Combination therapy using immune cell-associated proteins and immunomodulatory agents

Abstract

Provided herein are combinations or compositions comprising an immunomodulatory agent and an immune cell-associated protein, as well as methods of use thereof.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 158,721, filed March 9, 2021, which is incorporated herein 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 death in humans after coronary artery disease. Globally, millions of people die from cancer each year. In the United States alone, cancer claims the lives of well over 500,000 people each year, with approximately 1.4 million new cases diagnosed each year. While deaths from heart disease have declined significantly, deaths due to cancer in general are increasing. Early in the next century, cancer is predicted to become the leading cause of death.

[0004] Furthermore, even cancer patients who survive early primary cancers share a common experience of their lives being dramatically altered. Many cancer patients experience significant anxiety due to the awareness of the possibility of recurrence or treatment failure. Many cancer patients experience significant physical debilitation after treatment.

[0005] Generally speaking, a fundamental problem in the management of most deadly cancers is the lack of effective, non-toxic systemic therapies. Cancer is a complex disease characterized by genetic mutations that lead to uncontrolled cell proliferation. Cancer cells are present in all organisms, and under normal circumstances, their excessive proliferation is tightly controlled by various physiological factors.

[0006] The selective destruction of individual cells or specific cell types is often desired in various clinical settings. For example, the specific destruction of tumor cells while leaving healthy cells and tissues intact is a major goal of cancer treatment. One such approach is by eliciting an immune response against the tumor, inducing immune effector cells, such as natural killer (NK) cells or cytotoxic T lymphocytes (CTLs), to attack and destroy tumor cells. Summary of the Invention

[0007] Provided herein are combinations comprising an immunomodulatory agent and a half-life extending immune cell-associated protein. In some embodiments, the half-life extending immune cell-associated protein comprises an immune cell-associating domain. In some embodiments, the immune cell-associating domain comprises a natural killer (NK) cell-associating domain, a T cell-associating domain, a B cell-associating domain, a dendritic cell-associating domain, a macrophage cell-associating domain, or a combination thereof. In some embodiments, the immune cell-associating domain comprises a T cell-associating domain. In some embodiments, the T cell-associating domain binds to a CD3 molecule. In some embodiments, the CD3 molecule is at least one of a CD3γ molecule, a CD3δ molecule, or a CD3ε molecule.

[0008] In some embodiments, the immunomodulatory agent comprises an immunostimulatory antibody agonist of a costimulatory receptor. In some embodiments, the immunomodulatory agent comprises an immune checkpoint modulator. In some embodiments, the immune checkpoint modulator is programmed cell death 1 (PDCD1, PD1, PD-1), CD274 (CD274, PDL1, PD-L1), PD-L2, cytotoxic T-lymphocyte-associated protein 4 (CTLA4, CD152), CD276 (B7H3), V-set domain-containing inhibitor of T-cell activation 1 (VTCN1, B7H4), CD272 (B- and T-lymphocyte-associated (BTLA)), killer cell immunoglobulin-like receptor, three Ig domains and long stranded Cytoplasmic tail 1 (KIR, CD158E1), lymphocyte activation 3 (LAG3, CD223), hepatitis A virus cell receptor 2 (HAVCR2, TIMD3, TIM3), V-set immunoregulatory receptor (VSIR, B7H5, VISTA), T cell immunoreceptor with Ig domain and ITIM domain (TIGIT), programmed cell death 1 ligand 2 (PDCD1LG2, PD-L2, CD273), immunoglobulin superfamily member 11 (IGSF11, VSIG3), TNFRSF14 (HVE M, CD270), TNFSF14 (HVEML), PVR-related immunoglobulin domain containing (PVRIG, CD112R), galectin 9 (LGALS9), killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1), killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2), killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3), and killer cell immunoglobulin Ig-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1), killer cell lectin-like receptor C1 (KLRC1, NKG2A, CD159A), killer cell lectin-like receptor D1 (KLRD1, CD94), killer cell lectin-like receptor G1 (KLRG1, CLEC15A, MAFA, 2F1), sialic acid-binding Ig-like lectin 7 (SIGLEC7), SIGLEC, sialic acid-binding Ig-like lectin 9 (SIGLEC9), CEACAM (e.g., CEACAM-1, CEACAM-3,and / or CEACAM-5), VISTA, LAIR1, CD160, 2B4, CD80, CD86, B7-H1, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2AR, A2BR, MHC class I, MHC class II, GAL9, adenosine, TGFR (e.g., TGFRβ), CD94 / NKG2A, IDO, TDO, CD39, CD73, GARP, CD47, PVRIG, CSF1R, and NOX, or any combination thereof.

[0009] In some embodiments, the immune checkpoint modulator is an antagonist of PD-1, and is selected from the group consisting of pembrolizumab (humanized antibody), pidilizumab (CT-011, monoclonal antibody that binds to DLL1 and PD-1), spartalizumab (PDR001, monoclonal antibody), nivolumab (BMS-936558, MDX-1106, human IgG4 monoclonal antibody), MEDI0680 (AMP-514, monoclonal antibody), cemiplimab (REGN2810, monoclonal antibody), dostallimab (TSR-042, monoclonal antibody), and sasanlimab. (PF-06801591, monoclonal antibody), tislelizumab (BGB-A317, monoclonal antibody), BGB-108 (antibody), tislelizumab (BGB-A317, antibody), camrelizumab (INCSHR1210, SHR-1210), AMP-224, zimberelimab (AB122, GLS-010, WBP-3055, monoclonal antibody), AK-103 (HX-008, monoclonal antibody), AK-105 (anti-PD-1 antibody), CS1003 (monoclonal antibody), HLX10 (monoclonal antibody), retifanlimab (MGA-012, anti-PD-1monoclonal antibody), BI-754091 (antibody), balstilimab (AGEN2034, PD-1 antibody), toripalimab (JS-001, antibody), cetrelimab (JNJ-63723283, anti-PD-1 antibody), genolimzumab (CBT-501, anti-PD-1 antibody), LZM009 (anti-PD-1 monoclonal antibody), progolimab (BCD-100, anti-PD-1 monoclonal antibody), Sym021 (antibody), ABBV-181 (antibody), BAT-1306 (antibody), JTX-4014, sintilimab (s intilimab (IBI-308), tebotelimab (MGD013, PD-1 / LAG-3 bispecific), MGD-019 (PD-1 / CTLA4 bispecific antibody), KN-046 (PD-1 / CTLA4 bispecific antibody), MEDI-5752 (CTLA4 / PD-1 bispecific antibody), RO7121661 (PD-1 / TIM-3 bispecific antibody), XmAb20717 (PD-1 / CTLA4 bispecific antibody), and AK-104 (CTLA4 / PD-1 bispecific antibody).

[0010] In some embodiments, the immune checkpoint modulator is pembrolizumab. In some embodiments, the immune checkpoint modulator is an antibody that binds to PD-L1, such as atezolizumab (MPDL3280A, monoclonal antibody), avelumab (MSB0010718C, monoclonal antibody), durvalumab (MEDI-4736, human immunoglobulin G1 kappa (IgG1κ) monoclonal antibody), envafolimab (KN035, single-domain PD-L1 antibody), AUNP12, CA-170 (PD- Small molecule targeting PD-L1 and VISTA), BMS-986189 (macrocyclic peptide), BMS-936559 (anti-PD-L1 antibody), cosibelimab (CK-301, monoclonal antibody), LY3300054 (antibody), CX-072 (antibody), CBT-502 (antibody), MSB-2311 (antibody), BGB-A333 (antibody), SHR-1316 (antibody), CS1001 (WBP3155, antibody), HLX-20 (antibody), KL-A167 (HBM 9167, antibody), STI-A1014 (antibody), STI-A1015 (IMC-001, antibody), BCD-135 (monoclonal antibody), FAZ-053 (antibody), CBT-502 (TQB2450, antibody), MDX1105-01 (antibody), FS-118 (LAG-3 / PD-L1, bispecific antibody), M7824 (anti-PD-L1 / TGF-β receptor II fusion protein), CDX-527 (CD27 / PD-L1 bispecific antibody), LY3415244 (TIM3 / PD-L1 bispecific antibody), INBRX-105 (4-1BB / PD-L1 bispecific antibody).

[0011] In some embodiments, the immune checkpoint modulator is atezolizumab. In some embodiments, the immune checkpoint modulator is an anti-CD39 antibody. In some embodiments, the anti-CD39 antibody is IPH5201. In some embodiments, the immune checkpoint modulator is an anti-CD73 antibody. In some embodiments, the anti-CD73 antibody is IPH5301. In some embodiments, the immunomodulatory agent is an inhibitor of at least one of A2AR, CD39, or CD73. In some embodiments, the inhibitor is a small molecule inhibitor. In some embodiments, the immune checkpoint modulator comprises an immune checkpoint activator. In some embodiments, the immune checkpoint activator is CD27, CD70, CD40, CD40LG, TNF receptor superfamily member 4 (TNFRSF4, OX40), TNF superfamily member 4 (TNFSF4, OX40L), GITR (TNF receptor superfamily member 18, TNFRSF18, CD357), TNFSF18 (GITRL), CD137 (TNFRSF9, tumor necrosis factor receptor superfamily member 9, 4-1BB, ILA (induced by lymphocyte activation), In some embodiments, the agonist is an agonist of CD137L (TNFSF9), CD28, CD278 (inducible T cell costimulator, ICOS), inducible T cell costimulator ligand (ICOSLG, B7H2), CD80 (B7-1), nectin cell adhesion molecule 2 (NECTIN2, CD112), CD226 (DNAM-1), poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155), CD16, killer cell lectin-like receptor K1 (KLRK1, NKG2D, CD314), or SLAM family member 7 (SLAMF7). In some embodiments, the agonist is an antibody or antigen-binding fragment thereof.

[0012] In some embodiments, the half-life extended immune cell-associated protein comprises a first domain (A), a second domain (B), and a third domain (D), wherein (i) the first domain (A) is a T cell-associating domain and specifically binds to human CD3, (ii) the second domain (B) specifically binds to human serum albumin (HSA), and (iii) the third domain (C) specifically binds to a target antigen, and the domains are arranged in the following order: 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 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.

[0013] In some embodiments, the third domain is a target antigen in 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), 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), CD22 (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'-nucleotidin enzyme), CD39 (ENTPD1, ​​ectonucleotide triphosphate diphosphohydrolase-1), CD74 (HLA class II histocompatibility antigen gamma chain, HLA-DR antigen-associated invariant chain), CD79b (immunoglobulin-associated beta), CD80 (B7-1), CD86 (B7-2), CD123 (IL3RA, interleukin-3 receptor), CD133 (PROM1), CD137 (TNFRSF9, tumor necrosis factor receptor superfamily member 9, 4-1BB, ILA (induced by lymphocyte activation), 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 pyrophosphate Phosphatase / 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 (MUC1) and its splice variants (e.g., MUC1 / C, D, and Z), mucin 16 (MUC16), necdin (NDN), necdin cell adhesion molecule 4 (NECTIN4), SLIT and NTRK-like family member 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 (SIG LEC9), 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 (TNFRSF10 A, 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 beta-1 (TGFB1), trophoblast glycoprotein (TPBG, 5T4), trophinin (TRO, MAGED3), tumor-associated calcium signal transducer 2 (TACSTD2, TROP2,It specifically binds to a target antigen selected from the group consisting of EGP1, fucosyl GM1, sialyl Lewis adhesion molecule (sLe), ROR1, CD30, and Lewis Y antigen.

[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 the amino acid sequence of SEQ ID NO: 462, CDR2 comprises the amino acid sequence of SEQ ID NO: 473, and CDR3 comprises the 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 the amino acid sequence of SEQ ID NO: 489.

[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 the amino acid sequence of SEQ ID NO: 523, CDR2 comprises the amino acid sequence of SEQ ID NO: 562, and CDR3 comprises the 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 the amino acid sequence of SEQ ID NO: 647. 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.

[0016] 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 selected from the group consisting of SEQ ID NOs: 1751-2193, a CDR2 comprising an amino acid selected from the group consisting of SEQ ID NOs: 2194-2636, and a CDR3 comprising an amino acid selected from the group consisting of SEQ ID NOs: 2637-3080. In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 2182, CDR2 comprises the amino acid sequence of SEQ ID NO: 2625, and CDR3 comprises the 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 the amino acid sequence of SEQ ID NO: 1739. 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 selected from the group consisting of SEQ ID NOs: 651 to 699, a CDR2 comprising an amino acid selected from the group consisting of SEQ ID NOs: 700 to 748, and a CDR3 comprising an amino acid selected from the group consisting of SEQ ID NOs: 479 to 797. In some embodiments, the third domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 798 to 846.

[0017] 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, CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1150, 1152, 3497, and 3498, CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1226, 1228, 3499, and 3500, and 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 is a single-domain antibody that specifically binds to EpCAM. In some embodiments, the third domain comprises a CDR1 comprising an amino acid selected from the group consisting of SEQ ID NOs: 847-884, a CDR2 comprising an amino acid selected from the group consisting of SEQ ID NOs: 885-922, and a CDR3 comprising an amino acid selected from the group consisting of SEQ ID NOs: 923-960. In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 874 or 863, CDR2 comprises the amino acid sequence of SEQ ID NO: 885 or 901, and CDR3 comprises the amino acid sequence of SEQ ID NO: 923 or 939. In some embodiments, the third domain comprises a sequence selected from the group consisting of SEQ ID NOs: 961-1003. In some embodiments, the third domain comprises the amino acid sequence of SEQ ID NO: 999 or 1003. 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 variable heavy chain region (VH), a variable light chain region (VL), and a linker, wherein 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, HC CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3081 and 3087-3098, HC CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3082 and 3099-3109, and 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 an amino acid sequence selected from the group consisting of SEQ ID NOs: 3084 and 3120-3132, LC CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3085 and 3099-3109, and LC CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 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.

[0018] In some embodiments, the first domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 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: 3199).

[0019] 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, wherein 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 3182-3183. In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 3174, CDR2 comprises the amino acid sequence of SEQ ID NO: 3178, and CDR3 comprises the 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 the amino acid sequence of SEQ ID NO: 3190. In some embodiments, the linkers L1 and L2 are each independently (GS) n (SEQ ID NO: 3190), (GGS) n (SEQ ID NO: 3191), (GGGS) n (SEQ ID NO: 3192), (GGSG) n (SEQ ID NO: 3193), (GGSGG) n (SEQ ID NO: 3194), or (GGGGS) n (SEQ ID NO: 3195), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, linkers L1 and L2 are each independently (GGGGS)4 (SEQ ID NO: 3198) or (GGGGS)3 (SEQ ID NO: 3199).

[0020] In some embodiments, the immunomodulatory agent and the half-life extended immune cell-associated protein are in a single pharmaceutical composition. In some embodiments, the immunomodulatory agent and the half-life extended immune cell-associated protein are in separate pharmaceutical compositions.

[0021] Provided herein are compositions comprising an immunomodulatory agent and a half-life extended immune cell-associated protein.

[0022] Provided herein are methods for treating or ameliorating a disease, comprising administering a combination or composition provided herein to a subject in need thereof. In some embodiments, the disease is cancer.

[0023] Provided herein are methods for increasing the survival rate of a subject suffering from cancer, said methods comprising administering to the subject a combination or composition provided herein. Provided herein are methods for reducing tumor size, said methods comprising administering to a subject suffering from cancer a combination or composition provided herein.

[0024] In some embodiments, the cancer is selected from the group consisting of 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, 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), and / or esophageal cancer. SCLC), 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. In some embodiments, administering the combination provides an increased therapeutic benefit compared to administering the immunomodulatory agent alone without the half-life extended immune cell-associated protein. In some embodiments, administering the combination provides an increased therapeutic benefit compared to administering the half-life extended immune cell-associated protein alone without the immunomodulatory agent. In some embodiments, the half-life extended immune cell-associated protein and the immunomodulatory agent are administered simultaneously. In some embodiments, the half-life extended immune cell-associated protein and the immunomodulatory agent are administered sequentially.

[0025] Provided herein are methods for increasing a subject's sensitivity to a treatment comprising administering an immune checkpoint inhibitor, the method comprising administering to the subject a half-life extended immune cell-associated protein comprising: (i) a first domain (A) that specifically binds to human CD3; (ii) a second domain (B) that specifically binds to human serum albumin (HSA); and (iii) a third domain (C) that is specific for a target antigen. In some embodiments, administering the half-life extended immune cell-associated protein increases the concentration of the immune checkpoint protein targeted by the immune checkpoint inhibitor in the subject.

[0026] In some embodiments, the immune checkpoint protein is PD-1. In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of pembrolizumab (humanized antibody), pidilizumab (CT-011, monoclonal antibody that binds to DLL1 and PD-1), spartalizumab (PDR001, monoclonal antibody), nivolumab (BMS-936558, MDX-1106, human IgG4 monoclonal antibody), MEDI0680 (AMP-514, monoclonal antibody), cemiplimab (REGN2810, monoclonal antibody), dostallimab (TSR-042, monoclonal antibody), sasanlimab (PF-0680, monoclonal antibody), and pembrolizumab (humanized antibody). 1591, monoclonal antibody), tislelizumab (BGB-A317, monoclonal antibody), BGB-108 (antibody), tislelizumab (BGB-A317, antibody), camrelizumab (INCSHR1210, SHR-1210), AMP-224, zimberelimab (AB122, GLS-010, WBP-3055, monoclonal antibody), AK-103 (HX-008, monoclonal antibody), AK-105 (anti-PD-1 antibody), CS1003 (monoclonal antibody), HLX10 (monoclonal antibody), retifanlimab (MGA-012, anti-PD-1monoclonal antibody), BI-754091 (antibody), balstilimab (AGEN2034, PD-1 antibody), toripalimab (JS-001, antibody), cetrelimab (JNJ-63723283, anti-PD-1 antibody), genolimzumab (CBT-501, anti-PD-1 antibody), LZM009 (anti-PD-1 monoclonal antibody), progolimab (BCD-100, anti-PD-1 monoclonal antibody), Sym021 (antibody), ABBV-181 (antibody), BAT-1306 (antibody), JTX-4014, sintilimab ( sintilimab (IBI-308), tebotelimab (MGD013, PD-1 / LAG-3 bispecific), MGD-019 (PD-1 / CTLA4 bispecific antibody), KN-046 (PD-1 / CTLA4 bispecific antibody), MEDI-5752 (CTLA4 / PD-1 bispecific antibody), RO7121661 (PD-1 / TIM-3 bispecific antibody), XmAb20717 (PD-1 / CTLA4 bispecific antibody), and AK-104 (CTLA4 / PD-1 bispecific antibody). These include antibodies used in the treatment of leukemia, including pembrolizumab (humanized antibody), pidilizumab (CT-011, monoclonal antibody that binds to DLL1 and PD-1), spartalizumab (PDR001, monoclonal antibody), nivolumab (BMS-936558, MDX-1106, human IgG4 monoclonal antibody), MEDI0680 (AMP-514, monoclonal antibody), cemiplimab (REGN2810, monoclonal antibody), dostallimab (TSR-042, monoclonal antibody), and sasanlimab (PF-06801591, monoclonal antibody). antibody), tislelizumab (BGB-A317, monoclonal antibody), BGB-108 (antibody), tislelizumab (BGB-A317, antibody), camrelizumab (INCSHR1210, SHR-1210), AMP-224, zimberelimab (AB122, GLS-010, WBP-3055, monoclonal antibody), AK-103 (HX-008, monoclonal antibody), AK-105 (anti-PD-1 antibody), CS1003 (monoclonal antibody), HLX10 (monoclonal antibody), retifanlimab (MGA-012, anti-PD-1monoclonal antibody), BI-754091 (antibody), balstilimab (AGEN2034, PD-1 antibody), toripalimab (JS-001, antibody), cetrelimab (JNJ-63723283, anti-PD-1 antibody), genolimzumab (CBT-501, anti-PD-1 antibody), LZM009 (anti-PD-1 monoclonal antibody), progolimab (BCD-100, anti-PD-1 monoclonal antibody), Sym021 (antibody), ABBV-181 (antibody), BAT-1306 (antibody), JTX-4014, sintilimab (s intilimab (IBI-308), tebotelimab (MGD013, PD-1 / LAG-3 bispecific), MGD-019 (PD-1 / CTLA4 bispecific antibody), KN-046 (PD-1 / CTLA4 bispecific antibody), MEDI-5752 (CTLA4 / PD-1 bispecific antibody), RO7121661 (PD-1 / TIM-3 bispecific antibody), XmAb20717 (PD-1 / CTLA4 bispecific antibody), and AK-104 (CTLA4 / PD-1 bispecific antibody).

[0027] 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), 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), CD22 (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'-nucleotidin enzyme), CD39 (ENTPD1, ​​ectonucleotide triphosphate diphosphohydrolase-1), CD74 (HLA class II histocompatibility antigen gamma chain, HLA-DR antigen-associated invariant chain), CD79b (immunoglobulin-associated beta), CD80 (B7-1), CD86 (B7-2), CD123 (IL3RA, interleukin-3 receptor), CD133 (PROM1), CD137 (TNFRSF9, tumor necrosis factor receptor superfamily member 9, 4-1BB, ILA (induced by lymphocyte activation), 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 (E NPP3), 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 (MUC1) and its splice variants (e.g., MUC1 / C, D, and Z), mucin 16 (MUC16), necdin (NDN), necdin cell adhesion molecule 4 ( NECTIN4), SLIT and NTRK-like family member 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 transporter Family 34 (sodium phosphate), member 2 (SLC34A2), solute transporter 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 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,Specifically binds to a target antigen selected from the group consisting of sialyl Lewis adhesion molecule (sLe), ROR1, CD30, and Lewis Y antigen, wherein the target 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, CVID3). ID5, LEU-16, MS4A2, S7, transmembrane 4-domain A1), CD22 (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'-nucleotidin enzyme), CD39 (ENTPD1, ​​ectonucleotide triphosphate diphosphohydrolase-1), CD74 (HLA class II histocompatibility antigen gamma chain, HLA-DR antigen-associated invariant chain), CD79b (immunoglobulin-associated beta), CD80 (B7-1), CD86 (B7-2), CD123 (IL3RA, interleukin-3 receptor), CD133 (PROM1), CD137 (TNFRSF9, tumor necrosis factor receptor superfamily member 9, 4-1BB, ILA (induced by lymphocyte activation), 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), ectonuclease Enzyme 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), ...1 (MAGEA1), MAGE family member A2 (MAGEA2), MAGE family member B1 (MAGEB1), MAGE family member B2 (MAGEB2), MAGE family member B3 (MAGEB3), MAGE family member B4 (MAGEB4), MAGE family member B5 (MAGEB5 Sothelin (MSLN), mucin 1 (MUC1) and its splice variants (e.g., MUC1 / C, D, and Z), mucin 16 (MUC16), necdin (NDN), necdin cell adhesion molecule 4 (NECTIN4), SLIT and NTRK-like family member 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, LIV 1), 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 beta-1 (TGFB1), 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.

[0028] Provided herein are methods of improving the efficacy of a treatment, comprising administering an immunomodulatory agent to a subject, the method further comprising administering a half-life extended immune cell-associated protein to the subject. In some embodiments, the immunomodulatory agent comprises an immunostimulatory antibody agonist of a costimulatory receptor. In some embodiments, the immunomodulatory agent comprises an immune checkpoint modulator. In some embodiments, the immune checkpoint modulator is an antagonist of at least one of the following:

[0029] In some embodiments, the immune checkpoint modulator is an antagonist of PD-1, and is selected from the group consisting of pembrolizumab (humanized antibody), pidilizumab (CT-011, monoclonal antibody that binds to DLL1 and PD-1), spartalizumab (PDR001, monoclonal antibody), nivolumab (BMS-936558, MDX-1106, human IgG4 monoclonal antibody), MEDI0680 (AMP-514, monoclonal antibody), cemiplimab (REGN2810, monoclonal antibody), dostallimab (TSR-042, monoclonal antibody), and sasanlimab. (PF-06801591, monoclonal antibody), tislelizumab (BGB-A317, monoclonal antibody), BGB-108 (antibody), tislelizumab (BGB-A317, antibody), camrelizumab (INCSHR1210, SHR-1210), AMP-224, zimberelimab (AB122, GLS-010, WBP-3055, monoclonal antibody), AK-103 (HX-008, monoclonal antibody), AK-105 (anti-PD-1 antibody), CS1003 (monoclonal antibody), HLX10 (monoclonal antibody), retifanlimab (MGA-012, anti-PD-1monoclonal antibody), BI-754091 (antibody), balstilimab (AGEN2034, PD-1 antibody), toripalimab (JS-001, antibody), cetrelimab (JNJ-63723283, anti-PD-1 antibody), genolimzumab (CBT-501, anti-PD-1 antibody), LZM009 (anti-PD-1 monoclonal antibody), progolimab (BCD-100, anti-PD-1 monoclonal antibody), Sym021 (antibody), ABBV-181 (antibody), BAT-1306 (antibody), JTX-4014, sintilimab (s intilimab (IBI-308), tebotelimab (MGD013, PD-1 / LAG-3 bispecific), MGD-019 (PD-1 / CTLA4 bispecific antibody), KN-046 (PD-1 / CTLA4 bispecific antibody), MEDI-5752 (CTLA4 / PD-1 bispecific antibody), RO7121661 (PD-1 / TIM-3 bispecific antibody), XmAb20717 (PD-1 / CTLA4 bispecific antibody), and AK-104 (CTLA4 / PD-1 bispecific antibody). In some embodiments, the immune checkpoint modulator is pembrolizumab.

[0030] In some embodiments, the immune checkpoint modulator is an antibody that binds to PD-L1, and includes atezolizumab (MPDL3280A, a monoclonal antibody), avelumab (MSB0010718C, a monoclonal antibody), durvalumab (MEDI-4736, a human immunoglobulin G1 kappa (IgG1κ) monoclonal antibody), envafolimab (KN035, a single-domain PD-L1 antibody), AUNP12, CA-170 (PD- Small molecule targeting PD-L1 and VISTA), BMS-986189 (macrocyclic peptide), BMS-936559 (anti-PD-L1 antibody), cosibelimab (CK-301, monoclonal antibody), LY3300054 (antibody), CX-072 (antibody), CBT-502 (antibody), MSB-2311 (antibody), BGB-A333 (antibody), SHR-1316 (antibody), CS1001 (WBP3155, antibody), HLX-20 (antibody), KL-A167 (HBM 9167, antibody), STI-A1014 (antibody), STI-A1015 (IMC-001, antibody), BCD-135 (monoclonal antibody), FAZ-053 (antibody), CBT-502 (TQB2450, antibody), MDX1105-01 (antibody), FS-118 (LAG-3 / PD-L1, bispecific antibody), M7824 (anti-PD-L1 / TGF-β receptor II fusion protein), CDX-527 (CD27 / PD-L1 bispecific antibody), LY3415244 (TIM3 / PD-L1 bispecific antibody), INBRX-105 (4-1BB / PD-L1 bispecific antibody). In some embodiments, the immune checkpoint modulator is atezolizumab.

[0031] In some embodiments, the immune checkpoint modulator is an anti-CD39 antibody. In some embodiments, the anti-CD39 antibody is IPH5201. In some embodiments, the immune checkpoint modulator is an anti-CD73 antibody. In some embodiments, the anti-CD73 antibody is IPH5301. In some embodiments, the immunomodulatory agent is an inhibitor of at least one of A2AR, CD39, or CD73. In some embodiments, the inhibitor is a small molecule inhibitor.

[0032] In some embodiments, the immune checkpoint modulator comprises an immune checkpoint activator. In some embodiments, the immune checkpoint activator is selected from the group consisting of CD27, CD70, CD40, CD40LG, TNF receptor superfamily member 4 (TNFRSF4, OX40), TNF superfamily member 4 (TNFSF4, OX40L), GITR (TNF receptor superfamily member 18, TNFRSF18, CD357), TNFSF18 (GITRL), CD137 (TNFRSF9, tumor necrosis factor receptor superfamily member 9, 4-1BB, ILA (induced by lymphocyte activation), In some embodiments, the agonist is an agonist of CD137L (TNFSF9), CD28, CD278 (inducible T cell costimulator, ICOS), inducible T cell costimulator ligand (ICOSLG, B7H2), CD80 (B7-1), nectin cell adhesion molecule 2 (NECTIN2, CD112), CD226 (DNAM-1), poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155), CD16, killer cell lectin-like receptor K1 (KLRK1, NKG2D, CD314), or SLAM family member 7 (SLAMF7). In some embodiments, the agonist is an antibody. In some embodiments, the subject is a human.

[0033] Provided herein are kits comprising (a) an immunomodulatory agent and (b) a half-life extended immune cell-associated protein, and instructions for administering (a) and (b) sequentially or simultaneously to a subject. Provided herein are kits comprising a combination or composition provided herein, and instructions for administering an immunomodulatory agent and a half-life extended immune cell-associated protein, sequentially or simultaneously to a subject.

[0034] In some embodiments, the subject is suffering from cancer. In some embodiments, the cancer is selected from the group consisting of 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, soft tissue sarcoma, gastric cancer, digestive / gastrointestinal cancer, 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 (NSC), and / or ovarian cancer. In some embodiments, the cancer is selected from the group consisting of glioma, 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. In some embodiments, the subject is human. [Brief explanation of the drawings]

[0035] The novel features of the invention are set forth with particularity in the appended claims. For a better understanding of the features and advantages of the present invention, reference should be made to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Figure 1A]1A illustrates the molecular mechanism for combination therapy using an immunomodulatory drug and an immune cell-associated protein. Figure 1A illustrates the administration of an exemplary immune cell-associated protein (TriTAC protein) without an immunomodulatory drug, and Figure 1B illustrates the administration of an exemplary immune cell-associated protein (TriTAC protein) with an exemplary immunomodulatory drug (PD-1 and PD-L1 inhibitor). [Figure 1B] 1A illustrates the molecular mechanism for combination therapy using an immunomodulatory drug and an immune cell-associated protein. Figure 1A illustrates the administration of an exemplary immune cell-associated protein (TriTAC protein) without an immunomodulatory drug, and Figure 1B illustrates the administration of an exemplary immune cell-associated protein (TriTAC protein) with an exemplary immunomodulatory drug (PD-1 and PD-L1 inhibitor). [Figure 2A] Figure 2 illustrates FACS analysis of PD-1 and PD-L1 expression in T cells co-cultured with 22Rv1 prostate cancer cells after treatment with half-life-extended prostate-specific membrane antigen (PSMA)-binding immune cell-associated protein. Figure 2A illustrates PD-1 levels, and Figure 2B illustrates PD-L1 levels. [Figure 2B] Figure 2 illustrates FACS analysis of PD-1 and PD-L1 expression in T cells co-cultured with 22Rv1 prostate cancer cells after treatment with half-life-extended prostate-specific membrane antigen (PSMA)-binding immune cell-associated protein. Figure 2A illustrates PD-1 levels, and Figure 2B illustrates PD-L1 levels. [Figure 3] Figure 1 illustrates FACS analysis of PD-L1 expression in 22Rv1 prostate cancer cells after treatment with half-life extended prostate-specific membrane antigen (PSMA)-binding immune cell-associated protein or IFNγ. [Figure 4] Illustrates FACS analysis of PD-L1 expression in PC3-PSMA prostate cancer cells after treatment with half-life extended prostate-specific membrane antigen (PSMA)-binding immune cell-associated protein or IFNγ. [Figure 5] 2 illustrates a 22Rv1 prostate cancer tumor model treated with a half-life extended prostate-specific membrane antigen (PSMA)-binding immune cell-associated protein alone or in combination with pembrolizumab or atezolizumab. [Figure 6] Illustrates a PC3-PSMA prostate cancer tumor model treated with a half-life extended prostate-specific membrane antigen (PSMA)-binding immune cell-associated protein alone or in combination with pembrolizumab or atezolizumab. [Figure 7A] Figure 7 illustrates FACS analysis of PD-1 and PD-L1 expression in T cells cocultured with NCI-H292 lung cancer cells after treatment with mesothelin (MSLN)-binding immune cell-associated protein. Figure 7A illustrates PD-1 levels, and Figure 7B illustrates PD-L1 levels. [Figure 7B] Figure 7 illustrates FACS analysis of PD-1 and PD-L1 expression in T cells cocultured with NCI-H292 lung cancer cells after treatment with mesothelin (MSLN)-binding immune cell-associated protein. Figure 7A illustrates PD-1 levels, and Figure 7B illustrates PD-L1 levels. [Figure 8] Figure 1 illustrates FACS analysis of PD-L1 expression in NCI-H292 lung cancer cells after treatment with half-life-extended mesothelin (MSLN)-binding immune cell-associated protein or IFNγ. [Figure 9] 1 illustrates FACS analysis of PD-L1 expression in OVCAR8 cancer cells after treatment with IFNγ. [Figure 10] 1 illustrates the HCI-H292 lung cancer tumor model treated with half-life-extended mesothelin (MSLN)-binding immune cell-associated protein alone or in combination with atezolizumab or pembrolizumab. Two plots represent the results of two independent experiments. [Figure 11] Illustrates an OVCAR8 ovarian cancer tumor model treated with half-life extended mesothelin (MSLN)-binding immune cell-associated protein alone or in combination with atezolizumab. [Figure 12A] Figure 12 illustrates FACS analysis of PD-1 and PD-L1 expression in T cells co-cultured with SHP-77 small cell lung cancer cells after treatment with half-life-extended DLL3-binding immune cell-associated proteins. Figure 12A illustrates PD-1 levels, and Figure 12B illustrates PD-L1 levels. [Figure 12B]Figure 12 illustrates FACS analysis of PD-1 and PD-L1 expression in T cells co-cultured with SHP-77 small cell lung cancer cells after treatment with half-life-extended DLL3-binding immune cell-associated proteins. Figure 12A illustrates PD-1 levels, and Figure 12B illustrates PD-L1 levels. [Figure 13] Figure 1 illustrates FACS analysis of PD-L1 expression in SHP-77 small cell lung cancer cells after treatment with IFNγ. [Figure 14] 1 illustrates a SHP-77 small cell lung cancer tumor model treated with a half-life extended DLL3-binding immune cell-associated protein alone or in combination with pembrolizumab or atezolizumab. DETAILED DESCRIPTION OF THE INVENTION

[0036] Specific Definitions The terms used herein are for the purpose of describing particular instances only and are not intended to limit the invention. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including," "includes," "having," "has," "with," or variations thereof are used in either the detailed description and / or claims, such terms are intended to be included in a manner similar to the term "comprising."

[0037] The term "about" or "approximately" means within an acceptable error range of 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 1 or more than 1 standard deviation, according to convention, for any value. When particular values ​​are described in this application and in the claims, unless otherwise specified, the term "about" should be assumed to mean within an acceptable error range of the particular value.

[0038] The terms "individual," "patient," or "subject" are used interchangeably. None of the terms require or are limited to a condition characterized by or under the supervision (e.g., continuous or intermittent) of a health care professional (e.g., a physician, registered nurse, nurse practitioner, physician assistant, janitor, or hospice worker).

[0039] The term "antibody" typically refers to a Y-shaped tetrameric protein containing two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and noncovalent interactions. Human light chains contain a variable domain (VL) and a constant domain (CL), which can be easily classified as kappa or lambda based on amino acid sequence and gene locus. Each heavy chain contains one variable domain (VH) and a constant region, which in the case of IgG, IgA, and IgD, contains three domains called CH1, CH2, and CH3 (IgM and IgE have a fourth domain, CH4). In the classification of IgG, IgA, and IgD, the CH1 and CH2 domains are separated by a flexible hinge region, a proline- and cysteine-rich segment of variable length (usually 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 heavy chains having a "D" region of about 10 additional amino acids. Each class of antibody contains interchain and intrachain disulfide bonds formed by paired cysteine ​​residues. Immunoglobulin molecules have two types of natural disulfide bridges or bonds: interchain and intrachain disulfide bonds. The location and number of interchain disulfide bonds vary depending on the immunoglobulin class and species. Interchain disulfide bonds are located on the surface of the immunoglobulin, are solvent accessible, and are usually relatively easily reduced. In the human IgG1 isotype, there are four interchain disulfide bonds: one from each heavy chain to the light chain and two between heavy chains. Interchain disulfide bonds are not required for chain assembly. 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 comprise the interchain disulfide bonds (two heavy / heavy, two heavy / light), providing structural flexibility that facilitates Fab movement. The interchain 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 interchain disulfide bonds between the heavy chains are at positions C226 and C229 (all numbered according to the EU index according to Kabat, et al., below).

[0040] As used herein, the term "antibody" includes polyclonal antibodies, multiclonal 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, e.g., hcIgG, V-NAR, F v, Fd, Fab, F(ab')2, F(ab'), Fab2, Fab3 fragments, single chain fragments (e.g., di-scFv, scFv, scFvFc, scFv-zipper, scFab), disulfide-linked Fvs (sdFv), Fd fragments consisting of a VH and a CH1 domain, linear antibodies, single domain antibodies, e.g., nanobodies, or single variable domain antibodies comprising only one variable domain antibody (VH domain, VL domain, or VHH domain), such as sdAb, "r These include "IgG" ("half antibodies"), diabodies, single-chain diabodies, tandem diabodies (Tandabs), tandem di-scFvs, tandem tri-scFvs, "minibodies" exemplified in some examples by structures such as: (VH-VL-CH3)2, (scFv-CH3)2, ((scFv)2-CH3+CH3), ((scFv)2-CH3), or (scFv-CH3-scFv)2, multibodies such as triabodies or tetrabodies, and derivatives thereof containing Fc fusions and other modifications, and any other immunoreactive molecule so long as it contains a domain with a binding site for preferential association or binding to FLT3 protein. Furthermore, unless contextual constraints dictate otherwise, 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 corresponding to the various classes of antibodies are typically designated by the corresponding lowercase Greek letters α, δ, ε, γ, and μ, respectively.The light chains of antibodies of any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0041] As used herein, the term "variable region" or "variable domain" refers to the fact that certain portions of the variable domain vary significantly in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is not uniformly distributed throughout the variable domains of antibodies. It is concentrated in three segments or hypervariable regions called complementarity-determining regions (CDRs) in both the light- and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Naturally occurring heavy- and light-chain variable domains each contain four FR regions adopting a β-sheet structure, connected by three CDRs that form loops that connect, and in some cases form part of, the β-sheet structure. The CDRs of each chain are held together in close proximity by the FR regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institutes 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.The amino acid assignments for each domain, framework region, and CDR, in some embodiments, follow 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 Dubel, Ed. (2007) Handbook of Therapeutic Antibodies, 3rd Ed., Wily-VCH Verlag GmbH and Co. or AbM (Oxford Molecular / MSI Pharmacopeia), unless otherwise specified.

[0042] "Kabat-like variable domain residue numbering" or "Kabat-like amino acid position numbering," and variations thereof, refer to the numbering system used for the heavy or light chain variable domains of the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to omissions from, or insertions into, FRs or CDRs of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues can be determined for a given antibody by alignment of the homologous regions of the antibody's sequence with the "standard" Kabat-numbered sequence. The CDRs of this disclosure are not intended to necessarily correspond to the Kabat numbering convention.

[0043] The terms "framework" or "FR" residues (or regions) refer to variable domain residues other than the CDR or hypervariable region residues as defined herein. A "human consensus framework" is a framework representing the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences.

[0044] As used herein, the term "epitope" 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. An antigen may have more than one epitope. Thus, different antibodies may bind to different sites on the antigen and have different biological effects. Epitopes may be conformational or linear. Conformational epitopes are formed by spatially juxtaposed amino acids in different segments of a linear polypeptide chain. Linear epitopes are formed by adjacent amino acid residues within a polypeptide chain. In certain circumstances, epitopes may include sugar, phosphate, or sulfonyl moieties on the antigen.

[0045] As used herein, the term "percent (%) amino acid sequence identity" for 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 specific sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within 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 appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across 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 along with user documentation from the U.S. Copyright Office, Washington, DC 20559, where it 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.

[0046] 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 meant to encompass a quantitative measure 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 reach the concentration required for saturation of the elimination process. The rate of an exponential process can be measured by its rate constant, k, which represents the fractional rate of change per time unit, or by its half-life, t, which is the time required for 50% of the process to be completed. 1 / 2 The units of these two constants are hr-1 and hr, respectively. The first-order rate constant and the half-life of a reaction are simply related (k × t 1 / 2 =0.693), which may be exchanged accordingly. First-order elimination kinetics dictates that a constant fraction of the drug is lost per unit of time, so that a plot of the logarithm of drug concentration versus time is linear for all times after the initial distribution phase (i.e., after drug absorption and distribution are complete). Drug elimination half-life can be accurately determined from such a graph.

[0047] As used herein, the term "binding affinity" refers to the affinity of a protein described in this disclosure for a binding target and is expressed numerically using a "Kd" value. When two or more proteins are shown to have equivalent binding affinities for their binding targets, the Kd values ​​for the binding of each protein to those binding targets are within ±2-fold of each other. When two or more proteins are shown to have equivalent binding affinities for a single binding target, the Kd values ​​for the binding of each protein to the single binding target are within ±2-fold of each other. When proteins are shown to bind to two or more targets with equivalent binding affinities, the Kd values ​​for the binding of each 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" or "rate of association" or "k" and the "off-rate" or "rate of dissociation" or "k" are also determined with surface plasmon resonance technology using a BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ). In further embodiments, "K", "k", and "k" are measured using OCTET® Systems (Pall Life Sciences).In an exemplary method for measuring binding affinity using the OCTET® Systems, a ligand, e.g., biotinylated human or cynomolgus monkey FLT3, an immune cell-associated protein including a FLT3-binding single domain antibody, is immobilized on the OCTET® streptavidin capillary sensor tip. The streptavidin tip is then activated with approximately 20-50 μg / ml of human or cynomolgus monkey FLT3 protein according to the manufacturer's instructions. A PBS / casein solution is also introduced as a blocking agent. For association kinetic measurements, the FLT3-binding protein variant 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 FLT3-binding single domain protein is used at a concentration ranging from about 2 ng / mL to about 20 μg / mL. Complete dissociation is observed in the negative control, assay buffer without binding protein. The kinetic parameters of the binding reaction are then determined using an appropriate tool, for example, ForteBio software.

[0048] As used herein, in some embodiments, "treatment" or "treating" or "treated" refers to therapeutic treatment aimed at delaying (alleviating) an undesirable physiological disease, disorder, or condition, or to obtain a beneficial or desired clinical result. For purposes described herein, a beneficial or desired clinical result includes, but is not limited to, alleviation of symptoms, reduction in the extent of the disease, disorder, or condition, stabilization of the disease, disorder, or condition (i.e., not worsening), delay in the onset or progression of the disease, disorder, or condition, improvement of the disease, disorder, or disease state, and remission (whether partial or total), or enhancement or amelioration of the disease, disorder, or condition, whether detectable or undetectable. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment further includes prolonging survival compared to expected survival without treatment. In other embodiments, "treatment" or "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, for example, in an individual predisposed to the condition (e.g., an individual bearing a genetic marker for a disease such as breast cancer).

[0049] In general, it should be noted that the term "single domain antibody," as used herein in its broadest sense, is not limited to a specific biological source or a specific 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 that naturally lack light chains, single domain antibodies derived from traditional four-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. Single domain antibodies can be any of the single domain antibodies in the art or future. Single domain antibodies can be derived from any species, including, but not limited to, mouse, human, camel, llama, goat, rabbit, or cow. For example, in some embodiments, single domain antibodies of the present disclosure are obtained by: (1) isolating a VHH domain of a naturally occurring heavy chain antibody; (2) expressing a nucleotide sequence encoding the naturally occurring VHH domain; (3) "humanizing" a naturally occurring VHH domain or expressing a nucleic acid encoding such a humanized VHH domain; (4) "camelizing" a naturally occurring VH domain from any animal species, and in particular from a mammalian species such as human, or expressing a nucleic acid encoding such a camelized VH domain; (5) "camelizing" a "domain antibody" or "Dab" or expressing a nucleic acid encoding such a camelized VH domain; (6) using synthetic or semi-synthetic techniques to prepare proteins, polypeptides, or other amino acid sequences; (7) preparing a nucleic acid encoding a single domain antibody using techniques for nucleic acid synthesis known in the art, followed by expression of the nucleic acid obtained as above; and / or (8) any combination of one or more of the foregoing.

[0050] In some embodiments, immune cell-associated proteins as described herein include antibodies, such as single-domain antibodies targeting antigens such as PSMA, MSLN, BCMA, DLL3, EGFR, EpCAM, FLT3, etc., that comprise an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain, but that has been "humanized," e.g., by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH sequence (and in particular within the framework sequences) with one or more amino acid residues occurring at the corresponding positions in a VH domain from a conventional four-chain antibody of human origin (e.g., as shown above). This can be done in a manner known in the art that will be apparent to the skilled artisan, for example, based on the description further below. Again, such humanized single-domain antibodies of the present disclosure can be obtained in any suitable manner known per se (e.g., as shown in points (1) to (8) above) and are therefore not strictly limited to polypeptides obtained using a polypeptide comprising a naturally occurring VHH domain as starting material. In some further embodiments, single domain antibodies include single domain antibodies having an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain as described herein, but that has been "camelized," i.e., by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional four-chain antibody with one or more amino acid residues that occur at the corresponding positions in a VHH domain of a heavy-chain antibody. Such "camelizing" substitutions are preferably inserted at amino acid positions that form and / or are present at the VH-VL interface and / or residues that are prominent in so-called camelids (see, e.g., WO 94 / 04678 and Davies and Riechmann (1994 and 1996)). Preferably, the VH sequence used as starting material or starting point for generating or designing a camelized single domain is a VH sequence, preferably from a mammal, more preferably a human VH sequence, such as a VH3 sequence.However, it should be noted that in certain embodiments, such camelized anti-MSLN single domain antibodies of the present disclosure are obtained by any suitable method known in the art (i.e., as set forth in points (1) to (8) above) and are therefore not strictly limited to polypeptides obtained using a naturally occurring polypeptide comprising a VH domain as the starting material. For example, as further described herein, both "humanization" and "camelization" are carried out by providing a nucleotide sequence encoding a naturally occurring VHH domain or VH domain, and then altering one or more codons in said nucleotide sequence in such a way that the new nucleotide sequence encodes a "humanized" or "camelized" single domain antibody, respectively. This nucleic acid can then be expressed to obtain the desired single domain antibody of the present disclosure. Alternatively, in other embodiments, the amino acid sequence of the desired humanized or camelized single domain antibody of the present disclosure is designed based on the amino acid sequence of a naturally occurring VHH domain or VH domain, respectively, and then synthesized de novo using known techniques of peptide synthesis. In some embodiments, a nucleotide sequence encoding the desired humanized or camelized single domain antibody of the present disclosure is designed based on the amino acid or nucleotide sequence of a naturally occurring VHH domain or VH domain, respectively, and then synthesized de novo using known techniques for nucleic acid synthesis, and the nucleic acid thus obtained is then expressed using known expression techniques to obtain the desired single domain antibody of the present disclosure.

[0051] The term "patient" or "subject" refers to any single subject for whom treatment is desired or who is participating in a clinical trial, epidemiological study, or used as a control, and includes humans and mammalian veterinary patients, e.g., cattle, horses, dogs, and cats.

[0052] immune cell-associated proteins In some embodiments, the present specification describes a combination comprising an immunomodulatory agent and an immune cell-associated protein. In some embodiments, the immune cell-associated protein is a bispecific protein, a trispecific protein, or a multispecific protein. In some embodiments, the immune cell-associated protein is a half-life extending protein. In some embodiments, the immune cell-associated protein comprises a domain that binds to a bulk serum protein, such as serum albumin. In some embodiments, the serum albumin is human serum albumin (HSA).

[0053] In some embodiments, the immune cell-associating protein comprises an immune cell-associating domain. In some embodiments, "immune cell-associating 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 NK cells, B cells, dendritic cells, and macrophage cells. The immune cell-associating domain, in some embodiments, 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 embodiments, the immune cell-associating 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 association 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.

[0054] In some embodiments, the immune cell-associating domain comprises a natural killer (NK) cell-associating domain, a T cell-associating domain, a B cell-associating domain, a dendritic cell-associating domain, a macrophage cell-associating domain, or a combination thereof. In some embodiments, the immune cell-associated protein comprises a T cell-associating domain. In some embodiments, the T cell-associating domain binds to a CD3 molecule. In some embodiments, the CD3 molecule is at least one of a CD3γ molecule, a CD3δ molecule, or a CD3ε molecule.

[0055] In some embodiments, the immune cell-associated protein comprises an antigen-binding domain associated with the immune cell. In some examples, the antigen-binding domain comprises an antibody, single-chain antibody, Fab, Fv, T-cell receptor binding domain, ligand-binding domain, receptor-binding domain, domain antibody, single antibody, minibody, nanobody, peptibody, or various other antibody mimetics (such as affimers, affitins, alphabodies, atrimers, CTLA4-based molecules, adnectins, anticalins, Kunitz domain-based proteins, avimers, knottins, fynomers, darpins, affibodies, affilins, monobodies, and armadillo repeat protein-based proteins).

[0056] In some embodiments, the 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 In A1), CD22 (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'-nucleotidin enzyme), CD39 (ENTPD1, ​​ectonucleotide triphosphate diphosphohydrolase-1), CD74 (HLA class II histocompatibility antigen gamma chain, HLA-DR antigen-associated invariant chain), CD79b (immunoglobulin-associated beta), CD80 (B7-1), CD86 (B7-2), CD123 (IL3RA, interleukin-3 receptor), CD133 (PROM1), CD137 (TNFRSF9, tumor necrosis factor receptor superfamily member 9, 4-1BB, ILA (induced by lymphocyte activation), 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), 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 (MUC1) and its splice variants (e.g., MUC1 / C, D, and Z), mucin 16 (MUC16), necdin (NDN), necdin cell adhesion molecule 4 (NECTIN4), SLIT and NTRK-like family member 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 (SIG LEC9), 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 (TNFRSF10 A, 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 beta-1 (TGFB1), trophoblast glycoprotein (TPBG, 5T4), trophinin (TRO, MAGED3), tumor-associated calcium signal transducer 2 (TACSTD2, TROP2,In some embodiments, the immune cell-associated protein specifically binds to PSMA, MSLN, BCMA, DLL3, EGFR, FLT3, or EpCAM.

[0057] Described herein is an immune cell-associated protein comprising a first domain (A) that is a T cell-associating 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-associated protein are 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 a linker L1 and 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.

[0058] In some embodiments, immune cell-associated proteins described herein comprise polypeptides having sequences set forth in SEQ ID NOs: 3218-3462, and subsequences thereof. In some embodiments, immune cell-associated proteins comprise polypeptides having at least 70%-95% or more homology to the sequences set forth in SEQ ID NOs: 3218-3462. In some embodiments, immune cell-associated proteins comprise polypeptides having at least 70%, 75%, 80%, 85%, 90%, 95% or more homology to the sequences set forth in SEQ ID NOs: 3218-3462. In some embodiments, immune cell-associated proteins have sequences comprising at least a portion of the sequences set forth in SEQ ID NOs: 3218-3462. In some embodiments, immune cell-associated proteins comprise polypeptides comprising one or more of the sequences set forth in SEQ ID NOs: 3218-3462.

[0059] In some embodiments, immune cell-associated proteins described herein comprise polypeptides having sequences set forth in SEQ ID NOs: 3255, 3340, 3376, and 3462, and subsequences thereof. In some embodiments, immune cell-associated proteins comprise polypeptides having at least 70% to 95% or more homology to the sequences set forth in SEQ ID NOs: 3255, 3340, 3376, and 3462. In some embodiments, immune cell-associated proteins comprise polypeptides having at least 70%, 75%, 80%, 85%, 90%, 95% or more homology to the sequences set forth in SEQ ID NOs: 3255, 3340, 3376, and 3462. In some embodiments, immune cell-associated 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, immune cell-associated proteins comprise polypeptides comprising one or more of the sequences set forth in SEQ ID NOs: 3255, 3340, 3376, and 3462.

[0060] Prostate-specific membrane antigen (PSMA) binding protein Described herein are immune cell-associated proteins comprising a PMSA-binding domain, pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors, and host cells for making such proteins. Additionally, methods of using the disclosed proteins comprising the PMSA-binding domain of the present disclosure in the prevention and / or treatment of diseases, conditions, and disorders are also provided.

[0061] PSMA is a 100kD type II membrane glycoprotein expressed in prostate tissue that shares sequence identity with the transferrin receptor, which has NAALADase activity. PSMA is expressed in increased amounts in prostate cancer, and elevated PSMA levels can also be detected in the serum of these patients. PSMA expression increases with disease progression and is highest in metastatic hormone-refractory disease, for which there is currently no treatment.

[0062] The design of the PSMA-targeting immune cell-associated proteins described herein allows for flexibility in the binding domain to the PSMA protein, in that the PSMA-binding domain can be any type of binding domain, including, but not limited to, domains derived from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, and humanized antibodies. In some embodiments, the PSMA-binding domain is a single-chain variable fragment (scFv), a single-domain antibody, such as a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain of a camelid-derived single-domain antibody (VHH). In other embodiments, the PSMA-binding domain is a non-Ig binding domain, i.e., an antibody mimic such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, Kunitz domain peptides, and monobodies. In further embodiments, the binding domain for PSMA is a ligand or peptide that binds to or associates with PSMA. In yet further embodiments, the binding domain for PSMA is a knottin. In yet further embodiments, the binding domain for PSMA is a small molecule entity.

[0063] 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.

[0064] 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 HSA-binding protein comprises a polypeptide having at least 70%-95% or more homology to the sequence set forth in SEQ ID NOs: 462-489. In some embodiments, the HSA-binding domain comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or more homology to the sequence set forth in SEQ ID NOs: 462-489. In some embodiments, the HSA-binding domain has a sequence comprising at least a portion of the 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.

[0065] 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.

[0066] Mesothelin (MSLN) binding protein MSLN is a GPI-linked membrane-bound tumor antigen. It is overexpressed in ovarian, pancreatic, lung, and triple-negative breast cancers and mesothelioma. Normal tissue expression of MSLN is limited to the unicellular mesothelial layer lining the pleural, pericardial, and peritoneal cavities. MSLN overexpression is associated with poor prognosis in lung adenocarcinoma and triple-negative breast cancer. MSLN has been used as a cancer antigen for multiple modalities, including immunotoxins, vaccines, antibody-drug conjugates, and CAR-T cells. Early signs of clinical efficacy have validated MSLN as a target, but improved therapeutic efficacy is needed to treat MSLN-expressing cancers.

[0067] Mesothelin is a glycoprotein present on the surface of cells in the mesothelial lining of 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 was cloned from a library prepared from the HPC-Y5 cell line (Kojima et al. (1995) J. Biol. Chem. 270:21984-21990). The cDNA was further cloned using the monoclonal antibody K1, which recognizes mesothelioma (Chang and Pastan (1996) Proc. Natl. Acad. Sci. USA 93:136-40). 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 cancer, 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, weak expression seen in benign pancreatic tissue. Argani P, et al. Clin Cancer Res. 2001; 7(12):3862-3868. Epithelial malignant pleural mesothelioma (MPM) universally expresses mesothelin, whereas sarcomatoid MPM likely does not. Most serous epithelial ovarian cancers and related primary peritoneal carcinomas express mesothelin.

[0068] Because trace amounts of mesothelin can be detected in the blood of some patients with mesothelin-positive cancers, mesothelin may also be used as a marker for the diagnosis and prognosis of certain types of cancer (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 membrane-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 asbestos-exposed workers (Creaney and Robinson, Hematol. Oncol. Clin. North Am. 19:1025-1040, 2005). Furthermore, patients with ovarian, pancreatic, and lung cancer have also shown elevated serum levels of soluble mesothelin (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). Therefore, mesothelin is a suitable target for methods of disease prevention or treatment, and effective antibodies specific for mesothelin are needed.

[0069] Mature mesothelin on the cell surface has been shown to contain three distinct domains: region I (containing residues 296–390), region II (containing residues 391–486), and region 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 disrupt the interaction between mesothelin and CA-125. Phage display identified Fv SS, which was affinity-optimized and used to generate a recombinant immunotoxin, SS1P, that targets mesothelin. The MORAb-009 antibody amatuximab, which also uses SSI, recognizes a nonlinear epitope in the amino-terminal 64 amino acids of mesothelin within region I. SSI Fv was also used to generate chimeric antigen receptor-engineered T cells. Recently, new anti-mesothelin antibodies that recognize other regions of the mesothelin protein have been reported.

[0070] There remains a need for additional treatment options for solid tumor diseases associated with mesothelin overexpression, such as ovarian cancer, pancreatic cancer, mesothelioma, lung cancer, gastric cancer, and triple-negative breast cancer. In certain embodiments, the present disclosure provides MSLN-targeting immune cell-associated proteins that contain a binding domain that specifically binds to MSLN on the surface of tumor target cells.

[0071] The design of the MSLN-targeting immune cell-associated proteins described herein allows for flexibility in the binding domain for the MSLN protein, in that the binding domain for MSLN can be any type of binding domain, including, but not limited to, domains derived from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, and humanized antibodies. In some embodiments, the binding domain for MSLN is a single-chain variable fragment (scFv), a single-domain antibody, such as a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain of a camelid-derived single-domain antibody (VHH). In other embodiments, the binding domain for MSLN is a non-Ig binding domain, i.e., an antibody mimic such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, Kunitz domain peptides, and monobodies. In a further embodiment, the binding domain for MSLN is a ligand or peptide that binds to or associates with MSLN. In yet a further embodiment, the binding domain for MSLN is a knottin. In yet a further embodiment, the binding domain for MSLN is a small molecule entity.

[0072] In some embodiments, the MSLN-binding domain binds to a protein comprising the sequence of SEQ ID NO: 3204. In some embodiments, the MSLN-binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 3204.

[0073] In some embodiments, the MSLN-binding domain disclosed herein recognizes full-length mesothelin. In specific examples, the MSLN-binding domain disclosed herein recognizes an epitope in region I (comprising amino acid residues 296-390 of SEQ ID NO: 3204), region II (comprising amino acid residues 391-486 of SEQ ID NO: 3204), or region III (comprising amino acid residues 487-598 of SEQ ID NO: 3204) of mesothelin. It is contemplated that, in some embodiments, the MSLN-binding domain of the present disclosure may 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.

[0074] In some embodiments, the MSLN-binding domain is an anti-MSLN antibody or 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 anti-MSLN antibodies described herein are contemplated. For example, in certain embodiments, amino acid sequence variants of the anti-MSLN antibodies described herein are contemplated to improve the binding affinity and / or other biological properties of the antibody. Exemplary 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, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the antibody.

[0075] Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen binding. In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include CDRs and framework regions. Examples of such substitutions are described below. Amino acid substitutions may be introduced into an antibody of interest, and the products are screened for the desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved T-cell-mediated cytotoxicity (TDCC). Conservative and non-conservative amino acid substitutions are contemplated for preparing antibody variants.

[0076] In another example of substitutions to create a variant anti-MSLN antibody, one or more hypervariable region residues of the parent antibody are substituted. Generally, variants are selected based on a desired improved property compared to the parent antibody, such as increased affinity, decreased affinity, decreased immunogenicity, or increased pH-dependent binding.

[0077] In some embodiments, the MSLN-binding domain of the MSLN-targeting immune cell-associated protein is a single-domain antibody specific for mesothelin, such as a heavy chain variable domain (VH), a variable domain (VHH) of a llama-derived sdAb, a peptide, a ligand, or a small molecule. In some embodiments, the mesothelin-binding domain of the MSLN-targeting immune cell-associated protein described herein is any domain that binds to mesothelin, including, but not limited to, domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, and humanized antibodies. In certain embodiments, the MSLN-binding domain is a single-domain antibody. In other embodiments, the MSLN-binding domain is a peptide. In further embodiments, the MSLN-binding domain is a small molecule.

[0078] In one embodiment, the single domain antibody corresponds to a VHH domain of a naturally occurring heavy chain antibody against MSLN. As further described herein, such a VHH sequence can typically be generated or obtained by appropriately immunizing a species of llama with MSLN (i.e., to generate an immune response and / or heavy chain antibodies against MSLN), by obtaining a suitable biological sample (such as a blood sample, serum sample, or B cell sample) from said llama, and generating a VHH sequence against MSLN starting from said sample using any suitable technique known in the art.

[0079] In another embodiment, such naturally occurring VHH domains against MSLN are obtained from a naive library of Camelidae VHH sequences, for example by screening such a library with MSLN or by screening for at least one portion, 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 WO99 / 37681, WO01 / 90190, WO03 / 025020, and WO03 / 035694. Alternatively, improved synthetic or semi-synthetic libraries derived from naive VHH libraries are used, for example, VHH libraries obtained from naive VHH libraries by techniques such as random mutagenesis and / or CDR shuffling, as described in WO00 / 43507.

[0080] In a further embodiment, yet another technique for obtaining VHH sequences against MSLN involves appropriately immunizing a transgenic mammal capable of expressing heavy chain antibodies (i.e., to generate an immune response and / or heavy chain antibodies against MSLN), obtaining a suitable biological sample from said transgenic mammal (such as a blood sample, serum sample, or B cell sample), and generating VHH sequences against MSLN starting from said sample using any suitable technique known in the art. For example, for this purpose, heavy chain antibody-expressing rats or mice and methods and techniques such as those described in WO02 / 085945 and WO04 / 049794 can be used.

[0081] In some embodiments, anti-MSLN single domain antibodies of MSLN-targeting immune cell-associated proteins include single domain antibodies having an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain, but that has been "humanized", i.e., by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH sequence (and in particular within the framework sequences) with one or more amino acid residues occurring at the corresponding positions in a VH domain from a conventional four-chain antibody of human origin (e.g., as shown above).

[0082] Other suitable methods and techniques for obtaining an anti-MSLN single domain antibody of the present disclosure and / or a nucleic acid encoding the same, starting from a naturally occurring VH or VHH sequence, include, for example, combining, in a suitable manner, one or more portions of one or more naturally occurring VH sequences (such as one or more framework (FR) sequences and / or complementarity determining region (CDR) sequences), one or more portions of one or more naturally occurring VHH sequences (such as one or more FR sequences or CDR sequences), and / or one or more synthetic or semi-synthetic sequences to provide an anti-MSLN single domain antibody of the present disclosure or a nucleotide sequence or nucleic acid encoding the same.

[0083] In some embodiments, the MSLN-binding domain is an anti-MSLN-specific 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, the MSLN-binding domain includes any domain that binds to MSLN, including, but not limited to, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, or an antigen-binding fragment, such as a single-domain antibody (sdAb), Fab, Fab', F(ab)2, and Fv fragment, a fragment composed of one or more CDRs, a single-chain antibody (e.g., a single-chain Fv fragment (scFv)), a disulfide-stabilized (dsFv) Fv fragment, a heteroconjugate antibody (e.g., a bispecific antibody), a pFv fragment, a heavy chain monomer or dimer, a light chain monomer or dimer, and a dimer consisting of one heavy chain and one light chain. In some embodiments, the MSLN-binding domain is a single-domain antibody. In some embodiments, the anti-MSLN single-domain antibody comprises heavy chain variable complementarity-determining regions (CDRs): CDR1, CDR2, and CDR3.

[0084] 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 comprises an amino acid sequence selected from SEQ ID NOs: 607-650.

[0085] In some embodiments, CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 490, or a variant thereof 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 thereof 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 thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in SEQ ID NO: 3506.

[0086] In some embodiments, CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 518, or a variant thereof 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 thereof 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 thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in SEQ ID NO: 3508.

[0087] In some embodiments, CDR1 comprises the 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 the 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 the 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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 NOs: 3506, 3508, or 568-606.

[0092] In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 607. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 608. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 609. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 610. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 611. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 612. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 613. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 614. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 615. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 616. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 617. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 618. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 619.In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 620. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 621. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 622. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 623. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 624. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 625. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 626. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 627. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 628. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 629. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 630. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 631. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 632.In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 633. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 634. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a single domain antibody comprising the sequence of SEQ ID NO: 635.

[0093] In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a humanized single-domain antibody comprising the sequence of SEQ ID NO: 636. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a humanized single-domain antibody comprising the sequence of SEQ ID NO: 637. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a humanized single-domain antibody comprising the sequence of SEQ ID NO: 638. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a humanized single-domain antibody comprising the sequence of SEQ ID NO: 639. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a humanized single-domain antibody comprising the sequence of SEQ ID NO: 640. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a humanized single-domain antibody comprising the sequence of SEQ ID NO: 641. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a humanized single-domain antibody comprising the sequence of SEQ ID NO: 642. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a humanized single-domain antibody comprising the sequence of SEQ ID NO: 643. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a humanized single-domain antibody comprising the sequence of SEQ ID NO: 644. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a humanized single-domain antibody comprising the sequence of SEQ ID NO: 645. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a humanized single-domain antibody comprising the sequence of SEQ ID NO: 646. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a humanized single-domain antibody comprising the sequence of SEQ ID NO: 647. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a humanized single-domain antibody comprising the sequence of SEQ ID NO: 648.In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a humanized single-domain antibody comprising the sequence of SEQ ID NO: 649. In some embodiments, the MSLN-binding protein according to any one of the above embodiments is a humanized single-domain antibody comprising the sequence of SEQ ID NO: 650.

[0094] In some embodiments, the MSLN-binding domain is cross-reactive with human and cynomolgus monkey mesothelin. In some embodiments, the MSLN-binding domain is specific for human mesothelin. In certain embodiments, the MSLN-binding domain disclosed herein binds to human mesothelin with a human Kd (hKd). In certain embodiments, the MSLN-binding domain disclosed herein binds to cynomolgus monkey mesothelin with a cynomolgus monkey Kd (cKd). In certain embodiments, the MSLN-binding domain disclosed herein binds to both cynomolgus monkey mesothelin and human mesothelin with a cynomolgus monkey Kd (cKd) and a human Kd (hKd), respectively. In some embodiments, the MSLN-binding protein binds to human and cynomolgus monkey mesothelin with comparable binding affinity (i.e., the hKd and cKd values ​​do not differ by more than ±10%). In some embodiments, the hKd and cKd are in the range of about 0.1 nM to about 500 nM. In some embodiments, the hKd and cKd are in the range of about 0.1 nM to about 450 nM. In some embodiments, the hKd and cKd are in the range of about 0.1 nM to about 400 nM. In some embodiments, the hKd and cKd are in the range of about 0.1 nM to about 350 nM. In some embodiments, the hKd and cKd are in the range of about 0.1 nM to about 300 nM. In some embodiments, the hKd and cKd are in the range of about 0.1 nM to about 250 nM. In some embodiments, the hKd and cKd are in the range of about 0.1 nM to about 200 nM. In some embodiments, the hKd and cKd are in the range of about 0.1 nM to about 150 nM. In some embodiments, the hKd and cKd are in the range of about 0.1 nM to about 100 nM. In some embodiments, the hKd and cKd are in the range of about 0.1 nM to about 90 nM. In some embodiments, the hKd and cKd are in the range of about 0.2 nM to about 80 nM. In some embodiments, the hKd and cKd are in the range of about 0.3 nM to about 70 nM. In some embodiments, the hKd and cKd are in the range of about 0.4 nM to about 50 nM.In some embodiments, the hKd and cKd are in the range of about 0.5 nM to about 30 nM. In some embodiments, the hKd and cKd are in the range of about 0.6 nM to about 10 nM. In some embodiments, the hKd and cKd are in the range of about 0.7 nM to about 8 nM. In some embodiments, the hKd and cKd are in the range of about 0.8 nM to about 6 nM. In some embodiments, the hKd and cKd are in the range of about 0.9 nM to about 4 nM. In some embodiments, the hKd and cKd are in the range of about 1 nM to about 2 nM.

[0095] In some embodiments, any of the aforementioned MSLN-binding domains (e.g., the anti-MSLN single-domain antibodies of SEQ ID NOs: 607-646) are tagged with an affinity peptide to facilitate purification. In some embodiments, the affinity peptide tag is six consecutive histidine residues, also known as 6X-his (SEQ ID NO: 3503).

[0096] 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 mesothelin present in or on the cell membrane surface of cells that express mesothelin. Soluble mesothelin refers to mesothelin that is no longer present in or on the cell membrane surface of cells that express or have 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 or more than soluble mesothelin. In one embodiment, an MSLN-targeting immune cell-associated protein of the present disclosure preferentially binds membrane-bound mesothelin over soluble mesothelin by 30-fold or more. Determining 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.

[0097] B-cell maturation antigen (BCMA) binding protein B-cell maturation antigen (BCMA, TNFRSF17, CD269) is a transmembrane protein belonging to the tumor necrosis family of receptors (TNFR) superfamily 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 from peripheral naive B cells. BCMA is expressed on multiple myeloma (MM) cells, leukemia cells, and lymphoma cells.

[0098] BCMA was identified by molecular analysis of the t(4,16)(q26,p13) translocation found in human intestinal T-cell lymphoma, and the in-frame sequence was mapped to chromosomal band 16p13.1.

[0099] The human BCMA cDNA has a 552 bp open reading frame encoding a 184 amino acid polypeptide. The BCMA gene is organized into three exons separated by two introns, each flanked by a GT donor and AG receptor consensus splicing site, and encodes a 1.2 kb transcript. The BCMA protein structure comprises an integral transmembrane protein based on a central 24 amino acid hydrophobic region in an α-helical structure.

[0100] The mouse BCMA gene is located on chromosome 16, syntenic to the human 16p13 region, and contains three additional exons separated by two introns. The gene encodes a 185-amino acid protein. Mouse BCMA mRNA is expressed as a 404-bp transcript, with maximal levels in plasmacytoma cells (J558) and moderate levels in the A20 B-cell lymphoma line. Mouse BCMA mRNA transcripts were detected at lower levels in T-cell lymphoma (EL4, BW5147) and dendritic cell (CB1D6, D2SC1) lines, in contrast to human cell lines of T-cell and dendritic cell origin. The mouse BCMA cDNA sequence shares 69.3% nucleotide identity with the human BCMA cDNA sequence, with slightly higher identity (73.7%) when comparing the coding regions of 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 integral transmembrane segment. The N-terminal 40-amino acid domain of both mouse and human BCMA proteins contains six conserved cysteine ​​residues, consistent with the formation of a cysteine ​​repeat motif found in the extracellular domain of TNFRs. Similar to members of the TNFR superfamily, BCMA proteins contain conserved aromatic residues 4-6 residues C-terminal from the first cysteine.

[0101] 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 differentiation of the cell (highest in plasma cells).

[0102] BCMA is involved in B cell development and homeostasis through its interaction with its ligands BAFF (B cell-activating factor, also known as TALL-1 or TNFSF13B) and APRIL (A proliferation-inducing ligand). BCMA, along 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 various aspects of humoral immunity, B cell development, and homeostasis. BCMA expression appears somewhat 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 primarily known for its functional activity in mediating plasma cell survival, which maintains long-term humoral immunity.

[0103] There is a need to have treatment options for solid tumor diseases associated with overexpression of BCMA, such as cancer, multiple myeloma, leukemia, and lymphoma. The present disclosure provides, in certain embodiments, single domain proteins that specifically bind to BCMA on the surface of tumor target cells.

[0104] The design of the BCMA-targeting immune cell-associated proteins described herein allows for flexibility in the binding domain for the BCMA protein, in that the binding domain for BCMA can be any type of binding domain, including, but not limited to, domains derived from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, and humanized antibodies. In some embodiments, the binding domain for BCMA is a single-chain variable fragment (scFv), a single-domain antibody, such as a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain of a camelid-derived single-domain antibody (VHH). In other embodiments, the binding domain for BCMA is a non-Ig binding domain, i.e., an antibody mimic such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, Kunitz domain peptides, and monobodies. In a further embodiment, the binding domain for BCMA is a ligand or peptide that binds to or associates with BCMA. In yet a further embodiment, the binding domain for BCMA is a knottin. In yet a further embodiment, the binding domain for BCMA is a small molecule entity.

[0105] In some embodiments, the BCMA binding domain binds to a protein comprising the sequence of SEQ ID NO: 3201, 3202, or 3203. In some embodiments, the BCMA binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 3201, 3202, or 3203.

[0106] In some embodiments, the BCMA-binding domain is an anti-BCMA antibody or 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 anti-BCMA antibodies described herein are contemplated. For example, in certain embodiments, amino acid sequence variants of the anti-BCMA antibodies described herein are contemplated to improve the binding affinity and / or other biological properties of the antibody. Exemplary 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, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the antibody.

[0107] Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen binding. In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include CDRs and framework regions. Examples of such substitutions are described below. Amino acid substitutions may be introduced into an antibody of interest, and the products are screened for the desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved T-cell-mediated cytotoxicity (TDCC). Conservative and non-conservative amino acid substitutions are contemplated for preparing antibody variants.

[0108] In another example of substitutions to create variant anti-BCMA antibodies, one or more hypervariable region residues of the parent antibody are substituted. Generally, variants are selected based on a desired improved property compared to the parent antibody, e.g., increased affinity, decreased affinity, decreased immunogenicity, increased pH-dependent binding.

[0109] In some embodiments, the BCMA-binding domain of the BCMA-targeting immune cell-associated protein is a BCMA-specific single-domain antibody, such as a heavy chain variable domain (VH), a variable domain (VHH) of a llama-derived sdAb, a peptide, a ligand, or a small molecule entity. In some embodiments, the BCMA-binding domain of the BCMA-targeting immune cell-associated protein described herein is any domain that binds to BCMA, including, but not limited to, a domain from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, or a humanized antibody. In certain embodiments, the BCMA-binding domain is a single-domain antibody. In other embodiments, the BCMA-binding domain is a peptide. In some embodiments, the BCMA-binding domain is a small molecule.

[0110] In one embodiment, the single domain antibody corresponds to a VHH domain of a naturally occurring heavy chain antibody against BCMA. As further described herein, such a VHH sequence can typically be generated or obtained by suitably immunizing a species of llama with BCMA (i.e. to generate an immune response and / or heavy chain antibodies against BCMA), obtaining a suitable biological sample from said llama (such as a blood sample, serum sample or B cell sample), and generating a VHH sequence against BCMA starting from said sample using any suitable technique known in the art.

[0111] In another embodiment, such naturally occurring VHH domains against BCMA are obtained from a naive library of camelid VHH sequences, for example by screening such library with BCMA or by screening for at least one portion, fragment, antigenic determinant, or epitope thereof using at least one screening technique known in the art. Such libraries and techniques are described, for example, in WO99 / 37681, WO01 / 90190, WO03 / 025020, and WO03 / 035694. Alternatively, improved synthetic or semi-synthetic libraries derived from naive VHH libraries are used, for example, VHH libraries obtained from naive VHH libraries by techniques such as random mutagenesis and / or CDR shuffling, as described in WO00 / 43507.

[0112] In a further embodiment, yet another technique for obtaining VHH sequences against BCMA involves suitably immunizing a transgenic mammal capable of expressing heavy chain antibodies (i.e., to generate an immune response and / or heavy chain antibodies against BCMA), obtaining a suitable biological sample from said transgenic mammal (a blood sample, a serum sample, or a sample of B cells), and generating VHH sequences against BCMA starting from said sample using any suitable technique known in the art. For example, heavy chain antibody-expressing rats or mice and methods and techniques such as those described in WO02 / 085945 and WO04 / 049794 can be used for this purpose.

[0113] In some embodiments, anti-BCMA single domain antibodies of BCMA-targeting immune cell-associated proteins include single domain antibodies having an amino acid sequence which corresponds to the amino acid sequence of a naturally occurring VHH domain, but which has been "humanised", i.e. "humanised" by replacing one or more amino acid residues in the amino acid sequence (and in particular within the framework sequences) of said naturally occurring VHH sequence with one or more amino acid residues occurring at the corresponding position in a VH domain from a conventional four-chain antibody of human origin (e.g. as set out above).

[0114] Other suitable methods and techniques for obtaining an anti-BCMA single domain antibody of the disclosure and / or a nucleic acid encoding the anti-BCMA single domain antibody, starting from a naturally occurring VH or VHH sequence, include, for example, combining one or more parts of one or more naturally occurring VH sequences (such as one or more framework (FR) sequences and / or complementarity determining region (CDR) sequences), one or more parts of one or more naturally occurring VHH sequences (such as one or more FR sequences or CDR sequences), and / or one or more synthetic or semi-synthetic sequences in a suitable manner to provide an anti-BCMA single domain antibody of the disclosure or a nucleotide sequence or nucleic acid encoding the same.

[0115] In some embodiments, the BCMA binding domain is an anti-BCMA specific 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, the BCMA binding domain comprises any domain that binds to BCMA, including but not limited to, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, or an antigen-binding fragment such as a single domain antibody (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 consisting of one heavy chain and one light chain. In some embodiments, the BCMA binding domain is a single domain antibody. In some embodiments, the anti-BCMA single domain antibody comprises heavy chain variable complementarity determining regions (CDRs): CDR1, CDR2, and CDR3.

[0116] In some embodiments, a BCMA binding protein of the disclosure is a polypeptide comprising an amino acid sequence made up 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.

[0117] In some embodiments, an exemplary CDR1 comprises an amino acid sequence set forth in SEQ ID NOs: 1 to 115. In some embodiments, another exemplary CDR2 comprises an amino acid sequence set forth in SEQ ID NOs: 116 to 230. In some embodiments, another exemplary CDR3 comprises an amino acid sequence set forth in SEQ ID NOs: 231 to 345.

[0118] In various embodiments, the BCMA binding proteins of the disclosure have a CDR1 with 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.

[0119] In various embodiments, the BCMA binding proteins of the disclosure have a CDR2 with 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: 116-230.

[0120] In various embodiments, the complementarity determining regions of the BCMA binding proteins of the disclosure have a CDR3 with 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] The BCMA binding proteins described herein can bind to human BCMA with an hKd in the range of about 0.1 nM to about 500 nM. In some embodiments, the hKd range is about 0.1 nM to about 450 nM. In some embodiments, the hKd range is about 0.1 nM to about 400 nM. In some embodiments, the hKd range is about 0.1 nM to about 350 nM. In some embodiments, the hKd range is about 0.1 nM to about 300 nM. In some embodiments, the hKd range is about 0.1 nM to about 250 nM. In some embodiments, the hKd range is about 0.1 nM to about 200 nM. In some embodiments, the hKd range is about 0.1 nM to about 150 nM. In some embodiments, the hKd range is about 0.1 nM to about 100 nM. In some embodiments, the hKd range is about 0.1 nM to about 90 nM. In some embodiments, the hKd range is about 0.2 nM to about 80 nM. In some embodiments, the hKd range is about 0.3 nM to about 70 nM. In some embodiments, the hKd range is about 0.4 nM to about 50 nM. In some embodiments, the hKd range is about 0.5 nM to about 30 nM. In some embodiments, the hKd range is about 0.6 nM to about 10 nM. In some embodiments, the hKd range is about 0.7 nM to about 8 nM. In some embodiments, the hKd range is about 0.8 nM to about 6 nM. In some embodiments, the hKd range is about 0.9 nM to about 4 nM. In some embodiments, the hKd range is about 1 nM to about 2 nM.

[0134] In some embodiments, any of the aforementioned BCMA binding domains are tagged with an affinity peptide 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).

[0135] In certain embodiments, the BCMA-binding domain of the present disclosure preferentially binds membrane-bound BCMA over soluble BCMA. Membrane-bound BCMA refers to BCMA present in or on the cell membrane surface of cells that express 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 or more than soluble BCMA. In one embodiment, the BCMA-targeting immune cell-associated protein of the present disclosure preferentially binds membrane-bound BCMA over soluble BCMA by 30-fold or more. Determining the 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.

[0136] 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, humans (accession numbers NP_058637 and NP_982353), chimpanzees (accession number XP_003316395), mice (accession number NP_031892), and rats (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 generates two processed transcripts: one of 2389 bases (accession number NM_016941) and one of 2052 bases (accession number NM_203486). The former transcript encodes a 618-amino acid protein (accession number NP_058637), and the latter encodes a 587-amino acid protein (accession number 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. The EGF domains are generally recognized as occurring approximately at 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 at amino acid residues 176-215 of hDLL3, and the N-terminal domain occurs approximately at amino acid residues 27-175 of hDLL3. The EGF-like domain, DSL domain, and N-terminal domain each comprise portions of the DLL3 protein defined by distinct amino acid sequences. The EGF-like domains, in some embodiments, are referred to as EGF1-EGF6, with EGF1 being closest to the N-terminal portion of the protein.DSL ligands generally consist of a unique N-terminal domain followed by a series of structural domains: 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 unique E3 ubiquitin ligases. The DSL domain is a degenerate EGF domain that is necessary but not sufficient for interaction with Notch receptors. Furthermore, the first two EGF-like repeats of most DSL ligands contain a smaller protein sequence motif known as a DOS domain, which interacts cooperatively with the DSL domain to activate Notch signaling.

[0137] In some embodiments, the disclosed DLL3 immune cell-associated proteins of the present disclosure are generated, engineered, or selected to react with a selected domain, motif, or epitope within the DLL3 protein. In some embodiments, the DLL3-targeted immune cell-associated protein binds to the DSL domain, and in some embodiments, binds to an epitope within the DSL domain that includes G203, R205, and P206.

[0138] The DLL3-binding domain of the DLL3-targeting immune cell-associated proteins of the present disclosure is, in some embodiments, engineered and / or selected to react with both isoforms of DLL3 or a single isoform of the protein, or conversely, to 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, e.g., the DLL3-binding domain, is engineered, engineered, and / or selected to react with a domain (or epitope therein) that is only displayed by DLL3 or with a domain that is at least somewhat conserved across multiple or all DLL family members.

[0139] 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 the same extracellular region, including at least the N-terminal domain, the 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 of 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 the 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 the N-terminal domain or an epitope therein.

[0140] In some embodiments, the DLL3-binding protein of the present disclosure, e.g., the DLL3-binding domain of the immune cell-associated protein of the present disclosure, binds to a full-length DLL3 protein or a fragment thereof, e.g., an epitope-containing fragment within a full-length DLL3 protein, as described above. In some cases, the epitope-containing fragment includes an antigenic or immunogenic fragment of the DLL3 protein and derivatives thereof. In some embodiments, the epitope-containing fragment, including the antigenic or immunogenic fragment, is 12 or more amino acids, 20 or more amino acids, 50, or 100 or more amino acids. In some embodiments, the DLL3 fragment comprises 95% or more of the full-length protein, 90% or more, 75%, 50%, 25%, or 10% or more of the full-length protein. In some embodiments, the epitope-containing fragment of DLL3, including the antigenic or immunogenic fragment, can elicit a relevant immune response in a patient. Derivatives of DLL3, in some embodiments, include sequence variants in which one or more (e.g., 1 to 20, e.g., 15 amino acids, or up to 20%, e.g., 10%, 5%, or 1%, depending on the number of amino acids based on the full length of the protein) amino acid deletions, insertions, or substitutions have been made to the DLL3 sequence provided in SEQ ID NO: 3216 (UniProtKB Accession Q9NYJ7). In some embodiments, the substitutions include conservative substitutions. Derivatives and variants of DLL3, in some instances, have essentially the same biological function as the DLL3 protein from which they are derived. For example, derivatives and variants of DLL3, in some instances, are relatively antigenic or immunogenic to the protein from which they are derived, have either the ligand-binding activity or the active receptor-complex forming ability, or preferably both, of the protein from which they are derived, and have the same tissue distribution as DLL3.

[0141] The design of the DLL3-targeting immune cell-associated proteins described herein allows for flexibility in the binding domain for the DLL3 protein, in that the binding domain for DLL3 can be any type of binding domain, including, but not limited to, a domain derived from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, or a humanized antibody. In some embodiments, the binding domain for DLL3 is a single-chain variable fragment (scFv), a single-domain antibody, such as a heavy chain variable domain (VH), a light chain variable domain (VL), or a variable domain of a camelid-derived single-domain antibody (VHH). In other embodiments, the binding domain for DLL3 is a non-Ig binding domain, i.e., an antibody mimic such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, Kunitz domain peptides, and monobodies. In further embodiments, the binding domain for DLL3 is a ligand or peptide that binds to or associates with DLL3. In yet further embodiments, the binding domain for DLL3 is a knottin. In yet further embodiments, the binding domain for DLL3 is a small molecule entity.

[0142] 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 DLL3 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.

[0143] In some embodiments, the DLL3-binding domain is an anti-DLL3 antibody or 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 anti-DLL3 antibodies described herein are contemplated. For example, in certain embodiments, amino acid sequence variants of the anti-DLL3 antibodies described herein are contemplated to improve the binding affinity and / or other biological properties of the antibody. Exemplary 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, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the antibody.

[0144] Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, such as antigen binding. In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the CDRs and framework regions. Examples of such substitutions are described below. Amino acid substitutions may be introduced into an antibody of interest, and the products are screened for the desired activity, retained / improved antigen binding, reduced immunogenicity, or improved T-cell-mediated cytotoxicity (TDCC). Conservative and non-conservative amino acid substitutions are contemplated for preparing antibody variants.

[0145] In another example of substitutions to create variant anti-DLL3 antibodies, one or more hypervariable region residues of a parent antibody are substituted. Generally, variants are selected based on a desired improved property compared to the parent antibody, such as increased affinity, decreased affinity, decreased immunogenicity, or increased pH-dependent binding.

[0146] In some embodiments, the DLL3-binding domain of the DLL3-targeting immune cell-associated protein is a single-domain antibody specific to DLL3, such as a heavy chain variable domain (VH), a variable domain (VHH) of a llama-derived sdAb, a peptide, a ligand, or a small molecule entity. In some embodiments, the DLL3-binding domain of the DLL3-targeting immune cell-associated protein described herein is any domain that binds to DLL3, including, but not limited to, a domain from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, or a humanized antibody. In certain embodiments, the DLL3-binding domain is a single-domain antibody. In other embodiments, the DLL3-binding domain is a peptide. In further embodiments, the DLL3-binding domain is a small molecule.

[0147] In one embodiment, the single domain antibody corresponds to the VHH domain of a naturally occurring heavy chain antibody against DLL3. As further described herein, such VHH sequences can typically be generated or obtained by appropriately immunizing a species of llama with DLL3 (i.e., to generate an immune response and / or heavy chain antibodies against DLL3), by obtaining a suitable biological sample (such as a blood sample, serum sample, or B cell sample) from said llama, and generating VHH sequences against DLL3 starting from said sample using any suitable technique known in the art.

[0148] In another embodiment, such naturally occurring VHH domains against DLL3 are obtained from a naive library of camelid VHH sequences, for example, by screening such library with DLL3 or screening for at least one portion, fragment, antigenic determinant, or epitope thereof using at least one screening technique known in the art. Such libraries and techniques are described, for example, in WO99 / 37681, WO01 / 90190, WO03 / 025020, and WO03 / 035694. Alternatively, improved synthetic or semi-synthetic libraries derived from naive VHH libraries are used, for example, VHH libraries obtained from naive VHH libraries by techniques such as random mutagenesis and / or CDR shuffling, as described in WO00 / 43507.

[0149] In a further embodiment, yet another technique for obtaining VHH sequences against DLL3 involves appropriately immunizing a transgenic mammal capable of expressing heavy chain antibodies (i.e., to generate an immune response and / or heavy chain antibodies against DLL3), obtaining a suitable biological sample (blood sample, serum sample, or B cell sample) from said transgenic mammal, and generating VHH sequences against DLL3 starting from said sample using any suitable technique known in the art. For example, heavy chain antibody-expressing rats or mice and methods and techniques such as those described in WO02 / 085945 and WO04 / 049794 can be used for this purpose.

[0150] In some embodiments, anti-DLL3 single domain antibodies targeting immune cell-associated proteins include single domain antibodies having an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain, but that has been "humanized," i.e., by replacing one or more amino acid residues in the amino acid sequence (and in particular within the framework sequences) of said naturally occurring VHH sequence with one or more amino acid residues occurring at the corresponding positions in a VH domain from a conventional four-chain antibody of human origin (e.g., as shown above).

[0151] Other suitable methods and techniques for obtaining an anti-DLL3 single domain antibody of the present disclosure and / or a nucleic acid encoding the anti-DLL3 single domain antibody, starting from a naturally occurring VH or VHH sequence, include, for example, combining, in a suitable manner, one or more portions of one or more naturally occurring VH sequences (such as one or more framework (FR) sequences and / or complementarity determining region (CDR) sequences), one or more portions of one or more naturally occurring VHH sequences (such as one or more FR sequences or CDR sequences), and / or one or more synthetic or semi-synthetic sequences to provide an anti-DLL3 single domain antibody of the present disclosure or a nucleotide sequence or nucleic acid encoding the same.

[0152] In some embodiments, the DLL3-binding domain is an anti-DLL3-specific antibody comprising a heavy chain variable complementarity-determining region (CDR1), a heavy chain variable CDR2, a heavy chain variable CDR3, a light chain variable CDR1, a light chain variable CDR2, and a light chain variable CDR3. In some embodiments, the DLL3-binding domain includes any domain that binds to DLL3, including, but not limited to, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, or an antigen-binding fragment, such as a single-domain antibody (sdAb), Fab, Fab', F(ab)2, and Fv fragment, a fragment composed of one or more CDRs, a single-chain antibody (e.g., a single-chain Fv fragment (scFv)), a disulfide-stabilized (dsFv) Fv fragment, a heteroconjugate antibody (e.g., a bispecific antibody), a pFv fragment, a heavy chain monomer or dimer, a light chain monomer or dimer, and a dimer consisting of one heavy chain and one light chain. In some embodiments, the DLL3-binding domain is a single-domain antibody. In some embodiments, the anti-DLL3 single domain antibody comprises the heavy chain variable complementarity determining regions (CDRs): CDR1, CDR2, and CDR3.

[0153] 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 comprises an amino acid sequence selected from SEQ ID NOs: 1308-1750. In some embodiments, the CDR1 of the DLL3-binding domain comprises 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 substitutions relative to a sequence selected from SEQ ID NOs: 2637-3080.

[0154] In some embodiments, CDR1 comprises an amino acid sequence selected from SEQ ID NOs: 1751-2193, or a variant thereof 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 thereof 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 thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in a sequence selected from SEQ ID NOs: 2637-3080.

[0155] In some embodiments, CDR1 comprises an amino acid sequence selected from SEQ ID NOs: 1803-1836, or a variant thereof 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 thereof 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 thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in a sequence selected from SEQ ID NOs: 2689-2722.

[0156] In some embodiments, CDR1 comprises an amino acid sequence selected from SEQ ID NOs: 1837-2117, or a variant thereof 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 thereof 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 thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in a sequence selected from SEQ ID NOs: 2723-3003.

[0157] In some embodiments, CDR1 comprises an amino acid sequence selected from SEQ ID NOs: 2118-2193, or a variant thereof 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 thereof 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 thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in a sequence selected from SEQ ID NOs: 3004-3080.

[0158] 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.

[0159] In various embodiments, a 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.

[0160] 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.

[0161] 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.

[0162] In some embodiments, the DLL3-binding domain of a DLL3 targeting an immune cell-associated protein is cross-reactive with human and cynomolgus DLL3. In some embodiments, the DLL3-binding domain is specific for human DLL3. In certain embodiments, the DLL3-binding domain disclosed herein binds to human DLL3 with a human Kd (hKd). In certain embodiments, the DLL3-binding domain disclosed herein binds to cynomolgus DLL3 with a cynomolgus Kd (cKd). In certain embodiments, the DLL3-binding domain disclosed herein binds to both cynomolgus DLL3 and human DLL3 with a cynomolgus Kd (cKd) and a human Kd (hKd), respectively. In some embodiments, the DLL3-binding protein binds to human and cynomolgus DLL3 with comparable binding affinities (i.e., the hKd and cKd values ​​do not differ by more than ±10%). In some embodiments, the hKd and cKd range from about 0.001 nM to about 500 nM. In some embodiments, the hKd and cKd are in the range of about 0.001 nM to about 450 nM. In some embodiments, the hKd and cKd are in the range of about 0.001 nM to about 400 nM. In some embodiments, the hKd and cKd are in the range of about 0.001 nM to about 350 nM. In some embodiments, the hKd and cKd are in the range of about 0.001 nM to about 300 nM. In some embodiments, the hKd and cKd are in the range of about 0.001 nM to about 250 nM. In some embodiments, the hKd and cKd are in the range of about 0.001 nM to about 200 nM. In some embodiments, the hKd and cKd are in the range of about 0.001 nM to about 150 nM. In some embodiments, the hKd and cKd are in the range of about 0.001 nM to about 100 nM. In some embodiments, the hKd and cKd are in the range of about 0.1 nM to about 90 nM. In some embodiments, the hKd and cKd are in the range of about 0.2 nM to about 80 nM. In some embodiments, the hKd and cKd are in the range of about 0.3 nM to about 70 nM.In some embodiments, the hKd and cKd are in the range of about 0.4 nM to about 50 nM. In some embodiments, the hKd and cKd are in the range of about 0.5 nM to about 30 nM. In some embodiments, the hKd and cKd are in the range of about 0.6 nM to about 10 nM. In some embodiments, the hKd and cKd are in the range of about 0.7 nM to about 8 nM. In some embodiments, the hKd and cKd are in the range of about 0.8 nM to about 6 nM. In some embodiments, the hKd and cKd are in the range of about 0.9 nM to about 4 nM. In some embodiments, the hKd and cKd are in the range of about 1 nM to about 2 nM.

[0163] 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 DLL3 present in or on the cell membrane surface of a cell that expresses DLL3. Soluble DLL3 refers to DLL3 that is no longer present in or on the cell membrane surface of a cell that expresses or has 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 or more than soluble DLL3. In one embodiment, the antigen-binding protein of the present disclosure preferentially binds to membrane-bound DLL3 at least 30-fold over soluble DLL3. Determining the preferential binding of an antigen binding protein to membrane-bound DLL3 over soluble DLL3 can be readily determined using assays well known in the art.

[0164] In some embodiments, any of the aforementioned DLL3 binding domains (e.g., the anti-DLL3 single domain antibodies of SEQ ID NOs: 1308-1750) are tagged with an affinity peptide to facilitate purification. In some embodiments, the affinity peptide tag is six consecutive histidine residues, also known as 6X-his (SEQ ID NO: 3503).

[0165] In some embodiments, any of the aforementioned DLL3 binding domains (e.g., the anti-DLL3 single domain antibodies of SEQ ID NOs: 1308-1750) are tagged with an affinity peptide to facilitate purification. In some embodiments, the affinity peptide tag is six consecutive histidine residues, also known as 6X-his (SEQ ID NO: 3503).

[0166] Epidermal growth factor receptor (EGFR) binding proteins Epidermal growth factor receptor (EGFR) is causally involved in human malignancies. Abnormal activity of the Her family of receptors is involved in 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)). In particular, increased expression of EGFR has been observed in breast cancer, bladder cancer, lung cancer, head and neck cancer, and gastric cancer, as well as glioblastoma.

[0167] Increased EGFR receptor expression can be associated with increased production of the EGFR ligand, transforming growth factor alpha (TGF-α), by the same tumor cells, leading to receptor activation by an autocrine stimulatory pathway.

[0168] The anti-EGFR antibody cetuximab (Erbitux™) 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 a risk of interstitial lung disease (Sandler A, Oncology 20(5 Suppl 2):35-40 (2006)). Individual patients may be predisposed to certain types of complications, which influences the choice of drug therapy. A greater selection of treatment options is needed, allowing physicians to select therapeutic agents with the best side effect profile for each individual patient. The present disclosure provides novel polypeptide and protein therapeutic agents useful in methods of treatment, particularly for the treatment of diseases associated with aberrant expression of EGFR.

[0169] The 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.

[0170] RTKs, such as EGFR, function to enable cells to respond to diverse external stimuli. However, abnormal activation and / or overexpression of EGFR has been associated with the development and progression of several human cancers. Therefore, EGFR is a target for anticancer therapy. Approved drugs targeting EGFR include small molecule inhibitors such as gefitinib (Iressa®) and erlotinib (Tarceva®), as well as anti-EGFR antibodies such as cetuximab (Erbitux®) and panitumumab (Vectibix®). Anti-EGFR antibodies are described, for example, in U.S. Patent 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 for 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, colorectal 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. In certain embodiments, the present disclosure provides EGFR binding proteins that are EGFR-targeting immune cell-associated proteins containing an EGFR-binding domain that specifically binds to EGFR on the surface of tumor target cells.

[0171] The design of the EGFR-targeting immune cell-associated proteins described herein allows for flexibility in the binding domain to the EGFR protein, in that the binding domain to EGFR can be any type of binding domain, including, but not limited to, a domain derived from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, or a humanized antibody. In some embodiments, the binding domain to EGFR is a single-chain variable fragment (scFv), a single-domain antibody, such as a heavy chain variable domain (VH), a light chain variable domain (VL), or a variable domain of a camelid-derived single-domain antibody (VHH). In other embodiments, the binding domain to EGFR is a non-Ig binding domain, i.e., an antibody mimic such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, Kunitz domain peptides, and monobodies. In a further embodiment, the binding domain for EGFR is a ligand or peptide that binds to or associates with EGFR. In yet a further embodiment, the binding domain for EGFR is a knottin. In yet a further embodiment, the binding domain for EGFR is a small molecule entity.

[0172] In some embodiments, the EGFR-binding domain is an anti-EGFR antibody or 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 anti-EGFR antibodies described herein are contemplated. For example, in certain embodiments, amino acid sequence variants of the anti-EGFR antibodies described herein are contemplated to improve the binding affinity and / or other biological properties of the antibody. Exemplary 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, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the antibody.

[0173] Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen binding. In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include CDRs and framework regions. Examples of such substitutions are described below. Amino acid substitutions may be introduced into an antibody of interest, and the products are screened for the desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved T-cell-mediated cytotoxicity (TDCC). Conservative and non-conservative amino acid substitutions are contemplated for preparing antibody variants.

[0174] In another example of substitutions to create variant anti-EGFR antibodies, one or more hypervariable region residues of the parent antibody are substituted. Generally, variants are selected based on a desired improved property compared to the parent antibody, e.g., increased affinity, decreased affinity, decreased immunogenicity, or increased pH-dependent binding.

[0175] In some embodiments, the EGFR-binding domain of the EGFR-targeting immune cell-associated protein is an EGFR-specific single-domain antibody, such as a heavy chain variable domain (VH), a variable domain of a llama-derived sdAb (VHH), a peptide, a ligand, or a small molecule entity. In some embodiments, the EGFR-binding domain of the EGFR-targeting immune cell-associated protein described herein is any domain that binds to EGFR, including, but not limited to, a domain from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, or a humanized antibody. In certain embodiments, the EGFR-binding domain is a single-domain antibody. In other embodiments, the EGFR-binding domain is a peptide. In further embodiments, the EGFR-binding domain is a small molecule.

[0176] In one embodiment, the single domain antibody corresponds to the VHH domain of a naturally occurring heavy chain antibody against EGFR. As further described herein, such a VHH sequence can typically be generated or obtained by appropriately immunizing a species of llama with EGFR (i.e., to generate an immune response and / or heavy chain antibodies against EGFR), by obtaining a suitable biological sample from said llama (such as a blood sample, serum sample, or B cell sample), and generating a VHH sequence against EGFR starting from said sample using any suitable technique known in the art.

[0177] In another embodiment, such naturally occurring VHH domains against EGFR are obtained from a naive library of camelid VHH sequences, e.g., by screening such a library using EGFR or at least one portion, fragment, antigenic determinant, or epitope thereof using at least one screening technique known in the art. Such libraries and techniques are described, for example, in WO99 / 37681, WO01 / 90190, WO03 / 025020, and WO03 / 035694. Alternatively, improved synthetic or semi-synthetic libraries derived from naive VHH libraries are used, e.g., VHH libraries are obtained from naive VHH libraries by techniques such as random mutagenesis and / or CDR shuffling, as described in WO00 / 43507.

[0178] In a further embodiment, yet another technique for obtaining VHH sequences against EGFR involves appropriately immunizing a transgenic mammal capable of expressing heavy chain antibodies (i.e., to generate an immune response and / or heavy chain antibodies against EGFR), obtaining a suitable biological sample (blood sample, serum sample, or B cell sample) from said transgenic mammal, and generating VHH sequences against EGFR starting from said sample using any suitable technique known in the art. For example, for this purpose, heavy chain antibody-expressing rats or mice and methods and techniques such as those described in WO02 / 085945 and WO04 / 049794 can be used.

[0179] In some embodiments, an EGFR-binding domain of the present disclosure binds to a protein comprising the sequence of SEQ ID NO: 3205 (UniProt Accession No. Q504U8). In some embodiments, an EGFR-binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 3205. In some embodiments, an EGFR-binding domain binds to a protein comprising the sequence of SEQ ID NO: 3206 (UniProt Accession No. Q01279). In some embodiments, an EGFR-binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 3206. In some embodiments, an EGFR-binding domain binds to a protein comprising the sequence of SEQ ID NO: 3207 (UniProt Accession No. A0A2K5WK39). In some embodiments, an EGFR-binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 3207.

[0180] In some embodiments, the EGFR-binding domain disclosed herein recognizes full-length EGFR. In certain instances, the EGFR-binding domain disclosed herein recognizes an epitope within EGFR; for example, in some cases, the EGFR-targeting immune cell-associated protein interacts with one or more amino acids found within the extracellular domain of human EGFR (e.g., within extracellular domains I, II, III, and / or IV). 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 the extracellular domain of EGFR. Alternatively, the epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) located within the extracellular domain of EGFR.

[0181] In some embodiments, the EGFR binding proteins of the present disclosure bind to the full-length EGFR protein or fragments thereof, e.g., epitope-containing fragments within the full-length EGFR protein, as described above. In some cases, 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 or more amino acids, e.g., 20 or more amino acids, 50 or 100 or more amino acids. In some embodiments, the EGFR fragments comprise 95% or more of the full-length protein, 90% or more, 75%, 50%, 25%, or 10% or more of the full-length protein. In some embodiments, epitope-containing fragments of EGFR, including antigenic or immunogenic fragments, are capable of eliciting a relevant immune response in patients. Derivatives of EGFR, in some embodiments, include sequence variants in which one or more (e.g., 1-20, e.g., 15 amino acids, or up to 20%, e.g., 10%, or 5%, or 1%, depending on the number of amino acids based on the full length of the protein) deletions, insertions, or substitutions have been made to the EGFR sequence provided in SEQ ID NO: 3205, 3206, or 3207.

[0182] In some embodiments, substitutions include conservative substitutions. In some cases, the derivatives and variants thereof have essentially the same biological function as the proteins from which they are derived. For example, in some cases, the derivatives and variants of EGFR are relatively antigenic or immunogenic to the proteins from which they are derived, have either the ligand-binding activity or the active receptor-complex forming ability of the proteins from which they are derived, or preferably both, and have the same tissue distribution as EGFR.

[0183] In some embodiments, the EGFR-binding protein specifically binds to EGFR with affinity that is equal to or better than a reference EGFR-binding protein; in such embodiments, the EGFR-binding protein comprises an affinity-matured EGFR-binding molecule, which is derived from an EGFR-binding parent molecule and contains one or more amino acid mutations (e.g., stabilizing mutations, destabilizing mutations) relative to the EGFR-binding parent molecule. In some embodiments, the affinity-matured EGFR-binding molecule has superior stability against a selected destabilizing agent than the reference EGFR-binding parent molecule. In some embodiments, the affinity-matured EGFR-binding molecule is identified by a process comprising panning one or more pre-candidate EGFR-binding molecules derived from one or more EGFR-binding parent molecules expressed in a phage display library against an EGFR protein, such as a human EGFR protein. The pre-candidate EGFR-binding molecule, in some embodiments, contains amino acid substitutions in variable region, CDR, or framework residues relative to the parent molecule.

[0184] 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 large libraries of randomized protein variants can be rapidly and efficiently selected for sequences that bind to target molecules with high affinity. Displaying 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 fusing them to either gene III or gene VIII of filamentous phage. 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, resulting in phage particles displaying either one copy of the fusion protein or none at all. Because avidity effects are reduced compared to polyvalent phage, selection is based on affinity for endogenous ligands and phagemid vectors are used, which facilitates DNA manipulation. Lowman and Wells, Methods: A companion to Methods in Enzymology, 3:205-0216 (1991)

[0185] In some embodiments, panning involves using varying binding times and concentrations to identify EGFR-binding molecules with increased or decreased on-rates from pre-candidate EGFR-binding molecules. In some embodiments, panning involves using varying wash times to identify EGFR-binding molecules with increased or decreased off-rates from pre-candidate EGFR molecules. In some embodiments, panning involves using varying binding times and varying wash times. In some embodiments, one or more stabilizing mutations are combined to increase the stability of affinity-matured EGFR-binding molecules, for example, by shuffling to generate a second-stage combinatorial library from such mutants and performing a second round of panning and subsequent binding selection.

[0186] In some embodiments, affinity-matured EGFR-binding molecules have affinity for EGFR protein (e.g., human EGFR protein) that is comparable to or better than the EGFR-binding parent molecule, but have reduced or, in some embodiments, increased cross-reactivity with selected substances, e.g., ligands, proteins, antigens, etc., other than the EGFR epitope for which the EGFR-binding parent molecule is specific or designed to be specific. Regarding the latter, affinity-matured EGFR-binding molecules are, in some embodiments, more successfully tested in animal models when the affinity-matured EGFR-binding molecules are reacted with both human EGFR and the corresponding target in the animal model, e.g., mouse EGFR or cynomolgus EGFR. In some embodiments, the parent EGFR-binding molecule binds to human EGFR with an affinity of about 10 nM or less and to cynomolgus EGFR with an affinity of about 15 nM or less. In some embodiments, affinity-matured EGFR-binding molecules identified after one round of panning bind to human EGFR with an affinity of about 5 nM or less and to cynomolgus EGFR with an affinity of about 7.5 nM or less. In some embodiments, affinity-matured EGFR binding molecules identified after two rounds of panning bind to human EGFR with an affinity of about 2.5 nM or less and bind to cynomolgus monkey EGFR with an affinity of about 3.5 nM or less.

[0187] In some embodiments, the EGFR binding protein comprises an antigen-specific binding domain polypeptide that specifically binds to a target, e.g., a target on a diseased cell or a target on another cell that supports a disease state, e.g., a target on a stromal cell that supports tumor growth, or a target on an immune cell that supports disease-mediated immunosuppression. In some examples, the antigen-specific binding domain comprises an antibody, single-chain antibody, Fab, Fv, T-cell receptor binding domain, ligand-binding domain, receptor-binding domain, domain antibody, single-domain antibody, minibody, nanobody, peptibody, or various other antibody mimetics (such as affimers, affitins, alphabodies, atrimers, CTLA4-based molecules, adnectins, anticalins, Kunitz domain-based proteins, avimers, knottins, fynomers, darpins, affibodies, affilins, monobodies, and armadillo repeat protein-based proteins).

[0188] In some embodiments, anti-EGFR single domain antibodies of EGFR-targeting immune cell-associated proteins include single domain antibodies having an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain, but that has been "humanized", i.e., by replacing one or more amino acid residues in the amino acid sequence (and in particular within the framework sequences) of said naturally occurring VHH sequence with one or more amino acid residues occurring at the corresponding positions in a VH domain from a conventional four-chain antibody of human origin (e.g., as shown above).

[0189] Other suitable methods and techniques for obtaining an anti-EGFR single domain antibody of the present disclosure and / or a nucleic acid encoding the same, starting from a naturally occurring VH or VHH sequence, include, for example, combining one or more portions of one or more naturally occurring VH sequences (such as one or more framework (FR) sequences and / or complementarity determining region (CDR) sequences), one or more portions of one or more naturally occurring VHH sequences (such as one or more FR sequences or CDR sequences), and / or one or more synthetic or semi-synthetic sequences in a suitable manner to provide an anti-EGFR single domain antibody of the present disclosure or a nucleotide sequence or nucleic acid encoding the same.

[0190] In some embodiments, the EGFR-binding domain is an anti-EGFR-specific antibody comprising a heavy chain variable complementarity-determining region (CDR1), a heavy chain variable CDR2, a heavy chain variable CDR3, a light chain variable CDR1, a light chain variable CDR2, and a light chain variable CDR3. In some embodiments, the EGFR-binding domain comprises any domain that binds to EGFR, including, but not limited to, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, or an antigen-binding fragment, such as a single-domain antibody (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 consisting of one heavy chain and one light chain. In some embodiments, the EGFR-binding domain is a single-domain antibody. In some embodiments, the anti-EGFR single domain antibody comprises heavy chain variable complementarity determining regions (CDRs): CDR1, CDR2, and CDR3.

[0191] 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. The framework residues of the EGFR-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 EGFR-binding domain comprises an amino acid sequence selected from SEQ ID NOs: 798-846.

[0192] In some embodiments, the EGFR-binding domain described herein comprises a polypeptide having a sequence selected from SEQ ID NOs: 798-846, subsequences thereof, and variants thereof. In some embodiments, the EGFR-binding protein comprises at least 70%-95% or more homology to a sequence selected from SEQ ID NOs: 798-846, subsequences 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 homology to a sequence selected from SEQ ID NOs: 798-846, subsequences thereof, and variants thereof. In some embodiments, the EGFR-binding protein comprises at least 70%-95% or more identity to a sequence selected from SEQ ID NOs: 798-846, subsequences 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, subsequences thereof, and variants thereof.

[0193] In some embodiments, CDR1 comprises the 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 the 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 the 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: 749-797.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] In some embodiments, the EGFR-binding domain is cross-reactive with human, cynomolgus, and mouse EGFR. In some embodiments, the EGFR-binding domain is specific for human EGFR. In certain embodiments, the EGFR-binding domain disclosed herein binds to human EGFR with a human Kd (hKd). In certain embodiments, the EGFR-binding domain disclosed herein binds to cynomolgus EGFR with a cynomolgus Kd (cKd). In certain embodiments, the EGFR-binding domain disclosed herein binds to cynomolgus EGFR with a mouse Kd (mKd). In certain embodiments, the EGFR-binding domain disclosed herein binds to both cynomolgus EGFR and human EGFR with a cynomolgus Kd (cKd) and a human Kd (hKd), respectively. In certain embodiments, the EGFR-binding domains disclosed herein bind to cynomolgus EGFR, mouse EGFR, and human EGFR with cynomolgus Kd (cKd), mouse Kd (mKd), and human Kd (hKd), respectively. In some embodiments, the EGFR-binding proteins bind to human, mouse, and cynomolgus EGFR with comparable binding affinities (i.e., the hKd, mKd, and cKd values ​​do not differ by more than ±10%). In some embodiments, the hKd, mKd, and cKd range from about 0.1 nM to about 500 nM. In some embodiments, the hKd, mKd, and cKd range from about 0.1 nM to about 450 nM. In some embodiments, the hKd, mKd, and cKd range from about 0.1 nM to about 400 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.1 nM to about 350 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.1 nM to about 300 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.1 nM to about 250 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.1 nM to about 200 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.1 nM to about 150 nM.In some embodiments, the hKd, mKd, and cKd are in the range of about 0.1 nM to about 100 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.1 nM to about 90 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.2 nM to about 80 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.3 nM to about 70 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.4 nM to about 50 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.5 nM to about 30 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.6 nM to about 10 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.7 nM to about 8 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.8 nM to about 6 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.9 nM to about 4 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 1 nM to about 2 nM.

[0199] In some embodiments, any of the aforementioned EGFR binding domains (e.g., the anti-EGFR single domain antibodies of SEQ ID NOs: 798-846) are tagged with an affinity peptide to facilitate purification. In some embodiments, the affinity peptide tag is six consecutive histidine residues, also known as 6X-his (SEQ ID NO: 3503).

[0200] 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 EGFR present in or on the cell membrane surface of cells that express EGFR. Soluble EGFR refers to EGFR that is no longer present in or on the cell membrane surface of cells that express or have 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 or more than soluble EGFR. In one embodiment, the EGFR-targeting immune cell-associated protein of the present disclosure preferentially binds to membrane-bound EGFR at least 30-fold or more than soluble EGFR. The determination of the preferential binding of an antigen-binding protein to membrane-bound EGFR over soluble EGFR can be easily determined using a binding assay. In some embodiments, the EGFR-binding protein is quite small, and in some embodiments, it is contemplated that the EGFR-binding protein is 25 kDa or less, 20 kDa or less, 15 kDa or less, or 10 kDa or less. In a specific example, when the EGFR-binding protein is a peptide or small molecule entity, it is 5 kDa or less.

[0201] In other embodiments, the EGFR-binding proteins described herein comprise small molecular entity (SME) binders of EGFR. SME binders are small molecules, averaging approximately 500-2000 Da in size, that are attached to the EGFR-binding protein by known methods, such as sortase ligation or conjugation. In these examples, the EGFR-binding protein comprises a domain that includes a sortase recognition sequence, e.g., LPETG (SEQ ID NO: 3200). To attach 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 binding EGFR. Knottins are disulfide-stabilized peptides with a cysteine ​​knot scaffold and have an average size of approximately 3.5 kDa. Knottins have been contemplated for binding to specific tumor molecules, such as EGFR. In further embodiments, the EGFR-binding proteins described herein comprise natural EGFR ligands.

[0202] In some embodiments, the EGFR-binding protein comprises more than one domain and is a single polypeptide design with flexible domain connections. This allows for easy production and manufacturing of the EGFR-binding protein, as it can be encoded by a single cDNA molecule that can be easily incorporated into a vector. Furthermore, in some embodiments where the EGFR-binding protein described herein is a single monomeric polypeptide chain, there are no chain pairing issues and no requirement for dimerization. In such embodiments, it is contemplated that the EGFR-binding protein described herein has a reduced tendency to aggregate.

[0203] In EGFR-binding proteins containing more than one domain, the domains are linked by one or more internal linkers. In certain embodiments, the internal linker is "short," i.e., consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues. Thus, in certain examples, the internal linker consists of about 12 or fewer amino acid residues. In the case of 0 amino acid residues, the internal linker is a peptide bond. In certain embodiments, the internal linker is "long," i.e., consisting of 15, 20, or 25 amino acid residues. In some embodiments, the internal linker consists of about 3 to about 15, e.g., 8, 9, or 10, consecutive amino acid residues. Regarding the amino acid composition of the internal linker, the peptide is selected for its properties of providing flexibility to the EGFR-binding protein, not interfering with the binding domain, and resisting cleavage by proteases. For example, glycine and serine residues generally confer protease resistance. Examples of internal linkers suitable for linking domains in EGFR-binding proteins include, but are not limited to, (GS) n (SEQ ID NO: 3190), (GGS) n (SEQ ID NO: 3191), (GGGS) n (SEQ ID NO: 3192), (GGSG) n (SEQ ID NO: 3193), (GGSGG) n (SEQ ID NO: 3194), (GGGGS) n (SEQ ID NO: 3195), (GGGGG) n (SEQ ID NO: 3196), or (GGG) n (SEQ ID NO: 3197), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the linker is (GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 3198), (GGGGSGGGGSGGGGS) (SEQ ID NO: 3199), or (GGGGSGGGS) (SEQ ID NO: 3504).

[0204] In some cases, when an EGFR-binding protein contains more than one domain, domains within the EGFR-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 relatively pure enzymes in large quantities, its ease of use, and its lack of regulation by calcium or guanosine-5'-triphosphate (GTP) have made mTG the primary cross-linking enzyme used in the food industry and biotechnology. Currently, mTG is used in many applications to conjugate proteins and peptides to small molecules, polymers, surfaces, DNA, and other proteins. (See Pavel Strp, "Veracity of microbial transglutaminase," Bioconjugate Chem. 25, 5, 855-862.)

[0205] In some examples, an EGFR-binding protein is provided that includes more than one domain, wherein one of the domains includes an acceptor glutamine in the constant region, which can be conjugated to another domain via a lysine-based linker (e.g., any primary amine chain that is a substrate for TGase, including alkylamines, oxoamines), wherein conjugation occurs only at one or more acceptor glutamine residues present in the targeting portion outside the antigen-binding site (e.g., outside the variable region, within the constant region). Thus, conjugation does not occur at glutamines within the variable region, for example, at least partially surface-exposed glutamines. In some examples, the EGFR-binding protein is formed by reacting one of the domains with a lysine-based linker in the presence of TGase.

[0206] In some embodiments, when one or more domains within an EGFR-binding protein are directly linked, a hybrid vector is created in which the DNAs encoding the directly linked domains are themselves directly ligated to each other. In some embodiments in which 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 the linker portion, and the DNA encoding another domain is ligated to the other end of the linker portion.

[0207] In some embodiments, the EGFR-binding protein is a single-chain variable fragment (scFv), a single-domain antibody, e.g., a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain of a camelid-derived single-domain antibody (VHH). In other embodiments, the EGFR-binding protein is a non-Ig binding domain, i.e., an antibody mimic such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, Kunitz domain peptides, and monobodies. In further embodiments, the EGFR-binding protein is a ligand or peptide that binds to or associates with EGFR. In yet further embodiments, the EGFR-binding protein is a knottin. In yet further embodiments, the binding domain for EGFR is a small molecule entity.

[0208] In some embodiments, an EGFR binding protein as described above is fused to an Fc region from any species, including, but not limited to, a human immunoglobulin such as human IgG1, human IgG2, human IgG3, or human IgG4, resulting in an Fc-fusion protein. In some embodiments, the Fc-fusion proteins of the present 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 present disclosure contain, among other things, substitutions, mutations, and / or modifications of one or more additional amino acid residues in the Fc region, which result in the binding protein having favorable characteristics, including, but not limited to, altered pharmacokinetics, extended serum half-life, etc.

[0209] In some embodiments, such Fc-fusion EGFR-binding proteins have an extended half-life in mammals, e.g., 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. An increased half-life can, in some cases, result in a higher serum titer, thereby reducing the frequency of administration of the EGFR-binding protein and / or reducing the concentration of the administered antibody. The in vivo binding and serum half-life of human FcRn high-affinity-binding polypeptides to human FcRn are, in some examples, assayed in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which polypeptides having variant Fc regions are administered.

[0210] EGFR binding proteins are optionally 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 may be 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.

[0211] Various post-translational modifications of EGFR binding proteins encompassed by the present disclosure include, for example, N- or O-linked glycosylation, N- or C-terminal processing, conjugation of chemical moieties to the amino acid backbone, chemical modification of N- or O-linked glycosylation, and addition or deletion of N-terminal methionine residues upon expression in prokaryotic host cells. 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 modulators.

[0212] 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 a particular position or locus on a target molecule. If an antibody is monovalent, each binding site on the molecule specifically binds to one antigen position or epitope. If an antibody contains more than one target binding site (multivalent), each target binding site may specifically bind to the same molecule or different molecules (e.g., may bind to different ligands or different antigens, or different epitopes or positions on the same antigen).

[0213] FLT3-binding protein 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 kinase family. FLT3 is typically 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 by FLT3 ligand (FL) enhances the proliferation and survival of leukemic cells. Inhibition of FLT3 signaling leads to dendritic cell apoptosis and inhibits immune responses. The MAPK, PI3K, and Stat5 pathways have been identified as being involved in downstream signaling of activated FLT3 (see, e.g., Stirewalt DL and JP, Radich, JP. Nat Rev Cancer 3:650-665 (2003)).

[0214] Described herein are immune cell-associated proteins comprising a FLT3-binding domain, pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors, and host cells for producing such proteins. Additionally, methods of using the disclosed proteins comprising a FLT3-binding domain of the present disclosure in the prevention and / or treatment of diseases, conditions, and disorders are also provided. In some embodiments, the FLT3-binding domain of the present disclosure inhibits FL-induced phosphorylation of wild-type FLT3 and downstream kinases in the MPK, PI3K, and STAT5 pathways in diseases such as leukemia. In some embodiments, the FLT3-binding domain of the present disclosure has improved ability to activate downstream immune effector functions, such as antibody-dependent cellular cytotoxicity (ADCC).

[0215] 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).

[0216] In some embodiments, the FLT3-binding domain disclosed herein recognizes a full-length FLT3 (e.g., a FLT3 protein) comprising the sequence of SEQ ID NO: 3215 (UniProt ID: P36888). In particular examples, the FLT3-binding domain disclosed herein recognizes an epitope within FLT3; for example, 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, 20, or more) amino acids located within a domain of FLT3 (e.g., a 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 (UniProtID: P36888)).

[0217] In some embodiments, the FLT3 binding protein of the present disclosure binds to the full-length FLT3 protein or a fragment thereof, for example, an epitope-containing fragment within the full-length FLT3 protein as described above. In some cases, the epitope-containing fragment includes an antigenic or immunogenic fragment of the FLT3 protein and its derivatives. In some embodiments, the epitope-containing fragment, including the antigenic or immunogenic fragment, is 12 or more amino acids, e.g., 20 or more amino acids, 50 or 100 or more amino acids. In some embodiments, the FLT3 fragment comprises 95% or more of the full-length protein, 90% or more, 75%, 50%, 25%, or 10% or more of the full-length 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, a derivative of FLT3 includes a sequence variant in which one or more (e.g., 1 to 20, e.g., 15 amino acids, or up to 20%, e.g., 10%, 5%, or 1%) amino acids based on the total length of the protein have been deleted, inserted, or substituted in the FLT3 sequence (e.g., an FLT3 protein comprising the sequence of SEQ ID NO: 3215 (UniProtID: P36888)).

[0218] In some embodiments, substitutions include conservative substitutions. In some cases, the derivatives and variants thereof have essentially the same biological function as the proteins from which they are derived. For example, in some cases, the derivatives and variants of FLT3 are relatively antigenic or immunogenic to the proteins from which they are derived, have either the ligand-binding activity or the active receptor-complex forming ability of the proteins from which they are derived, or preferably both, and have the same tissue distribution as FLT3.

[0219] In some embodiments, the FLT3-binding protein specifically binds to FLT3 with affinity that is equal to or better than that of a reference FLT3-binding protein. In such embodiments, the FLT3-binding protein comprises an affinity-matured FLT3-binding molecule, which is derived from a FLT3-binding parent molecule and contains one or more amino acid mutations (e.g., stabilizing mutations, destabilizing mutations) relative to the FLT3-binding parent molecule. In some embodiments, the affinity-matured FLT3-binding molecule has superior stability against a selected destabilizing agent than the reference FLT3-binding parent molecule. In some embodiments, the affinity-matured FLT3-binding molecule is identified by a process comprising panning one or more pre-candidate FLT3-binding molecules derived from one or more FLT3-binding parent molecules expressed in a phage display library against a FLT3 protein, such as a human FLT3 protein. The pre-candidate FLT3-binding molecule, in some embodiments, contains amino acid substitutions in variable region, CDR, or framework residues relative to the parent molecule.

[0220] As used herein, "phage display" refers to a technique in which mutant polypeptides are displayed as fusion proteins to at least a portion of a coat protein on the surface of phage, e.g., filamentous phage, particles. The utility of phage display lies in the fact that large libraries of randomized protein variants can be rapidly and efficiently selected for sequences that bind to target molecules with high affinity. Displaying 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 proteins by fusing them to either gene III or gene VIII of filamentous phage. See, e.g., 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, resulting in phage particles displaying one copy of the fusion protein or none at all. Because avidity effects are reduced compared to polyvalent phage, selection is based on affinity for endogenous ligands and phagemid vectors are used, which facilitate DNA manipulation. See, e.g., Lowman and Wells, Methods: A companion to Methods in Enzymology, 3:205-0216 (1991).

[0221] In some embodiments, panning involves using varying binding times and concentrations to identify FLT3-binding molecules with increased or decreased on-rates from pre-candidate FLT3-binding molecules. In some embodiments, panning involves using varying wash times to identify FLT3-binding molecules with increased or decreased off-rates from pre-candidate FLT3-binding molecules. In some embodiments, panning involves using varying binding times and varying wash times. In some embodiments, one or more stabilizing mutations are combined to increase the stability of affinity-matured FLT3-binding molecules, for example, by shuffling to generate a second-stage combinatorial library from such mutants and performing a second round of panning and subsequent binding selection.

[0222] In some embodiments, the affinity-matured FLT3 binding molecule has affinity for FLT3 protein (such as human FLT3 protein) that is comparable to or better than the parent FLT3-binding molecule, but has reduced, or in some embodiments, increased, cross-reactivity with selected substances, e.g., 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, affinity-matured FLT3 binding molecules are, in some embodiments, more successfully tested in animal models when they are reacted with both human FLT3 and the corresponding target in the animal model, e.g., mouse FLT3 or cynomolgus monkey FLT3.

[0223] In some embodiments, the FLT3-binding domain is an anti-FLT3 antibody or antigen-binding fragment thereof, or a variant of an anti-FLT3 or antigen-binding fragment thereof. As used herein, the term "variant" refers to variants and derivatives of an antibody or antigen-binding fragment thereof, as described herein. In certain embodiments, amino acid sequence variants of the anti-FLT3 antibodies or antigen-binding fragments thereof described herein are contemplated. For example, in certain embodiments, amino acid sequence variants of the anti-FLT3 antibodies or antigen-binding fragments thereof described herein are contemplated to improve their binding affinity and / or other biological properties. Exemplary methods for preparing amino acid variants include, but are not limited to, introducing appropriate modifications into the nucleotide sequence encoding the antibody or antigen-binding fragment thereof, or peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of, residues within the amino acid sequence of the antibody or antigen-binding fragment thereof.

[0224] Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics (e.g., antigen-binding). In certain embodiments, variants with one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the CDRs and framework regions. Examples of such substitutions are described below. Amino acid substitutions may be introduced into an antibody or antigen-binding fragment of interest, and the products may be screened for the desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, altered antibody-dependent cellular cytotoxicity (ADCC), or improved T-cell-mediated cytotoxicity (TDCC). Conservative and non-conservative amino acid substitutions are contemplated for preparing variants.

[0225] In another example of substitutions to create a variant anti-FLT3 antibody or antigen-binding fragment thereof, one or more hypervariable region residues of a parent antibody or antigen-binding fragment thereof are substituted. Generally, variants are selected based on a desired improved property compared to the parent antibody, e.g., increased affinity, decreased affinity, decreased immunogenicity, or increased pH-dependent binding.

[0226] In some embodiments, the FLT3-binding domain is a single-domain antibody (sdAb), such as a heavy chain variable domain (VH), a variable domain (VHH) of a llama-derived sdAb, a peptide specific for FLT3, a ligand, or a small molecule entity. In some embodiments, the FLT3-binding domain described herein is any domain that binds to FLT3, including, but not limited to, a domain derived from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, or a humanized antibody. In certain embodiments, the FLT3-binding domain is a single-domain antibody. In other embodiments, the FLT3-binding domain is a peptide. In further embodiments, the FLT3-binding domain is a small molecule.

[0227] In one embodiment, the single domain antibody corresponds to the VHH domain of a naturally occurring heavy chain antibody against FLT3. As further described herein, such VHH sequences can typically be generated or obtained by suitably immunizing a species of llama with FLT3 (i.e., to generate an immune response and / or heavy chain antibodies against FLT3), by obtaining a suitable biological sample from said llama (such as a blood sample, serum sample, or B cell sample), and generating VHH sequences against FLT3 starting from said sample using any suitable technique.

[0228] In another embodiment, such naturally occurring VHH domains against FLT3 are obtained from a naive library of Camelidae VHH sequences, e.g., by screening such a library with FLT3 or by screening for at least one portion, 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 WO99 / 37681, WO01 / 90190, WO03 / 025020, and WO03 / 035694. Alternatively, an improved synthetic or semi-synthetic library derived from a naive VHH library is used, e.g., a VHH library is obtained from a naive VHH library by techniques such as random mutagenesis and / or CDR shuffling, as described in WO00 / 43507.

[0229] In a further embodiment, yet another technique for obtaining VHH sequences against FLT3 involves appropriately immunizing a transgenic mammal capable of expressing heavy chain antibodies (i.e., to generate an immune response and / or heavy chain antibodies against FLT3), obtaining a suitable biological sample from said transgenic mammal (such as a blood sample, serum sample, or B cell sample), and generating VHH sequences against FLT3 starting from said sample using any suitable technique known in the art. For example, heavy chain antibody-expressing rats or mice and methods and techniques such as those described in WO02 / 085945 and WO04 / 049794 can be used for this purpose.

[0230] In some embodiments, the anti-FLT3 single domain antibodies of the present disclosure include single domain antibodies having an amino acid sequence that corresponds to the amino acid sequence of a non-human antibody and / or naturally occurring VHH domain, e.g., a llama anti-FLT3 antibody, but that has been "humanized", i.e., humanized by replacing one or more amino acid residues in the amino acid sequence (particularly in the framework sequences) of said non-human anti-FLT3 and / or naturally occurring VHH sequence with one or more amino acid residues occurring at the corresponding positions in a VH domain from a conventional four-chain antibody of human origin (e.g., as shown above).

[0231] Other suitable methods and techniques for obtaining an anti-FLT3 single domain antibody of the present disclosure and / or a nucleic acid encoding the anti-FLT3 single domain antibody, starting from a naturally occurring VH or VHH sequence, include, for example, combining one or more portions of one or more naturally occurring VH sequences (such as one or more framework (FR) sequences and / or complementarity determining region (CDR) sequences), one or more portions of one or more naturally occurring VHH sequences (such as one or more FR sequences or CDR sequences), and / or one or more synthetic or semi-synthetic sequences in a suitable manner to provide an FLT3 single domain antibody of the present disclosure or a nucleotide sequence or nucleic acid encoding the anti-FLT3 single domain antibody.

[0232] In some embodiments, the FLT3-binding domain is an anti-FLT3-specific antibody comprising a heavy chain variable complementarity-determining region (CDR1), a heavy chain variable CDR2, a heavy chain variable CDR3, a light chain variable CDR1, a light chain variable CDR2, and a light chain variable CDR3. In some embodiments, the FLT3-binding domain comprises any domain that binds to FLT3, including, but not limited to, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, or an antigen-binding fragment such as a single domain antibody (sdAb), Fab, Fab', F(ab)2, and Fv fragment, a fragment composed of one or more CDRs, a single-chain antibody (e.g., a single-chain Fv fragment (scFv)), a disulfide-stabilized (dsFv) Fv fragment, a heteroconjugate antibody (e.g., a bispecific antibody), a pFv fragment, a heavy chain monomer or dimer, a light chain monomer or dimer, and a dimer consisting of one heavy chain and one light chain. In some embodiments, the FLT3 binding domain is a single domain antibody. In some embodiments, the anti-FLT3 single domain antibody comprises heavy chain variable complementarity determining regions (CDRs): CDR1, CDR2, and CDR3.

[0233] 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. The framework residues of the FLT3 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.

[0234] In some embodiments, the binding proteins described herein comprise polypeptides having a sequence selected from SEQ ID NOs: 1004-1079, subsequences thereof, and variants thereof. In some embodiments, the FLT3 binding protein comprises at least 60%-95% or more homology to a sequence selected from SEQ ID NOs: 1004-1079, subsequences thereof, and variants thereof. In some embodiments, the FLT3 binding protein comprises at least 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to a sequence selected from SEQ ID NOs: 1004-1079, subsequences thereof, and variants thereof. In some embodiments, the FLT3 binding protein comprises at least 60% to 95% or more identity to a sequence selected from SEQ ID NOs: 1004 to 1079, subsequences thereof, and variants thereof. In some embodiments, the FLT3 binding protein comprises at least 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to a sequence selected from SEQ ID NOs: 1004 to 1079, subsequences thereof, and variants thereof.

[0235] 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.

[0236] In various embodiments, the FLT3 binding domain of the present disclosure has a sequence similar to at least about 60%, about 61%, at least about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, 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%, about 100%, about 101%, about 102%, about 103%, about 104%, about 105%, about 106%, about 107%, about 108%, about 109%, about 110%, about 111, about 112%, about 113%, about 114%, about 115%, about 116%, about 117%, about 118, about 119%, about 120%, about 121%, about 122%, about 123%, about 124%, about 125%, about 126%, about 127%, about 128%, about 129%, about 130%, about 131%, about 132%, about 133%, about 134%, about 135%, about 136%, about 137%, about 138%, about 139%, about 140%, about 141%, about 142%, about 143%, about 144%, about 145%, about 146%, about 147%, %, about 74%, 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.

[0237] 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.

[0238] 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 SEQ ID NOs: 1156-1231 and 3499-3500.

[0239] 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 SEQ ID NOs: 1232-1307 and 3501-3502.

[0240] 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.

[0241] In some embodiments, the FLT3 binding domain is cross-reactive with human, cynomolgus monkey (cyno), and mouse FLT3. In some embodiments, the FLT3 binding domain is specific for human FLT3. In certain embodiments, the FLT3 binding domain disclosed herein binds to human FLT3 with a human Kd (hKd). In certain embodiments, the FLT3 binding domain disclosed herein binds to cynomolgus monkey FLT3 with a cynomolgus monkey Kd (cKd). In certain embodiments, the FLT3 binding domain disclosed herein binds to mouse FLT3 with a mouse Kd (mKd). In certain embodiments, the FLT3 binding domain disclosed herein binds to both cynomolgus monkey FLT3 and human FLT3 with a cynomolgus monkey Kd (cKd) and a human Kd (hKd), respectively. In certain embodiments, the FLT3-binding domain disclosed herein binds to cynomolgus monkey FLT3, mouse FLT3, and human FLT3 with cynomolgus monkey Kd (cKd), mouse Kd (mKd), and human Kd (hKd), respectively. In some embodiments, the FLT3-binding protein binds to human, mouse, and cynomolgus monkey FLT3 with comparable binding affinities (i.e., the hKd, mKd, and cKd values ​​do not differ by more than ±10%). In some embodiments, the hKd, mKd, and cKd range from about 0.1 nM to about 500 nM. In some embodiments, the hKd, mKd, and cKd range from about 0.1 nM to about 450 nM. In some embodiments, the hKd, mKd, and cKd range from about 0.1 nM to about 400 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.1 nM to about 350 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.1 nM to about 300 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.1 nM to about 250 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.1 nM to about 200 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.1 nM to about 150 nM.In some embodiments, the hKd, mKd, and cKd are in the range of about 0.1 nM to about 100 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.1 nM to about 90 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.2 nM to about 80 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.3 nM to about 70 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.4 nM to about 50 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.5 nM to about 30 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.6 nM to about 10 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.7 nM to about 8 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.8 nM to about 6 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.9 nM to about 4 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 1 nM to about 2 nM.

[0242] In some embodiments, any of the aforementioned FLT3-binding domains (e.g., anti-FLT3 single-domain antibodies of SEQ ID NOS: 1004-1079) are tagged with an affinity peptide to facilitate purification. In some embodiments, the affinity peptide tag is six consecutive histidine residues, also known as 6X-his (SEQ ID NO: 3503). In certain embodiments, the FLT3-binding domain of the present disclosure preferentially binds membrane-bound FLT3 over soluble FLT3. Membrane-bound FLT3 refers to FLT3 present within or on the cell membrane surface of cells that express FLT3. Soluble FLT3 refers to FLT3 that is no longer present within 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 or more more than soluble FLT3. In one embodiment, the FLT3-binding protein of the present disclosure preferentially binds to membrane-bound FLT3 at least 30-fold more than soluble FLT3. Determining the preferential binding of an antigen-binding protein to membrane-bound FLT3 over soluble FLT3 can be readily determined using binding assays.

[0243] In some embodiments, the FLT3 binding protein is fairly small, and in some embodiments, it is contemplated that the FLT3 binding protein is 25 kDa or less, 20 kDa or less, 15 kDa or less, or 10 kDa or less. In particular examples, when the FLT3 binding protein is a peptide or small molecule entity, it is 5 kDa or less.

[0244] In other embodiments, the FLT3 binding proteins described herein comprise a small molecular entity (SME) binder of FLT3. SME binders are small molecules, averaging approximately 500-2000 Da in size, that are 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 containing a sortase recognition sequence, e.g., LPETG (SEQ ID NO: 3200). To bind the SME binder to a FLT3 binding protein containing 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 binding FLT3. Knottins are disulfide-stabilized peptides with a cysteine ​​knot scaffold and have an average size of approximately 3.5 kDa. Knottins have been proposed for binding to specific tumor molecules, such as FLT3. In further embodiments, the FLT3 binding proteins described herein comprise a natural FLT3 ligand.

[0245] In some embodiments, the FLT3-binding protein comprises more than one domain and is a single polypeptide design with flexible domain connections. This allows for easy production and manufacturing of the FLT3-binding protein, since it can be encoded by a single cDNA molecule that can be easily incorporated into a vector. Furthermore, in some embodiments, the FLT3-binding protein described herein is a single monomeric polypeptide chain, eliminating the problem of chain pairing and eliminating the requirement for dimerization. In such embodiments, it is contemplated that the FLT3-binding protein described herein has a reduced tendency to aggregate.

[0246] In FLT3 binding proteins containing more than one domain, the domains are linked by one or more internal linkers. In certain embodiments, the internal linker is "short," i.e., consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues. Thus, in certain examples, the internal linker consists of about 12 or fewer amino acid residues. In the case of 0 amino acid residues, the internal linker is a peptide bond. In certain embodiments, the internal linker is "long," i.e., consisting of 15, 20, or 25 amino acid residues. In some embodiments, the internal linker consists of about 3 to about 15, e.g., 8, 9, or 10, consecutive amino acid residues. Regarding the amino acid composition of the internal linker, the peptide is selected for its properties of providing flexibility to the FLT3 binding protein, not interfering with the binding domain, and resisting cleavage by proteases. For example, glycine and serine residues generally confer protease resistance. Examples of internal linkers suitable for linking domains in FLT3 binding proteins include, but are not limited to, (GS) n (SEQ ID NO: 3190), (GGS) n (SEQ ID NO: 3191), (GGGS) n (SEQ ID NO: 3192), (GGSG) n (SEQ ID NO: 3193), (GGSGG) n (SEQ ID NO: 3194), (GGGGS) n (SEQ ID NO: 3195), (GGGGG) n (SEQ ID NO: 3196), or (GGG) n (SEQ ID NO: 3197), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the linker is (GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 3198), (GGGGSGGGGSGGGGS) (SEQ ID NO: 3199), or (GGGGSGGGS) (SEQ ID NO: 3504).

[0247] In some cases, when FLT3-binding proteins contain more than one domain, domains within the FLT3-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 relatively pure enzymes in large quantities, its ease of use, and its lack of regulation by calcium or guanosine-5'-triphosphate (GTP) have made mTG the primary cross-linking enzyme used in the food industry and biotechnology. Currently, mTG is used in many applications to conjugate proteins and peptides to small molecules, polymers, surfaces, DNA, and other proteins. See Pavel Strp, "Veracity of microbial transglutaminase," Bioconjugate Chem. 25, 5, 855-862.

[0248] In some examples, FLT3 binding proteins are provided that contain more than one domain, wherein one of the domains contains an acceptor glutamine in the constant region, which can be conjugated to another domain via a lysine-based linker (e.g., any primary amine chain that is a substrate for TGase, including alkylamines, oxoamines), wherein conjugation occurs only at one or more acceptor glutamine residues present in the targeting portion outside the antigen-binding site (e.g., outside the variable region, within the constant region). Thus, conjugation does not occur at glutamines within the variable region, for example, at least partially surface-exposed glutamines. 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.

[0249] In some embodiments, when one or more domains within the FLT3 binding protein are directly linked, a hybrid vector is created in which the DNAs encoding the directly linked domains are themselves directly ligated to each other. In some embodiments in which 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 the linker portion, and the DNA encoding another domain is ligated to the other end of the linker portion.

[0250] In some embodiments, the FLT3-binding protein is a single-chain variable fragment (scFv), a single-domain antibody, such as a heavy chain variable domain (VH), a light chain variable domain (VL), or a variable domain of a camelid-derived single-domain antibody (VHH). In other embodiments, the FLT3-binding protein is a non-Ig binding domain, i.e., an antibody mimic such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, Kunitz domain peptides, and monobodies. In further embodiments, the FLT3-binding protein is a ligand or peptide that binds to or associates with FLT3. In yet further embodiments, the FLT3-binding protein is a knottin. In yet further embodiments, the binding domain for FLT3 is a small molecule entity.

[0251] In certain embodiments, the FLT3-binding proteins of the present disclosure can be incorporated into immune cell-associated proteins. In some embodiments, the immune cell-associated protein comprises a CD3-binding domain, a half-life extending domain, and a FLT3-binding domain of the present disclosure. In some embodiments, the FLT3-binding trispecific protein comprises a trispecific antibody.

[0252] EpCAM-binding protein Described herein are immune cell-associated proteins that bind to EpCAM, pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors, and host cells for producing such proteins. Also provided are methods of using the disclosed EpCAM-binding proteins in the prevention and / or treatment of diseases, conditions, and disorders. In some embodiments, the EpCAM-binding protein is part of an immune cell-associated protein that includes an EpCAM-binding domain as described herein.

[0253] Epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein expressed in most normal human epithelia and overexpressed in most carcinomas. This molecule is involved in cell-cell adhesion and also in signal transduction, cell migration, proliferation, and differentiation. Therefore, EpCAM has become an immunotherapeutic target in clinical trials for several solid tumors. It has been shown to play an important role in the detection and isolation of circulating tumor cells (CTCs). EpCAM has been shown to be beneficial in the diagnosis and treatment of various carcinomas in various studies. Furthermore, tumor cells often express EpCAM to a much higher extent than their parent epithelium or less invasive forms of the cancers mentioned above. For example, EpCAM expression was shown to be significantly higher in tumor tissue and adenocarcinomas than in normal prostate epithelium (n=76, p<0.0001), suggesting that increased EpCAM expression represents an early event in the development of prostate cancer. See Poczatek, J Urol., 1999, 162, 1462-1644. Additionally, in the majority of both squamous cell carcinomas and cervical adenocarcinomas, strong EpCAM expression has been shown to correlate with increased proliferation and loss of markers of terminal differentiation. See Litvinov, Am. J. Pathol. 1996, 148, 865-75. One example is breast cancer, where overexpression of EpCAM on tumor cells is a predictor of survival. See Gastl, Lancet. 2000, 356, 1981-1982. Furthermore, EpCAM has been described as a marker for detecting disseminated tumor cells in patients with squamous cell carcinoma of the head and 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.

[0254] EpCAM is thought to help epithelial cells adhere in an oriented, highly ordered manner. See Litvinov, J Cell Biol. 1997, 139, 1337-1348. It is believed that with malignant transformation of epithelial cells, rapidly growing tumor cells abandon the high cellular order of the epithelium. As a result, the surface distribution of EpCAM is thought to become less restricted, making the molecule more readily exposed to tumor cells. Because of their epithelial cell origin, tumor cells in most cancers are expected to express EpCAM on their surface.

[0255] EpCAM is a 314-amino acid, 40-kDa membrane-bound glycoprotein differentially expressed in certain epithelia and many human cancers. (See Balzar, J. Mol. Med. 1999, 77, 699-712.) EpCAM was discovered and subsequently cloned through recognition by the murine monoclonal antibody 17-1A / edrecolomab. (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 carcinoma cells. (See Koprowski, Somatic Cell Genet. 1979, 5, 957-971.) The EGF-like repeats of EpCAM have been shown to mediate lateral and reciprocal interactions in homophilic cell adhesion. See, for example, Balzar, Mol. Cell. Biol. 2001, 21, 2570-2580), which is why they are predominantly located among 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).

[0256] EpCAM is also known by the following alternative names: epithelial cell adhesion molecule, tumor-associated calcium signal transduction signal, 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 (35 kD glycoprotein), 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 Nos. P16422 and B5MCA4. In some embodiments, an EpCAM binding protein of the disclosure binds to the EpCAM sequence provided in UniProtkB ID Nos. P16422 (SEQ ID NO: 478) or B5MCA4 (SEQ ID NO: 475).

[0257] 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.

[0258] In some embodiments, the EpCAM-binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 3208. In some embodiments, the EPCAM-binding domain binds to a protein comprising the sequence of SEQ ID NO: 3208. In some embodiments, the EpCAM-binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 3209. In some embodiments, the EPCAM-binding domain binds to a protein comprising the sequence of SEQ ID NO: 3209. In some embodiments, the EpCAM-binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 3210. In some embodiments, the EpCAM-binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 3210. In some embodiments, the EpCAM-binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 478. In some embodiments, the EpCAM-binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 3211. In some embodiments, the EpCAM-binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 3212. In some embodiments, the EpCAM-binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 3212. In some embodiments, the EpCAM-binding domain binds to a protein comprising 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.

[0259] 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 within EpCAM; for example, in some cases, the EpCAM-binding protein interacts with one or more amino acids found within a domain of human EpCAM. 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, 20, or more) amino acids located within a domain of EpCAM. Alternatively, the epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) located within a domain of EpCAM.

[0260] In some embodiments, the EpCAM binding proteins of the present disclosure bind to the full-length EpCAM protein or fragments thereof, such as epitope-containing fragments within the full-length EpCAM protein, as described above. In some cases, the epitope-containing fragments include antigenic or immunogenic fragments of the EpCAM protein and derivatives thereof. In some embodiments, epitope-containing fragments, including antigenic or immunogenic fragments, are 12 or more amino acids, e.g., 20 or more amino acids, 50 or 100 or more amino acids. In some embodiments, the EpCAM fragments comprise 95% or more of the full-length protein, 90% or more, 75%, 50%, 25%, or 10% or more of the full-length protein. In some embodiments, epitope-containing fragments of EpCAM, including antigenic or immunogenic fragments, can induce a relevant immune response in patients. In some embodiments, derivatives of EpCAM include sequence variants in which one or more (e.g., 1 to 20, e.g., 15 amino acids, or up to 20%, e.g., 10%, or 5%, or 1%) amino acids have been deleted, inserted, or substituted in the EpCAM sequence provided in SEQ ID NOs: 3208-3214.

[0261] In some embodiments, substitutions include conservative substitutions. In some cases, the derivatives and variants thereof have essentially the same biological function as the proteins from which they are derived. For example, in some cases, the derivatives and variants of EpCAM have antigenicity or immunogenicity comparable to that of the proteins from which they are derived, have either the ligand-binding activity or the active receptor-complex forming ability of the proteins from which they are derived, or preferably both, and have the same tissue distribution as EpCAM.

[0262] In some embodiments, the EpCAM-binding protein specifically binds to EpCAM with an affinity that is equal to or better than that of a reference EpCAM-binding protein; in such embodiments, the EpCAM-binding protein comprises an affinity-matured EpCAM-binding molecule, which is derived from an EpCAM-binding parent molecule and contains one or more amino acid mutations (e.g., stabilizing mutations, destabilizing mutations) relative to the EpCAM-binding parent molecule. In some embodiments, the affinity-matured EpCAM-binding molecule has superior stability against a selected destabilizing agent than the reference EpCAM-binding parent molecule. In some embodiments, the affinity-matured EpCAM-binding molecule is identified by a process comprising panning one or more pre-candidate EpCAM-binding molecules derived from one or more EpCAM-binding parent molecules expressed in a phage display library against an EpCAM protein, such as human EpCAM protein. The pre-candidate EpCAM-binding molecule, in some embodiments, contains amino acid substitutions in variable region, CDR, or framework residues relative to the parent molecule.

[0263] 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 large libraries of randomized protein variants can be rapidly and efficiently selected for sequences that bind to target molecules with high affinity. Displaying 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 fusing them to either gene III or gene VIII of filamentous phage. See, e.g., 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, resulting in phage particles displaying either one copy of the fusion protein or none at all. Because avidity effects are reduced compared to polyvalent phage, selection is based on affinity for endogenous ligands and phagemid vectors are used, which facilitate DNA manipulation. See, e.g., Lowman and Wells, Methods: A companion to Methods in Enzymology, 3:205-0216 (1991).

[0264] In some embodiments, panning involves using varying binding times and concentrations to identify EpCAM-binding molecules with increased or decreased on-rates from pre-candidate EpCAM-binding molecules. In some embodiments, panning involves using varying wash times to identify EpCAM-binding molecules with increased or decreased on-rates from pre-candidate EpCAM-binding molecules. In some embodiments, panning involves using varying binding times and varying wash times. In some embodiments, one or more stabilizing mutations are combined to increase the stability of the affinity-matured EpCAM-binding molecules, for example, by shuffling to generate a second-stage combinatorial library from such mutants and performing a second round of panning and subsequent binding selection.

[0265] In some embodiments, the affinity-matured EpCAM-binding molecule has affinity for EpCAM protein (such as human EpCAM protein) that is equal to or better than the EpCAM-binding parent molecule, but it has reduced, or in some embodiments, increased, cross-reactivity with selected substances, such as ligands, proteins, or antigens, other than the EpCAM epitope for which the EpCAM-binding parent molecule is specific or designed to be specific. Regarding the latter, affinity-matured EpCAM-binding molecules are, in some embodiments, more successfully tested in animal models when they are reacted with both human EpCAM and the corresponding target in the animal model, e.g., mouse EpCAM or cynomolgus EpCAM. In some embodiments, the parent EpCAM-binding molecule binds to human 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, 10 nM or less, and binds to 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, or 10 nM or less. In some embodiments, the affinity-matured EpCAM-binding molecule identified after a single round of panning binds to human EpCAM with an affinity of about 5 nM or less, such as 1 nM or less, and binds to cynomolgus EpCAM with an affinity of about 7.5 nM or less, such as 1 nM or less. In some embodiments, affinity-matured EpCAM-binding molecules identified after two rounds of panning bind to human EpCAM with an affinity of about 2.5 nM or less and bind to cynomolgus EpCAM with an affinity of about 3.5 nM or less.

[0266] In some embodiments, the EpCAM-binding protein comprises an antigen-specific binding domain polypeptide that specifically binds to a target, e.g., a target on a diseased cell or a target on another cell that supports a disease state, e.g., a target on a stromal cell that supports tumor growth, or a target on an immune cell that supports disease-mediated immunosuppression. In some examples, the antigen-specific binding domain comprises an antibody, single-chain antibody, Fab, Fv, T-cell receptor binding domain, ligand-binding domain, receptor-binding domain, domain antibody, single-domain antibody, minibody, nanobody, peptibody, or various other antibody mimetics (such as affimers, affitins, alphabodies, atrimers, CTLA4-based molecules, adnectins, anticalins, Kunitz domain-based proteins, avimers, knottins, fynomers, darpins, affibodies, affilins, monobodies, and armadillo repeat protein-based proteins).

[0267] In some embodiments, the EpCAM-binding domain is an anti-EpCAM antibody or antigen-binding fragment thereof, or an antibody variant of the EpCAM-binding domain or antigen-binding fragment thereof. As used herein, the term "antibody variant" refers to variants and derivatives of the antibodies or antigen-binding fragments thereof described herein. In certain embodiments, amino acid sequence variants of the anti-EpCAM antibodies or antigen-binding fragments thereof are contemplated as described herein. For example, in certain embodiments, amino acid sequence variants of the anti-EpCAM antibodies or antigen-binding fragments thereof described herein are contemplated to improve their binding affinity and / or other biological properties. Exemplary methods for preparing amino acid variants include, but are not limited to, introducing appropriate modifications into the nucleotide sequence encoding the antibody or antigen-binding fragment thereof, or peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequence of the antibody or antigen-binding fragment thereof.

[0268] Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics (e.g., antigen-binding). In certain embodiments, variants with one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the CDRs and framework regions. Examples of such substitutions are described below. Amino acid substitutions may be introduced into an antibody or antigen-binding fragment of interest, and the products may be screened for the desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, altered antibody-dependent cellular cytotoxicity (ADCC), or improved T-cell-mediated cytotoxicity (TDCC). Conservative and non-conservative amino acid substitutions are contemplated for the preparation of antibody variants.

[0269] In another example of substitutions to create a variant anti-EpCAM antibody or antigen-binding fragment thereof, one or more hypervariable region residues of the parent antibody are substituted. Generally, variants are selected based on a desired improved property compared to the parent antibody or antigen-binding fragment thereof, such as increased affinity, decreased affinity, decreased immunogenicity, or increased pH-dependent binding.

[0270] In some embodiments, the EpCAM-binding domain is a single-domain antibody (sdAb), e.g., a heavy chain variable domain (VH), a variable domain (VHH) of a llama-derived sdAb, a peptide specific for EpCAM, a ligand, or a small molecule entity. In some embodiments, the EpCAM-binding domain described herein is any domain that binds to EpCAM, including, but not limited to, domains derived from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, and humanized antibodies. In certain embodiments, the EpCAM-binding domain is a single-domain antibody. In other embodiments, the EpCAM-binding domain is a peptide. In further embodiments, the EpCAM-binding domain is a small molecule.

[0271] In one embodiment, the single domain antibody corresponds to the VHH domain of a naturally occurring heavy chain antibody against EpCAM. As further described herein, such a VHH sequence can typically be generated or obtained by appropriately immunizing a species of llama with EpCAM (i.e., to generate an immune response and / or heavy chain antibodies against EpCAM), by obtaining a suitable biological sample from said llama (such as a blood sample, serum sample, or B cell sample), and generating a VHH sequence against EpCAM starting from said sample using any suitable technique known in the art.

[0272] In another embodiment, such naturally occurring VHH domains against EpCAM are obtained from a naive library of Camelidae VHH sequences, for example, by screening such a library with EpCAM or by screening for at least one portion, 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 WO99 / 37681, WO01 / 90190, WO03 / 025020, and WO03 / 035694. Alternatively, improved synthetic or semi-synthetic libraries derived from naive VHH libraries are used, for example, VHH libraries obtained from naive VHH libraries by techniques such as random mutagenesis and / or CDR shuffling, as described in WO00 / 43507.

[0273] In a further embodiment, yet another technique for obtaining VHH sequences against EpCAM involves appropriately immunizing a transgenic mammal capable of expressing heavy chain antibodies (i.e., to generate an immune response and / or heavy chain antibodies against EpCAM), obtaining a suitable biological sample from said transgenic mammal (such as a blood sample, serum sample, or B cell sample), and generating VHH sequences against EpCAM starting from said sample using any suitable technique known in the art. For example, heavy chain antibody-expressing rats or mice and methods and techniques such as those described in WO02 / 085945 and WO04 / 049794 can be used for this purpose.

[0274] In some embodiments, the anti-EpCAM single domain antibodies of the present disclosure include single domain antibodies having an amino acid sequence that corresponds to the amino acid sequence of a non-human antibody and / or a naturally occurring VHH domain, e.g., a llama anti-EpCAM antibody, but that has been "humanized", i.e., humanized by replacing one or more amino acid residues in the amino acid sequence (particularly in the framework sequences) of said non-human anti-EpCAM and / or naturally occurring VHH sequence with one or more amino acid residues occurring at the corresponding positions in a VH domain from a conventional four-chain antibody of human origin (e.g., as shown above).

[0275] Other suitable methods and techniques for obtaining an anti-EpCAM single domain antibody of the present disclosure and / or a nucleic acid encoding the anti-EpCAM single domain antibody, starting from a naturally occurring VH or VHH sequence, include, for example, combining, in a suitable manner, one or more portions of one or more naturally occurring VH sequences (such as one or more framework (FR) sequences and / or complementarity determining region (CDR) sequences), one or more portions of one or more naturally occurring VHH sequences (such as one or more FR sequences or CDR sequences), and / or one or more synthetic or semi-synthetic sequences to provide an EpCAM single domain antibody of the present disclosure or a nucleotide sequence or nucleic acid encoding the anti-EpCAM single domain antibody.

[0276] In some embodiments, the EpCAM-binding domain is an anti-EpCAM-specific antibody comprising heavy chain variable complementarity-determining regions 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, the EpCAM-binding domain comprises any domain that binds to EpCAM, including, but not limited to, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, or an antigen-binding fragment, such as a single-domain antibody (sdAb), Fab, Fab', F(ab)2, and Fv fragment, a fragment composed of one or more CDRs, a single-chain antibody (e.g., a single-chain Fv fragment (scFv)), a disulfide-stabilized (dsFv) Fv fragment, a heteroconjugate antibody (e.g., a bispecific antibody), a pFv fragment, a heavy chain monomer or dimer, a light chain monomer or dimer, and a dimer consisting of one heavy chain and one light chain. In some embodiments, the EpCAM-binding domain is a single-domain antibody. In some embodiments, the anti-EpCAM single domain antibody comprises the heavy chain variable complementarity determining regions (CDRs): CDR1, CDR2, and CDR3.

[0277] 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. The framework residues of the EpCAM-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 EpCAM-binding domain comprises an amino acid sequence selected from SEQ ID NOs: 961-1003.

[0278] In some embodiments, the EpCAM binding protein comprises at least 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to a sequence selected from SEQ ID NOs: 961-1003, subsequences thereof, and variants thereof. In some embodiments, the EpCAM binding protein comprises at least 70%-95% or more identity to a sequence selected from SEQ ID NOs: 961-1003, subsequences thereof, and variants thereof. In some embodiments, the EpCAM binding protein comprises at least 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to a sequence selected from SEQ ID NOs: 961-1003, subsequences thereof, and variants thereof.

[0279] In some embodiments, CDR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 847 to 884, or a sequence that contains one or more substitutions compared to a sequence selected from the group consisting of SEQ ID NOs: 847 to 884. In some embodiments, CDR2 comprises the sequence set forth in any one of SEQ ID NOs: 885 to 922, or a sequence that contains one or more substitutions compared to a sequence selected from the group consisting of SEQ ID NOs: 885 to 922. In some embodiments, CDR3 comprises the sequence set forth in any one of SEQ ID NOs: 923 to 960, or a sequence that contains one or more substitutions compared to a sequence selected from the group consisting of SEQ ID NOs: 923 to 960.

[0280] In various embodiments, the EpCAM-binding domain of the present disclosure is at least about 60%, about 61%, at least about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, 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: 961-1003.

[0281] 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.

[0282] 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.

[0283] 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: 923-960.

[0284] In some embodiments, the EpCAM-binding domain is cross-reactive with human, cynomolgus, and mouse EpCAM. In some embodiments, the EpCAM-binding domain is specific for human EpCAM. In certain embodiments, the EpCAM-binding domain disclosed herein binds to human EpCAM with a human Kd (hKd). In certain embodiments, the EpCAM-binding domain disclosed herein binds to cynomolgus EpCAM with a cynomolgus Kd (cKd). In certain embodiments, the EpCAM-binding domain disclosed herein binds to cynomolgus EpCAM with a mouse Kd (mKd). In certain embodiments, the EpCAM-binding domain disclosed herein binds to both cynomolgus EpCAM and human EpCAM with cynomolgus Kd (cKd) and human Kd (hKd), respectively. In certain embodiments, the EpCAM-binding domain disclosed herein binds to cynomolgus EpCAM, mouse EpCAM, and human EpCAM with a cynomolgus Kd (cKd), mouse Kd (mKd), and human Kd (hKd), respectively. In some embodiments, the EpCAM-binding protein binds to human, mouse, and cynomolgus EpCAM with comparable binding affinities (i.e., the hKd, mKd, and cKd values ​​do not differ by more than ±10%). In some embodiments, the hKd, mKd, and cKd range from about 0.001 nM to about 500 nM. In some embodiments, the hKd, mKd, and cKd range from about 0.001 nM to about 450 nM. In some embodiments, the hKd, mKd, and cKd range from about 0.001 nM to about 400 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.001 nM to about 350 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.001 nM to about 300 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.001 nM to about 250 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.001 nM to about 200 nM.In some embodiments, the hKd, mKd, and cKd are in the range of about 0.001 nM to about 150 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.001 nM to about 100 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.1 nM to about 90 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.2 nM to about 80 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.3 nM to about 70 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.4 nM to about 50 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.5 nM to about 30 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.6 nM to about 10 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.7 nM to about 8 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.8 nM to about 6 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.9 nM to about 4 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 1 nM to about 2 nM.

[0285] In some embodiments, any of the aforementioned EpCAM-binding domains (e.g., the anti-EpCAM single domain antibodies of SEQ ID NOs: 961-1003) are tagged with an affinity peptide to facilitate purification. In some embodiments, the affinity peptide tag is six consecutive histidine residues, also known as 6X-his (SEQ ID NO: 3503).

[0286] 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 EpCAM present 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 lymphatic 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 or more than soluble EpCAM. In one embodiment, the EpCAM-binding protein of the present disclosure preferentially binds to membrane-bound EpCAM at least 30-fold or more than soluble EpCAM. Determining the preferential binding of an antigen binding protein to membrane-bound EpCAM over soluble EpCAM can be readily determined using binding assays.

[0287] In some embodiments, the EpCAM binding protein is fairly small, contemplated in some embodiments to be 25 kDa or less, 20 kDa or less, 15 kDa or less, or 10 kDa or less. In particular examples, the EpCAM binding protein, when a peptide or small molecule entity, is 5 kDa or less.

[0288] In other embodiments, the EpCAM-binding proteins described herein comprise small molecular entity (SME) binders of EpCAM. SME binders are small molecules, averaging approximately 500-2000 Da in size, that are attached to the EpCAM-binding protein by known methods, such as sortase ligation or conjugation. In these examples, the EpCAM-binding protein comprises a domain containing a sortase recognition sequence, e.g., LPETG (SEQ ID NO: 3200). To bind the SME binder to an EpCAM-binding protein containing 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 EpCAM-binding proteins described herein comprise knottin peptides for binding EpCAM. Knottins are disulfide-stabilized peptides with a cysteine ​​knot scaffold and have an average size of approximately 3.5 kDa. Knottins have been proposed for binding to specific tumor molecules, such as EpCAM. In further embodiments, the EpCAM-binding proteins described herein comprise a natural EpCAM ligand.

[0289] In some embodiments, the EpCAM-binding protein comprises more than one domain and is a single polypeptide design with flexible domain connections. This allows for easy production and manufacturing of the EpCAM-binding protein, as it can be encoded by a single cDNA molecule that can be easily incorporated into a vector. Furthermore, in some embodiments in which the EpCAM-binding protein described herein is a single monomeric polypeptide chain, there is no problem with chain pairing and no requirement for dimerization. In such embodiments, it is contemplated that the EpCAM-binding protein described herein has a reduced tendency to aggregate.

[0290] In EpCAM-binding proteins containing more than one domain, the domains are linked by one or more internal linkers. In certain embodiments, the internal linker is "short," i.e., consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues. Thus, in certain examples, the internal linker consists of about 12 or fewer amino acid residues. In the case of 0 amino acid residues, the internal linker is a peptide bond. In certain embodiments, the internal linker is "long," i.e., consisting of 15, 20, or 25 amino acid residues. In some embodiments, the internal linker consists of about 3 to about 15, e.g., 8, 9, or 10, consecutive amino acid residues. Regarding the amino acid composition of the internal linker, the peptide is selected for its properties of providing flexibility to the EpCAM-binding protein, not interfering with the binding domain, and resisting cleavage by proteases. For example, glycine and serine residues generally confer protease resistance. Examples of internal linkers suitable for linking domains in EpCAM-binding proteins include, but are not limited to, (GS) n (SEQ ID NO: 3190), (GGS) n (SEQ ID NO: 3191), (GGGS) n (SEQ ID NO: 3192), (GGSG) n (SEQ ID NO: 3193), (GGSGG) n (SEQ ID NO: 3194), (GGGGS) n (SEQ ID NO: 3195), (GGGGG) n (SEQ ID NO: 3196), or (GGG) n (SEQ ID NO: 3197), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the linker is (GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 3198), (GGGGSGGGGSGGGGS) (SEQ ID NO: 3199), or (GGGGSGGGS) (SEQ ID NO: 3504).

[0291] In some cases, when an EpCAM-binding protein contains more than one domain, 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 relatively pure enzymes in large quantities, its ease of use, and its lack of regulation by calcium or guanosine-5'-triphosphate (GTP) have made mTG the primary cross-linking enzyme used in the food industry and biotechnology. Currently, mTG is used in many applications to conjugate proteins and peptides to small molecules, polymers, surfaces, DNA, and other proteins. See Pavel Strp, "Veracity of microbial transglutaminase," Bioconjugate Chem. 25, 5, 855-862.

[0292] In some examples, an EpCAM-binding protein is provided that includes more than one domain, wherein one of the domains includes an acceptor glutamine in the constant region, which can be conjugated to another domain via a lysine-based linker (e.g., any primary amine chain that is a substrate for TGase, including alkylamines, oxoamines), wherein conjugation occurs only at one or more acceptor glutamine residues present in the targeting portion outside the antigen-binding site (e.g., outside the variable region, within the constant region). Thus, conjugation does not occur at glutamines within the variable region, for example, at least partially surface-exposed glutamines. 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.

[0293] In some embodiments, when one or more domains within an EpCAM-binding protein are directly linked, a hybrid vector is created in which the DNAs encoding the directly linked domains are themselves directly ligated to each other. In some embodiments in which 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 the linker portion and the DNA encoding another domain is ligated to the other end of the linker portion.

[0294] In some embodiments, the EpCAM-binding protein is a single-chain variable fragment (scFv), a single-domain antibody, e.g., a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain of a camelid-derived single-domain antibody (VHH). In other embodiments, the EpCAM-binding protein is a non-Ig binding domain, i.e., an antibody mimic such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, Kunitz domain peptides, and monobodies. In further embodiments, the EpCAM-binding protein is a ligand or peptide that binds to or associates with EpCAM. In yet further embodiments, the EpCAM-binding protein is a knottin. In yet further embodiments, the binding domain for EpCAM is a small molecule entity.

[0295] In certain embodiments, the EpCAM-binding proteins of the present disclosure may be incorporated into an immune cell-associated protein. In some embodiments, the immune cell-associated protein comprises a CD3-binding domain, a half-life-extending domain, and an EpCAM-binding domain of the present disclosure. In some embodiments, the immune cell-associated protein comprises a trispecific antibody.

[0296] CD3-binding domain The immune cell-associated proteins described herein comprise an immune cell-associated domain. In some embodiments, the immune cell-associated domain comprises a natural killer (NK) cell-associated domain, a T cell-associated domain, a B cell-associated domain, a dendritic cell-associated domain, a macrophage cell-associated domain, or a combination thereof. In some embodiments, the immune cell-associated protein comprises a T cell-associated domain. In some embodiments, the T cell-associated domain is a CD3-binding domain.

[0297] The specificity of T cell responses is mediated by the recognition of antigens (referred to in the context of the major histocompatibility complex, MHC) by the T cell receptor complex. As part of the T cell receptor complex, CD3 is a protein complex present on the cell surface that includes the CD3γ (gamma) chain, the CD3δ (delta) chain, and two CD3ε (epsilon) chains. To comprise the T cell receptor complex, CD3 associates with the α (alpha) and β (beta) chains of the T cell receptor (TCR), as well as CD3ζ (zeta). Clustering of CD3 on T cells, such as by immobilized anti-CD3 antibodies, triggers T cell activation, similar to T cell receptor engagement but independent of the specificity typical of that clone.

[0298] In one embodiment, the single-chain variable fragment CD3 binding proteins described herein comprise a domain that specifically binds to CD3. In one embodiment, the single-chain variable fragment CD3 binding proteins described herein comprise a domain that specific...

Claims

1. 1. A pharmaceutical composition comprising a half-life extended immune cell associated protein for use in combination with an immunomodulatory agent, wherein the half-life extended immune cell associated protein comprises an immune cell binding domain.

2. 2. The pharmaceutical composition of claim 1, wherein the immune cell-associated domain comprises a T cell-associated domain, a natural killer (NK) cell-associated domain, a B cell-associated domain, a dendritic cell-associated domain, a macrophage cell-associated domain, or a combination thereof.

3. The pharmaceutical composition of claim 2 , wherein the T cell-associated domain binds to a CD3 molecule.

4. The pharmaceutical composition of claim 3 , wherein the CD3 molecule is at least one of a CD3γ molecule, a CD3δ molecule, or a CD3ε molecule. (i) the immunomodulatory agent comprises an immunostimulatory antibody agonist of a costimulatory receptor; (ii) the immunomodulatory agent comprises an immune checkpoint modulator; The pharmaceutical composition according to any one of claims 1 to 4.

6. The immune checkpoint modulators include programmed cell death 1 (PDCD1, PD1, PD-1), CD274 (CD274, PDL1, PD-L1), PD-L2, cytotoxic T-lymphocyte-associated protein 4 (CTLA4, CD152), CD276 (B7H3), V-set domain-containing inhibitor of T-cell activation 1 (VTCN1, B7H4), CD272 (B-lymphocyte- and T-lymphocyte-associated (BTLA)), killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1), lymphocyte activation 3 (LAG3, CD 223), Hepatitis A virus cell receptor 2 (HAVCR2, TIMD3, TIM3), V-set immunoregulatory receptor (VSIR, B7H5, VISTA), T cell immunoreceptor with Ig and ITIM domains (TIGIT), programmed cell death 1 ligand 2 (PDCD1LG2, PD-L2, CD273), immunoglobulin superfamily member 11 (IGSF11, VSIG3), TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML), PVR-related immunoglobulin domain containing (PVRIG, CD112R), and GA Killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1), killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2), killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3), and killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1), killer cell lectin-like receptor C1 (KLRC1, NKG2A, CD159A), killer cell lectin-like receptor C2 (KLRC2, NKG2A, CD159A), killer cell lectin-like receptor C3 (KLRC3, NKG2A, CD159A), killer cell lectin-like receptor C4 (KLRC4, NKG2A, CD159A), killer cell lectin-like receptor C5 (KLRC5, NKG2A, CD159A), killer cell lectin-like receptor C6 (KLRC6, NKG2A, CD159A), killer cell lectin-like receptor C7 (KLRC7, NKG2A, CD159A), killer cell lectin-like receptor C8 (KLRC8, NKG2A, CD159A), killer cell lectin-like receptor C9 (KLRC9, NKG2A, CD159A), killer cell lectin-like receptor C1 ...2 (KLRC9, NKG2A, CD159A), killer cell lect Cutin-like receptor D1 (KLRD1, CD94), killer cell lectin-like receptor G1 (KLRG1, CLEC15A, MAFA, 2F1), sialic acid-binding Ig-like lectin 7 (SIGLEC7), SIGLEC, sialic acid-binding Ig-like lectin 9 (SIGLEC9), CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), VISTA, LAIR1, CD160, 2B4, CD80, CD86, B7-H1, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270),6. The pharmaceutical composition of claim 5, which is an antagonist of at least one of KIR, A2AR, A2BR, MHC class I, MHC class II, GAL9, adenosine, TGFR (e.g., TGFRβ), CD94 / NKG2A, IDO, TDO, CD39, CD73, GARP, CD47, PVRIG, CSF1R, and NOX, or any combination thereof.

7. (i) The immune checkpoint modulator is a PD-1 antagonist, and is selected from the group consisting of pembrolizumab (humanized antibody), pidilizumab (CT-011, monoclonal antibody that binds to DLL1 and PD-1), spartalizumab (PDR001, monoclonal antibody), nivolumab (BMS-936558, MDX-1106, human IgG4 monoclonal antibody), MEDI0680 (AMP-514, monoclonal antibody), cemiplimab (REGN2810, monoclonal antibody), dostallimab (TSR-042, monoclonal antibody), and sasunlimab (PF -06801591, monoclonal antibody), tislelizumab (BGB-A317, monoclonal antibody), BGB-108 (antibody), tislelizumab (BGB-A317, antibody), camrelizumab (INCSHR1210, SHR-1210), AMP-224, zimberelimab (AB122, GLS-010, WBP-3055, monoclonal antibody), AK-103 (HX-008, monoclonal antibody), AK-105 (anti-PD-1 antibody), CS1003 (monoclonal antibody), HLX10 (monoclonal antibody), retifanlimab (MGA-012, anti-PD-1monoclonal antibody), BI-754091 (antibody), balstilimab (AGEN2034, PD-1 antibody), toripalimab (JS-001, antibody), cetrelimab (JNJ-63723283, anti-PD-1 antibody), genolimzumab (CBT-501, anti-PD-1 antibody), LZM009 (anti-PD-1 monoclonal antibody), progolimab (BCD-100, anti-PD-1 monoclonal antibody), Sym021 (antibody), ABBV-181 (antibody), BAT-1306 (antibody), JTX-4014, sintilimab (s intilimab (IBI-308), tebotelimab (MGD013, PD-1 / LAG-3 bispecific), MGD-019 (PD-1 / CTLA4 bispecific antibody), KN-046 (PD-1 / CTLA4 bispecific antibody), MEDI-5752 (CTLA4 / PD-1 bispecific antibody), RO7121661 (PD-1 / TIM-3 bispecific antibody), XmAb20717 (PD-1 / CTLA4 bispecific antibody), and AK-104 (CTLA4 / PD-1 bispecific antibody); (ii) The immune checkpoint modulator is an antibody that binds to PD-L1, and examples thereof include atezolizumab (MPDL3280A, monoclonal antibody), avelumab (MSB0010718C, monoclonal antibody), durvalumab (MEDI-4736, human immunoglobulin G1 kappa (IgG1κ) monoclonal antibody), envafolimab (KN035, single domain PD-L1 antibody), AUNP12, CA-170 (PD-L1 and and VISTA-targeting small molecules), BMS-986189 (macrocyclic peptide), BMS-936559 (anti-PD-L1 antibody), Cosibelimab (CK-301, monoclonal antibody), LY3300054 (antibody), CX-072 (antibody), CBT-502 (antibody), MSB-2311 (antibody), BGB-A333 (antibody), SHR-1316 (antibody), CS1001 (WBP3155, antibody), HLX-20 (antibody), KL-A167 (HBM 9167, antibody), STI-A1014 (antibody), STI-A1015 (IMC-001, antibody), BCD-135 (monoclonal antibody), FAZ-053 (antibody), CBT-502 (TQB2450, antibody), MDX1105-01 (antibody), FS-118 (LAG-3 / PD-L1, bispecific antibody), M7824 (anti-PD-L1 / TGF-β receptor II fusion protein), CDX-527 (CD27 / PD-L1 bispecific antibody), LY3415244 (TIM3 / PD-L1 bispecific antibody), INBRX-105 (4-1BB / PD-L1 bispecific antibody), or (iii) the immune checkpoint modulator comprises an immune checkpoint activator; The pharmaceutical composition according to claim 6.

8. 8. The pharmaceutical composition of claim 7, wherein the immune checkpoint modulator is pembrolizumab.

9. The half-life extending immune cell associated protein comprises a first domain (A), a second domain (B), and a third domain (C); (i) the first domain (A) is a T cell-associated domain and specifically binds to human CD3; (ii) the second domain (B) specifically binds to human serum albumin (HSA); (iii) a third domain (C) that specifically binds to a target antigen; The domains are in the following order: ２ N-(C)-(B)-(A)-COOH, H ２ N-(A)-(B)-(C)-COOH, H ２ N-(B)-(A)-(C)-COOH, H ２ N-(C)-(A)-(B)-COOH, H ２ N-(A)-(C)-(B)-COOH, H ２ N-(B)-(C)-(A)-COOH or in the following order via linkers L1 and L2: ２ N-(C)-L1-(B)-L2(A)-COOH, H ２ N-(A)-L1-(B)-L2-(C)-COOH, H ２ N-(B)-L1-(A)-L2-(C)-COOH, H ２ N-(C)-L1-(A)-L2-(B)-COOH, H ２ N-(A)-L1-(C)-L2(B)-COOH, H ２ 9. The pharmaceutical composition according to any one of claims 2 to 8, wherein said compound is linked at one of the following positions: N-(B)-L1-(C)-L2-(A)-COOH. (i) the target antigen is a tumor antigen; (ii) the third domain is selected from the group consisting of DLL3 (delta-like ligand 3), 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), 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 CD22 (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′-nucleotidyltransferase, and CD80), CD90, CD100, CD110, CD120, CD130, CD140, CD150, CD160, CD170, CD180, CD200, CD210, CD220, CD230, CD240, CD250, CD300, CD310, CD320, CD330, CD340, CD350, CD350, CD360, CD370, CD380, CD390, CD400, CD410, CD420, CD430, CD440, CD440, CD450, CD460, CD470, CD480, CD490, CD500, CD510, CD520, CD530, CD540, CD550, CD560, CD570, CD580, CD590, CD600, CD610, CD620, CD630, CD640, CD650, CD650, CD660, CD670, CD680, CD690, CD700, CD700, CD710, CD720, CD730, CD740, CD750, CD750, CD760, CD770, CD780, CD790, CD810, CD enzyme), CD39 (ENTPD1, ​​ectonucleotide triphosphate diphosphohydrolase-1), CD74 (HLA class II histocompatibility complex gamma chain, HLA-DR antigen-associated invariant chain), CD79b (immunoglobulin-associated beta), CD80 (B7-1), CD86 (B7-2), CD123 (IL3RA, interleukin-3 receptor), CD133 (PROM1), CD137 (TNFRSF9, tumor necrosis factor receptor superfamily member 9, 4-1BB, ILA (induced by lymphocyte activation), 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 (ENPP 3), 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 (MUC1) and its splice variants (e.g., MUC1 / C, D, and Z), mucin 16 (MUC16), necdin (NDN), necdin cell adhesion molecule 4 (NECTIN4), SLIT, and NTR K-like family member 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 um), 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 family 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, sialyl Lewis adhesion molecule (sLe), ROR1, CD30, and Lewis Y antigen,Specifically binds to a target antigen selected from the group consisting of: The pharmaceutical composition according to claim 9.

11. 11. The pharmaceutical composition of claim 9 or claim 10, wherein the third domain is a single domain antibody that specifically binds to DLL3.

12. The pharmaceutical composition of claim 11, wherein the third domain comprises a CDR1 comprising an amino acid selected from the group consisting of SEQ ID NOs: 1751-2193, a CDR2 comprising an amino acid selected from the group consisting of SEQ ID NOs: 2194-2636, and a CDR3 comprising an amino acid selected from the group consisting of SEQ ID NOs: 2637-3080.

13. The pharmaceutical composition of claim 12, wherein the CDR1 comprises the amino acid sequence of SEQ ID NO: 2182, the CDR2 comprises the amino acid sequence of SEQ ID NO: 2625, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 3069.

14. The pharmaceutical composition of any one of claims 11 to 13, wherein the third domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1308-1750.

15. The pharmaceutical composition of any one of claims 11 to 14, wherein the third domain comprises the amino acid sequence of SEQ ID NO: 1739.

16. the first domain comprises a single chain variable fragment (scFv) specific for human CD3; The pharmaceutical composition according to any one of claims 9 to 15, wherein the scFv specific for human CD3 comprises a variable heavy region (VH), a variable light region (VL), and a linker, wherein the VH comprises the complementarity determining regions HC CDR1, HC CDR2, and HC CDR3, and the VL comprises the complementarity determining regions LC CDR1, LC CDR2, and LC CDR3. (i) 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; (ii) the LC CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3084, and 3120-3132, the LC CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3085, and 3099-3109, and the LC CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3086, and 3146-3152; 17. The pharmaceutical composition of claim 16.

18. 18. The pharmaceutical composition of claim 17, wherein the HC CDR1 comprises the amino acid sequence of SEQ ID NO: 3097, the HC CDR2 comprises the amino acid sequence of SEQ ID NO: 3108, the HC CDR3 comprises the amino acid sequence of SEQ ID NO: 3110, the LC CDR1 comprises the amino acid sequence of SEQ ID NO: 3120, the LC CDR2 comprises the amino acid sequence of SEQ ID NO: 3145, and the LC CDR3 comprises the amino acid sequence of SEQ ID NO: 3146.

19. The pharmaceutical composition of any one of claims 9 to 18, wherein the first domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3153 to 3169.

20. The pharmaceutical composition of any one of claims 9 to 19, wherein the first domain comprises the amino acid sequence of SEQ ID NO: 3153.

21. The pharmaceutical composition of any one of claims 16 to 20, wherein the linker comprises the amino acid sequence GGGGSGGGGSGGGGGS (SEQ ID NO: 3199).

22. The pharmaceutical composition of any one of claims 9 to 21, wherein the second domain comprises a single domain antibody (sdAb) that specifically binds to HSA.

23. The pharmaceutical composition of claim 22, wherein the sdAb specifically binds to HSA comprises complementarity determining regions CDR1, CDR2, and CDR3, wherein 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 3182-3183.

24. 24. The pharmaceutical composition of claim 23, wherein the CDR1 comprises the amino acid sequence of SEQ ID NO: 3174, the CDR2 comprises the amino acid sequence of SEQ ID NO: 3178, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 3183.

25. The pharmaceutical composition of any one of claims 9 to 24, wherein the second domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3184 to 3193.

26. 26. The pharmaceutical composition of claim 25, wherein the second domain comprises the amino acid sequence of SEQ ID NO: 3190.

27. The linkers L1 and L2 are each independently (GS) ｎ (SEQ ID NO: 3190), (GGS) ｎ (SEQ ID NO: 3191), (GGGS) ｎ (SEQ ID NO: 3192), (GGSG) ｎ (SEQ ID NO: 3193), (GGSGGG) ｎ (SEQ ID NO: 3194), or (GGGGS) ｎ (SEQ ID NO: 3195), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The pharmaceutical composition of any one of claims 9 to 26.

28. The linkers L1 and L2 are each independently (GGGGS). ４ (SEQ ID NO: 3198) or (GGGGS) ３ 28. The pharmaceutical composition of claim 27, wherein the nucleic acid is (SEQ ID NO: 3199).

29. A pharmaceutical composition described in any one of claims 9 to 28, wherein the half-life extended immune cell-associated protein comprises the amino acid sequence of SEQ ID NO: 3461.

30. 30. The pharmaceutical composition of any one of claims 1 to 29, wherein the immunomodulatory agent and the half-life extending immune cell associated protein are in a single pharmaceutical composition.

31. 30. The pharmaceutical composition of any one of claims 1 to 29, wherein the immunomodulatory agent and the half-life extending immune cell associated protein are in separate pharmaceutical compositions.

32. A pharmaceutical composition comprising an immunomodulatory agent according to any one of claims 1 to 31 and an extended half-life immune cell-associated protein according to any one of claims 1 to 31.

33. Use of a pharmaceutical composition according to any one of claims 1 to 31 or a pharmaceutical composition according to claim 32 in the manufacture of a medicament for the treatment of cancer.

34. The cancers include 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, 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, 34. The use of claim 33, wherein the cancer is selected from the group consisting of 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.

35. (i) administration of the combination provides an increased therapeutic benefit compared to administration of the immunomodulatory agent alone, without the half-life extending immune cell associated protein; or (ii) administration of the combination results in an increased therapeutic benefit compared to administration of the half-life extended immune cell associated protein alone without the immunomodulatory agent; 35. The use according to claim 33 or claim 34.

36. The use according to any one of claims 33 to 35, wherein the half-life extended immune cell associated protein and the immunomodulatory drug are administered simultaneously.

37. The use according to any one of claims 33 to 35, wherein the half-life extended immune cell associated protein and the immunomodulatory drug are administered sequentially.

38. A pharmaceutical composition comprising a half-life extended immune cell associated protein for use in increasing the sensitivity of a subject in need of treatment comprising an immune checkpoint inhibitor, the half-life extended immune cell associated protein comprising: (i) a first domain (A) that specifically binds to human CD3; (ii) a second domain (B) that specifically binds to human serum albumin (HSA); and (iii) a third domain (C) that provides specificity for the target antigen; 13. A pharmaceutical composition comprising:

39. The immune checkpoint inhibitors include pembrolizumab (humanized antibody), pidilizumab (CT-011, monoclonal antibody that binds to DLL1 and PD-1), spartalizumab (PDR001, monoclonal antibody), nivolumab (BMS-936558, MDX-1106, human IgG4 monoclonal antibody), MEDI0680 (AMP-514, monoclonal antibody), cemiplimab (REGN2810, monoclonal antibody), dostallimab (TSR-042, monoclonal antibody), sasunlimab (PF-06801591, monoclonal antibody), and sasunlimab (PF-06801592, monoclonal antibody). monoclonal antibody), tislelizumab (BGB-A317, monoclonal antibody), BGB-108 (antibody), tislelizumab (BGB-A317, antibody), camrelizumab (INCSHR1210, SHR-1210), AMP-224, zimberelimab (AB122, GLS-010, WBP-3055, monoclonal antibody), AK-103 (HX-008, monoclonal antibody), AK-105 (anti-PD-1 antibody), CS1003 (monoclonal antibody), HLX10 (monoclonal antibody), retifanlimab (MGA-012, anti-PD-1monoclonal antibody), BI-754091 (antibody), balstilimab (AGEN2034, PD-1 antibody), toripalimab (JS-001, antibody), cetrelimab (JNJ-63723283, anti-PD-1 antibody), genolimuzumab (CBT-501, anti-PD-1 antibody), LZM009 (anti-PD-1 monoclonal antibody), progolimab (BCD-100, anti-PD-1 monoclonal antibody), Sym021 (antibody), ABBV-181 (antibody), BAT-1306 (antibody), JTX-4014, sintilimab (IBI-3 08), tebotelimab (MGD013, PD-1 / LAG-3 bispecific), MGD-019 (PD-1 / CTLA4 bispecific antibody), KN-046 (PD-1 / CTLA4 bispecific antibody), MEDI-5752 (CTLA4 / PD-1 bispecific antibody), RO7121661 (PD-1 / TIM-3 bispecific antibody), XmAb20717 (PD-1 / CTLA4 bispecific antibody), and AK-104 (CTLA4 / PD-1 bispecific antibody), and monoclonal antibody), pidilizumab (CT-011, monoclonal antibody that binds to DLL1 and PD-1), spartalizumab (PDR001, monoclonal antibody), nivolumab (BMS-936558, MDX-1106, human IgG4 monoclonal antibody), MEDI0680 (AMP-514, monoclonal antibody), cemiplimab (REGN2810, monoclonal antibody), dostallimab (TSR-042, monoclonal antibody), sasunlimab (PF-06801591, monoclonal antibody), tisleri izumab (BGB-A317, monoclonal antibody), BGB-108 (antibody), tislelizumab (BGB-A317, antibody), camrelizumab (INCSHR1210, SHR-1210), AMP-224, zimberelimab (AB122, GLS-010, WBP-3055, monoclonal antibody), AK-103 (HX-008, monoclonal antibody), AK-105 (anti-PD-1 antibody), CS1003 (monoclonal antibody), HLX10 (monoclonal antibody), retifanlimab (MGA-012, anti-PD-1monoclonal antibody), BI-754091 (antibody), balstilimab (AGEN2034, PD-1 antibody), toripalimab (JS-001, antibody), cetrelimab (JNJ-63723283, anti-PD-1 antibody), genolimuzumab (CBT-501, anti-PD-1 antibody), LZM009 (anti-PD-1 monoclonal antibody), progolimab (BCD-100, anti-PD-1 monoclonal antibody), Sym021 (antibody), ABBV-181 (antibody), BAT-1306 (antibody), JTX-4014, sintilimab (IBI 39. The pharmaceutical composition of claim 38, wherein the bispecific antibody is selected from the group consisting of tebotelimab (MGD013, PD-1 / LAG-3 bispecific), MGD-019 (PD-1 / CTLA4 bispecific antibody), KN-046 (PD-1 / CTLA4 bispecific antibody), MEDI-5752 (CTLA4 / PD-1 bispecific antibody), RO7121661 (PD-1 / TIM-3 bispecific antibody), XmAb20717 (PD-1 / CTLA4 bispecific antibody), and AK-104 (CTLA4 / PD-1 bispecific antibody).

40. The third domain is a member selected from the group consisting of DLL3 (delta-like ligand 3), 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), 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 A 1), CD22 (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′-nucleotidyltransferase, and CD80), CD90, CD100, CD110, CD120, CD130, CD140, CD150, CD160, CD170, CD180, CD200, CD210, CD220, CD230, CD240, CD250, CD300, CD310, CD320, CD330, CD340, CD350, CD350, CD360, CD370, CD380, CD390, CD400, CD410, CD420, CD430, CD440, CD440, CD450, CD460, CD470, CD480, CD490, CD500, CD510, CD520, CD530, CD540, CD550, CD560, CD570, CD580, CD590, CD600, CD610, CD620, CD630, CD640, CD650, CD650, CD660, CD670, CD680, CD690, CD700, CD700, CD710, CD720, CD730, CD740, CD750, CD750, CD760, CD770, CD780, CD790, CD810, CD enzyme), CD39 (ENTPD1, ​​ectonucleotide triphosphate diphosphohydrolase-1), CD74 (HLA class II histocompatibility complex gamma chain, HLA-DR antigen-associated invariant chain), CD79b (immunoglobulin-associated beta), CD80 (B7-1), CD86 (B7-2), CD123 (IL3RA, interleukin-3 receptor), CD133 (PROM1), CD137 (TNFRSF9, tumor necrosis factor receptor superfamily member 9, 4-1BB, ILA (induced by lymphocyte activation), 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 (ENPP 3), 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 (MUC1) and its splice variants (e.g., MUC1 / C, D, and Z), mucin 16 (MUC16), necdin (NDN), necdin cell adhesion molecule 4 (NECTIN4), SLIT, and NTR K-like family member 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 um), 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 family 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, sialyl Lewis adhesion molecule (sLe), ROR1, CD30, and Lewis Y antigen,and wherein the target antigen is selected from the group consisting of DLL3 (delta-like ligand 3), 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), EGFR (epidermal growth factor receptor), FLT3 (FMS-like tyrosine kinase 3), CD20 (B-lymphocyte antigen CD20, MS4A1, B1, Bp35, CVID5, LEU-16, MS4A2, S 7, transmembrane 4-domain A1), CD22 (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′-nucleotidyltransferase, and CD80), CD90, CD100, CD110, CD120, CD130, CD140, CD150, CD160, CD170, CD180, CD200, CD210, CD220, CD230, CD240, CD250, CD300, CD310, CD320, CD330, CD340, CD350, CD350, CD360, CD370, CD380, CD390, CD400, CD410, CD420, CD430, CD440, CD440, CD450, CD460, CD470, CD480, CD490, CD500, CD510, CD520, CD530, CD540, CD550, CD560, CD570, CD580, CD590, CD600, CD610, CD620, CD630, CD640, CD650, CD650, CD660, CD670, CD680, CD690, CD700, CD700, CD710, CD720, CD730, CD740, CD750, CD750, CD760, CD770, CD780, CD790, CD810, CD enzyme), CD39 (ENTPD1, ​​ectonucleotide triphosphate diphosphohydrolase-1), CD74 (HLA class II histocompatibility complex gamma chain, HLA-DR antigen-associated invariant chain), CD79b (immunoglobulin-associated beta), CD80 (B7-1), CD86 (B7-2), CD123 (IL3RA, interleukin-3 receptor), CD133 (PROM1), CD137 (TNFRSF9, tumor necrosis factor receptor superfamily member 9, 4-1BB, ILA (induced by lymphocyte activation), 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), 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 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), necdin cell adhesion molecule 4 (NECTIN4), SLIT and NTRK-like family member 6 (SLITRK6), promyelocytic leukemia (PML, TRIM19), protein tyrosine kinase 7 (inactive) (PTK7), CD40, CD50, CD60, CD80, CD90, CD100, CD120, CD140, CD160, CD180, CD190, CD200, CD210, CD220, CD230, CD240, CD250, CD260, CD300, CD310, CD320, CD410, CD310, CD420, CD310, CD410, CD320, CD420, CD410, CD190, CD250, CD310, CD410, CD190, CD260, CD310, CD410, CD190, CD260, CD310, CD410, CD190, CD270, CD280, CD310, CD310, CD410, CD410, CD190, CD280, CD310, CD410, CD410, CD190, CD290, CD41 ... 352 (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, LI V1), 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 40. The pharmaceutical composition of claim 38 or 39, wherein the antigen is selected from the group consisting of: 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, sialyl Lewis adhesion molecule (sLe), ROR1, CD30, and Lewis Y antigen.

41. 33. A kit comprising the pharmaceutical composition of any one of claims 1 to 31, or the composition of claim 32, and instructions for sequentially or simultaneously administering an immunomodulatory agent and a half-life extended immune cell associated protein to a subject in need thereof having cancer.

42. The cancers include 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, soft tissue sarcoma, gastric cancer, digestive / gastrointestinal cancer, 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, 42. The kit of claim 41, wherein the cancer is selected from the group consisting of: 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.

43. A pharmaceutical composition comprising an extended half-life immune cell-associated protein comprising the amino acid sequence of SEQ ID NO: 3461, in combination with pembrolizumab or atezolizumab for use in the treatment of cancer in a subject in need thereof.

44. A pharmaceutical composition comprising pembrolizumab in combination with an extended half-life immune cell-associated protein comprising the amino acid sequence of SEQ ID NO:3461 for use in treating cancer in a subject in need thereof.