 34.9 ± 0.5 0.9999 51 Antibody 2-12 3.3 ± 0.2 0.9962 5 Antibody 2-13 14.2 ± 0.2 0.9998 twenty one

Figure 10B shows the binding of various versions of antibody 2 (excluding antibodies 2-10 with non-cleavable linkers) in the presence of protease (ie activation). The unmasked parent antibody 2-6 was included as a control and was not exposed to protease. Protease digestion was performed according to the protocol provided in Example 6. The data shown in Figure 10B indicates that the proteolytic cleavage of the cleavable peptide completely rescues the binding of the antibody to human CTLA4 by restoring the binding of the antibody at a level similar to that of the unmasked parent antibody (antibody 2-6). The binding data and activation data of the tested antibodies that were "activated" by the presence of proteases are provided in Table 8 below. The activation level is calculated by the following formula: activation level = 1-(the EC50 of the activated masked antibody-the EC50 of the unmasked parent antibody (antibody 2-6)) / (the unactivated masked antibody The EC50 of the parent antibody (antibody 2-6)). Table 8 The activated antibody binding to CTLA4 of Antibody EC ₅₀ ± SE (nM) R ² Activation level Antibody 2-6 0.69 ± 0.03 0.9967 Antibody 2-7 (activated) 0.58 ± 0.02 0.9971 1.00 Antibody 2-8 (activated) 0.67 ± 0.04 0.9951 1.00 Antibody 2-9 (activated) 0.67 ± 0.02 0.9984 1.00 Antibody 2-11 (activated) 0.62 ± 0.03 0.9973 1.00 Antibody 2-12 (activated) 0.62 ± 0.02 0.9962 1.03 Antibody 2-13 (activated) 0.57 ± 0.02 0.9985 1.01 Example 9 : In Vitro Characterization of Protease Cleavage of Masked Anti- CTLA4 Antibody: Enzyme Kinetics, Protein Cleavage and Radar Chart

Binding studies were performed in the presence of an exemplary protease using a masked form of Antibody 2, each including a masked peptide (which includes the amine sequence of SEQ ID NO: 5 or 19), and a first spacer Linker and second spacer linker and cleavable peptide. Antibody 2-15 includes a non-cleavable cleavable peptide sequence for use as a negative control. In some studies, unmasked parent antibodies (antibodies 2-6) were also tested for comparison. Examples of antibodies tested (including sequences of masking peptides, spacer linkers and cleavable peptides) are provided in Table 9. Table 9 Antibody Masking peptide Spacer linker 1 Cleavable peptide Spacer linker 2 Heavy chain Light chain + masking component Antibody 2-14 CPGKGLPSC (SEQ ID NO: 5) GGGSSGGSG (SEQ ID NO: 96) VPLSLY (SEQ ID NO: 86) SGG (SEQ ID NO: 102) SEQ ID NO: 421 SEQ ID NO: 358 Antibody 2-15 CPGKGLPSC (SEQ ID NO: 5) GGGSSGGSG (SEQ ID NO: 96) GSGGSG (SEQ ID NO: 414) SGG (SEQ ID NO: 102) SEQ ID NO: 421 SEQ ID NO: 423 Antibody 2-16 CPFPALELC (SEQ ID NO: 19) SSGGSGP (SEQ ID NO: 420) DSGGFMLT (SEQ ID NO: 50) SGG (SEQ ID NO: 102) SEQ ID NO: 421 SEQ ID NO:422 Antibody 2-17 CPFPALELC (SEQ ID NO: 19) SSPSPSGG (SEQ ID NO: 418) GGIGQLTA (SEQ ID NO: 48) SGG (SEQ ID NO: 102) SEQ ID NO: 421 SEQ ID NO: 428 Antibody 2-18 CPFPALELC (SEQ ID NO: 19) GGPGSSP (SEQ ID NO: 416) MPYDLYHP (SEQ ID NO:47) SGG (SEQ ID NO: 102) SEQ ID NO: 421 SEQ ID NO: 425 Antibody 2-19 CPFPALELC (SEQ ID NO: 19) GGSSPP (SEQ ID NO:417) HEQLTV (SEQ ID NO: 57) SGG (SEQ ID NO: 102) SEQ ID NO: 421 SEQ ID NO: 426 Antibody 2-20 CPFPALELC (SEQ ID NO: 19) GGSSPP (SEQ ID NO:417) RAAAVKSP (SEQ ID NO: 72) SGG (SEQ ID NO: 102) SEQ ID NO: 421 SEQ ID NO: 429 Antibody 2-21 CPFPALELC (SEQ ID NO: 19) GGSSPP (SEQ ID NO:417) TSVLMAAP (SEQ ID NO: 51) SGG (SEQ ID NO: 102) SEQ ID NO: 421 SEQ ID NO: 430

The comparison of the in vitro sensitivity of the cleavable peptide sequence selection protease is shown in the form of a radar chart in Figure 11A-H. Use the radar chart to depict the activation of each antibody by each protease on the radar chart, where the activation is depicted in the range of 0 (inactive) to 1.0 (fully activated). The activation level is calculated by the following formula: activation level = 1-(the EC50 of the activated masked antibody-the EC50 of the unmasked parent antibody (antibody 2-6)) / (the unactivated masked antibody The EC50 of the parent antibody (antibody 2-6)). The occlusion of each masked antibody is shown in Table 12 below, and is calculated by dividing the EC50 of the masked antibody by the EC50 of the unmasked parent antibody (antibody 2-6). The binding was carried out according to the schemes provided in Example 7 and Example 8, and the proteolytic treatment of the antibody was carried out according to the schemes provided in Example 6 and Example 8. A masked solution of anti-CTLA4 antibody (1000 nM) was prepared in a suitable lysis buffer. Divide the two antibody samples into two 0.5 mL Eppendorf tubes. The working solution of lysis buffer was added to the corresponding antibody sample (see Table 11) and the lysis buffer was added to the control (no protease) sample. Table 10 Protease Protease source Lysis buffer ¹ Protease in each reaction (ng) Working solution (ng/uL) Reaction time (min) Adam17 R&D Systems A 50 5 60 MMP1 Anaspec B 234 23.4 15 MMP2 R&D Systems B 8 0.8 15 MMP7 ProspecBio B 15 1.5 15 MMP9 ProspecBio B 50 5 15 MMP10 R&D Systems B 382 38.2 15 MMP14 R&D Systems C 201 20.1 15 NapsinA R&D Systems B 400 40 300 ^{1 For the} lysis buffer, see Table 11. Table 11 Lysis buffer Lysis buffer Buffer chemical composition A 100 mM NaOAc, 200 mM NaCl, pH 3.5 B 50 mM Tris, 50 mM NaCl, 0.01% Tween-20, pH 9 C 50 mM Tris, 3 mM CaCl2, 1 μM ZnCl2, pH 8.5

The radar charts of antibodies with various cleavable peptide sequences are shown in Figures 11A-G. Figure 11H shows that the binding of antibody 2-15 (which contains a non-cleavable cleavable peptide for use as a negative control) is not affected by the addition of various exemplary proteases. Figure 11A shows the radar chart of antibody 2-14.

As previously described, the SDS-PAGE analysis of protease cleavage on day 2 (d2), day 4 (d4) or day 7 (d7) was evaluated in healthy and MC38 tumor-bearing mice, and is shown in the figure 11I-11M. As shown in Figure 11I, the cleavage of the cleavable peptide in antibodies 2-14 was evaluated in the plasma of healthy mice. Except for mouse 1* (upper figure, 150 µg of antibody 2-14 was administered to it), 200 µg of antibody 2-14 was administered to a group of 5 healthy mice in each group. Standards were treated with protease (right lane) or without protease (left lane). As shown in Figure 11J, the cleavage of the cleavable peptide in antibodies 2-16 was evaluated in the plasma of healthy mice. The standard used in Figure 11J is antibody 2-18. Except for mouse 4* (to which 150 µg of antibody 2-14 was administered), 200 µg of antibody 2-16 was administered to healthy mice.

As shown in Figure 11K-11M, the cleavable peptide in antibody 2-14, the non-cleavable peptide in antibody 2-15, and the cleavable peptide in antibody 2-16 were evaluated in the plasma of mice bearing MC38 tumors. Cracked. 200 µg of each antibody was administered to mice bearing MC38 tumors. In Figure 11M, the day 2 (d2) sample of mouse 2 seems to have an error and should be ignored.

The binding of Antibody 2-6, Antibody 2-14, and Antibody 2-15 to CTLA4 in the presence or absence of protease is shown in Figure 12A, and EC50, occlusion and activation are provided in Table 12. Unmasked antibodies 2-6 showed binding unaffected by the presence of proteases (Figure 12A). In the absence of protease, compared to the unmasked parent antibody 2-6 that was not rescued by the addition of protease, the masked antibody 2-15, which includes an uncleavable cleavable peptide sequence, showed reduced binding Affinity (Figure 12A). In the absence of protease, the masked antibody 2-14 including the cleavable peptide showed reduced binding affinity and a encapsulation value of 101, but this reduced binding affinity was rescued when activated by the addition of protease (Figure 12A) ), where the activation value is 1.0 (Table 12). The EC50 of Antibody 2-6, Antibody 2-14, and Antibody 2-15 in the presence or absence of protease are provided in Table 12 below. Table 12 An antibody binding to CTLA4 masked and the activation of Antibody Protease EC ₅₀ (nM) R ² Conceal activation Antibody 2-6 - 0.39 ± 0.01 0.9979 Antibody 2-6 + 0.39 ± 0.01 0.9981 1.0 Antibody 2-14 - 39.4 ± 1.8 0.9989 101 Antibody 2-14 + 0.41 ± 0.01 0.9982 1.1 1.00 Antibody 2-15 - 24.3 ± 0.5 0.9997 62 Antibody 2-15 + 17.3 ± 0.7 0.9982 44 0.29

The binding of Antibody 2-6, Antibody 2-7, and Antibody 2-10 to CTLA4 in the presence or absence of protease is shown in Figure 12B, and EC50, occlusion and activation are provided in Table 13. Antibody 2-10 includes a non-cleavable cleavable peptide sequence for use as a negative control. Antibodies 2-7, which were masked and included a cleavable peptide, showed a encapsulation value of 49 in the absence of protease and an activation value of 1.0 after activation with protease (Figure 12B; Table 13). In the absence of protease, the masked antibody 2-7 showed similar binding to the masked antibody 2-10, and after activation by the addition of protease, the antibody 2-7 showed the same as the unmasked parent Antibodies 2-6 bind similarly. The EC50 of Antibody 2-6, Antibody 2-7, and Antibody 2-10 are provided in Table 13 below. Table 13 An antibody binding to CTLA4 masked and the activation of Antibody Protease EC ₅₀ (nM) R ² Conceal activation Antibody 2-6 - 0.69 0.9967 Antibody 2-7 - 33.9 0.9997 49 Antibody 2-7 + 0.58 0.9971 0.8 1.00 Antibody 2-10 - 29.4 0.9985 43

The data shown in Figure 12A, Figure 12B, Table 12 and Table 13 confirm that the exemplary masking peptides included in antibody 2-7 (SEQ ID NO: 19) and antibody 2-14 (SEQ ID NO: 5) can be reversible. Ways to mask the binding activity. Example 10 : Enterotoxin analysis method SEB analysis method

Staphylococcal enterotoxin B (SEB) assay was used to test the ability of various antibodies to promote IL-2 production by peripheral blood mononuclear cells (PBMC). For the SEB analysis method, a dilution is prepared from the selected antibody in a pre-warmed medium (RPMI + 10% heat-inactivated FBS + 1% HEPES + 1% MEM NEAA + 1% sodium pyruvate). Coat the antibody solution in triplicate and add medium only to the "PBMC+SEB" wells, "PBMC only" wells and the outermost row. Prepare SEB solution in pre-warmed medium and add it to all experimental wells except "PBMC only". PBMC (BioIVT) was thawed in a 37°C water bath. Transfer the cells dropwise to a conical tube and add 20x pre-warmed medium to wash the cells. Centrifuge the cells and aspirate the medium. Resuspend the cells in 20 mL of pre-warmed medium and remove an aliquot for counting. The remaining cells were centrifuged and the cells were resuspended in a volume of ^{1×10 6 cells/ml.} The cells were coated at 100 μl/well, and the culture plate was cultured in a 37°C, 5% CO ₂ incubator for five days. On the fifth day, the culture plate was rotated at 1000 RPM for five minutes. Transfer 250 µL of cells from each well to a new 96-well plate. The culture plate was rotated once again, and 225 µL of cells were transferred to the PCR tube. The samples were stored at -80°C until ELISA analysis. IL-2 ELISA

The analysis was performed using the Human IL-2 ELISA MAX combination (BioLegend, catalog number 431806) to determine the IL-2 content in the cell supernatant produced using the protocol described above. Dilute the cell supernatant sample with assay buffer to fall within the standard curve. The samples were analyzed using GraphPad Prism and Tukey's multiple comparisons test (one-way ANOVA) to determine the statistical significance between the treatment groups. result

Various forms of antibody 1 and antibody 2 are tested in the SEB analysis method. The data of IL-2 content produced in the presence or absence of antibody 1-1 or antibody 1-2 (as described in Example 7) is shown in Figure 13A. Data on the amount of IL-2 produced in the presence of antibody 2-1, antibody 2-2, antibody 2-3, antibody 2-4, and antibody 2-5 (as described in Example 7) or in the absence of antibody Shown in Figure 13C. Compared with the control without antibody, all tested antibodies showed the ability to increase IL-2 content. The IL-2 content of each antibody is higher than that of the no-antibody control and the comparison with the isotype control is shown in Fig. 13B and Fig. 13. Example 11 : Immune function of masked and activated anti- CTLA4 antibody

The SEB analysis method according to the protocol of Example 10 was performed to test IL-2 production in response to treatment with various forms of Antibody 2. The analysis method is also performed in the absence of antibody (ie, no antibody) or isotype control antibody as a control substance. The unmasked parent antibodies (antibodies 2-6) were also tested for comparison.

As shown in Figures 14A and 14B, in contrast to the unmasked antibody 2-6, which promotes large amounts of IL-2 production, the masked form of antibody 2 is generally ineffective in promoting IL-2 production. As shown in Figure 14B, antibody 2-12 was the only masked antibody that caused a significant increase in IL-2 production compared to the isotype control, but this increase was significantly less than when using the unmasked antibody 2-6 parent. The increase observed with this antibody. In contrast, antibody 2-7, antibody 2-8, antibody 2-9, antibody 2-10, antibody 2-11, and antibody 2-13 did not promote a significant increase in IL-2 production compared to the isotype control (Figure 14B). Statistical analysis was performed using one-way ANOVA comparison.

Figures 14C and 14D show the effect of protease treatment according to the protocol of Example 6 on the ability of Antibody 2-7, Antibody 2-8, Antibody 2-9, and Antibody 2-10 to promote IL-2 production. Antibody 2-10 includes a non-cleavable cleavable peptide sequence for use as a negative control. Test the unmasked parent antibody 2-6 as a positive control, and test the isotype control antibody and the control without antibody as the negative control. The statistical analysis shown in Figure 14D was performed using a one-way ANOVA comparison with antibodies 2-6.

The results shown in Figure 14C indicate that treatment with protease rescued the ability of antibody 2-7, antibody 2-8, and antibody 2-9 to promote IL-2 production at a level similar to that of the unmasked parent antibody 2-6. The curve shown in Figure 14B compares the fold increase in IL-2 production higher than the control without antibody. Treatment with the masked but not protease-activated antibody 2-10 caused IL-2 production similar to the isotype control and similar fold changes higher than the control without antibody (Figure 14C and Figure 14D). Example 12: The in vivo cleavage in vivo anti-CTLA4 antibody of the anti-masking method of the plasma of healthy mice masked cleavage of CTLA4 antibodies

On day 0, healthy, tumor-free female C57BL/6J mice of seven to eight weeks of age were injected intraperitoneally with 200 µg of the masked form of antibody 2. On the second and fourth days, blood was collected through the RO sinus and treated with LiHep for plasma separation. On the seventh day, blood was collected via cardiac puncture and treated with LiHep for plasma separation. The plasma was aliquoted and stored at -80°C until western blot analysis. Include a standard sample of the masked antibody treated in vitro in the presence or absence of protease as a positive control. For Western blot analysis, the plasma sample was diluted in PBS, followed by Laemmli sample buffer and βME. The sample was heated at 95°C and then loaded on a Criterion TGX non-staining precast gel. SDS-PAGE was performed at 200 V for 42 minutes. The protein was transferred to a nitrocellulose membrane and blocked with 1% casein blocker in 1x TBS for one hour at room temperature with shaking. Under shaking, the membrane was probed with the HRP-bound Rb mAb for human kappa light chain [pure EPR5367-8] diluted 1:10,000 in casein blocker at 4°C overnight. The membrane was washed three times with PBS-Tween and studied with SuperSignal ELISA Pico chemiluminescence substrate. Use ImageLab software to analyze the raw data. The activation percentage is calculated as follows: the intensity of the activated band/(the intensity of the activated band + the intensity of the masked unactivated band)×100%.

In vivo experiments were performed over two weeks in two groups, and antibodies 2-14 were included in both groups. The antibodies tested include antibody 2-14, antibody 2-15, antibody 2-16, antibody 2-17, antibody 2-18, antibody 2-19, antibody 2-20, and antibody 2-21. Antibody 2-15 includes a non-cleavable cleavable peptide sequence for use as a negative control. result

Antibody 2-14 (Figure 15A) and antibody 2-20 (Figure 15C) are both activated in vivo. The activation of antibody 2-19 was not detected in vivo (Figure 15B). The in vivo activation of antibody 2-16, antibody 2-17, antibody 2-18, and antibody 2-21 was not detected (data not shown), and the in vivo activation of negative control antibody 2-15 was also not detected . The increase in activation percentage of antibody 2-14 and antibody 2-20 from day 2 to day 7 is shown in Figure 15D. Example 13 : In vivo lysis of masked anti-CTLA4 antibodies in the plasma of tumor-bearing mice

MC38 cells (1×10 ⁶ cells) were injected subcutaneously into seven- to eight-week-old female C57BL/6J mice. On day 0, mice were randomly divided into groups based on tumor volume measurements and injected intraperitoneally with 200 µg of antibody 2 construct. On the second and fourth days, blood was collected through the RO sinus and treated with LiHep for plasma separation. On the seventh day, blood was collected via cardiac puncture and treated with LiHep for plasma separation. The plasma was aliquoted and stored at -80°C until western blot analysis. Include a standard sample of the masked antibody treated in vitro in the presence or absence of protease as a positive control. For Western blot analysis, the plasma sample was diluted in PBS, followed by Laemmli sample buffer and βME. The sample was heated at 95°C and then loaded on a Criterion TGX non-staining precast gel. SDS-PAGE was performed at 200 V for 42 minutes. The protein was transferred to a nitrocellulose membrane and blocked with a 1% casein blocker in 1x TBS for one hour at room temperature with shaking. Under shaking, the membrane was probed with the HRP-bound Rb mAb for human kappa light chain [pure EPR5367-8] diluted 1:10,000 in casein blocker at 4°C overnight. The membrane was washed three times with PBS-Tween and studied with SuperSignal ELISA Pico chemiluminescent substrate. Use ImageLab software to analyze the raw data.

In vivo experiments were performed over two weeks in two groups, and antibodies 2-14 were included in both groups. The antibodies tested include antibody 2-14, antibody 2-15, antibody 2-16, antibody 2-17, antibody 2-18, antibody 2-19, antibody 2-20, and antibody 2-21. Antibody 2-15 includes a non-cleavable cleavable peptide sequence for use as a negative control.

In another set of experiments, MC38 cells (1×10 ⁶ cells) were injected subcutaneously into a mouse model using human CTLA4 for gene insertion. A single injection of 200 µg of test antibody (eg IgG control, antibody 2-1, antibody 2-10, Ipelizumab or Ipelizumab without trehalosylation is administered to mice bearing MC38 tumors Form (Ipelizumab-aFuc)), and CD45+ splenocytes and CD45+ intratumoral cells were subjected to immunophenotyping analysis on the fifth day after injection. In some studies, the tumor volume was measured over time after a single injection of 20 µg, 7 µg, or 2 µg of the test antibody. Evaluation of CD45+ cells for 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+ selectivity, and the relative proportion of identified CD45+ cells with these markers. result

In mice carrying MC38, antibody 2-14 and antibody 2-20 were activated in vivo (Figures 16A and 16C). The activation of antibody 2-19 was not detected in vivo (Figure 16B). The activation of antibody 2-16, antibody 2-17, antibody 2-18, and antibody 2-21 was also not detected (data not shown), and activation of the negative control antibody 2-15 was also not detected. Compared with healthy, tumor-free mice, the activation percentage of antibody 2-14 in MC38-bearing mice was significantly greater, as determined by 2-way ANOVA (adjusted P value = 0.0113) (Figure 16D) ).

The results of the study using MC38 tumor-bearing mice are depicted in Figures 19A-19D, where CD3+/ICOS+, CD3+ T cells, CD4+/Ki67+, CD3+/Ki67+, CD4+/ICOS+, CD4+ T cells, CD8+/ICOS+, CD8+ T The markers of cells, Treg+/ICOS+, CD8+/Ki67+, Treg, Treg+/Ki67+ selectively provide the relative ratio of splenic CD45+ (Figures 19A and 19B) or intratumoral CD45+ (Figures 19C and 19D). Statistics are provided in Figure 19E (spleen cells) and 19F (intratumoral cells). Group 1: IgG, Group 2: Antibody 2-1; Group 3: Antibody 2-10; Group 4: Ipelizumab; Group 5: Ipelizumab-aFuc.

The results of studies using MC38 tumor-bearing mice that received a single injection of 20 µg, 7 µg, or 2 µg of the test antibody are depicted in Figures 20A and 20B. ^{Figure 20A shows the tumor volume (mm 3} ) over time for antibody 2-6, antibody 2-10, and RSV antibody with S239D and I332E mutations in the Fc domain (RSV-m). As shown in Figure 20A, the unmasked parent antibody 2-6 inhibited tumor growth compared to the masked but non-cleavable antibody 2-10. Figure 20B depicts the tumor average of tumor volume over time, which indicates that tumor growth after antibody 2-6 administration is slower compared to antibody 2-10 or RSV-m control. Example 14 : ADCC activity achieved by anti- CTLA4 antibody and CTLA4 blocking method reporter bioanalysis method

The FcγRIIIa reporter bioanalysis method was performed using various anti-CTLA4 antibodies. The antibody was diluted in pre-warmed complete medium (FBS with RPMI1640). The CTLA4 effector cells (target cell: Promega J158A) were thawed and transferred to a conical tube containing complete medium. The cells are mixed and counted, and the density of the cells is adjusted to 1×10 ⁶ cells/ml. Add target cells to each well of a 96-well plate (Corning, catalog number: 3917). The diluted antibody is added to the appropriate wells and tested in duplicate. The contents of each well were mixed gently and the plate was incubated at 37°C for fifteen minutes. The effector cells (FcγRIIIa that express Jurkat cells) were thawed and transferred to a conical tube containing complete medium. The cells were mixed and counted, and the density of the cells was adjusted to 3×10 ⁶ cells/ml. Immediately afterwards, the effector cells are distributed to each well and mixed gently. The pan was covered with a lid and kept at 37°C for six hours. One hour before the measurement, the Bio-Glo substrate and Bio-Glo buffer were removed at 4°C. Transfer the Bio-Glo buffer to the Bio-Glo substrate to produce the Bio-Glo reagent, and mix gently by inversion. The bottle is kept at room temperature. After incubation, the analysis tray was removed from the incubator and kept at room temperature for ten minutes. Bio-Glo reagent is added to each well, and the plate is incubated at room temperature for five to fifteen minutes. Next, the disc is read with a photometer.

The antibodies tested included antibody 2-6, antibody 2-14, and antibody 2-15, as well as isotype controls. Antibody 2-6 is an unmasked parent antibody against antibody 2-14 and antibody 2-15. Antibody 2-15 includes a non-cleavable cleavable peptide sequence for use as a negative control. Antibody 2-6, Antibody 2-14, and Antibody 2-15 were each tested without prior exposure to protease, and also tested after exposure to protease, which is activated by cleavage of cleavable peptide. The occlusion degree of each antibody was calculated by dividing the EC50 of the antibody by the EC50 of the unmasked parent antibody (antibody 2-6). result

The unmasked parent antibody 2-6 showed similar reporter activation curves in the presence and absence of protease (Figure 17). When tested without prior exposure to protease, the masked antibodies (antibody 2-14 and antibody 2-15) showed reduced reporter activation compared to the unmasked parent antibody 2-6 (Figure 17). The reporter activation of antibody 2-15 (which cannot be activated by protease) was not rescued by the pre-addition of protease (ie, did not return to an activation level similar to that associated with the unmasked parent antibody 2-6) (Figure 17). The reporter activation of antibody 2-14 (which can be activated by protease) was rescued to a level similar to that associated with the unmasked parent antibody (antibody 2-6) (Figure 17). The EC50 values and occlusion values of the tested antibodies are provided in Table 14. Table 14 Antibody Protease EC ₅₀ (ng/mL) ± SE R ² Occlusion Antibody 2-6 - 8.0 × 10 ^-3 ± 1.8 × 10 ^-3 0.927 Antibody 2-6 + 4.9 × 10 ^-3 ± 1.3 × 10 ^-3 0.8759 0.6 Antibody 2-14 - 33 ± 6 0.9906 4,125 Antibody 2-14 + 2.8 × 10 ^-3 ± 0.5 × 10 ^-3 0.8953 0.4 Antibody 2-15 - 19 ± 2 0.9942 2,375 Antibody 2-15 + 10 ± 2 0.9722 1,250 CTLA4 blocking bioassay

The CTLA4 blocking bioassay was used to test the ability of anti-CTLA4 antibodies (including masked anti-CTLA4 antibodies) to block the interaction between CTLA4 and its ligands CD80 and CD86. The antibody is diluted in pre-warmed complete medium. The CTLA4 expressing Jurkat cells (effector cells) was thawed and transferred to a conical tube containing complete medium. Mix the cells and count. Add cells to each well of a 96-well plate (Corning, #3917). The diluted antibody is added to the appropriate wells and tested in duplicate. Mix the plates gently and incubate at 37°C for 15 minutes. The APC cells (CD80/CD86 expressing Raji cells) were thawed and transferred to a conical tube containing complete medium. The cells are mixed gently, counted and immediately distributed to each well and mixed gently. Cover the pan and keep at 37°C for 6 hours. Mix the Bio-Glo reagents gently by inverting and keep the bottle at room temperature. After six hours of incubation, the analysis tray was removed from the incubator and kept at room temperature for ten minutes. Bio-Glo reagent is added to each well and the plate is incubated at room temperature for five to fifteen minutes. Next, the disc was read with a photometer and analyzed using GraphPad Prism. A one-way ANOVA comparison was used to perform statistical analysis of the fold change in the ability to block binding.

The antibodies tested included antibody 2-2 and antibody 2-14 and isotype controls. Antibody 2-2 is the unmasked form of Antibody 2. Antibody 2-14 was tested without prior exposure to protease (ie, in a masked, unactivated form), and also after exposure to protease (ie, in an activated form). The analysis method was also performed without the use of antibodies (that is, as a no-antibody control). result

As shown in Figure 18A, when in an unactivated form due to the absence of protease, antibody 2-14 did not show the ability to effectively block the binding of CTLA4 to its ligand. For blocking the binding of CTLA4 to its ligand, the analysis of the fold change higher than that of the control without antibody did not show a significant difference between the ability of the antibody 2-14 in the unactivated form and the ability of the isotype control ( Figure 18B). However, when antibody 2-14 was in an activated form due to pre-exposure to protease, it showed the ability to effectively block the binding of CTLA4 to its ligand at a concentration within a certain range (Figure 18A). The ability of the activated antibody 2-14 to block the binding of CTLA4 to its ligand is similar to that of the unmasked antibody 2-2 (Figures 18A and 18B). The fact is that the analysis of the fold change in blocking the binding of CTLA4 to its ligand is higher than that of the non-antibody control did not show a significant difference between the ability of the activated antibody 2-14 and the unmasked antibody 2-2 Difference (Figure 18B). Example 15: Efficacy of anti-CTLA4 antibody and pharmacodynamics method MB49 murine bladder tumor model

The MB49 murine bladder tumor model was used to evaluate the efficacy and pharmacodynamics (PD) of anti-CTLA4 antibodies. Efficacy was evaluated by administering each test antibody (RSV-m control antibody, Ipelizumab, antibody 2-6, antibody 2-14, and antibody 2-15) and then evaluating tumor volume and body weight. The anti-RSV control antibody contains S239D and I332E mutations (RSV-m control antibody). Peripheral immunophenotyping was also performed on the 5th day, which evaluated the percentage of CD4+Ki67+ cells and the percentage of CD4+ICOS+ cells in the peripheral blood on the 5th day after administration. Fourteen groups of mice were evaluated for efficacy studies, where the dosage (mg/kg) is shown in Table 15. As used herein, the dose in mg/kg is also referred to as "mpk". MB49 cells were subcutaneously inoculated into C57/BL6-huCTLA4 mice. Treatment begins when the tumor reaches about 350 mm ^3. One-way ANOVA and Dunnett's post-test were performed to determine the statistical significance of the treatment group and the control group (RSV-m control). *P<0.05;**P<0.01;***P<0.001;****P<0.0001. Table 15 Group Antibody Dose (mg/kg) 1 RSV-m control 10 2 Ipelizumab 0.3 3 Ipelizumab 1 4 Ipelizumab 3 5 Antibody 2-6 0.3 6 Antibody 2-6 1 7 Antibody 2-6 3 8 Antibody 2-6 10 9 Antibody 2-14 1 10 Antibody 2-14 3 11 Antibody 2-14 10 12 Antibody 2-15 1 13 Antibody 2-15 3 14 Antibody 2-15 10

The pharmacodynamics was evaluated by administering the antibody to each of the five mouse groups, where the dose (mg/kg) is shown in Table 16. Perform immunophenotyping and other readings in tumors, liver, spleen and blood. T cells are evaluated by measuring Foxp3+CD25+ cells as a percentage of CD4+ cells, and CD8+ cells are measured as a percentage of CD45+ cells. The lysis and drug content in tumor, liver, kidney, spleen and plasma are also evaluated. Table 16 Group Antibody Dose (mg/kg) 1 RSV-m control 10 2 Ipelizumab 3 3 Antibody 2-6 3 4 Antibody 2-14 3 5 Antibody 2-15 3 result

Figure 23A shows the results of an efficacy study on the percentage of CD4+Ki67+ cells (left) and CD4+ICOS+ cells (right) in peripheral blood on the 5th day after administration, which represents the level of T cell activation. The results showed that the masked anti-CTLA4 antibody (antibody 2-14) exhibited T cell activation at a dose of 10 mg/kg lower than that of the anti-CTLA4 antibody Ipelizumab at a 3.3-fold lower dose (3 mg/kg). ) T cell activation presented below. This indicates that in the MB49 murine model, the masked antibody 2-14 is safer than Ipelizumab.

As shown in Figure 23B, in the small group treated with 10 mg/kg (RSV-m control) or 3 mg/kg (Ipelizumab, antibody 2-6, antibody 2-14, antibody 2-15) In mice, tumor weight and CD8/Treg ratio were evaluated on the 7th day. On day 7, at 3 mg/kg, mice treated with antibody 2-6 or antibody 2-14 showed the lowest tumor weight and the highest CD8/Treg ratio in the tumor. No changes in body weight, spleen weight, kidney weight, or liver weight were observed under all treatment conditions (data not shown).

As shown in Figure 23C, regulatory T cell depletion and CD8+ T cell activation were observed in the case of antibody 2-14 but non-Ipelizumab.

As shown in Figure 23D, strong anti-tumor activity was observed after treatment with anti-CTLA4 antibody. At a dose of 0.3 mg/kg, antibody 2-6 showed better anti-tumor activity compared with ipelizumab. Example 16: In vivo lysis detection method of anti-CTLA4 antibody in tumor, liver, kidney, spleen and plasma

B-hCTLA-4 gene transgenic female mice were purchased from Biocytogen and were 8-10 weeks old at the beginning of the study. Subcutaneously subcutaneously MC38 colorectal tumor cells (5×10 ⁵ cells/mouse), MB49 bladder cancer cells (1×10 ⁶ cells/mouse) or MCA205 fibrosarcoma cells (1×10 ⁶ cells/mouse) Inoculated into the right abdomen of each mouse. When the tumor size reached approximately 250-500 mm ³ (day 0), the mice received a single 10 mg/kg intraperitoneal administration of the indicated antibody. Tumor, liver, kidney, spleen and plasma were collected on the 4th day for lysis analysis by Western blotting method.

For plasma samples, use the direct western blotting method by separating an equal volume of denatured plasma (1-2 µL input) on a gradient regular precast gel (BioRad). Next, the separated protein was transferred to the nitrocellulose membrane using the Turbo Transblot system (BioRad). Next, the membrane was studied for the anti-human kappa light chain reactivity of the antibody (ab202549) bound to HRP using standard methods. The cleavage was evaluated visually because the migration of the cleaved light chain was lower than that of the uncleaved light chain at a mass difference of 2 kD.

In RIPA buffer (Pierce, 87788) supplemented with protease and phosphatase inhibitors (Thermo Fisher, 78442), use Tissue Lyser II (Qiagen) bead homogenizer to produce protein lysates from tumor, liver, spleen and kidney . The protein concentration was determined by BCA analysis and 3 mg of tissue lysate was combined with biotin anti-human kappa light chain antibody (LSBio, LS-C351451-500) and streptavidin magnetic beads (Pierce, 88816) to undergo immunization together precipitation. The protein was eluted in 50 µL of 10 mM glycine (pH 2) and neutralized with 1 M Tris (pH 8), followed by Western blot detection of human kappa light chain as described above.

In another study, the CTLA4 ELISA assay was used to evaluate the in vitro lysis of healthy monkey (cynomolgus macaque) tissue (N=2) and plasma (N=3). The tissues tested included kidney, spleen, liver, bladder, skin, colon and lung.

In another study, in vivo lysis in the plasma of healthy cynomolgus monkeys was evaluated at 1 hour, 1 day, and 7 days after administration. The cleavage was identified by capillary electrophoresis (CE) and mass spectrometry (MS). Calculate the abundance of the cleavage product at the expected cleavage site (VPLS**LY) in antibody 2-14. The cleavage at another site (VPLSLY**SGG) was also calculated for comparison. The abundance of the lysate of antibody 2-16 was also calculated.

In another study, the CTLA4 ELISA assay was used to assess ex vivo lysis in murine tumors, tissues, and plasma. This includes the following murine models: C57/Bl6 (N=9), MC38 (N=9) in wild-type mice, MC38 (N=5) in hCTLA4 mice, MB49 (N=5) in wild-type mice =6), MCA205 in wild-type mice (N=4) and B16 in wild-type mice (N=4). Assess lysis in tumor, spleen, liver, kidney, and plasma. Pool the plasma samples. Calculate the incidence of cleavage and the percentage of cleavage molecules (median value).

In another study, human conditioned media derived from human tumors (colon tumors, lung tumors) were used for ex vivo lysis. Prior to analysis, protein A/G beads were used in two rounds of immunoprecipitation to remove human IgG from human samples. SDS-PAGE analysis was performed using anti-hkLC antibody to detect the lysed and uncleaved form of the tested antibody. ELISA analysis was performed to calculate the percentage of lysis (%). result

As shown in Figure 24A, for the study using MCA205 fibrosarcoma cells, in the case of antibody 2-14 but not antibody 2-16, a large amount of lysate was detected in tumor and liver tissues. The positive control (control*) includes samples from antibody 2-14 with 50% unlysed and 50% lysed products.

As shown in Figure 24B, for the study using MB49 bladder cancer cells, in the case of antibody 2-14 but not antibody 2-16, a large amount of lysate was detected in liver tissue. The positive control (control*) includes samples from antibody 2-14 with 50% unlysed and 50% lysed products.

As shown in Figure 24C, for the study using MC38 colorectal cancer cells, in the case of antibody 2-14 but not antibody 2-16, a large amount of lysate was detected in liver tissue. The positive control (control*) includes samples from antibody 2-14 with 50% unlysed and 50% lysed products.

The following study was also carried out: the IgG content of each antibody was quantified to mice carrying MB49 by ELISA analysis, and then on the MSD plate coated with CTLA4, 1 µg/mL (spleen) or 0.04 µg/ mL (kidney and liver tissue) began to analyze each sample in a series of 3-fold dilutions to assess CTLA4 binding. These studies include unmasked parent antibodies (antibody 2-6), masked and cleavable antibodies (e.g., antibody 2-14 and antibody 2-16), and masked but non-cleavable antibodies (antibody 2-10). ). Figure 24D is an SDS-PAGE analysis showing the percentage (%) of the lysate of antibody 2-14. Calculate the percentage of fragmentation based on the sensitivity of the photosensitivity (percentage of fragmentation=low band/(low band+high band)). The first lane (left) shows 28% lysis in plasma, 60% lysis in kidney, 59% lysis in liver, and 73% lysis in spleen. The second lane (middle) shows 39% lysis in plasma, 54% lysis in kidney, 77% lysis in liver, and 72% lysis in spleen. The third lane (right) shows 35% lysis in plasma, 64% lysis in kidney, 75% lysis in liver, and 69% lysis in spleen. The arrow indicates the sample used to assess binding to CTLA4, and the results are shown in Figure 24E. Figure 24E shows the binding between each antibody and CTLA4, in which each antibody was isolated from plasma, kidney, liver, or spleen after administration of MB49-carrying B-hCTLA4 gene transgenic mice. As shown in Figure 24E, the non-cleavable masked antibody 2-10 showed reduced binding compared to the unmasked parent antibody 2-6, while the cleavable masked antibody 2-14 was found in the liver and spleen. It showed similar binding to antibody 2-6 and showed more binding than antibody 2-10 in plasma, kidney, liver and spleen. In the kidney, liver, and spleen, antibody 2-16 showed stronger binding than antibody 2-10. Figure 24F depicts a heat map of in vitro cleavage of cleavable peptide substrates by a set of exemplary proteases.

The study on the in vitro lysis of antibodies 2-14, 2-15, 2-10, 2-16, and 1334 in the organs and plasma of cynomolgus monkeys using CTLA4 ELISA assay is summarized as follows. Antibody 2-14 showed high percentage of lysis in lung (88%), bladder (37%) and colon (36%). Antibodies 2-16 showed a moderate percentage of lysis in the lung (30%) and spleen (15%). Antibody 1334 showed a moderate percentage of lysis in the lung (22%). Antibody 2-14 showed a low percentage of lysis in plasma (4%). No lysis of antibody 2-16 was detected in the plasma. Antibody 1334 showed a low percentage of lysis in plasma (5%).

For C57/Bl6-wt, MC38-wt, MC38-hCTLA4, M49-wt, MCA205-wt and B16 mice, the CTLA4 ELISA assay is used to evaluate certain antibodies (such as antibody 2-14, antibody 2-15) , Antibody 2-10, Antibody 2-16 and Antibody 1334) The results of the study of ex vivo lysis in murine tumors, tissues and plasma are shown in Figures 25A-25F. The percentages of lysed molecules as calculated by ELISA analysis are summarized in Table 17. Table 17. Percentage of lysed molecules (%) Rodent model organize Antibody 2-14 Antibody 2-15 Antibody 2-10 Antibody 2-16 Antibody 1334 C57/Bl6-wt Spleen (n=9) 3% 2% 2% 3% 4% Liver (n=9) 2% 2% 3% 2% 2% Kidney (n=9) 7% 4% 3% 2% 40% Plasma (n=2) 6% 3% 5% 19% 0% MC38-wt Tumor (n=9) 1% 0% 4% Spleen (n=9) 4% 3% 0% Liver (n=9) 2% 1% 5% Kidney (n=9) 8% 1% 3% Plasma (n=1) 1% 0% 0% 0% 0% MC38-hCTLA4 Tumor (n=5) 0% 0% 0% 7% 1% Spleen (n=5) 7% 0% 0% 6% 0% Liver (n=5) 2% 0% 2% 2% 6% Kidney (n=5) 25% 4% 3% 5% 72% Plasma (n=1) 1% 0% 0% 0% 30% MB49-wt Tumor (n=6) 1% 5% 15% 2% 0% Spleen (n=6) 3% 0% 0% 1% 0% Liver (n=6) 0% 6% 7% 1% 4% Kidney (n=6) 9% 2% 1% 5% 84% Plasma (n=1) 0% 4% MCA205-wt Tumor (n=4) 2% 2% 0% 0% Spleen (n=4) 0% 12% 0% 2% Liver (n=4) 0% 2% 0% 0% Kidney (n=4) 10% 4% 1% 0% Plasma (n=1) 0% 0% 0% B16-wt Tumor (n=4) 0% 0% 0% 0% 1% Spleen (n=4) 2% 0% 2% 1% 3% Liver (n=4) 2% 0% 1% 1% 0% Kidney (n=4) 16% 3% 3% 2% 38% Plasma (n=1) 0% 0% 0% 0% 1%

The incidence of cracking is displayed in the form of a radar chart, as shown in Figures 25A-25F. Regarding the incidence of lysis in tumor conditioned medium, antibody 2-14 showed a 33% incidence in MC38-wt tumor conditioned medium, a 17% incidence in MB49-wt tumor conditioned medium, and an MCA205-wt tumor 25% incidence in conditioned medium; and antibody 2-16 showed 100% incidence in MC38-wt tumor conditioned medium, 40% incidence in MC38-hCTLA4 tumor conditioned medium and MB49-wt tumor The conditioned medium showed a 17% incidence. Regarding the incidence of lysis in the kidney-conditioned medium, antibody 2-14 showed a 100% incidence in the kidney-conditioned medium of MC38-wt, MC38-hCTLA4, MB49-wt, MCA205-wt and B16-wt; and antibody 2-16 It exhibited a 67% incidence in MC38-wt kidney conditioned medium, an 80% incidence in MC38-hCTLA4 kidney conditioned medium, a 17% incidence in MB49-wt kidney conditioned medium, and a 17% incidence in B16-wt. The incidence of 100% in kidney conditioned medium.

Figures 26A and 26B depict the results of an in vivo lysis study using antibodies 2-14 in the plasma of healthy cynomolgus monkeys. As shown in Figure 26A, the calculated lysis of antibody 2-14 using capillary electrophoresis (CE) is 0% (all three individuals, the first hour) and 8% (the first individual, the first day) , 6% (Individual No. 3, Day 1) and 4% (Individual No. 5, Day 1); and 35% (Individual No. 1, Day 7), 32% (Individual No. 3, Day 7) and 31% (Individual No. 5, Day 7). Mass spectrometry (MS) was also used to calculate the cleavage of antibody 2-14 of individual No. 3, which was calculated as 0% (1st hour), 18% (1st day) and 32.3% (7th day). The abundance of the intact (ie, uncleaved) antibody 2-14 and the cleavage product at the expected cleavage site (VPLS**LY) or another site (VPLSLY**SGG) was quantified. The calculated abundance of intact antibody 2-14 is 100% (1st hour), 82.1% (24th hour) and 67.7% (72nd hour); the calculated abundance of cleavage product VPLS**LY is 0 % (1st hour), 17% (24th hour) and 32.3% abundance (72nd hour); and the calculated abundance of cleavage product VPLSLY**SGG is 0% (1st hour and 72nd hour) And 1% (the 24th hour).

Figure 26C depicts the results of an in vivo lysis study using antibodies 2-16 in the plasma of healthy cynomolgus monkeys. As shown in Figure 26C, under the test conditions, lysis of antibody 2-16 was not identified using CE or MS. The calculated abundance of intact (ie, uncleaved) antibody 2-16 is 100% abundance (hour 1, 24, and 72).

Figures 27A and 27B depict the results of ex vivo lysis studies that used media conditioned with human lung or colon tumor samples. Figure 27A depicts SDS-PAGE analysis, which demonstrates the removal of human IgG by performing two rounds of immunoprecipitation (IP), thereby enabling lysis analysis. Figure 27B depicts the results of SDS-PAGE analysis, which shows that after culturing in a culture medium conditioned by a human tumor sample, there is a test antibody in a lysed state, an unlysed state, or two states. The displayed lysis percentage (%) of the selected sample depicting lysis was also used to calculate the result using the ELISA analysis method (see Figure 28E). As shown in Figure 27B, it was confirmed that antibody 2-14 showed lysis in the tumor sample, and calculated to show 2.7% lysis in the case of lung tumor samples, 100% lysis in the case of colon 28 aug colon tumor samples, and 28 aug in colon The colon tumor sample showed 75% lysis and the colon 2 4sept colon tumor sample showed 100% lysis. As shown in Figure 27B, it was confirmed that antibody 2-16 showed lysis in the tumor sample, and it was calculated to show 21% lysis in the case of lung tumor samples, 98% lysis in the case of colon 28 aug colon tumor samples, and 28 aug in colon. The colon tumor sample showed 45% lysis and the colon 2 4sept colon tumor sample showed 100% lysis. This indicates that the results of the ELISA binding analysis method are related to lysis, as shown for antibody 2-14 and antibody 2-16 in the SDS-PAGE analysis.

The colon tumor supernatant (colon 2 4 sept colon tumor sample) was also used to analyze the percentage (%) of lysed molecules by mass spectrometry and compared with the results of ELISA analysis. Calculate the abundance of the intact (ie, uncleaved) antibody at each point in the cleavable peptide and the cleavage product produced by the cleavage. The results are shown in Figures 28A-28D. As shown in Figure 28A, when cultured in colon tumor conditioned medium, the abundance of intact antibody 2-14 was 4.8%, compared to 99.6% when cultured in RPMI as a control. . When antibody 2-14 was cultured in colon tumor conditioned medium, the abundance of L*SLY lysate was 69%, the abundance of S*LY lysate was 5.9%, and the abundance of L*YSGG lysate was 2.7%. The abundance of Y*SGG cleavage product was 11.3%, and the abundance of S*GG cleavage product was 6.3%. As shown in Figure 28B, when cultured in colon tumor conditioned medium or RMPI as a control, the abundance of intact antibodies 2-15 was 98.8%. As shown in Figure 28C, when cultured in colon tumor conditioned medium, the abundance of intact antibodies 2-16 was 0.2%, in contrast, when cultured in RMPI as a control, the abundance was 99.8% . When antibody 2-16 was cultured in colon tumor conditioned medium, the abundance of T*SGG lysate was 96.8%. As shown in Figure 28D, when cultured in colon tumor conditioned medium, the abundance of intact antibodies 2-10 was 94.3%, compared to 97.5% when cultured in RMPI as a control. .

Use the masked version of Antibody 2-6, Antibody 2-14, Antibody 2-15, Antibody 2-10, Antibody 2-16, and Ipelizumab containing the S239D and I332E mutations in the Fc domain (masked Ipelizumab-m) was subjected to ELISA analysis, in which each tested antibody was cultured in colon tumor conditioned medium (colon 24 sept colon tumor sample) or RPMI as a control. The results are shown in Figure 28E. The calculated antibody 2-14 shows 100% molecular lysis, the calculated antibody 2-15 shows 0% molecular lysis, the calculated antibody 2-10 shows 4% molecular lysis, and the calculated antibody 2-16 shows 100% Molecular cleavage.

For the percentage (%) of lysed molecules, the MS data shown in Figures 28A-28D correlates with the ELISA data. For antibodies 2-14, in the case of this colon tumor sample, ELISA analysis showed 100% lysis, compared to 95.2% based on MS data. For antibodies 2-15, in the case of this colon tumor sample, the ELISA analysis showed 0% lysis, compared to 1.2% based on MS data. For antibodies 2-10, in the case of this colon tumor sample, the ELISA analysis showed 4% lysis, compared to 5.7% based on MS data. For antibodies 2-16, in the case of this colon tumor sample, ELISA analysis showed 100% lysis, compared to 99.8% based on MS data.

Anti-CTLA4 ELISA analysis was performed using human tumor tissues and normal tissue adjacent to tumor tissues (NAT) obtained from the Cooperative Human Tissue Network (CHTN). The antibodies tested included antibody 2-14, antibody 2-16, and antibody 1334. Table 18 summarizes the results. As shown in Table 18, the overall incidence of lysis of human tumor tissues by antibody 2-14 (65%) is higher than antibody 2-16 (22%). Antibody 2-16 (26%) has a slightly higher percentage of molecules that are lysed by tumors than antibody 2-14 (22%). Antibody 2-14 (24%) has a higher total percentage of molecules cleaved by NAT than antibody 2-16 (7%). Table 18 Incidence of lysis Cracking percentage (%) Antibody organize NAT Tumor NAT Tumor Antibody 2-14 Melanoma 1/1 3/3 100% 18% Kidney cells 19/25 15/26 10% 25% Ovaries 1/1 4/9 4% 5% bladder 3/3 4/5 56% 7% Lymph nodes 1/1 2% colon 8/8 7/7 34% 46% lung 5/6 4/7 3% 4% breast 3/4 3/4 64% 37% liver 4/5 3/6 28% 2% total 44/53 = 83% 44/68 = 65% twenty four% twenty two% Antibody 2-16 Melanoma 1/1 1/3 9% 1% Kidney cells 9/25 4/26 2% 26% Ovaries 0/1 2/9 0% 5% bladder 0/3 0/5 0% 0% Lymph nodes 0/1 0% colon 3/8 5/7 3% 50% lung 1/6 1/7 2% twenty one% breast 1/4 1/4 5% 6% liver 1/5 1/6 71% 2% total 16/53 = 30% 15/68 = 22% 7% 26% Antibody 1334 Melanoma 1/1 2/3 32% 4% Kidney cells 16/24 17/25 8% 15% Ovaries 1/1 3/9 5% 6% bladder 2/3 4/5 57% 8% Lymph nodes 1/1 14% colon 2/6 4/6 41% 39% lung 1/6 1/7 18% 6% breast 2/4 2/3 20% 11% liver 4/5 2/6 28% 4% total 26/50 = 52% 33/65 = 51% 18% 14% Example 17 : Enzyme and proteomic analysis of MC38 and H228 cell lines using patient tumors and normal adjacent tissues (NAT)

According to the standard protocol (Thermo Scientific #23225), the total protein concentration in the sample was evaluated by the bicinchoninic acid (BCA) analysis method. The concentration of tumor/NAT to sample is about 1 mg/mL, while the concentration of cell line sample is about 10 times lower. Therefore, two replicate cell line samples (MC38-1 and H2228-1) were concentrated 10-fold using a 10 kDa cut-off membrane. The sample is incubated with the A MSP-MS library at a final reaction concentration of 50 μg/mL protein. A. Cytotoxicity

LDH assay (Thermo Scientific #88953) was used to measure the activity of lactate dehydrogenase (LDH) in conditioned medium samples according to the manufacturer's protocol to measure cytotoxicity. The background LDH activity was also measured in serum-free DMEM medium as a control for MC38 and RPMI with or without serum and NAT/tumor samples as a control for H2228. The cytotoxicity is expressed relative to the cytotoxicity of the corresponding background medium control.

The activity of LDH in the conditioned medium sample was used as a representative of necrosis, as shown in Figure 29. Figure 29 shows the cytotoxicity measured using the LDH release assay done in serum-free or supplemented with fetal calf serum (FCS) medium, which was adjusted with the indicated cell line or tumor/NAT tissue. The absorbance value is expressed as the percentage of baseline activity measured in the corresponding medium of each sample. Although MC38 cells have LDH cytotoxicity similar to previous cell viability data, H2228 cells have higher than expected cytotoxicity (about 2.6 times higher than MC38).

When cultured in serum-free medium, tumors and NAT tissues exhibit increased cell death. Tumor samples are more sensitive to serum deficiency than NAT samples, potentially reflecting higher growth rates in tumor samples. As shown in Figure 29, tissue samples cultured in complete medium have the lowest level of cytotoxicity compared to tissues or cultured cell lines in serum-free medium. B. Protease specificity analysis

By means of mass spectrometry ( A MSP-MS), Alaunus Multiplex Substrate Profiling was used for protease-specific screening. This method uses a physicochemically diverse peptide library as a substrate for proteases, and monitors the reaction over time by mass spectrometry detection of the cleavage product. The resulting lysis was evaluated for specific lysis in the enzyme-treated sample by comparison with the results from a control culture without enzyme.

For the difference analysis of the specific profile, use iceLogo software (v.1.2) to generate a matrix of the Z scores of each amino acid at each position. These Z scores calculate the number of deviations between the occurrence rate of the specific amino acid and the average value (that is, the occurrence rate of the specific amino acid at the specific position in the negative reference set). Then, using the gplot and RColorBrewer software packages, the difference Z scores between the sample pairs are used to generate the heat map in the R environment.

The time-dependent peptide cleavage products from the A MSP-MS library were identified via LC-MS/MS. The two cell line samples MC38 and H228 each showed a good level of activity against the library. The total number of lysis observed during the time course is 40 (15 minutes), 107 (60 minutes) and 306 (240 minutes), for MC38; 149 (15 minutes), 170 (60 minutes) and 371 (240 minutes), for H2228. The profile of the substrate specificity was extrapolated from the cumulative list of 453 (for MC38) and 690 (for H2228) lyses observed across the entire time course, and reported in Figure 30 using iceLogo notation. IceLogo considers the cleavage and uncleaved positions in the peptide library, and is used to observe the multiple enrichment and de-enrichment of amino acids flanking the cleavage site at (P1-P1'). Figure 30 shows the substrate lysis of MC38 and H2228 cell lines in the form of iceLogo diagram. The letter height measures the percentage difference in the amino acid occurrence rate of the cleaved peptide relative to all possible peptide cleavage in the library, plotted at each position P4 to P4'. The residues above the center line are favorable; the residues below the center line are unfavorable. The statistically significant residues ( p ≤0.05) are colored by physical and chemical properties (black: hydrophobic; red: acidic; blue: basic; purple: aminated; green: small). Gray residues are observed at p >0.05.

In this paper, the specific profile identified by A MSP-MS shows the endopeptidase activity, and the hydrophobic and basic amino acids in P1 shared by the two cell lines are highly preferred. MC38 cells display a preference for R and Y in P1' and other hydrophobic specificities at P3, P2, and P4'. H2228 cells display a preference for H, S and Y at P1' and other hydrophobic specificities at P2' and P4'. C. NAT and tumor difference analysis

The time-dependent peptide cleavage products from the MSP-MS library were identified as described above in Example 17.B. Two patient tissue samples showed high levels of activity against the library. The total number of lysis observed during the time course is 134 (15 minutes), 270 (60 minutes) and 475 (240 minutes), for NAT; 63 (15 minutes), 184 (60 minutes) and 447 (240 minutes), For tumor tissue. There is a cumulative list of 879 (for NAT) and 694 (for tumor) lysates observed across the entire time course to extrapolate the profile of the substrate specificity and report using the iceLogo notation shown in Figure 31A. Similar to Fig. 2, Figs. 31A and 31B show the lysis of NAT and tumor (TUM) tissue in iceLogo format.

In addition, the activity of the two patient samples against the library was monitored at a longer 24 hour time point to reveal other differences in the specificity of the substrate. The observed lysis at this additional time point was 719 (for NAT) and 636 (for tumor). The overview of the entire time course (including the 24-hour time point) is shown in Figure 31B. Leucine (n) is a substitute for Met in the library. The NAT samples showed a strong preference for basic amino acids (mainly R and K) in P1 and R, H, and S in P1'. The tumor samples showed a preference for basic (R and K) and hydrophobic amino acids in P1, and a strong preference for H in P1'. The tumor samples also showed a preference for the hydrophobic amino acid at P2', while the patient samples shared a preference for the hydrophobic amino acid at P4'.

In order to quantify the difference in overall substrate specificity between NAT and tumor samples, the cleavage Z score from each sample was also used to generate a difference map derived from the residue preference at each subsite, as shown in Figure 31C exhibit. Figure 31C shows the difference in Z scores at P4-P4' positions in the form of a heat map to highlight residues that are conducive to NAT or tumor-specific lysis. Leucine (n) is a substitute for Met in the library. This confirms the main specificity of lysine and arginine at P1 in the NAT sample, and highlights other smaller preferences for other locations, especially in tumor samples (such as G and F in P1).

The inclusion of cell line H2228 can provide an opportunity to compare immortalized cell lines with major tissues of similar origin in terms of specificity profiles. In this article, non-small cell lung cancer (NSCLC) is selected. Compared with tumor samples, H2228 cells showed a more significant difference in specificity from NAT. In addition, when compared with NAT (left panel), this difference analysis shows some preferences in H2228 cells, which are similar to the profile of tumor tissues (including G, H, and F at P1) compared with NAT in a similar manner. However, a specific difference (albeit significantly lower) was also detected between H2228 cells and the tumor tissue shown in Figure 31D, providing support for patient heterogeneity in terms of protease activity in the tumor microenvironment. Figure 31D shows the difference in Z scores at the P4-P4' position in the form of a heat map to highlight the residues that are conducive to the specific cleavage of H2228 compared to NAT or tumor samples. Leucine (n) is a substitute for Met in the library. D. Kinetic analysis of peptide sequence

The ten sequences shown in Table 19 (AK10) were analyzed by A MSP-MS. These sequences were slightly modified from the original sequences to produce mass spectrometry compatible peptides (in some cases, basic residues were added to increase the positive charge) and spacer residues (GG) to produce longer approximately 14-mer peptides. In the A MSP-MS reaction, peptides were prepared at a final concentration of 500 nM. The AK10 library was also analyzed separately by LC-MS/MS for chemical verification of peptide substances. Table 19 Subject name Related sequence Modified sequence for analysis AK-01 ISSGLLSGRSDNH (SEQ ID NO:464) KISSGLLSGRSDNH (SEQ ID NO:470) AK-02 AVGLLAPPGGLSGRSDNH (SEQ ID NO:465) RAVGLLAPPGGLSGRSDNH (SEQ ID NO:471) AK-03 VPLSLYSG (SEQ ID NO: 466) RGGVPLSLYSGGGK (SEQ ID NO:472) AK-04 MPYDLYHP RGGMPYDLYHPGGK (SEQ ID NO:473) AK-05 DSGGFMLT RGGDSGGFMLTGGK (SEQ ID NO: 474) AK-06 HEQLTV RGSGHEQLTVGGSK (SEQ ID NO:475) AK-07 RAAAVKSP GSGRAAAVKSPGSK (SEQ ID NO: 476) AK-08 RQARVVG (SEQ ID NO:467) GSGRQARVVGGGSK (SEQ ID NO: 477) AK-09 LSGRSNAMPYDLYHP (SEQ ID NO: 468) N/A (unmodified) AK-10 MPYDLYHPRQARVVG (SEQ ID NO: 469) N/A (unmodified)

The A MSP-MS analysis was performed under standard substrate-specific analysis conditions, and the sampling time points during the reaction culture period were 0, 15, 60, 240, and 1200 minutes. From the MS1 precursor ion peak area of each peptide substance, the peptide was quantified by mass spectrometry unlabeled quantification. In GraphPad Prism software v 8.0, the enzyme process curve is modeled and nonlinear least square fitting is used to fit the data to the first-order kinetic equation: 𝑌 = 𝑒 ^{( — [ K} _obs ^{] t )} where Y = product formation percentage, K _obs is the observed ratio, and t = time. In Michaelis-Menten kinetics, the observed ratio _Kobs is a function of enzyme concentration and catalytic efficiency (k _cat /K _M ). In this analysis of the enzyme mixture, the cleavage of the product was fractionated _{using Kobs.}

To represent the overall picture of the AK10 peptide in the A MSP-MS reaction, the normalized relative abundance of each substrate peptide is plotted over time. The MC38 serum-free conditioned medium was used to monitor the relative abundance of the substrate peptides in the AK10 library via AMSP-MS. It was observed that for the MC38 serum-free conditioned medium response, due to a combination of enzymatic and non-enzymatic processes (such as Met oxidation), the abundance of each substrate decreased at different ratios. To evaluate enzymatic cleavage, product formation analysis is also required.

In order to evaluate whether the substrate is depleted by the enzymatic treatment, the product formation rate is evaluated for the fitting quality of a single-phase decay model that ideally reaches saturation. Figures 32A and 32B show the individual progression curves of the selected peptides, which were significantly degraded in the case of the MC38 cell line. Figures 32A and 32B show that the selected substrates formed curves in the MC38 conditioned medium; AK10-01 (Figure 32A, left), AK10-02 (Figure 32A, right); AK10-04 (Figure 32B, left) ) And AK10-05 (Figure 32B, right side). In the case of AK10-02, it is obvious that a transparent product is formed with a very small curve, and examples of non-specific degradation of the "product" formed by AK10-01, AK10-4 and AK10-05. AK10-01 is basically linear, AK10-04 exists at time zero, and the product of AK10-05 continues to degrade due to chemical processes (processes observed by LC-MS/MS).

The best substrates for MC38 are AK-09 and AK-10, in which multiple cleavage products of these peptides are observed. Fit its progress curve and first-order kinetic ratio, and then grade the lysis by its relative efficiency, as shown in Figure 33. Figure 33 shows the relative efficiency of the main cleavage products from the MC38 AMSP-MS reaction using AK10 library peptides. The fastest cleavage occurs at PRQA|RVVG in peptide AK-10, where "|" indicates an easily cleavable bond. In the peptide AK-09, multiple cleavages were observed within the narrow multiple range of the most effective substrate, and the cleavages were located at: LS|GRSNAM, LSG|RSNAM and LSGR|SNAM. It also shows the inefficient lysis of AK-02.

The H2228 cell line produces cleavage in peptides AK10-01, -02, -04, -09 and -10. The formation of individual substrates is shown in Figures 34A and 34B. Figures 34A and 34B show that in the case of H2228 conditioned medium, the selected substrate products form curves; AK10-01 (Figure 34A, upper left), AK10-02 (Figure 34A, upper right), AK10-04 (Figure 34A) , Lower part), AK10-09 (Figure 34B, left) and AK10-10 (Figure 34B, right). The highest substrate lysed by H2228 conditioned medium and the calculated relative efficiency are shown in Figure 35.

Fit the ratio of the highest cleavage substrate to first-order kinetics in the AK10 library, and the most effective cleavage motifs are: YDLY|HP and LSGR|S. The motif LSGR|S is cleaved in the following order in the three peptides: AK-01>AK-09>AK-02. The YDLY|HP motif is cleaved in the following order in the case of two peptides: AK-04>AK-09. In the peptide AK-10, the easily cleavable bond shifts to HPRQ|AR, but this peptide is still cleaved within 5 times the efficiency of the highest substrate.

The patient sample produced a broader lysed set than any cell line sample in the AMSP-MS analysis of AK10 library peptides. Individual product formation traces are shown in Figures 36A-36C. These diagrams depict the curve formed by the selected substrate in the case of tumor-conditioned media; AK10-01 (Figure 36A, upper left), AK10-02 (Figure 36A, upper middle), 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). As mentioned earlier, several degradation products of peptide AK-09 (in both non-oxidized and oxidized peptide species) were observed. However, the only significant cleavage of peptide AK-10 is located at PRQA|RVV, and the product progression curve in Figure 36C, on the right, supports the secondary reaction to reduce product signal.

The highest substrate of the AK10 library in the tumor conditioned medium is shown in Figure 36D. The highest cleavage is located in the LSLY|SG motif of AK-03 and YDLY|HP of AK-04. NSCLC samples from H2228 and tumor tissues shared peptide AK-04 (at YDLY|HP) and significant cleavage of AK-09 (LSG|R|SNAMPYDLY|HP) at multiple sites.

NAT-conditioned medium also produced specific lysis. The monitoring of substrate abundance showed that the overall trend of degradation of the AK10 library was significant in the experimental situation. Individual products exhibited one of the series of characteristics depicted in Figures 37A-37D. Figures 37A-37D depict curves formed by the selected substrates in the case of 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). The better-performing substrates are AK-01, -04, -09, and -10, and the weaker substrates are AK-02 and AK-05.

The kinetic analysis of the most effective NAT catalyzed substrate is shown in Figure 37E. The most effective NAT substrate is AK-10 (LSGR|SDNH), which is shared with H2228 but not tumor samples. Other effective substrates are AK-09 (LS|GR|SNAMPYDLY|HP) at multiple sites, some of which are shared with H2228 and tumors, and AK-04 (at YDLY|HP).

This analysis indicates that normal adjacent tissues contain site-specific enzyme activities shared with their matched tumor tissues. Specifically, for the tumor conditioned medium shown in Figure 31A, the right panel, YDL Y | HP cleavage is obvious in the iceLogo motif. The specificity towards the LSG R | S motif is also evident in Figure 31A, the tumor-conditioned medium iceLogo produced after 4 hours of reaction in the right image, and even in Figure 31B, the same established 24 hours incubation time in the left image , Which is more clearly detected in the NAT samples.

MC38 only shares cleavage with NSCLC samples within the LSGRS motif found in peptides AK-09 and AK-02. The cleavage of AK-10 by MC38 at HPRQ|AR motif is shared with H2228 but not tumor samples, or shared with NAT at HPRQA|R site. The MC38 cleavage of AK-02 at GLSG| RS is consistent with its strong P1' preference for Arg, as seen in the iceLogo motif depicted in Figure 30, on the left.

In conclusion, Table 20 supports the conclusion that AK10 library peptides have first-order kinetic properties consistent with enzymatic cleavage. Table 20 indicates whether the peptide substrate is lysed by the conditioned medium sample (yes/no/not determined). No determination indicates that it has not been detected in the form of intact or lysed material in the sample treated with the conditioned medium. Table 20 Was it lysed from the conditioned medium sample? Hostage MC38 H2228 NAT Tumor AK-01 no Yes Yes Yes AK-02 no Yes no Yes AK-03 no no no Yes AK-04 no Yes Yes Yes AK-05 no no no Yes AK-06 no no no no AK-07 no no no no AK-08 Not determined Not determined Not determined Not determined AK-09 Yes Yes Yes Yes AK-10 Yes Yes Yes Yes E. Proteome analysis

The protein concentration in the sample was quantified by BCA analysis, and 5 μg total protein was used for each digestion reaction. The sample was denatured with 4 M urea and reduced with 5 mM dithiothreitol (DTT) by reacting at 56°C for 30 minutes. The sample was alkylated with iodoacetamide (IAM) at 10 mM by reacting in the dark at room temperature for 1 hour. The excess IAM was quenched with 5 mM DTT, and then the reaction was diluted to 1 M final urea. The samples were digested with sequencing grade porcine trypsin (Promega, V5111) at a trypsin:sample mass ratio of 1:50 at 37°C overnight. On the second day, the resulting peptide digestion sample was desalted with a C18 pipette tip (C18 zip tip, Millipore-Sigma) and then freeze-dried. The sample was resuspended in HPLC grade water containing 0.1% formic acid for LC-MS/MS analysis.

A QExactive Plus mass spectrometer (Thermo) equipped with a nanoACQUITY (Waters) ultra-efficient LC (UPLC) system and an EASY-Spray ion source (Thermo) was used for peptide sequencing by LC-MS/MS. EASY-Spray PepMap C18 column (Thermo, ES800; 3 μm bead size, 75 μm×150 mm) was used for reverse phase chromatography. During the 14-minute sample loading period, chromatography was performed at a flow rate of 600 nl/min, followed by a linear gradient of 0.1% formic acid containing 2 to 35% (volume/volume) acetonitrile, at 400 nl for peptide separation. Chromatography took 90 minutes at a flow rate of /min. With a minimum of 2,000 counts, a separation width of 2.0 m/z, and a minimum normalized collision energy of 25, peptide fragmentation was performed by high energy collision dissociation (HCD) on the six most severe precursor ions.

MSConvert ^{6 was used to} generate the MS peak list, and Protein Prospector software, v.5.22.1 (UCSF) was used to analyze the data. (November 1, 2017 Downloads) to retrieve data for SwissProt human or mouse libraries were a total of 20,240 or 16,942 Clause. Analyze MC38, a mouse cell line, independently of human NSCLC samples. The SwissProt bovine library was also searched to identify FCS proteins. The decoy database search method is used to evaluate the false discovery rate (FDR), and the score threshold for reporting protein identification is selected to achieve <1% FDR. The mass tolerance is set to 20 ppm (parent ion) and 30 ppm (fragment ion), and trypsin specificity is selected under the condition that one non-specific cleavage and one missed cleavage are allowed. The aminomethylation on Cys was used as a fixed modification. The following standard variable modifications were used: protein N-terminal acetylation, protein N-terminal acetylation + oxidation, N-terminal pyroglutamic acid formation from glutamic acid, Met loss, and Met oxidation. In addition, in a predetermined experiment spanning repeated samples, at least two unique peptides are required for each protein identification that requires one LC-MS/MS.

Output data as an approximation of relative protein abundance by spectral count (peptide count). To correct for potential differences in protein loading between operations, the peptide counts were normalized by the sum of the total counts from the operations. The search is then processed in Perseus, and the data is _{normalized by log 2} , and then if the protein does not have a peptide count> 1 in at least 2 of the 3 copies, these proteins are excluded. Then, replace the missed value with a random number estimated from the normal distribution. A Welch's t-test is performed between the two replica sets, and a volcano map is generated in R.

Protein identification includes 47 proteases or protease inhibitors, including matrix metalloproteinases (Mmp2, 7 and 12), MMP inhibitors (Timp2), proteasome subunits (Psma), serine protease inhibitors (serpins) and cystatin Protein, carboxypeptidase (Cpe and Cpd), cathepsin (Ctsd, b, h, z and l), aminopeptidase (Rnpep, Lap3), calcium activated neutral protease (Cpns), proline-directed peptide Enzymes (Dpp3, Xpnpep1 and Pepd) serine protease (Prss23), tripeptidyl-peptidase (Tpp1), Adam10 and Casp3.

Protease and inhibitor abundances do not necessarily total the observed activity, but protein identification provides an understanding of the motifs identified in the activity data. The presence of cathepsin can explain P1 nLeu preference (Ctsd), or for example P1 Arg, Gly (Ctsb, Ctsl). The strong preference for basic residues at P1 in the substrate-specific profile can also be related to background serum proteins from the coagulation cascade, such as the detection of bovine thrombin and plasmin in the proteomic data.

Combine the analysis of human NSCLC-related samples to achieve comparison between samples; this includes the H2228 cell line, patient tumors and normal adjacent tissues. In this article, proteomic analysis resulted in the confident identification of 2243 unique proteins across all three samples.

The identification includes similar lists of proteases and their inhibitors: proteasome subunits; aminopeptidases B and N; calpain subunits; carboxypeptidases E and A; caspase 1, 3, 6, 8; tissue Protease B, D, E, L1, L2, S and Z; DPP1, 2 and 3; ADAM10; MMP14 and 9; MMP inhibitors 1 and 2; serine protease inhibitor; serine protease HTRA1; proline Endopeptidase, including FAP, proline amino peptidase, tripeptidyl peptidase TPP1 and TPP2; and coagulation factor and complement protein.

The Perseus software was used to analyze the difference of the examples of the protein list detected in the conditioned medium from the NAT and tumor samples to realize the two-sided Weibull's t test (based on peptide count) of the differential performance between the two sets of duplicate samples. Observing the results in the volcano map in Fig. 38, proteins that are significantly enriched in the tumor (upper right, part 2) or NAT (upper left, part 1) are present in these parts. Three related proteins are identified in this list: Serpin H1, α-1-antichymotrypsin (SERPINA3) and Macrophage Mannose Receptor 1 (MRC1).

In summary, the unbiased activity analysis performed using A MSP-MS showed the cleavage profile of each sample and the overall strong preference for basic residues. MC38 and H2228 cell lines have different protease specificity profiles, while tumors and NAT tissues from the same patient share similar protease specificities, but the difference analysis shows that compared with P1 Leu, Phe or Gly in tumors, NAT samples Stronger preference for P1 Arg/Lys. Comparative kinetic analysis showed that the two specific motifs were stably cleaved in multiple samples and individual substrates (including YDLY|HP and LSGR|S). The mouse MC38 cell line and another human NSCLC-related sample have different biological sources, and the collection of proteases and inhibitors identified in MC38 conditioned medium is obviously similar to the NSCLC collection. The proteases identified by the proteomic analysis included candidates with specificity consistent with the observed AMSP-MS cleavage, including cathepsins. Example 18: In vivo evaluation of masked anti-CTLA4 antibodies in cynomolgus monkeys

In the first set of experiments, cynomolgus monkeys were administered ipelizumab, antibody 2-6, antibody 2-10, or isotype control, and the percentage (%) of Ki67+ cells in CD4+ cells was measured. Evaluation of pharmacodynamic (PD) effects. In the second set of experiments, antibody 2-6, antibody 2-10, antibody 2-14, antibody 2-16 or isotype control were administered to cynomolgus monkeys, and the percentage of Ki67+ cells in CD4+ cells was measured (%) to evaluate the pharmacodynamic effect.

The results of the first and second sets of PD experiments are shown in FIG. 21. Compared with Ipelizumab, the parental unmasked antibody 2-6 is more effective in inducing peripheral PD effects, as shown by the increase in the percentage of Ki67+ cells in CD4+ cells compared with Ipelizumab. Show. Antibody 2-10 (which is a masked but non-cleavable version of Antibody 2-6) blocks the PD effects associated with Antibody 2-6. As shown in the second set of experiments (Experiment 2), compared with the unmasked parent antibody 2-6, the masked and cleavable antibodies (antibody 2-14 and antibody 2-16) showed reduced PD effects .

Cynomolgus monkeys were also used to test antibodies (RSV-m control, Ipelizumab, antibody 2-6, antibody 2-10, antibody 2-14, antibody 2-16 or Ipelizumab containing Fc The pharmacokinetics of the masked version of the S239D and I332D mutations in the domain (masked Ipelizumab-m)) were evaluated after intravenous administration of 10 mg/kg. Measure the content of each antibody in the plasma, and measure the half-life (days), Cmax (µg/mL) and area under the curve (AUC) (days × micrograms/ml) in two studies.

The results of the pharmacokinetic study are shown in Figure 22. Figure 22 shows the level of each antibody administered during the 14-day period. This data indicates that antibody 2-14 and antibody 2-16 show favorable pharmacokinetics in cynomolgus monkeys after intravenous administration of 10 mg/kg.

Figures 1A and 1B are a series of curves showing in vitro analysis of anti-CTLA4 antibody encapsulation by masking peptides. A surface plasmon resonance (SPR) was performed to examine the A) kinetics and B) affinity between the 9D9 antibody (ligand) and the masking peptide CNLIVEGHC (analyte). At 37°C, analysis was performed with 2-fold serial dilutions of increasing concentrations from 0.1 to 50 μM. Plot the response minus the reference. SPR was performed three times and a representative trace from one experiment was shown.

Figures 2A-2C are a series of curves showing the protease activation of masked murine anti-CTLA4 antibodies. Figure 2A ) Compared with the parental anti-CTLA4 antibody (9D9, filled circles), the activatable masked anti-CTLA4 antibody exhibited 90-fold lower binding to murine CTLA4-Fc (masked 9D9, filled squares) . At a level similar to the parental anti-CTLA4 antibody, the protease activation of the activatable masked anti-CTLA4 antibody completely restored the binding to murine CTLA4-Fc (protease activation, asterisk). The X axis indicates the amount of antibody tested. Figure 2B) and parental anti-CTLA4 antibody (9D9, filled circle), activatable masked anti-CTLA4 antibody (masked 9D9, solid inverted triangle) and non-cleavable masked anti-CTLA4 antibody (NC masked 9D9, open squares) showed that the binding to murine CTLA4 was about 156 times lower and about 218 times lower, respectively. The X axis indicates the amount of antibody tested. Figure 2C) In the absence or presence of recombinant MMP2, the activatable masked anti-CTLA4 antibody (masked 9D9) and the non-cleavable masked anti-CTLA4 antibody (NC masked 9D9) were determined by ELISA. And the EC50 of the parental anti-CTLA4 antibody (9D9). Each group was performed at least three times and the average EC50+/-SE was reported.

Figures 3A-3D are a series of curves showing that the efficacy of the protease-activated masked anti-CTLA4 antibody (masked 9D9) in reducing tumor volume is equivalent to the efficacy of the parental anti-CTLA4 antibody (9D9 IgG2a). The MC38 syngeneic tumor was implanted in C57BI/6 mice. After the tumor size reached 60-120 mm3, a single 200 μg dose was administered to mice intraperitoneally . Figure 3A) muIgG2a control antibody, Figure 3B) parental anti-CTLA4 antibody with IgG2a isotype (9D9 IgG2a), Figure 3C) The activatable masked anti-CTLA4 antibody (masked 9D9), in the MC38 murine tumor model, compared with mice treated with 3B) the parental anti-CTLA4 antibody (9D9 IgG2a) or 3A) muIgG2a control antibody, Figure 3D) Humanized IgG1 anti-CTLA4 antibody 1 (antibody 1) showed similar in vivo efficacy in reducing tumor volume.

Figure 4 shows the depletion of equivalent regulatory T lymphocytes (Treg) by protease-activated masked anti-CTLA4 antibody (masked 9D9) and parental anti-CTLA4 antibody (9D9 IgG2a) in tumor infiltrates picture. The control indicates treatment with muIgG2a control antibody.

Figures 5A and 5B are a series of curves showing that in the spleen of treated mice, the protease-activated masked anti-CTLA4 antibody (masked 9D9) significantly reduced the ratio of A) CD4+ T cells and B) CD8+ T cells proliferation. The arrow points to the average value between the treatment groups using the parental anti-CTLA4 antibody (9D9 IgG2a) and the protease-activated masked anti-CTLA4 antibody (masked 9D9).

Figures 6A-6D are a series of curves showing the efficacy and safety of masked anti-CTLA4 antibodies. Figure 6A) Isotype control antibody (IgG2a control), Figure 6B) non-cleavable masked anti-CTLA4 antibody (NC masked 9D9), Figure 6A) Isotype control antibody (IgG2a control), Figure 6B) In C57BL/6 mice (n=10) carrying MC38 tumors 6C) The tumor suppressor activity of the parental anti-CTLA4 antibody (9D9) and Figure 6D) activatable masked anti-CTLA4 antibody (masked 9D9).

Figures 7A and 7B are a series of curves showing A) depletion of regulatory T lymphocytes (Treg) in tumors, and B) isotype control antibody (IgG2a control), parental anti-CTLA4 antibody (9D9), Treg proliferation in the spleen of mice (n=6) treated with activated masked anti-CTLA4 antibody (masked 9D9) and non-cleavable masked anti-CTLA4 antibody (NC masked 9D9).

Figure 8 is a graph showing the protease activation of masked humanized anti-CTLA4 antibody 1. Compared with the parental humanized anti-CTLA4 antibody 1 (parent, open circles), the masked humanized anti-CTLA4 antibody 1 exhibited lower binding to human CTLA4-Fc (masked, closed squares). At a level similar to that of the parental humanized anti-CTLA4 antibody 1, the protease activation of the masked humanized anti-CTLA4 antibody completely restored the binding to human CTLA4-Fc (protease activation, asterisk). The X axis indicates the amount of antibody tested. The molecular RLU/parental RLU on the y-axis indicates the fraction of relative light units compared to the parental humanized anti-CTLA4 antibody 1.

Figures 9A and 9B are graphs showing the binding of unmasked anti-CTLA4 antibody to human CTLA4-Fc under a certain range of antibody concentration. Figure 9A shows the binding of various forms of Antibody 1 (Antibody 1-1 and Antibody 1-2) to human CTLA4-Fc at a range of antibody concentrations, which shows similar binding of the displayed antibodies. Figure 9B shows the relationship between various forms of Antibody 2 (Antibody 2-1, Antibody 2-2, Antibody 2-3, Antibody 2-4, and Antibody 2-5) and human CTLA4-Fc under a certain range of antibody concentration Binding, which shows similar binding of the displayed antibody.

Figures 10A and 10B are curves showing the binding of a masked anti-CTLA4 antibody to human CTLA4-Fc under a certain range of antibody concentrations. Figure 10A shows the binding of masked antibodies in the absence of protease activation. Figure 10B shows the binding of 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 antibody showed similar binding characteristics to the unmasked parent antibody, antibody 2-6.

Figures 11A-11H show a radar chart of the masked version of Antibody 2 containing various cleavable peptide sequences. Antibodies 2-15 include non-cleavable cleavable peptide sequences. The radar chart is used to depict the activation of each antibody shown in FIGS. 11A-11H by each protease shown on the radar chart, where activation is depicted in the range of 0 (inactive) to 1.0 (fully activated). Figures 11I-11M depict the SDSP-PAGE Western blot analysis of protease cleavage with selected antibodies in the plasma of healthy mice ( Figures 11I and 11J ) and the plasma of mice bearing MC38 tumors ( Figures 11K-11M). SDSP-PAGE Western blot analysis) results.

Figures 12A and 12B are curves depicting the binding of a masked anti-CTLA4 antibody 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 a non-activatable isotype control and a non-activatable masked antibody (antibody 2-15 because its cleavable peptide sequence is not cleavable) as controls. Figure 12B includes isotype controls and non-activatable masked antibodies (antibodies 2-10 because their cleavable peptide sequence is not cleavable) as controls. At a level similar to that of the unmasked parent antibody, antibody 2-6, the activation of antibody 2-14 completely restored its binding to human CTLA4-Fc ( Figure 12B ).

Figures 13A-13D depict the unmasked form of antibody 1 and antibody 2, such as the IL-2 content (pg/mL) determined by the Staphylococcal enterotoxin B (SEB) analysis method ( Figure 13A and 13C ) or the fold change of IL-2 content ( Figure 13B and 13D ). Using the SEB analysis method, test the unmasked form of antibody 1 (antibody 1-1 and 1-2) ( Figure 13A and 13B ) and the unmasked form of antibody 2 (antibody 2-1, antibody 2-2, Antibody 2-3, Antibody 2-4, and Antibody 2-5) ( Figure 13C and 13D ) promote the ability of peripheral monocytes to produce IL-2. Compared with the control without antibody, all the tested antibody 1 and antibody 2 forms showed the ability to increase IL-2 content ( Figure 13A and 13C ).

Figures 14A-14D depict the masked form of antibody 2, such as the IL-2 content (pg/mL) ( Figures 14A and 14C ) or the IL-2 content determined by the Staphylococcal Enterotoxin B (SEB) analysis method The curve of multiple change ( Figure 14B and 14D). Test the unmasked form of antibody 2 (antibody 2-6) and the masked form 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 depict the effect of antibody on IL-2 content ( Figure 14A ) and the fold change of IL-2 content ( Figure 14B ) in an unactivated state (ie, without protease exposure). For the unmasked parent antibody, antibody 2-6 showed the ability to significantly increase IL-2 content, and only one masked antibody (antibody 2-12) showed a significant increase in IL-2 content, but compared with antibody 2-6 The comparison level is significantly lower ( Figure 14A and 14B ). Figures 14C and 14D depict the effect of antibodies on IL-2 content ( Figure 14C ) and the fold change of IL-2 content ( Figure 14D ) in the activated state (ie, after exposure to protease). Figures 14C and 14D show the ability of antibody 2-7, antibody 2-8, and antibody 2-9 to promote IL-2 production at a level similar to that of the unmasked parent antibody, antibody 2-6, exposure to protease.

Figures 15A-15D depict the degree of in vivo lysis of masked anti-CTLA4 antibodies in the plasma of healthy mice, as shown by Western blot analysis. The masked form of antibody 2 (antibody 2-14, antibody 2-19, and antibody 2-20) was shown in vivo lysis in healthy mice on days 2, 4, and 7 after the intraperitoneal administration of the antibody. A standard sample in which the masked antibody is treated in vitro in the presence or absence of protease is used as a control for lysis. Antibody 2-14 ( Figure 15A ) and Antibody 2-20 ( Figure 15C ) are activated in vivo. Figure 15B shows insufficient activation of antibody 2-19. The increase in activation percentage of antibody 2-14 and antibody 2-20 from day 2 to day 7 is shown in Figure 15D.

Figures 16A-16D depict the degree of in vivo lysis of masked anti-CTLA4 antibodies in the plasma of tumor-bearing mice, as shown by Western blot analysis. The masked form of antibody 2 (antibody 2-14, antibody 2-19, and antibody 2-20) was displayed in vivo lysis of the masked form of antibody 2 (antibody 2-14, antibody 2-19, and antibody 2-20) in mice bearing MC38 tumors on days 2, 4, and 7 after the intraperitoneal administration of the antibody. A standard sample in which the masked antibody is treated in vitro in the presence or absence of protease is used as a control for lysis. Antibody 2-14 ( Figure 16A ) and antibody 2-20 ( Figure 16C ) are both activated in vivo. Figure 16B shows insufficient activation of antibodies 2-19. The increase in activation percentage of antibody 2-14 and antibody 2-20 from day 2 to day 7 is shown in Figure 16D . Compared with healthy mice without tumors, the activation percentage of antibodies 2-14 in MC38 tumor-bearing mice was significantly higher, as determined by 2-way ANOVA (adjusted P value = 0.0113) ( Figure 16D ).

Figure 17 is a graph showing the ability of the masked anti-CTLA4 antibody to promote ADCC activity in a masked state and an "activated" state as a result of pre-exposure to protease, as determined by the ADCC reporter bioanalysis method. Figure 17 depicts the extent of reporter activation by the unmasked parent antibody (antibody 2-6) and the masked version of antibody 2-6 (antibody 2-14 and antibody 2-15). Antibody 2-15 includes a non-cleavable cleavable peptide sequence for use as a negative control. Isotype controls were also tested. Antibodies pre-exposed to protease are depicted as "activated". When tested without pre-exposure to protease, the masked antibodies (antibody 2-14 and antibody 2-15) showed reduced reporters compared to the unmasked parent antibody (antibody 2-6) Activation ( Figure 17 ). The reduced reporter activation of antibody 2-15 (which cannot be activated by protease) was not rescued by pre-exposure to protease ( Figure 17 ). The reporter activation of antibody 2-14 (which can be activated by protease) was rescued to a level similar to that induced by the unmasked parent antibody (antibody 2-6) ( Figure 17 ).

Figures 18A and 18B are curves depicting the ability of anti-CTLA4 antibodies to block the interaction between CTLA4 and its ligands CD80 and CD86, as determined by the CTLA4 blocking bioassay method. Figure 18A depicts the ability of the unmasked form of Antibody 2 (Antibody 2-2) and the masked form of Antibody 2 (Antibody 2-14) to block the binding of CTLA4 to its ligand. Antibodies 2-14 were tested in a masked state (ie, no pre-exposure to protease) and an "activated" state caused by pre-exposure to protease. As a comparison, an isotype control was also used for the analysis method and the analysis method was also performed without antibody (that is, as a control without antibody). Figure 18A shows that when in the masked (unactivated) form caused by the absence of protease, antibody 2-14 did not show the ability to effectively block the binding of CTLA4 to its ligand. For blocking the binding of CTLA4 to its ligand, the analysis of the fold change higher than that of the control without antibody did not show the difference between the ability of the antibody 2-14 in the masked (unactivated) form and the ability of the isotype control Significant difference ( Figure 18B ). However, Figure 18A shows that when antibody 2-14 is in the "activated" form caused by pre-exposure to protease, it shows the ability to effectively block the binding of CTLA4 to its ligand at a concentration within a certain range. The ability of "activating" antibody 2-14 to block the binding of CTLA4 to its ligand is similar to that of unmasked antibody 2-2 ( Figures 18A and 18B ). For blocking the binding of CTLA4 to its ligand, the analysis of the fold change higher than that of the control without antibody did not show a significant difference between the ability of the "activated" antibody 2-14 and the unmasked antibody 2-2 (" ns”) ( Figure 18B ).

Figures 19A-19D depict the results of immunophenotyping studies. The evaluation performance of these studies includes the ratio of CD45+ cells with the following markers: CD3+/ICOS+, CD3+ T cells, CD4+/Ki67+, CD3+/Ki67+, CD4+/ICOS+, CD4+ T Cells, CD8+/ICOS+, CD8+ T cells, Tregs+/ICOS+, CD8+/Ki67+, Tregs, Tregs+/Ki67+. Figures 19E and 19F provide comparisons of the tested antibodies (Group 1: IgG, Group 2: Antibody 2-1; Group 3: Antibody 2-10; Group 4: Ipelizumab; Group 5: Iraq Statistics of Pelimumab-aFuc).

Figures 20A and 20B depict curves showing the tumor volume over time after a single injection of 20 µg, 7 µg, or 2 µg test antibody was administered.

Figure 21 depicts a curve showing the results of two sets of experiments (experiment 1 and experiment 2), which are evaluated by the administration of antibody 2-6, antibody 2-10, antibody 2-14, antibody 2-16 or isotype control The percentage of Ki67+ cells in the total CD4+ cells after the test was used to evaluate the pharmacodynamic effect in cynomolgus monkeys.

Figure 22 depicts the results of experiments that evaluated the pharmacokinetics in cynomolgus monkeys after administration of one of the following test antibodies: RSV-m control, Ipelizumab, antibody 2-6, antibody 2-10, Antibody 2-14, Antibody 2-16, or masked Ipelizumab-m. Figure 22 depicts a curve showing the amount (μg/mL) of each antibody administered over a 14-day period.

Figure 23A depicts the results of an efficacy study on the percentage of CD4+Ki67+ cells (left) and CD4+ICOS+ cells (right) in peripheral blood on the 5th day after administration, which represents the level of T cell activation. Figure 23B depicts the curve, which shows that the treatment with 10 mg/kg (RSV-m control) or 3 mg/kg (Ipelizumab, Antibody 2-6, Antibody 2-14, Antibody 2-15) In mice, tumor weight (left) and CD8/Treg ratio (right) assessed on day 7. Figure 23C depicts a curve that shows the regulation of the tumor microenvironment after administration of RSV-m control, Ipelizumab, antibody 2-6, antibody 2-14, or antibody 2-15 in mice T cells (left) and CD8+ T cells in the tumor microenvironment (right). Figure 23D depicts a curve, which shows that the antibody (including RSV-m control, Ipelizumab, antibody 2-6, After antibody 2-14 and antibody 2-15), tumor volume (mm ³ ) in mice over time.

Figure 24A depicts the results of SDS-PAGE Western blot analysis using MCA205 fibrosarcoma cells, which evaluates the results of antibody 2-6, antibody 2-14, antibody 2-16, and antibody 2-10 in plasma, kidney, liver, and tumor tissues Cracked. Figure 24B depicts the results of SDS-PAGE Western blot analysis using MB49 cells, which evaluates the lysis of antibody 2-6, antibody 2-14, antibody 2-16, and antibody 2-10 in plasma, kidney and liver tissues. Figure 24C depicts the results of SDS-PAGE Western blot analysis using MC38 cells to evaluate the lysis of antibodies 2-6, 2-14, 2-16, and 2-10 in plasma, kidney, liver, and tumor tissues. Figure 24D depicts the results of SDS-PAGE Western blot analysis using MC38 cells to assess lysis, and provides the percentage of lysis of selected samples. Calculate the percentage of fragmentation based on the sensitivity of the photosensitivity (percentage of fragmentation=low band/(low band+high band)). Figure 24E depicts a curve showing the binding between each antibody and CTLA4 after administration of MB49-carrying B-hCTLA4 gene transgenic mice, where each antibody system is isolated from plasma, kidney, liver, or spleen. Figure 24F depicts a heat map of in vitro cleavage of cleavable peptide substrates by a set of exemplary proteases.

Figures 25A-25F depict a radar chart showing the lysis in the conditioned medium using the identified murine model (C57/Bl6-wt, MC38-wt, MC38-hCTLA4, MB49-wt, MCA205-wt, B16-wt) The incidence rate. Use tissue conditioning media as indicated, such as spleen, liver, kidney, plasma or tumor.

Figures 26A and 26B depict the results of an in vivo lysis study using antibodies 2-14 in the plasma of healthy cynomolgus monkeys. As depicted in FIG. 26A, using a capillary electrophoresis (CE) and mass spectrometry (MS) calculated 2-14 cleavage of antibodies, as indicated. Figure 26B depicts a graph showing the ratio of intact antibody 2-14 and its lysate at 1 hour, 24 hours, and 72 hours. Figure 26C depicts the results of an in vivo lysis study using antibodies 2-16 in the plasma of healthy cynomolgus monkeys, including the calculated lysis using capillary electrophoresis (CE) and mass spectrometry (MS) evaluation, as indicated.

Figures 27A and 27B depict the results of ex vivo lysis studies using media conditioned with human lung or colon tumor samples.

Figures 28A-28D depict the use of colon tumor supernatants, using mass spectrometry lysis molecules (antibody 2-14, Figure 28A ; antibody 2-15, Figure 28B ; antibody 2-16, Figure 28C ; antibody 2-10, Figure 28D ) The analysis result of the percentage (%). Figure 28E depicts the results of an ELISA assay using antibody 2-6, antibody 2-14, antibody 2-15, antibody 2-10, antibody 2-16, and masked ipelizumab-m, in which the colon Each tested antibody was cultured in tumor conditioned medium (colon 24 sept colon tumor sample) or RPMI as a control.

Figure 29 depicts the cytotoxicity measured using the LDH release assay done in serum-free or supplemented with fetal calf serum (FCS) medium, which is adjusted with the indicated cell line or tumor/NAT tissue.

Figure 30 depicts the substrate lysis of MC38 and H2228 cell lines in the form of an iceLogo diagram.

Figures 31A and 31B depict the lysis of NAT and tumor (TUM) tissue in iceLogo format. Figure 31C depicts the difference in Z scores at P4-P4' positions in the form of a heat map to highlight residues that are conducive to NAT or tumor-specific lysis. Leucine (n) is a substitute for Met in the library. Figure 31D depicts the difference in Z scores at P4-P4' positions in the form of a heat map to highlight the residues that are conducive to the specific cleavage of H2228 compared to NAT or tumor samples. Leucine (n) is a substitute for Met in the library.

Figures 32A and 32B depict the formation curve of the selected substrate under MC38 conditioned medium; AK10-01 ( Figure 32A , left), AK10-02 ( Figure 32A , right); AK10-04 ( Figure 32B , left) ) And AK10-05 ( Figure 32B , right side).

Figure 33 depicts the relative efficiency of the main cleavage products from the MC38 AMSP-MS reaction using AK10 library peptides.

Figures 34A and 34B depict the formation of curves of selected substrate products in the case of H2228 conditioned medium; AK10-01 ( Figure 34A , top left), AK10-02 ( Figure 34A , top right), AK10-04 ( Figure 34A) , Lower part), AK10-09 ( Figure 34B , left) and AK10-10 ( Figure 34B , right).

Figure 35 depicts the highest substrate lysed by H2228 conditioned medium and its relative efficiency monitored by AMSP-MS.

Figures 36A-36C depict curves formed by the selected substrates in the case of tumor-conditioned media; AK10-01 ( Figure 36A , upper left), AK10-02 ( Figure 36A , upper middle), 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 36D depicts the highest substrate lysed by tumor conditioned medium displayed via AMSP-MS and its relative efficiency.

Figures 37A-37D depict the formation of curves of selected substrates in the case of 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 37E depicts the highest lysis and relative efficiency produced by NAT-conditioned medium in the case of an AK10 library of peptides.

Figure 38 depicts proteins in different ways in tumor and NAT tissue samples in the form of a volcano map. A statistically significant protein has a threshold fold difference of ≥2 fold change. Proteins that are significantly enriched in tumors (upper right, part 2) or NAT (upper left, part 1) are present in these parts.

Claims (40)

A masked antibody comprising: a) an anti-CTLA4 antibody or antigen-binding fragment thereof, which comprises a variable light (VL) domain and a variable heavy (VH) domain; and b) A masking peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-46; The masking peptide is connected to the amino or carboxyl end of the VL domain or the VH domain via a linker containing a cleavable peptide. The masked antibody of claim 1, wherein the linker comprising a cleavable peptide comprises a cleavable peptide comprising an amino acid selected from the group consisting of SEQ ID NO: 47-88, 464-469 and 479-508 sequence. The masked antibody of claim 2, wherein the cleavable peptide comprises an amino terminal and a carboxy terminal, and the linker comprising the cleavable peptide comprises a first spacer linker and a second spacer linker, wherein the first spacer The linker is connected to the amino end of the cleavable peptide and includes an amino acid sequence selected from the group consisting of SEQ ID NO: 89-112 and 415-420, and the second spacer linker is connected to the cleavable peptide The carboxy-terminal and includes an amino acid sequence selected from the group consisting of SEQ ID NO: 89-112 and 415-420. The masked antibody of claim 1, wherein the linker comprising the cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 454-462. The masked antibody of claim 1, wherein the masked antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 113-231 and 444-453. The masked antibody of claim 1, wherein the anti-CTLA4 antibody or antigen-binding fragment thereof is a humanized antibody, a chimeric antibody, or a human antibody. Such as the masked antibody of claim 1, where: a) The VL domain includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 402, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 403, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 404; and/or the VH domain includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 405, (ii) CDR-H2 , Which includes the amino acid sequence of SEQ ID NO: 406, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 407; or b) The VL domain includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 408, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 409, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 410; and/or the VH domain includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 411, (ii) CDR-H2 , Which includes the amino acid sequence of SEQ ID NO: 412, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 413; or c) The VL domain includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 432, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 433, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 434; and/or the VH domain includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 435, (ii) CDR-H2 , Which includes the amino acid sequence of SEQ ID NO: 436, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 437; or d) The VL domain includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 438, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 439, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 440; and/or the VH domain includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 441, (ii) CDR-H2 , Which includes the amino acid sequence of SEQ ID NO: 442, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 443. Such as the masked antibody of claim 1, where: a) The VL domain includes the amino acid sequence of SEQ ID NO: 321, and the VH domain includes the amino acid sequence of SEQ ID NO: 323; or b) The VL domain includes the amino acid sequence of SEQ ID NO: 322, and the VH domain includes the amino acid sequence of SEQ ID NO: 324. The masked antibody of claim 1, wherein the VL domain is contained in a light chain, the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 327-341, and the VH domain is contained in a heavy chain Inside, the heavy chain includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 366-380 and 421. The masked antibody of claim 1, wherein 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 NO: 358 and 422-431. The masked antibody of claim 1, wherein the anti-CTLA4 antibody or antigen-binding fragment thereof binds to a drug. The masked antibody of claim 11, wherein the agent is a tubulin polymerization inhibitor, a DNA damaging agent, or a DNA synthesis inhibitor. The masked antibody of claim 11, wherein the agent is maytansinoid, auristatin, pyrrolobenzodiazepine (PBD) dimer, calicheamicin , Duocarmycin, indo-linobenzodiazepine dimer or exatecan derivative Dxd. A masked bispecific antibody comprising: a) It specifically binds to the first pair of light and heavy chains of CTLA4; b) The second pair of light and heavy chains that specifically bind to the antigen; and c) a masking peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-46, The masking peptide is connected to the amino or carboxyl end of the light chain or the heavy chain of the first pair via a linker containing a cleavable peptide. The masked bispecific antibody of claim 14, wherein the linker comprising a cleavable peptide comprises a cleavable peptide, which comprises a group selected from the group consisting of SEQ ID NOs: 47-88, 464-469 and 479-508 Amino acid sequence. The masked bispecific antibody of claim 15, wherein the cleavable peptide comprises an amino terminal and a carboxy terminal, and the linker comprising the cleavable peptide comprises a first spacer linker and a second spacer linker, wherein the The first spacer linker is connected to the amino end of the cleavable peptide and includes an amino acid sequence selected from the group consisting of SEQ ID NO: 89-112 and 415-420, and the second spacer linker is connected to the cleavable peptide The carboxy terminus of the cleavage peptide includes an amino acid sequence selected from the group consisting of SEQ ID NO: 89-112 and 415-420. The masked bispecific antibody of claim 14, wherein the linker comprising the cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 454-462. The masked bispecific antibody of claim 14, wherein the masked bispecific antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 113-231 and 444-453. The masked bispecific antibody of claim 14, wherein the light chain of the first pair comprises a VL domain, and the heavy chain of the first pair comprises a VH domain, wherein: a) The VL domain includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 402, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 403, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 404; and/or the VH domain includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 405, (ii) CDR-H2 , Which includes the amino acid sequence of SEQ ID NO: 406, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 407; or b) The VL domain includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 408, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 409, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 410; and/or the VH domain includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 411, (ii) CDR-H2 , Which includes the amino acid sequence of SEQ ID NO: 412, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 413; or c) The VL domain includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 432, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 433, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 434; and/or the VH domain includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 435, (ii) CDR-H2 , Which includes the amino acid sequence of SEQ ID NO: 436, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 437; or d) The VL domain includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 438, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 439, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 440; and/or the VH domain includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 441, (ii) CDR-H2 , Which includes the amino acid sequence of SEQ ID NO: 442, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 443. Such as the masked bispecific antibody of claim 14, wherein: a) The VL domain includes the amino acid sequence of SEQ ID NO: 321, and the VH domain includes the amino acid sequence of SEQ ID NO: 323; or b) The VL domain includes the amino acid sequence of SEQ ID NO: 322, and the VH domain includes the amino acid sequence of SEQ ID NO: 324. The masked bispecific antibody of claim 14, wherein the light chain of the first pair comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 327-341, and the heavy chain of the first pair comprises a selected Free amino acid sequence of the group consisting of SEQ ID NO: 366-380 and 421. The masked bispecific antibody of claim 14, wherein the masked bispecific antibody comprises the amino acid sequence of SEQ ID NO: 421, and comprises the group selected from the group consisting of SEQ ID NO: 358 and 422-431 The amino acid sequence. A masked chimeric receptor comprising: a) The ligand binding domain that binds to CTLA4, which includes the VL domain and the VH domain; b) A masking peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-46; c) Transmembrane domain; and d) Intracellular signal transduction domain, which contains signal transduction domain, The masking peptide is connected to the amino or carboxyl end of the VL domain or the VH domain via a linker containing a cleavable peptide. The masked chimeric receptor of claim 23, wherein the linker comprising a cleavable peptide comprises a cleavable peptide, which comprises a group selected from the group consisting of SEQ ID NO: 47-88, 464-469 and 479-508 Amino acid sequence. The masked chimeric receptor of claim 23, wherein the cleavable peptide comprises an amino terminal and a carboxy terminal, and the linker comprising the cleavable peptide comprises a first spacer linker and a second spacer linker, wherein the The first spacer linker is connected to the amino end of the cleavable peptide and includes an amino acid sequence selected from the group consisting of SEQ ID NO: 89-112 and 415-420, and the second spacer linker is connected to the cleavable peptide The carboxy terminus of the cleavage peptide includes an amino acid sequence selected from the group consisting of SEQ ID NO: 89-112 and 415-420. The masked chimeric receptor of claim 23, wherein the linker comprising the cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 454-462. The masked chimeric receptor of claim 23, wherein the masked chimeric receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 113-231 and 444-453. Such as the masked chimeric receptor of claim 23, in which: a) The VL domain includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 402, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 403, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 404; and/or the VH domain includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 405, (ii) CDR-H2 , Which includes the amino acid sequence of SEQ ID NO: 406, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 407; or b) The VL domain includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 408, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 409, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 410; and/or the VH domain includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 411, (ii) CDR-H2 , Which includes the amino acid sequence of SEQ ID NO: 412, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 413; or c) The VL domain includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 432, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 433, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 434; and/or the VH domain includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 435, (ii) CDR-H2 , Which includes the amino acid sequence of SEQ ID NO: 436, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 437; or d) The VL domain includes (i) CDR-L1, which includes the amino acid sequence of SEQ ID NO: 438, (ii) CDR-L2, which includes the amino acid sequence of SEQ ID NO: 439, and (iii) CDR-L3, which includes the amino acid sequence of SEQ ID NO: 440; and/or the VH domain includes (i) CDR-H1, which includes the amino acid sequence of SEQ ID NO: 441, (ii) CDR-H2 , Which includes the amino acid sequence of SEQ ID NO: 442, and (iii) CDR-H3, which includes the amino acid sequence of SEQ ID NO: 443. Such as the masked chimeric receptor of claim 23, in which: a) The VL domain includes the amino acid sequence of SEQ ID NO: 321, and the VH domain includes the amino acid sequence of SEQ ID NO: 323; or b) The VL domain includes the amino acid sequence of SEQ ID NO: 322, and the VH domain includes the amino acid sequence of SEQ ID NO: 324. A nucleic acid encoding the masked antibody of claim 1. A vector comprising the nucleic acid of claim 30. A host cell comprising the nucleic acid of claim 30. A method for preparing a masked antibody, which comprises culturing a host cell according to claim 32 under conditions for producing the masked antibody. A masked antibody prepared by the method described in claim 33. A composition comprising the masked antibody as claimed in claim 1. A pharmaceutical composition comprising the masked antibody as in claim 1 and a pharmaceutically acceptable carrier. A kit comprising the masked antibody as in claim 1. A use of the masked antibody as in claim 1, which is used to manufacture a medicament for the treatment or prevention of neoplastic diseases. A masked antibody comprising: a) an anti-CTLA4 antibody or antigen-binding fragment thereof, which comprises a variable light (VL) domain and a variable heavy (VH) domain; and b) Masking peptide, which includes the amino acid sequence of SEQ ID NO: 5; Wherein the masking peptide is connected to the amino end of the VL domain via a linker comprising a cleavable peptide; Wherein the linker comprising a cleavable peptide comprises a cleavable peptide, which comprises the amino acid sequence of SEQ ID NO: 86; Wherein (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 the amino group comprising SEQ ID NO: 410 Acid sequence of CDR-L3; and the VH domain includes CDR-H1 including the amino acid sequence of SEQ ID NO: 411, CDR-H2 including the amino acid sequence of SEQ ID NO: 412, and includes SEQ ID NO: CDR-H3 of the amino acid sequence of 413; or (b) the VL domain includes CDR-L1 including the amino acid sequence of SEQ ID NO: 438, and CDR- including the amino acid sequence of SEQ ID NO: 439 L2 and CDR-L3 including the amino acid sequence of SEQ ID NO: 440; and the VH domain includes CDR-H1 including the amino acid sequence of SEQ ID NO: 441, including the amino acid of SEQ ID NO: 442 The sequence of CDR-H2 and the CDR-H3 including the amino acid sequence of SEQ ID NO:443. A masked antibody comprising: a) an anti-CTLA4 antibody or antigen-binding fragment thereof, which comprises a variable light (VL) domain and a variable heavy (VH) domain; and b) Masking peptide, which includes the amino acid sequence of SEQ ID NO: 19; Wherein the masking peptide is connected to the amino end of the VL domain via a linker comprising a cleavable peptide; Wherein the linker comprising the cleavable peptide comprises a cleavable peptide, which comprises the amino acid sequence of SEQ ID NO: 50; Wherein (a) the VL domain includes CDR-L1 including the amino acid sequence of SEQ ID NO: 408, CDR-L2 including the amino acid sequence of SEQ ID NO: 409, and the amino group including SEQ ID NO: 410 Acid sequence of CDR-L3; and the VH domain includes CDR-H1 including the amino acid sequence of SEQ ID NO: 411, CDR-H2 including the amino acid sequence of SEQ ID NO: 412, and includes SEQ ID NO: CDR-H3 of the amino acid sequence of 413; or (b) the VL domain includes CDR-L1 including the amino acid sequence of SEQ ID NO: 438, and CDR- including the amino acid sequence of SEQ ID NO: 439 L2 and CDR-L3 including the amino acid sequence of SEQ ID NO: 440; and the VH domain includes CDR-H1 including the amino acid sequence of SEQ ID NO: 441, including the amino acid of SEQ ID NO: 442 The sequence of CDR-H2 and the CDR-H3 including the amino acid sequence of SEQ ID NO:443.

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