Sustained release immune cell binding proteins and methods of treatment

Abstract

Provided herein is a description of sustained release immune cell binding proteins comprising a masking peptide and a cleavable linker, and methods of using same. Also described are pharmaceutical compositions comprising the proteins comprising a masking peptide and a cleavable linker, and methods of using same.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 214,963, filed June 25, 2021, U.S. Provisional Patent Application No. 63 / 276,804, filed November 8, 2021, and U.S. Provisional Patent Application No. 63 / 328,603, filed April 7, 2022, each of which is incorporated by reference in its entirety.

[0002] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Background technology]

[0003] Cancer is the second leading cause of human death after coronary artery disease. Millions of people worldwide die from cancer each year. In the United States alone, cancer kills more than 500,000 people each year, and approximately 1.4 million new cases are diagnosed each year. Deaths from heart disease have declined significantly, but those attributable to cancer overall are rising. Cancer is projected to remain the leading cause of death early in the next century.

[0004] Furthermore, even for cancer patients who initially survive the primary cancer, daily experiences have shown that their lives change dramatically. Many cancer patients suffer from intense anxiety driven by the realization that recurrence or treatment failure may be possible. Many cancer patients suffer significant physical debilitation after treatment.

[0005] Generally, a fundamental problem in the management of deadly cancers is the lack of effective and non-toxic systemic therapies. Cancer is a complex disease characterized by genetic mutations that lead to uncontrolled cell growth. Cancer cells exist in all organisms, and under normal circumstances, their excessive growth is tightly regulated by various physiological factors. Controlling toxicity of various cancer treatments remains a significant and unsolved challenge. Summary of the Invention

[0006] Provided herein is a pharmaceutical composition comprising a sustained release binding protein comprising a half-life extended immune cell engaging protein, a masking peptide, and a cleavable linker, wherein the masking peptide is covalently attached to the N-terminus or C-terminus of the half-life extended immune cell binding protein via the cleavable linker, and the cleavable linker is cleaved in substantial amounts in the systemic circulation. In some embodiments, the sustained release binding protein has a higher therapeutic index than the corresponding half-life extended immune cell binding protein without the masking peptide. In some embodiments, administration of the sustained release binding protein results in a lower Cmax / Cmin ratio of the active form of the sustained release binding protein in the systemic circulation than the corresponding half-life extended immune cell binding protein without the masking peptide is administered. In some embodiments, multiple administrations of the sustained release binding protein result in an incremental increase in the level of the active form of the sustained release binding protein in the systemic circulation than the corresponding half-life extended immune cell binding protein without the masking peptide is administered. In some embodiments, administration of the sustained release binding protein results in lower levels of cytokine release syndrome (CRS) than is observed when a corresponding extended half-life immune cell binding protein that does not contain a masking peptide is administered.

[0007] In some embodiments, the extended half-life immune cell binding protein comprises an immune cell binding domain. In some embodiments, the immune cell binding domain comprises a natural killer (NK) cell binding domain, a T cell binding domain, a NK-T cell binding domain, a B cell binding domain, a dendritic cell binding domain, a macrophage cell binding domain, or a combination thereof. In some embodiments, the immune cell binding domain comprises a T cell binding domain. In some embodiments, the T cell binding domain binds to a CD3 molecule. In some embodiments, the CD3 molecule is at least one of a CD3 gamma molecule, a CD3 delta molecule, or a CD3 epsilon molecule.

[0008] In some embodiments, the extended half-life immune cell binding protein comprises a first domain (A), a second domain (B), and a third domain (C), where (i) the first domain (A) is a T cell binding domain and specifically binds human CD3, (ii) the second domain (B) specifically binds human serum albumin (HSA), and (iii) the third domain (C) specifically binds a target antigen, the domains being arranged in the following order: H2N-(C)-(B)-(A)-COOH, H2N-(A)-(B)-(C)-COOH, H2N-(B)-(A)-(C)-COOH, H2N- or linked by linkers L1 and L2 in one of the following orders: H2N-(C)-L1-(B)-L2(A)-COOH, H2N-(A)-L1-(B)-L2-(C)-COOH, H2N-(B)-L1-(A)-L2-(C)-COOH, H2N-(B)-L1-(A)-L2-(C)-COOH, H2N-(C)-L1-(A)-L2-(B)-COOH, H2N-(A)-L1-(C)-L2(B)-COOH, H2N-(B)-L1-(C)-L2-(A)-COOH. In some embodiments, the third domain comprises a single domain antibody (sdAb), a single chain variable fragment (scFv), a heavy chain variable domain (VH), a light chain variable domain (VL), an antigen binding fragment (Fab), a DARPin, or a peptide. In some embodiments, the masking peptide inhibits or reduces binding of the first domain (A) to human CD3. In some embodiments, the masking peptide inhibits or reduces binding of the first domain (A) to the N-terminus of human CD3ε. In some embodiments, the masking peptide comprises an amino acid sequence having at least 80% homology to QDGNEE (SEQ ID NO: 3633). In some embodiments, the masking peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3633-3652. In some embodiments, the masking peptide inhibits or reduces binding of the third domain (C) to the target antigen. In some embodiments, the cleavable linker comprises a cleavable site.In some embodiments, the cleavable site is recognized by a protease. In some embodiments, the protease is selected from the group consisting of serine proteases, cysteine ​​proteases, aspartate proteases, threonine proteases, glutamic acid proteases, metalloproteinases, gelatinases, and asparagine peptide lyases. In some embodiments, the protease is present in the blood circulation. In some embodiments, the protease is cathepsin, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin H, cathepsin S, cathepsin K, cathepsin L, kallikrein, hK1, hK10, hK15, plasmin, collagenase, collagen type IV, stromelysin, factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidain, bromelain, calpain, caspase, caspase-3, Mir1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidase, melanin, erythropoietin ... The protease is selected from the group consisting of thaloendopeptidase, matrix metalloprotease (MMP), MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, ADAM10, ADAM12, urokinase plasminogen activator (uPA), enterokinase, prostate specific target (PSA, hK3), interleukin-1β converting enzyme, thrombin, FAP (FAP-α), type II transmembrane serine protease (TTSP), neutrophil elastase, cathepsin G, proteinase 3, neutrophil serine protease 4, mast cell chymase, mast cell tryptase, dipeptidyl peptidase, and dipeptidyl peptidase IV (DPPIV / CD26).

[0009] In some embodiments, the cleavable linker comprises an amino acid sequence having at least 80% homology to SEQ ID NOs: 3688-3770 and 3878. In some embodiments, the cleavable linker comprises an amino acid sequence of SEQ ID NOs: 3688-3770 and 3878. In some embodiments, the target antigen is a tumor antigen. In some embodiments, the third domain is selected from the group consisting of CD19 (B-lymphocyte antigen CD19, B-lymphocyte surface antigen B4, T-cell surface antigen Leu-12, CVID3), PSMA (prostate-specific membrane antigen), MSLN (mesothelin), BCMA (B-cell maturation antigen), DLL3 (delta-like ligand 3), FLT3 (FMS-like tyrosine kinase 3), CD20 (B-lymphocyte antigen CD20, MS4A1, B1, Bp35, CVID5, LEU-16, MS4A2, S7, transmembrane 4-domain A1), C D22 (SIGLEC-2, SIGLEC2), CD25 (IL2RA, interleukin-2 receptor α chain), CD27 (S152, S152.LPFS2, T14, TNFRSF7, Tp55), CD30 (TNFRSF8), CD33 (Siglec-3, sialic acid-binding Ig-like lectin 3, SIGLEC3, SIGLEC-3, gp67, p67), CD37 (GP52-40, TSPAN26), CD38 (cyclic ADP-ribose hydrolase, ADPRC1, ADPRC 1), CD40 (Bp50, CDW40, TNFRSF5, p50), CD44 (HCAM, homing cell adhesion molecule), Pgp-1 (phagocyte glycoprotein-1), Hermes antigen, lymphocyte homing receptor, ECM-III, and HUTCH-1), CD48 (BLAST-1, B lymphocyte activation marker, SLAMF2, signaling lymphocyte activation molecule 2), CD52 (CAMPATH-1 antigen), CD70, CD73 (NT5E, ecto-5 '-nucleotidase), CD39 (ENTPD1, ​​ectonucleoside triphosphate diphosphohydrolase-1), CD74 (HLA class II histocompatibility antigen gamma chain, invariant chain associated with HLA-DR antigen), CD79b (immunoglobulin-associated beta), CD80 (B7-1), CD86 (B7-2), CD123 (IL3RA, interleukin-3 receptor), CD133 (PROM1), CD137 (induced by lymphocyte activation, TNFRSF9,Tumor necrosis factor receptor superfamily member 9, 4-1BB, ILA), CD138 (SDC1), alpha-fetoprotein (AFP), c-Met, c-Kit, CD371 (CLEC12A, C-type lectin domain family 12 member A, CLL1), CD370 (CLEC9A, C-type lectin domain-containing 9A), cadherin 3 (CDH3, p-cadherin, PCAD), carbonic anhydrase 6 (CA6), carbonic anhydrase 9 (CA9, CAIX), carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), CD66c (CEACAM6, carcinoembryonic antigen-related cell adhesion molecule 6), chorionic somatomammotropic hormone 1 (CSH1, CS1), coagulation factor III, tissue factor (F3, TF), collectin subfamily member 10 (COLEC10), delta-like positive Associated Notch ligand 3 (DLL3), ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3), ephrin A1 (EFNA1), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), EPH receptor A2 (EPHA2), epithelial cell adhesion molecule (EPCAM), erb-b2 receptor tyrosine kinase 2 (ERBB2, HER2), fibroblast activation protein alpha (FAP), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), folate hydrolase 1 (FOLH1, PSMA), folate receptor 1 (FOLR1, FRa), GD2 ganglioside, glycoprotein NMB (GPNMB, osteoactivin), guanylate cyclase 2C (GUCY2C, GCC), human papillomavirus (HPV) E6, HPV E7, major histocompatibility complex (MHC) class I-presented neoantigen, major histocompatibility complex (MHC) class II-presented neoantigen, major histocompatibility complex, class I, E (HLA-E), major histocompatibility complex, class I, F (HLA-F), major histocompatibility complex, class I, G (HLA-G, MHC-G), integrin subunit β7 (ITGB7), leukocyte immunoglobulin-like receptor B1 (LILRB1, ILT2), leukocyte immunoglobulin-like receptor B2 (LILRB2, ILT4), LY6 / PLAUR domain-containing 3 (LYPD3, C4.4A), glypican 3 (GPC3),KRAS proto-oncogene, GTPase (KRAS), MAGE family member A1 (MAGEA1), MAGE family member A3 (MAGEA3), MAGE family member A4 (MAGEA4), MAGE family member A11 (MAGEA11), MAGE family member C1 (MAGEC1), MAGE family member C2 (MAGEC2), MAGE family member D1 (MAGED1), MAGE family member D2 (MAGED2), mesothelin (MSLN), mucin 1 (MUC 1) and its splice variants (e.g., MUC1 / C, D, and Z), mucin 16 (MUC16), necdin (NDN), nectin cell adhesion molecule 4 (NECTIN4), SLIT and NTRK-like family 6 (SLITRK6), promyelocytic leukemia (PML, TRIM19), protein tyrosine kinase 7 (inactive) (PTK7), CD352 (SLAMF6, SLAM family member 6), CD319 (SLAMF7, SLAM family member 7, 19A, CRACC, CS1), sialic acid-binding Ig-like lectin 7 (SIGLEC7), sialic acid-binding Ig-like lectin 9 (SIGLEC9), solute carrier family 34 (sodium phosphate) member 2 (SLC34A2), solute carrier family 39 member 6 (SLC39A6, LIV1), STEAP family member 1 (STEAP1), STEAP family member 2 (STEAP2), CD134 (TNFRSF4, TNF receptor superfamily member 4, OX40), CD137L (TNFSF9, TNF superfamily member 9, 4-1BB- L), CD261 (TNFRSF10A, TNF receptor superfamily member 10a, DR4, TRAILR1), CD262 (TNFRSF10B, TNF receptor superfamily member 10b, DR5, TRAILR2), CD267 (TNFRSF13B, TNF receptor superfamily member 13B, TACI, IGAD2), CD269 (TNFRSF17, TNF receptor superfamily member 17, BCMA), CD357 (TNFRSF18, TNF receptor superfamily member 18 GITR), transferrin (TF), transforming growth factor β1 (TGFB1),Specifically binds to a target antigen selected from the group consisting of trophoblast glycoprotein (TPBG, 5T4), trophinin (TRO, MAGED3), tumor-associated calcium signal transducer 2 (TACSTD2, TROP2, EGP1), fucosyl-GM1, sialyl Lewis adhesion molecule (sLe), ROR1, CD30, and Lewis Y antigen.

[0010] In some embodiments, the third domain specifically binds to CD19. In some embodiments, the third domain comprises an amino acid sequence having at least 80% homology to SEQ ID NOs: 3771-3792. In some embodiments, the third domain comprises an amino acid sequence having at least 90% homology to SEQ ID NOs: 3771-3792. In some embodiments, the third domain comprises an amino acid sequence of SEQ ID NO: 3771-3792. In some embodiments, the third domain comprises an amino acid sequence of SEQ ID NO: 3771. In some embodiments, the domains of the half-life extended immune cell binding protein are linked in one of the following orders: H2N-(C)-(B)-(A)-COOH, H2N-(C)-(A)-(B)-COOH, or linked by linkers L1 and L2 in one of the following orders: H2N-(C)-L1-(B)-L2-(A)-COOH, H2N-(C)-L1-(A)-L2-(B)-COOH. In some embodiments, the half-life extended immune cell binding protein comprises the amino acid sequence of SEQ ID NOs: 3839-3843. In some embodiments, the half-life extended immune cell binding protein comprises the amino acid sequence of SEQ ID NOs: 3839-3843.

[0011] In some embodiments, the third domain specifically binds to CD20. In some embodiments, the third domain comprises an amino acid sequence having at least 80% homology to SEQ ID NOs: 3793-3808 and 3880. In some embodiments, the third domain comprises an amino acid sequence having at least 90% homology to SEQ ID NOs: 3793-3808 and 3880. In some embodiments, the third domain comprises the amino acid sequence of SEQ ID NO: 3793-3808 and 3880. In some embodiments, the third domain comprises the amino acid sequence of SEQ ID NO: 3793. In some embodiments, the domains of the extended half-life immune cell binding protein are linked in the following order: H2N-(A)-(B)-(C)-COOH or linked by linkers L1 and L2 in the following order: H2N-(A)-L1-(B)-L2-(C)-COOH.

[0012] In some embodiments, the third domain specifically binds to CD33. In some embodiments, the third domain comprises an amino acid sequence having at least 80% homology to SEQ ID NOs: 3809-3823. In some embodiments, the third domain comprises an amino acid sequence having at least 90% homology to SEQ ID NOs: 3809-3823. In some embodiments, the third domain comprises an amino acid sequence of SEQ ID NO: 3809-3823. In some embodiments, the third domain comprises an amino acid sequence of SEQ ID NO: 3809. In some embodiments, the domains of the extended half-life immune cell binding proteins are linked in one of the following orders: H2N-(C)-(B)-(A)-COOH, H2N-(A)-(B)-(C)-COOH, H2N-(C)-(A)-(B)-COOH, H2N-(A)-(C)-(B)-COOH, or are linked by linkers L1 and L2 in one of the following orders: H2N-(C)-L1-(B)-L2(A)-COOH, H2N-(A)-L1-(B)-L2-(C)-COOH, H2N-(C)-L1-(A)-L2-(B)-COOH, H2N-(A)-L1-(C)-L2(B)-COOH. In some embodiments, the half-life extended immune cell binding protein comprises the amino acid sequence of SEQ ID NOs: 3833-3837. In some embodiments, the half-life extended immune cell binding protein comprises the amino acid sequence of SEQ ID NOs: 3834-3837.

[0013] In some embodiments, the third domain specifically binds to FLT3. In some embodiments, the third domain is a single domain antibody that specifically binds to FLT3. In some embodiments, the third domain comprises a CDR1 comprising an amino acid selected from the group consisting of SEQ ID NOs: 1080-1155 and 3497-3498, a CDR2 comprising an amino acid selected from the group consisting of SEQ ID NOs: 1156-1231 and 3499-3500, and a CDR3 comprising an amino acid selected from the group consisting of SEQ ID NOs: 1232-1307 and 3501-3502. In some embodiments, the CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1150, 1152, 3497, and 3498, the CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1226, 1228, 3499, and 3500, and the CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1302, 1304, 3501, and 3502. In some embodiments, the third domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1004-1079 and 3495-3496. In some embodiments, the third domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1074, 1076, 3495, and 3496. In some embodiments, the third domain comprises the amino acid sequence of SEQ ID NO: 1074. In some embodiments, the domains of the extended half-life immune cell binding proteins are linked in one of the following orders: H2N-(C)-(B)-(A)-COOH, H2N-(A)-(B)-(C)-COOH, H2N-(C)-(A)-(B)-COOH, H2N-(A)-(C)-(B)-COOH, or are linked by linkers L1 and L2 in one of the following orders: H2N-(C)-L1-(B)-L2(A)-COOH, H2N-(A)-L1-(B)-L2-(C)-COOH, H2N-(C)-L1-(A)-L2-(B)-COOH, H2N-(A)-L1-(C)-L2(B)-COOH. In some embodiments, the half-life extended immune cell binding protein comprises the amino acid sequence of SEQ ID NOs: 3844 to 3849. In some embodiments, the half-life extended immune cell binding protein comprises the amino acid sequence of SEQ ID NOs: 3846 to 3849.In some embodiments, the third domain comprises the amino acid sequence of SEQ ID NO: 1076. In some embodiments, the domains of the extended half-life immune cell binding protein are linked in the following order: H2N-(C)-(B)-(A)-COOH, or linked by linkers L1 and L2 in the following order: H2N-(C)-L1-(B)-L2(A)-COOH. In some embodiments, the extended half-life immune cell binding protein comprises the amino acid sequence of SEQ ID NO: 3850-3855. In some embodiments, the extended half-life immune cell binding protein comprises the amino acid sequence of SEQ ID NO: 3854.

[0014] In some embodiments, the third domain is a single domain antibody that specifically binds to PSMA. In some embodiments, the third domain comprises a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 462-465, a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 466-472, and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 474-475. In some embodiments, CDR1 comprises an amino acid sequence of SEQ ID NO: 462, CDR2 comprises an amino acid sequence of SEQ ID NO: 473, and CDR3 comprises an amino acid sequence of SEQ ID NO: 474. In some embodiments, the third domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 476-489. In some embodiments, the third domain comprises an amino acid sequence of SEQ ID NO: 489. In some embodiments, the extended half-life immune cell-binding protein comprises an amino acid sequence of SEQ ID NOs: 3824-3831.

[0015] In some embodiments, the third domain is a single domain antibody that specifically binds to MSLN. In some embodiments, the third domain comprises a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 490-528, a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 529-567, and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 568-606. In some embodiments, CDR1 comprises an amino acid sequence of SEQ ID NO: 523, CDR2 comprises an amino acid sequence of SEQ ID NO: 562, and CDR3 comprises an amino acid sequence of SEQ ID NO: 601. In some embodiments, the third domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 607-650. In some embodiments, the third domain comprises an amino acid sequence of SEQ ID NO: 647. In some embodiments, the extended half-life immune cell-binding protein comprises an amino acid sequence of SEQ ID NOs: 3856-3858.

[0016] In some embodiments, the third domain is a single domain antibody that specifically binds to BCMA. In some embodiments, the third domain comprises a CDR1 comprising an amino acid selected from the group consisting of SEQ ID NOs: 1-115, a CDR2 comprising an amino acid selected from the group consisting of SEQ ID NOs: 116-230, and a CDR3 comprising an amino acid selected from the group consisting of SEQ ID NOs: 231-345. In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 73, CDR2 comprises the amino acid sequence of SEQ ID NO: 188, and CDR3 comprises the amino acid sequence of SEQ ID NO: 303. In some embodiments, the third domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 346-461. In some embodiments, the third domain comprises the amino acid sequence of SEQ ID NO: 383.

[0017] In some embodiments, the third domain is a single domain antibody that specifically binds to DLL3. In some embodiments, the third domain comprises a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1751-2193, a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2194-2636, and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2637-3080. In some embodiments, CDR1 comprises an amino acid sequence of SEQ ID NO: 2182, CDR2 comprises an amino acid sequence of SEQ ID NO: 2625, and CDR3 comprises an amino acid sequence of SEQ ID NO: 3069. In some embodiments, the third domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1308-1750. In some embodiments, the third domain comprises an amino acid sequence of SEQ ID NO: 1739.

[0018] In some embodiments, the third domain is a single domain antibody that specifically binds to EGFR. In some embodiments, the third domain comprises a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 651-699, a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 700-748, and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 479-797. In some embodiments, the third domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 798-846.

[0019] In some embodiments, the third domain is a single domain antibody that specifically binds to EpCAM. In some embodiments, the third domain comprises a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 847-884, a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 885-922, and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 923-960. In some embodiments, CDR1 comprises an amino acid sequence of SEQ ID NO: 874 or 863, CDR2 comprises an amino acid sequence of SEQ ID NO: 885 or 901, and CDR3 comprises an amino acid sequence of SEQ ID NO: 923 or 939. In some embodiments, the third domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 961-1003. In some embodiments, the third domain comprises an amino acid sequence of SEQ ID NO: 999 or 1003.

[0020] In some embodiments, the first domain comprises a single chain variable fragment (scFv) specific for human CD3. In some embodiments, the scFv specific for human CD3 comprises a heavy chain variable region (VH), a light chain variable region (VL), and a linker, where the VH comprises complementarity determining regions HC CDR1, HC CDR2, and HC CDR3, and the VL comprises complementarity determining regions LC CDR1, LC CDR2, and LC CDR3. In some embodiments, the HC CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3081 and 3087-3098, the HC CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3082 and 3099-3109, and the HC CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3083 and 3110-3119. In some embodiments, HC CDR1 comprises the amino acid sequence of SEQ ID NO: 3097, HC CDR2 comprises the amino acid sequence of SEQ ID NO: 3108, and HC CDR3 comprises the amino acid sequence of SEQ ID NO: 3110. In some embodiments, LC CDR1 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 3084 and 3120-3132, LC CDR2 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 3085 and 3099-3109, and LC CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 3086 and 3146-3152. In some embodiments, LC CDR1 comprises the amino acid sequence of SEQ ID NO: 3120, LC CDR2 comprises the amino acid sequence of SEQ ID NO: 3145, and LC CDR3 comprises the amino acid sequence of SEQ ID NO: 3146. In some embodiments, the first domain comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 3153-3169. In some embodiments, the first domain comprises the amino acid sequence of SEQ ID NO: 3153. In some embodiments, the linker comprises the amino acid sequence GGGGSGGGSGGGGS (SEQ ID NO: 3868).

[0021] In some embodiments, the second domain comprises a single domain antibody (sdAb) that specifically binds to HSA. In some embodiments, the sdAb that specifically binds to HSA comprises complementarity determining regions CDR1, CDR2, and CDR3, where CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3170 and 3173-3175, CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3171 and 3176-3181, and CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3172 and 8182-3183. In some embodiments, CDR1 comprises an amino acid sequence of SEQ ID NO: 3174, CDR2 comprises an amino acid sequence of SEQ ID NO: 3178, and CDR3 comprises an amino acid sequence of SEQ ID NO: 3183. In some embodiments, the second domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3184-3193. In some embodiments, the second domain comprises an amino acid sequence of SEQ ID NO: 3190. In some embodiments, the linkers L1 and L2 are each independently (GS) n (SEQ ID NO: 3859), (GGS) n (SEQ ID NO: 3860), (GGGS) n (SEQ ID NO:3861), (GGSG) n (SEQ ID NO: 3862), (GGSGG) n (SEQ ID NO: 3863), (GGGGS) n (SEQ ID NO:3864), (GGGGG) n (SEQ ID NO: 3865), or (GGG) n (SEQ ID NO:3866), (GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO:3867), (GGGGSGGGGSGGGGS) (SEQ ID NO:3868), LPETG (SEQ ID NO:3869), (GGGGSGGGS) (SEQ ID NO:3871), or SGGG (SEQ ID NO:3872), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, linkers L1 and L2 are each independently GGGGSGGGS (SEQ ID NO:3871).

[0022] Provided herein is a method for the treatment or amelioration of a disease, comprising administering to a subject in need of such treatment or amelioration a pharmaceutical composition as described herein. In some embodiments, the disease is cancer. Provided herein is a method for increasing survival of a subject suffering from cancer, comprising administering to the subject a pharmaceutical composition as described herein. Provided herein is a method for reducing tumor size, comprising administering to a subject suffering from cancer a pharmaceutical composition as described herein. In some embodiments, the cancer is mesothelioma, prostate cancer, breast cancer, brain cancer, bladder cancer, pancreatic cancer, kidney cancer, solid tumors, liver cancer, leiomyosarcoma, endometrial cancer, breast cancer, female reproductive system cancer, ovarian cancer, soft tissue sarcoma, gastric cancer, digestive / gastrointestinal cancer. In some embodiments, the cancer is selected from the group consisting of colorectal cancer, glioblastoma multiforme, head and neck cancer, squamous cell carcinoma, colon cancer, gastric cancer, rhabdomyosarcoma, adrenal cancer, lung cancer, esophageal cancer, colon cancer, lung cancer, non-small cell lung cancer (NSCLC), neuroblastoma, melanoma, glioblastoma multiforme, ovarian cancer, endocrine cancer, respiratory / thoracic cancer, anal cancer, gastroesophageal cancer, thyroid cancer, cervical cancer, endometrial cancer, blood cancer, leukemia, lymphocytic leukemia, multiple myeloma, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, lymphocytic leukemia, anaplastic large cell lymphoma (ALCL), or myeloid leukemia. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is mesothelioma. In some embodiments, the cancer is lung cancer.

[0023] Provided herein is a pharmaceutical composition comprising a sustained release binding protein comprising an antigen binding protein, a masking peptide, and a cleavable linker, where after administration to a subject, the sustained release binding protein slowly releases an active form of the sustained release binding protein when the cleavable linker is cleaved in the systemic circulation. In some embodiments, the antigen binding protein comprises an antigen binding domain that binds to a target antigen. In some embodiments, the masking peptide inhibits or reduces binding of the antigen binding protein to the target antigen. In some embodiments, the antigen binding protein comprises an immune cell binding domain. In some embodiments, the immune cell binding domain comprises a T cell binding domain. In some embodiments, the T cell binding domain binds to a CD3 molecule. In some embodiments, the masking peptide inhibits or reduces binding of the antigen binding protein to a CD3 molecule. In some embodiments, the antigen binding protein comprises a half-life prolonging domain. In some embodiments, the half-life prolonging domain binds to human serum albumin (HSA). In some embodiments, the cleavable linker comprises a cleavable site. In some embodiments, the cleavable site is recognized by a protease. In some embodiments, the masking peptide is covalently attached to the N-terminus or C-terminus of the antigen binding protein via a cleavable linker.

[0024] Provided herein is a method of making a sustained release binding protein, comprising adding a cleavable linker and a masking peptide to an antigen binding protein, where after administration to a subject, the sustained release binding protein slowly releases an active form of the sustained release binding protein when the cleavable linker is cleaved in the systemic circulation. In some embodiments, the antigen binding protein comprises an antigen binding domain that binds to a target antigen. In some embodiments, the masking peptide inhibits or reduces binding of the antigen binding protein to the target antigen. In some embodiments, the antigen binding protein comprises an immune cell binding domain. In some embodiments, the immune cell binding domain comprises a T cell binding domain. In some embodiments, the T cell binding domain binds to a CD3 molecule. In some embodiments, the masking peptide inhibits or reduces binding of the antigen binding protein to a CD3 molecule. In some embodiments, the antigen binding protein comprises a half-life prolonging domain. In some embodiments, the half-life prolonging domain binds to human serum albumin (HSA). In some embodiments, the cleavable linker comprises a cleavable site. In some embodiments, the cleavable site is recognized by a protease. In some embodiments, the masking peptide is covalently attached to the N-terminus or C-terminus of the antigen binding protein via a cleavable linker.

[0025] Provided herein is a method of increasing the therapeutic index of an antigen binding protein, comprising adding a cleavable linker and a masking peptide to an antigen binding protein to form a sustained release binding protein, where after administration to a subject, the sustained release binding protein gradually releases the active form of the sustained release binding protein when the cleavable linker is cleaved in the systemic circulation. Provided herein is a method of reducing CRS levels in a subject, comprising adding a cleavable linker and a masking peptide to an antigen binding protein to form a sustained release binding protein, where after administration to a subject, the sustained release binding protein gradually releases the active form of the sustained release binding protein when the cleavable linker is cleaved in the systemic circulation. Provided herein is a method of gradually increasing the concentration of an active drug in the systemic circulation of a subject, comprising administering to a subject a sustained release binding protein, where the sustained release binding protein comprises an antigen binding protein, a masking peptide, and a cleavable linker, where the sustained release binding protein releases the active drug in the systemic circulation after cleavage of the cleavable linker.

[0026] Provided herein is a pharmaceutical composition comprising a protein comprising a binding moiety, a masking peptide, and a cleavable linker, wherein the masking peptide is covalently attached to the binding moiety via the cleavable linker, and the cleavable linker is cleaved in substantial amounts in the systemic circulation.

[0027] Provided herein is a pharmaceutical composition comprising a protein comprising a binding moiety, a masking peptide, and a cleavable linker, wherein the masking peptide is covalently attached to the binding moiety via the cleavable linker, and wherein the half-life of the protein in systemic circulation is longer than a control protein that does not contain the masking peptide.

[0028] Provided herein is a pharmaceutical composition comprising a protein comprising a binding moiety, a masking peptide, and a cleavable linker, wherein the masking peptide is covalently attached to the binding moiety via the cleavable linker, and a comparator protein not comprising the masking peptide has non-linear pharmacokinetics (PK) over the dose range evaluated, and the protein has improved PK linearity over the dose range evaluated compared to the comparator protein.

[0029] Provided herein is a pharmaceutical composition comprising a protein comprising a binding moiety, a masking peptide, and a cleavable linker, wherein the masking peptide is covalently attached to the binding moiety via the cleavable linker, and the binding moiety specifically binds to a target in a subject, and wherein the molar amount of the binding moiety of the protein that binds to the target when administered to a subject is less than the molar amount of the binding moiety of a comparative protein that does not contain the masking peptide when administered to a subject at the same dose level.

[0030] Provided herein is a pharmaceutical composition comprising a protein comprising a binding moiety, a masking peptide, and a cleavable linker, wherein the masking peptide is covalently attached to the binding moiety via the cleavable linker, the binding moiety specifically binds to a target in a subject, and when administered to a subject, a first binding rate between the binding moiety of the protein and the target is lower compared to a second binding rate between the binding moiety of a comparative protein that does not include the masking peptide and the target.

[0031] In some embodiments, the binding moiety specifically binds to ICOS (inducible T cell costimulatory factor, CD278), OX40 (CD134, TNFRSF4, tumor necrosis factor receptor superfamily member 4), CD40 (TNFRSF5, tumor necrosis factor receptor superfamily member 5), DR5 (death receptor 5, TRAIL receptor 2), GITR (glucocorticoid-inducible TNFR-related protein, TNFRSF18, tumor necrosis factor receptor superfamily member 18), 4-1BB (CD137, TNFRSF9, tumor necrosis factor receptor superfamily member 9), or any combination thereof. In some embodiments, the protein comprises an antibody selected from the group consisting of GSK3359609, PF-8600, JNJ-64457107, CP-870,893, SGN-40 (dacetuzumab), MEDI3039, ABBV-621, MEDI1873, AMG228, PF-05082566 (utomirumab), and urelumab.

[0032] Provided herein is a method of treating or ameliorating a disease, comprising administering to a subject in need of such treatment or amelioration a pharmaceutical composition described herein. [Brief description of the drawings]

[0033] The novel features of the invention are set forth with particularity in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings.

[0034] [Figure 1A] FIG. 1 shows an exemplary construct of a sustained release binding protein. [Figure 1B] FIG. 1 shows an exemplary construct of an active form of a sustained release binding protein. [Figure 1C] FIG. 1 shows predicted concentrations of intact and active forms in the systemic circulation after multiple administration events. [Figure 1D]FIG. 1 shows the predicted concentration of the active form of the corresponding antigen binding protein without the masking peptide in the systemic circulation after multiple administration events. [Figure 2A] Figure 2 illustrates additional sustained release binding protein constructs with various configurations of anti-target, anti-albumin, and anti-CD3 binding domains. Figure 2A illustrates the intact form after cleavage of the linker, where the linker amino acids remain after protease cleavage (as described herein, these remaining amino acids are referred to as "stubs"). [Figure 2B] Figure 2A illustrates additional sustained release binding protein constructs with various configurations of anti-target, anti-albumin, and anti-CD3 binding domains. Figure 2B illustrates the intact form after cleavage of the linker, where the linker amino acids remain after protease cleavage (as described herein, these remaining amino acids are referred to as "stubs"). [Figure 2C] Figure 2A illustrates additional sustained release binding protein constructs with various configurations of anti-target, anti-albumin, and anti-CD3 binding domains. Figure 2C illustrates the intact form after cleavage of the linker, where the linker amino acids remain after protease cleavage (as described herein, these remaining amino acids are referred to as "stubs"). [Figure 2D] Figure 2A illustrates additional sustained release binding protein constructs with various configurations of anti-target, anti-albumin, and anti-CD3 binding domains. Figure 2D illustrates the active form after cleavage of the linker, where the linker amino acids remain after protease cleavage (as described herein, these remaining amino acids are referred to as "stubs"). [Figure 2E] Figure 2A illustrates additional sustained release binding protein constructs with various configurations of anti-target, anti-albumin, and anti-CD3 binding domains. Figure 2E illustrates the active form after cleavage of the linker, where the linker amino acids remain after protease cleavage (as described herein, these remaining amino acids are referred to as "stubs"). [Figure 2F]Figure 2A illustrates additional sustained release binding protein constructs with various configurations of anti-target, anti-albumin, and anti-CD3 binding domains. Figure 2F illustrates the active form after cleavage of the linker, where the linker amino acids remain after protease cleavage (as described herein, these remaining amino acids are referred to as "stubs"). [Figure 2G] Figure 2A illustrates additional sustained release binding protein constructs with various configurations of anti-target, anti-albumin, and anti-CD3 binding domains. Figure 2G illustrates the intact form after cleavage of the linker, where the linker amino acids remain after protease cleavage (as described herein, these remaining amino acids are referred to as "stubs"). [Figure 2H] Figure 2A illustrates additional sustained release binding protein constructs with various configurations of anti-target, anti-albumin, and anti-CD3 binding domains. Figure 2H illustrates the intact form after cleavage of the linker, where the linker amino acids remain after protease cleavage (as described herein, these remaining amino acids are referred to as "stubs"). [Figure 2I] Figure 2 illustrates additional sustained release binding protein constructs with various configurations of anti-target, anti-albumin, and anti-CD3 binding domains. Figure 2I illustrates the intact form after cleavage of the linker, where the linker amino acids remain after protease cleavage (as described herein, these remaining amino acids are referred to as "stubs"). [Figure 2J] Figure 2A illustrates additional sustained release binding protein constructs with various configurations of anti-target, anti-albumin, and anti-CD3 binding domains. Figure 2J illustrates the active form after cleavage of the linker, where the linker amino acids remain after protease cleavage (as described herein, these remaining amino acids are referred to as "stubs"). [Figure 2K]Figure 2 illustrates additional sustained release binding protein constructs with various configurations of anti-target, anti-albumin, and anti-CD3 binding domains. Figure 2K illustrates the active form after cleavage of the linker, where the linker amino acids remain after protease cleavage (as described herein, these remaining amino acids are referred to as "stubs"). [Figure 2L] Figure 2A illustrates additional sustained release binding protein constructs with various configurations of anti-target, anti-albumin, and anti-CD3 binding domains. Figure 2L illustrates the active form after cleavage of the linker, where the linker amino acids remain after protease cleavage (as described herein, these remaining amino acids are referred to as "stubs"). [Diagram 3] FIG. 1 shows analytical cation exchange chromatography demonstrating the purity of the three PSMA binding proteins. [Figure 4] FIG. 1 demonstrates gradual activation of peptide-masked PSMA-targeted sustained release binding proteins in a TDCC assay as measured in the presence of HSA. [Diagram 5] FIG. 1 demonstrates the masking effect of various peptide masks engineered into PSMA binding proteins, as measured in the presence of HSA. [Figure 6A] FIG. 1 demonstrates the masking effect of various peptide masks engineered into MSLN binding proteins. [Figure 6B] FIG. 1 demonstrates the masking effect of various peptide masks engineered into MSLN binding proteins. [Figure 7] FIG. 1 demonstrates the results of a TDCC assay for various CD19-targeted proteins, measured in the presence of HSA. [Figure 8] FIG. 1 demonstrates the results of a TDCC assay for various FLT3-targeted proteins, measured in the presence of HSA. [Figure 9] FIG. 1 demonstrates the results of a TDCC assay for various FLT3-targeted proteins, measured in the presence of HSA. [Figure 10]FIG. 1 demonstrates the results of a TDCC assay for various CD33-targeting proteins, measured in the presence of HSA. [Figure 11] FIG. 1 demonstrates the results of a TDCC assay for CD19-targeted proteins in T:C:A or T:A:C configurations measured in the presence of HSA. [Figure 12] FIG. 1 demonstrates the results of a TDCC assay for FLT3-targeted proteins in T:C:A or T:A:C configurations, measured in the presence of HSA. [Figure 13A] FIG. 1 illustrates binding of TriTAC-XR non-cleavable prodrug, cleavable prodrug, and active drug to human CD3ε by ELISA. [Figure 13B] FIG. 1 illustrates binding of TriTAC-XR non-cleavable prodrug, cleavable prodrug, and active drug to human T cells as measured by flow cytometry. [Figure 14A] FIG. 1 illustrates the pharmacokinetics of FLT3 TriTAC-XR-L001 in cynomolgus monkeys after a single iv dose of 300 μg / kg. Two assays were used to quantify intact, active, and total amounts of TriTAC-XR. Mean values ​​measured in plasma samples collected from two subjects per dose group are plotted. [Figure 14B] Figure 1 illustrates the pharmacokinetics of FLT3 TriTAC-XR-L001 in cynomolgus monkeys after a single iv dose of 1000 μg / kg. Two assays were used to quantify intact, active, and total amounts of TriTAC-XR. Mean values ​​measured in plasma samples collected from two subjects per dose group are plotted. [Figure 14C] Figure 1 illustrates the pharmacokinetics of FLT3 TriTAC-XR-L085 after a single iv dose of 1000 μg / kg. Two assays were used to quantify intact, active, and total amounts of TriTAC-XR. Mean values ​​measured in plasma samples collected from two subjects per dose group are plotted. [Figure 15]FIG. 1 illustrates the amount of soluble FLT3L present in serum samples collected from cynomolgus monkeys following a single iv dose of FLT3 TriTAC-XR-L001 (SEQ ID NO: 3873) or TriTAC-XR-L085 (SEQ ID NO: 3874). Data are plotted individually for each subject per dose group. [Figure 16] Figure 1 illustrates the amount of FLT3 transcript present in RNA prepared from bone marrow collected from cynomolgus monkeys after a single iv dose of FLT3 TriTAC-XR-L001 (SEQ ID NO: 3873) or TriTAC-XR-L085 (SEQ ID NO: 3874). Technical replicates are plotted. Data is plotted for individual subjects by treatment group. [Figure 17] FIG. 1 illustrates the pharmacokinetics of FLT3 TriTAC-XR-L001 (SEQ ID NO: 3873) or constitutively active FLT3 TriTAC (SEQ ID NO: 3875) in non-human primates (NHPs). [Figure 18] FIG. 1 illustrates the amount of FLT3 transcript present in RNA prepared from bone marrow collected from cynomolgus monkeys after a single iv dose of 1000 μg / kg FLT3 TriTAC-XR-L001 (SEQ ID NO: 3873) or constitutively active FLT3 TriTAC (SEQ ID NO: 3875). Technical replicates are plotted. Data are plotted individually for each subject per treatment group. [Figure 19] FIG. 1 illustrates the predicted pharmacokinetics of repeated administration of TriTAC-XR or TriTAC to NHPs. [Figure 20] FIG. 1 illustrates the empirical pharmacokinetics of repeated administration of CD20 TriTAC-XR to cynomolgus monkeys. [Figure 21A] FIG. 21A illustrates peak cytokine levels (IL-2 in FIG. 21A) following iv administration of 300 and 1000 μg / kg FLT3 TriTAC-XR (FLT3 TriTAC-XR-L001, SEQ ID NO: 3873), or 10, 100, and 1000 μg / kg constitutively active FLT3 TriTAC (SEQ ID NO: 3875). Mean values ​​measured in plasma samples collected from two subjects per treatment group are plotted. [Figure 21B]FIG. 21B illustrates peak cytokine levels (IL-6 in FIG. 21B) following iv administration of 300 and 1000 μg / kg FLT3 TriTAC-XR (FLT3 TriTAC-XR-L001, SEQ ID NO: 3873), or 10, 100, and 1000 μg / kg constitutively active FLT3 TriTAC (SEQ ID NO: 3875). Mean values ​​measured in plasma samples pooled from two subjects per treatment group are plotted. [Figure 22] FIG. 1 illustrates the amount of soluble FLT3L present in serum samples collected from cynomolgus monkeys following iv administration of 300 and 1000 μg / kg FLT3 TriTAC-XR-L001 (SEQ ID NO: 3873), or 10, 100, and 1000 μg / kg constitutively active FLT3 TriTAC (SEQ ID NO: 3875). Mean values ​​measured in serum samples collected from two subjects per treatment group are plotted. [Figure 23] FIG. 1 illustrates the efficacy of FLT3 TriTAC-XR on a mouse tumor model. [Figure 24] FIG. 1 illustrates the pharmacokinetics of CD19 TriTAC-XR or constitutively active CD19 TriTAC in NHPs. [Figure 25A] FIG. 25A illustrates peak cytokine levels (IL-2 in FIG. 25A) following iv administration of CD19 TriTAC-XR or constitutively active CD19 TriTAC. [Figure 25B] FIG. 25B illustrates peak cytokine levels (IL-6 in FIG. 25B) following iv administration of CD19 TriTAC-XR or constitutively active CD19 TriTAC. [Figure 26] FIG. 1 illustrates that target cell depletion is comparable between CD19 TriTAC and CD19 TriTAC-XR. [Figure 27] FIG. 1 illustrates the pharmacokinetics of CD20 TriTAC-XR or constitutively active CD20 TriTAC in NHPs. [Figure 28A]FIG. 28A illustrates peak cytokine levels (IL-2 in FIG. 28A) following iv administration of CD20 TriTAC-XR or constitutively active CD20 TriTAC. [Figure 28B] FIG. 28B illustrates peak cytokine levels (IL-6 in FIG. 28B) following iv administration of CD20 TriTAC-XR or constitutively active CD20 TriTAC. [Figure 29] FIG. 1 illustrates that target cell depletion is comparable between CD20 TriTAC and CD20 TriTAC-XR. [Diagram 30] FIG. 1 illustrates an exemplary construct of a protein without a masking peptide as described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Specific Definitions The terms used herein are intended to describe only the specific cases and are not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Furthermore, terms such as "including," "includes," "having," "has," "with," or variations thereof, to the extent used in either the detailed description and / or the claims, are intended to be inclusive in the same manner as the term "comprising."

[0036] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within one or more standard deviations per run for a given value. When a particular value is described in this application and claims, unless otherwise specified, the term "about" should be assumed to mean an acceptable error range for the particular value.

[0037] The terms "individual," "patient," or "subject" are used interchangeably herein. None of these terms require or are limited to a situation characterized by the supervision (e.g., regular or intermittent) of a medical professional (e.g., a physician, registered nurse, clinical nurse, paramedic, orderly or hospice worker).

[0038] An "antibody" typically refers to a Y-shaped tetrameric protein that contains two polypeptide heavy (H) chains and two light (L) chains held together by covalent disulfide bonds and non-covalent interactions. Human light chains contain a variable domain (VL) and a constant domain (CL), which can be readily classified as kappa or lambda based on amino acid sequence and locus. Each heavy chain contains one variable domain (VH) and one constant region, which in the case of IgG, IgA, and IgD contain domains designated CH1, CH2, and CH3 (IgM and IgE have a fourth domain, CH4). In the IgG, IgA, and IgD classes, the CH1 and CH2 domains are separated by a flexible hinge region, which is a proline- and cysteine-rich segment of variable length (generally about 10 to about 60 amino acids in IgG). The variable domains of both the light and heavy chains are connected to the constant domains by a "J" region of about 12 or more amino acids, with the heavy chains having an additional "D" region of about 10 amino acids. Each class of antibody further contains intrachain and interchain disulfide bonds formed by paired cysteine ​​residues. There are two types of native disulfide bridges or bonds in immunoglobulin molecules: intrachain disulfide bonds and interchain disulfide bonds. The location and number of intrachain disulfide bonds vary among immunoglobulin classes and species. Intrachain disulfide bonds are located on the surface of the immunoglobulin, are solvent accessible, and are usually relatively easy to reduce. There are four intrachain disulfide bonds in the human IgG1 isotype, one from each heavy chain to the light chain and two between heavy chains. Intrachain disulfide bonds do not require chain association. As is well known, the cysteine-rich IgG1 hinge region of the heavy chain is generally maintained as consisting of three parts: the upper hinge, the core hinge, and the lower hinge. Those skilled in the art will appreciate that the IgG1 hinge region contains cysteines in the heavy chain that contain intrachain disulfide bonds (two heavy / heavy, two heavy / light), providing structural flexibility that facilitates the movement of Fab.The intrachain disulfide bond between the light and heavy chains of IgG1 is formed between C214 of the kappa or lambda light chain and C220 in the upper hinge region of the heavy chain. The intrachain disulfide bond between the heavy chains is at positions C226 and C229 (all numbering hereafter is based on the EU index according to Kabat et al.).

[0039] As used herein, the term "antibody" includes polyclonal antibodies, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, deimmunized antibodies, humanized and primatized antibodies, CDR-grafted antibodies, human antibodies, recombinantly produced antibodies, intrabodies, multispecific antibodies, bispecific antibodies, monovalent antibodies (e.g., monovalent IgG), multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies including muteins and variants thereof, immunospecific antibody fragments, such as Single domain antibodies, such as nanobodies or single variable domain antibodies, which contain only one variable domain (VH, VL, or VHH domain), e.g., hcIgG, V-NAR, Fv, Fd, Fab, F(ab')2, F(ab'), Fab2, Fab3 fragments, single chain fragments (e.g., di-scFv, scFv, scFvFc, scFv-zipper, scFab), disulfide-linked Fv (sdFv), Fd fragments consisting of a VH domain and a CH1 domain, linear antibodies, sdAbs, etc. These include "IgG" ("half antibodies"), diabodies, single chain diabodies, tandem diabodies, tandem tri-scFvs, "minibodies", exemplified in some instances by the structures (VH-VL-CH3)2, (scFv-CH3)2, ((scFv)2-CH3+CH3), ((scFv)2-CH3), or (scFv-CH3-scFv)2, multibodies such as triabodies and tetrabodies, and derivatives thereof, including Fc fusions and other modifications, as well as any other immunoreactive molecule so long as it contains a domain that has a binding site for preferential association or binding to the FLT3 protein. Moreover, unless otherwise dictated by contextual constraints, the term further includes all classes of antibodies (i.e., IgA, IgD, IgE, IgG, and IgM), and all subclasses (i.e., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The heavy chain constant domains that correspond to the different classes of antibodies are typically represented by the corresponding lowercase Greek letters α, δ, ε, γ, and μ, respectively.The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on their constant domain amino acid sequences.

[0040] As used herein, "variable region" or "variable domain" refers to the fact that certain portions of the variable domain vary widely in order among antibodies and are used in the binding and specificity of each particular antibody to its particular antigen. However, variability is not evenly distributed throughout the variable domain of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in both the light and heavy chain variable domains. The more highly conserved portions of the variable domain are called frameworks (FRs). Native heavy and light chain variable domains each contain four FR regions that mostly adopt a β-sheet configuration and are connected by three CDRs that form loop connections and sometimes form part of the β-sheet structure. The CDRs of each chain are held together in close proximity by the FR regions, and the CDRs from the other chain contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.In some embodiments, the assignment of amino acids for each domain, framework region, and CDR follows one of the numbering schemes provided by Kabat et al. (1991) Sequences of Proteins of Immunological Interest (5th Ed.), US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, Chothia et al. (1987) PMID: 3681981, Chothia et al. (1989) PMID: 2687698, MacCallum et al. (1996) PMID: 8876650, or Handbook of Therapeutic Antibodies, 3rd Ed., Wily-VCH Verlag GmbH and Co or AbM (Oxford Molecular / MSI Pharmacopeia), edited by Dubel (2007).

[0041] "Numbering of variable domain residues as in Kabat" or "numbering of amino acid positions as in Kabat", and variations thereof, refer to the numbering system used for the heavy or light chain variable domains of the compilation of antibodies in Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) by Kabat et al. Using this numbering system, the actual linear amino acid sequence may include fewer or additional amino acids corresponding to shortening or insertion into the FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat numbering of residues may be determined by alignment of regions of homology in the sequence of the antibody with the "standard" Kabat numbered sequence for a given antibody. It is not intended that the CDRs of this disclosure must conform to the Kabat numbering convention.

[0042] The term "framework" or "FR" residues (or regions) refers to variable domain residues other than the CDR or hypervariable region residues as defined herein. A "human consensus framework" is a framework that represents the amino acid residues most commonly occurring in a selection of human immunoglobulin VL or VH framework sequences.

[0043] The term "epitope" as used herein refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as the paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on the antigen and exhibit different biological effects. Epitopes may be conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes generated by adjacent amino acid residues in a polypeptide chain. In certain circumstances, epitopes may also include portions of sugars, phosphoryl groups, or sulfonyl groups on the antigen.

[0044] As used herein, the term "percent amino acid sequence identity" with respect to a sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular sequence, after aligning the sequences and introducing gaps as necessary, to achieve the maximum percent sequence identity, but not considering conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the scope of those skilled in the art, for example, using publicly available computer software such as EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared. Alignment for the purpose of determining percent amino acid sequence identity can be achieved, for example, using the publicly available sequence comparison computer program ALIGN-2. The source code for the ALIGN-2 sequence comparison computer program is available by user documentation at the U.S. Copyright Office, Washington, DC 20559, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program can be compiled for use on UNIX operating systems, such as Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0045] As used herein, "elimination half-life" is used in its ordinary sense as described in Goodman and Gillman, The Pharmaceutical Basis of Therapeutics 21-25 (Alfred Goodman Gilman, Louis S. Goodman, and Alfred Gilman, eds., 6th ed. 1980). Briefly, the term is intended to encompass a quantitative measurement of the time course of drug elimination. The elimination of most drugs is exponential (i.e., follows first-order kinetics) because the drug concentration usually does not approximate the concentration required for saturation of the elimination process. The rate of an exponential process is determined by the rate constant k, which represents the fractional change per unit of time, or by the half-life t, which is the time required for 50% of the process to be completed. 1 / 2 These two constants have units of time-1 and time, respectively. The first-order reaction constant and the half-life of a reaction are simply related (k × t 1 / 2 = 0.693) and may be exchanged accordingly. First-order elimination kinetics represent the loss of a constant proportion of drug per unit time, so a plot of the logarithm of drug concentration versus time is always linear after the initial distribution phase (i.e., after drug absorption and distribution are complete). Drug elimination half-lives can be accurately determined from such graphs.

[0046] As used herein, the term "binding affinity" refers to the affinity of a protein described in this disclosure to its target, and is expressed numerically using a "Kd" value. When two or more proteins are shown to have comparable binding affinity to their binding targets, the Kd values ​​of the binding of each protein to the binding target are within ±2-fold of each other. When two or more proteins are shown to have comparable binding affinity to a single binding target, the Kd values ​​of the binding of each protein to the single binding target are within ±2-fold of each other. When a protein is shown to bind to two or more targets with comparable binding affinity, the Kd values ​​of the binding of the protein to the two or more targets are within ±2-fold of each other. Generally, a higher Kd value corresponds to weaker binding. In some embodiments, "Kd" is measured by radiolabeled antigen binding assay (RIA) or surface plasmon resonance assay using a BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ). In certain embodiments, the "on rate", "rate of association", "on rate", and "off rate", "rate of dissociation", or "koff" are also determined by surface plasmon resonance technology using a BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ). In additional embodiments, the "Kd", "koff", and "koff" are measured using OCTETR Systems (Pall Life Sciences). In a typical method for measuring binding affinity using OCTET® Systems, a ligand, e.g., in the case of immune cell-binding proteins including FLT3-binding single domain antibodies, biotinylated human or cynomolgus FLT3 is immobilized on the chip surface of an OCTET® streptavidin capillary sensor, where the streptavidin chip is activated using about 20-50 μg / ml of human or cynomolgus FLT3 protein according to the manufacturer's instructions. A PBS / casein solution is also introduced as a blocking agent.In the association kinetics measurement, the variant of FLT3 binding protein is introduced at a concentration ranging from about 10 ng / mL to about 100 μg / mL, about 50 ng / mL to about 5 μg / mL, or about 2 ng / mL to about 20 μg / mL. In some embodiments, the single domain protein that binds to FLT3 is used at a concentration ranging from about 2 ng / mL to about 20 μg / mL. In the case of the negative control, i.e., assay buffer without binding protein, complete dissociation is observed. The kinetic parameters of the binding reaction are then determined using an appropriate tool, for example, ForteBio software.

[0047] As used herein, in some embodiments, "treatment," "treating," or "treated" refers to a therapeutic treatment aimed at slowing (reducing) an undesired physiological disease, disorder, or condition, or to obtain a beneficial or desired clinical outcome. For purposes herein, beneficial or desired clinical outcome includes, but is not limited to, alleviation of symptoms, reduction in the extent of the disease, disorder, or condition, stabilization (i.e., prevention of deterioration) of the disease, disorder, or condition, delay in onset or slowing of progression of the disease, disorder, or condition, remission of the disease, disorder, or condition, and remission, improvement, or amelioration of the disease, disorder, or condition, whether detectable or undetectable. Treatment includes eliciting a clinically significant response without causing excessive levels of side effects. Treatment further includes prolonging survival beyond that expected in the absence of treatment. In other embodiments, "treatment," "treating," or "treated" refers to a preventative measure, the purpose of which is to delay the onset of or reduce the severity of an unwanted physiological disease, disorder, or condition, such as in an individual who is predisposed to the condition (e.g., an individual who carries a genetic marker for a disease such as breast cancer).

[0048] In general, it should be noted that the term single domain antibody as used herein in its broad sense is not limited to a particular biological source or a particular preparation method. A single domain antibody is an antibody whose complementarity determining region is part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally lacking light chains, single domain antibodies derived from traditional four-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. A single domain antibody can be any of the art or any future single domain antibody. A single domain antibody can be derived from any species, including, but not limited to, mouse, human, camel, llama, goat, rabbit, cow. For example, in some embodiments, a single domain antibody of the present disclosure is obtained by: (1) isolating a VHH domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) by "humanizing" a naturally occurring VHH domain or by expression of a nucleic acid encoding such a humanized VHH domain; (4) by "camelizing" a naturally occurring VH domain from any animal species, in particular a mammalian species such as human, or by expression of a nucleic acid encoding such a camelized VH domain; (5) by "camelizing" a "domain antibody" or "Dab" or by expression of a nucleic acid encoding such a camelized VH domain; (6) by using synthetic or semi-synthetic techniques to prepare proteins, polypeptides or other amino acid sequences; (7) by preparing a nucleic acid encoding a single domain antibody using techniques for nucleic acid synthesis well known in the art, followed by expressing the resulting nucleic acid; and / or (8) by any combination of one or more of the foregoing.

[0049] In some embodiments, the immune cell-binding proteins described herein include antibodies, such as single domain antibodies targeting antigens such as PSMA, MSLN, BCMA, DLL3, EGFR, EpCAM, FLT3, that comprise an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain, but that has not been "humanized" by replacing one or more amino acid residues in the amino acid sequence (specifically in the framework sequence) of the aforementioned naturally occurring VHH sequence by one or more of the amino acid residues that occur at the corresponding positions in the VH domain from a conventional four-chain antibody of human origin (e.g. as shown above). This can be done as known in the art and will be clear to the skilled person, for example from the further description herein. Again, it should be noted that such humanized single domain antibodies of the present disclosure are obtained by any suitable method known per se (e.g. as shown in (1) to (8) above), and thus are not strictly limited to polypeptides obtained using a polypeptide comprising a naturally occurring VHH domain as starting material. In some additional embodiments, the single domain antibodies described herein include single domain antibodies having an amino acid sequence which corresponds to that of a naturally occurring VH domain, but which has not been "camelized", e.g. by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VH domain from a conventional four-chain antibody by one or more of the amino acid residues occurring at the corresponding positions in the VHH domain of a heavy chain antibody. Such "camelized" substitutions are preferably inserted at amino acid positions which occur and / or are present at the VH-VL interface and / or at the so-called Camelidae hallmark residues (see, e.g., WO 94 / 04678, Davies and Riechmann (1994, 1996)). Preferably, the VH sequence used as starting material or starting point for generating or designing a camelized single domain is a mammalian VH sequence, more preferably a human VH sequence, such as a VH3 sequence.However, it should be noted that such camelized anti-MSLN single domain antibodies of the present disclosure are obtained in certain embodiments (i.e., as shown in (1)-(8) above) by any suitable method known in the art, and thus are not strictly limited to polypeptides obtained using a naturally occurring VH domain-containing polypeptide as starting material. For example, as described further herein, both "humanization" and "camelization" are performed by providing a nucleotide sequence encoding a naturally occurring VHH domain or VH domain, respectively, and then modifying one or more codons in said nucleotide sequence such that the new nucleotide sequence encodes a "humanized" or "camelized" single domain antibody, respectively. This nucleic acid can then be expressed to provide the desired single domain antibody of the present disclosure. Alternatively, in other embodiments, based on the amino acid sequence of a naturally occurring VHH domain or VH domain, respectively, the amino acid sequence of the desired humanized or camelized single domain antibody of the present disclosure is designed and synthesized de novo using known peptide synthesis techniques. In some embodiments, based on the amino acid or nucleotide sequence of a naturally occurring VHH domain or VH domain, respectively, a nucleic acid sequence encoding the desired humanized or camelized single domain antibody of the present disclosure is designed and synthesized de novo using known nucleic acid synthesis techniques, and then the nucleic acid thus obtained is expressed using known expression techniques to yield the desired single domain antibody of the present disclosure.

[0050] The term "patient" or "subject" refers to any subject for whom treatment is desired or who is participating in or used as a control in a clinical trial or epidemiological study, including humans, and mammalian patients such as bovine, equine, canine, feline, etc.

[0051] The term "therapeutic index" refers to the TD determined from a quantitative dose-response curve. 50 (or L.D. 50 ) and ED 50 This is the ratio of ED 50is the effective dose of a drug for 50% of subjects. TD 50 is the toxic dose of a drug for 50% of human subjects. LD 50 is the lethal dose of a drug for 50% of an animal population. Therapeutic index demonstrates a quantitative measure of the relative safety of a drug. A higher therapeutic index is preferable to a lower one. To reach the toxic threshold, a patient must take a much higher dose of such a drug than would be taken to elicit a therapeutic effect.

[0052] The term "substantially cleaved" means that greater than 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the molecule is cleaved.

[0053] Sustained Release Binding Proteins Described herein is a pharmaceutical composition comprising a sustained release binding protein comprising an antigen binding protein / immune cell binding protein, a masking peptide, and a cleavable linker, where after administration to a subject, the sustained release binding protein slowly releases an active form of the sustained release binding protein after the cleavable linker is cleaved in the systemic circulation.

[0054] Without being bound by any theory, the masking peptide, when connected to the antigen binding protein / immune cell binding protein via a cleavable linker, confers sustained release properties to the antigen binding protein / immune cell binding protein. The extended binding protein is converted to the active form by the gradual cleavage of the cleavable linker, thereby removing the masking effect of the masking peptide and releasing the active form. The gradual release of the active form from the masked form allows the concentration of the active form to be maintained constant over an extended period of time compared to direct administration of the unmasked form. It is contemplated that the sustained release binding protein has a higher therapeutic index than the corresponding antigen binding protein without the masking peptide. Figure 1C shows the predicted concentrations of the intact and active forms in the systemic circulation after multiple administration events. Figure 1D shows the predicted concentrations of the active form of the corresponding antigen binding protein without the masking peptide in the systemic circulation after multiple administration events. The concentration of the active form of the sustained release binding protein is relatively constant compared to the concentration of the active form of the corresponding antigen binding protein without the masking peptide.

[0055] Cytokine release syndrome (CRS) is the major dose-limiting toxicity, and it is hypothesized that there are two factors that contribute to CRS: 1) immediate exposure to high concentrations of active drug, and 2) exposure of active drug that greatly exceeds Cmin. The sustained release concept addresses both the problem of an initial slow release of active drug and by minimizing the difference between Cmax and Cmin. A relatively constant concentration of active drug in serum is expected to result in a relatively constant drug exposure to tissues. Thus, administration of a sustained release binding protein results in lower levels of CRS than those observed when the corresponding antigen binding protein without the masking peptide is administered.

[0056] In some embodiments, the masking peptide is linked to the N-terminus or C-terminus of the antigen binding protein / immune cell binding protein via a cleavable linker. Various exemplary configurations are provided in Figures 2A-2L. In some embodiments, the masking peptide is linked to the N-terminus of the domain that specifically binds to HSA, and the domain that specifically binds to HSA is connected to the target antigen binding domain or the T cell binding domain via its C-terminus (e.g., via a linker). In some embodiments, the masking peptide is linked to the N-terminus of the target antigen binding domain, and the target antigen binding domain is connected to the domain that specifically binds to HSA, or the T cell binding domain via its C-terminus (e.g., via a linker). In some embodiments, the masking peptide is linked to the C-terminus of the target antigen binding domain, and the target antigen binding domain is connected to the domain that specifically binds to HSA, or the T cell binding domain via its N-terminus (e.g., via a linker). In some embodiments, the masking peptide is linked to the N-terminus of the T cell binding domain. In some embodiments, the masking peptide is linked to the C-terminus of the T cell binding domain. In some embodiments, the masking peptide is linked to the VH or VL domain of a T cell binding domain that is a single chain variable fragment (e.g., an scFv specific for human CD3, such as CD3ε). masking peptide-cleavable linker-target antigen binding domain-serum albumin binding domain-T cell binding domain, masking peptide-cleavable linker-target antigen binding domain-T cell binding domain-serum albumin binding domain, masking peptide-cleavable linker-T cell binding domain-serum albumin binding domain-target antigen binding domain, masking peptide-cleavable linker-T cell binding domain-target antigen binding domain-serum albumin binding domain, masking peptide-cleavable linker-serum albumin binding domain-T cell binding domain-target antigen binding domain, masking peptide-cleavable linker-serum albumin binding domain-target antigen binding domain-T cell binding domain, target antigen binding domain-serum albumin binding domain-T cell binding domain-cleavable linker-masking peptide, target antigen binding domain-T cell binding domain-serum albumin binding domain-cleavable linker-masking peptide, T cell binding domain-serum albumin binding domain-target antigen binding domain-cleavable linker-masking peptide, T cell binding domain-target antigen binding domain-serum albumin binding domain-cleavable linker-masking peptide, Serum albumin binding domain-T cell binding domain-target antigen binding domain-cleavable linker-masking peptide, Serum albumin binding domain-target antigen binding domain-T cell binding domain-cleavable linker-masking peptide Sustained release binding proteins include, but are not limited to,

[0057] In some embodiments, the antigen binding proteins / sustained release immune cell binding proteins of the disclosure comprise an amino acid sequence that is at least about 60% to about 100% identical to the sequence of SEQ ID NOs: 3600, 3824-3858, or 3882.

[0058] Immune cell-binding proteins In some embodiments herein, a pharmaceutical composition is described that includes an immune cell-binding protein, a masking peptide, and a sustained release binding protein that includes a cleavable linker. In some embodiments, the immune cell-binding protein is a half-life extended protein. In some embodiments, the masking peptide is covalently attached to the N-terminus or C-terminus of the half-life extended immune cell-binding protein via a cleavable linker. In some embodiments, the cleavable linker is substantially cleaved in the systemic circulation. In some embodiments, the immune cell-binding protein is a bispecific protein, a trispecific protein, or a multispecific protein. In some embodiments, the immune cell-binding protein includes a domain that binds to a bulk serum protein, such as serum albumin. In some embodiments, the serum albumin is human serum albumin (HSA).

[0059] In some embodiments, the sustained release binding protein has a higher therapeutic index than the corresponding half-life extended immune cell binding protein without the masking peptide. In some embodiments, administration of the sustained release binding protein results in a lower Cmax / Cmin ratio of the active form of the sustained release binding protein in the systemic circulation than the Cmax / Cmin ratio when the corresponding half-life extended immune cell binding protein without the masking peptide is administered. In some embodiments, multiple administrations of the sustained release binding protein result in an incremental increase in the level of the active form of the sustained release binding protein in the systemic circulation than when the corresponding half-life extended immune cell binding protein without the masking peptide is administered. In some embodiments, administration of the sustained release binding protein results in a lower level of cytokine release syndrome (CRS) than observed when the corresponding half-life extended immune cell binding protein without the masking peptide is administered.

[0060] In some embodiments, the immune cell binding protein comprises an immune cell binding domain. In some embodiments, "immune cell binding domain" as used herein refers to one or more binding specificities that bind to and / or activate immune cells, e.g., cells involved in an immune response. In some embodiments, the immune cells are selected from natural killer (NK) cells, B cells, dendritic cells, macrophage cells. In some embodiments, the immune cell binding domain is an antibody or antigen-binding fragment thereof, a receptor molecule (e.g., a full-length receptor, a receptor fragment, or a fusion thereof (e.g., a receptor-Fc fusion)), or a ligand molecule (e.g., a full-length ligand, a ligand fragment, or a fusion thereof (e.g., a ligand-Fc fusion)) that binds to an immune cell antigen (e.g., an NK cell antigen, a B cell antigen, a dendritic cell antigen, and / or a macrophage cell antigen). In some embodiments, the immune cell binding domain specifically binds to a target immune cell, e.g., preferentially binds to a target immune cell. For example, in some embodiments, the immune cell binding domain is an antibody or antigen-binding fragment thereof that binds to an immune cell antigen (e.g., an NK cell antigen, a B cell antigen, a dendritic cell antigen, and / or a macrophage cell antigen) with a dissociation constant of less than about 10 nM.

[0061] In some embodiments, the immune cell binding domain comprises a NK cell binding domain, a T cell binding domain, a B cell binding domain, a dendritic cell binding domain, a macrophage cell binding domain, or a combination thereof. In some embodiments, the immune cell binding domain comprises a T cell binding domain. In some embodiments, the T cell binding domain binds to a CD3 molecule. In some embodiments, the CD3 molecule is at least one of a CD3 gamma molecule, a CD3 delta molecule, or a CD3 epsilon molecule.

[0062] In some embodiments, the immune cell binding protein comprises an antigen binding domain. In some examples, the antigen binding domain comprises an antibody, a single chain antibody, a Fab, an Fv, a T cell receptor binding domain, a ligand binding domain, a receptor binding domain, a domain antibody, a single domain antibody, a minibody, a nanobody, a peptibody, or various other antibody mimetics (affimers, affitins, alphabodies, atrimers, CTLA4-based molecules, adnectins, anticalins, Kunitz domain-based proteins, avimers, knottins, finomers, darpins, affibodies, affilins, monobodies, and armadillo repeat protein-based proteins).

[0063] In some embodiments, the target antigen is a tumor antigen. In some embodiments, the antigen is CD19 (B-lymphocyte antigen CD19, B-lymphocyte surface antigen B4, T-cell surface antigen Leu-12, CVID3), PSMA (prostate-specific membrane antigen), MSLN (mesothelin), BCMA (B-cell maturation antigen), DLL3 (delta-like ligand 3), EGFR (epidermal growth factor receptor), FLT3 (FMS-like tyrosine kinase 3), CD20 (B-lymphocyte antigen CD20, MS4A1, B1, Bp35, CVID5, LEU-16, MS4A2, S7, transmembrane 4-domain A1), CD 22 (SIGLEC-2, SIGLEC2), CD25 (IL2RA, interleukin-2 receptor α chain), CD27 (S152, S152.LPFS2, T14, TNFRSF7, Tp55), CD30 (TNFRSF8), CD33 (Siglec-3, sialic acid-binding Ig-like lectin 3, SIGLEC3, SIGLEC-3, gp67, p67), CD37 (GP52-40, TSPAN26), CD38 (cyclic ADP ribose hydrolase, ADPRC1, ADPRC 1), CD40 (Bp50, CDW40, TNFRSF5, p50), CD44 (HCAM, homing cell adhesion molecule), Pgp-1 (phagocyte glycoprotein-1), Hermes antigen, lymphocyte homing receptor, ECM-III, and HUTCH-1), CD48 (BLAST-1, B lymphocyte activation marker, SLAMF2, signaling lymphocyte activation molecule 2), CD52 (CAMPATH-1 antigen), CD70, CD73 (NT5E, ecto-5'-nucleotidase), CD39 (ENTPD1, ​​ectonucleoside triphosphate diphosphohydrolase-1), CD74 (HLA class II histocompatibility antigen gamma chain, HLA-DR antigen-related invariant chain), CD79b (immunoglobulin-associated beta), CD80 (B7-1), CD86 (B7-2), CD123 (IL3RA, interleukin-3 receptor), CD133 (PROM1), CD137 (inducible upon lymphocyte activation, TNFRSF9, tumor necrosis factor receptor superfamily member 9, 4-1BB, ILA), CD138 (SDC1), alpha-fetoprotein (AFP), c-Met, c-Kit, CD371 (CLEC12A, C-type lectin domain family 12 member A, CLL1)),CD370 (CLEC9A, C-type lectin domain-containing 9A), cadherin 3 (CDH3, p-cadherin, PCAD), carbonic anhydrase 6 (CA6), carbonic anhydrase 9 (CA9, CAIX), carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), CD66c (CEACAM6, carcinoembryonic antigen-related cell adhesion molecule 6), chorionic somatomammotropic hormone 1 (CSH1, CS1), coagulation factor III, tissue factor (F3, TF), collectin subfamily member 10 (COLEC10), delta-like canonical Notch ligand 3 (DLL3), ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3), ephri EFNA1, epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), EPH receptor A2 (EPHA2), epithelial cell adhesion molecule (EpCAM), erb-b2 receptor tyrosine kinase 2 (ERBB2, HER2), fibroblast activation protein alpha (FAP), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), folate hydrolase 1 (FOLH1, PSMA), folate receptor 1 (FOLR1, FRa), GD2 ganglioside, glycoprotein NMB (GPNMB, osteoactivin), guanylate cyclase 2C (GUCY2C, GCC), human papillomavirus (HPV) E6, HPV E7, major histocompatibility complex (MHC) class I-presented neoantigen, major histocompatibility complex (MHC) class II-presented neoantigen, major histocompatibility complex, class I, E (HLA-E), major histocompatibility complex, class I, F (HLA-F), major histocompatibility complex, class I, G (HLA-G, MHC-G), integrin subunit beta 7 (ITGB7), leukocyte immunoglobulin-like receptor B1 (LILRB1, ILT2), leukocyte immunoglobulin-like receptor B2 (LILRB2, ILT4), LY6 / PLAUR domain-containing 3 (LYPD3, C4.4A), glypican 3 (GPC3), KRAS proto-oncogene, GTPase (KRAS), MAGE family member A1 (MAGEA1), MAGE family member A3 (MAGEA3), MAGE family member A4 (MAGEA4),MAGE family member A11 (MAGEA11), MAGE family member C1 (MAGEC1), MAGE family member C2 (MAGEC2), MAGE family member D1 (MAGED1), MAGE family member D2 (MAGED2), mesothelin (MSLN), mucin 1 (MUC1) and its splice variants (e.g., MUC1 / C, D, and Z), mucin 16 (MUC16), necdin (NDN), nectin cell adhesion molecule 4 (NECTIN4), SLIT and NTRK-like family 6 (SLITRK6), promyelocytic leukemia (PML, TRIM19), protein tyrosine kinase 7 (inactive) (PTK7), CD352 (SLAMF6, SLAM family member 6), CD319 (SLAMF7, SLAM family member 7, 19A, CRACC, CS1), sialic acid-binding Ig-like lectin 7 (SIGLEC7), sialic acid-binding Ig-like lectin 9 (SIGLEC9), solute carrier phagocytosis Myristyl 34 (sodium phosphate) member 2 (SLC34A2), solute carrier family 39 member 6 (SLC39A6, LIV1), STEAP family member 1 (STEAP1), STEAP family member 2 (STEAP2), CD134 (TNFRSF4, TNF receptor superfamily member 4, OX40), CD137L (TNFSF9, TNF superfamily member 9, 4-1BB-L), CD261 (TNFRSF10A, TNF receptor Receptor superfamily member 10a, DR4, TRAILR1), CD262 (TNFRSF10B, TNF receptor superfamily member 10b, DR5, TRAILR2), CD267 (TNFRSF13B, TNF receptor superfamily member 13B, TACI, IGAD2), CD269 (TNFRSF17, TNF receptor superfamily member 17, BCMA), CD357 (TNFRSF18, TNF receptor superfamily member 18 GITR), transferrin (TF), transforming growth factor beta 1 (TGFB1), trophoblast glycoprotein (TPBG, 5T4), trophinin (TRO, MAGED3), tumor-associated calcium signal transducer 2 (TACSTD2, TROP2, EGP1), fucosyl-GM1,In some embodiments, the immune cell-associated protein specifically binds to PSMA, MSLN, BCMA, DLL3, EGFR, FLT3, or EpCAM.

[0064] In some embodiments herein, an immune cell binding protein is described that includes a first domain (A) that is a T cell binding domain and specifically binds human CD3, a second domain (B) that specifically binds human serum albumin (HSA), and a third domain (C) that specifically binds a target antigen. In some embodiments, the domains of the immune cell binding protein are linked in one of the following orders: H2N-(C)-(B)-(A)-COOH, H2N-(A)-(B)-(C)-COOH, H2N-(B)-(A)-(C)-COOH, H2N-(C)-(A)-(B)-COOH, H2N-(A)-(C)-(B)-COOH, H2N-(B)-(C)-(A)-COOH, or linked by linkers L1 and L2. and L2 in one of the following orders: H2N-(C)-L1-(B)-L2(A)-COOH, H2N-(A)-L1-(B)-L2-(C)-COOH, H2N-(B)-L1-(A)-L2-(C)-COOH, H2N-(C)-L1-(A)-L2-(B)-COOH, H2N-(A)-L1-(C)-L2(B)-COOH, H2N-(B)-L1-(C)-L2-(A)-COOH. In some embodiments, linkers L1 and L2 are each independently selected from the amino acid sequences of SEQ ID NOs: 3190-3200.

[0065] The design of the immune cell binding proteins described herein allows the binding domain for the target antigen, i.e. the third domain (C) described above, to be flexible in that it can be any type of binding domain, including but not limited to domains derived from monoclonal, polyclonal, recombinant, human or humanized antibodies. In some embodiments, the binding domain for the target antigen is a single domain antibody, such as a single chain variable fragment (scFv), a heavy chain variable domain (VH), a light chain variable domain (VL), a variable domain of a llama-derived sdAb (VHH), or a variable domain of a camelid-derived single domain antibody (VHH). In other embodiments, the binding domain for the target antigen is a non-Ig binding domain, e.g. an antibody mimic, such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, finomers, kunitz domain peptides, and monobodies. In further embodiments, the binding domain for the target antigen is a ligand or a protein that binds to or associates with the target antigen. In yet further embodiments, the binding domain for the target antigen is a knottin.In yet further embodiments, the binding domain for the target antigen is a small molecule entity.

[0066] In some embodiments, the target antigen binding domain is an antibody or an antibody variant. As used herein, the term "antibody variant" refers to variants and derivatives of the antibodies described herein. In certain embodiments, amino acid sequence variants of the antibodies described herein are contemplated. For example, in certain embodiments, the amino acid sequence variants described herein are contemplated to improve the binding affinity and / or other biological properties of the antibody. Typical methods for preparing amino acid variants include, but are not limited to, introducing appropriate modifications into the nucleotide sequence encoding the antibody or peptide synthesis. Such modifications include, for example, deletions from, insertions into, and / or substitutions of residues within the amino acid sequence of the antibody.

[0067] Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct has the desired characteristics, such as antigen binding. In certain embodiments, antibody variants are provided with one or more amino acid substitutions. Sites of interest for substitution mutagenesis include CDR and framework regions. Examples of such substitutions are described below. Amino acid substitutions can be introduced into the antibody of interest and the products screened for the desired activity, such as retaining / improving antigen binding, reducing immunogenicity, or improving T-cell-mediated cytotoxicity (TDCC). Both conservative and non-conservative amino acid substitutions are contemplated for the preparation of antibody variants.

[0068] Another example of a substitution to create a variant antibody is to replace one or more hypervariable regions of a parent antibody. In general, variants are then selected based on the desired improved properties compared to the parent antibody, such as increased affinity, decreased affinity, decreased immunogenicity, or increased pH-dependence of binding.

[0069] In one embodiment, the single domain antibody corresponds to the VHH domain of a naturally occurring heavy chain antibody directed against the target antigen. As further described herein, such a VHH sequence can generally be generated or obtained by suitable immunization of a llama species with the target antigen (i.e. to raise an immune response and / or heavy chain antibodies directed against the target antigen), by obtaining a suitable biological sample from said llama (such as a blood sample, a serum sample or a B cell sample) and by generating the VHH sequence directed against the target antigen starting from said antibody using any suitable technique known in the art.

[0070] In another embodiment, such naturally occurring VHH domains against the target antigen are obtained from a naive library of camelid VHH sequences, for example by screening such a library with the target antigen, at least a part, fragment, antigenic determinant or epitope thereof, using one or more screening techniques known in the art. Such libraries and techniques are described, for example, in WO 99 / 37681, WO 01 / 90190, WO 03 / 025020 and WO 03 / 035694. Alternatively, improved synthetic or semi-synthetic libraries derived from naive VHH libraries are used, including VHH libraries obtained from naive VHH libraries by techniques such as random mutagenesis and / or CDR shuffling, as described, for example, in WO 00 / 43507.

[0071] In a further embodiment, another technique for obtaining VHH sequences directed against a target antigen comprises suitably immunizing a transgenic mammal capable of expressing heavy chain antibodies (i.e. to raise an immune response and / or heavy chain antibodies directed against the target antigen), obtaining a suitable biological sample from this transgenic mammal (such as a blood sample, a serum sample or a B cell sample) and generating VHH sequences directed against the target antigen starting from the previous antibody using any suitable technique known in the art. For this purpose, for example rats or mice expressing heavy chain antibodies can be used, as well as the methods and techniques described in WO 02 / 085945 and WO 04 / 049794.

[0072] In some embodiments, single domain antibodies of immune cell binding proteins specific for a target antigen include antibodies, e.g. single domain antibodies, that have an amino acid sequence which corresponds to the amino acid sequence of a naturally occurring VHH domain, but which have not been "humanized" by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH sequence (particularly in the framework sequences) by one or more of the amino acid residues that occur at the corresponding positions in a VH domain from a conventional four-chain antibody of human origin (e.g. as shown above).

[0073] Other suitable methods and techniques for obtaining a target antigen-binding single domain antibody of the present disclosure, and / or a nucleic acid encoding same, starting from a naturally occurring VH or VHH sequence, include, for example, obtaining a target antigen-binding single domain antibody of the present disclosure, or a nucleotide sequence or nucleic acid encoding same, by suitable combination of one or more naturally occurring VH sequences (such as one or more framework (FR) sequences and / or complementarity determining regions (CDRs)), one or more parts of one or more naturally occurring VHH sequences (one or more FR sequences or CDR sequences), and / or one or more synthetic or semi-synthetic sequences.

[0074] In some embodiments, the target antigen binding domain is an antibody comprising heavy chain variable complementarity determining region CDR1, heavy chain variable CDR2, heavy chain variable CDR3, light chain variable CDR1, light chain variable CDR2, and light chain variable CDR3. In some embodiments, examples of target antigen binding domains include, but are not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, humanized antibodies, humanized antibodies, or antigen binding fragments such as single domain antibodies (sdAb), Fab, Fab', F(ab)2, and Fv fragments, fragments composed of one or more CDRs, single chain antibodies (e.g., single chain Fv fragments (scFv)), disulfide stabilized (dsFv) Fv fragments, heteroconjugate antibodies (e.g., bispecific antibodies), pFv fragments, heavy chain monomers or dimers, light chain monomers or dimers, and dimers composed of one heavy chain and one light chain. In some embodiments, the target antigen binding domain is a single domain antibody. In some embodiments, a single domain antibody comprises heavy chain variable complementarity determining regions (CDRs): CDR1, CDR2, and CDR3.

[0075] In some embodiments, the immune cell-binding protein comprising the target antigen-binding domain specifically binds to a target with equal or greater affinity compared to the immune cell-binding protein comprising a reference target antigen-binding domain, and the target antigen-binding domain in such embodiments is derived from a parent molecule comprising an affinity-matured target antigen-binding domain and comprising one or more amino acid mutations (e.g., stabilizing or destabilizing mutations) relative to the parent molecule. In some embodiments, the affinity-matured target antigen-binding molecule has greater stability against a selected destabilizing agent than the reference parent molecule specific for the same antigen. In some embodiments, the affinity-matured target antigen-binding molecule is identified in a process comprising panning against a target protein one or more pre-candidate target antigen-binding molecules expressed in a library derived from one or more parent molecules. The pre-candidate target antigen-binding molecule, in some embodiments, comprises amino acid substitutions in the variable region, CDR, or framework residues compared to the parent molecule.

[0076] As used herein, "phage display" refers to a technique in which variant polypeptides are displayed as fusion proteins to at least a portion of a coat protein on the surface of a phage, e.g., a filamentous phage particle. The utility of phage display lies in the fact that a large library of randomized protein variants can be rapidly and efficiently selected for sequences that bind to a target molecule with high affinity. Display of peptide and protein libraries on phage has been used to screen millions of polypeptides for those with specific binding properties. Multivalent phage display methods have been used to display small random peptides and small proteins by fusion to either gene III or gene VIII of filamentous phage. See, for example, Wells and Lowman, Curr. Opin. Struct. Biol, 3:355-362 (1992), and references cited therein. In monovalent phage display, a protein or peptide library is fused to gene III or a portion thereof and expressed at low levels in the presence of wild-type gene III protein such that the phage particle displays one or no copy of the fusion protein. Avidity effects are reduced compared to polyvalent phage, as selection is based on intrinsic ligand affinity and phagemid vectors are used that simplify DNA manipulations. See, e.g., Lowman and Wells, Methods: A companion to Methods in Enzymology, 3:205-0216 (1991).

[0077] In some embodiments, panning includes using different binding times and concentrations to identify target antigen-binding molecules with increased or decreased on-rates from the preliminary candidate target antigen-binding molecules. In some embodiments, panning includes using different washing times to identify target antigen-binding molecules with increased or decreased off-rates from the preliminary candidate target antigen-binding molecules. In some embodiments, panning includes using both different binding times and different washing times. In some embodiments, one or more stabilizing mutations are combined to increase the stability of the affinity-matured target antigen-binding molecules, for example, by shuffling such mutations to create a second combinatorial library, and performing a second round of panning followed by binding selection.

[0078] In some embodiments, the affinity matured target antigen binding molecule comprises an affinity for a target antigen protein (such as a human target antigen protein) that is equal to or greater than that of the target antigen binding parent molecule, but has reduced or, in some embodiments, increased cross-reactivity to selected substances, such as ligands, proteins, antigens, other than the target antigen epitope for which the target antigen binding parent molecule is specific or designed to be specific. Regarding the latter, the affinity matured target antigen binding molecule is, in some embodiments, more successfully tested against animal models when it is reacted with both a human target and a corresponding animal model target, such as a mouse target or a cynomolgus target. In some embodiments, the parent target antigen binding molecule binds to the human target antigen with an affinity of about 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 50 nM or less, 10 nM or less, and to the cynomolgus EpCAM with an affinity of about 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 50 nM or less, 15 nM or less, 10 nM or less. In some embodiments, the affinity matured target antigen binding molecule identified after the first round of panning binds to the human target antigen with an affinity of about 5 nM or less, such as 1 nM or less, and to the cynomolgus target antigen with an affinity of about 7.5 nM or less, such as 1 nM or less. In some embodiments, the affinity matured target antigen binding molecule identified after the second round of panning binds to the human target antigen with an affinity of 2.5 nM or less, and to the cynomolgus target antigen with an affinity of about 3.5 nM or less.

[0079] In some embodiments, the domains of the immune cell binding protein are linked by one or more internal linkers. In certain embodiments, the internal linker is "short", i.e., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues. Thus, in certain examples, the internal linker is about 12 or less amino acid residues. If there are 0 amino acid residues, the internal linker is a peptide bond. In certain embodiments, the internal linker is "long", i.e., 15-25 amino acid residues. In some embodiments, the internal linker is about 3 to about 15, e.g., 8, 9, or 10 adjacent amino acid residues. With regard to the amino acid composition of the internal linker, peptides are selected that have properties that provide flexibility to the target antigen binding protein and do not interfere with the binding domain or resist cleavage from proteases. For example, glycine and serine residues generally provide protease resistance. Examples of internal linkers suitable for linking domains in the target antigen binding protein include (GS) n (SEQ ID NO: 3859), (GGS) n (SEQ ID NO: 3860), (GGGS) n (SEQ ID NO:3861), (GGSG) n (SEQ ID NO: 3862), (GGSGG) n (SEQ ID NO: 3863), (GGGGS) n (SEQ ID NO:3864), (GGGGG) n (SEQ ID NO: 3865), or (GGG) n (SEQ ID NO:3866), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the linker is (GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO:3867), (GGGGSGGGGSGGGGS) (SEQ ID NO:3868), LPETG (SEQ ID NO:3869), (GGGGSGGGS) (SEQ ID NO:3871), or SGGG (SEQ ID NO:3872).

[0080] In some embodiments, the immune cell binding proteins described herein include polypeptides having sequences set forth in SEQ ID NOs: 3218-3462 and subsequences thereof. In some embodiments, the immune cell binding proteins include polypeptides having at least 70%-95% or more identity to sequences set forth in SEQ ID NOs: 3218-3462. In some embodiments, the immune cell binding proteins include polypeptides having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to sequences set forth in SEQ ID NOs: 3218-3462. In some embodiments, the immune cell binding proteins have sequences comprising at least a portion of sequences set forth in SEQ ID NOs: 3218-3462. In some embodiments, the immune cell binding proteins include polypeptides comprising one or more of the sequences set forth in SEQ ID NOs: 3218-3462.

[0081] In some embodiments, the immune cell binding proteins described herein include polypeptides having sequences set forth in SEQ ID NOs: 3255, 3340, 3376, and 3462, as well as subsequences thereof. In some embodiments, the immune cell binding proteins include polypeptides having at least 70% to 95% or more identity to the sequences set forth in SEQ ID NOs: 3255, 3340, 3376, and 3462. In some embodiments, the immune cell binding proteins include polypeptides having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to the sequences set forth in SEQ ID NOs: 3255, 3340, 3376, and 3462. In some embodiments, the immune cell binding proteins have sequences comprising at least a portion of the sequences set forth in SEQ ID NOs: 3255, 3340, 3376, and 3462. In some embodiments, the immune cell binding protein comprises a polypeptide comprising one or more of the sequences set forth in SEQ ID NOs: 3255, 3340, 3376, and 3462.

[0082] CD19-binding domain Described herein are immune cell-binding proteins that contain a CD19-binding domain as a target antigen-binding protein, pharmaceutical compositions thereof, and nucleic acids, recombinant expression vectors, and host cells for producing such proteins. Also provided are methods of using the proteins that contain the CD19-binding domain of the present disclosure in the prevention and / or treatment of diseases, illnesses, and disorders.

[0083] The CD19 binding domain is specific for CD19 expressed on cell surface in some embodiments. The anti-CD19 antibody disclosed herein in some examples shows higher binding affinity to CD19 (e.g., cell surface CD19), higher stability, and / or binds to a different CD19 epitope compared to anti-CD19 scFv FMC63. CD19 is a 95 kDa transmembrane glycoprotein that is expressed mainly on B lineage cells and follicular dendritic cells. It is a member of the immunoglobulin superfamily. CD19 molecules from various species are well known in the art. For example, the amino acid sequence of human CD19 can be found under GenBank accession number AAA69966. CD19 plays an essential role in B cell malignancies and autoimmunity. It has been reported that CD19 is expressed on the surface of cancer cells in 90% of patients with acute lymphoblastic leukemia (ALL), as well as on cancer cells from patients with B-cell non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Therefore, CD19 is considered as a promising target in the immunotherapy of B-cell lineage cancers. See, for example, Stanciu-Herrera et al., Leuk Res. 2008, 32:625-32, and Le Gall et al., FEBS Lett. 1999, 453:164-8.

[0084] In some embodiments, the CD19 binding domain comprises a sequence that is at least about 70% to about 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 3771-3792. In some embodiments, the CD19 binding domain comprises a sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 3771-3792. In some embodiments, the CD19 binding domain comprises a sequence selected from the group consisting of SEQ ID NOs: 3771-3792.

[0085] CD20 binding domain Described herein are immune cell binding proteins that contain a CD20 binding domain as a target antigen binding protein, pharmaceutical compositions thereof, and nucleic acids, recombinant expression vectors, and host cells for producing such proteins. Also provided are methods of using the proteins that contain the CD20 binding domain of the present disclosure in the prevention and / or treatment of diseases, illnesses, and disorders.

[0086] The CD20 molecule is a non-glycosylated phosphoprotein that is specifically labeled on the surface of human lymphocyte subpopulations (B cells). It consists of 297 amino acids with a molecular weight of 33-37 kDa and is expressed on the surface of more than 95% of B cells. CD20 molecules are present on both normal B cells and malignant cells, and are expressed in more than 90% of B cell non-Hodgkin's lymphomas. The CD20 molecule has four transmembrane domains, with the amino and carboxy termini located on the inner side of the plasma membrane. Between the third and fourth transmembrane domains is a loop domain consisting of 43 amino acid residues that constitutes the major epitope. The CD20 antigen molecule is relatively exposed and accessible. When CD20 are brought into close proximity to each other under the action of antibodies, the polymers formed by cross-linking or even hyper-cross-linking function as calcium ion channels, allowing extracellular calcium ions to flow into the cell. In addition, tyrosine protein kinases of the Src family activate each other by their proximity. Signaling pathways are initiated to mobilize endogenous calcium stores, both of which increase intracellular calcium ion concentrations, which then affect cell cycle operation, regulate cell proliferation and differentiation, and even lead to the occurrence of apoptosis. Although the actual role of CD20 in promoting B cell proliferation and differentiation is unclear, CD20 provides an important target for antibody-mediated therapy, which can be used to control B cells involved in cancer and autoimmune diseases.

[0087] In some embodiments, the CD20 binding domain comprises a sequence that is at least about 70% to about 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 3793-3808 and 3880. In some embodiments, the CD20 binding domain comprises a sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 3793-3808 and 3880. In some embodiments, the CD20 binding domain comprises a sequence of SEQ ID NOs: 3793-3808 and 3880.

[0088] CD33-binding domain Described herein are immune cell-binding proteins comprising a CD33 binding domain as a target antigen-binding protein, pharmaceutical compositions thereof, and nucleic acids, recombinant expression vectors, and host cells for producing such proteins. Also provided are methods of using the proteins comprising the CD33 binding domain of the present disclosure in the prevention and / or treatment of diseases, illnesses, and disorders.

[0089] CD33 (also known as Siglec-3, SIGLEC3, gp67, p67) is a 67 kDa plasma membrane protein that binds sialic acid and is a member of the sialic acid-binding Ig-related lectin (SIGLEC) family of proteins. Siglec proteins are thought to be involved in a variety of biological processes, including hematopoiesis, nervous system development, and immunity (Vinson et al., J. Biol. Chem. 271:9267-9272 (1996)). Further studies suggest that Siglec proteins mediate cell adhesion / cell signaling through recognition of sialylated cell surface glycans (Kelm et al., Glycoconj. J. 13:913-926 (1996); Kelm et al., Eur. J. Biochem. 255:663-672 (1998); Vinson et al., J Biol. Chem. 271:9267-9272 (1996)). The extracellular portion of CD33 contains two immunoglobulin domains, one IgV domain and one IgC2 domain. The IgV domain is distal to the lamina propria, whereas the IgC2 domain is proximal to the lamina propria. The intracellular portion of CD33 contains immunoreceptor tyrosine-based inhibitory motifs (ITIMs). In immune responses, CD33 can act as an inhibitory receptor upon ligand-induced tyrosine phosphorylation by recruiting cytoplasmic phosphatases that block signaling by dephosphorylating signaling molecules.

[0090] CD33 is known to be expressed on myeloid cells. CD33 expression has also been reported on many malignant cells. Anti-CD33 agents are generally assigned to four groups: naked antibodies, antibody toxin conjugates, radionuclide conjugates, and bispecific antibodies. Although CD33 has been targeted for the treatment of cancer, such as acute myeloid leukemia, no effective CD33-targeted treatments are currently on the market. Existing anti-CD33 agents suffer from, among other things, low tumor antigen binding activity and short in vivo half-life.

[0091] In some embodiments, the CD33 binding domain comprises a sequence that is at least about 70% to about 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 3809-3823. In some embodiments, the CD33 binding domain comprises a sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 3809-3823. In some embodiments, the CD33 binding domain comprises a sequence selected from the group consisting of SEQ ID NOs: 3809-3823.

[0092] Prostate-specific membrane antigen (PSMA) binding protein Described herein are immune cell-binding proteins that contain a PSMA-binding domain as a target antigen-binding protein, pharmaceutical compositions thereof, and nucleic acids, recombinant expression vectors, and host cells for making such proteins. Also provided are methods of using the proteins that contain the PSMA-binding domain of the present disclosure in the prevention and / or treatment of diseases, illnesses, and disorders.

[0093] PSMA is a 100 kD type II membrane glycoprotein expressed in prostate tissue with sequence identity to the transferrin receptor, which has NAALADase activity. PSMA is abundantly expressed in prostate cancer, and high levels of PSMA are also detectable in the serum of these patients. PSMA expression increases with disease progression and is highest in metastatic, hormone-refractory disease, for which no treatment currently exists.

[0094] In some embodiments, the PSMA binding domain comprises the following formula: f1-r1-f2-r2-f3-r3-f4, where r1, r2, and r3 are complementarity determining regions CDR1, CDR2, and CDR3, respectively; f1, f2, f3, and f4 are framework residues; r1 comprises SEQ ID NO:462, SEQ ID NO:463, SEQ ID NO:464, or SEQ ID NO:465; r2 comprises SEQ ID NO:466, SEQ ID NO:467, SEQ ID NO:468, SEQ ID NO:469, SEQ ID NO:470, SEQ ID NO:471, SEQ ID NO:472, or SEQ ID NO:473; and r3 comprises SEQ ID NO:474 or SEQ ID NO:475.

[0095] In some embodiments, the PSMA binding domain described herein comprises a polypeptide having a sequence set forth in SEQ ID NOs: 462-489 and subsequences thereof. In some embodiments, the PSMA binding domain comprises a polypeptide having at least 70%-95% or more homology to a sequence set forth in SEQ ID NOs: 462-489. In some embodiments, the PSMA binding domain comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to a sequence set forth in SEQ ID NOs: 462-489. In some embodiments, the PSMA binding domain has a sequence comprising at least a portion of a sequence set forth in SEQ ID NOs: 462-489. In some embodiments, the HSA binding domain comprises a polypeptide comprising one or more of the sequences set forth in SEQ ID NOs: 462-489.

[0096] In some embodiments, the PSMA binding domain described herein comprises a single domain antibody having a CDR1 comprising SEQ ID NOs: 462-465. In some embodiments, the PSMA binding domain described herein comprises a single domain antibody having a CDR2 comprising SEQ ID NOs: 466-473. In some embodiments, the PSMA binding domain described herein comprises a single domain antibody having a CDR3 comprising SEQ ID NOs: 474 and 475.

[0097] Mesothelin (MSLN) binding protein Described herein are immune cell-binding proteins comprising MSLN-binding domains as target antigen-binding proteins, pharmaceutical compositions thereof, and nucleic acids, recombinant expression vectors, and host cells for producing such proteins.Also provided are methods of using the proteins comprising the MSLN-binding domains of the present disclosure in the prevention and / or treatment of diseases, illnesses, and disorders.

[0098] MSLN is a GPI-linked thin-film-bound tumor antigen. MSLN is overexpressed in ovarian, pancreatic, lung, and triple-negative breast cancers, as well as mesothelioma. Normal tissue expression of MSLN is restricted to the mesothelial layer of isolated cells lining the pleura, pericardium, and peritoneal cavity. Overexpression of MSLN is associated with poor prognosis in lung adenocarcinoma and triple-negative breast cancer. MSLN has been used as a cancer antigen in a number of modalities, including immunotoxins, vaccines, antibody-drug conjugates, and CAR-T cells. Early signs of clinical efficacy have enabled targeting MSLN, but therapies with improved efficacy are needed to treat cancers that express MSLN.

[0099] Mesothelin is a glycoprotein present on the surface of mesothelial cells lining the peritoneal, pleural, and pericardial cavities. The mesothelin gene (MSLN) encodes a 71 kD precursor protein that is processed to a 40 kD protein called mesothelin, a glycosyl-phosphatidylinositol-anchored glycoprotein present on the cell surface (Chang et al., Proc Natl Acad Sci USA (1996) 93:136-40). Mesothelin cDNA is cloned from a library prepared from the HPC-Y5 cell line (Kojima et al., J. Biol. Chem. 270:21984-21990 (1995)). cDNA is also cloned using monoclonal antibody K1, which recognizes mesothelioma (Chang and Pastan, Proc. Natl. Acad. Sci. USA 93:136-40 (1996)). Mesothelin is a differentiation antigen whose expression in normal human tissues is restricted to mesothelial cells lining body cavities such as the pleura, pericardium, and peritoneum. Mesothelin is also highly expressed in several different human cancers, including mesothelioma, pancreatic adenocarcinoma, ovarian carcinoma, gastric adenocarcinoma, and lung adenocarcinoma (Hassan et al., Eur J Cancer (2008) 44:46-53) (Ordonez, Am J Surg Pathol (2003) 27:1418-28; Ho et al., Clin Cancer Res (2007) 13:1571-5). Mesothelin is overexpressed in the majority of primary pancreatic adenocarcinomas, with rare and weak expression seen in benign pancreatic tissue. Argani P et al., Clin Cancer Res. 2001, 7(12):3862-3868. Epithelial malignant pleural mesothelioma (MPM) widely expresses mesothelin, whereas sarcoma MPM is unlikely to express mesothelin. Most serous epithelial ovarian cancers and associated primary peritoneal cancers express mesothelin.

[0100] Mesothelin can also be used as a marker for the diagnosis and prognosis of certain types of cancer, since trace amounts of mesothelin can be detected in the blood of some patients with mesothelin-positive cancers (Cristaudo et al., Clin. Cancer Res. 13:5076-5081, 2007). It has been reported that mesothelin can be released into serum by deletion at its carboxyl terminus or by proteolytic cleavage from its mesothelin-bound form (Hassan et al., Clin. Cancer Res. 10:3937-3942, 2004). Increases in the soluble form of mesothelin were detectable several years before the development of malignant mesothelioma among workers exposed to asbestos (Creaney and Robinson, Hematol. Oncol. Clin. North Am. 19:1025-1040, 2005). In addition, soluble mesothelin is elevated in serum from patients with ovarian, pancreatic, and lung cancer (Cristaudo et al., Clin. Cancer Res. 13:5076-5081, 2007; Hassan et al., Clin. Cancer Res. 12:447-453, 2006; Croso et al., Cancer Detect. Prev. 30:180-187, 2006). Thus, mesothelin is a suitable target for methods to prevent or treat disease, and effective antibodies specific for mesothelin are needed.

[0101] Mature mesothelin on the cell surface contains three distinct domains: region I (containing residues 296-390), II (containing residues 391-486), and III (containing residues 487-598) (Tang et al., A human single-domain antibody elicits potent antitumor activity by targeting an epitope in mesothelin close to the cancer cell surface, Mol. Can. Therapeutics, 12(4):416-426, 2013). The first antibody generated against mesothelin for therapeutic intervention was designed to interfere with the interaction of mesothelin with CA-125. Fv SS was identified by phage display and its affinity was optimized to generate a recombinant immunotoxin that targets mesothelin, i.e., SS1P. The MORAb-009 antibody, amatuximab, also uses SS1 and recognizes a nonlinear epitope within region I at the amino-terminal 64 amino acids of mesothelin. SS1 Fv was also used to generate T cells engineered with chimeric antigen receptors. Recently, new anti-mesothelin antibodies that recognize other regions of the mesothelin protein have been reported. In certain embodiments, the present disclosure provides MSLN-targeting immune cell-binding proteins that contain a binding domain that specifically binds to MSLN on the surface of tumor target cells.

[0102] In some embodiments, the MSLN-binding domain binds to a protein that includes the sequence of SEQ ID NO: 3204. In some embodiments, the MSLN-binding domain binds to a protein that includes a truncated sequence compared to SEQ ID NO:3204.

[0103] In some embodiments, the MSLN-binding domain disclosed herein recognizes full-length mesothelin. In certain examples, the MSLN-binding domain disclosed herein recognizes an epitope in region I (including amino acid residues 296-390 of SEQ ID NO: 3204), region II (including amino acid residues 391-486 of SEQ ID NO: 3204), or region III (amino acid residues 487-598 of SEQ ID NO: 3204) of mesothelin. In some embodiments, the MSLN-binding domain of the present disclosure is contemplated to recognize and bind to an epitope located outside of region I, II, or III of mesothelin. In yet other embodiments, MSLN-binding domains are disclosed that recognize and bind to an epitope distinct from that of the MORAb-009 antibody.

[0104] In some embodiments, the MSLN binding domain is a polypeptide comprising an amino acid sequence composed of four framework regions / sequences (f1-f4) interrupted by three complementarity determining regions / sequences, as represented by the formula: f1-r1-f2-r2-f3-r3-f4, where r1, r2, and r3 are complementarity determining regions CDR1, CDR2, and CDR3, respectively, and f1, f2, f3, and f4 are framework residues. The framework residues of the MSLN-binding proteins of the present disclosure include, for example, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, or 94 amino acid residues, and the complementarity determining regions include, for example, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 amino acid residues. In some embodiments, the MSLN-binding domain includes an amino acid sequence selected from SEQ ID NOs: 607-650, or a sequence selected from the group consisting of SEQ ID NOs: 605-670 that includes at least 75% to about 95% or more (e.g., 96%, 97%, 98%, 99%, or more) identity to an amino acid sequence selected from SEQ ID NOs: 607-650, or a sequence selected from the group consisting of SEQ ID NOs: 605-670.

[0105] In some embodiments, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:490, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in SEQ ID NO:490. In some embodiments, CDR2 comprises the sequence set forth in SEQ ID NO:3505, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in SEQ ID NO:3505. In some embodiments, CDR3 comprises the sequence set forth in SEQ ID NO:3506, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in SEQ ID NO:3506.

[0106] In some embodiments, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:518, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in SEQ ID NO:518. In some embodiments, CDR2 comprises the sequence set forth in SEQ ID NO:3507, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in SEQ ID NO:3507. In some embodiments, CDR3 comprises the sequence set forth in SEQ ID NO:3508, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in SEQ ID NO:3508.

[0107] In some embodiments, CDR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 490-528, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in any one of SEQ ID NOs: 490-528. In some embodiments, CDR2 comprises a sequence set forth in any one of SEQ ID NOs: 529-567, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in any one of SEQ ID NOs: 529-567. In some embodiments, CDR3 comprises a sequence set forth in any one of SEQ ID NOs: 568-606, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in any one of SEQ ID NOs: 568-606.

[0108] In various embodiments, the MSLN binding domain of the present disclosure is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOs: 607-650.

[0109] In various embodiments, the complementarity determining regions of the MSLN binding domain of the present disclosure are at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:490, SEQ ID NO:518, or any one of SEQ ID NOs:490-528.

[0110] In various embodiments, the complementarity determining regions of the MSLN binding domain of the present disclosure are at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 3505, SEQ ID NO: 3507, or any one of SEQ ID NOs: 529-567.

[0111] In various embodiments, the complementarity determining regions of the MSLN binding domain of the present disclosure are at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in any one of SEQ ID NO: 3506, SEQ ID NO: 3508, or SEQ ID NOs: 568-606.

[0112] In some embodiments, all of the aforementioned MSLN binding domains (e.g., anti-MSLN single domain antibodies of SEQ ID NOs: 607-650) are affinity peptide tagged to facilitate purification. In some embodiments, the affinity peptide tag is six consecutive histidine residues designated 6X-his (SEQ ID NO: 3503).

[0113] In certain embodiments, the MSLN-binding domain of the present disclosure preferentially binds to membrane-bound mesothelin over soluble mesothelin. Membrane-bound mesothelin refers to the presence of mesothelin in or on the cell membrane surface of cells expressing mesothelin. Soluble mesothelin refers to mesothelin that is no longer present in or on the cell membrane surface of cells expressing or having expressed mesothelin. In certain examples, soluble mesothelin is present in the blood and / or lymphatic circulation of a subject. In one embodiment, the MSLN-binding domain binds to membrane-bound mesothelin at least 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, or 1000-fold higher than soluble mesothelin. In one embodiment, the MSLN-targeting immune cell-binding protein of the present disclosure preferentially binds to membrane-bound mesothelin 30-fold higher than soluble mesothelin. The preferential binding of an antigen binding protein to membrane-bound MSLN over soluble MSLN can be readily determined using assays well known in the art.

[0114] B-cell maturation antigen (BCMA) binding protein Described herein are immune cell binding proteins comprising a BCMA binding domain as a target antigen binding protein, pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors, and host cells for making such proteins. Also provided are methods of using the proteins comprising the BCMA binding domain of the present disclosure in the prevention and / or treatment of diseases, conditions, and disorders.

[0115] B cell maturation antigen (BCMA, TNFRSF17, CD269) is a transmembrane protein belonging to the superfamily of tumor necrosis family receptors (TNFRs) that is expressed primarily on terminally differentiated B cells. BCMA expression is restricted to the B cell lineage, present primarily on plasma cells and plasmablasts, and to some extent on memory B cells, but is virtually absent on peripheral naive B cells. BCMA is also expressed on multiple myeloma (MM) cells, leukemia cells, and lymphoma cells.

[0116] BCMA was identified by molecular analysis of the t(4,16)(q26,p13) translocation found in human intestinal T-cell lymphomas, with in-frame sequences mapping to the 16p13.1 chromosomal band.

[0117] The human BCMA cDNA has an open reading frame of 552 bp encoding a 184 amino acid polypeptide. The BCMA gene is organized into three exons separated by two introns and flanked by GT donor and AG acceptor consensus splice sites, respectively, and encodes a 1.2 kb transcript. The structure of the BCMA protein is based on a central 24 amino acid hydrophobic region in an α-helical structure, containing an essential transmembrane protein.

[0118] The mouse BCMA gene is located on chromosome 16, syntenic to the human 16p13 site, and further contains three exons separated by two introns. The gene encodes a 185 amino acid protein. Mouse BCMA mRNA is expressed as a 404 bp transcript, with highest levels in plasmacytoma cells (J558) and at moderate levels in the A20 B cell lymphoma line. Mouse BCMA mRNA transcripts were also detected at lower levels in T cell lymphoma (EL4, BW5147) and dendritic cell (CB1D6, D2SC1) lines, in contrast to human cell lines derived from T cells and dendritic cells. The mouse BCMA cDNA sequence has 69.3% nucleotide identity to the human BCMA cDNA sequence, with slightly higher identity (73.7%) when comparing the coding regions between these two cDNA sequences. The mouse BCMA protein is 62% identical to the human BCMA protein, and like human BCMA, contains one hydrophobic region that may be an internal transmembrane segment. The N-terminal 40 amino acid domain of both mouse and human BCMA proteins has six conserved cysteine ​​residues, consistent with the formation of a cysteine ​​repeat motif found in the extracellular domain of TNFRs. Like members of the TNFR superfamily, BCMA proteins contain one conserved aromatic residue, 4-6 residues C-terminal from the first cysteine.

[0119] BCMA is not expressed on the cell surface, but rather is located in the Golgi apparatus. The amount of BCMA expression is proportional to the stage of cell differentiation (highest in plasma cells).

[0120] BCMA is involved in B cell development and homeostasis through interaction with its ligands BAFF (B cell activating factor, also designated TALL-1 or TNFSF13B) and APRIL (A proliferation-inducing ligand). BCMA, together with its family members TACI (transmembrane activator and cyclophilin ligand interactor) and BAFF-R (B cell activating factor receptor, also known as tumor necrosis factor receptor superfamily member 13C), regulates different aspects of humoral immunity, B cell development and homeostasis. BCMA expression appears relatively late in B cell differentiation and contributes to the long-term survival of plasmablasts and plasma cells in the bone marrow. BCMA also supports the growth and survival of multiple myeloma (MM) cells. BCMA is mostly known for its functional activity in mediating the survival of plasma cells that maintain long-term humoral immunity.

[0121] The present disclosure provides, in certain embodiments, single domain proteins that specifically bind to BCMA on the surface of tumor target cells.

[0122] In some embodiments the BCMA binding domain binds to a protein comprising the sequence of SEQ ID NO: 3201, 3203, or 3203. In some embodiments the BCMA binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 3201, 3203, or 3203.

[0123] In some embodiments, a BCMA binding protein of the disclosure is a polypeptide comprising an amino acid sequence composed of four framework regions / sequences (f1-f4) interrupted by three complementarity determining regions / sequences, as represented by the formula: f1-r1-f2-r2-f3-r3-f4, where r1, r2, and r3 are complementarity determining regions CDR1, CDR2, and CDR3, respectively, and f1, f2, f3, and f4 are framework residues. The r1 residues of BCMA binding proteins of the disclosure include, for example, amino acid residues 26, 27, 28, 29, 30, 31, 32, 33, and 34; the r2 residues of BCMA binding proteins of the disclosure include, for example, amino acid residues 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63; and the r3 residues of BCMA binding proteins of the disclosure include, for example, amino acid residues 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, and 108. In some embodiments, the BCMA binding protein comprises an amino acid sequence selected from SEQ ID NOs: 346-460, or a sequence comprising at least 75% to about 95% or more (e.g., 96%, 97%, 98%, 99%, or more) identity to a sequence selected from the group consisting of SEQ ID NOs: 346-460.

[0124] In some embodiments, an exemplary CDR1 comprises an amino acid sequence set forth in SEQ ID NOs: 1-115. In some embodiments, another exemplary CDR2 comprises an amino acid sequence set forth in SEQ ID NOs: 116-230. In some embodiments, another exemplary CDR3 comprises an amino acid sequence set forth in SEQ ID NOs: 231-345.

[0125] In various embodiments, the BCMA binding proteins of the disclosure have a CDR1 having an amino acid sequence that is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOs: 1-115.

[0126] In various embodiments, the BCMA binding proteins of the disclosure have a CDR2 having an amino acid sequence that is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOs: 1116-230.

[0127] In various embodiments, the complementarity determining region of a BCMA binding protein of the disclosure has a CDR3 having an amino acid sequence that is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOs:231-345.

[0128] In various embodiments, the BCMA binding proteins of the disclosure have an amino acid sequence that is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOs: 346-460.

[0129] In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 346. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 347. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 348. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 349. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 350. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 351. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 352. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 353. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 354. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 355. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 356. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 357. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 358. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 359.

[0130] In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 360. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 361. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 362. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 363. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 364. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 365. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 366. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 367. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 368. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 369.

[0131] In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 370. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 371. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 372. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 373. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 374. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 375. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 376. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 377. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 378. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 379.

[0132] In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 380. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 381. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 382. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 383. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 384. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 385. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 386. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 387. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 388. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 389.

[0133] In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 390. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 391. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 392. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 393. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 394. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 395. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 396. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 397. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 398. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 399.

[0134] In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 400. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 401. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 402. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 403. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 404. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 405. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 406. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 407. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 408. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 409.

[0135] In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 410. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 411. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 412. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 413. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 414. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 415. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 416. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 417. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 418. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 419.

[0136] In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 420. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 421. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 422. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 423. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 424. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 425. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 426. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 427. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 428. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 429.

[0137] In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 430. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 431. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 432. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 433. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 434. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 435. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 436. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 437. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 438. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 439.

[0138] In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 440. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 441. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 442. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 443. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 444. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 445. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 446. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 447. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 448. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 449.

[0139] In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 450. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 451. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 452. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 453. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 454. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 455. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 456. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 457. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 458. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 459. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 460.

[0140] In some embodiments, all of the foregoing BCMA binding domains are affinity peptide tagged to facilitate purification. In some embodiments, the affinity peptide tag is six consecutive histidine residues, also referred to as a His tag or 6X-his (His-His-His-His-His-His, SEQ ID NO: 3503).

[0141] In certain embodiments, the BCMA binding domain of the present disclosure preferentially binds membrane-bound BCMA over soluble BCMA. Membrane-bound BCMA refers to the presence of BCMA in or on the cell membrane surface of cells expressing BCMA. Soluble BCMA refers to BCMA that is no longer present in or on the cell membrane surface of cells that express or have expressed BCMA. In certain examples, soluble BCMA is present in the blood and / or lymphatic circulation of a subject. In one embodiment, the BCMA binding domain binds membrane-bound BCMA at least 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, or 1000-fold more than soluble BCMA. In one embodiment, the BCMA-targeted immune cell-associated protein of the present disclosure preferentially binds membrane-bound BCMA 30-fold more than soluble BCMA. Preferential binding of an antigen-binding protein to membrane-bound BCMA over soluble BCMA can be readily determined using assays well known in the art.

[0142] Delta-like ligand 3 (DLL3) binding protein DLL3 (also known as delta-like ligand 3 or SCDO1) is a member of the delta-like family of Notch DSL ligands. Representative DLL3 protein orthologs include, but are not limited to, human (accession numbers NP_058637 and NP_982353), chimpanzee (accession number XP_003316395), mouse (accession number NP_031892), and rat (accession number NP_446118). In humans, the DLL3 gene consists of eight exons spanning 9.5 kbp located on chromosome 19q13. Alternative splicing within the last exon gives rise to two processed transcripts, one of 2389 bases (accession number NM_016941) and another of 2052 bases (accession number NM_203486). The former transcript encodes a 618 amino acid protein (accession no. NP_058637), while the latter encodes a 587 amino acid protein (accession no. NP_982353). These two protein isoforms of DLL3 share 100% overall identity across their extracellular and transmembrane domains, differing only in that the longer isoform contains an extended cytoplasmic tail containing 32 additional residues at the carboxy terminus of the protein. The extracellular region of the DLL3 protein contains six EGF-like domains, a single DSL domain, and an N-terminal domain. Generally, the EGF domains are recognized as occurring approximately amino acid residues 216-249 (domain 1), 274-310 (domain 2), 312-351 (domain 3), 353-389 (domain 4), 391-427 (domain 5), and 429-465 (domain 6), the DSL domain occurs approximately amino acid residues 176-215, and the N-terminal domain occurs approximately amino acid residues 27-175 of hDLL3. Each of the EGF-like domains, the DSL domain, and the N-terminal domain comprises a portion of the DLL3 protein defined by a distinct amino acid sequence. The EGF-like domains are, in some embodiments, referred to as EGF1-EGF6, with EGF1 being most proximal to the N-terminal portion of the protein.In general, DSL ligands are composed of a series of structural domains: a unique N-terminal domain, followed by a conserved DSL domain, multiple tandem epidermal growth factor (EGF)-like repeats, a transmembrane domain, and a cytoplasmic domain that is not highly conserved across ligands but contains multiple lysine residues that are potential sites for ubiquitination by a unique E3 ubiquitin ligase. The DSL domain is a degenerate EGF domain that is essential but not sufficient for interaction with Notch receptors. In addition, the first two EGF-like repeats of most DSL ligands contain a smaller protein sequence motif known as the DOS domain that interacts cooperatively with the DSL domain in activating Notch signaling.

[0143] In some embodiments, the DLL3 immune cell binding proteins of the present disclosure are generated, engineered, or selected to react with selected domains, motifs, or epitopes within the DLL3 protein. In some embodiments, the DLL3 targeting immune cell binding proteins bind to the DSL domain, and in some embodiments, to epitopes within the DSL domain including G203, R205, P206.

[0144] The DLL3 binding domain of the DLL3-targeting immune cell binding proteins of the present disclosure, in some embodiments, are engineered, created, and / or selected to react with both isoforms of DLL3 and isoforms of the protein, or conversely, comprise a pan-DLL binding domain that reacts with or associates with at least one additional DLL family member in addition to DLL3. In some embodiments, the DLL3 binding domain, such as a DLL3 binding domain, is engineered, created, and / or selected to react with a domain (or an epitope therein) that is displayed only by DLL3, or a domain that is at least somewhat conserved across multiple or all DLL family members.

[0145] In some embodiments, the DLL3 binding domain associates with or binds to a specific epitope, portion, motif, or domain of DLL3. Both DLL3 isoforms incorporate identical extracellular regions that include at least one N-terminal domain, a DSL (Delta / Serrate / lag-2) domain, and six EGF-like domains (i.e., EGF1-EGF6). Thus, in certain embodiments, the DLL3 binding domain associates with or binds to the N-terminal domain of DLL3 (amino acids 27-175 in the mature protein), while in other embodiments, the DLL3 binding domain associates with the DSL domain (amino acids 176-215) or an epitope therein. In other aspects of the present disclosure, the DLL3 binding domain associates with or binds to a specific epitope located in a particular EGF-like domain of DLL3. In some embodiments, the DLL3 binding domain associates with or binds to an epitope located in EGF1 (amino acids 216-249), EGF2 (amino acids 274-310), EGF3 (amino acids 312-351), EGF4 (amino acids 353-389), EGF5 (amino acids 391.427), or EGF6 (amino acids 429-465). In some embodiments, each of the aforementioned domains comprises more than one epitope, and / or more than one bin. In some embodiments, the DLL3 binding domain binds to, reacts with, or associates with a DSL domain or an epitope therein. In other embodiments, the DLL3 binding domain binds to, reacts with, or associates with a specific EGF-like domain or an epitope therein. In some embodiments, the DLL3 binding domain binds to, reacts with, or associates with an N-terminal domain or an epitope therein.

[0146] In some embodiments, the DLL3 binding proteins of the present disclosure, such as the DLL3 binding domain of the immune cell binding proteins of the present disclosure, bind to full-length DLL3 or to fragments, such as epitope-containing fragments, within the full-length DLL3 protein, as described above. In certain cases, the epitope-containing fragments include antigenic or immunogenic fragments of the DLL3 protein and derivatives thereof. Epitope-containing fragments, including antigenic or immunogenic fragments, are in some embodiments 12 amino acids or more, 20 amino acids or more, 50 or 100 amino acids or more. In some embodiments, the DLL3 fragments include 95% or more, 90% or more, 75%, 50%, 25%, or 10% or more of the length of the full protein. In some embodiments, epitope-containing fragments of DLL3, including antigenic or immunogenic fragments, are capable of eliciting a relevant immune response in a patient. In some embodiments, derivatives of DLL3 include variants in sequence that have one or more (e.g., 1-20, e.g., 15 amino acids, or up to 20%, such as up to 10%, 5%, or 1% of the amino acid number based on the full length of the protein) deletions, insertions, or substitutions relative to the DLL3 sequence provided in SEQ ID NO: 3216 (UniProtKB Accession Q9NYJ7). In some embodiments, the substitutions include conservative substitutions. In some examples, derivatives and variants of DLL3 have essentially the same biological function as the DLL3 protein from which they are derived. For example, derivatives and variants of DLL3 are, in some cases, antigenic or immunogenic equivalent to the protein from which they are derived, have either the ligand binding activity or the ability to form an active receptor complex, or preferably both, of the protein from which they are derived, and have the same tissue distribution as DLL3.

[0147] In some embodiments, the DLL3 binding domain binds to a protein comprising the sequence of SEQ ID NO: 3216 (UniProtKB Accession Q9NYJ7). In some embodiments, the BCMA binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 3216 (UniProtKB Accession Q9NYJ7). In some embodiments, the DLL3 binding domain binds to a protein comprising the sequence of SEQ ID NO: 3509 or SEQ ID NO: 3217 (which is the mature extracellular domain of the DLL3 protein). In some embodiments, the DLL3 binding domain binds to a protein comprising amino acids 47-492 of SEQ ID NO: 3509. In some embodiments, the DLL3 binding domain recognizes an epitope within amino acids 47-4492 of SEQ ID NO: 3509.

[0148] Another example of a substitution to create a variant anti-DLL3 antibody is to substitute one or more hypervariable regions of a parent antibody. Generally, variants are then selected based on the improvement of a desired property compared to the parent antibody, such as increased affinity, decreased affinity, decreased immunogenicity, or increased pH-dependence of binding.

[0149] In some embodiments, the DLL3 binding domain is a polypeptide comprising an amino acid sequence composed of four framework regions / sequences (f1-f4) interrupted by three complementarity determining regions / sequences, as represented by the formula: f1-r1-f2-r2-f3-r3-f4, where r1, r2, and r3 are complementarity determining regions CDR1, CDR2, and CDR3, respectively, and f1, f2, f3, and f4 are framework residues. The framework residues of the DLL3-binding proteins of the present disclosure include, for example, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, or 94 amino acid residues, and the complementarity determining regions include, for example, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 amino acid residues. In some embodiments, the DLL3-binding domain includes an amino acid sequence selected from SEQ ID NOs: 1308-1750. In some embodiments, the CDR1 of the DLL3-binding domain includes a sequence selected from SEQ ID NOs: 1751-2193, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID NOs: 1751-2193. In some embodiments, CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 2194-2636, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID NOs: 2194-2636. In some embodiments, CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 2637-3080, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID NOs: 2637-3080.

[0150] In some embodiments, CDR1 comprises an amino acid sequence selected from SEQ ID NOs: 1751-2193, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in an amino acid sequence selected from SEQ ID NOs: 1751-2193. In some embodiments, CDR2 comprises an amino acid sequence selected from SEQ ID NOs: 2194-2636, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in an amino acid sequence selected from SEQ ID NOs: 2194-2636. In some embodiments, CDR3 comprises an amino acid sequence selected from SEQ ID NOs: 2637-3080, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in an amino acid sequence selected from SEQ ID NOs: 2637-3080.

[0151] In some embodiments, CDR1 comprises an amino acid sequence selected from SEQ ID NOs: 1803-1836, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in an amino acid sequence selected from SEQ ID NOs: 1803-1836. In some embodiments, CDR2 comprises an amino acid sequence selected from SEQ ID NOs: 2246-2279, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in an amino acid sequence selected from SEQ ID NOs: 2246-2279. In some embodiments, CDR3 comprises an amino acid sequence selected from SEQ ID NOs: 2689-2722, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in an amino acid sequence selected from SEQ ID NOs: 2689-2722.

[0152] In some embodiments, CDR1 comprises an amino acid sequence selected from SEQ ID NOs: 1837-2117, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in an amino acid sequence selected from SEQ ID NOs: 1837-2117. In some embodiments, CDR2 comprises an amino acid sequence selected from SEQ ID NOs: 2280-2560, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in an amino acid sequence selected from SEQ ID NOs: 2280-2560. In some embodiments, CDR3 comprises an amino acid sequence selected from SEQ ID NOs: 2723-3003, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in an amino acid sequence selected from SEQ ID NOs: 2723-3003.

[0153] In some embodiments, CDR1 comprises an amino acid sequence selected from SEQ ID NOs: 2118-2193, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in an amino acid sequence selected from SEQ ID NOs: 2118-2193. In some embodiments, CDR2 comprises an amino acid sequence selected from SEQ ID NOs: 2561-2636, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in an amino acid sequence selected from SEQ ID NOs: 2561-2636. In some embodiments, CDR3 comprises an amino acid sequence selected from SEQ ID NOs: 3004-3080, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in an amino acid sequence selected from SEQ ID NOs: 3004-3080.

[0154] In various embodiments, the DLL3 binding domain of the present disclosure is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOs: 1308-1750. In various embodiments, the DLL3 binding domain of the present disclosure is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOs: 1360-1393.

[0155] In various embodiments, the DLL3 binding domain of the present disclosure is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOs: 1394-1674.

[0156] In various embodiments, the DLL3 binding domain of the present disclosure is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID NO:1375, or a sequence derived from SEQ ID NO:1375.

[0157] In various embodiments, the DLL3 binding domain of the present disclosure is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID NO:1382, or a sequence derived from SEQ ID NO:1382.

[0158] In certain embodiments, the DLL3 binding domain of the present disclosure preferentially binds to membrane-bound DLL3 over soluble DLL3. Membrane-bound DLL3 refers to the presence of DLL3 in or on the cell membrane surface of cells expressing DLL3. Soluble DLL3 refers to DLL3 that is no longer present in or on the cell membrane surface of cells that express or have expressed DLL3. In certain examples, soluble DLL3 is present in the blood and / or lymphatic circulation of a subject. In one embodiment, the DLL3 binding protein binds to membrane-bound DLL3 at least 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, or 1000-fold higher than soluble DLL3. In one embodiment, the antigen binding protein of the present disclosure preferentially binds to membrane-bound DLL3 30-fold higher than soluble DLL3. Preferential binding of an antigen binding protein to membrane-bound DLL3 over soluble DLL3 can be easily determined using assays well known in the art.

[0159] In some embodiments, all of the aforementioned DLL3 binding domains (e.g., anti-DLL3 single domain antibodies of SEQ ID NOs: 1308-1750) are affinity peptide tagged to facilitate purification. In some embodiments, the affinity peptide tag is six consecutive histidine residues designated 6X-his (SEQ ID NO: 3503).

[0160] In some embodiments, all of the aforementioned DLL3 binding domains (e.g., anti-DLL3 single domain antibodies of SEQ ID NOs: 1308-1750) are affinity peptide tagged to facilitate purification. In some embodiments, the affinity peptide tag is six consecutive histidine residues designated 6X-his (SEQ ID NO: 3503).

[0161] Epidermal growth factor receptor (EGFR) binding proteins Described herein are immune cell-binding proteins that contain an EGFR-binding domain as a target antigen-binding protein, pharmaceutical compositions thereof, and nucleic acids, recombinant expression vectors, and host cells for making such proteins. Also provided are methods of using the proteins that contain the disclosed EGFR-binding domain in the prevention and / or treatment of diseases, illnesses, and disorders.

[0162] The epidermal growth factor receptor (EGFR) has been implicated in human malignancies. Aberrant activity of the Her family of receptors has been associated with breast cancer. EGFR, Her-3, and Her-4 are frequently expressed in ovarian granulosa cell tumors (Leibl, S. et al., Gynecol Oncol 101:18-23 (2005)). Notably, increased expression of EGFR has been observed in breast, bladder, lung, head, neck, and gastric cancers, as well as glioblastoma.

[0163] Increased EGFR receptor expression may be associated with increased production of the EGFR ligand, transforming growth factor alpha (TGF-α), by the same tumor cells leading to receptor activation via an autocrine stimulatory pathway.

[0164] Cetuximab (Erbitux™), an anti-EGFR antibody, is associated with potentially life-threatening infusion reactions (Thomas, M, Clin J Oncol Nurs. 9(3):332-8 (2005)). Gefitinib (Iressa™) and erlotinib (Tarceva™), both EGFR-specific small molecule inhibitors, are associated with risk of interstitial lung disease (Sandler A, Oncology 20(5 Suppl 2):35-40 (2006)). Individual patients may be susceptible to certain types of complications that affect the choice of drug therapy. There is a need for more treatment options that allow physicians to select the therapy with the best side effect profile for individual patients. The present disclosure provides novel polypeptide and protein therapeutics that are useful in treatment methods, particularly for the treatment of diseases associated with aberrant expression of EGFR.

[0165] Epidermal growth factor receptor (EGFR, also known as HER1 or ErbB1) is a member of the ErbB / HER family of type 1 receptor tyrosine kinases (RTKs). Other members of this family include ErbB2 (HER2 or Neu), ErbB3 (HER3), and ErbB4 (HER4). Known ligands for EGFR include epidermal growth factor (EGF) and transforming growth factor alpha (TGF-α). Ligand binding to EGFR is known to induce tyrosine phosphorylation and receptor dimerization with other ErbB family members.

[0166] RTKs such as EGFR function to enable cells to respond to various external stimuli. However, aberrant activation and / or overexpression of EGFR is associated with the development and progression of several human cancers. Thus, EGFR is a target for anti-cancer therapy. Approved drugs targeting EGFR include small molecule inhibitors such as gefitinib (Iressa®) and erlotinib (Tarceva®), and anti-EGFR antibodies such as cetuximab (Erbitux®) and panitumumab (Vectibix®). Anti-EGFR antibodies are mentioned, for example, in U.S. Pat. Nos. 4,943,533, 5,844,093, 7,060,808, 7,247,301, 7,595,378, 7,723,484, and 7,939,072. There remains a need to have additional options available in the treatment of diseases associated with overexpression of EGFR, including, but not limited to, renal cell carcinoma, pancreatic cancer, breast cancer, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colon cancer, gastric cancer (e.g., gastric cancer with MET amplification), malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer (e.g., EGFR-dependent non-small cell lung cancer), synovial sarcoma, thyroid cancer, or melanoma. The present disclosure provides, in certain embodiments, EGFR-binding proteins, i.e., EGFR-targeted immune cell-binding proteins that contain an EGFR-binding domain that specifically binds to EGFR on the surface of tumor target cells.

[0167] In some embodiments, the EGFR binding domain of the present disclosure binds to a protein comprising a sequence of SEQ ID NO: 3205 (UniProt Accession No. Q504U8). In some embodiments, the EGFR binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 3205. In some embodiments, the EGFR binding domain of the present disclosure binds to a protein comprising a sequence of SEQ ID NO: 3206 (UniProt Accession No. Q01279). In some embodiments, the EGFR binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 3206. In some embodiments, the EGFR binding domain of the present disclosure binds to a protein comprising a sequence of SEQ ID NO: 3207 (UniProt Accession No. A0A2K5WK3). In some embodiments, the EGFR binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 3207.

[0168] In some embodiments, the EGFR binding domain disclosed herein recognizes full-length EGFR. In certain examples, the EGFR binding domain disclosed herein recognizes an epitope within EGFR, for example, the EGFR-targeting immune cell binding protein optionally interacts with one or more amino acids found within the extracellular domain of human EGFR (e.g., within extracellular domain I, II, III, and / or IV). The epitope that the antibody binds to may consist of a single contiguous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more) amino acids located within the extracellular domain of EGFR. Alternatively, the epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) within the extracellular domain of EGFR.

[0169] In some embodiments, the EGFR binding proteins of the present disclosure bind to the full-length EGFR protein or to fragments thereof, such as epitope-containing fragments within the full-length EGFR protein, as described above. Optionally, the epitope-containing fragments include antigenic or immunogenic fragments of the EGFR protein and derivatives thereof. Epitope-containing fragments, including antigenic or immunogenic fragments, in some embodiments, are 12 amino acids or more, such as 20 amino acids or more, 50 or 100 amino acids or more. In some embodiments, the EGFR fragments include 95% or more, 90% or more, 75%, 50%, 25%, or 10% or more of the length of the full protein. In some embodiments, epitope-containing fragments of EGFR, including antigenic or immunogenic fragments, are capable of eliciting a relevant immune response in patients. In some embodiments, derivatives of EGFR include sequence variants having one or more (e.g., 1-20, e.g., 15 amino acids, or up to 20%, such as up to 10%, 5%, or 1% of the number of amino acids based on the total length of the protein) deletions, insertions, or substitutions relative to the EGFR sequence provided in SEQ ID NO: 3205, 3206, or 3207.

[0170] In some embodiments, the EGFR binding domain is a polypeptide comprising an amino acid sequence composed of four framework regions / sequences (f1-f4) interrupted by three complementarity determining regions / sequences, as represented by the formula: f1-r1-f2-r2-f3-r3-f4, where r1, r2, and r3 are complementarity determining regions CDR1, CDR2, and CDR3, respectively, and f1, f2, f3, and f4 are framework residues. Framework residues of the EGFR binding proteins of the disclosure include, for example, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, or 94 amino acid residues, and complementarity determining regions include, for example, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 amino acid residues. In some embodiments, the EGFR binding domain comprises an amino acid sequence selected from SEQ ID NOs: 798-846.

[0171] In some embodiments, the EGFR binding domain described herein comprises a polypeptide having a sequence selected from SEQ ID NOs: 798-846, sequences thereof, and variants thereof. In some embodiments, the EGFR binding domain comprises at least 70%-95% or more identity to a sequence selected from SEQ ID NOs: 798-846, sequences thereof, and variants thereof. In some embodiments, the EGFR binding protein comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to a sequence selected from SEQ ID NOs: 798-846, sequences thereof, and variants thereof.

[0172] In some embodiments, CDR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 651-699, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in any one of SEQ ID NOs: 651-699. In some embodiments, CDR2 comprises a sequence set forth in any one of SEQ ID NOs: 700-748, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in any one of SEQ ID NOs: 700-748. In some embodiments, CDR3 comprises a sequence set forth in any one of SEQ ID NOs: 148-196, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in any one of SEQ ID NOs: 148-196.

[0173] In various embodiments, the EGFR binding domain of the present disclosure is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOs:798-846.

[0174] In various embodiments, the complementarity determining regions of the EGFR binding domain of the present disclosure are at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs:651-699.

[0175] In various embodiments, the complementarity determining regions of the EGFR binding domain of the present disclosure are at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs:700-748.

[0176] In various embodiments, the complementarity determining regions of the EGFR binding domain of the present disclosure are at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs:749-797.

[0177] In some embodiments, all of the aforementioned EGFR binding domains (e.g., anti-EGFR single domain antibodies of SEQ ID NOs: 798-846) are affinity peptide tagged to facilitate purification. In some embodiments, the affinity peptide tag is six consecutive histidine residues designated 6X-his (SEQ ID NO: 3503).

[0178] In certain embodiments, the EGFR binding domain of the present disclosure preferentially binds to membrane-bound EGFR over soluble EGFR. Membrane-bound EGFR refers to the presence of EGFR in or on the cell membrane surface of cells expressing EGFR. Soluble EGFR refers to EGFR that is no longer present in or on the cell membrane surface of cells expressing or having expressed EGFR. In certain examples, soluble EGFR is present in the blood and / or lymphatic circulation of a subject. In one embodiment, the EGFR binding domain binds to membrane-bound EGFR at least 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, or 1000-fold higher than soluble EGFR. In one embodiment, the EGFR-targeting immune cell-binding protein of the present disclosure preferentially binds to membrane-bound EGFR 30-fold higher than soluble EGFR. Preferential binding of an antigen binding protein to membrane-bound EGFR over soluble EGFR can be easily determined using binding assays well known in the art. In some embodiments, it is contemplated that the EGFR binding protein is fairly small, 25 kDa or less, 20 kDa or less, 15 kDa or less, or in some embodiments, 10 kDa or less. In particular examples, the EGFR binding protein is 5 kDa or less when it is a peptide or small molecule entity.

[0179] In other embodiments, the EGFR binding proteins described herein comprise small molecular entity (SME) binders to EGFR. SME binders are small molecules averaging about 500-2000 Da in size that are attached to the EGFR binding proteins by known methods such as sortase ligation or conjugation. In these examples, the EGFR binding proteins comprise a domain that includes a sortase recognition sequence, e.g., LPETG) (SEQ ID NO: 3200). To bind the SME binder to an EGFR binding protein that includes a sortase recognition sequence, the protein is incubated with sortase and the SME binder, whereby the sortase binds the SME binder to the recognition sequence. In yet other embodiments, the EGFR binding proteins described herein comprise knottin peptides for EGFR binding. Knottins are disulfide-stabilized peptides with a cysteine ​​knot scaffold and have an average size of about 3.5 kDa. Knottins are intended to bind to certain tumor molecules such as EGFR. In further embodiments, the EGFR binding proteins described herein comprise natural EGFR ligands.

[0180] In some embodiments, the EGFR binding protein comprises more than one domain and is in a single polypeptide design with flexible linkage of the domains. This allows for easy production and manufacture of the EGFR binding protein, since it can be encoded by a single cDNA molecule that is easy to incorporate into a vector. Furthermore, in some embodiments where the EGFR binding protein described herein is a monomeric single polypeptide chain, there is no challenge or requirement for chain pairing for dimerization. In such embodiments, it is contemplated that the EGFR binding protein described herein is less prone to aggregation.

[0181] In some cases, EGFR binding proteins contain more than one domain, and domains within the EGFR binding proteins are conjugated using enzymatic, site-specific conjugation methods, including the use of mammalian or bacterial transglutaminase enzymes. Microbial transglutaminase (mTG) is a versatile tool in modern research and biotechnology. The availability of large amounts of relatively pure enzyme, ease of use, and lack of regulation by calcium and guanosine-5'-triphosphate (GTP) have propelled mTG to become the primary cross-linking enzyme used in both the food industry and biotechnology. mTG is currently used in many applications to conjugate proteins and peptides to small molecules, polymers, surfaces, DNA, as well as other proteins. See, for example, Veracity of microbial transglutaminase by Pavel Strp, Bioconjugate Chem. 25, 5, 855-862.

[0182] In some examples, EGFR binding proteins are provided that include more than one domain, one of which includes an acceptor glutamine in the constant region that can then be conjugated to another domain via a lysine-based linker (e.g., any primary amine chain that is a substrate for TGase, including, for example, alkylamines and oxoamines), with the conjugation occurring exclusively to one or more acceptor glutamine residues present in a targeting moiety that is external to the antigen binding site (e.g., outside the variable region, within the constant region). Thus, conjugation does not occur to glutamines within the variable region, e.g., glutamines that are at least partially surface exposed. In some examples, the EGFR binding protein is formed by reacting one of the domains with the lysine-based linker in the presence of TGase.

[0183] In some embodiments where one or more domains in an EGFR binding protein are directly linked, a hybrid vector is created in which the DNA encoding the directly linked domains are directly ligated to each other themselves. In some embodiments where a linker is used, a hybrid vector is created in which the DNA encoding one domain is ligated to the DNA encoding one end of a linker portion, and the DNA encoding another domain is ligated to the other end of the linker portion.

[0184] In some embodiments, the EGFR binding proteins described above are fused to an Fc region from any species, including but not limited to, human immunoglobulins such as human IgG1, human IgG2, human IgG3, human IgG4, etc., to generate an Fc fusion protein. In some embodiments, the Fc fusion proteins of the disclosure have an extended half-life compared to an otherwise identical EGFR binding protein. In some embodiments, the Fc fusion EGFR binding proteins of the disclosure include substitutions, mutations, and / or modifications, e.g., in the Fc region, inter alia, of one or more additional amino acid residues, resulting in a binding protein with favorable characteristics, including but not limited to, modified pharmacokinetics and extended serum half-life.

[0185] In some embodiments, such Fc-fusion EGFR binding proteins provide an increased half-life for mammals, such as humans, of more than 5 days, more than 10 days, more than 15 days, more than 20 days, more than 25 days, more than 30 days, more than 35 days, more than 40 days, more than 45 days, more than 2 months, more than 3 months, more than 4 months, or more than 5 months. In some cases, the increased half-life reduces the frequency of administration of the EGFR binding protein and / or increases the serum titer, which reduces the concentration of the antibody to be administered. The binding to human FcRn in vivo and the serum half-life of human FcRn high affinity binding polypeptides are analyzed in some examples for genetically engineered mice or transfected human cell lines expressing human FcRn, or primates to which the polypeptides having variant Fc regions are administered.

[0186] In some cases, the EGFR binding proteins are differentially modified during or after production, for example, by glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to antibody molecules or other cellular ligands, etc. Any of a number of chemical modifications are performed by techniques including, but not limited to, specific chemical cleavage with cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.

[0187] Various post-translational modifications of EGFR binding proteins further encompassed by the present disclosure include, for example, N- or O-linked hydrocarbon chains, N- or C-terminal processing, conjugation of chemical moieties to the amino acid backbone, chemical modifications of N- or O-linked hydrocarbon chains, and addition or deletion of an N-terminal methionine residue as a result of expression in a prokaryotic host cell. Additionally, EGFR binding proteins are optionally modified with a detectable label, such as an enzymatic, fluorescent, radioisotope, or affinity label, to allow for detection and isolation of the modulators.

[0188] In some embodiments, the EGFR binding proteins of the present disclosure are monovalent or multivalent (bivalent, trivalent, etc.). As used herein, the term "valency" refers to the number of potential target binding sites associated with an antibody. Each target binding site specifically binds to one target molecule, or to a specific position or locus on a target molecule. If an antibody is monovalent, each binding site on the molecule will specifically bind to a single antigen position or epitope. If an antibody contains more than one target binding site (multivalent), the target binding sites may each specifically bind to the same or different molecules (e.g., different ligands or different antigens, or different epitopes or positions on the same antigen).

[0189] FLT3-binding protein Described herein are immune cell-binding proteins that contain FLT3-binding domains as target antigen-binding proteins, pharmaceutical compositions thereof, and nucleic acids, recombinant expression vectors, and host cells for producing such proteins.Also provided are methods of using the proteins that contain the FLT3-binding domains of the present disclosure in the prevention and / or treatment of diseases, illnesses, and disorders.

[0190] FLT3, also known as fetal liver kinase 2 (FLK-2), stem cell tyrosine kinase 1 (STK-1), and CD135, is a member of the class III receptor tyrosine kinases. Normally, FLT3 is expressed on immature myeloid lymphoid progenitors and dendritic cell precursors, but rarely on mature adult cells. FLT3 is overexpressed in approximately 90% of acute myeloid leukemias (AML), the majority of acute lymphoblastic leukemias (ALL), and the blast crisis phase of chronic myeloid leukemia (BC-CML). Stimulation with FLT3 ligand (FL) enhances the proliferation and survival of leukemic cells. Inhibition of FLT3 signaling leads to apoptosis in dendritic cells and inhibition of immune responses. The MAPK, PI3K, and Stat5 pathways have been identified as involved in downstream signaling of activated FLT3 (see, e.g., Stirewalt DL and JP, Radich, JP, Nat Rev Cancer 3:650-665 (2003)).

[0191] Described herein are immune cell-binding proteins comprising FLT3 binding domains, pharmaceutical compositions thereof, and nucleic acids, recombinant expression vectors, and host cells for producing such proteins. Also provided are methods of using proteins comprising the FLT3 binding domains of the present disclosure in the prevention and / or treatment of diseases, illnesses, and disorders. In some embodiments, the FLT3 binding domains of the present disclosure inhibit FL-induced phosphorylation of wild-type FLT3 and downstream kinases of the MPK, PI3K, and STAT5 pathways in diseases such as leukemia. In some embodiments, the FLT3 binding domains of the present disclosure have improved ability to activate downstream immune effector functions such as antibody-dependent cellular cytotoxicity (ADCC).

[0192] In some embodiments, the FLT3 binding domain binds to a human FLT3 protein comprising the sequence set forth in SEQ ID NO: 3215 (UniProt ID: P36888). In some embodiments, the FLT3 binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 3215 (UniProt ID: P36888).

[0193] In some embodiments, the FLT3 binding domain disclosed herein recognizes full-length FLT3 (e.g., an FLT3 protein comprising the sequence of SEQ ID NO: 3215 (UniProt ID: P36888)). In certain examples, the FLT3 binding domain disclosed herein recognizes an epitope within FLT3, e.g., in some cases, the FLT3 binding protein interacts with one or more amino acids found within a domain of human FLT3. The epitope to which the antibody binds may consist of a single contiguous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more) amino acids located within a domain of FLT3 (e.g., an FLT3 protein comprising the sequence of SEQ ID NO: 3215 (UniProt ID: P36888)). Alternatively, an epitope may consist of multiple non-contiguous amino acids (or a sequence of amino acids) located within a domain of FLT3 (eg, an FLT3 protein comprising the sequence of SEQ ID NO: 3215 (UniProt ID: P36888)).

[0194] In some embodiments, the FLT3 binding protein of the present disclosure binds to the full-length FLT3 protein or to a fragment thereof, such as an epitope containing fragment within the full-length FLT3 protein, as described above. Optionally, the epitope-containing fragment comprises an antigenic or immunogenic fragment of FLT3 protein and its derivatives. In some embodiments, the epitope-containing fragment, including the antigenic or immunogenic fragment, is 12 amino acids or more, such as 20 amino acids or more, 50 or 100 amino acids or more. In some embodiments, the FLT3 fragment comprises 95% or more, 90% or more, 75%, 50%, 25%, or 10% or more of the length of the full protein. In some embodiments, the epitope-containing fragment of FLT3, including the antigenic or immunogenic fragment, can induce a relevant immune response in a patient. In some embodiments, derivatives of FLT3 include sequence variants in which one or more (e.g., 1-20, e.g., 15 amino acids, or up to 20%, such as up to 10%, 5%, or 1% of the number of amino acids based on the total length of the protein) have been deleted, inserted, or substituted relative to the FLT3 sequence (e.g., an FLT3 protein comprising the sequence of SEQ ID NO: 3215 (UniProt ID: P36888)).

[0195] In some embodiments, the affinity matured FLT3 binding molecule comprises an affinity for FLT3 protein (such as human FLT3 protein) that is equal to or greater than that of the parent FLT3 binding molecule, but has reduced or, in some embodiments, increased cross-reactivity to selected substances, such as ligands, proteins, antigens, etc., other than the FLT3 epitope for which the parent FLT3 binding molecule is specific or designed to be specific. Regarding the latter, the affinity matured FLT3 binding molecule is, in some embodiments, more successfully tested against animal models when it is reacted with both human FLT3 and the corresponding animal model target, e.g., mouse FLT3 or cynomolgus monkey FLT3.

[0196] Another example of a substitution to create a variant anti-FLT3 antibody or antigen-binding fragment thereof is to replace one or more hypervariable regions of a parent antibody or antigen-binding fragment thereof. In general, variants are subsequently selected based on the improvement of a desired property compared to the parent antibody, such as increased affinity, decreased affinity, decreased immunogenicity, or increased pH-dependence of binding.

[0197] In some embodiments, the FLT3 binding domain is a polypeptide comprising an amino acid sequence composed of four framework regions / sequences (f1-f4) interrupted by three complementarity determining regions / sequences, as represented by the formula: f1-r1-f2-r2-f3-r3-f4, where r1, r2, and r3 are complementarity determining regions CDR1, CDR2, and CDR3, respectively, and f1, f2, f3, and f4 are framework residues. Framework residues of the FLT3 binding proteins of the disclosure include, for example, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, or 94 amino acid residues, and complementarity determining regions include, for example, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 amino acid residues.

[0198] In some embodiments, the binding proteins described herein include polypeptides having a sequence selected from SEQ ID NOs: 1004-1079 and 3495-3496, their subsequences, and variants thereof. In some embodiments, the FLT3 binding domain comprises at least 75%-95% or more homology to a sequence selected from SEQ ID NOs: 1004-1079 and 3495-3496, their subsequences, and variants thereof. In some embodiments, the FLT3 binding protein comprises at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to a sequence selected from SEQ ID NOs: 1004-1079 and 3495-3496, their subsequences, and variants thereof. In some embodiments, the FLT3 binding domain comprises at least 60%-95% or more identity to a sequence selected from SEQ ID NOs: 1004-1079 and 3495-3496, their subsequences, and variants thereof. In some embodiments, the FLT3 binding protein comprises at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to a sequence selected from SEQ ID NOs: 1004-1079, sequels thereof, and variants thereof.

[0199] In some embodiments, CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1080-1155 and 3497-3498, or a sequence comprising one or more amino acid substitutions in a sequence selected from the group consisting of SEQ ID NOs: 1080-1155 and 3497-3498. In some embodiments, CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1156-1231 and 3499-3500, or a sequence comprising one or more amino acid substitutions in a sequence selected from the group consisting of SEQ ID NOs: 1156-1231 and 3499-3500. In some embodiments, CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1232-1307 and 3501-3502, or a sequence comprising one or more amino acid substitutions in a sequence selected from the group consisting of SEQ ID NOs: 1232-1307 and 3501-3502. In some embodiments, CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1150, 1152, 3497, and 3498, or a sequence comprising one or more amino acid substitutions in a sequence selected from the group consisting of SEQ ID NOs: 1150, 1152, 3497, and 3498. In some embodiments, CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1226, 1228, 3499, and 3500, or a sequence comprising one or more amino acid substitutions in a sequence selected from the group consisting of SEQ ID NOs: 1226, 1228, 3499, and 3500. In some embodiments, CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1302, 1304, 3501, and 3502, or a sequence comprising one or more amino acid substitutions in a sequence selected from the group consisting of SEQ ID NOs: 1293 or 1302.

[0200] In various embodiments, the FLT3 binding domain of the present disclosure is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOs: 1004-1079 and 3495-3496.

[0201] In various embodiments, the complementarity determining regions of the FLT3 binding domain of the present disclosure are at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1080-1155 and 3497-3498.

[0202] In various embodiments, the complementarity determining regions of the FLT3 binding domain of the present disclosure are at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1156-1231 and 3499-3500.

[0203] In various embodiments, the complementarity determining regions of the FLT3 binding domain of the present disclosure are at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1232-1307 and 3501-3502.

[0204] In various embodiments, the complementarity determining regions of the FLT3 binding domain of the present disclosure are at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1074, 1076, 3495, and 3496, and the FLT3 binding domain comprises a humanized FLT3 binding domain.

[0205] In some embodiments, all of the aforementioned FLT3 binding domains (e.g., anti-FLT3 single domain antibodies of SEQ ID NOs: 1004-1079) are affinity peptide tagged to facilitate purification. In some embodiments, the affinity peptide tag is six consecutive histidine residues referred to as 6X-his (SEQ ID NO: 3503). In certain embodiments, the FLT3 binding domains of the present disclosure preferentially bind membrane-bound FLT3 over soluble FLT3. Membrane-bound FLT3 refers to the presence of FLT3 in or on the cell membrane surface of cells that express FLT3. Soluble FLT3 refers to FLT3 that is no longer present in or on the cell membrane surface of cells that express or have expressed FLT3. In certain examples, soluble FLT3 is present in the blood and / or lymphatic circulation of a subject. In one embodiment, the FLT3 binding domain binds to membrane-bound FLT3 at least 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, or 1000-fold higher than soluble FLT3. In one embodiment, the FLT3 binding protein of the present disclosure preferentially binds to membrane-bound FLT3 30-fold higher than soluble FLT3. The preferential binding of an antigen binding protein to membrane-bound FLT3 over soluble FLT3 can be easily determined using binding assays well known in the art.

[0206] In some embodiments, it is contemplated that the FLT3 binding protein is fairly small, 25 kDa or less, 20 kDa or less, 15 kDa or less, or in some embodiments, 10 kDa or less. In certain instances, the FLT3 binding protein is 5 kDa or less when it is a peptide or small molecule entity.

[0207] In other embodiments, the FLT3 binding proteins described herein comprise a small molecular entity (SME) binder for FLT3. The SME binder is a small molecule averaging about 500-2000 Da in size and is attached to the FLT3 binding protein by known methods such as sortase ligation or conjugation. In these examples, the FLT3 binding protein comprises a domain that includes a sortase recognition sequence, e.g., LPETG (SEQ ID NO: 3200). To bind the SME binder to the FLT3 binding protein that includes a sortase recognition sequence, the protein is incubated with sortase and the SME binder, whereby the sortase binds the SME binder to the recognition sequence. In yet other embodiments, the FLT3 binding proteins described herein comprise a knottin peptide for FLT3 binding. Knottins are disulfide-stabilized peptides with a cysteine ​​knot scaffold and have an average size of about 3.5 kDa. Knottins are intended to bind to certain tumor molecules such as FLT3. In further embodiments, the FLT3 binding proteins described herein comprise a naturally occurring FLT3 ligand.

[0208] In some embodiments, the FLT3 binding protein comprises more than one domain and is in a single polypeptide design with flexible linkage of the domains. This allows for easy production and manufacturing of the FLT3 binding protein, because it can be encoded by a single cDNA molecule that is easy to incorporate into a vector. Furthermore, in some embodiments where the FLT3 binding protein described herein is a monomeric single polypeptide chain, there is no challenge or requirement for chain pairing in dimerization. In such embodiments, it is contemplated that the FLT3 binding protein described herein is less prone to aggregation.

[0209] In some cases, FLT3 binding proteins contain more than one domain, and domains within the FLT3 binding proteins are conjugated using enzymatic, site-specific conjugation methods, including the use of mammalian or bacterial transglutaminase enzymes. Microbial transglutaminase (mTG) is a versatile tool in modern research and biotechnology. The availability of large amounts of relatively pure enzyme, ease of use, and lack of regulation by calcium and guanosine-5'-triphosphate (GTP) have propelled mTG to become the primary cross-linking enzyme used in both the food industry and biotechnology. Currently, mTG is used in many applications to conjugate proteins and peptides to small molecules, polymers, surfaces, DNA, as well as other proteins. See, for example, Veracity of microbial transglutaminase by Pavel Strp, Bioconjugate Chem. 25, 5, 855-862.

[0210] In some examples, FLT3 binding proteins are provided that contain more than one domain, one of which contains an acceptor glutamine in the constant region that can then be conjugated to another domain via a lysine-based linker (e.g., any primary amine chain that is a substrate for TGase, including, for example, alkylamines and oxoamines), with the conjugation occurring exclusively to one or more acceptor glutamine residues present in the targeting moiety that is external to the antigen binding site (e.g., outside the variable region, within the constant region). Thus, conjugation does not occur to glutamines within the variable region, e.g., glutamines that are at least partially surface exposed. In some examples, the FLT3 binding protein is formed by reacting one of the domains with a lysine-based linker in the presence of TGase.

[0211] In some embodiments where one or more domains in the FLT3 binding protein are directly linked, a hybrid vector is created in which the DNA encoding the directly linked domains are directly ligated to each other themselves. In some embodiments where a linker is used, a hybrid vector is created in which the DNA encoding one domain is ligated to the DNA encoding one end of a linker portion, and the DNA encoding another domain is ligated to the other end of the linker portion.

[0212] In certain embodiments, the FLT3 binding protein according to the present disclosure may be incorporated into an immune cell binding protein. In some embodiments, the immune cell binding protein comprises a CD3 binding domain, a half-life extension domain, and an FLT3 binding domain according to the present disclosure. In some embodiments, the FLT3 binding trispecific protein comprises a trispecific antibody.

[0213] EpCAM-binding protein Described herein is an immune cell-binding protein that binds to EpCAM, its pharmaceutical composition, and the nucleic acid, recombinant expression vector, and host cell for producing such protein.Also provided is a method of using the EpCAM-binding protein of the present disclosure in the prevention and / or treatment of disease, illness, and disorder.In some embodiments, the EpCAM-binding protein is a part of an immune cell-binding protein that comprises the EpCAM-binding domain described herein.

[0214] Epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein expressed in the majority of normal human epithelia and overexpressed in most carcinomas. This molecule is responsible for cell-cell adhesion, as well as signal transduction, cell migration, proliferation, and differentiation. EpCAM has therefore been a target of immunotherapy in clinical trials for several solid tumors. EpCAM has been found to play an important role in the detection and isolation of circulating tumor cells (CTCs). EpCAM has been found to be beneficial in the diagnosis and treatment of various carcinomas in various studies. Furthermore, in many cases, tumor cells have been observed to express EpCAM to a much higher extent than their parental epithelium or to a lower extent than the aggressive forms of the aforementioned cancers. For example, EpCAM expression has been shown to be significantly higher on neoplastic tissues and in adenocarcinomas than in normal prostate epithelium (n=76, p<0.0001), supporting that increased EpCAM expression represents an early event in the development of the prostate. See Poczatek, J Urol., 1999, 162, 1462-1644. Furthermore, in the majority of both squamous and cervical adenocarcinomas, strong EpCAM expression has been shown to correlate with increased proliferation and loss of markers for terminal differentiation. See Litvinov, Am. J. Pathol. 1996, 148, 865-75. One example in which overexpression of EpCAM on tumor cells is a predictor of survival is breast cancer. See Gastl, Lancet. 2000, 356, 1981-1982. Furthermore, EpCAM has been described as a marker for the detection of disseminated tumor cells in patients with squamous cell carcinoma of the head, neck, and lung. See Chaubal, Anticancer Res 1999, 19, 2237-2242; Piyathilake, Hum Pathol. 2000, 31, 482-487. Normal squamous epithelium, such as that found in the epidermis, oral cavity, epiglottis, pharynx, larynx, and esophagus, did not significantly express EpCAM. See Quak, Hybridoma, 1990, 9, 377-387.

[0215] EpCAM is expected to play a role in adhering epithelial cells in an oriented and highly ordered manner. See Litvinov, J Cell Biol. 1997, 139, 1337-1348. After malignant transformation of epithelial cells, the rapidly growing tumor cells are expected to discard the high cell order of the epithelium. Accordingly, the surface distribution of EpCAM is expected to be less restricted and the molecule is better expressed on tumor cells. Due to their epithelial cell origin, tumor cells from most carcinomas are expected to express EpCAM on their surface.

[0216] EpCAM is a 314 amino acid, 40 kDa membrane-integrated glycoprotein with specific expression in certain epithelia and on many human carcinomas. See, for example, Balzar, J. Mol. Med. 1999, 77, 699-712. EpCAM was discovered by recognition by mouse monoclonal antibody 17-1A / edrecolomab and subsequently cloned. See Goettlinger, Int J Cancer. 1986, 38, 47-53, and Simon, Proc. Natl. Acad. Sci. USA. 1990, 87, 2755-2759. Monoclonal antibody 17-1A was generated by immunization of mice with human colon cancer cells. See Koprowski, Somatic Cell Genet. 1979, 5, 957-971. The EGF-like repeats of EpCAM have been found to mediate lateral and alternating interactions in homophilic cell adhesion (see, e.g., Balzar, Mol. Cell. Biol. 2001, 21, 2570-2580) and are therefore primarily located between epithelial cells (Litvinov, J Cell Biol. 1997, 139, 1337-1348; Balzar, J Mol Med. 1999, 77, 699-712; and Trebak, J Biol Chem. 2001, 276, 2299-2309).

[0217] EpCAM is also known by the following aliases: epithelial cell adhesion molecule, tumor-associated calcium signal transducer, major gastrointestinal tumor-associated protein GA733-2, adenocarcinoma-associated antigen, cell surface glycoprotein Trop-1, epithelial glycoprotein 314, TACSTD1, EGP314, MIC18, TROP1, M4S1, KSA, membrane component chromosome 4 surface marker (glycoprotein of 35 kD), antigen identified by monoclonal antibody AUA-1, human epithelial glycoprotein-2, epithelial cell surface antigen, epithelial glycoprotein, KS 1 / 4 antigen, CD326 antigen, GA722-2, HEGP314, HNPCC8, Ep-CAM, DIAR5, EGP-2, EGP40, KS1 / 4, MK-1, M1S2, ESA, and EGP. Exemplary protein sequences for EpCAM are provided in UniProtkB ID numbers P16422 and B5MCA4. In some embodiments, the EpCAM binding protein of the disclosure binds to the EpCAM sequence provided in UniProtkB ID number P16422 (SEQ ID NO: 478) or B5MCA4 (SEQ ID NO: 475).

[0218] In some embodiments, the EpCAM-binding domain binds to the extracellular domain of the mature EpCAM protein. The human extracellular domain sequence is provided in SEQ ID NO: 3212, the cynomolgus monkey extracellular domain sequence is provided in SEQ ID NO: 3213, and the mouse extracellular domain sequence is provided in SEQ ID NO: 3214.

[0219] In some embodiments, the EpCAM binding domain binds to a protein that comprises a truncated sequence compared to SEQ ID NO: 3208. In some embodiments, the EpCAM binding domain binds to a protein that comprises the sequence of SEQ ID NO: 3208. In some embodiments, the EpCAM binding domain binds to a protein that comprises a truncated sequence compared to SEQ ID NO: 3209. In some embodiments, the EpCAM binding domain binds to a protein that comprises the sequence of SEQ ID NO: 3209. In some embodiments, the EpCAM binding domain binds to a protein that comprises a truncated sequence compared to SEQ ID NO: 3210. In some embodiments, the EpCAM binding domain binds to a protein that comprises a truncated sequence compared to SEQ ID NO: 3210. In some embodiments, the EpCAM binding domain binds to a protein that comprises a truncated sequence compared to SEQ ID NO: 478. In some embodiments, the EpCAM binding domain binds to a protein that comprises a truncated sequence compared to SEQ ID NO: 3211. In some embodiments, the EpCAM binding domain binds to a protein that comprises a truncated sequence compared to SEQ ID NO: 3212. In some embodiments, the EpCAM binding domain binds to a protein that comprises a truncated sequence compared to SEQ ID NO: 3212. In some embodiments, the EpCAM binding domain binds to a protein that comprises a truncated sequence compared to SEQ ID NO: 3213. In some embodiments, the EpCAM binding domain binds to a protein that comprises a truncated sequence compared to SEQ ID NO: 3213. In some embodiments, the EpCAM binding domain binds to a protein that comprises a truncated sequence compared to SEQ ID NO: 3214. In some embodiments, the EpCAM binding domain binds to a protein that comprises a truncated sequence compared to SEQ ID NO: 3214.

[0220] In some embodiments, the EpCAM binding domain disclosed herein recognizes full-length EpCAM.In certain examples, the EpCAM binding domain disclosed herein recognizes an epitope in EpCAM, for example, in some cases, the EpCAM binding protein interacts with one or more amino acids found in the domain of human EpCAM.The epitope that the antibody binds to can consist of a single continuous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more) amino acids located in the domain of EpCAM.Alternatively, the epitope can consist of multiple non-contiguous amino acids (or amino acid sequences) located in the domain of EpCAM.

[0221] In some embodiments, the EpCAM binding protein of the present disclosure binds to the full-length EpCAM protein or to a fragment thereof, such as an epitope containing fragment within the full-length EpCAM protein, as described above. Optionally, the epitope-containing fragment comprises an antigenic or immunogenic fragment of the EpCAM protein and its derivatives. In some embodiments, the epitope-containing fragment, including the antigenic or immunogenic fragment, is 12 amino acids or more, such as 20 amino acids or more, 50 or 100 amino acids or more. In some embodiments, the EpCAM fragment comprises 95% or more, 90% or more, 75%, 50%, 25%, or 10% or more of the length of the full protein. In some embodiments, the epitope-containing fragment of EpCAM, including the antigenic or immunogenic fragment, can induce a relevant immune response in a patient. In some embodiments, derivatives of EpCAM include sequence variants in which one or more (e.g., 1-20, e.g., 15 amino acids, or up to 20%, such as up to 10%, 5%, or 1% of the number of amino acids based on the total length of the protein) have been deleted, inserted, or substituted relative to the EpCAM sequence provided in SEQ ID NOs: 3208-3214.

[0222] Another example of a substitution to create a variant anti-EpCAM antibody or antigen-binding fragment thereof is to substitute one or more hypervariable regions of a parent antibody or antigen-binding fragment thereof. In general, variants are subsequently selected based on the improvement of a desired property compared to the parent antibody or antigen-binding fragment thereof, such as increased affinity, decreased affinity, decreased immunogenicity, or increased pH-dependence of binding.

[0223] In some embodiments, the EpCAM-binding domain is a polypeptide comprising an amino acid sequence composed of four framework regions / sequences (f1-f4) interrupted by three complementarity determining regions / sequences, as represented by the formula: f1-r1-f2-r2-f3-r3-f4, where r1, r2, and r3 are complementarity determining regions CDR1, CDR2, and CDR3, respectively, and f1, f2, f3, and f4 are framework residues. Framework residues of the EpCAM-binding proteins of the disclosure include, for example, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, or 94 amino acid residues, and complementarity determining regions include, for example, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 amino acid residues. In some embodiments, the EpCAM-binding domain includes an amino acid sequence selected from SEQ ID NOs: 961-1003.

[0224] In some embodiments, the EpCAM-binding protein comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to a sequence selected from SEQ ID NOs: 961-1003, sequels and variants thereof. In some embodiments, the EpCAM-binding domain comprises at least 70%-95% or more identity to a sequence selected from SEQ ID NOs: 961-1003, sequels and variants thereof.

[0225] In some embodiments, CDR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 847-884, or a sequence containing one or more substitutions compared to a sequence selected from the group consisting of SEQ ID NOs: 847-884. In some embodiments, CDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 885-922, or a sequence containing one or more substitutions compared to a sequence selected from the group consisting of SEQ ID NOs: 885-922. In some embodiments, CDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 923-960, or a sequence containing one or more substitutions compared to a sequence selected from the group consisting of SEQ ID NOs: 923-960.

[0226] In various embodiments, the complementarity determining regions of the EpCAM binding domain of the present disclosure are at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NOs:847-884.

[0227] In various embodiments, the complementarity determining regions of the EpCAM binding domain of the present disclosure are at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NOs:885-922.

[0228] In various embodiments, the complementarity determining regions of the EpCAM binding domain of the present disclosure are at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequences set forth in SEQ ID NOs:923-960.

[0229] In some embodiments, all of the aforementioned EpCAM binding domains (e.g., anti-EpCAM single domain antibodies of SEQ ID NOs: 961-1003) are affinity peptide tagged to facilitate purification. In some embodiments, the affinity peptide tag is six consecutive histidine residues, designated 6X-his (SEQ ID NO: 3503).

[0230] In certain embodiments, the EpCAM-binding domain of the present disclosure preferentially binds to membrane-bound EpCAM over soluble EpCAM. Membrane-bound EpCAM refers to the presence of EpCAM in or on the cell membrane surface of cells that express EpCAM. Soluble EpCAM refers to EpCAM that is no longer present in or on the cell membrane surface of cells that express or have expressed EpCAM. In certain examples, soluble EpCAM is present in the blood and / or lymph circulation of a subject. In one embodiment, the EpCAM-binding domain binds to membrane-bound EpCAM at least 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, or 1000-fold higher than soluble EpCAM. In one embodiment, the EpCAM-binding protein of the present disclosure preferentially binds to membrane-bound EpCAM 30-fold higher than soluble EpCAM. The preferential binding of an antigen-binding protein to membrane-bound EpCAM over soluble EpCAM can be easily determined using binding assays well known in the art.

[0231] In some embodiments, it is contemplated that the EpCAM binding protein is fairly small, 25 kDa or less, 20 kDa or less, 15 kDa or less, or in some embodiments, 10 kDa or less. In certain examples, the EpCAM binding protein is 5 kDa or less when it is a peptide or small molecule entity.

[0232] In other embodiments, the EpCAM binding proteins described herein comprise small molecular entity (SME) binders for EpCAM. SME binders are small molecules averaging about 500-2000 Da in size that are attached to the EpCAM binding proteins by known methods such as sortase ligation or conjugation. In these examples, the EpCAM binding proteins comprise a domain that includes a sortase recognition sequence, e.g., LPETG (SEQ ID NO: 3200). To bind the SME binder to an EpCAM binding protein that includes a sortase recognition sequence, the protein is incubated with a sortase and an SME binder, whereby the sortase binds the SME binder to the recognition sequence. In yet other embodiments, the EpCAM binding proteins described herein comprise a knottin peptide for EpCAM binding. Knottins are disulfide-stabilized peptides with a cysteine ​​knot scaffold and have an average size of about 3.5 kDa. Knottins are contemplated for binding to certain tumor molecules, such as EpCAM. In further embodiments, the EpCAM binding proteins described herein comprise a natural EpCAM ligand.

[0233] In some embodiments, the EpCAM-binding protein comprises more than one domain and is a single polypeptide design with flexible linkage of the domains. This allows for easy production and manufacture of the EpCAM-binding protein, because it can be encoded by a single cDNA molecule that is easy to incorporate into a vector. Furthermore, in some embodiments where the EpCAM-binding protein described herein is a monomeric single polypeptide chain, there is no challenge or requirement for chain pairing in dimerization. In such embodiments, it is contemplated that the EpCAM-binding protein described herein is less prone to aggregation.

[0234] In EpCAM-binding proteins that contain more than one domain, the domains are linked by one or more internal linkers. In certain embodiments, the internal linker is "short", i.e., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues. Thus, in certain examples, the internal linker is about 12 or less amino acid residues. If the amino acid residue is 0, the internal linker is a peptide bond. In certain embodiments, the internal linker is "long", i.e., 15, 20, or 25 amino acid residues. In some embodiments, the internal linker is about 3 to about 15, e.g., 8, 9, or 10 adjacent amino acid residues. With regard to the amino acid composition of the internal linker, peptides are selected that have properties that provide flexibility to the EpCAM-binding protein and do not interfere with the binding domains or resist cleavage from proteases. For example, glycine and serine residues generally provide protease resistance. Examples of internal linkers suitable for linking domains in EpCAM-binding proteins include (GS) n (SEQ ID NO: 3859), (GGS) n (SEQ ID NO: 3860), (GGGS) n (SEQ ID NO:3861), (GGSG) n (SEQ ID NO: 3862), (GGSGG) n (SEQ ID NO: 3863), (GGGGS) n (SEQ ID NO:3864), (GGGGG) n(SEQ ID NO: 3865), or (GGG) n (SEQ ID NO:3866), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the linker is (GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO:3867), (GGGGSGGGGSGGGGS) (SEQ ID NO:3868), LPETG (SEQ ID NO:3869), (GGGGSGGGS) (SEQ ID NO:3871), or SGGG (SEQ ID NO:3872).

[0235] In some cases, the EpCAM-binding protein contains more than one domain, and domains within the EpCAM-binding protein are conjugated using enzymatic, site-specific conjugation methods, including the use of mammalian or bacterial transglutaminase enzymes. Microbial transglutaminase (mTG) is a versatile tool in modern research and biotechnology. The availability of large amounts of relatively pure enzyme, ease of use, and lack of regulation by calcium and guanosine-5'-triphosphate (GTP) have propelled mTG to become the primary cross-linking enzyme used in both the food industry and biotechnology. mTG is currently used in many applications to conjugate proteins and peptides to small molecules, polymers, surfaces, DNA, as well as other proteins. See, for example, Veracity of microbial transglutaminase by Pavel Strp, Bioconjugate Chem. 25, 5, 855-862.

[0236] In some examples, an EpCAM-binding protein is provided that includes more than one domain, one of which includes an acceptor glutamine in the constant region that can then be conjugated to another domain via a lysine-based linker (e.g., any primary amine chain that is a substrate for TGase, including, for example, alkylamines and oxoamines), with the conjugation occurring exclusively to one or more acceptor glutamine residues present in a targeting moiety that is external to the antigen-binding site (e.g., outside the variable region, within the constant region). Thus, conjugation does not occur to glutamines within the variable region, e.g., glutamines that are at least partially surface exposed. In some examples, the EpCAM-binding protein is formed by reacting one of the domains with a lysine-based linker in the presence of TGase.

[0237] In some embodiments where one or more domains in the EpCAM-binding protein are directly linked, a hybrid vector is created in which the DNA encoding the directly linked domains is directly ligated to itself. In some embodiments where a linker is used, a hybrid vector is created in which the DNA encoding one domain is ligated to the DNA encoding one end of a linker portion, and the DNA encoding another domain is ligated to the other end of the linker portion.

[0238] In certain embodiments, the EpCAM-binding protein according to the present disclosure may be incorporated into an immune cell-binding protein. In some embodiments, the immune cell-binding protein comprises a CD3-binding domain, a half-life extending domain, and an EpCAM-binding domain according to the present disclosure. In some embodiments, the immune cell-binding protein comprises a trispecific antibody.

[0239] CD3-binding domain The immune cell-binding proteins described herein comprise an immune cell-binding domain. In some embodiments, the immune cell-binding domain comprises a natural killer (NK) cell-binding domain, a T cell-binding domain, a B cell-binding domain, a dendritic cell-binding domain, a macrophage cell-binding domain, or a combination thereof. In some embodiments, the immune cell-binding protein comprises a T cell-binding domain. In some embodiments, the T cell-binding domain is a CD3-binding domain.

[0240] The specificity of T cell responses is mediated by the recognition of antigens (displayed in the context of the major histocompatibility complex, or MHC) by the T cell receptor complex. As part of the T cell receptor complex, CD3 is a protein complex that includes the CD3γ (gamma) chain, the CD3δ (delta) chain, and two CD3ε (epsilon) chains present on the cell surface. CD3 integrally associates with the α (alpha) and β (beta) chains of the T cell receptor (TCR), and the CD3ζ (zeta) chain, to comprise the T cell receptor complex. Clustering of CD3 on T cells, such as by immobilized anti-CD3 antibodies, results in T cell activation similar to T cell receptor engagement but independent of the specificity typical of that clone.

[0241] In one aspect, the single chain variable fragment CD3 binding proteins described herein comprise a domain that specifically binds to CD3. In one aspect, the single chain variable fragment CD3 binding proteins described herein comprise a domain that specifically binds to human CD3. In one aspect, the single chain variable fragment CD3 binding proteins described herein comprise a domain that specifically binds to cynomolgus CD3. In one aspect, the single chain variable fragment CD3 binding proteins described herein comprise a domain that specifically binds to human CD3 and cynomolgus CD3. In some embodiments, the single chain variable fragment CD3 binding proteins described herein comprise a domain that specifically binds to CD3γ. In some embodiments, the single chain variable fragment CD3 binding proteins described herein comprise a domain that specifically binds to CD3δ. In some embodiments, the single chain variable fragment CD3 binding proteins described herein comprise a domain that specifically binds to CD3ε.

[0242] In another aspect, an immune cell binding protein is provided that comprises a single-chain variable fragment CD3 binding protein according to the present disclosure. In some embodiments, the immune cell binding protein comprises a single-chain variable fragment CD3 binding protein according to the present disclosure specifically binds to a T cell receptor (TCR). In a particular example, the immune cell binding protein comprises a single-chain variable fragment CD3 binding protein according to the present disclosure binds to the α chain of the TCR. In a particular example, the immune cell binding protein comprises a single-chain variable fragment CD3 binding protein according to the present disclosure binds to the β chain of the TCR.

[0243] In certain embodiments, the CD3 binding domain of the immune cell binding protein described herein not only exhibits strong CD3 binding affinity to human CD3, but also exhibits excellent cross-reactivity with the respective cynomolgus CD3 protein. In some examples, the CD3 binding domain of the immune cell binding protein binding protein is cross-reactive to cynomolgus CD3. In certain examples, the Kd for binding to human CD3 (hKd) is approximately the same as the Kd for binding to cynomolgus CD3 (cKd). In certain examples, the ratio of hKd to cKd (hKd:cKd) is between about 20:1 and about 1:2.

[0244] In some embodiments, the CD3 binding domain of the immune cell binding protein can be any domain that binds to CD3, including but not limited to domains derived from monoclonal, polyclonal, recombinant, human, or humanized antibodies. In some instances, it is beneficial for the CD3 binding domain to be derived from the same species in which the immune cell binding protein will ultimately be used. For example, for human use, it may be beneficial for the CD3 binding domain to include human or humanized residues derived from the antigen binding domain of an antibody or antibody fragment.

[0245] Thus, in one aspect, the antigen binding domain comprises a human or humanized antibody or antibody fragment, or a murine antibody or antibody fragment. In one embodiment, the humanized or human anti-CD3 binding domain comprises one or more (e.g., all three) of the light chain complementarity determining region 1 (LC CDR1), the light chain complementarity determining region 2 (LC CDR2), and the light chain complementarity determining region 3 (LC CDR3) of a humanized or human anti-CD3 binding domain described herein, and / or one or more (e.g., all three) of the heavy chain complementarity determining region 1 (HC CDR1), the heavy chain complementarity determining region 2 (CDR2), and the heavy chain complementarity determining region 3 (CDR3) of a humanized or human anti-CD3 binding domain described herein, e.g., the humanized or human anti-CD3 binding domain comprises one or more, e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs.

[0246] In some embodiments, the humanized or human anti-CD3 binding domain comprises a humanized or human light chain variable region specific for CD3, where the light chain variable region specific for CD3 comprises human or non-human light chain CDRs in a human light chain framework region. In a particular example, the light chain framework region is a λ (lambda) light chain framework. In another example, the light chain framework region is a κ (kappa) light chain framework.

[0247] In some embodiments, the humanized or human anti-CD3 binding domain comprises a humanized or human heavy chain variable region specific for CD3, where the heavy chain variable region specific for CD3 comprises human or non-human heavy chain CDRs in a human heavy chain framework region.

[0248] In particular examples, the complementarity determining regions of the heavy and / or light chains are selected from the group consisting of, for example, muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), SP34, TR-66 or X35-3, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5 , F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, and WT-31 were derived from known anti-CD3 antibodies.

[0249] In one embodiment, the anti-CD3 binding domain is a single chain variable fragment (scFv) comprising a light chain and a heavy chain of the amino acid sequence provided herein. As used herein, "single chain variable fragment" or "scFv" refers to an antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, where the variable regions of the light and heavy chains are contiguously linked via a short, flexible polypeptide linker and can be expressed as a single polypeptide chain, and the scFv retains the specificity of the intact antibody from which it is derived. In one embodiment, the anti-CD3 binding domain comprises a light chain variable region comprising an amino acid sequence having one, two, or three or more and not more than 30, 20, or 10 modifications (e.g., substitutions) relative to the amino acid sequence of the light chain variable region provided herein, or a sequence having 95-99% identity to the amino acid sequence provided herein, and / or a heavy chain variable region comprising an amino acid sequence having one, two, or three or more and not more than 30, 20, or 10 modifications (e.g., substitutions) relative to the amino acid sequence of the heavy chain variable region provided herein, or a sequence having 95-99% identity to the amino acid sequence provided herein. In one embodiment, the humanized or human anti-CD3 binding domain is an scFv, and the light chain variable region comprising the amino acid sequence described herein is linked to the heavy chain variable region comprising the amino acid sequence described herein via an scFv linker. The light chain variable region and heavy chain variable region of the scFv can be in either the configuration of light chain variable region-scFv linker-heavy chain variable region or heavy chain variable region-scFv linker-heavy chain variable region.

[0250] In some examples, scFvs that bind to CD3 are prepared by known methods. For example, scFv molecules can be produced by linking together VH and VL regions using a flexible polypeptide linker. The scFv molecules comprise scFv linkers that are optimized in length (e.g., Ser-Gly linkers) and / or amino acid composition. Thus, in some embodiments, the length of the scFv linker is such that the VH or VL domain can intermolecularly associate with other variable domains to form a CD3 binding site. In certain embodiments, such scFv linkers are "short", i.e., comprise 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues. Thus, in certain examples, the scFv linker comprises about 12 or less amino acid residues. When the amino acid residue is 0, the scFv linker is a peptide bond. In some embodiments, these scFv linkers comprise about 3 to about 15, e.g., 8, 10, or 15, adjacent amino acid residues. Regarding the amino acid composition of the scFv linker, a peptide is selected that provides flexibility but does not interfere with the variable domains, and yet allows interchain folding to bring the two variable domains together to form a functional CD3 binding site. For example, scFv linkers containing glycine and serine residues generally provide protease resistance. In some embodiments, the linker in the scFv contains glycine and serine residues. The amino acid sequence of the scFv linker can be optimized, for example, by phage display methods to improve the CD3 binding and production yield of the scFv. An example of a peptide scFv linker suitable for linking the light chain variable domain and the heavy chain variable domain in an scFv is (GS) n (SEQ ID NO: 3859), (GGS) n (SEQ ID NO: 3860), (GGGS) n (SEQ ID NO:3861), (GGSG) n (SEQ ID NO: 3862), (GGSGG) n (SEQ ID NO: 3863), (GGGGS) n (SEQ ID NO:3864), (GGGGG) n (SEQ ID NO: 3865), or (GGG) n(SEQ ID NO:3866), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the linker is (GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO:3867), (GGGGSGGGGSGGGGS) (SEQ ID NO:3868), LPETG (SEQ ID NO:3869), (GGGGSGGGS) (SEQ ID NO:3871), or SGGG (SEQ ID NO:3872). Variation in linker length may retain or improve activity, resulting in superior efficacy in activity testing.

[0251] In some embodiments, the CD3 binding domain of the immune cell binding protein has an affinity for CD3 on CD3 expressing cells with a Kd of 1000 nM or less, 500 nM or less, 200 nM or less, 100 nM or less, 80 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, or 0.5 nM or less. In some embodiments, the CD3 binding domain of the single chain variable fragment CD3 binding protein has an affinity for CD3 epsilon, gamma, or delta with a Kd of 1000 nM or less, 500 nM or less, 200 nM or less, 100 nM or less, 80 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, or 0.5 nM or less. In further embodiments, the CD3 binding domain of the single chain variable fragment CD3 binding protein has a low affinity for CD3, i.e., about 100 nM or more.

[0252] In certain embodiments, the single chain variable fragment CD3 binding proteins described herein bind to human CD3 having a human Kd (hKd) and to cynomolgus CD3 having a cynomolgus Kd (cKd). In some embodiments, the hKd and cKd are between about 1 nM and about 2 nM, between about 3 nM and about 5 nM, between about 6 nM and about 10 nM, between about 11 nM and about 20, between about 25 nM and about 40 nM, between about 40 nM and about 60 nM, between about 70 nM and about 90 nM, between about 100 nM and about 120 nM, between about 125 nM and about 140 nM, between about 145 nM and about 160 nM, between about 170 nM and about 200 nM, between about 210 nM and about 250 nM, or between about 260 nM and about 300 nM.

[0253] In some embodiments, the hKd and cKd of the single chain variable fragment CD3 binding protein are approximately the same as the Kd of the CD3 binding protein having the sequence set forth in SEQ ID NO: 3167. In some embodiments, the hKd and cKd of the single chain variable fragment CD3 binding protein are about 1.1-fold to about 1.5-fold the Kd of the CD3 binding protein having the sequence set forth in SEQ ID NO: 3167. In some embodiments, the hKd and cKd of the single chain variable fragment CD3 binding protein are about 1.5-fold to about 2-fold the Kd of the CD3 binding protein having the sequence set forth in SEQ ID NO: 3167. In some embodiments, the hKd and cKd of the single chain variable fragment CD3 binding protein are about 2.5-fold to about 3-fold the Kd of the CD3 binding protein having the sequence set forth in SEQ ID NO: 3167. In some embodiments, the hKd and cKd of the single chain variable fragment CD3 binding protein are about 3-fold to about 5-fold higher than the Kd of the CD3 binding protein having the sequence set forth in SEQ ID NO: 3167. In some embodiments, the hKd and cKd of the single chain variable fragment CD3 binding protein are about 6-fold to about 15-fold higher than the Kd of the CD3 binding protein having the sequence set forth in SEQ ID NO: 3167. In some embodiments, the hKd and cKd of the single chain variable fragment CD3 binding protein are about 15-fold to about 20-fold higher than the Kd of the CD3 binding protein having the sequence set forth in SEQ ID NO: 3167. In some embodiments, the hKd and cKd of the single chain variable fragment CD3 binding protein are about 20-fold to about 50-fold higher than the Kd of the CD3 binding protein having the sequence set forth in SEQ ID NO: 3167. In some embodiments, the hKd and cKd of the single chain variable fragment CD3 binding protein are about 55-fold to about 70-fold higher than the Kd of the CD3 binding protein having the sequence set forth in SEQ ID NO: 3167. In some embodiments, the hKd and cKd of the single chain variable fragment CD3 binding protein are about 75-fold to about 100-fold higher than the Kd of the CD3 binding protein having the sequence set forth in SEQ ID NO: 3167.In some embodiments, the hKd and cKd of the single chain variable fragment CD3 binding protein is about 120-fold to about 200-fold higher than the Kd of a CD3 binding protein having the sequence set forth in SEQ ID NO:3167.

[0254] In some embodiments, the ratio of hKd to cKd (hKd:cKd) ranges from about 20:1 to about 1:2. The affinity of binding to CD3 can be determined by the ability of the single chain variable fragment CD3 binding protein itself or its CD3 binding domain to bind to CD3, e.g., coated on an assay plate, displayed on a microbial cell surface, in solution, etc. The binding activity of the single chain variable fragment CD3 binding protein itself or its CD3 binding domain of the present disclosure to CD3 can be analyzed by immobilizing the ligand (e.g., CD3), the single chain variable fragment CD3 binding protein, or its CD3 binding domain to beads, substrates, cells, etc. The agent can be added to the binding partner incubated for a period of time at a given temperature in an appropriate buffer. After washing to remove unbound material, the bound protein can be released, e.g., by SDS, high or low pH buffers, etc., and analyzed, e.g., by surface plasmon resonance (SPR).

[0255] In some embodiments, the single chain variable fragment CD3 binding protein has an amino acid sequence selected from SEQ ID NOs: 3153-3169. In various embodiments, the single chain variable fragment CD3 binding protein comprises an amino acid sequence that is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOs: 3153-3169.

[0256] Single Domain Serum Albumin Binding Protein The immune cell binding protein described herein is a half-life extending protein. In some embodiments, the immune cell binding protein comprises a domain that binds to serum albumin. In some embodiments, the serum albumin is human serum albumin (HSA).

[0257] Serum albumin is the most abundant blood protein in mammals, produced by the liver and dissolved in plasma. Albumin is essential for maintaining the tonic pressure required for proper fluid distribution between blood vessels and body tissues; without albumin, high pressure in blood vessels would pump more fluid to tissues. Albumin also acts as a plasma carrier by non-specifically binding some hydrophobic steroid hormones, and as a transport protein for hemin and fatty acids. Human serum albumin (HSA) (molecular weight about 67 kDa) is the most abundant in plasma, present at about 50 mg / ml (600 μM), and has a half-life of about 20 days in humans. HSA is responsible for maintaining plasma pH, contributes to colloidal blood pressure, functions as a carrier for many metabolites, and serves as the major drug transport protein in plasma. In some embodiments, the single domain serum albumin binding protein binds to HSA. In some embodiments, the single domain serum albumin binding protein binds to serum albumin protein of cynomolgus monkeys. In some embodiments, the single domain serum albumin binding protein binds to HSA and cynomolgus serum albumin protein. In some embodiments, the single domain serum albumin binding protein also binds to mouse serum albumin protein. In some embodiments, the binding affinity to mouse serum albumin is about 1.5-fold to about 20-fold weaker than that to human or cynomolgus serum albumin.

[0258] Non-covalent association with albumin extends the excretion half-life of short-lived proteins. For example, recombinant fusion of an albumin-binding domain with a Fab fragment resulted in a 25- and 58-fold reduction in in vivo clearance and a 26- and 37-fold increase in half-life when administered intravenously to mice and rabbits, respectively, compared to administration of the Fab fragment alone. In another example, when insulin was acylated with fatty acids to promote albumin association, a sustained effect was observed upon subcutaneous injection in rabbits or pigs. Collectively, these studies demonstrate the association between albumin binding and sustained action / serum half-life.

[0259] In some embodiments, the single domain serum albumin binding proteins described herein are single domain antibodies, such as a heavy chain variable domain (VH) or a variable domain (VHH) of a camelid-derived sdAb, peptide, ligand, or small molecule entity specific for serum albumin. In some embodiments, the single domain serum albumin binding proteins described herein are single domain antibodies, such as a heavy chain variable domain (VH) or a variable domain (VHH) of a camelid-derived sdAb, peptide, ligand, or small molecule entity specific for HSA. In some embodiments, the serum albumin binding domain of the single domain serum albumin binding proteins described herein is any domain that binds to serum albumin, including but not limited to domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, and humanized antibodies. In certain embodiments, the serum albumin binding domain is a single domain antibody. In other embodiments, the serum albumin binding domain is a peptide. In further embodiments, the serum albumin binding domain is a small molecule. It is contemplated that single domain serum albumin binding proteins are fairly small, being 25 kD or less, 20 kD or less, 15 kD or less, or in some embodiments 10 kD or less. In certain instances, single domain serum albumin binding proteins are 5 kD or less when they are peptides or small molecule entities.

[0260] In some embodiments, the single domain serum albumin binding protein described herein is a half-life prolonging domain that results in the modification of the pharmacodynamics and pharmacokinetics of the single domain serum albumin binding protein itself. As described above, the half-life prolonging domain extends the excretion half-life. The half-life prolonging domain also modifies the pharmacodynamic properties, including the modification of the tissue distribution, penetration, and diffusion of the single domain serum albumin binding protein. In some embodiments, the half-life prolonging domain improves tissue (including tumor) targeting, tissue distribution, tissue penetration, tissue diffusion in tissues, and improves efficacy, compared to proteins without the half-life prolonging domain. In one embodiment, the treatment method effectively and efficiently utilizes a reduced amount of the single domain serum albumin binding protein, resulting in reduced side effects, such as reduced non-tumor cytotoxicity.

[0261] Furthermore, the binding affinity of the single domain serum albumin binding protein to its binding target can be selected to target a specific excretion half-life in a particular single domain serum albumin binding protein. Thus, in some embodiments, the single domain serum albumin binding protein has a high binding affinity to its binding target. In other embodiments, the single domain serum albumin binding protein has a moderate binding affinity to its binding target. In yet other embodiments, the single domain serum albumin binding protein has a low or slight binding affinity to its binding target. Exemplary binding affinities include KD of 10 nM or less (high), between 10 nM and 100 nM (moderate), and above 100 nM (low). As described above, the binding affinity of the single domain serum albumin binding protein to its binding target is determined by known methods, such as surface plasmon resonance (SPR).

[0262] In some embodiments, the single domain serum albumin binding protein has an amino acid sequence selected from SEQ ID NOs: 3185-3193. In various embodiments, the single chain variable fragment CD3 binding protein comprises an amino acid sequence that is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOs: 3185-3193.

[0263] In some embodiments, the single domain serum albumin binding protein has an excretion half-life of at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 12 hours, at least 20 hours, at least 25 hours, at least 30 hours, at least 35 hours, at least 40 hours, at least 45 hours, at least 50 hours, or at least 100 hours.

[0264] Modification of immune cell-associated proteins The immune cell binding proteins described herein, including antigen binding domains and immune cell binding domains, encompass derivatives or analogs in which (i) amino acids are substituted with amino acid residues that are not encoded by the genetic code, (ii) the mature polypeptide is fused to another compound, such as polyethylene glycol, or (iii) additional amino acids are fused to the protein, such as a leader sequence, secretion sequence, or sequence for protein purification.

[0265] Exemplary modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0266] Modifications may occur anywhere in the immune cell-binding proteins described herein, including the peptide backbone, amino acid side chains, and amino or carboxyl termini.Specific common peptide modifications that are useful for modifying FLT3-binding proteins include glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, blocking of amino or carboxyl groups or both in polypeptides, covalent modification, and ADP-ribosylation.

[0267] In some embodiments, derivatives of the immune cell binding proteins described herein include immunoreactive modulator derivatives and antigen binding molecules that contain one or more modifications.

[0268] In some embodiments, the immune cell-binding proteins of the present disclosure are monovalent or multivalent (bivalent, trivalent, etc.). As used herein, the term "valency" refers to the number of potential target binding sites associated with an antibody. Each target binding site specifically binds to one target molecule, or to a specific position or locus on a target molecule. If an antibody is monovalent, each binding site on the molecule will specifically bind to a single antigen position or epitope. If an antibody contains more than one target binding site (multivalent), the target binding sites may each specifically bind to the same or different molecules (e.g., different ligands or different antigens, or different epitopes or positions on the same antigen).

[0269] In some embodiments, the immune cell binding proteins described above are fused to an Fc region from any species, including but not limited to human immunoglobulins such as human IgG1, human IgG2, human IgG3, human IgG4, etc., to generate an Fc-fusion FLT3 binding protein. In some embodiments, the Fc-fusion immune cell binding proteins of the disclosure have an extended half-life compared to an otherwise identical immune cell binding protein. In some embodiments, the Fc-fusion immune cell binding proteins of the disclosure include substitutions, mutations, and / or modifications, e.g., in the Fc region, inter alia, of one or more additional amino acid residues, resulting in a binding protein with favorable characteristics, including but not limited to, improved pharmacokinetics and extended serum half-life.

[0270] In some embodiments, such Fc-fusion immune cell binding proteins provide an increased half-life for mammals, such as humans, of more than 5 days, more than 10 days, more than 15 days, more than 20 days, more than 25 days, more than 30 days, more than 35 days, more than 40 days, more than 45 days, more than 2 months, more than 3 months, more than 4 months, or more than 5 months. In some cases, the increased half-life reduces the frequency of administration of the immune cell binding protein and / or increases the serum titer, which reduces the concentration of the antibody to be administered. The binding to human FcRn in vivo and the serum half-life of the human FcRn high affinity binding polypeptides are analyzed in some examples for genetically engineered mice or transfected human cell lines expressing human FcRn, or primates to which the polypeptides having variant Fc regions are administered.

[0271] In some cases, immune cell binding proteins are differentially modified during or after production, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to antibody molecules or other cellular ligands, etc. Any of a number of chemical modifications are carried out by techniques including, but not limited to, specific chemical cleavage with cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.

[0272] Various post-translational modifications of immune cell-binding proteins further encompassed by the present disclosure include, for example, N- or O-linked hydrocarbon chains, N- or C-terminal processing, attachment of chemical moieties to the amino acid backbone, chemical modifications of N- or O-linked hydrocarbon chains, and addition or deletion of N-terminal methionine residues as a result of expression in a prokaryotic host cell.Furthermore, the FLT3 binding proteins are optionally modified with a detectable label, such as an enzymatic, fluorescent, radioisotope, or affinity label, to allow for detection and isolation of the modulators.

[0273] Peptide Mask The sustained release binding proteins described herein, in some embodiments, comprise a masking peptide. When bound to the antigen binding domain of an immune cell binding protein, the masking peptide, in some embodiments, blocks, occludes, inhibits (e.g., reduces), or otherwise prevents (e.g., masks) the activity or binding of the antigen binding domain to a target. In some embodiments, the masking peptide interferes with binding of the antigen binding domain to a target antigen. In some embodiments, the immune cell binding protein comprises a CD3 binding domain, and the masking peptide is specific for the CD3 binding domain and interferes with binding of the CD3 binding domain to its target.

[0274] In particular examples, the masking peptide is covalently attached to the N-terminus or C-terminus of the immune cell binding protein, for example by a cleavable linker. In some embodiments, the masking peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 3663-3682, or a sequence comprising one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID NOs: 3663-3682.

[0275] In some embodiments, the masking peptide is a peptide that is 5 amino acids long, 6 amino acids long, 7 amino acids long, 8 amino acids long, 9 amino acids long, 10 amino acids long, 11 amino acids long, 12 amino acids long, 13 amino acids long, 14 amino acids long, or longer. In some embodiments, the masking peptide is at least 16 amino acids long. In some embodiments, the masking peptide is 3-12 amino acids long, 4-15 amino acids long, 5-20 amino acids long, or 6-25 amino acids long. In some embodiments, the masking peptide is 6 amino acids long. In some embodiments, the masking peptide is a linear peptide or a cyclic peptide. In some embodiments, 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 ​​located at or near the N-terminus and / or C-terminus of the masking peptide. In some embodiments, a disulfide bond connects the N-terminal cysteine ​​to the C-terminal cysteine.

[0276] In some embodiments, the masking peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 3663-3682, or a sequence containing one or more amino acid substitutions (e.g., two or three amino acid substitutions) relative to a sequence selected from the group consisting of SEQ ID NOs: 3663-3682.

[0277] Cleavable Linkers As described above, in some embodiments, the masking peptide and the half-life extended immune cell binding protein are connected by a cleavable linker. In some embodiments, the cleavable linker facilitates the release of the active immune cell binding protein in cells. Examples of cleavable linkers include, but are not limited to, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers (see, for example, Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020). In some embodiments, the cleavable linker comprises a sequence recognized by a protease. Examples of proteases include ABHD12, ADAM12, ABHD12B, ABHD13, ABHD17A, ADAM 19, ADAM20, ADAM21, ADAM28, ADAM30, ADAM33, ADAM8, ABHD17A, ADAMDEC1, AD AMTS 1, AD AMTS 10, AD AMTS 12, AD AMTS 13, AD AMTS 14, AD AMTS 15, AD AMTS 16, AD AMTS 17, AD AMTS 18, AD AMTS 19, ADAMTS2, ADAMTS20, AD AMTS 3, AD AMTS 4, ABHD17B, AD AMTS 5, AD AMTS 6, ADAMTS 7, ADAMTS 8, ADAMTS 9, ADAMTSL1, ADAMTSL2, ADAMTSL3, ABHD17C, ADAMTSL5, ASTL, BMP1, CELA1, CELA2A, CELA2B, CELA3A, CELA3B, ADAM 10, ADAM 15, ADAM 17, ADAM9, ADAMTS4, CTSE, CTSF, ADAMTSL4, CMA1, CTRB 1, CTRC, CTSO, CTR1, 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, MMP 16, MMP17, MMP 19, 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, PRSS 1.PRSS 12, PRSS2, PRSS21, PRSS3, PRSS33, PRSS4, PRSS55, PRSS57, MMP 12, PRSS8, PRSS9, PRTN3, MMP13, MMP14, ST14, TMPRSS10, TMPRSS11A, TMPRSS11D, TMPRSS11E, TMPRSS11F, TMPRSS12, TMPRSS13, MMP15, TMPRSS15, MMP2, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS5, TMPRSS6, TMPRSS7, TMPRSS9, NRDC, OVCH1, PAMR1, PCSK3, PHEX, TINAG, TPSAB1, TPSD1, and TPSGE. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of ADAM 17, HTRA1, PRSS1, FAP, GZMK, NAPSA, MMP1, MMP2, MMP9, MMP10, MMP7, MMP12, MMP28, AD AMTS 9, HGFAC, and HTRA3. Exemplary protease recognition sequences are shown in Table 1.

[0278] [Table 1-1]

[0279] [Table 1-2]

[0280] In some embodiments, the linkers described herein comprise a sequence selected from the sequences provided in Table 2. In some embodiments, the linkers described herein comprise flanking sequences on the N-terminus and / or C-terminus. In some embodiments, the flanking sequences can be (without limitation) GGGG, GGGS, GGGT, GGGGG, GGGGS, and / or GGGGT.

[0281] [Table 2]

[0282] Polynucleotides encoding sustained release binding proteins Also provided in some embodiments are polynucleotide molecules that encode the sustained release binding proteins described herein. In some embodiments, the polynucleotide molecules are provided as DNA constructs. In other embodiments, the polynucleotide molecules are provided as messenger RNA transcripts.

[0283] The polynucleotide molecule is constructed by known methods, such as combining genes encoding immune cell binding proteins, or genes encoding various domains of immune cell binding proteins that contain more than one domain. In some embodiments, the genes encoding the domains are separated into a single gene construct operably linked by a peptide linker to a suitable promoter and optionally a suitable transcription terminator, or in other embodiments, linked to the gene construct by a peptide bond to express it in a suitable expression system, such as bacteria or, for example, CHO cells. Depending on the vector system and host utilized, any number of suitable transcription and translation elements may be used, including constitutive and inducible promoters. The promoter is selected to induce expression of the polynucleotide in the corresponding host cell.

[0284] In some embodiments, the polynucleotide encoding the sustained release binding protein described herein is inserted into a vector, preferably an expression vector, which represents a further embodiment.This recombinant vector can be constructed by known methods.Vector of particular interest includes plasmid, phagemid, phage derivative, virus (e.g., retrovirus, adenovirus, adeno-associated virus, herpes virus, lentivirus, etc.), and cosmid.

[0285] A variety of expression vector / host systems can be utilized to contain and express the polynucleotides encoding the polypeptides of the described immune cell binding proteins. An example of an expression vector for expression in E. coli is pSKK (Le Gall et al., J Immunol Methods. (2004) 285(1):111-27), or for expression in mammalian cells is pcDNA5 (Invitrogen).

[0286] Thus, the sustained release binding proteins described herein may, in some embodiments, be produced by introducing a vector encoding the above-described protein into a host cell and culturing the host cell under conditions in which the protein domain is expressed, isolated, and optionally further purified.

[0287] Proteins with masking peptides The present specification describes a pharmaceutical composition comprising a protein that comprises a binding moiety, a masking peptide, and a cleavable linker.Without being bound by any theory, the protein with the masking peptide described herein can reduce target-mediated drug elimination when administered to a subject by gradually releasing the active form of the binding moiety when in systemic circulation.

[0288] In some embodiments, the cleavable linker is cleaved in a substantial amount in the systemic circulation. In some embodiments, the half-life of the protein described herein in the systemic circulation is longer than that of a control protein without a masking peptide. In some embodiments, the equivalent protein without a masking peptide has non-linear pharmacokinetics (PK) over the dose range evaluated, and the protein described herein has improved linearity over the dose range evaluated compared to the control protein. In some embodiments, the molar amount of the binding moiety of the protein that is bound to the target when administered to a subject is less than the molar amount of the binding moiety of a control protein that does not include a masking peptide when administered to a subject at the same dose level. In some embodiments, the binding rate of the binding moiety of the protein to the target is less than that of a control protein that does not include a masking peptide when administered to a subject. Figure 30 provides an exemplary construct of a peptide with a masking peptide described herein.

[0289] target The binding portion of the protein with the masking peptide described herein can bind to various targets, including, in a non-exhaustive list, ICOS (inducible T cell costimulatory factor, CD278), OX40 (CD134, TNFRSF4, tumor necrosis factor receptor superfamily member 4), CD40 (TNFRSF5, tumor necrosis factor receptor superfamily member 5), DR5 (death receptor 5, TRAIL receptor 2), GITR (glucocorticoid-inducible TNFR-related protein, TNFRSF18, tumor necrosis factor receptor superfamily member 18), and 4-1BB (CD137, TNFRSF9, tumor necrosis factor receptor superfamily member 9).

[0290] In some embodiments, the target can be ICOS.ICOS is a T cell specific CD28 superfamily costimulatory molecule and immune checkpoint protein.ICOS is normally expressed on certain activated T cells and plays an important role in the proliferation and activation of T cells.

[0291] In some embodiments, the target may be OX40. OX40 is a cell surface glycoprotein and member of the tumor necrosis factor receptor superfamily (TNFRSF). OX40 is expressed on T lymphocytes and plays an essential role in T cell activation. Co-stimulation of activated T cells with agonist monoclonal antibodies (mAbs) against tumor necrosis factor receptor superfamily member OX40 represents a new immunotherapeutic approach to cancer. OX40 engagement may co-stimulate effector T cells and deplete regulatory T cells, thereby enhancing tumor immunity.

[0292] In some embodiments, the target may be CD40. CD40 is a stimulatory receptor and a member of the tumor necrosis factor (TNF) receptor superfamily. CD40 is expressed on various immune cells, such as macrophages, dendritic cells, various tumor cell types, including many B-cell malignancies, and some solid tumors. CD40 plays an important role in activating the immune system, mediating both indirect tumor cell killing through immune system activation and direct tumor cell apoptosis. CD40 is highly expressed on most B-lineage hematological malignancies, including multiple myeloma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, Hodgkin's disease, and acute lymphoblastic leukemia.

[0293] In some embodiments, the target may be DR5. DR5, also known as TRAIL receptor 2 (TRAILR2) and tumor necrosis factor receptor superfamily member 10B (TNFRSF10B), is a cell surface receptor of the TNF receptor superfamily that binds TRAIL and mediates apoptosis. DR5 contains an intracellular death domain. DR5 can be activated by tumor necrosis factor-related apoptosis-inducing ligand (TNFSF10 / TRAIL / APO-2L) to transduce apoptotic signals. TRAIL, a member of the TNF superfamily of cytokines, plays an important role in inducing apoptosis via the TRAIL-mediated death receptor pathway.

[0294] In some embodiments, the target may be GITR (glucocorticoid-induced tumor necrosis factor receptor, tumor necrosis factor superfamily member 18, TNFRSF18). GITR is a TNF receptor superfamily costimulatory molecule expressed primarily by regulatory T cells (Tregs), effector T cells, and natural killer cells that inhibit the suppressive activity of Tregs. Agonistic antibodies or GITR ligands that bind to GITR in cooperation with T cell receptor (TCR) stimulation result in activation of the MAPK / ERK pathway and NFkB, resulting in increased T cell proliferation and inflammatory cytokine production, enhanced anti-tumor effector function, and resistance of CD4+ and CD8+ T cells to Treg suppression. In tumor models, signaling through GITR has been shown to suppress Treg proliferation, induce Treg depletion, and cause tumor regression.

[0295] In some embodiments, the target may be 4-1BB. 4-1BB is a member of the tumor necrosis factor (TNF) / nerve growth factor (NGF) family of receptors and is expressed by activated T and B lymphocytes and monocytes. 4-1BB ligand has been found to play an important role in regulating immune responses.

[0296] antibody The protein having the masking peptide described herein may include antibodies.A non-exhaustive list of antibodies is GSK3359609 (GSK609, ferazilimab), PF-8600 (PF-04518600, ivuxolimab), JNJ-64457107 (JNJ-107, JNJ7107, ADC-1013, mitazarimab), CP-870,893, SGN-40 (huS2C6, dacetuzumab), MEDI3039, ABBV-621 (eftozanermin alfa, APG880), MEDI1873 (efgivanermin alfa), AMG228, PF-05082566 (utomirumab, uto) and urelumab (BMS-663513).

[0297] In some embodiments, the antibody may be GSK3359609 (GSK609, feragilimab). GSK3359609 is an agonistic antibody against inducible T cell costimulator (ICOS, CD278), which may be associated with immune checkpoint inhibition and antitumor activity. After administration, GSK3359609 targets and binds to ICOS expressed on tumor infiltrating CD4 positive T cells. This stimulates the proliferation of ICOS positive T cells, enhances the survival of cytotoxic T lymphocytes (CTL), and increases the CTL-mediated immune response against tumor cells.

[0298] In some embodiments, the antibody may be PF-8600 (PF-04518600, ivuxolimab). PF-8600 is a fully human agonistic IgG2 mAb that targets the costimulatory receptor OX40 (CD134, TNFRSF4) and may be associated with immune stimulatory activity. After administration, PF-8600 selectively binds to and activates OX40, which induces the proliferation of memory and effector T lymphocytes. In the presence of tumor-associated antigens (TAA), this may promote T cell-mediated immune responses against TAA-expressing tumor cells.

[0299] In some embodiments, the antibody may be JNJ-64457107 (JNJ-107, JNJ7107, ADC-1013, mitazarimab). JNJ-64457107 is a human immunoglobulin (Ig) G1 monoclonal antibody directed against the cell surface receptor CD40, with potential immunostimulatory and antineoplastic activity. Following intratumoral administration, JNJ-64457107 binds to CD40 on antigen-presenting dendritic cells, which results in the activation and proliferation of effector and memory T cells, enhancing the immune response against tumor cells. Additionally, the agent induces antibody-dependent cellular cytotoxicity (ADCC) by binding to the CD40 antigen present on the surface of tumor cells, which ultimately inhibits the proliferation of CD40-expressing tumor cells.

[0300] In some embodiments, the antibody may be CP-870,893. CP-870,893 is a fully human monoclonal antibody (mAb) agonist of the cell surface receptor CD40, potentially associated with immunostimulatory and antineoplastic activity. Similar to CD40 ligand (CD40L or CD154), CP-870,893 binds to CD40 on various immune cell types, inducing cell proliferation and activation of antigen-presenting cells (APCs), activating B cells and T cells, and enhancing immune responses. In addition, the agent may activate CD40 present on the surface of some solid tumor cells, resulting in apoptosis and reducing tumor growth.

[0301] In some embodiments, the antibody may be SGN-40 (huS2C6, dacetuzumab). SGN-40 is a humanized monoclonal antibody directed against the CD40 receptor, and may be associated with anti-neoplastic activity. SGN-40 specifically binds to and inhibits the CD40 receptor, thereby inducing apoptosis and inhibiting cell proliferation in cells that overexpress this receptor through antibody-dependent cellular cytotoxicity (ADCC).

[0302] In some embodiments, the antibody may be MEDI3039. MEDI3039 is a highly potent multivalent DR5 agonist. MEDI3039 is a modified protein derived from the third fibronectin type III domain of the glycoprotein tenascin-C, with a region (rgion) that resembles the variable region characteristics of antibodies. The optimized multivalent DR5 agonist is highly potent in inducing cell death in multiple TRAIL-sensitive strains, one to two orders of magnitude more potent than TRAIL, and has shown promising results in multiple cancer cells (colon cancer, lung cancer, leukemia, liver cancer) and in vivo colon cancer models.

[0303] In some embodiments, the antibody may be ABBV-621 (eftozanermin alfa, APG880). ABBV-621 is a fusion protein composed of a tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptor agonist consisting of six receptor binding domains (RBDs) of TRAIL fused to the Fc domain of a human immunoglobulin G1 (IgG1) antibody, with potential pro-apoptotic and antineoplastic activity. After administration, ABBV-621 binds to the TRAIL receptors expressed on tumor cells, the pro-apoptotic death receptors (DRs) TRAIL-R1 (death receptor 4, DR4), and TRAIL-R2 (death receptor 5, DR5), thereby inducing tumor cell apoptosis. ABBV-621 is designed to maximize receptor clustering for optimal efficacy.

[0304] In some embodiments, the antibody may be MEDI1873 (efgivanermin alfa). MEDI1873 is a homogenous hexameric agonistic fusion protein composed of the extracellular domain (ECD) of the T cell costimulatory receptor human GITR ligand (GITRL) and the immunoglobulin (Ig) G1 Fc domain, with potential immunomodulatory and antineoplastic activity. After administration, MEDI1873 binds to and activates GITR found on multiple types of T cells, thereby inducing both activation and proliferation of tumor antigen-specific T-effector cells. This abrogates the suppression of T effector cells induced by inappropriately activated regulatory T cells (Tregs), suppresses Tregs to reduce tumor infiltration, and activates the immune system, aiding in the eradication of tumor cells.

[0305] In some embodiments, the antibody may be AMG228. AMG228 is an agonistic human IgG1 monoclonal antibody that binds to human GITR in a similar manner to MEDI1873.

[0306] In some embodiments, the antibody may be PF-05082566 (utomirumab, uto). PF-05082566 is a humanized agonistic IgG2 monoclonal antibody against tumor necrosis factor superfamily receptor 4-1BB. PF-05082566 binding to human 4-1BB results in activation of NF-κB and downstream cytokine production in cell lines and primary lymphocytes. PF-05082566 can induce human leukocyte proliferation and has demonstrated significant antitumor activity as a single agent in human peripheral blood lymphocyte (PBL) SCID xenograft tumor models.

[0307] In some embodiments, the antibody can be urelumab. Urelumab is a humanized agonistic monoclonal antibody that targets 4-1BB and may be associated with immune stimulatory and anti-neoplastic activity. Urelumab specifically binds to and activates 4-1BB-expressing immune cells, stimulating immune responses against tumor cells, particularly cytotoxic T cell responses.

[0308] Treatment Also provided herein in some embodiments are methods and uses for stimulating the immune system of an individual in need thereof, comprising administering a protein having a sustained release binding protein and a masking peptide as described herein. In some examples, the administration induces and / or sustains cytotoxicity against cells expressing the target antigen. In some examples, the cells expressing the target antigen are cancer or tumor cells, virus-infected cells, bacteria-infected cells, autoreactive T or B cells, damaged red blood cells, arterial plaque, or fibrotic tissue. In some embodiments, the target antigen is an immune checkpoint protein.

[0309] Also provided herein are methods and uses for treating a disease, disorder, or condition associated with a target antigen, comprising administering to an individual in need of treatment a protein having a sustained release binding protein and a masking peptide as described herein. The disease, disorder, or condition associated with the target antigen includes, but is not limited to, a viral infection, a bacterial infection, an autoimmune disease, a transplant rejection, atherosclerosis, or a fibrosis. In other embodiments, the disease, disorder, or condition associated with the target antigen is a proliferative disease, a neoplastic disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic response, a parasitic response, a graft versus host disease, or a host versus graft disease. In one embodiment, the disease, disorder, or condition associated with the target antigen is a cancer. In one embodiment, the cancer is a blood cancer. In one embodiment, the cancer is a melanoma. In one embodiment, the cancer is a lung cancer. In one embodiment, the cancer is an ovarian cancer. In one embodiment, the cancer is a prostate cancer. In one embodiment, the cancer is a pancreatic cancer. In one embodiment, the cancer is a mesothelioma. In one embodiment, the cancer is a neuroendocrine cancer. In one embodiment, the cancer is multiple myeloma. In one embodiment, the cancer is breast cancer.

[0310] As used herein, in some embodiments, "treatment," "treating," or "treated" refers to a therapeutic treatment aimed at slowing (reducing) an undesired physiological disease, disorder, or condition, or to obtain a beneficial or desired clinical outcome. For purposes herein, beneficial or desired clinical outcome includes, but is not limited to, alleviation of symptoms, reduction in the extent of the disease, disorder, or condition, stabilization (i.e., prevention of deterioration) of the disease, disorder, or condition, delay in onset or slowing of progression of the disease, disorder, or condition, remission of the disease, disorder, or condition, and remission, improvement, or amelioration of the disease, disorder, or condition, whether detectable or undetectable. Treatment includes eliciting a clinically significant response without causing excessive levels of side effects. Treatment further includes prolonging survival beyond that expected in the absence of treatment. In other embodiments, "treatment," "treating," or "treated" refers to a preventative measure, the purpose of which is to delay the onset of or reduce the severity of an unwanted physiological disease, disorder, or condition, such as in an individual who is predisposed to the condition (e.g., an individual who carries a genetic marker for a disease such as breast cancer).

[0311] In some embodiments of the methods described herein, the sustained release binding proteins and proteins with masking peptides described herein are administered in combination with an agent for treating a particular disease, disorder, or condition. Agents include, but are not limited to, therapeutic agents including antibodies, small molecules (e.g., chemotherapeutic agents), hormones (steroids, peptides, etc.), radiotherapy agents (directed delivery of gamma rays, X-rays, and / or radioisotopes, microwave, UV radiation, etc.), gene therapy agents (e.g., antisense, retroviral therapy agents, etc.), and other immunotherapeutics. In some embodiments, the sustained release binding proteins described herein are administered in combination with antidiarrheal agents, antiemetic agents, analgesics, opioids, and / or nonsteroidal anti-inflammatory agents. In some embodiments, the conditionally active binding proteins described herein are administered before, during, or after surgery.

[0312] Administration of the sustained release binding proteins and proteins having masking peptides described herein can be accomplished by a variety of methods, for example, intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration. In some embodiments, the route of administration depends on the type of therapeutic agent and the type of compound contained in the pharmaceutical composition. In some embodiments, the sustained release binding proteins are injected or injectable. EXAMPLES

[0313] The following examples further illustrate the described embodiments without limiting the scope of the disclosure.

[0314] Example 1: Exemplary constructs of sustained release binding proteins An exemplary construct of the sustained release binding protein is shown in FIG. 1. TriTAC-XR is a trispecific binding protein with anti-target binding, anti-albumin binding, and anti-CD3 binding capabilities. The intact form of the sustained release binding protein (FIG. 1A) contains a CD3 mask linked to the binding protein by a cleavable linker. The cleavable linker, when gradually cleaved in the subject, releases the active form of the sustained release binding protein (FIG. 1B). FIG. 1C shows the predicted concentration of the intact and active forms in the systemic circulation after multiple administration events. FIG. 1D shows the predicted concentration of the active form of the corresponding antigen binding protein without the masking peptide in the systemic circulation after multiple administration events.

[0315] Additional sustained release binding protein constructs are shown in Figures 2A-2L with various configurations of the anti-target, anti-albumin, and anti-CD3 binding domains, with Figures 2A-2C and 2G-2I illustrating intact forms and Figures 2D-2F and 2J-2L illustrating active forms after linker cleavage.

[0316] Example 2: Production and purification of sustained release binding proteins used in this study Protein production: The sequence of an exemplary TriTAC-XR molecule was cloned into the mammalian expression vector pcDNA3.4 (Invitrogen) preceded by a leader and followed by a 6x histidine tag. Expi293F cells (Life Technologies A14527) were cultured at 0.2–8×10 6 The cells were maintained in suspension at 1000x cells / ml. Purified plasmid DNA was transfected into Expi293 cells according to the protocol of the Expi293 Expression System Kit (Life Technologies, A14635) and maintained for 4-6 days post-transfection. The amount of exemplary TriTAC XR proteins in conditioned media was quantified using an Octet RED96 instrument equipped with a Protein A chip (ForteBio / Sartorius) using control trispecific proteins for the standard curve.

[0317] Protein purification: Conditioned medium from either host cell was filtered and partially purified by affinity and desalting chromatography. TriTAC-XR protein was then polished by ion exchange and formulated in a vehicle containing a neutral buffer after pooling fractions. Final purity was assessed by SDS-PAGE and analytical SEC using an Acquity BEH SEC 200 1.7u 4.6×150mm column (Waters Corporation), separated with an aqueous / organic mobile phase on a 1290LC system with a neutral pH vehicle, and peaks were integrated with Chemstation CDS software (Agilent).

[0318] Example 3: T cell-dependent cytotoxicity assay induced by PSMA-targeted sustained release binding protein The binding proteins used in this study are summarized in Table 3. Stub:T:A:C is a PSMA binding protein without a masking domain. As described herein, stub refers to the residual linker remaining in the active drug fragment after protease cleavage. Peptide:NCLV is a PSMA binding protein with a peptide mask and a non-cleavable linker. Peptide:L001 is a PSMA-targeted sustained release binding protein with a peptide mask and linker L001, a non-cleavable linker. The ion exchange chromatography profile (Figure 3) demonstrates the purity of the PSMA-targeted protein and further observes that little or no active drug (stub:T:A:C) is present in the purified Peptide:L001 protein.

[0319] [Table 3]

[0320] TDCC Assay: For functional potency in the T-cell dependent cytotoxicity assay, soluble test molecules shown in Table 3 were incubated in multi-well plates with purified human T cells (effector cells) and MDAPCa2b cancer cells at an effector:target cell ratio of 10:1 in the presence of 15 mg / ml human serum albumin (HSA) for 48 hours at 37°C. MDAPCa2b cells were stably transduced with a luciferase reporter gene to allow measurement of specific T-cell mediated cell death by Steady-Glo (Promega). The results shown in Figure 4 demonstrate that the slow activation of the peptide masked the PSMA-targeted sustained release binding protein in the TDCC assay. EC 50 Values ​​are shown in Table 3. EC of peptide:NCLV (SEQ ID NO:3601) or peptide:L001 (SEQ ID NO:3600) against stub:T:A:C (SEQ ID NO:3609) 50 The efficiency of masking was quantified by the fold increase in (Table 3).

[0321] Example 4: T cell-dependent cytotoxicity assay induced by PSMA-targeted sustained release binding proteins with different peptide mask sequences The binding proteins with various masking peptides used in this study are summarized in Table 4. The configurations of the anti-target binding domain, anti-albumin binding domain, and anti-CD3 binding domain are according to Figure 2A (SEQ ID NO: 3601-3608) and Figure 2D (SEQ ID NO: 3609), except for SEQ ID NO: 3610, which is the ProTriTAC molecule. A description of the structure of the ProTriTAC molecule can be found in WO2019222283, which is incorporated herein by reference.

[0322] [Table 4]

[0323] TDCC Assay: For functional potency in the T-cell dependent cytotoxicity assay, soluble test molecules shown in Table 4 were incubated in multi-well plates with purified human T cells (effector cells) and LNCaP cancer cells at a 10:1 effector:target cell ratio for 48-72 h at 37 °C in the presence of human serum albumin. Cancer target cell lines were stably transduced with a luciferase reporter gene to allow measurement of specific T-cell mediated cell death by Steady-Glo (Promega). The results shown in Figure 5 demonstrate the masking effect of the various peptide masks. EC 50 The values ​​are shown in Table 4. EC of SEQ ID NO: 3601-3608 and 3610 relative to SEQ ID NO: 3609 (Table 4) 50 The efficiency of masking is quantified by the fold increase in the number of nucleotides. As a result, it is found that masking of the sequences QDGNEE, QDANEE, and QEGNEE results in the most efficient masking effect.

[0324] Example 5: T cell-dependent cytotoxicity assay induced by MSLN-targeted sustained release binding proteins with different peptide mask sequences The binding proteins with various masking peptides used in this study are summarized in Table 5. The configurations of the anti-target binding domain, anti-albumin binding domain, and anti-CD3 binding domain are according to Figure 2A (SEQ ID NO: 3659-3661) or Figure 2D (SEQ ID NO: 3662).

[0325] [Table 5]

[0326] TDCC Assay: For functional potency in the T-cell dependent cytotoxicity assay, soluble test molecules shown in Table 5 were incubated in multi-well plates with purified human T cells (effector cells) and OVCAR8 cancer cells at an effector:target cell ratio of 10:1 for 48 hours at 37°C. Bovine serum albumin (BSA) or HSA was present at 15mg / ml. Cancer target cell lines were stably transduced with a luciferase reporter gene to allow measurement of specific T-cell mediated cell death by Steady-Glo (Promega). The results shown in Figure 6A-B demonstrate the masking effect of the different peptide masks. EC 50 The values ​​are shown in Table 5. EC of SEQ ID NO: 3659-3661 relative to SEQ ID NO: 3663 (Table 5) 50 The efficiency of masking is quantified by the fold increase in

[0327] Example 6: T cell-dependent cytotoxicity assays induced by CD19-targeted sustained release binding proteins with different domain configurations The binding proteins used in this study are summarized in Table 6. The anti-target binding domain, anti-albumin binding domain, anti-CD3 binding domain are according to Figures 2A-2C and 2G-2I (SEQ ID NOs: 3627-3631) and Figures 2D-2F and 2J-2L (SEQ ID NOs: 3632-3636). SEQ ID NOs: 3627-3631 contain a masking domain and a non-cleavable linker. SEQ ID NOs: 3632-3636 are engineered to code for products after protease cleavage when the molecule is made with a non-cleavable linker (stub: active molecule).

[0328] [Table 6]

[0329] TDCC Assay: For functional potency in the T-cell dependent cytotoxicity assay, soluble test molecules as shown in Table 6 were incubated in multi-well plates with purified human T cells (effector cells) and Raji cancer cells at a 10:1 effector:target cell ratio for 72 hours at 37°C. HSA was present. Cancer target cell lines were stably transduced with a luciferase reporter gene to allow measurement of specific T-cell mediated cell death by Steady-Glo (Promega). Results shown in Figure 7 demonstrate the results of the TDCC assay for various CD19-targeted proteins. EC 50 The values ​​are shown in Table 6. stub: EC of NCLV proteins against active protein 50 The efficiency of masking is quantified by the fold increase in stub:activity (Table 6). The results show that the T:A:C and T:C:A configurations have two orders of magnitude lower picomolar stub:activity EC50 and are inactive when masked with the NCLV linker, while the A:C:T and A:T:C configurations are less effective at masking.

[0330] Example 7: T cell-dependent cytotoxicity assay induced by FLT3-targeting sustained release binding proteins with different domain configurations The binding proteins used in this study are summarized in Table 7. The anti-target binding domain, anti-albumin binding domain, anti-CD3 binding domain are according to Figures 2A-2C and 2G-2I (SEQ ID NOs: 3648-3653) and Figures 2D-2F and 2J-2L (SEQ ID NOs: 3654-3658). SEQ ID NOs: 3648-3653 contain a masking domain and a non-cleavable linker. SEQ ID NOs: 3654-3658 are engineered to encode products after protease cleavage when the molecule is made with a non-cleavable linker (stub: active molecule). The FLT3 binder used in this study is 19C (SEQ ID NO: 1076).

[0331] [Table 7]

[0332] TDCC Assay: For functional potency in the T-cell dependent cytotoxicity assay, soluble test molecules shown in Table 7 were incubated in multi-well plates with purified human T cells (effector cells) and MOLM13 cancer cells at a 10:1 effector:target cell ratio for 48-72 hours at 37°C. HSA was present. Cancer target cell lines were stably transduced with a luciferase reporter gene to allow measurement of specific T-cell mediated cell death by Steady-Glo (Promega). Results shown in Figure 8 demonstrate the results of the TDCC assay for various FLT3-targeted proteins. EC 50 Values ​​are shown in Table 7. stub: EC of NCLV proteins against active protein 50 The efficiency of masking is quantified by the fold increase in EC2 activity (Table 7). As a result, the configuration of TAC, when masked with the NCLV linker, is stub:active form of EC2 activity. 50 is about 50 picomolar and has no activity, while the A:C:T and A:T:C configurations are found to have weak masking effects.

[0333] Example 8: T cell-dependent cytotoxicity assay induced by FLT3-targeting sustained release binding proteins with different domain configurations The binding proteins used in this study are summarized in Table 8. The anti-target binding domain, anti-albumin binding domain, anti-CD3 binding domain are according to Figures 2A-2C and 2G-2I (SEQ ID NOs: 3637-3642) and Figures 2D-2F and 2J-2L (SEQ ID NOs: 3643-3647). SEQ ID NOs: 3637-3642 contain a masking domain and a non-cleavable linker. SEQ ID NOs: 3643-3647 are engineered to encode products after protease cleavage when the molecule is made with a non-cleavable linker (stub: active molecule). The FLT3 binder used in this study is 107 (SEQ ID NO: 1074).

[0334] [Table 8]

[0335] TDCC Assay: For functional potency in the T-cell dependent cytotoxicity assay, soluble test molecules shown in Table 8 were incubated in multi-well plates with purified human T cells (effector cells) and MOLM13 cancer cells at a 10:1 effector:target cell ratio for 48 hours at 37°C. HSA was present. Cancer target cell lines were stably transduced with a luciferase reporter gene to allow measurement of specific T-cell mediated cell death by Steady-Glo (Promega). Results shown in Figure 9 demonstrate the results of the TDCC assay for various FLT3-targeted proteins. EC 50 The values ​​are shown in Table 8. stub: EC of NCLV proteins against active protein 50 The efficiency of masking is quantified by the fold increase in EC50 / EC50 / A (Table 8). As a result, four configurations (C:A:T, C:T:A, T:A:C, and T:C:A) have single-digit picomolar activity EC50 / EC50 / A in the active form. 50 and is inactive when masked with the NCLV linker, while the A:T:C configuration provides weak masking. The stub_A:C:T configuration was not expressed, so the masking efficiency of the A:C:T configuration could not be assessed.

[0336] Example 9: T cell-dependent cytotoxicity assay induced by CD33-targeted sustained release binding proteins with different domain configurations The binding proteins used in this study are summarized in Table 9. The anti-target binding domain, anti-albumin binding domain, anti-CD3 binding domain are according to Figures 2A-2C and 2G-2I (SEQ ID NOs: 3614-3619) and Figures 2D-2F and 2J-2L (SEQ ID NOs: 3620-3625). SEQ ID NOs: 3614-3619 contain a masking domain and a non-cleavable linker. SEQ ID NOs: 3620-3625 are engineered to encode products after protease cleavage when the molecule is made with a non-cleavable linker (stub: active molecule).

[0337] [Table 9]

[0338] TDCC Assay: For functional potency in the T-cell dependent cytotoxicity assay, soluble test molecules shown in Table 9 were incubated in multi-well plates with purified human T cells (effector cells) and MOLM13 cancer cells at a 10:1 effector:target cell ratio for 48 hours at 37°C. HSA was present. Cancer target cell lines were stably transduced with a luciferase reporter gene to allow measurement of specific T-cell mediated cell death by Steady-Glo (Promega). Results shown in Figure 10 demonstrate the results of the TDCC assay for various CD33-targeted proteins. EC 50 The values ​​are shown in Table 9. stub: EC of NCLV proteins against active protein 50 The efficiency of masking is quantified by the fold increase in EC50 / EC50 / A (Table 9). As a result, four configurations (C:A:T, C:T:A, T:A:C, and T:C:A) have single-digit picomolar activity EC50 / EC50 / A in the active form. 50 and has no activity when masked with the NCLV linker, while the A:C:T and A:T:C configurations show weak masking effects.

[0339] Example 10: TDCC activity of protease-activated sustained release binding proteins CD19-targeted sustained release binding proteins in the configurations T:A:C or T:C:A (FIGS. 2A and 2B) were expressed and purified. For each configuration, four different forms of the protein were produced. The first form (SEQ ID NOs: 3842 and 3843) contained a protease cleavage site L001 (SEQ ID NO: 3688) between the masking domain (SEQ ID NO: 3663) and the CD19 targeting domain. The second form was identical to the first form, except that the protein was treated with ST14 protease. The third form (SEQ ID NOs: 3630 and 3631) contained a non-cleavable linker (SEQ ID NO: 3687) between the masking domain and the targeting domain. A fourth form (SEQ ID NO: 3611 and 3636) was engineered with the amino acid sequence VVGGGG (SEQ ID NO: 3879), designated "stub", on the N-terminus of the target binding domain to produce a sustained release binding protein after protease cleavage (FIGS. 2D and 2E). These proteins were tested in the Raji TDCC assay described in Example 6, and the Raji cell viability data versus the concentration of CD19 protein added is plotted in FIG. 11. As expected, protease-activated CD19 binding protein and stub CD19 protein potently and efficiently induced T cells to kill Raji cells. CD19 binding protein with a non-cleavable linker was unable to induce T cells to kill Raji cells. CD19 protein with a non-cleavable linker but not treated with protease was partially able to induce T cells to kill Raji cells. It is hypothesized that during this 72 hour period, these proteins became partially activated by proteases produced by T cells and / or Raji cells.

[0340] The FLT3-targeted sustained release binding protein in the configuration T:A:C (FIG. 2A) was expressed and purified. Four different forms of the protein were produced. The first form (SEQ ID NO: 3854) contained a protease-cleavable linker L001 (SEQ ID NO: 3688) (as well as adjacent sequences at the N- and C-termini of L001) between the masking domain (SEQ ID NO: 3633) and the FLT3-targeting domain. The second form was identical to the first form, except that the protein was treated with ST14 protease. The third form (SEQ ID NO: 3652) contained a non-cleavable linker (SEQ ID NO: 3687) between the masking domain and the targeting domain. The fourth form (SEQ ID NO: 3613) was engineered with the amino acid sequence VVGGGG (SEQ ID NO: 3879), referred to as "stub", on the N-terminus of the target binding domain to the product of the sustained release binding protein after protease cleavage (FIG. 2D). These proteins were tested in the MOLM13 TDCC assay described in Example 6, and the MOLM13 cell viability data versus added FLT3 protein concentration is plotted in FIG. 12. As expected, protease-activated FLT3 binding protein and stub FLT3 protein potently and efficiently induced T cells to kill MOLM13 cells. FLT3 binding protein with a non-cleavable linker was unable to induce T cells to kill MOLM13 cells. FLT3 protein with a non-cleavable linker but not treated with protease was able to induce T cells to kill MOLM13 cells, but less potently than stub or protease-activated proteins. It is hypothesized that these proteins became partially activated by proteases produced by T cells and / or MOLM13 cells during this 72-hour period.

[0341] E...

Claims

【Claim 1】 A pharmaceutical composition comprising a sustained-release binding protein comprising a half-life extended immune cell-binding protein, a masking peptide, and a cleavable linker, wherein the masking peptide is covalently bound to the N-terminus or C-terminus of the half-life extended immune cell-binding protein via the cleavable linker, and the cleavable linker is substantially cleaved in the systemic circulation.