41BB single domain antibodies and therapeutic compositions

Abstract

Provided herein are binding molecules that specifically bind 41BB, a member of the TNF receptor superfamily (TNFRSF). This disclosure further provides fusion proteins, including multivalent and / or multispecific polypeptides, that bind at least 41BB. The disclosure also provides nucleic acid molecules encoding the polypeptides and vectors and cells thereof. Also provided are methods of using the provided 41BB-binding molecules for treating diseases and conditions, such as cancer.

Description

Attorney Docket No. 01202-0066-00PCT 41BB SINGLE DOMAIN ANTIBODIES AND THERAPEUTIC COMPOSITIONSCross-Reference to Related Applications

[0001] This application claims the benefit of priority of US Provisional Application No. 63 / 790,937, filed April 18, 2025, which is incorporated by reference herein in its entirety for any purpose.Incorporation by Reference of Sequence Listing

[0002] The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on April 10, 2026, is named “01202-0066-00PCT.xml” and is 100,968 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.Field

[0003] This disclosure generally provides molecules that specifically bind 4 IBB, a member of the TNF receptor superfamily (TNFRSF). This disclosure further provides fusion proteins, including multivalent and / or multispecific polypeptides, that bind at least 4 IBB. The disclosure also provides nucleic acid molecules encoding the polypeptides and vectors and cells thereof. Also provided are methods of using the provided 41BB-binding molecules for treating diseases and conditions, such as cancer.Background

[0004] 41BB, (also known as 4-1BB; tumor necrosis factor receptor superfamily, member 9 (TNFRSF9); and / or CD137) is a potent co-stimulatory molecule of the TNFRSF that is expressed by activated T cells and NK cells. 4 IBB is of interest as a therapeutic target both as a co-stimulatory immune checkpoint molecule and as a target in cancer immunotherapy. Improved therapeutic molecules and agents targeting and modulating 4 IBB activity are needed. Provided herein are embodiments that meet such needs.Summary

[0005] The disclosure provides 41BB-binding molecules, comprising at least one heavy chain only variable domain (referred to herein as a “4 IBB VHH domain”, or simply a “4 IBB VHH”) that specifically bind at least 4 IBB. This disclosure further provides 41BB-binding molecules which are multivalent and / or multispecific molecules, that bind at least 4 IBB. The disclosure also provides nucleic acid molecules encoding the molecules and vectors and cells thereof, and methods of use and uses of the provided 41BB-binding molecules for treating diseases and conditions, such as cancer, immune and / orAttorney Docket No. 01202-0066-00PCT inflammatory disease. In some embodiments, the 41BB-binding molecule is a multivalent molecule. In some embodiments, the 41BB-binding molecule is a multispecific molecule. In some embodiments, the 41BB-binding molecule is a multivalent and multispecific molecule. Some additional embodiments are provided below.Embodiment 1. A 4 IBB-binding molecule, comprising at least one heavy chain only variable domain that specifically binds 4 IBB (4 IBB VHH), wherein at least one 4 IBB VHH comprises a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 47, 53, 60, 68, and 75; a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 51, 57, 64, 72, and 79; and a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:52, 58, 66, 73, 81.Embodiment 2. The 41BB-binding molecule of embodiment 1, wherein at least one 41BB VHH comprises CDR1, CDR2, and CDR3 sequences selected from: SEQ ID NOs: 47, 51 and 52; SEQ ID NOs: 53, 57, and 58; SEQ ID NOs: 60, 64, and 66; SEQ ID NOs: 68, 72, and 73.Embodiment 3. The 41BB-binding molecule of embodiment 1, wherein at least one 41BB VHH comprises CDR1, CDR2, and CDR3 sequences selected from: SEQ ID NOs: 47, 49, and 52; SEQ ID NOs: 47, 50, and 52; SEQ ID NOs: 53, 55, 58; SEQ ID NOs: 53, 56, 58; SEQ ID NOs: 60, 62, and 66; SEQ ID NOs: 60, 63, and 66; SEQ ID NOs: 68, 70, and 73; SEQ ID NOs: 68, 71, and 73.Embodiment 4. The 4 IBB-binding molecule of any one of embodiments 1-3, wherein at least one 4 IBB VHH comprises the sequence of any of SEQ ID NO: 44, 45, 98, and 99, or an amino acid sequence that exhibits at least an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 44, 45, 98, and 99.Embodiment 5. A 4 IBB-binding molecule, comprising at least one heavy chain only variable domain that specifically binds 4 IBB (4 IBB VHH), wherein at least one 4 IBB VHH comprises the sequence of any of SEQ ID NO: 44, 45, 98, and 99, or an amino acid sequence that exhibits at least an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 44, 45, 98, and 99.Embodiment 6. The 4 IBB-binding molecule of any one of embodiments 1-5, wherein the 4 IBB is a human 4 IBB.Embodiment 7. The 41BB-binding molecule of any one of embodiments 1-6, comprising two 4 IBB VHHs.Embodiment 8. The 41BB-binding molecule of any one of embodiments 1-7, comprising three 4 IBB VHHs.Embodiment 9. The 41BB-binding molecule of any one of embodiments 1-8, wherein the molecule comprises one or more additional binding domains that bind to a target other than 41BB.Attorney Docket No. 01202-0066-00PCT Embodiment 10. The 41BB-binding molecule of embodiment 9, wherein the additional binding domain, or independently each of the additional binding domains is a domain that binds to an antigen selected from the group consisting of: 1-92-LFA-3, 5T4, ADAM-9, Alpha-4 integrin, Alpha-V integrin, alpha4betal integrin, alpha4beta7 integrin, AGR2, Anti-Lewis-Y, Apelin J receptor, APRIL, B7-H3, B7-H4, B7-H6, BAFF, BCMA, BTLA, C5 complement, C-242, CA9, CAI 9-9, (Lewis a), Carbonic anhydrase 9, CD2, CD3, CD6, CD9, CDlla, CD16, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD138, CD166, CD172A, CD248, CDH6, CDH17, CEACAM5 (CEA), CEACAM6 (NCA-90), CLAUDIN-3, CLAUDIN-4, CLAUDIN-18.2, cMet, Collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, Endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, Folate receptor alpha (FRa), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GP Ilb / IIIa receptors, GPC3, Gpl30, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, Hyaluronidase, ICOS, IFNalpha, IFNbeta, IFNgamma, IgE, IgE Receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rbetal, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R (wsxl), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, Insulin Receptor, lagged Ligands, lagged 1, lagged 2, KISSl-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPDl, MCSP, Mesothelin, MRP4, MUC1, Mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, NKp46, Nicastrin, Notch Receptors, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, 0X40, PAR2, PDGF-AA, PDGF-BB, PDGFRalpha, PDGFRbeta, PD-1, PD-L1, PD-L2, Phosphatidyl-serine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, R0R1, SIGLEC-6, SLC44A4, Sphingosine 1 Phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFbeta, TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFalpha, TNFR, TNFR2, NFRS12A, TRAIL-R1, TRAIL-R2, Transferrin, Transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, and WISP-3.Embodiment 11. The 41BB-binding molecule of embodiment 9, wherein at least one of the one or more additional binding domains binds to an activating receptor on an immune cell.Embodiment 12. The 41BB-binding molecule of embodiment 11, wherein the immune cell is a T cell.Embodiment 13. The 41BB-binding molecule of embodiment 11 or 12, wherein the activating receptor is CD3, optionally wherein the activating receptor is CD3e.Embodiment 14. The 41BB-binding molecule of embodiment 11 or 12, wherein the activating receptor is a T cell receptor (“TCR”) comprising an alpha, beta, delta, or gamma chain, optionally wherein the activating receptor is a gamma / delta (y5) TCR.Attorney Docket No. 01202-0066-00PCT Embodiment 15. The 41BB-binding molecule of embodiment 11, wherein the immune cell is an NK cell.Embodiment 16. The 41BB-binding molecule of embodiment 11 or 15, wherein the activating receptor is CD 16a.Embodiment 17. The 41BB-binding molecule of embodiment 11 or 15, wherein the activating receptor is NKp46.Embodiment 18. The 41BB-binding molecule of any one of embodiments 11-17, wherein the molecule further comprises at least one additional binding domain for an antigen on the surface of a target cell (a “TCSA-binding domain”).Embodiment 19. The 41BB-binding molecule of embodiment 18, wherein the TCSA-binding domain, or independently each of the TCSA-binding domains, is a domain that binds antigen on the surface of a target cell is selected from the group consisting of: 1-92-LFA-3, 5T4, ADAM-9, Alpha-4 integrin, Alpha-V integrin, alpha4betal integrin, alpha4beta7 integrin, AGR2, Anti-Lewis-Y, Apelin J receptor, APRIL, B7-H3, B7-H4, B7-H6, BAFF, BCMA, BTLA, C5 complement, C-242, CA9, CA19-9, (Lewis a), Carbonic anhydrase 9, CD2, CD3, CD6, CD9, CDlla, CD16, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD138, CD166, CD172A, CD248, CDH6, CDH17, CEACAM5 (CEA), CEACAM6 (NCA-90), CLAUDIN-3, CLAUDIN-4, CLAUDIN-18.2, cMet, Collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, Endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, Folate receptor alpha (FRa), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GP Ilb / IIIa receptors, GPC3, Gpl30, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, Hyaluronidase, ICOS, IFNalpha, IFNbeta, IFNgamma, IgE, IgE Receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rbetal, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R (wsxl), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, Insulin Receptor, Jagged Ligands, Jagged 1, Jagged 2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPDl, MCSP, Mesothelin, MRP4, MUC1, Mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, NKp46, Nicastrin, Notch Receptors, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, 0X40, PAR2, PDGF-AA, PDGF-BB, PDGFRalpha, PDGFRbeta, PD-1, PD-L1, PD-L2, Phosphatidyl-serine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, R0R1, SIGLEC-6, SLC44A4, Sphingosine 1 Phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFbeta, TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFalpha, TNFR, TNFR2, NFRS12A, TRAIL-Rl, TRAIL-R2, Transferrin, Transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF,Atorney Docket No. 01202-0066-00PCT VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, and WISP-3.Embodiment 20. The 41BB-binding molecule of any one of embodiments 9-19, wherein at least one of the additional binding domains comprises an antibody or antigen-binding fragment thereof.Embodiment 21. The 41BB-binding molecule of embodiment 20, wherein each of the additional binding domains comprises an antibody or antigen-binding fragment thereof.Embodiment 22. The 41BB-binding molecule of embodiment 20 or 21, wherein the antibody or antigen-binding fragment thereof, or independently each antibody or antigen-binding fragment thereof, is an Fv, a disulfide-stabilized Fv (dsFv), scFv, a Fab, a single domain antibody (sdAb).Embodiment 23. The 41BB-binding molecule of any one of embodiments 11-22, wherein the molecule comprises one binding domain that binds to an activating receptor on an immune cell.Embodiment 24. The 41BB-binding molecule of any one of embodiments 11-22, wherein the molecule comprises two binding domains that binds to an activating receptor on an immune cell.Embodiment 25. The 41BB-binding molecule of embodiment 24, wherein the domains that binds to an activating receptor on an immune cell bind the same epitope of an activating receptor.Embodiment 26. The 41BB-binding molecule of embodiment 24, wherein the domains that binds to an activating receptor on an immune cell bind to different activating receptors, or different epitopes of the same activating receptor.Embodiment 27. The 41BB-binding molecule of any one of embodiments 18-26, wherein the molecule comprises one TCSA-binding domain.Embodiment 28. The 41BB-binding molecule of any one of embodiments 18-26, wherein the molecule comprises two or more TCSA-binding domains.Embodiment 29. The 41BB-binding molecule of embodiment 28, wherein at least two of the two or more TCSA-binding domains bind to the same epitope of a TCSA.Embodiment 30. The 41BB-binding molecule of embodiment 28, wherein at least two of the two or more TCSA-binding domains bind to different TCSA, or different epitopes of the same TCSA.Embodiment 31. The 41BB-binding molecule of any one of embodiments 1-30, wherein the molecule comprises an Fc region.Embodiment 32. The 41BB-binding molecule of any one of embodiments 9-31, wherein the molecule comprises an immunoglobulin Fc region that links the at least one 41BB VHH and the one or more additional binding domain.Embodiment 33. The 41BB-binding molecule of any one of embodiments 1-32, wherein the molecule comprises at least two polypeptide chains each comprising an Fc region polypeptide.Embodiment 34. The 41BB-binding molecule of any one of embodiments 31-33, wherein the Fc region is a homodimeric Fc region,Attorney Docket No. 01202-0066-00PCT Embodiment 35. The 41BB-binding molecule of any one of embodiments 31-34, wherein the Fc region comprises the sequence of any of SEQ ID NO: 1-4, 12-17, or 30-32, or an amino acid that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOS: 1-4, 12-17, or 30-32.Embodiment 36. The 41BB-binding molecule of any one of embodiments 31-33, wherein the Fc region is a heterodimeric Fc region.Embodiment 37. The 41BB-binding molecule of embodiment 36, where the heterodimeric Fc region comprises paired or complementary amino acid modifications.Embodiment 38. The 41BB-binding molecule of embodiment 36 or 37, wherein the heterodimeric Fc region comprises “knob” and “hole” modifications.Embodiment 39. The 41BB-binding molecule of any one of embodiments 36-38, wherein the heterodimeric Fc region comprises a first Fc polypeptide and a second Fc polypeptide, wherein the first Fc polypeptide comprises any of SEQ ID NOs: 18-23, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 18-23, and the second Fc polypeptide comprises any of SEQ ID NOs: 24-29 or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs:24-29.Embodiment 40. The 41BB-binding molecule of any one of embodiments 1-14 and 18-39, wherein the molecule comprises least one TCSA-binding domain, and at least one binding domain capable of binding to an activating receptor expressed on a T cell.Embodiment 41. The 41BB-binding molecule of embodiment 40, wherein the activating receptor is CD3.Embodiment 42. The 41BB-binding molecule of embodiment 41, wherein the CD3 is CD3e. Embodiment 43. The 41BB-binding molecule of embodiment 40, wherein the activating receptor is a TCR comprising an alpha, beta, delta, or gamma chain.Embodiment 44. The 41BB-binding molecule of embodiment 40, wherein the activating receptor is a gamma / delta (y5) TCR.Embodiment 45. The 41BB-binding molecule of any one of embodiments 1-11, and 15-39, wherein the molecule comprises least one TCSA-binding domain, and at least one binding domain capable of binding to an activating receptor expressed on an NK cell.Embodiment 46. The 41BB-binding molecule of any one of embodiments 1-14, and 18-44, wherein the molecule comprises a first component containing an immunoglobulin Fc region and a second component comprising a CD3 -binding region, wherein the first and second components are coupled by a linker, the Fc region is positioned N-terminal to the CD3-binding region; and one or both of the first and second components comprises (1) a TCSA-binding domain and (2) a 41BB-binding domain.Attorney Docket No. 01202-0066-00PCT Embodiment 47. The 41BB-binding molecule of any one of embodiments 1-46, wherein at least one 4 IBB VHH comprises the sequence of SEQ ID NO: 44.Embodiment 48. The 41BB-binding molecule of any one of embodiments 1-46, wherein at least one 4 IBB VHH comprises the sequence of SEQ ID NO: 45.Embodiment 49. The 41BB-binding molecule of any one of embodiments 1-46, wherein at least one 41BB VHH comprises the sequence of SEQ ID NO: 98.Embodiment 50. The 41BB-binding molecule of any one of embodiments 1-46, wherein at least one 4 IBB VHH comprises the sequence of SEQ ID NO: 99.Embodiment 51. A polynucleotide(s) encoding the 41BB-binding molecule of any one of embodiments 1-50.Embodiment 52. A vector(s) comprising the polynucleotide(s) of embodiment 51.Embodiment 53. A cell comprising the polynucleotide (s) of embodiment 51 or the vector of embodiment 52.Embodiment 54. A method of producing a 41BB-binding molecule, the method comprising introducing into a cell a polynucleotide or polynucleotides of embodiment 51 or a vector or vectors of embodiment 52 and culturing the cell under conditions to produce the 41BB-binding molecule.Embodiment 55. The method of embodiment 54, further comprising isolating or purifying the 41BB-binding molecule from the cell.Embodiment 56. A 41BB-binding molecule produced by the method of embodiment 54 or 55. Embodiment 57. A pharmaceutical composition comprising the 41BB-binding molecule of any of embodiments 1-50 and 56.Embodiment 58. The pharmaceutical composition of embodiment 57, comprising a pharmaceutically acceptable carrier.Embodiment 59. A method of increasing T cell proliferation or activation comprising contacting T cells with the 41BB-binding molecule of any of embodiments 1-14, 18-44, 47-50 and 56, or the pharmaceutical composition of embodiment 57 or 58.Embodiment 60. The method of embodiment 59, wherein the T cells are in vitro.Embodiment 61. The method of embodiment 59, wherein the T cells are in vivo.Embodiment 62. Use of the 41BB-binding molecule of any of embodiments 1-14, 18-50 and 56, or the pharmaceutical composition of claim 57 or 58 in the manufacture of a medicament for increasing T cell proliferation or activation, wherein the medicament is provided for contact with the T cells Embodiment 63. A method of increasing NK cell proliferation or activation comprising contacting NK cells with the 41BB-binding molecule of any of embodiments 1-11, 15-39, 45, 47-50 and 56, or the pharmaceutical composition of embodiment 57 or 58.Embodiment 64. The method of embodiment 63, wherein the NK cells are in vitro.Embodiment 65. The method of embodiment 63, wherein the NK cells are in vivo.Attorney Docket No. 01202-0066-00PCT Embodiment 66. Use of the 41BB-binding molecule of any of embodiments 1-11, 15-39, 45, 47-50 and 56, or the pharmaceutical composition of claim 57 or 58 in the manufacture of a medicament for increasing NK cell proliferation or activation, wherein the medicament is provided for contact with the NK cellsEmbodiment 67. A method of stimulating or inducing an immune response in a subject, the method comprising administering, to a subject in need thereof, the 41BB-binding molecule of any of embodiments 1-50 and 56, orthe pharmaceutical composition of embodiment 57 or 58.Embodiment 68. The method of embodiment 67, wherein the immune response is increased against a target cell.Embodiment 69. The method of embodiment 67 or 68, wherein the method treats a disease or condition in the subject.Embodiment 70. The method of embodiment 69, wherein the disease or condition is a tumor or a cancer, an immune disease or an inflammatory disease.Embodiment 71. The method of any one of embodiments 62-65, wherein the subject is a human. Embodiment 72. Use of the 41BB-binding molecule of any of embodiments 1-50 and 56, orthe pharmaceutical composition of embodiment 57 or 58, in the manufacture of a medicament for stimulating or inducing an immune response.Embodiment 73. The use of embodiment 72, wherein the immune response is increased against a target cell.Embodiment 74. The use of embodiment 72 or 73, wherein the use treats a disease or condition in the subject.Embodiment 75. The use of embodiment 74, wherein the disease or condition is a tumor or a cancer, an immune disorder or an inflammatory disease.Brief Description of the Drawings

[0006] FIGS. 1A-B show the in vivo pharmacokinetic (PK) profde of 4 IBB VHH Fc fusion protein comprising the previously described humanized 41BB VHH designated “hzRH3v5-l” over 504 hours. FIG. 1A shows the serum PK profde for each rat dosed with 30 mg / kg of cx7999 as determined by ELISA, while FIG. IB shows the serum PK profde for each rat dosed with 0.3 mg / kg of cx7999 as determined by ELISA.

[0007] FIG. 2 shows an alignment of the parental RH3p, and the optimized variants, the CDRs (AbM definition) are underlined. Residues identical to the parental VHH are indicated with a

[0008] FIGS. 3A-3C show the binding of 4 IBB VHH Fc fusion proteins comprising the parental (cxl04) or optimized VHHs (cxl 1318 and cxl 1317) targeting 41BB to bind cell surface 41BB. BindingAttorney Docket No. 01202-0066-00PCT was assessed by flow cytometry on HEK-293 freestyle cells overexpressing human 4 IBB (FIG. 2A), cynomolgus 4 IBB (FIG. 2B), and untransfected cells (FIG. 2C).

[0009] FIGS. 4A-4B show the in vivo pharmacokinetic (PK) profde of 4 IBB VHH Fc fusion proteins comprising the optimized 41BB VHHs designated “RH3v23” and “RHv24” (cxl 1318, and cxl 1317, respectively) over 168 hours. FIG. 3A shows the serum PK profde in mice dosed with 5 or 50 mg / kg (mpk) of cxl 1318 as determined by ELISA, while FIG. 3B shows the serum PK profde in mice dosed with 5 or 50 mg / kg of cxl 1317 as determined by ELISA.

[0010] FIG. 5 is a schematic of the basic components of 41BB-binding molecules of the present disclosure having constrained CD3 binding. The antigen binding domain(s) are positioned at the amino and / or carboxy termini. The Fc region, such as a heterodimeric Fc region, is positioned N-terminal to the CD3 binding region. The linker between the Fc region and the CD3 binding region can be a non-cleavable linker, or a cleavable linker.Detailed Description

[0011] Provided herein are molecules that specifically bind to 4 IBB, hereinafter also called 41BB-binding molecules and their use in targeting and / or modulating 4 IBB. A variety of 41BB-binding molecule formats are provided.

[0012] 4 IBB is a member of the TNF receptor superfamily that is predominately expressed on activated T-cells and NK cells and serves as a co-stimulatory molecule. Agonizing 4 IBB enhances T cell proliferation and survival, cytolytic activity and cytokine secretion (e.g., IL-2, TNFa and INFy). In mice, 4 IBB engagement has been shown to enhance anti-tumor immunity. (Croft, 2009, Nat Rev Immunol 9:271-285; Lynch, 2008, Immunol Rev. 22: 277-286). Importantly, tumor infiltrating cytotoxic T-cells (CTLs), have been shown to express 4 IBB and it is these 4 IBB positive CTLs that have the highest antitumor cytotoxic activity (Ye et al Clin Cancer Res; 20(1): 44-55). 41BB has been found to be upregulated in cancer cell lines, and targeted stimulation of 4 IBB is being examined as an anti -cancer therapy.

[0013] The ligand for 4 IBB, 41BBL, naturally forms a homotrimer and thereby suggests that signaling is mediated by higher order clustering of 4 IBB. Interest in exploiting 4 IBB signaling for antitumor immunotherapy has prompted the development of therapeutic 4 IBB antibodies. However, the capacity of bivalent 4 IBB antibodies to induce signaling is weak in absence of an exogenous clustering event. This can be achieved to some degree through the interaction with Fcy-receptors (FcyRs), yet this can also lead to depletion of the 41BB-expressing cell through effector mechanisms (e.g. ADCC and ADCP). Therefore, there exists a need for optimized 41BB-binding molecules that may be used as agonist molecules capable of mediating signaling in the absence of an exogenous crosslinking agent or FcyR interaction. The 41BB-binding molecules of the present disclosure have improved binding and pharmacokinetics and are capable of mediating potent 4 IBB signaling 1) without any additionalAttorney Docket No. 01202-0066-00PCT interactions when formatted as a multivalent fusion protein or 2) conditionally when engaged with at least a second antigen interaction when formatted as a multispecific fusion protein. The fusion proteins of the present disclosure are capable of standalone (multivalent) or conditional (multispecific) costimulatory activity on T-cell and NK cells.

[0014] Provided herein are 41BB-binding molecules that comprise at least one VHH that specifically binds to 4 IBB. In some embodiments, the VHH binds human 4 IBB. In some of any of the provided embodiments, the VHH binds 4 IBB having the sequence set forth in SEQ ID NO: 41 or a mature form thereof lacking the signal sequence.

[0015] In some embodiments, the provided binding molecules comprise at least one VHH that binds 4 IBB. In some embodiments, a 41BB-binding molecule provided herein comprises one, two, three, four, five, six, seven, or eight VHHs that each individually binds 41BB. In some embodiments, a 41BB-binding molecule provided herein comprises one, two, three, or four VHHs that bind 41BB. In some embodiments, the 41BB-binding molecules are monospecific. In such embodiments, the 41BB-binding molecule may incorporate multiple copies of the same VHH. In some embodiments, the 41BB-binding molecules may incorporate multiple copies of a VHH that are different but that recognize the same epitope on 4 IBB.

[0016] In some embodiments, the 41BB-binding molecules are multispecific. For example, provided 41BB-binding molecules include molecules that may comprise at least one VHH that binds 4 IBB and one or more additional binding domains, that bind one or more antigens other than 4 IBB. In some embodiments, such multispecific 41BB-binding molecules exhibit multispecific binding. In some embodiments, the binding to the additional antigen is capable of providing the additional crosslinking function and 4 IBB activation can be achieved with only one or two 41BB-binding domains. In some embodiments, 41BB signaling is enhanced and focused by the presence of the additional antigen. In some embodiments, such multispecific molecules exhibit greatly enhanced 41BB-dependent signaling when the multispecific molecule is bound to a cell expressing the additional antigen.

[0017] In some embodiments, a 41BB-binding molecule comprises at least one VHH that binds 4 IBB and an Fc region. In some embodiments, a 41BB-binding molecule provided herein comprises one, two, three, or four VHHs that bind 4 IBB and an Fc region. In some embodiments, an Fc region mediates dimerization of the 41BB-binding molecule at physiological conditions such that a dimer is formed that doubles the number of 41BB-binding sites. For example, a 41BB-binding molecule comprising three VHH that bind 4 IBB and an Fc region is trivalent as a monomer, but at physiological conditions, the Fc region may mediate dimerization, such that the 41BB-binding molecule exists as a hexavalent dimer under such conditions.

[0018] The 41BB-binding molecules can be formatted in a number of formats, including, but not limited to any as described in Section III below.Attorney Docket No. 01202-0066-00PCT

[0019] In some embodiments, the provided 41BB-binding molecules can be used to modulate an immune response in a subject, which, in some embodiments, treats a disease or disorder, such as a cancer, or an immune and / or inflammatory disease or disorder, in the subject.

[0020] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0021] The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (2003)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc ): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987));Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney), ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G.Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds I),' Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRE Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer:Principles and Practice of Oncology (V. T. DeVita et al., eds., J.B. Lippincott Company, 1993); and updated versions thereof.

[0022] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.Attorney Docket No. 01202-0066-00PCT I. DEFINITIONS

[0023] Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context or expressly indicated, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art. For any conflict in definitions between various sources or references, the definition provided herein will control.

[0024] It is understood that embodiments of the invention described herein include “consisting” and / or “consisting essentially of’ embodiments. As used herein, the singular form “a”, “an”, and “the” includes plural references unless indicated otherwise. Use of the term “or” herein is not meant to imply that alternatives are mutually exclusive.

[0025] In this application, the use of “or” means “and / or” unless expressly stated or understood by one skilled in the art. In the context of a multiple dependent claim, the use of “or” refers back to more than one preceding independent or dependent claim.

[0026] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.

[0027] In this disclosure, “comprises,” “comprising,” “containing,” “having,” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like; the terms “consisting essentially of’ or “consists essentially” likewise have the meaning ascribed in U.S. Patent law and these terms are open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited are not changed by the presence of more than that which is recited, but excludes prior art embodiments.

[0028] The terms “nucleic acid molecule”, “nucleic acid” “and “polynucleotide” may be used interchangeably, and refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and / or non-natural nucleotides, and include, but are not limited to, DNA, RNA, and PNA. “Nucleic acid sequence” refers to the linear sequence of nucleotides comprised in the nucleic acid molecule or polynucleotide.

[0029] The term “isolated polynucleotide” as used herein shall mean a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin (1) is not associated with all or a portion of a polynucleotide found in nature, (2) is operably linked to a polynucleotide that it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence.Attorney Docket No. 01202-0066-00PCT

[0030] The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length and macromolecules comprising one or more polymers of amino acid residues, a multimer (e.g., dimer, trimer, or tetramer) formed by a plurality of the same or different polymers of amino acids. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins, and fragments thereof are encompassed by the definition. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like.Furthermore, for purposes of the present disclosure, a “polypeptide” refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

[0031] “4 IBB” as used herein refers to any native, mature polypeptide that results from processing of an 4 IBB precursor in a cell. The use of the term “4 IBB” is intended to cover any variation thereof, such as, by way of non-limiting example, 41-BB and / or 4-1BB, and all variations are used herein interchangeably. The term includes 4 IBB from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus or rhesus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated. The term also includes naturally-occurring variants of 4 IBB, such as splice variants or allelic variants. A nonlimiting representative human 41BB amino acid sequence is shown, e.g., in NCBI Reference Sequence: NP_001552.2. See, the sequence set forth in SEQ ID NO: 41 or a mature form thereof lacking the signal sequence.

[0032] As used herein a “41BB-binding molecule” comprising at least one heavy chain only variable domain that specifically binds 4 IBB and encompasses fusion proteins of the disclosure, including, but not limited to, multivalent fusion proteins, monospecific fusion proteins, and multispecific fusion proteins. 41BB-binding molecules may also be referred to herein as “41BB-binding polypeptides” or “41BB-binding fusions” or “41BB-binding proteins” or “41BB-binding fusion polypeptides” or “41BB-binding fusion proteins.” As used herein, the term “fusion protein” or “fusion polypeptide” or “41BB-binding fusion protein” or “41BB-binding fusion polypeptide,” unless otherwise specifically denoted, refers to any fusion protein embodiment of the disclosure, including, but not limited to, multivalent fusion proteins, multispecific fusion proteins, or multivalent and multispecific fusion proteins.

[0033] The term “isolated” as used herein refers to a molecule that has been separated from at least some of the components with which it is typically found in nature or produced. For example, a molecule is referred to as “isolated” when it is separated from at least some of the components of the cell in which it was produced. Where a molecule is secreted by a cell after expression, physically separating theAttorney Docket No. 01202-0066-00PCT supernatant containing the molecule from the cell that produced it is considered to be “isolating” the molecule . Similarly, a polynucleotide is referred to as “isolated” when it is not part of the larger polynucleotide (such as, for example, genomic DNA or mitochondrial DNA, in the case of a DNA polynucleotide) in which it is typically found in nature, or is separated from at least some of the components of the cell in which it was produced, for example, in the case of an RNA polynucleotide. Thus, a DNA polynucleotide that is contained in a vector inside a host cell may be referred to as “isolated”.

[0034] As used herein, “substantially pure” means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, for example, in some embodiments, more than about 85%, 90%, 95%, and 99%. In some embodiments, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.

[0035] The term “operably linked” as used herein refers to positions of components so described are in a relationship permitting them to function in their intended manner. A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0036] The term “specifically binds” to an antigen or epitope is a term that is well understood in the art, and methods to determine such specific binding are also well known in the art. A molecule is said to exhibit “specific binding” or “preferential binding” if it reacts or associates more frequently, more rapidly, with greater duration and / or with greater affinity with a particular cell or substance than it does with alternative cells or substances. A single-domain antibody (sdAb) or VHH-containing molecule “specifically binds” or “preferentially binds” to a target if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances. For example, a sdAb or VHH-containing molecule that specifically or preferentially binds to a 4 IBB epitope is a sdAb or VHH-containing molecule that binds this epitope with greater affinity, avidity, more readily, and / or with greater duration than it binds to other 4 IBB epitopes or non-41BB epitopes. It is also understood by reading this definition that; for example, a sdAb or VHH-containing molecule that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding. “Specificity” refers to the ability of a binding protein to selectively bind an antigen.Attorney Docket No. 01202-0066-00PCT

[0037] As used herein, the term “epitope” refers to a site on a target molecule (for example, an antigen, such as a protein, nucleic acid, carbohydrate or lipid) to which an antigen-binding molecule (e.g., an IgG based antibody), or an antigen-binding fragment there of (for example, a sdAb or VHH-containing molecule) binds. Epitopes often include a chemically active surface grouping of molecules such as amino acids, polypeptides or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be formed both from contiguous and / or juxtaposed noncontiguous residues (for example, amino acids, nucleotides, sugars, lipid moiety) of the target molecule. Epitopes formed from contiguous residues (for example, amino acids, nucleotides, sugars, lipid moiety) typically are retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding typically are lost on treatment with denaturing solvents. An epitope may include but is not limited to at least 3, at least 5 or 8-10 residues (for example, amino acids or nucleotides). In some embodiments, an epitope is less than 20 residues (for example, amino acids or nucleotides) in length, less than 15 residues or less than 12 residues. Two antibodies may bind the same epitope within an antigen if they exhibit competitive binding for the antigen. In some embodiments, an epitope can be identified by a certain minimal distance to a CDR residue on the antigen-binding molecule. In some embodiments, an epitope can be identified by the above distance, and further limited to those residues involved in a bond (for example, a hydrogen bond) between a residue of the antigen-binding molecule and an antigen residue. An epitope can be identified by various scans as well, for example an alanine or arginine scan can indicate one or more residues that the antigen-binding molecule can interact with. Unless explicitly denoted, a set of residues as an epitope does not exclude other residues from being part of the epitope for a particular antigen-binding molecule. Rather, the presence of such a set designates a minimal series (or set of species) of epitopes. Thus, in some embodiments, a set of residues identified as an epitope designates a minimal epitope of relevance for the antigen, rather than an exclusive list of residues for an epitope on an antigen.

[0038] A “nonlinear epitope” or “conformational epitope” comprises noncontiguous polypeptides, amino acids and / or sugars within the antigenic protein to which an antigen-binding molecule specific to the epitope binds. In some embodiments, at least one of the residues will be noncontiguous with the other noted residues of the epitope; however, one or more of the residues can also be contiguous with the other residues.

[0039] A “linear epitope” comprises contiguous polypeptides, amino acids and / or sugars within the antigenic protein to which an antigen-binding molecule specific to the epitope binds. It is noted that, in some embodiments, not every one of the residues within the linear epitope need be directly bound (or involved in a bond) by the antigen-binding molecule. In some embodiments, linear epitopes can be from immunizations with a peptide that effectively consisted of the sequence of the linear epitope, or from structural sections of a protein that are relatively isolated from the remainder of the protein (such that the antigen-binding molecule can interact, at least primarily), just with that sequence section.Attorney Docket No. 01202-0066-00PCT

[0040] The terms “antibody” and “antigen-binding molecule” are used interchangeably in the broadest sense and encompass various polypeptides that comprise antibody-like antigen-binding domains, including but not limited to conventional antibodies (typically comprising at least one heavy chain and at least one light chain), single-domain antibodies (sdAbs, comprising just one chain, which is typically similar to a heavy chain), VHH-containing molecules (molecules comprising at least one heavy chain only antibody variable domain, or VHH), and fragments of any of the foregoing so long as they exhibit the desired antigen-binding activity. In some embodiments, an antibody comprises a dimerization domain. Such dimerization domains include, but are not limited to, heavy chain constant domains (comprising CHI, hinge, CH2, and CH3, where CHI typically pairs with a light chain constant domain, CL, while the hinge mediates dimerization) and Fc regions (comprising hinge, CH2, and CH3, where the hinge mediates dimerization).

[0041] The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies of various species such as camelid (including llama), shark, mouse, human, cynomolgus monkey, etc.

[0042] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable regions of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen -binding specificity, e.g. a single domain antibody, such as a VHH.Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0043] An “antibody fragment” or “antigen-binding fragment” or “antigen-binding domain” refers to a a portion of an conventional or intact antibody containing at least a variable region that binds an antigen. Examples of antibody fragments include but are not limited to Fv, single chain Fvs (sdFvs), Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; and single-domain antibodies comprising only the VH region (VHH).

[0044] As used herein, “monovalent” with reference to a binding molecule refers to binding molecules that have a single antigen recognition site that is specific for an epitope on a target antigen. Examples of monovalent binding molecules include, for example, a monovalent antibody fragment, a proteinaceous binding molecule with antibody-like binding properties or an MHC molecule. Examples of monovalent antibody fragments include, but are not limited to, a VHH, a Fab fragment, an Fv fragment, and a single- chain Fv fragment (scFv).

[0045] As used herein, “multivalent” with reference to a binding molecule refers to binding molecules comprising multiple antigen recognition sites. Each antigen recognition sites may recognizeAttorney Docket No. 01202-0066-00PCT the same epitope (“monospecific”) or different epitopes (“multispecific”). Examples of multivalent binding molecules include, for example, a bispecific antibody, or a proteinaceous binding molecule comprising multiple antigen-binding fragments (e.g., multiple VHHs).

[0046] The terms “single domain antibody”, “sdAb,” “VHH” are used interchangeably herein to refer to an antibody having a single monomeric domain antigen binding / recognition domain. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, alpaca, vicuna, guanaco, shark, goat, rabbit, and / or bovine. In some embodiments, a single domain antibody as used herein is a naturally occurring single domain antibody known as heavy chain antibody devoid of light chains. For clarity reasons, this variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH to distinguish it from the conventional VH of four chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca, vicuna and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; such VHHs are within the scope of the disclosure. A VHH domain is an antibody fragment that is a single monomeric variable antibody domain that is able to bind selectively to a specific antigen. With a molecular weight of only 12-15 kDa, VHH domains (also called single-domain antibodies) are much smaller than common antibodies (150-160 kDa) which are composed of two heavy protein chains and two light chains, and even smaller than Fab fragments (~50 kDa, one light chain and half a heavy chain) and single-chain variable fragments (~25 kDa, two variable domains, one from a light and one from a heavy chain).

[0047] In some embodiments, a VHH comprises three CDRs and four framework regions, designated FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, a VHH may be truncated at the N-terminus or C-terminus such that it comprises only a partial FR1 and / or FR4, or lacks one or both of those framework regions, so long as the VHH substantially maintains antigen binding and specificity.

[0048] The term “VHH-containing molecule” refers to a molecule that comprises at least one VHH domain. In some embodiments, a VHH molecule comprises two, three, or four or more VHH domains, wherein each VHH domain may be the same or different. In some embodiments, a VHH-containing molecule comprises an Fc region. In some such embodiments, the VHH molecule may form a dimer. Nonlimiting structures of VHH-containing molecules include VHHi-Fc, VHHi-VHFE-Fc. and VHHi-VHH2-VHH3-FC, wherein VHHi, VHH2, and VHH3 may be the same or different. In some embodiments of such structures, one VHH may be connected to another VHH by a linker, or one VHH may be connected to the Fc by a linker. In some such embodiments, the linker comprises 1-20 amino acids, preferably 1-20 amino acids predominantly composed of glycine and, optionally, serine. In someAtorney Docket No. 01202-0066-00PCT embodiments, when a VHH-containing molecule comprises an Fc, it forms a dimer. Thus, the structure VHH1-VHH2-FC, if it forms a dimer, is considered to be tetravalent (i.e., the dimer has four VHH domains). Similarly, the structure VHH1-VHH2-VHH3-FC, if it forms a dimer, is considered to be hexavalent (i.e., the dimer has six VHH domains).

[0049] As used herein, a 41BB-binding molecule is a molecule that specifically binds 4 IBB.Typically, a 41BB-binding molecule herein is a VHH-containing molecule containing at least one VHH domain that binds 41BB. A 41BB-binding molecule includes conjugates, including fusion proteins. A 41BB-binding molecule includes fusion proteins, including those containing an Fc region. In some embodiments, a 41BB-binding molecule comprises a single polypeptide chain. In some embodiments, a 41BB-binding molecule includes at least two polypeptide chains. In some embodiments, a 41BB-binding molecule contains two, three, or four or more VHH domains that each specifically bind to 4 IBB, wherein each VHH domain may be the same or different. In some embodiments, a 41BB-binding molecule is multivalent. In some embodiments, a 41BB-binding molecule is multispecific. In some cases, a 41BB-binding molecule may contain one or more additional domains that bind to one or more further or additional antigens other than 4 IBB.

[0050] The term “monoclonal antibody” refers to an antibody (including an sdAb or VHH-containing polypeptide) of a substantially homogeneous population of antibodies, that is, the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Thus, a sample of monoclonal antibodies can bind to the same epitope on the antigen. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or may be made by recombinant DNA methods such as described in U.S. Pat. No.4,816,567. The monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554, for example.

[0051] The term “CDR” denotes a complementarity determining region as defined by at least one manner of identification to one of skill in the art. The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed.Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol.262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J.Atorney Docket No. 01202-0066-00PCT Mol. Biol. 262, 732-745.” (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 Jan;27(l):55-77 (“IMGT” numbering scheme); Honegger A and Pltickthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun 8;309(3):657-70, (“Aho” numbering scheme); and Martin et al., “Modeling antibody hypervariable loops: a combined algorithm,” PNAS, 1989, 86(23):9268-9272, (“AbM” numbering scheme).

[0052] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion leters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. The AbM scheme is a compromise between Kabat and Chothia definitions based on that used by Oxford Molecular’s AbM antibody modeling software.

[0053] In some embodiments, CDRs can be defined in accordance with any of the Chothia numbering schemes, the Kabat numbering scheme, a combination of Kabat and Chothia, the AbM definition, and / or the contact definition. A VHH comprises three CDRs, designated CDR1, CDR2, and CDR3. Table 1, below, lists exemplary position boundaries of CDR-H1, CDR-H2, CDR-H3 as identified by Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1, residue numbering is listed using both the Kabat and Chothia numbering schemes. FRs are located between CDRs, for example, with FR-H1 located before CDR-H1, FR-H2 located between CDR-H1 and CDR-H2, FR-H3 located between CDR-H2 and CDR-H3 and so forth. It is noted that because the shown Kabat numbering scheme places insertions at H35A and H35B, the end of the Chothia CDR-H1 loop when numbered using the shown Kabat numbering convention varies between H32 and H34, depending on the length of the loop.Table 1. Boundaries of CDRs according to various numbering schemes.CDR Kabat Chothia AbM Contact CDR-H1(Kabat Numbering1) H31-H35B H26— H32..34 H26-H35B H30-H35B CDR-H1(Chothia Numbering2) H31-H35 H26-H32 H26-H35 H30-H35 CDR-H2 H50-H65 H52-H56 H50-H58 H47-H58 CDR-H3 H95-HI02 H95-HI02 H95-HI02 H93-HI0II - Kabat et al. (1991), “Sequences of Proteins of mmunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD2 - Al-Lazikani et al., (1997) JMB 273,927-948Attorney Docket No. 01202-0066-00PCT

[0054] Thus, unless otherwise specified, a “CDR” or “complementary determining region,” or individual specified CDRs (e.g., CDR-H1, CDR-H2, CDR-H3), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) complementary determining region as defined by any of the aforementioned schemes. For example, where it is stated that a particular CDR (e.g., a CDR-H3) contains the amino acid sequence of a corresponding CDR in a given VHH amino acid sequence, it is understood that such a CDR has a sequence of the corresponding CDR (e.g., CDR-H3) within the VHH, as defined by any of the aforementioned schemes. In some embodiments, specific CDR sequences are specified. Exemplary CDR sequences of provided antibodies are described using various numbering schemes (see e.g. Table 1), although it is understood that a provided antibody can include CDRs as described according to any of the other aforementioned numbering schemes or other numbering schemes known to a skilled artisan.

[0055] As used herein, “conjugate,” “conjugation” or grammatical variations thereof refers the joining or linking together of two or more compounds resulting in the formation of another compound, by any joining or linking methods known in the art. It can also refer to a compound which is generated by the joining or linking together two or more compounds. For example, a VHH domain linked directly or indirectly to one or more agent, or polypeptide is an exemplary conjugate. Such conjugates include fusion proteins, those produced by chemical conjugates and those produced by any other methods.

[0056] The term “agent” is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, and / or an extract made from biological materials. The term “pharmaceutical agent or drug” as used herein refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

[0057] The term “antineoplastic agent” is used herein to refer to agents that have the functional property of inhibiting a development or progression of a neoplasm in a human, particularly a malignant (cancerous) lesion, such as a carcinoma, sarcoma, lymphoma, or leukemia. Inhibition of metastasis is frequently a property of antineoplastic agents. Exemplary classes of such agents in include, but are not limited to, chemotherapeutic agents, anti -cancer biologies (such as cytokines, receptor extracellular domain-Fc fusions, and antibodies), radiation therapy, CAR-T therapy, therapeutic oligonucleotides (such as antisense oligonucleotides and siRNAs) and oncolytic viruses.

[0058] The term “heavy chain constant region” as used herein refers to a region comprising at least three heavy chain constant domains, CHI, hinge, CH2, and CH3. Of course, non-fimction-altering deletions and alterations within the domains are encompassed within the scope of the term “heavy chain constant region,” unless designated otherwise. Nonlimiting exemplary heavy chain constant regions include y, 5, and a. Nonlimiting exemplary heavy chain constant regions also include 8 and p. Each heavy constant region corresponds to an antibody isotype. For example, an antibody comprising a y constant region is an IgG antibody, an antibody comprising a 5 constant region is an IgD antibody, and an antibody comprising an a constant region is an IgA antibody. Further, an antibody comprising a pAtorney Docket No. 01202-0066-00PCT constant region is an IgM antibody, and an antibody comprising an a constant region is an IgE antibody. Certain isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgGl (comprising a yi constant region), IgG2 (comprising a 72 constant region), IgG3 (comprising a 73 constant region), and IgG4 (comprising a 74 constant region) antibodies; IgA antibodies include, but are not limited to, IgAl (comprising an ai constant region) and IgA2 (comprising an 0.3 constant region) antibodies; and IgM antibodies include, but are not limited to, IgMl and IgM2.

[0059] A “Fc region” as used herein refers to a portion of a heavy chain constant region comprising CH2 and CH3. In some embodiments, an Fc region comprises a hinge (or portion thereof), CH2, and CH3. In some embodiments, when an Fc region comprises a hinge (or portion thereof), the hinge and / or CH3 mediates dimerization between two Fc-containing polypeptides. An Fc region may be of any antibody heavy chain constant region isotype discussed herein. In some embodiments, an Fc region is an IgGl, IgG2, IgG3, or IgG4.

[0060] A “functional Fc region” possesses an “effector function” of a native sequence Fc region. Exemplary “effector functions” include Fc receptor binding; Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (for example B-cell receptor); and B-cell activation, etc. Such effector functions generally require the Fc region to be combined with a binding domain (for example, an antibody variable domain) and can be assessed using various assays. Nonlimiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'lAcad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I etal., Proc. Nat'lAcad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96™ non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). Clq binding assays may also be carried out to confirm that the multispecific polypeptide construct or cleaved components thereof is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, M. S. et al., Blood 101: 1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12): 1759-1769 (2006))Atorney Docket No. 01202-0066-00PCT

[0061] A “native sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include a native sequence human IgGl Fc region (e.g., non-A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region as well as naturally occurring variants thereof. Nonlimiting representative human IgGl, IgG2, IgG3 and IgG4 amino acid sequences are set forth in SEQ ID NOs: 1-4, and representative human hinge amino acid sequences are set forth in SEQ ID NOs: 5, 7-10.

[0062] A “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification. In some embodiments, a “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification, yet retains at least one effector function of the native sequence Fc region, or has at least one effector function reduced or eliminated. In some embodiments, a variant Fc region comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification and has an extended serum half-life. In some embodiments, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent Fc region, for example, from about one to about ten amino acid substitutions, and preferably, from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent Fc region. In some embodiments, the variant Fc region herein will possess at least about 80% sequence identity with a native sequence Fc region and / or with an Fc region of a parent Fc region, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity therewith.

[0063] In general, the numbering of the residues in an immunoglobulin heavy chain or portion thereof, such as an Fc region, is by EU numbering system. “EU Numbering” refers to the residue numbering of the human IgGl EU antibody according to the most recently updated IMGT Scientific Chart (IMGT®, the international ImMunoGeneTics information system®, htp: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html (created: 17 May 2001, last updated: 10 Jan 2013) and the EU index as reported in Kabat, E.A. et al. Sequences of Proteins of Immunological interest. 5th ed. US Department of Health and Human Services, NIH publication No. 91-3242 (1991).

[0064] An immunoglobulin Fc fusion (“Fc-fusion”), such as VHH-Fc, is a molecule comprising one or more VHH domains operably linked to an Fc region of an immunoglobulin. An immunoglobulin Fc region may be linked indirectly or directly to one or more VHH domains. Various linkers are known in the art and can optionally be used to link an Fc to a fusion partner to generate an Fc-fusion. In some such embodiments, the linker comprises 1-20 amino acids, preferably 1-20 amino acids predominantly composed of glycine and, optionally, serine. Fc-fusions of identical species can be dimerized to form Fc-Attorney Docket No. 01202-0066-00PCT fusion homodimers, or using non-identical species to form Fc-fusion heterodimers. In some embodiments, the Fc is a mammalian Fc such as human Fc.

[0065] ‘ ‘Fc receptor” or “FcR” describes a receptor that binds to the Fc region of an antibody. In some embodiments, an FcyR is a native human FcR. In some embodiments, an FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of those receptors. FcyRII receptors include FcyRIIA (an “activating receptor”) and FcyRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, for example, Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein. For example, the term “Fc receptor” or “FcR” also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and regulation of homeostasis of immunoglobulins. Methods of measuring binding to FcRn are known (see, for example, Ghetie and Ward, Immunol. Today 18( 12): 592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton etal., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004 / 92219 (Hinton etal.).

[0066] An “acceptor human framework” as used herein is a framework comprising the amino acid sequence of a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as discussed herein. An acceptor human framework derived from a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence thereof, or it can contain amino acid sequence changes. In some embodiments, the number of amino acid changes are fewer than 10, or fewer than 9, or fewer than 8, or fewer than 7, or fewer than 6, or fewer than 5, or fewer than 4, or fewer than 3, across all of the human frameworks in a single antigen binding domain, such as a VHH.

[0067] As used herein, a “chimeric antigen receptor” or “CAR” refers to an engineered receptor, which introduces an antigen specificity, via an antigen binding domain, onto cells to which it is engineered (for example T cells such as naive T cells, central memory T cells, effector memory T cells or combination thereof) thus combining the antigen binding properties of the antigen binding domain with the T cell activity (e.g. lytic capacity and self renewal) of T cells. A CAR typically includes an extracellular antigen-binding domain (ectodomain), a transmembrane domain and an intracellular signaling domain. The intracellular signaling domain generally contains at least one ITAM signaling domain, e.g. derived from CD3zeta, and optionally at least one costimulatory signaling domain, e.g.Attorney Docket No. 01202-0066-00PCT derived from CD28 or 4-1BB. In a CAR provided herein, a VHH domain forms the antigen binding domain and is located at the extracellular side when expressed in a cell.

[0068] “Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (for example, an antibody or VHH-containing molecule) and its binding partner (for example, an antigen). The affinity or the apparent affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD) or therespectively, wherein a smaller KDrepresents a greater affinity. The affinity may be determined by measuring the rates of antigen-binding site / antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions. Thus, both the “on rate constant” (kon) and the “off rate constant” (koff) can be determined by calculation of the concentrations and the actual rates of association and dissociation. (See Nature 361: 186-87 (1993)). The ratio of kOfr / konenables the cancellation of all parameters not related to affinity, and is equal to the dissociation constant Kd. (See, generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). Affinity can be measured by common methods known in the art (such as, for example, ELISA KD, KinExA, flow cytometry, and / or surface plasmon resonance devices), including those described herein. Such methods include, but are not limited to, methods involving BIAcore®, Octet®, or flow cytometry.

[0069] The term “KD”, as used herein, refers to the equilibrium dissociation constant of an antigenbinding molecule / antigen interaction. When the term “KD” is used herein, it includes KD and

[0070] In some embodiments, the KD of the antigen-binding molecule is measured by flow cytometry using an antigen-expressing cell line and fitting the mean fluorescence measured at each antibody concentration to a non-linear one-site binding equation (Prism Software graphpad). In some such embodiments, the KD is KD

[0071] The term “biological activity” refers to any one or more biological properties of a molecule (whether present naturally as found in vivo, or provided or enabled by recombinant means). Biological properties include, but are not limited to, binding a ligand, inducing or increasing cell proliferation (such as T cell proliferation), and inducing or increasing expression of cytokines.

[0072] An “affinity matured” VHH-containing molecule refers to a VHH-containing molecule with one or more alterations in one or more CDRs compared to a parent VHH-containing molecule that does not possess such alterations, such alterations resulting in an improvement in the affinity of the VHH-containing molecule for antigen.

[0073] A “humanized VHH” as used herein refers to a VHH in which one or more framework regions have been substantially replaced with human framework regions. In some instances, certain framework region (FR) residues of the human immunoglobulin are replaced by corresponding nonhuman residues. Furthermore, the humanized VHH can comprise residues that are found neither in the original VHH nor in the human framework sequences, but are included to further refine and optimizeAtorney Docket No. 01202-0066-00PCT VHH or VHH-containing molecule performance. In some embodiments, a humanized VHH-containing molecule comprises a human Fc region. As will be appreciated, a humanized sequence can be identified by its primary sequence and does not necessarily denote the process by which the antibody was created. Humanized antibodies and methods of making them are reviewed, for example, in Almagro and Fransson, (2008) Front. Biosci. 13: 1619-1633, and are further described, for example, in Riechmann et al., (1988) Nature 332:323-329; Queen etal., (1989) roc. Natl Acad. Sci. USA 86: 10029-10033; US Patent Nos. 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., (2005) Methods 36:25-34; Padlan, (1991) Mol. Immunol. 28:489-498 (describing “resurfacing”); DallAcqua et al., (2005) Methods 36:43-60 (describing “FR shuffling”); and Osbourn etal., (2005) Methods 36:61-68 and Klimka etal., (2000) Br. J. Cancer, 83:252-260 (describing the “guided selection” approach to FR shuffling). Human framework regions that can be used for humanization include but are not limited to: framework regions selected using the “best-fit” method (see, for example, Sims et al. (1993) J. Immunol. 151 :2296); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of heavy chain variable regions (see, for example, Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; and Presta et al. (1993) J. Immunol, 151:2623); human mature (somatically mutated) framework regions or human germline framework regions (see, for example, Almagro and Fransson, (2008) Front. Biosci.13: 1619-1633); and framework regions derived from screening FR libraries (see, for example, Baca et al., (1997) J. Biol. Chem. 272: 10678-10684 and Rosok et al., (1996) J. Biol. Chem. 271 :22611-22618). Typically, the FR regions of a VHH are replaced with human FR regions to make a humanized VHH. In some embodiments, certain FR residues of the human FR are replaced in order to improve one or more properties of the humanized VHH. VHH domains with such replaced residues are still referred to herein as “humanized.”

[0074] The term “substantially similar” or “substantially the same,” as used herein, denotes a sufficiently high degree of similarity between two or more numeric values such that one of skill in the art would consider the difference between the two or more values to be of litle or no biological and / or statistical significance within the context of the biological characteristic measured by said value. In some embodiments the two or more substantially similar values differ by no more than about any one of 5%, 10%, 15%, 20%, 25%, or 50%.

[0075] A polypeptide “variant” means a biologically active polypeptide having at least about 80% amino acid sequence identity with the native or parental sequence polypeptide after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Such variants include, for instance, polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the polypeptide. In some embodiments, a variant will have at least about 80% amino acid sequence identity. In some embodiments, a variant will have at least about 90% amino acid sequenceAtorney Docket No. 01202-0066-00PCT identity. In some embodiments, a variant will have at least about 95% amino acid sequence identity with the native or parental sequence polypeptide.

[0076] As used herein, “percent (%) amino acid sequence identity” and “homology” with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0077] An amino acid substitution may include but are not limited to the replacement of one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 2. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, for example, retained / improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.Table 2Original Residue Exemplary SubstitutionsAla (A) Vai; Leu; HeArg (R) Lys; Gin; AsnAsn (N) Gin; His; Asp, Lys; ArgAsp (D) Glu; AsnCys (C) Ser; AlaGln (Q) Asn; GluGlu (E) Asp; GinGly (G) AlaHis (H) Asn; Gin; Lys; ArgHe (I) Leu; Vai; Met; Ala; Phe; NorleucineLeu (L) Norleucine; He; Vai; Met; Ala; PheLys (K) Arg; Gin; AsnMet (M) Leu; Phe; liePhe (F) Trp; Leu; Vai; He; Ala; TyrPro (P) AlaSer (S) ThrThr (T) Vai; SerTrp (W) Tyr; PheTyr (Y) Trp; Phe; Thr; SerVai (V) He; Leu; Met; Phe; Ala; Norleucine

[0078] Amino acids may be grouped according to common side-chain properties:Attorney Docket No. 01202-0066-00PCT (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;(3) acidic: Asp, Glu;(4) basic: His, Lys, Arg;(5) residues that influence chain orientation: Gly, Pro;(6) aromatic: Trp, Tyr, Phe.

[0079] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0080] The term “vector” is used to describe a polynucleotide that can be engineered to contain a cloned polynucleotide or polynucleotides that can be propagated in a host cell. A vector can include one or more of the following elements: an origin of replication, one or more regulatory sequences (such as, for example, promoters and / or enhancers) that regulate the expression of the polypeptide of interest, and / or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that can be used in colorimetric assays, for example, [3-galactosidase). The term “expression vector” refers to a vector that is used to express a polypeptide of interest in a host cell.

[0081] A “host cell” refers to a cell that may be or has been a recipient of a vector or isolated polynucleotide. Host cells may be prokaryotic cells or eukaryotic cells. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast; plant cells; and insect cells. Nonlimiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell), and 293 and CHO cells, and their derivatives, such as 293-6E, CHO-DG44, CH0-K1, CHO-S, and CHO-DS cells. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) a provided herein.

[0082] The terms “individual” and “subject” are used interchangeably herein to refer to an animal; for example a mammal. The term patient includes human and veterinary subjects. In some embodiments, methods of treating mammals, including, but not limited to, humans, rodents, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets, are provided. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some examples, an “individual” or “subject” refers to an individual or subject in need of treatment for a disease or disorder. In some embodiments, the subject to receive the treatment can be a patient, designating the fact that the subject has been identified as having a disorder of relevance to the treatment, or being at adequate risk of contracting the disorder. In particular embodiments, the subject is a human, such as a human patient.Attorney Docket No. 01202-0066-00PCT

[0083] A “disease” or “disorder” as used herein refers to a condition where treatment is needed and / or desired.

[0084] The term “tumor cell”, “cancer cell”, “cancer”, “tumor”, and / or “neoplasm”, unless otherwise designated, are used herein interchangeably and refer to a cell (or cells) exhibiting an uncontrolled growth and / or abnormal increased cell survival and / or inhibition of apoptosis which interferes with the normal functioning of bodily organs and systems. Included in this definition are benign and malignant cancers, polyps, hyperplasia, as well as dormant tumors or micrometastases.

[0085] The terms “cancer” and “tumor” encompass solid and hematological / lymphatic cancers and also encompass malignant, pre-malignant, and benign growth, such as dysplasia. Also, included in this definition are cells having abnormal proliferation that is not impeded (e.g. immune evasion and immune escape mechanisms) by the immune system (e.g. virus infected cells). Exemplary cancers include, but are not limited to: basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.

[0086] The term “non-tumor cell” as used herein refers to a normal cells or tissue. Exemplary nontumor cells include, but are not limited to: T-cells, B-cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, monocytes, macrophages, epithelial cells, fibroblasts, hepatocytes, interstitial kidney cells, fibroblast-like synoviocytes, osteoblasts, and cells located in the breast, skeletal muscle, pancreas, stomach, ovary, small intestines, placenta, uterus, testis, kidney, lung, heart, brain,Attorney Docket No. 01202-0066-00PCT liver, prostate, colon, lymphoid organs, bone, and bone-derived mesenchymal stem cells. The term “a cell or tissue located in the periphery” as used herein refers to non-tumor cells not located near tumor cells and / or within the tumor microenvironment.

[0087] The term “cells or tissue within the tumor microenvironment” as used herein refers to the cells, molecules, extracellular matrix and / or blood vessels that surround and / or feed a tumor cell.Exemplary cells or tissue within the tumor microenvironment include, but are not limited to: tumor vasculature; tumor-infdtrating lymphocytes; fibroblast reticular cells; endothelial progenitor cells (EPC); cancer-associated fibroblasts; pericytes; other stromal cells; components of the extracellular matrix (ECM); dendritic cells; antigen presenting cells; T-cells; regulatory T-cells (Treg cells); macrophages; neutrophils; myeloid-derived suppressor cells (MDSCs) and other immune cells located proximal to a tumor. Methods for identifying tumor cells, and / or cells / tissues located within the tumor microenvironment are well known in the art, as described herein, below.

[0088] The term “pathogenic B cell” is used to refer to a B cell that is dysfunctional and / or misregulated such that it contributes to immune and / or inflammatory disease. Pathogenic B cells may for example produce autoantibodies, and / or mediate aberrant co-stimulatory and / or antigen presentation functions.

[0089] The term “auto-reactive T cells” is used to refer to a T cell that responds to self-antigens such that it contributes to immune and / or inflammatory disease. Auto-reactive T-cells may when activated damage tissues directly or help B cells produce harmful autoantibodies.

[0090] The terms “immune disease” and “inflammatory disease” encompass a range of diseases and disorders, characterized by dysregulation of the immune system including diseases associated with autoimmunity and / or autoinflammation for example Systemic Lupus Erythematosus (SLE), rheumatoid arthritis (RA), Graves disease, Myasthenia Gravis (gMG), Hashimoto's Thyroiditis, Type 1 Diabetes, multiple sclerosis (MS), psoriasis, inflammatory bowel disease (IBD), autoimmune hepatitis, Sjogren’s syndrome, antiphospholipid syndrome, pemphigus vulgaris, among others.

[0091] In some embodiments, an “increase” or “decrease” refers to a statistically significant increase or decrease, respectively. As will be clear to the skilled person, “modulating” can also involve effecting a change (which can either be an increase or a decrease) in affinity, avidity, specificity and / or selectivity of a target or antigen, for one or more of its ligands, binding partners, partners for association into a homomultimeric or heteromultimeric form, or substrates; effecting a change (which can either be an increase or a decrease) in the sensitivity of the target or antigen for one or more conditions in the medium or surroundings in which the target or antigen is present (such as pH, ion strength, the presence of cofactors, etc.),- and / or cellular proliferation or cytokine production, compared to the same conditions but without the presence of a test agent. This can be determined in any suitable manner and / or using any suitable assay known per se or described herein, depending on the target involved.Attorney Docket No. 01202-0066-00PCT

[0092] As used herein, “an immune response” is meant to encompass cellular and / or humoral immune responses that are sufficient to inhibit or prevent onset or ameliorate the symptoms of disease (for example, cancer or cancer metastasis). “An immune response” can encompass aspects of both the innate and adaptive immune systems.

[0093] As used herein, the terms “treating,” “treatment,” or “therapy” of a disease, disorder or condition is an approach for obtaining beneficial or desired clinical results. “Treatment” as used herein, covers any administration or application of a therapeutic for disease in a mammal, including a human. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, any one or more of: alleviation of one or more symptoms, diminishment of extent of disease, preventing or delaying spread (for example, metastasis, for example metastasis to the lung or to the lymph node) of disease, preventing or delaying recurrence of disease, delay or slowing of disease progression, amelioration of the disease state, inhibiting the disease or progression of the disease, inhibiting or slowing the disease or its progression, arresting its development, and remission (whether partial or total). Also encompassed by “treatment” is a reduction of pathological consequence of a proliferative disease. The methods provided herein contemplate any one or more of these aspects of treatment. In-line with the above, the term treatment does not require one-hundred percent removal of all aspects of the disorder.

[0094] As used herein in the context of cancer, the terms “treatment” or, “inhibit,” “inhibiting” or “inhibition” of cancer refers to at least one of: a statistically significant decrease in the rate of tumor growth, a cessation of tumor growth, or a reduction in the size, mass, metabolic activity, or volume of the tumor, as measured by standard criteria such as, but not limited to, the Response Evaluation Criteria for Solid Tumors (RECIST), or a statistically significant increase in progression free survival (PFS) or overall survival (OS).

[0095] “Ameliorating” means a lessening or improvement of one or more symptoms as compared to not administering a therapeutic agent. “Ameliorating” also includes shortening or reduction in duration of a symptom.

[0096] “Preventing,” “prophylaxis,” or “prevention” of a disease or disorder refers to administration of a pharmaceutical composition, either alone or in combination with another compound, to prevent the occurrence or onset of a disease or disorder or some or all of the symptoms of a disease or disorder or to lessen the likelihood of the onset of a disease or disorder.

[0097] The terms “inhibition” or “inhibit” refer to a decrease or cessation of any phenotypic characteristic or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic. To “reduce” or “inhibit” is to decrease, reduce or arrest an activity, function, and / or amount as compared to a reference. In some embodiments, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 10% or greater. In some embodiments, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 50% or greater. In some embodiments, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater. In some embodiments, theAttorney Docket No. 01202-0066-00PCT amount noted above is inhibited or decreased over a period of time, relative to a control over the same period of time.

[0098] As used herein, “delaying development of a disease” means to defer, hinder, slow, retard, stabilize, suppress and / or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and / or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.

[0099] “Preventing,” as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease. Unless otherwise specified, the terms “reduce”, “inhibit”, or “prevent” do not denote or require complete prevention over all time, but just over the time period being measured.

[0100] The term “biological sample” means a quantity of a substance from a living thing or formerly living thing. Such substances include, but are not limited to, blood, (for example, whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes and spleen.

[0101] The term “control” or “reference” refers to a composition known to not contain an analyte (“negative control”) or to contain an analyte (“positive control”). A positive control can comprise a known concentration of analyte.

[0102] The terms “effective amount” or “therapeutically effective amount” refer to a quantity and / or concentration of a composition containing an active ingredient (e.g. sdAb or VHH-containing molecule) that when administered into a patient either alone (i.e., as a monotherapy) or in combination with additional therapeutic agents, yields a statistically significant decrease in disease progression as, for example, by ameliorating or eliminating symptoms and / or the cause of the disease. An effective amount may be an amount that relieves, lessens, or alleviates at least one symptom or biological response or effect associated with a disease or disorder, prevents progression of the disease or disorder, or improves physical functioning of the patient. A therapeutically effective amount of a composition containing an active agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the active agent to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the active agent are outweighed by the therapeutically beneficial effects. A therapeutically effective amount may be delivered in one or more administrations. A therapeutically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic and / or prophylactic result.Atorney Docket No. 01202-0066-00PCT

[0103] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, nonaqueous or any combination thereof.

[0104] The terms “pharmaceutical formulation” and “pharmaceutical composition” refer to a preparation which is in such form as to permit the biological activity of the active ingredient(s) to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Hence, it is a composition suitable for pharmaceutical use in a mammalian subject, often a human. A pharmaceutical composition typically comprises an effective amount of an active agent (e.g., sdAb or VHH-containing molecule) and a carrier, excipient, or diluent. The carrier, excipient, or diluent is typically a pharmaceutically acceptable carrier, excipient or diluent, respectively. Such formulations may be sterile.

[0105] A “pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional in the art for use with a therapeutic agent that together comprise a “pharmaceutical composition” for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients of the formulation. The pharmaceutically acceptable carrier is appropriate for the formulation employed.

[0106] Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and sequential administration in any order.

[0107] The term “concurrently” is used herein to refer to administration of two or more therapeutic agents, where at least part of the administration overlaps in time, or where the administration of one therapeutic agent falls within a short period of time relative to administration of the other therapeutic agent, or wherein the therapeutic effect of both agents overlap for at least a period of time.

[0108] The term “sequentially” is used herein to refer to administration of two or more therapeutic agents that does not overlap in time, or wherein the therapeutic effects of the agents do not overlap.

[0109] As used herein, “in conjunction with” refers to administration of one treatment modality in addition to another treatment modality. As such, “in conjunction with” refers to administration of one treatment modality before, during, or after administration of the other treatment modality to the individual.

[0110] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contain directions and / or warnings concerning the use of such therapeutic products.

[0111] An “article of manufacture” is any manufacture (for example, a package or container) or kit comprising at least one reagent, for example, a medicament for treatment of a disease or disorder (for example, cancer), or a probe for specifically detecting a biomarker described herein. In someAttorney Docket No. 01202-0066-00PCT embodiments, the manufacture or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

[0112] The terms “label” and “detectable label” mean a moiety attached, for example, to an antibody or antigen to render a reaction (for example, binding) between the members of the specific binding pair, detectable. The labeled member of the specific binding pair is referred to as “detectably labeled.” Thus, the term “labeled binding protein” refers to a protein with a label incorporated that provides for the identification of the binding protein. In some embodiments, the label is a detectable marker that can produce a signal that is detectable by visual or instrumental means, for example, incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (for example,3H,14C,35S,90Y, "Tc,inIn,125I,131I,177Lu,166Ho, or153Sm); chromogens, fluorescent labels (for example, FITC, rhodamine, lanthanide phosphors), enzymatic labels (for example, horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by a secondary reporter (for example, leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags); and magnetic agents, such as gadolinium chelates. Representative examples of labels commonly employed for immunoassays include moieties that produce light, for example, acridinium compounds, and moieties that produce fluorescence, for example, fluorescein. In this regard, the moiety itself may not be detectably labeled but may become detectable upon reaction with yet another moiety.II. VHH DOMAINS BINDING 41BB

[0113] Provided herein are optimized 4 IBB VHH domains (generally referred to herein simply as “4 IBB VHH” (singular) or “4 IBB VHHs” (plural)) that may be incorporated in 41BB-binding molecules including those provided herein. The 41BBVHHs can be formatted in a variety of formats, including any as described in Section III below.

[0114] In some embodiments, an optimized VHH that binds 4 IBB (a “4 IBB VHH”) comprises a CDR1, CDR2 and CDR3. In some embodiments, the 41BB-binding molecules herein comprise at least one 41BB VHH that comprises the CDR1, CDR2, and CDR3 sequences, under any CDR definition (e.g., AbM, Kabat, IMGT, Chothia, Contact, etc.) as provided in Table 3. A name that ends in “con” represents a consensus sequence.Table 3: CDRs of optimized 4 IBB VHH according to the indicated definitionsDefinition CDR Sequence Fragment SEQ IDNOAbM CDR1 GFTFDINAMG 47Attorney Docket No. 01202-0066-00PCT Definition CDR Sequence Fragment SEQ IDNO CDR2 AIESGGNTV 49CDR2 AIESGGSTV 50CDR2 con AIESGGXiTV 51wherein Xi is N or SCD3 LKGNRWSPSVAY 52CDR1 INAMG 53CDR2 AIESGGNTVYAESVKG 55CDR2 AIESGGSTVYAESVKGKabat 56CDR2 con AIESGGXiTVYAESVKG 57wherein Xi is N or SCD3 LKGNRWSPSVAY 58CDR1 GFTFDINA 60CDR2 IESGGNT 62I CDR2 IESGGSTMGT 63CDR2 con IESGGXIT 64wherein Xi is N or SCD3 ALLKGNRWSPSVAY 66CDR1 GFTFDIN 68CDR2 ESGGN 70Chothi CDR2 ESGGSa 71CDR2 con ESGGXi 7?wherein Xi is N or SCD3 LKGNRWSPSVAY 73CDR1 D INAMG 75CDR2 FVAAIESGGNTV 77CD FVAAIESGGSTVContact R2 78CDR2 con FVAAIESGGXiTV 79wherein Xi is N or SCD3 ALLKGNRWSPSVA 81

[0115] In some embodiments, the 4 IBB VHH comprises a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 47, 53, 60, 68, and 75; a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 51, 57, 64, 72, and 79; and a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:52, 58, 66, 73, 81. In some aspects, the CDRs are AbM CDRs. In some embodiments, the CDRs are Kabat CDRs. In some embodiments, the CDRs are IMGT CDRs. In some embodiments, the CDRs are Chothia CDRs. In some embodiments, the CDRs are Contact CDRs.

[0116] In some embodiments, the 4 IBB VHH comprises a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 47, 53, 60, 68, and 75; a complementarity determining region 2 (CDR2) comprising an amino acid sequenceAttorney Docket No. 01202-0066-00PCT selected from the group consisting of SEQ ID NOs: 49, 50, 55, 56, 62, 63, 70, 71, 77, and 78; and a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:52, 58, 66, 73, 81. In some aspects, the CDRs are AbM CDRs. In some embodiments, the CDRs are Kabat CDRs. In some embodiments, the CDRs are IMGT CDRs. In some embodiments, the CDRs are Chothia CDRs. In some embodiments, the CDRs are Contact CDRs.

[0117] In some embodiments, the 41BB VHH comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 47, 51, and 52. In some embodiments, the 41BB VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 47, 49, and 52. In some embodiments, the 4 IBB VHH comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 47, 50 and 52. In some embodiments, the 41BB VHH comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 53, 57, and 58. In some embodiments, the 41BB VHH comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 53, 55, and 58. In some embodiments, the 41BB VHH comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 53, 56, and 58. In some embodiments, the 41BB VHH comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 60, 64, and 66. In some embodiments, the 41BB VHH comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 60, 62, and 66. In some embodiments, the 41BB VHH comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 60, 63, and 66. In some embodiments, the 41BB VHH comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 68, 72, and 73. In some embodiments, the 41BB VHH comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 68, 70, and 73. In some embodiments, the 4 IBB VHH comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 68, 71, and 72. In some embodiments, the 41BB VHH comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 75, 79, 81. In some embodiments, the 41BB VHH comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 75, 77, 81. In some embodiments, the 41BB VHH comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 75, 78, 81.

[0118] In some embodiments, the 4 IBB VHH has an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a VHH of any one of SEQ ID NOs: 43-45, 98, and 99. In some embodiments, the 41BB VHH comprises a CDR1, CDR2 and CDR3 sequences, under any CDR definition provided in Table 3 above, and has an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a VHH of any one of SEQ ID NOs: 43-45, 98, and 99. In some embodiments, the 4 IBB VHH has the amino acid sequence of any one of SEQ ID NOS: 43-45, 98, and 99.

[0119] In some embodiments, the 41BB VHH comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, the 4 IBB VHH comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the 4 IBB VHH comprises the amino acid sequence of SEQ ID NO: 45. In some embodiments, the 41BB VHH comprises the amino acid sequence of SEQ ID NO: 98. In some embodiments, the 41BB VHH comprises the amino acid sequence of SEQ ID NO: 99.Atorney Docket No. 01202-0066-00PCT

[0120] In some embodiments, a 4 IBB VHH comprises a glutamate (E), aspartate (D), lysine (K), or arginine (R) at position 11 according to Kabat numbering. In certain embodiments the 4 IBB VHH may comprise a modification at position 11 within framework 1 to substitute the existing residues for an E, D, K or R. In some embodiments, a 4 IBB VHH comprises a lysine (K), arginine (R) glutamate (E), or aspartate (D) at position 112 according to Kabat numbering. In certain embodiments the 4 IBB VHH may comprise a modification at position 112 within framework 4 to substitute the existing residue for a K, R, E, or D. It will be understood that Kabat positions 11, and 112 correspond to residues 11, 120, respectively of SEQ ID NOs: 43-45, 98 and 99. In some embodiments, a VHH domain that binds IgE comprises a Glu at position 11 and a Lys or Arg at position 112. In some embodiments, a 4 IBB VHH comprises a C-terminal extension of one or more glycine residues.

[0121] In any of the embodiments provided herein, a 4 IBB VHH can be any as described above.III. FUSION PROTEINS AND CONJUGATES CONTAINING 41BB-BINDING VHH DOMAINS

[0122] Provided herein are 41BB-binding molecules containing at least one VHH domain that specifically binds 4 IBB (“4 IBB VHH”) linked, directly or indirectly, to one or more additional domains or moieties. In some embodiments, the 41BB-binding molecules of the present disclosure are composed of a single polypeptide. In other embodiments, the 41BB-binding molecules of the present disclosure are composed of more than one polypeptide. In some embodiments, the 41BB-binding molecules of the present disclosure incorporate at least one 4 IBB VHH. In some embodiments, the 41BB-binding molecules are multivalent. In some embodiments, the 41BB-binding molecules include two or more copies of a 4 IBB VHH, for example, three or more, four or more, five or more, or six or more copies of a 4 IBB VHH. In certain embodiments, the 41BB-binding molecules are multispecific. For example, in some cases, the one or more additional domain may be one or more additional binding domain that binds to one or more further antigen.A. Einkers

[0123] In some embodiments, the 41BB-binding molecules of the present disclosure include two or more polypeptide sequences that are operably linked via amino acid linkers. Various polypeptide linkers for use in fusion proteins are known (see e.g. Chen et al. (2013) Adv. Drug. Deliv. 65: 1357-1369; and International PCT publication No. WO 2014099997, W0200024884; U.S. Pat. No. 5,258,498; U.S. Pat. No. 5,525,491; U.S. Pat. No. 5,525,491, U.S. Pat. No. 6,132,992).

[0124] The polypeptide linker can be a flexible linker or a rigid linker or a combination of both. In some embodiments, the linker is a short, medium or long linker. In some embodiments, the linker is up to 40 amino acids in length. In some embodiments, the linker is up to 25 amino acids in length. In some embodiments, the linker is at least or is at least about 2 amino acids in length. In some embodiments, aAttorney Docket No. 01202-0066-00PCT suitable length is, e.g., a length of at least one and typically fewer than about 40 amino acid residues, such as 2-25 amino acid residues, 5-20 amino acid residues, 5-15 amino acid residues, 8-12 amino acid . In some embodiments, the linker is from or from about 2 to 24 amino acids, 2 to 20 amino acids, 2 to 18 amino acids, 2 to 14 amino acids, 2 to 12 amino acids, 2 to 10 amino acids, 2 to 8 amino acids, 2 to 6 amino acids, 6 to 24 amino acids, 6 to 20 amino acids, 6 to 18 amino acids, 6 to 14 amino acids, 6 to 12 amino acids, 6 to 10 amino acids, 6 to 8 amino acids, 8 to 24 amino acids, 8 to 20 amino acids, 8 to 18 amino acids, 8 to 14 amino acids, 8 to 12 amino acids, 8 to 10 amino acids, 10 to 24 amino acids, 10 to 20 amino acids, 10 to 18 amino acids, 10 to 14 amino acids, 10 to 12 amino acids, 12 to 24 amino acids, 12 to 20 amino acids, 12 to 18 amino acids, 12 to 14 amino acids, 14 to 24 amino acids, 14 to 20 amino acids, 14 to 18 amino acids, 18 to 24 amino acids, 18 to 20 amino acids or 20 to 24 amino acids. In some embodiments, the linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length.

[0125] The linkers can be naturally-occurring, synthetic or a combination of both. Particularly suitable linker polypeptides predominantly include amino acid residues selected from Glycine (Gly), Serine (Ser), Alanine (Ala), and Threonine (Thr). For example, the linker may contain at least 75% (calculated on the basis of the total number of residues present in the peptide linker), such as at least 80%, at least 85%, or at least 90% of amino acid residues selected from Gly, Ser, Ala, and Thr. The linker may also consist of Gly, Ser, Ala and / or Thr residues only. In some embodiments, the linker contains 1-25 glycine residues, 5-20 glycine residues, 5-15 glycine residues, or 8-12 glycine residues. In some embodiments, suitable peptide linkers typically contain at least 50% glycine residues, such as at least 75% glycine residues. In some embodiments, a peptide linker comprises glycine residues only. In some embodiments, a peptide linker comprises glycine and serine residues only.

[0126] In some embodiments, the linker is a flexible linker comprising predominately, or solely Glycine residues, such as, by way of non-limiting example, GG, GGG, GGGG (SEQ ID NO: 33), etc. The glycine linkers of the fusion proteins of the present disclosure can be of various lengths, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in length. In some embodiments, the glycine linker comprises 1-6 amino acids, 1-12 amino acids, 6-12 amino acids, 6-18 amino acids, 12-18 amino acids, 12 to 20 amino acids, or 18-20 amino acids. In some embodiments, these linkers are composed predominately of the amino acids Glycine and Serine, denoted as GS-linkers herein. The GS-linkers of the fusion proteins of the present disclosure can be of various lengths, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in length. In some embodiments, the GS-linker comprises 1-6 amino acids, 1-12 amino acids, 6-12 amino acids, 6-18 amino acids, 12-18 amino acids, 12 to 20 amino acids, or 18-20 amino acids. In some embodiments, the linker contains (GGS)n, wherein n is 1 to 10, or n is 1 to 5, or n is 1 to 3. In some embodiments, the linker contains the sequence (GGGGS)n (SEQ ID NO: 82), wherein n is 1 to 10 or n is 1 to 5, or n is 1 to 3.Attorney Docket No. 01202-0066-00PCT

[0127] In some embodiments, the GS-linker comprises an amino acid sequence selected from the group consisting of GSGGGS (SEQ ID NO: 34), GGSGGS, i.e., (GGS)2(SEQ ID NO: 35);GGSGGSGGS, i.e., (GGS)3(SEQ ID NO: 36); GGSGGSGGSGGS, i.e., (GGS)4(SEQ ID NO: 37); and GGSGGSGGSGGSGGS, i.e., (GGS)5(SEQ ID NO: 38). In some embodiments, the linker is (GGGGS)n, wherein n is 1 to 5 (SEQ ID NO: 82); (GGGGGS)n, wherein n is 1 to 4 (SEQ ID NO: 83) . In some embodiments, the 41BB-binding molecule includes a combination of a GS-linker and a Glycine linker.

[0128] In some cases, it may be desirable to provide some rigidity into the peptide linker. This may be accomplished by including proline residues in the amino acid sequence of the peptide linker. Thus, in some embodiments, a linker comprises at least one proline residue in the amino acid sequence of the peptide linker. For example, a peptide linker can have an amino acid sequence wherein at least 25% (e.g., at least 50% or at least 75%) of the amino acid residues are proline residues. In one particular embodiment, the peptide linker comprises proline residues only. In some embodiments, the linker comprises PGGGG (SEQ ID NO: 84).B. Fc Fusions

[0129] Provided herein is a 41BB-binding molecule that is a fusion protein containing at least one 4 IBB VHH provided herein and an Fc region. In some embodiments, a 41BB-binding molecule provided herein comprises an Fc region and a polypeptide derived from an immunoglobulin hinge region. In some embodiments, a 41BB-binding molecule provided herein comprises one, two, three, or four 4 IBB VHHs and an Fc region and optionally may further comprise one or more additional binding domains that bind to one or more additional antigen other than 4 IBB.

[0130] In some embodiments, incorporation of an immunoglobulin Fc region into the fusion protein can, in some embodiments, be composed of two polypeptides that together form a dimer. In some embodiments, an Fc region mediates dimerization of the 41BB-binding molecule at physiological conditions, such as when expressed from a cell, such that a dimer is formed that doubles the number of 41BB-binding sites. For example, a 41BB-binding molecule comprising three 4 IBB VHHs and an Fc region is trivalent as a monomer, but the Fc region may mediate dimerization, such that the 41BB-binding molecule exists as a hexavalent dimer under such conditions. In some embodiments, a 4 IBB VHH is fused to an IgG Fc region and in these embodiments, the fusion protein is bivalent having two 4 IBB VHHs per molecule. In some embodiments, two 4 IBB VHHs (2x) are fused to an IgG Fc region and in these embodiments, the fusion protein is tetravalent having four 4 IBB VHHs per molecule. In some embodiments, three 4 IBB VHHs (3x) are fused to an IgG Fc region and in these embodiments, the fusion protein is hexavalent having six 4 IBB VHHs per molecule.

[0131] In some embodiments, the multivalent 41 BB-binding molecule is bivalent. In some embodiments, the bivalent 41 BB-binding molecule of the disclosure includes two polypeptides having the following structure: (41BB VHH)-Linker-Fc and / or Fc-Linker-(41BB VHH). In some embodiments,Attorney Docket No. 01202-0066-00PCT the multivalent 41 BB-binding molecule is tetravalent. In some embodiments, the tetravalent 41BB-binding molecule of the disclosure includes two polypeptides having the following structure: (41BB VHH)-Linker-(41BB VHH)-Linker- Fc and / or (41BB VHH)-Linker-Fc-Linker-(41BB VHH). In some embodiments, the multivalent 4 IBB -binding molecule is hexavalent. In some embodiments, the hexavalent 41 BB-binding molecule of the disclosure includes two polypeptides having the following structure: (4 IBB VHH)-Linker-(41BB VHH)-Linker-(41BB VHH)-Linker- Fc; (4 IBB VHH) -Linker-(4 IBB VHH)-Linker- Fc-Linker-(41BB VHH); and / or (4 IBB VHH)-Linker- Fc-Linker-(41BB VHH)-Linker-(41BB VHH).

[0132] In some cases, the CH3 domain of the Fc region can be used as homodimerization domain, such that the resulting fusion protein is formed from two identical polypeptides. In other cases, the CH3 dimer interface region of the Fc region can be mutated so as to enable heterodimerization. For example, a heterodimerization domain can be incorporated into the fusion protein such that the construct is an asymmetric fusion protein.

[0133] In some embodiments, an Fc region included in a 41BB-binding molecule is a human Fc region, or is derived from a human Fc region. In some embodiments, the 41BB-binding molecule contains an immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region is an IgG isotype selected from the group consisting of IgG 1 isotype, IgG2 isotype, IgG3 isotype, and IgG4 isotype. In some embodiments, the Fc region is derived from a human Fc region and lacks the C-terminal lysine residue. In some embodiments, the Fc region is derived from a human Fc region and comprises the C-terminal lysine residue. In some embodiments, the C-terminal amino acid of the Fc region is an amino acid other than lysine. In some embodiments, the Fc region is derived from a human Fc region and lacks the C-terminal glycine and lysine residues.

[0134] In some embodiments, the immunoglobulin Fc region or immunologically active fragment thereof comprises a human IgGl, IgG2, IgG3 or IgG4 polypeptide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1-4. In some embodiments, the Fc region or a variant thereof such as any described below can be from any IgG allotype, e.g., a G1 ml or G1 m3 allotype. In some embodiments, the Fc region can contain amino acids of the human G1 ml allotype, such as residues containing Asp (D) and Leu (L) at positions 356 and 358 by EU numbering. In other embodiments, the Fc region can contain amino acids of the human G1 m3 allotype, such as residues Glu (E) and Met (M) at positions 356 and 358 by EU numbering.

[0135] In some embodiments where the 41BB-binding molecule of the disclosure includes an Fc region, the Fc region is mutated or modified at one or more position. In some cases, the mutations include one or more amino acid substitutions to reduce an effector function of the Fc region. Various examples of mutations to Fc regions to alter, such as reduce, effector function are known, including anyAtorney Docket No. 01202-0066-00PCT as described below. Unless described with reference to a position within a specific SEQ ID NO. reference to an amino acid position in an Fc region is according to the EU Numbering.

[0136] In some embodiments, an Fc region that exhibits reduced effector functions may be a desirable candidate for applications in which 4 IBB binding is desired yet certain effector functions (such as CDC and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be conducted to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the fusion proteins and / or cleaved components thereof lack FcyR binding (hence likely lacking ADCC activity), but retain FcRn binding ability.

[0137] In some embodiments, the Fc is an Fc that exhibits immune effector activity, such as one or more effector functions such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC).

[0138] In some embodiments, the human IgG Fc region is modified to alter antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), e.g., the amino acid modifications described in Natsume et al., 2008 Cancer Res, 68(10): 3863-72; Idusogie et al., 2001 J Immunol, 166(4): 2571-5; Moore et al., 2010 mAbs, 2(2): 181-189; Lazar et al., 2006 PNAS, 103(11): 4005-4010, Shields et al., 2001 JBC, 276(9): 6591-6604; Stavenhagen et al., 2007 Cancer Res, 67(18): 8882-8890; Stavenhagen et al., 2008 Advan. Enzyme Regul., 48: 152-164; Alegre et al, 1992 J Immunol, 148: 3461-3468; Reviewed in Kaneko and Niwa, 2011 Biodrugs, 25(1): 1-11.

[0139] Examples of mutations that enhance ADCC include modification at Ser239 and Ile332, for example Ser239Asp and Ile332Glu (S239D, I332E). Examples of mutations that enhance CDC include modifications at Lys326 and Glu333. In some embodiments, the Fc region is modified at one or both of these positions, for example Lys326Ala and / or Glu333Ala (K326A and E333A) using the EU numbering system.

[0140] In some embodiments, the Fc region of the fusion protein is altered at one or more of the following positions to reduce Fc receptor binding: 234, 235, 265, 270, 298, 297, 325, 327 or 329. For example, Leu234Ala (L234A), Leu235Ala (L235A), Leu235Glu (L235E), Asp265Asn (D265N), Asp265Ala (D265A), Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Pro329Ala (P329A) or Pro239Gly (P329G), Asn325Glu (N325E) orAla327Ser (A327S) (EU numbering). In preferred embodiments, modifications within the Fc region reduce binding to Fc-receptor-gamma receptors while have minimal impact on binding to the neonatal Fc receptor (FcRn).

[0141] In some embodiments, the human IgGl Fc region is modified at amino acid Asn297 (EU Numbering) to prevent glycosylation of the fusion protein, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the Fc region of the fusion protein is modified at position 235 (EU Numbering) to alter Fc receptor interactions, e.g., Leu235Glu (L235E) or Leu235Ala (L235A). In some embodiments, the Fc region of the fusion protein is modified at position 234 (EU Numbering) to alter Fc receptor interactions, e.g., Leu234Ala (L234A). In some embodiments, the Fc region of the fusionAttorney Docket No. 01202-0066-00PCT protein is modified at position 235 (EU Numbering) to alter Fc receptor interactions, e.g., Leu235Glu (L235E). In some embodiments, the Fc region of the fusion protein is altered at both positions 234 and 235, e.g., Leu234Ala and Leu235Ala (L234A / L235A) or Leu234Val and Leu235Ala (L234V / L235A). In some embodiments, the Fc region of the fusion protein is altered at positions at 234, 235, and 297, e.g., Leu234Ala, Leu235Ala, Asn297Ala (L234A / L235A / N297A). In some embodiments, the Fc region of the fusion protein is altered at positions at 234, 235, and 329, e.g., Leu234Ala, Leu235Ala, Pro239Ala (L234A / L235A / P329A). In some embodiments, the Fc region of the fusion protein is modified at position 265 (EU Numbering) to alter Fc receptor interactions, e.g Asp265Ala (D265A). In some embodiments, the Fc region of the fusion protein is modified at position 329 (EU Numbering) to alter Fc receptor interactions, e.g Pro329Ala (P329A) or Pro329Gly (P329G). In some embodiments, the Fc region of the fusion protein is altered at both positions 265 and 329, e.g., Asp265Ala and Pro329Ala (D265A / P329A) or Asp265Ala and Pro329Gly (D265A / P329G). In some embodiments, the Fc region of the fusion protein is altered at positions at 234, 235, and 265, e.g., Leu234Ala, Leu235Ala, Asp265Ala (L234A / L235A / D265A). In some embodiments, the Fc region of the fusion protein is altered at positions at 234, 235, and 329, e.g., Leu234Ala, Leu235Ala, Pro329Gly (L234A / L235A / P329G). In some embodiments, the Fc region of the fusion protein is altered at positions at 234, 235, 265 and 329, e.g., Leu234Ala, Leu235Ala, Asp265Ala, Pro329Gly (L234A / L235A / D265A / P329G). In some embodiments, the human IgGl Fc region is modified at amino acid Gly236 to enhance the interaction with CD32A, e.g., Gly236Ala (G236A).

[0142] In some embodiments, the Fc region of the fusion protein is lacking an amino acid at one or more of the following positions to reduce Fc receptor binding: 233, 234, and 235 (EU Numbering). In some embodiments, the Fc region of the fusion protein is lacking an amino acid at one or more of the following positions 233, 234, and 235 and is modified at one or more of the positions 2, 297, or 329 (EU numbering) to reduce Fc receptor binding. For example, an Fc region included in a 41BB-bindng molecule is derived from a human Fc region, and comprises a three amino acid deletion corresponding to IgGl E233, L234, and L235, IgG2 P233, V234, V235, IgG3 E233, E234, L235, or IgG4 E233, F234, L235. In some embodiments, such Fc regions do not engage FcyRs and thus are referred to as “effector silent” or “effector null.” For example, Fc deletion of these three amino acids reduces the complement protein Clq binding. In some embodiments, a polypeptide with an Fc region with Fc deletion of these three amino acids retains binding to FcRn and therefore has extended half-life and transcytosis associated with FcRn mediated recycling. Such a modified Fc region is referred to as “Fc xELL” (see, e.g., SEQ ID NOs: 12-29).

[0143] In some embodiments, the human IgG Fc region is modified to enhance FcRn binding and / or serum half-life. Examples of Fc mutations that enhance binding to FcRn are Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S254T, T256E, respectively), referred herein as “YTE”) (EU Numbering,Atorney Docket No. 01202-0066-00PCT Dall’Acqua et al 2006, J. Biol Chem Vol. 281(33) 23514-23524), Met428Leu and Asn434Ser (M428L, N434S) (Zalevsky et al 2010 Nature Biotech, Vol. 28(2) 157-159), or Met252Ile, Thr256Asp, Met428Leu (M252I, T256D, M428L, respectively), (EU Numbering).

[0144] In some embodiments, the Fc region included in a 41BB-bindng molecule is derived from a human Fc region and comprises mutations M252Y and M428V (EU Numbering), herein referred to as “YV” (see, e.g., SEQ ID NOs: 14, 17, 20, 23, 26, 29, and 32). In some embodiments, the mutated or modified Fc region includes the following mutations: M252Y and M428L (EU Numbering), herein referred to as “YL” (see e.g., SEQ ID NOs: 15, 16, 21, 22, 26, and 28). In some embodiments, such mutations enhance binding to FcRn at the acidic pH of the endosome (near 6.5), while losing detectable binding at neutral pH (about 7.2), allowing for enhanced FcRn mediated recycling and extended half-life.

[0145] In some embodiments, the Fc region included in a 41BB-binding molecule is derived from a human Fc region and comprises mutations to induce heterodimerization. Various methods are known for promoting heterodimerization of complementary Fc polypeptides, see e.g. Ridgway et al, Protein Eng.9:617-621 (1996); Merchant et al, Nat. Biotechnol. 16(7): 677-81 (1998); Moore et al. (2011) MAbs, 3:546-57; Von Kreudenstein et al. MAbs, (2013) 5:646-54; Gunasekaran et al. (2010) J. Biol. Chem., 285: 19637-46; Leaver-Fay et al. (2016) Structure, 24:641-51; Ha et al. (2016) Frontiers in Immunology, 7:1; Davis et al. (2010) Protein Eng Des Sei, 23:195-202; published international PCT Appl. No. WO 1998 / 050431, WO 2009 / 089004, WO2011143545 WO 2014 / 067011, WO 2012 / 058768, W02018027025; published U.S. patent Appl. No. US20140363426, US20150307628, US20180016354, US20150239991; and U.S. patent Nos. US5731168, US7183076, US9701759, US9605084, and US9650446. Methods to promote heterodimerization of Fc chains include mutagenesis of the Fc region, such as by including a set of “knob-into-hole” mutations or including mutations to effect electrostatic steering of the Fc to favor atractive interactions among different polypeptide chains. For example, in some embodiments, the Fc polypeptides of a heterodimer includes a mutation to alter charge polarity across the Fc dimer interface such that coexpression of electrostatically matched Fc chains support favorable atractive interactions thereby promoting desired Fc heterodimer formation, whereas unfavorable repulsive charge interactions suppress unwanted Fc homodimer formation (Guneskaran et al. (2010) JBC, 285: 19637-19646). When co-expressed in a cell, association between the chains is possible but the chains do not substantially self-associate due to charge repulsion. Other strategies for generating a heterodimeric Fc include mixing human IgG and IgA CH3 domain segments to create a complementary CH3 heterodimer, which is referred to as a SEED Fc.

[0146] Methods and variants for heterodimerization also include those described in published international PCT App. WO2014145806, including “knobs and holes” mutations (also called “skew” variants), mutations that relate to “electrostatic steering” or “charge pairs,” and pl variants.Heterodimeric variants also include any as described in U.S. published Appl. No. US2012 / 0149876 or US2018 / 011883.Atorney Docket No. 01202-0066-00PCT

[0147] In some embodiments, to promote heterodimerization both polypeptides of the heterodimeric Fc region contain paired or complementary amino acid modifications. Exemplary paired amino acid modification of polypeptides of an Fc fusion are set forth in Table 4.Table 4: Paired amino acids of Heterodimeric FcFirst Fc polypeptide Second Fc PolypeptideT366W (Knob) T366S / L368W / Y407V (Hole) T366W / S354C (Knob-Cys) T366S / L368A / Y407V7Y349C (Hole-Cys) S364H / F405A Y349T / Y349FT350V / L351Y / F405A / Y407V T35 OV7T366L / K392L / T394W K360D / D399M / Y407A E345R / Q347R / T366V / K409V K409D / K392D D399K / E356KK360E / K409W Q347R / D399V / F405T L360E / K409W / Y349C Q347R / 399V / F405T / S354CK370E / K409W E357N / D399V / F405T

[0148] Additional examples of variants that can facilitate the promotion of heterodimers are any combination or pair of steric variants (e.g. skew variants) of a first Fc polypeptide and a second Fc polypeptide from among: S364K / E357Q and L368D / K370S; L368D / K370S and S364K; L368E / K370S and S364K; T411T / E360E / Q362E and D401K; L368D / K370S and S364K / E357L; K370S and S364K / E357Q; and T366S / L368A / Y407V and T366W or 366S / L368A / Y407V / Y349C and T366W / S354C), where each pair represents mutations in the first Fc polypeptide and second Fc polypeptide. In particular embodiments, a provided molecule contains a first and second Fc polypeptide containing the pair of mutations L368D / K370S and S364K and E357Q.

[0149] An additional mechanism that can be used in the generation of heterodimers is sometimes referred to as “electrostatic steering” as described in Gunasekaran et al., J. Biol. Chem. 285(25): 19637 (2010). This is sometimes referred to herein as “charge pairs”. In this embodiment, electrostatics are used to skew the formation towards heterodimerization. As those in the art will appreciate, these may also have an effect on pl, and thus on purification, and thus could in some cases also be considered pl variants. However, as these were generated to force heterodimerization and were not used as purification tools, they are classified as “steric variants”. In some embodiments, a first Fc polypeptide can contain mutations D221E / P228E / L368E and a second Fc polypeptide can contain mutations D221R / P228R / K409R. In another embodiments, a first Fc polypeptide can contain mutations C220E / P228E / 368E and a second Fc polypeptide can contain mutations C220R / E224R / P228R / K409R.

[0150] In some embodiments, heterodimerization can be facilitated by pl variants. In some embodiments, a pl variant can include those that increase the pl of the protein (basic changes). In other embodiments, the pl variant can include those that decrease the pl of the protein (acidic changes). In some cases, all combinations of these variants can be done, including combinations in which one Fc polypeptide may be wild type, or a variant that does not display a significantly different pl from wildtype, and the other Fc polypeptide can be either more basic or more acidic. Alternatively, each FcAttorney Docket No. 01202-0066-00PCT polypeptide can be changed, one to more basic and one to more acidic. In some embodiments, at least one Fc polypeptide is a negative pl variant Fc containing mutations Q295E / N384D / Q418E / N421D.

[0151] In some embodiments, one member of a heterodimeric Fc pair comprises the “knob” mutation (see, e.g., SEQ ID NOs: 18-23) and the other member of a heterodimeric Fc pair comprises the “hole” mutations (see, e.g., SEQ ID NOs: 24-29). In some embodiments, one member of a heterodimeric Fc pair comprises the modification H435R or H435K to prevent protein A binding while maintaining FcRn binding (see, e.g., SEQ ID NOs: 22, 23, 28, and 29). In some embodiments, one member of a heterodimeric Fc pair comprises the modification H435R or H435K, while the second member of the heterodimeric Fc pair is not modified at H435. In various embodiments, the hole Fc region comprises the modification H435R or H435K (referred to as “hole-R” in some instances when the modification is H435R), while the knob Fc region does not. In some instances, the hole-R mutation improves purification of the heterodimer over homodimeric hole Fc regions that may be present.

[0152] In some embodiments, the Fc region comprises a first Fc polypeptide and a second Fc polypeptide. In some embodiments, the first Fc polypeptide comprises any of SEQ ID NOs: 18-23, or comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 18-23. In some embodiments, the second Fc polypeptide comprises any of SEQ ID NOs: 24-29, or or comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs:24-29.

[0153] In some embodiments, the human IgG Fc region included in a 41BB-binding molecule is modified to prevent dimerization. In these embodiments, the fusion proteins of the present disclosure are monomeric. For example modification at residue Thr366 to a charged residue, e.g. Thr366Lys, Thr366Arg, Thr366Asp, or Thr366Glu (T366K, T366R, T366D, or T366E, respectively), prevents CH3-CH3 dimerization.

[0154] In some embodiments, the immunoglobulin Fc region or immunologically active fragment thereof included in a 41BB-binding molecule comprises a human IgGl polypeptide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the human IgGl Fc region lacks Lys447 (EU Numbering).

[0155] In some embodiments, the immunoglobulin Fc region or immunologically active fragment thereof included in a 41BB-binding molecule comprises a human IgG2 polypeptide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the human IgG2 Fc region lacks Lys447 (EU Numbering).

[0156] In some embodiments, the immunoglobulin Fc region or immunologically active fragment thereof included in a 41BB-binding molecule comprises a human IgG3 polypeptide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the human IgG3 Fc region is modified at amino acid 435 toAtorney Docket No. 01202-0066-00PCT extend the half-life, e.g., Arg435His (R435H). In some embodiments, the human IgG3 Fc region lacks Lys447 (EU Numbering). In some embodiments, the human IgG3 Fc region lacks Lys447 (EU Numbering).

[0157] In some embodiments, the immunoglobulin Fc region or immunologically active fragment thereof included in a 41BB-binding molecule comprises a human IgG4 polypeptide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the human IgG4 Fc region is modified at amino acid 235 to alter Fc receptor interactions, e.g., Leu235Glu (L235E). In some embodiments, the human IgG4 Fc region lacks Lys447 (EU numbering).

[0158] In some embodiments, the Fc region included in a 41BB-binding molecule comprises a polypeptide derived from an immunoglobulin hinge region. The hinge region can be derived from any of the human IgG subclasses (see, e.g., SEQ ID NOs: 5-11). For example, the Fc region may contain a modified IgGl hinge having the sequence of EPKSSDKTHTCPPC (SEQ ID NO: 6), where in the Cys220 that forms a disulfide with the C-terminal cysteine of the light chain is mutated to serine, e.g., Cys220Ser (C220S). In other embodiments, the fusion protein contains a truncated hinge having a sequence DKTHTCPPC (SEQ ID NO: 7). Unless noted the Fc regions provided in the Table of Certain Sequences comprise a truncated hinge having the sequence SEQ ID NO: 7.

[0159] In some embodiments, the Fc region included in a 41BB-binding molecule has a modified hinge from IgG4, which is modified to prevent or reduce strand exchange, e.g., Ser228Pro (S228P), having the sequence ESKYGPPCPPC (SEQ ID NO: 11). In some embodiments, the Fc region included in a 41BB-binding molecule contains linker polypeptides. In other embodiments, the Fc region included in a 41BB-binding molecule contains linker and hinge polypeptides.

[0160] In some embodiments, the Fc region included in a 41BB-binding molecule comprises any of SEQ ID NOs: 11-32, or a polypeptide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 11-32.

[0161] In some embodiments, the Fc region included in a 41BB-binding molecule lacks or has reduced Fucose atached to the N-linked glycan-chain at N297. There are numerous ways to prevent fucosylation, including but not limited to production in a FUT8 deficient cell line; additional inhibitors to the mammalian cell culture media, for example castanospermine; and metabolic engineering of the production cell line.

[0162] In some embodiments, the Fc regions included in a 41BB-binding molecule are operably linked to the other domains (e.g., VHH) via amino acid linkers, such as any described above.Attorney Docket No. 01202-0066-00PCT C. Conjugates

[0163] Provided herein are 41BB-binding molecules containing at least one 4 IBB VHH provided herein conjugated to one or more further moiety referred to herein as “41BB-binding conjugates.” The further moiety can be a therapeutic agent or a detection agent. In some embodiments, the moiety can be a targeting moiety, a small molecule drug (non-polypeptide drug of less than 500 Daltons molar mass), a toxin, a cytostatic agent, a cytotoxic agent, an immunosuppressive agent, a radioactive agent suitable for diagnostic purposes, a radioactive metal ion for therapeutic purposes, a prodrug-activating enzyme, an agent that increases biological half-life, or a diagnostic or detectable agent.

[0164] In some embodiments, the 41BB-binding conjugate is an antibody drug conjugate (ADC, also called immunoconjugates) containing one or more 41BB-binding molecules provided herein conjugated to a therapeutic agent, which is either cytotoxic, cytostatic or otherwise provides some therapeutic benefit. In some embodiments, the cytotoxic agent is a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). In some embodiments, provided antibody drug conjugates of the present disclosure allow targeted-delivery of the drug moiety to tumors. In some cases, this can result in targeted killing of the tumor cell.

[0165] In some embodiments, there is provided a 41BB-binding molecule conjugated with a therapeutic agent. In some embodiments, the therapeutic agent includes, for example, daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., Cancer Immunol. Immunother. 21: 183-187, 1986). In some embodiments, the therapeutic agent has an intracellular activity. In some embodiments, the 41BB-binding conjugate is internalized and the therapeutic agent is a cytotoxin that blocks the protein synthesis of the cell, therein leading to cell death. In some embodiments, the therapeutic agent is a cytotoxin comprising a polypeptide having ribosome-inactivating activity including, for example, gelonin, bouganin, saporin, ricin, ricin A chain, bryodin, diphtheria toxin, restrictocin, Pseudomonas exotoxin A and variants thereof. In some embodiments, where the therapeutic agent is a cytotoxin comprising a polypeptide having a ribosome-inactivating activity, the 41BB-binding conjugate must be internalized upon binding to the target cell in order for the protein to be cytotoxic to the cells.

[0166] In some embodiments, there is provided a 41BB-binding molecule conjugated with a toxin. In some embodiments, the toxin includes, for example, bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Mandler et al., J. Nat. Cancer Inst. 92(19): 1573-1581 (2000); Mandler et al., Bioorganic & Med. Chem. Letters 10:1025- 1028 (2000); Mandler et al., Bioconjugate Chem. 13:786-791 (2002)), maytansinoids (EP 1391213; Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996)), and calicheamicin (Lode et al., Cancer Res. 58:2928 (1998); Hinman et al., Cancer Res. 53:3336-3342 (1993)). The toxins may exert their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.Attorney Docket No. 01202-0066-00PCT

[0167] In some embodiments, the 41BB-binding conjugate is radionucleotide antibody conjugate (RAC, also called radioconjugates) containing one or more 41BB-binding molecule provided herein conjugated radioactive metal ion for therapeutic purposes. In some embodiments, the radioactive metal ion is an alpha particle emitter such as actinium -225 or radium -223, or a beta particle emitter such as lutetium-177. In some embodiments, 41BB-binding molecule is conjugated to a chelating agent capable of chelating a radioactive metal ion, such as DOTA (also known as tetraxetan, see, e.g., CAS No: 60239-18-1), DTPA (see e.g., CAS No: 67-43-6), CHX-A”-DTPA (see, e.g., CAS No: 142434-84-2), PEPA, see e.g., CAS No: ), Oxo-D03A (see, e.g., CAS No: 112193-74-5), and derivatives of the same.

[0168] In some embodiments, there is provided a 41BB-binding molecule conjugated with a label, which can generate a detectable signal, indirectly or directly. These 41BB-binding conjugates can be used for research or diagnostic applications, such as for the in vivo detection of cancer. The label is preferably capable of producing, either directly or indirectly, a detectable signal. For example, the label may be radio-opaque or a radioisotope, such as 3H, 14C, 32P, 35S, 1231, 1251, 1311; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, [3-galactosidase or horseradish peroxidase; an imaging agent; or a metal ion. In some embodiments, the label is a radioactive atom for scintigraphic studies, for example 99Tc or 1231, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as zirconium-89, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.Zirconium-89 may be complexed to various metal chelating agents and conjugated to antibodies, e.g., for PET imaging (WO 2011 / 056983).

[0169] The 41BB-binding conjugates may be prepared using any methods known in the art. See, e.g., WO 2009 / 067800, WO 2011 / 133886, and U.S. Patent Application Publication No. 2014322129, incorporated by reference herein in their entirety.

[0170] In some embodiments, the attachment can be covalent or non-covalent, e.g., via a biotinstreptavidin, or chelator non-covalent interaction. In some embodiments, 1, 2, 3, 4, 5 or more moieties, which can be the same or different, are conjugated, linked or fused to a 41BB-binding molecule to form a 41BB-binding conjugate. In some embodiments, such moieties can be attached to the 41BB-binding molecule using various molecular biological or chemical conjugation and linkage methods known in the art and described below. In some embodiments, linkers such as peptide linkers, cleavable linkers, non-cleavable linkers or linkers that aid in the conjugation reaction, can be used to link or conjugate the effector moieties the 41BB-binding molecule.

[0171] In some embodiments, a 41BB-binding molecule is conjugated to one or more moieties, e.g. about 1 to about 20 moieties per molecule, through a linker (L). In some embodiments, the 41BB-binding conjugate comprises the following components: (VHH domain), (L)qand (moiety)m, wherein the VHH domain is any of the described VHH domains capable of specifically binding 4 IBB as described; LAttorney Docket No. 01202-0066-00PCT is a linker for linking the protein or polypeptide to the moiety; m is at least 1; q is 0 or more; and the resulting 41BB-binding conjugate binds to 41BB. In particular embodiments, m is 1 to 4 and q is 0 to 8.

[0172] The linker may be composed of one or more linker components. For covalent attachment of the antibody and the drug moiety the linker typically has two reactive functional groups, i.e. bivalency in a reactive sense. Bivalent linker reagents which are useful to attach two or more functional or biologically active moieties, such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups are known, and methods have been described their resulting conjugates (Hermanson, G. T. (1996) Bioconjugate Techniques; Academic Press: New York, p 234-242).

[0173] Non-limiting examples of linker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”), valine -citrulline (“val-cif ’), a alanine -phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-Succinimidyl 4-(2-pyridylthio)pentanoate (“SPP”), N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-I carboxylate (“SMCC”), and N-Succinimidyl (4-iodo-acetyl)aminobenzoate (“SIAB”).

[0174] In some embodiments, the linker may comprise amino acid residues. Exemplary amino acid linker components include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptides include: valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include: glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acid residues which comprise an amino acid linker component include those occurring naturally, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. Amino acid linker components can be designed and optimized in their selectivity for enzymatic cleavage by particular enzymes, for example, a tumor-associated protease, cathepsin B, C and D, at a plasmin protease.

[0175] Conjugates of a 41BB-binding molecule and cytotoxic agent can be made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2 -pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl substrate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene).

[0176] The 41BB-binding conjugates can be prepared by a variety of methods, such as organic chemistry reactions, conditions, and reagents known to those skilled in the art. In one embodiments, methods include: (1) reaction of a nucleophilic group of a 41BB-binding molecule with a bivalent linker reagent, to form VHH-L, via a covalent bond, followed by reaction with a drug moiety D; and (2) reaction of a nucleophilic group of a drug moiety with a bivalent linker reagent, to form D-L, via a covalent bond, followed by reaction with the nucleophilic group of a 41BB-binding molecule.Atorney Docket No. 01202-0066-00PCT

[0177] Nucleophilic groups on antibodies, including 41BB-binding molecules, include, but are not limited to: (i) N-terminal amine groups, (ii) side chain amine groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated.Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups. Additional nucleophilic groups can be introduced into antibodies through the reaction of lysines with 2-iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol. Reactive thiol groups may be introduced into the antibody (or fragment thereof) by introducing one, two, three, four, or more cysteine residues (e.g., preparing mutant antibodies comprising one or more non-native cysteine amino acid residues).

[0178] Conjugates, such as antibody drug conjugates, may also be produced by modification of an antibody, such as a 41BB-binding molecule, to introduce electrophilic moieties, which can react with nucleophilic substituents on the linker reagent or drug. The sugars of glycosylated antibodies may be oxidized, e.g., with periodate oxidizing reagents, to form aldehyde or ketone groups which may lead with the amine group of linker reagents or drug moieties. The resulting imine Schiff base groups may form a stable linkage, or may be reduced, e.g., by borohydride reagents to form stable amine linkages. In one embodiment, reaction of the carbohydrate portion of a glycosylated antibody with either glactose oxidase or sodium meta-periodate may yield carbonyl (aldehyde and ketone) groups in the protein that can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, proteins containing N-terminal serine or threonine residues can react with sodium meta-periodate, resulting in production of an aldehyde in place of the first amino acid. Such aldehyde can be reacted with a drug moiety or linker nucleophile.

[0179] Likewise, nucleophilic groups on a drug moiety include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBi esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups.

[0180] Alternatively, a fusion protein containing a VHH domain and cytotoxic agent may be made, e.g., by recombinant techniques or peptide synthesis. The length of DNA may comprise respective regions encoding the two portions of the conjugate either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate.Attorney Docket No. 01202-0066-00PCT D. Multispecific Formats

[0181] Provided herein are 41BB-binding molecules that are multispecific comprises at least one 4 IBB VHH and one or more additional binding domains that bind one or more additional antigens. In some embodiments, such multispecific 41BB-binding molecules exhibit multispecific binding. Typically, the one or more additional binding domains bind to one or more additional antigen other than 4 IBB. In some embodiments, an additional binding domain is an antibody or antigen-binding fragment specific for the one or more additional antigens. In some embodiments, an additional binding domain is an antigenbinding fragment such as, a Fab fragment, a F(ab')2 fragment, an Fv fragment, a scFv, disulfide stabilized Fv fragment (dsFv), a scAb, a dAb, a single domain heavy chain antibody (VHH), or a single domain light chain antibody. In some embodiments, an additional binding domain is an scFv. In some embodiments, an addition binding domain is a Fab fragment, or a F(ab’)2 fragment. In some embodiments, an additional binding domain is a VHH. In some embodiments, 41BB-binding molecules that are multispecific comprises at least one 4 IBB VHH and at least two additional binding domains that bind an antigen other than 4 IBB. In some embodiments, some or all of the additional binding domains bind the same epitope. In some embodiments, some or all of the additional binding domains bind different antigens or different epitopes on the same antigen. In some embodiments, 41BB-binding molecules that are multispecific comprises at least one 4 IBB VHH and one or more additional binding domains that bind one or more additional antigens and may further comprise an Fc region or other dimerization domain.

[0182] In some embodiments, an additional binding domain binds an antigen selected from the group consisting of: 1-92-LFA-3, 5T4, ADAM-9, Alpha-4 integrin, Alpha-V integrin, alpha4betal integrin, alpha4beta7 integrin, AGR2, Anti-Lewis-Y, Apelin J receptor, APRIL, B7-H3, B7-H4, B7-H6, BAFF, BCMA, BTLA, C5 complement, C-242, CA9, CAI 9-9, (Lewis a), Carbonic anhydrase 9, CD2, CD3, CD6, CD9, CDlla, CD16, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD138, CD166, CD172A, CD248, CDH6, CDH17, CEACAM5 (CEA), CEACAM6 (NCA-90), CLAUDIN-3, CLAUDIN-4, CLAUDIN-18.2, cMet, Collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, Endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, Folate receptor alpha (FRa), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GP Ilb / IIIa receptors, GPC3, Gpl30, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, Hyaluronidase, ICOS, IFNalpha, IFNbeta, IFNgamma, IgE, IgE Receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rbetal, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R (wsxl), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, Insulin Receptor, Jagged Ligands,Attorney Docket No. 01202-0066-00PCT Jagged 1, Jagged 2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPDl, MCSP, Mesothelin, MRP4, MUC1, Mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, NKp46, Nicastrin, Notch Receptors, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, 0X40, PAR2, PDGF-AA, PDGF-BB, PDGFRalpha, PDGFRbeta, PD-1, PD-L1, PD-L2, Phosphatidyl-serine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, R0R1, SIGLEC-6, SLC44A4, Sphingosine 1 Phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFbeta, TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFalpha, TNFR, TNFR2, NFRS12A, TRAIL-R1, TRAIL-R2, Transferrin, Transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, and WISP-3.

[0183] In some embodiments, a 41BB-binding molecule comprises at least one 4 IBB VHH and at least one additional binding domain that binds an additional antigen. In some embodiments, the binding to the additional antigen is capable of providing the additional crosslinking function and 4 IBB activation can be achieved with only one or two 41BB-binding domains. In some embodiments, 4 IBB signaling is enhanced and focused by the presence of the additional antigen. In some embodiments, such multispecific molecules exhibit greatly enhanced 41BB-dependent signaling when the multispecific molecule is bound to a cell expressing the additional antigen.

[0184] In some embodiments, an additional antigen is a tumor associated antigen (TAA) or tumor microenvironment associated antigen (TMEAA) (e.g., Folate Receptor Alpha, FRa). In some embodiments, the binding to the TAA or TMEAA antigen is capable of providing the additional crosslinking function and 41BB activation can be achieved with only one or two 41BB-binding domains.

[0185] In some embodiments, an additional antigen is an immunomodulatory antigen. In some embodiments, the immunomodulatory antigen is involved with enhancing or dampening a signaling pathway in an immune cell. In some embodiments, the immunomodulatory antigen is an immune checkpoint antigen (e.g., PD-L1, CTLA-4). In some embodiments, binding to the immune checkpoint antigen inhibits the immune checkpoint antigen leading to enhanced immune cell activation. In some embodiments, such 41BB-binding molecule modulates immune cells leading to enhanced tumor destruction. In some embodiments, the binding to the immunomodulatory antigen is capable of providing the additional crosslinking function and 4 IBB activation can be achieved with only one or two 41 BB -binding domains .

[0186] In some embodiments, an additional antigen is a additional TNFRSF member for example GITR, 0X40, CD27, TNFR2 and / or CD40. In some embodiments, such 41BB-binding molecules are capable of modulating immune cells leading to enhanced tumor destruction. In other embodiments, such 41BB-binding molecules have utility in treating inflammatory conditions. In these embodiments, such 41BB-binding molecules modulate immune cells leading to dampening of the inflammatory insult. For example, specifically agonizing TNFR2 can enhance Treg proliferation leading to immune suppression.Attorney Docket No. 01202-0066-00PCT In some embodiments, the binding to the additional TNFR is capable of providing the additional crosslinking function and 41BB activation can be achieved with only one or two 41BB-binding domains.

[0187] In some embodiments, a 41BB-binding molecule further comprises at least one binding domain that binds to an activating receptor on an immune cell. In some embodiments the immune cell is a T cell and / or an NK cell. In some embodiments the immune cell is a T cell and the activating receptor is CD3 or a CD3 complex. In some embodiments the immune cells is a T cell and the activating receptor is a T cell receptor (“TCR”) component, or a TCR complex. In some embodiments, the additional binding domain binds an alpha (a), beta (P), delta (5), and / or gamma (y) chain of a TCR. In some embodiments, the additional binding domain that binds an activating receptor binds a gamma and / or delta chain of a TCR. In some embodiments the immune cell is an NK cell and the activating receptor is CD16a or NKp46. In some embodiments, a 41BB-binding molecule comprises one binding domain that binds to an activating receptor on an immune cell. In some embodiments, a 41BB-binding molecule comprises two binding domains that bind to an activating receptor on an immune cell. In some embodiments, some or all of the binding domains that bind to an activating receptor on an immune cell bind the same epitope. In some embodiments, some or all of the binding domains that bind to an activating receptor on an immune cell bind different antigens or different epitopes on the same antigen. In some embodiments, 41BB-binding molecules that are multispecific comprises at least one 4 IBB VHH and one or more additional binding domains that bind to an activating receptor on an immune cell and may further comprise an Fc region or other dimerization domain.

[0188] In some embodiments, a 41BB-binding molecule further comprises at least one binding domain for an antigen expressed on the surface of a target cell (also referred to herein as a target cell surface antigen or a “TCSA”), which may be for example an antigen expressed on a cancer cells or an antigen expressed on pathogenic B cells, or auto-reactive T cells and at least one binding domain for an antigen present on the surface of an immune effector cell (e.g. , a T cell or an NK cell). In some embodiments, the antigen present on the surface of a immune effector cell is an activating receptor. In some embodiments, such 41BB-binding molecules are capable of engaging or activating immune effectors cells at the site of expression of the TCSA (e.g., a tumor, pathogenic B cells, or auto-reactive T cells) upon binding of the TCSA. In some embodiments, such 41BB-binding molecules bind 4 IBB, a TCSA and a T cell antigen, such as CD3 or TCR. In some embodiments, 41BB-binding molecules bind a TCSA and CD3 and exhibit constrained CD3 binding. In some embodiments, such 41BB-binding molecules are capable of engaging or activating T cells at the site of expression of the TCSA (e.g., a tumor, pathogenic B cells, or auto-reactive T cells) upon binding of the TCSA. In some embodiments, the 41BB-binding molecule mediates enhanced T cell-mediated cell killing of TCSA expressing cells by cosimulating 41BB. In some embodiments, the 41BB-binding molecules bind 41BB, a TCSA, and an NK cell antigen, such as NKp46 or CD 16. In some embodiments, such multispecific molecules are capable of engaging or activating NK cells at the site of a tumor upon binding of the TCSA. In some embodiments,Attorney Docket No. 01202-0066-00PCT the 41BB-binding molecule mediates enhanced NK cell-mediated cell killing of TCSA expressing cells by co-simulating 4 IBB.

[0189] In some embodiments, a TCSA-binding domain binds an antigen selected from the group consisting of: 1-92-LFA-3, 5T4, ADAM-9, Alpha-4 integrin, Alpha-V integrin, alpha4betal integrin, alpha4beta7 integrin, AGR2, Anti-Lewis-Y, Apelin J receptor, APRIL, B7-H3, B7-H4, B7-H6, BAFF, BCMA, BTLA, C5 complement, C-242, CA9, CAI 9-9, (Lewis a), Carbonic anhydrase 9, CD2, CD3, CD6, CD9, CDlla, CD16, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD138, CD166, CD172A, CD248, CDH6, CDH17, CEACAM5 (CEA), CEACAM6 (NCA-90), CLAUDIN-3, CLAUDIN-4, CLAUDIN-18.2, cMet, Collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, Endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, Folate receptor alpha (FRa), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GP Ilb / IIIa receptors, GPC3, Gpl30, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, Hyaluronidase, ICOS, IFNalpha, IFNbeta, IFNgamma, IgE, IgE Receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rbetal, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R (wsxl), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, Insulin Receptor, Jagged Ligands, Jagged 1, Jagged 2, KISSl-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPDl, MCSP, Mesothelin, MRP4, MUC1, Mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, NKp46, Nicastrin, Notch Receptors, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, 0X40, PAR2, PDGF-AA, PDGF-BB, PDGFRalpha, PDGFRbeta, PD-1, PD-L1, PD-L2, Phosphatidyl-serine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, R0R1, SIGLEC-6, SLC44A4, Sphingosine 1 Phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFbeta, TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFalpha, TNFR, TNFR2, NFRS12A, TRAIL-R1, TRAIL-R2, Transferrin, Transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, and WISP-3.

[0190] In some embodiments, a 41BB-binding molecule can further contain an Fc region, such as any described above. In some embodiments, a multispecific 41BB-binding molecule provided herein at least one VHH that binds 4 IBB, at least one additional binding domain that binds a second antigen, and an Fc region. In some embodiments, an Fc region mediates dimerization of the multispecific 41BB-binding molecule at physiological conditions such that a dimer is formed that doubles the number of binding sites for 41BB and for the additional antigen.

[0191] Non-limiting exemplary multispecific 41BB-binding molecules are described herein.Attorney Docket No. 01202-0066-00PCT

[0192] In one embodiment, the 41BB-binding molecule is a multispecific single-domain antibody-linked Fab (S-Fab) containing at least one 4 IBB VHH linked, directly or indirectly to at least one Fab specific for an additional antigen. In some embodiments, the 4 IBB VHH is linked to the C-terminus of the VH or VL of the Fab specific for the additional antigen. In some embodiments, the S-Fab can be further modified, such as by conjugation with polyethylene glycol (PEG), N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers, proteins (such as albumin), polyglutamic acid or PASylation (Pan et al. (2018) International Journal of Nanomedicine, 2018:3189-3201).

[0193] In another embodiment, the multispecific molecule is a scFv-single domain antibody in which the molecule comprises at least one 4 IBB VHH as described linked, directly or indirectly, to at least one scFv containing a VH and a VL of an antigen binding domain specific for an additional antigen. In some embodiments, the VHH domain and the scFv are connected by a linker, such as a peptide linker. In some embodiments, the peptide linker can be a peptide linker as described herein. In some embodiments, the VHH domain and the scFv are each connected, optionally through a hinge region or a linker (e.g. peptide linker), to an Fc region, such as the N-terminus and / or C-terminus of an Fc region. In some embodiments, the Fc region can be an Fc region as described herein. In particular examples, the Fc region is formed by variant Fc regions, e.g. variant human IgGl domains, that are mutated or modified to promote heterodimerization in which different Fc polypeptides can be dimerized to yield a heterodimer.

[0194] In another embodiment, the multispecific molecule is a single domain antibody molecule comprising at least one 4 IBB VHH as described linked, directly or indirectly, to at least one VHH specific for the additional antigen. For formatting the molecules, in some cases, each VHH domain is connected, optionally through a hinge region or linker (e.g. peptide linker), to an Fc region, such as the N-terminus and / or C-terminus of an Fc region. The Fc region can be any described herein, such as a human Fc region or a variant thereof, e.g. a human IgGl Fc region or a variant thereof. In particular examples, the Fc region is formed by variant Fc regions, e.g. variant human IgGl domains, that are mutated or modified to promote heterodimerization in which different Fc polypeptides can be dimerized to yield a heterodimer.1. T cells Engager

[0195] In some embodiments, the 41BB-binding molecule is a multispecific molecule that comprises at least one 4 IBB VHH provided herein, at least one additional binding domain capable of binding a target cell surface antigen (“TCSA”), and at least one additional binding domain capable of binding to a surface molecule expressed on a T cell. In some embodiments, the T cell surface molecule is an activating component of a T cell, such as a component of the T cell receptor complex. In some embodiments, the T cell surface molecule is an activating T cell antigen that is expressed on a T cell and is capable of inducing T cell activation upon interaction with the TCSA. For example, in some embodiments, interaction of a TCSA with an activating T cell antigen may induce T cell activation byAttorney Docket No. 01202-0066-00PCT triggering the signaling cascade of the T cell receptor complex. Suitable assays to measure T cell activation are known, and include any assay to measure or assess proliferation, differentiation, cytokine secretion, cytotoxic activity and / or expression of one or more activation marker. In some embodiments, the simultaneous or near simultaneous binding of such a 41BB-binding molecule to both of its targets, 41BB expressed on target cell and a T cell molecule expressed on a T cell, e.g. activating T cell antigen, can result in a temporary interaction between the target cell and T cell, thereby resulting in activation, e.g. cytotoxic activity, of the T cell and subsequent lysis of the target cell. In some embodiments, the further engagement of 4 IBB mediates enhanced T cell-mediated cell killing of TCSA expressing cells by co-simulating 4 IBB.

[0196] In some embodiments, the T cell surface molecule, such as activating T cell antigen, is CD3, T cell receptor (“TCR” e.g., y5 TCR, V|3 TCR), or is CD2. Specifically, a provided bispecific 41BB-binding molecule is capable of specifically binding an activating T cell antigen expressed on a human T cell, such as human CD3, human y5 TCR, or CD2. In some embodiments, the T cell binding domain that is specific to the activating T cell antigen (e.g. CD3, y5 TCR, or CD2) is an antibody or antigen-binding fragment. In some embodiments, a 41BB-binding molecule can be a multispecific antibody T cellengager containing at least one 4 IBB VHH that specifically binds to 4 IBB, at least one additional binding domain specific for a TCSA (also referred to herein as a “TCSA-binding domain”) and at least one additional binding domain that is an antibody or antigen-binding fragment specific for an activating component of a T cell (e.g. a T cell surface molecule, e.g. CD3, y5 TCR, or CD2). In some embodiments, the binding domain specific to an activating T cell antigen is an antigen-binding fragment selected from a Fab fragment, a F(ab')2 fragment, an Fv fragment, a scFv, disulfide stabilized Fv fragment (dsFv), a scAb, a dAb, a single domain heavy chain (VHH), or a single domain light chain. In some embodiments, the additional binding domain is monovalent for binding the activating T cell antigen, such as CD2 or CD3.a. Multispecific T cell engagers

[0197] Numerous bispecific antibody T cell-engagers have been described and include bispecific T cell engager (BiTE) molecules, which contain tandem scFv molecules fused by a flexible linker (see e.g. Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011); tandem scFv molecules fused to each other via, e.g. a flexible linker, and that further contain an Fc region composed of a first and a second subunit capable of stable association (WO2013026837); diabodies and derivatives thereof, including tandem diabodies (Holliger et al, Prot Eng 9, 299-305 (1996); Kipriyanov et al, J Mol Biol 293, 41-66 (1999)); dual affinity retargeting (DART) molecules that can include the diabody format with a C-terminal disulfide bridge; or triomabs that include whole hybrid mouse / rat IgG molecules (Seimetz et al, Cancer Treat Rev 36, 458-467 (2010); various heterodimeric antibodies that bind CD3 and tumor antigens comprising a Fab domain and an scFv or two Fab domains, optionally comprising an Fc regionAtorney Docket No. 01202-0066-00PCT (WO2016086189; WO2017223111, WO2012143524); scFv molecules fused to each other via abridging domain (W02013101909); multispecific molecules containing a constrained CD3 binding domain, which is not able to, or is not substantially able to, bind or engage cell surface CD3 unless at least one target cell expressed antigen binding domain is bound to its antigen (WO2018191438, W02019200022); and multispecific molecules that bind 4 IBB, CD3, and a third antigen comprising Fab domains, which do not bind CD3 and 4 IBB at the same time (W02020067419). Multispecific antibodies that bind the constant or variable regions of alpha, beta, delta and / or gamma TCR chains have been described, and include, multispecific antibodies that bind TCR Vy9V52 (e.g. , WO2022093888, WO2023242319); the TCR gamma / delta constant region (e.g., WO2022093888); TCR alpha / beta (e.g., WO2016180969); TCR VP regions (e.g., W02020010250, WO2021138474) and multispecific antibodies comprising single domain antibodies that bind y5 TCR (e.g., WO2023133394). Multispecific T cell engagers that further bind 41BB can be generated by incorporating at least one 41BB-binding domain into the formats of any of the above molecules using any of the 4 IBB VHHs provided herein. In some embodiments, engagement of 4 IBB enhances T cell activation.

[0198] In some embodiments, the binding domain specific to an activating receptor on a T cell is capable of binding to CD3 or a CD3 complex. A CD3 complex is a complex of at least five membranebound polypeptides in mature T-lymphocytes that are non-covalently associated with one another and with the T-cell receptor. The CD3 complex includes the gamma, delta, epsilon, zeta, and eta chains (also referred to as subunits). In some embodiments, the additional binding domain is an antibody or antigenbinding fragment capable of specifically binding to CD3 or a CD3 complex, also called a CD3 -binding domain. In some embodiments, the CD3-binding domain capable of binding CD3 or a CD3 complex includes one or more copies of an anti-CD3 Fab fragment, an anti-CD3 F(ab')2 fragment, an anti-CD3 Fv fragment, an anti-CD3 scFv, an anti-CD3 dsFv, an anti-CD3 diabody, an anti-CD3 scAb, an anti-CD3 dAb, an anti-CD3 single domain heavy chain antibody (VHH), and an anti-CD3 single domain light chain antibody. In some embodiments, the anti-CD3 binding domain is monovalent for binding CD3. In some cases, the CD3-binding domain recognizes the CD3s-chain.

[0199] In some embodiments, the binding domain specific to an activating receptor on a T cell is capable of binding to a T cell receptor (“TCR”) component or a TCR complex. A TCR complex typically comprises alpha and beta chains, or delta, and gamma chains. In some embodiments, the additional binding domain is an antibody or antigen-binding fragment capable of specifically binding to a TCR component or a TCR complex, also called a TCR-binding domain. In some embodiments, the TCR-binding domain capable of binding TCR or a TCR complex includes one or more copies of an anti -TCR Fab fragment, an anti-TCR F(ab')2 fragment, an anti- TCR Fv fragment, an anti-TCR scFv, an anti-TCR dsFv, an anti-TCR diabody, an anti-TCR scAb, an anti-TCR dAb, an anti-TCR single domain heavy chain antibody (VHH), and an anti-TCR single domain light chain antibody. In some embodiments, theAttorney Docket No. 01202-0066-00PCT anti-TCR binding domain is monovalent for binding TCR. In some cases, the TCR-binding domain recognizes the gamma and / or delta chain of a TCR.b. Constrained CD3 Multispecific Molecules

[0200] Multispecific molecules containing a constrained CD3 binding domain, which are not able to, or are not substantially able to, bind or engage cell surface CD3 unless at least one target cell surface antigen binding domain is bound to its antigen have been described (WO2018191438, W02019200022, and W02020023553). In particular, W02020023553 describes such constrained CD3 multispecific molecules that further comprise a co-stimulatory receptor binding region such as a 41BB-binding domain (e.g., a 4 IBB VHH). In some embodiments, a 41BB-binding molecule is a constrained T-cell engaging fusion protein that binds 4 IBB, an activating T cell antigen such as CD3 and a target cell surface antigen (“TCSA”).

[0201] Such 41BB-binding molecules comprise a first component containing an immunoglobulin Fc region and a second component comprising a CD3 -binding region, wherein the first and second components are coupled by a linker, the Fc region is positioned N-terminal to the CD3-binding region; and one or both of the first and second components comprises (1) at least one “TCSA-binding domain” and (2) at least one 41BB-binding domain.

[0202] In some embodiments, the 41BB-binding molecule contains in order, from N-terminus to C-terminus: an immunoglobulin Fc region; a linker; a CD3 binding region that binds CD3 (CD3e); and at least two binding domains where one is TCSA-binding domain, and the other is a 4 IBB VHH.

[0203] In some embodiments, the 41BB-binding molecule contains in order, from N-terminus to C-terminus: at least two binding domains where one is TCSA-binding domain, and the other is a 4 IBB VHH; an immunoglobulin Fc region; a linker; and a CD3 binding region that binds CD3 (CD3e).

[0204] In some embodiments, the 41BB-binding molecule contains, in order from N-terminus to C-terminus: at least one binding domain that binds a TCSA or 4 IBB; an immunoglobulin Fc region; a linker; a CD3 binding region that binds CD3 (CD3s); and at least one binding domain that binds the other of a TCSA or 41BB. In some embodiments, the N- or C-terminal portion of the 41BB-binding molecule contains one TCSA-binding domain. In some embodiments, the N- or C-terminal portion of the 41BB-binding molecule contains at least two TCSA-binding domains. In some embodiments, at least two of the TCSA-binding domains are the same. In some embodiments, each of the TCSA-binding domains is the same. In some embodiments, each of the TCSA-binding domains bind the same epitope of the TCSA. In some embodiments, at least two of the TCSA-binding domains bind to different TCSAs or to different epitopes of the same TCSA.

[0205] In some embodiments, the 41BB-binding molecule contains, in order, from N-terminus to C-terminus: a 41BB VHH; an immunoglobulin Fc region; a linker; a CD3 binding region that binds CD3 (CD3e); and at least one TCSA-binding domain. In some embodiments, the C-terminal portion of theAttorney Docket No. 01202-0066-00PCT 41BB-binding molecule contains one TCSA-binding domain. In some embodiments, the C-terminal portion of the 41BB-binding molecule contains two or more TCSA-binding domains. In some embodiments, at least two of the TCSA-binding domains are the same. In some embodiments, each of the TCSA-binding domain is the same. In some embodiments, each of the TCSA-binding domains bind the same epitope of the TCSA. In some embodiments, at least two of the TCSA-binding domains bind to different TCSAs or to a different epitope of the same TCSA.

[0206] In some embodiments, the 41BB-binding molecule contains, in order, from N-terminus to C-terminus: at least one TCSA-binding domain; an immunoglobulin Fc region; a linker; a CD3 binding region that binds CD3 (CD3e); and a 4 IBB VHH. In some embodiments, the N-terminal portion of the 41BB-binding molecule contains one TCSA-binding domain. In some embodiments, the N-terminal portion of the 41BB-binding molecule contains two or more TCSA-binding domains. In some embodiments, at least two of the TCSA-binding domains are the same. In some embodiments, each of the TCSA-binding domains is the same. In some embodiments, each of the TCSA-binding domains bind the same epitope of the TCSA. In some embodiments, at least two of the TCSA-binding domains bind to a different TCSAs or to a different epitope of the same TCSA.

[0207] In some embodiments, the 41BB-binding molecule contains in order, from N-terminus to C-terminus: at least one TCSA-binding domain; an immunoglobulin Fc region; a linker; a CD3 binding region that binds CD3 (CD3e); and at least two binding domains where at least one is a TCSA-binding domain and at least one is a 4 IBB VHH. In some embodiments, the C-terminal portion of the 41BB-binding molecule contains at least two TCSA-binding domains. In some embodiments, at least two of the TCSA-binding domains are the same. In some embodiments, each of the TCSA-binding domains is the same. In some embodiments, each of the TCSA-binding domains bind the same epitope of the TCSA. In some embodiments, at least two of the TCSA-binding domains bind to a different TCSAs or to a different epitope of the same TCSA.

[0208] In some embodiments, the 41BB-binding molecule contains in order, from N-terminus to C-terminus: at least two binding domains where one is TCSA-binding domain and the other is a 41BB-binding domain; an immunoglobulin Fc region; a linker; a CD3 binding region that binds CD3 (CD3s); and at least one TCSA-binding domain. In some embodiments, the N-terminal portion of the 41BB-binding molecule contains two TCSA-binding domains. In some embodiments, at least two of the TCSA-binding domains are the same. In some embodiments, each of the TCSA-binding domains is the same. In some embodiments, each of the TCSA-binding domains bind the same epitope of the TCSA. In some embodiments, at least two of the TCSA-binding domains bind to a different TCSAs or to a different epitope of the same TCSA.

[0209] In some embodiments, the 41BB-binding molecule is a dimer, in which dimerization is formed by covalent or non-covalent interactions between two polypeptide chains. In some embodiments, the two polypeptide chains are covalently bonded to each other by, for example, interchain disulfideAttorney Docket No. 01202-0066-00PCT bonds. In some embodiments, the Fc region mediates dimerization via interchain disulfide bonds. In some embodiments, the 41BB-binding molecule contains a homodimeric Fc region in which, in some cases, both polypeptide chains of the multispecific polypeptide construct are identical (homodimer). In some embodiments, the 41BB-binding molecule contains a heterodimeric Fc region in which, in some cases, the polypeptide chains of the 41BB-binding molecule are different (heterodimer). In some embodiments, the 41BB-binding molecule, the CD3-binding region is a two chain polypeptide containing a VH and a VL, such as is an Fv antibody fragment containing the VH and VL. In some embodiments, the Fv antibody fragment includes a disulfide stabilized anti-CD3 binding Fv fragment (dsFv).

[0210] In some embodiments, the 41BB-binding molecule is formed from or includes at least two polypeptide chains, including a first polypeptide comprising a first Fc polypeptide of a heterodimeric Fc region, a linker, a VH of an anti-CD3 antibody or antigen binding fragment (e.g. Fv); and a second polypeptide comprising a second Fc polypeptide of the heterodimeric Fc region, the linker, a VL of the anti-CD3 antibody or antigen binding fragment (e.g. Fv). In some embodiments, the first polypeptide contains at least one TCSA-binding domain. In some embodiments, the second polypeptide contains at least one TCSA-binding domain. In some embodiments, at least one TCSA-binding domain is located N-terminally to the Fc polypeptide and / or C-terminally to the chain of the CD3-binding region. In some embodiments, the first polypeptide or second polypeptide or both the first and second polypeptide further includes a 41BB-binding domain, such that the 41BB-binding molecule contains at least one 41BB-binding domain and at least one TCSA binding domain. In some embodiments, the 41BB-binding molecule contains two 41BB-binding domains. In some embodiments, the 41BB-binding domain of the first and / or second polypeptide can be located N-terminally to the Fc polypeptide and / or C-terminally to the chain of the CD3-binding region.

[0211] In some embodiments, the 41BB-binding molecule contains at least two TCSA- binding domains and at least one 41BB-binding domain. In some such embodiments, the 41BB-binding molecule contains (1) a first polypeptide comprising in order of N-terminus to C-terminus: a first TCSA binding domain, the first Fc polypeptide of a heterodimeric Fc region, a linker (e.g. a cleavable linker), a chain (e.g. VH or VL) of an anti-CD3 antibody or antigen binding fragment (e.g. Fv or dsFv); and (2) a second polypeptide comprising in order of N-terminus to C-terminus: a second TCSA binding domain, the second Fc polypeptide of the heterodimeric Fc region, the same linker (e.g. same cleavable linker), the other chain (other of the VH or VL) of the anti-CD3 antibody or antigen binding fragment, and a 41BB-binding domain. In some such embodiments, the 41BB-binding molecule contains (1) a first polypeptide comprising in order of N-terminus to C-terminus: a first TCSA-binding domain, the first Fc polypeptide of a heterodimeric Fc region, a linker (e.g. a cleavable linker), a chain (e.g. VH or VL) of an anti-CD3 antibody or antigen binding fragment (e.g. Fv or dsFv), and a second TCSA-binding domain; and (2) a second polypeptide comprising in order of N-terminus to C-terminus: a 41BB-binding domain, the second Fc polypeptide of the heterodimeric Fc region, the same linker (e.g. same cleavable linker), andAttorney Docket No. 01202-0066-00PCT the other chain (other of the VH or VL) of the anti-CD3 antibody or antigen binding fragment. In some such embodiments, the 41BB-binding molecule contains (1) a first polypeptide comprising in order of N-terminus to C-terminus: a first TCSA-binding domain, the first Fc polypeptide of a heterodimeric Fc region, a linker (e.g. a cleavable linker), a chain (e.g. VH or VL) of an anti-CD3 antibody or antigen binding fragment (e.g. Fv or dsFv), and a second TCSA-binding domain; and (2) a second polypeptide comprising in order of N-terminus to C-terminus: the second Fc polypeptide of the heterodimeric Fc region, the same linker (e.g. same cleavable linker), the other chain (other of the VH or VL) of the anti-CD3 antibody or antigen binding fragment, and a 41BB-binding domain.

[0212] In some embodiments, the 41BB-binding molecule contains at least two 4 IBB- binding domains and at least one TCSA-binding domain. In some such embodiments, the 41BB-binding molecule contains (1) a first polypeptide comprising in order of N-terminus to C-terminus: a first 41BB-binding domain, the first Fc polypeptide of a heterodimeric Fc region, a linker (e.g. a cleavable linker), a chain (e.g. VH or VL) of an anti-CD3 antibody or antigen binding fragment (e.g. Fv or dsFv); and (2) a second polypeptide comprising in order of N-terminus to C-terminus: a 41BB- binding domain, the second Fc polypeptide of the heterodimeric Fc region, the same linker (e.g. same cleavable linker), the other chain (other of the VH or VL) of the anti-CD3 antibody or antigen binding fragment, and a TCSA-binding domain. In some such embodiments, the 41BB-binding molecule contains (1) a first polypeptide comprising in order of N-terminus to C-terminus: a first 41 BB-binding domain, the first Fc polypeptide of a heterodimeric Fc region, a linker (e.g. a cleavable linker), a chain (e.g. VH or VL) of an anti-CD3 antibody or antigen binding fragment (e.g. Fv or dsFv), and a second 41BB-binding domain; and (2) a second polypeptide comprising in order of N-terminus to C-terminus: a TCSA-binding domain, the second Fc polypeptide of the heterodimeric Fc region, the same linker (e.g. same cleavable linker), and the other chain (other of the VH or VL) of the anti-CD3 antibody or antigen binding fragment. In some such embodiments, the 41BB-binding molecule contains (1) a first polypeptide comprising in order of N-terminus to C-terminus: a first 41 BB-binding domain, the first Fc polypeptide of a heterodimeric Fc region, a linker (e.g. a cleavable linker), a chain (e.g. VH or VL) of an anti-CD3 antibody or antigen binding fragment (e.g. Fv or dsFv), and a second 41BB-binding domain; and (2) a second polypeptide comprising in order of N-terminus to C-terminus: the second Fc polypeptide of the heterodimeric Fc region, the same linker (e.g. same cleavable linker), the other chain (other of the VH or VL) of the anti-CD3 antibody or antigen binding fragment, and a TCSA-binding domain.

[0213] It is provided that in any of the above embodiments of 41BB-binding molecules exhibiting constrained CD3 binding, the first and / or the second polypeptide may further comprise one or more additional TCSA-binding domains, which may be positioned N-terminal to the Fc region and / or C-terminal to the CD3-binding domain. It is further provided that in any of the above embodiments, the first and / or the second polypeptide may comprise one or more additional 41 BB-binding domains, which may be positioned N-terminal to the Fc region and / or C-terminal to the CD3-binding domain. In someAtorney Docket No. 01202-0066-00PCT embodiments, each of the TCSA-binding domains is the same. In some embodiments, at least two of the TCSA-binding domains bind to a different TCSAs or to a different epitope of the same TCSA. FIG. 5 depicts representative formats of 41BB-binding molecules exhibiting constrained CD3 binding.

[0214] In some of the provided embodiments, the 41BB-binding molecule is a T cell engager comprising at least at least one 4 IBB VHH provided herein. In some embodiments, the 4 IBB VHH comprises a CDR1, CDR2 and CDR3 sequences, under any CDR definitions provided in Table 3 above. In some embodiments, the 4 IBB VHH comprises a CDR1, CDR2 and CDR3 sequences, under any CDR definition provided in Table 3 above and has an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a VHH of any one of SEQ ID NOs: 43-45, 98 and 99. In some embodiments, the 41BB VHH has the amino acid sequence of any one of SEQ ID NOS: 43-45, 98, and 99. Additional embodiments of 41BB VHHs which may be incorporated into a 41BB-binding molecule that is a T cell engager are provided herein.

[0215] In some of the provided embodiments, a 41BB-binding molecule that is a T cell engager comprises a TCSA-binding domain that binds an antigen selected from any of those provided here.

[0216] In some of the provided embodiments, a 41BB-binding molecule is a T cell engager comprising an Fc region, wherein the Fc region comprises at least one of the Fc region sequences provided herein. In some such embodiments the 41BB-binding molecule comprises a homodimer Fc region. In some such embodiments the homodimer Fc region comprises the sequence of any of SEQ ID NO: 1-4, 12-17, or 30-32, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1-4, 12-17, and 30-32. In some such embodiments the 41BB-binding molecule comprises a heterodimeric Fc region comprising any of the paired or complementary amino acid modifications provided herein. In some embodiments, the heterodimeric Fc region comprises “knob” and “hole” modifications. In some embodiments, the first Fc region comprises any of SEQ ID NOs: 18-23, or comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 18-23. In some embodiments, the second Fc region comprises any of SEQ ID NOs: 24-29, or comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs:24-29. Additional embodiments of Fc regions which may be incorporated into a 41BB-binding molecule that is a T cell engager are provided herein.

[0217] In some of the provided embodiments, a 41BB-binding molecule that is a T cell engager comprises a linker, wherein the linker comprises at least one of the linker sequences provided herein. In some embodiments, the TCSA-binding domain, the 4 IBB VHH, and / or the CD3 -binding domain are linked, directly or indirectly via a linker. In some embodiments, the 4 IBB VHH, TCSA-binding domain, and / or the CD3-binding domain are linked, directly or indirectly via a linker, to the Fc region. In some embodiments, the linker is a linking peptide (LP), which can include any flexible or rigid linker including any of the linkers described herein. In some embodiments, the linker is selected from the groupAtorney Docket No. 01202-0066-00PCT consisting of SEQ ID NOs: 33-40 and 82-84. Additional embodiments of linking peptides which may be incorporated into a 41BB-binding molecule that is a T cell engager are provided herein.i. CD3-Binding Domains

[0218] Exemplary monoclonal antibodies against CD3 or a CD3 complex which may be incorporated into a 41BB-binding molecule that is a T-cell engager include, but are not limited to, OKT3, SP34, UCHT1 or 64.1, or an antigen-binding fragment thereof (See e.g., June, et al., J. Immunol.136:3945-3952 (1986); Yang, et al., J. Immunol. 137: 1097-1100 (1986); and Hayward, et al., Immunol.64:87-92 (1988)). In one embodiment, the CD3-binding domain specifically binds a CD3 antigen, and is derived from OKT3 (ORTHOCLONE-OKT3™ (muromonab-CD3); humanized OKT3 (U.S. Pat. No. 7,635,475 and published international application No. W02005040220); SP34 (Pessano et ai. The EMBO Journal. 4: 337-344, 1985); humanized variant of SP34 (W02015001085); Teplizumab™ (MGA031, Eli Lilly); an anti-CD3 binding molecule described in US2011 / 0275787; UCHT1 (Pollard et al. 1987 J Histochem Cytochem. 35(11): 1329-38; W02000041474); NI0401 (W02007 / 033230); visilizumab (US Patent No. 5,834,597); BC-3 (Anaseti et al., Transplantation 54: 844 (1992); H2C (described in PCT publication no. W02008 / 119567); V9 (described in Rodrigues et al., Int J Cancer Suppl 7, 45-50 (1992) and U.S. Pat. No. 6,054,297)) single domain antibodies, including VHH domains (see e.g. published U.S. patent application No. US20160280795). Other anti-CD3 antibodies also can be used in the molecules provided herein, including any described in International published PCT application Nos. WO199404679; W02008119567; WO2015095392; WO2016204966; WO2017053856; W02018052503; WO2019133761; W02019160904; W02019200022; WO2019244107;WO2019224715; WO2022171192; WO2022266660; W02023014809; WO2023061417;WO2023061419; WO2023125611; W02025002014; published patent application Nos. US20170369563, US20180194842, US20180355038, US20190270810; U.S. Patent Nos. 7,728,114, 7,381,803, 7,994,289, 11,472,880.

[0219] In some embodiments, the CD3-binding domain has a disulfide stabilized linkage between the VH and VL regions. In some embodiments, CD3-binding domain is disulfide stabilized Fv (dsFv) in which the VH-VL heterodimer is stabilized by an interchain disulfide bond. In some embodiments, the disulfide stabilized anti-CD3 Fv comprises an anti-CD3 VH with the mutation 44 to Cys and an anti-CD3 VL with the mutation 100 to Cys by Kabat numbering. In some embodiments, the disulfide stabilized anti-CD3 Fv comprises an anti-CD3 VH with the mutation G44C and an anti-CD3 VL with the mutation G100C by Kabat numbering. In some embodiments, the disulfide stabilized anti-CD3 Fv comprises an anti-CD3 VH with the mutation at position 105 to Cys and an anti-CD3 VL with the mutation position 43 to Cys by Kabat numbering.

[0220] In some embodiments, the CD3 -binding domain contains a VH sequence set forth in SEQ ID NO: 85 or 87 and / or a VL sequence set forth in SEQ ID NO: 86 or 88 or VH and / or VL sequencesAttorney Docket No. 01202-0066-00PCT having at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% identity with these sequences and specifically binds CD3. In some embodiments, the CD3-binding domain contains a VH sequence set forth in SEQ ID NO: 85 and a VL sequence set forth in SEQ ID NO: 86 or VH and / or VL sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% identity with these sequences and specifically binds CD3. In some embodiments, the CD3-binding domain contains a VH sequence set forth in SEQ ID NO: 87 and a VL sequence set forth in SEQ ID NO: 88 or VH and / or VL sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% identity with these sequences and specifically binds CD3.

[0221] In some embodiments, the CD3 binding domain includes a VH CDR1 sequence that includes at least the amino acid sequence GFTFNTYAMN (SEQ ID NO: 89); a VH CD2 sequence that includes at least the amino acid sequence RIRSKYNNYATY (SEQ ID NO: 90); a VH CDR3 sequence that includes at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 91), a VL CDR1 sequence that includes at least the amino acid sequence GSSTGAVTTSNYAN (SEQ ID NO: 93); a VL CDR2 sequence that includes at least the amino acid sequence GTNKRAP (SEQ ID NO: 94); and a VL CDR3 sequence that includes at least the amino acid sequence ALWYSNHWV (SEQ ID NO: 96). In some embodiments, the CD3-binding domain includes a VH CDR1 sequence that includes at least the amino acid sequence GFTFNTYAMN (SEQ ID NO: 89); a VH CD2 sequence that includes at least the amino acid sequence RIRSKYNNYATY (SEQ ID NO: 90); a VH CDR3 sequence that includes at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 91), a VL CDR1 sequence that includes at least the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 92); a VL CDR2 sequence that includes at least the amino acid sequence GTNKRAP (SEQ ID NO: 94); and a VL CDR3 sequence that includes at least the amino acid sequence ALWYSNLWV (SEQ ID NO: 95).

[0222] In some embodiments, the CD3 -binding domain contains a VH that comprises CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:89, 90, and 91, respectively, and has an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a VH of any one of SEQ ID NOs: 85 or 87. In some embodiments, the CD3-binding domain contains a VL that comprises CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: SEQ ID NOs: 93, 94 and 96, respectively, and has an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a VL of any one of SEQ ID NOs: 88. In some embodiments, the CD3 -binding domain contains a VL that comprises CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 92, 94, and 95, respectively and has an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a VL of any one of SEQ ID NOs: 86.2. NK cell recruitment

[0223] In some embodiments, the 41BB-binding molecule is a multispecific molecule that is or comprises at least one 4 IBB VHH provided herein, at least one binding domain for an antigen expressedAtorney Docket No. 01202-0066-00PCT on the surface of a target cell (“TCSA”) and at least one additional binding domain capable of binding to a surface molecule expressed on a Natural Killer (NK) cells and / or recruiting NK cells, such as CD 16 or NKp46. In some embodiments, the 41BB-binding molecule mediates enhanced NK cell-mediated cell killing of TCSA expressing cells by co-simulating 4 IBB.

[0224] In some embodiments, the NK cell surface molecule is CD16 (FcyRIII). CD16, a low affinity receptor for the Fc portion of some IgGs known to be involved in antibody-dependent cellular cytotoxicity (ADCC), is the best-characterized membrane receptor responsible for triggering of target cell lysis by NK cells (Mandelboim et al., 1999, PNAS 96:5640-5644). Human FcyRIII exists as two isoforms, CD 16a (FcyRIIIA) and CD 16b (FcyRIIIB), that share 96% sequence identity in their extracellular immunoglobulin-binding regions (van de Winkel and Capel, 1993, Immunol. Today 14(5):215-221). In particular embodiments, the additional binding domain is capable of specifically binding CD 16a. CD 16a is expressed on macrophages, mast cells, and NK cells as a transmembrane receptor. Engagement of CD 16a, such as with an agonist comprising a binding domain capable of specifically binding CD16a can result in activating ofNK cells expressing CD16a, thereby eliciting a biological response, in particular a signaling response. In some cases, the CD16abinding molecule is capable of triggering cell killing, in a manner analogous to antibody-dependent cellular cytotoxicity (ADCC), by virtue of its binding to such cells.

[0225] In some embodiments, the NK cell surface molecule is NKp46. NKp46, also known as CD335, LY94-homolog, or NCR1, is an activating cell-surface receptor expressed on NK cell but not on CD4+ or CD8+ T-cells, B-cells, monocytes, or granulocytes. Ligation and clustering of NKp46 drives activating signals through its immunoreceptor tyrosine-based activation motif (ITAM) containing coreceptors Fc epsilon RI and CD3 zeta, which induce the expression of interferon-gamma and NK-mediated cytotoxicity.

[0226] In some embodiments, 41BB-binding molecules that can specifically bind to 4 IBB, a TCSA and to CD 16a or NKp46 may target NK cells to target cells bearing the TCSA, so that the cell bearing the TCSA may be eradicated via NK cell mediated cell killing. For example, a 41BB-binding molecule that specifically binds TCSA expressed on a tumor cell and CD 16a or NKp46 may target NK-cells to the tumor cell. In some cases, activation of the NK cell caused by the 41BB-binding molecule binding to CD 16a or NKp46 can lead to killing of the tumor cells. In some embodiments, the 41BB-binding molecule enhances NK cell mediated cell killing by co-simulating 4 IBB.

[0227] In some embodiments, the additional binding domain specific to an activating NK cell receptor (also referred to herein as an “NKCR-binding domain”), such as CD 16a or NKp46, is an antigen-binding fragment selected from a Fab fragment, a F(ab')2 fragment, an Fv fragment, a scFv, disulfide stabilized Fv fragment (dsFv), a scAb, a dAb, a single domain heavy chain antibody (VHH), or a single domain light chain antibody. In some embodiments, the additional binding domain is monovalent for binding the activating NK cell receptor, such as CD 16a or NKp46.Attorney Docket No. 01202-0066-00PCT

[0228] Antibodies and antigen-binding fragments thereof specific for CD 16a are known and include, for example, NM3E2 (McCall et al. (1999) Mol. Immunol., 36:433-045. Other anti-CD16a antibodies also can be used in the molecules provided herein, including any described in published U.S. patent application No. US10160280795; U.S. Patent No. 9,701,750; Behar etal. (2008) Protein Eng Des Sei. 21:1-10; Arndt et al., (1999) Blood 94:2562-2568. In particular examples, the anti-CD16a is an antiCD 16a scFv. In some embodiments, the anti-CD16a is an anti-CD16a antibody included in a TandAb molecule (see e.g. Reush et al. (2014) Mabs, 6:727-738). In some aspects, the anti-CD16a is an anti-CD16a or antigen binding fragment, such as an scFv, described in U.S. Patent No. 9,035,026.

[0229] Antibodies and antigen-binding fragments thereof specific for NKp46 are known and include for example, any of those described in WO2021205438, US20240141038, W02023034740, WO2024131711, WO2024155044, W02024165500.

[0230] In some of the provided embodiments, the 41BB-binding molecule is capable ofNK cell recruitment and comprises at least at least one 4 IBB VHH provided herein. In some embodiments, the 4 IBB VHH comprises a CDR1, CDR2 and CDR3 sequences, under any CDR definitions provided in Table 3 above. In some embodiments, the 41BB VHH comprises a CDR1, CDR2 and CDR3 sequences, under any CDR definition provided in Table 3 above and has an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a VHH of any one of SEQ ID NOs: 43-45, 98, and 99. Additional embodiments of 4 IBB VHHs which may be incorporated into a 41BB-binding molecule that is capable of NK cell recruitment are provided herein.

[0231] In some of any of the provided embodiments, a 41BB-binding molecule that is a capable of NK cell recruitment comprises a TCSA-binding domain that binds an antigen selected from any of those provided here.

[0232] In some of the provided embodiments, the 41BB-binding molecule is capable of NK cell recruitment and comprises an Fc region, wherein the Fc region comprises at least one of the Fc region sequences provided herein. In some such embodiments the 41BB-binding molecule comprises a homodimer Fc region. In some such embodiments the homodimer Fc region comprises a polypeptide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1-4, 12-17, and 30-32. In some such embodiments the 41BB-binding molecule comprises a heterodimeric Fc region comprising any of the paired or complementary amino acid modifications provided herein. In some embodiments, the heterodimeric Fc region comprises “knob” and “hole” modifications. In some embodiments, the first Fc polypeptide is selected from the group consisting of SEQ ID NOs: 18-23. In some embodiments, the second Fc polypeptide is selected from the group consisting of SEQ ID NOs: 24-29. Additional embodiments of Fc regions which may be incorporated into a 41BB-binding molecule that is capable of NK cell recruitment are provided herein.Attorney Docket No. 01202-0066-00PCT

[0233] In some of the provided embodiments, a 41BB-binding molecule is capable of NK cell recruitment and comprises a linker, wherein the linker comprises at least one of the linker sequences provided herein. In some embodiments, the 4 IBB VHH, the TCSA-binding domain, and / or the NKCR-binding domain are linked, directly or indirectly via a linker. In some embodiments, the 4 IBB VHH, the TCSA-binding domain, and / or the NKCR-binding domain are linked, directly or indirectly via a linker, to the Fc region. In some embodiments, the linker is a linking peptide (LP), which can include any flexible or rigid linker including any of the linkers described herein. In some embodiments, the linker is selected from the group consisting of SEQ ID NOs: 33-40 and 82-84. Additional embodiments of linking peptides which may be incorporated into a 41BB-binding molecule that is capable of NK cell recruitment are provided herein.

[0234] The provided 41BB-binding molecules capable ofNK cell recruitment can be formatted in any of a number of formats containing at least one 4 IBB VHH, at least one TCSA-binding domain and at least one NKCR-binding domain, such as a CD16a-binding domain or a NKp46-binding domain, including any of the fusion protein and multispecific formats provided herein.IV. POLYPEPTIDE EXPRESSION AND PRODUCTION

[0235] Nucleic acid molecules comprising polynucleotides that encode any of the provided sdAb and 41BB-binding molecules are provided. In some embodiments, the provided nucleic acid sequences and particularly DNA sequences encode fusion proteins as provided herein. In any of the foregoing embodiments, the nucleic acid molecule may also encode a leader sequence that directs secretion of the 41BB-binding molecule, which leader sequence is typically cleaved such that it is not present in the secreted polypeptide. The leader sequence may be a native heavy chain (or VHH) leader sequence, or may be another heterologous leader sequence.

[0236] Nucleic acid molecules can be constructed using recombinant DNA techniques conventional in the art. In some embodiments, a nucleic acid molecule is an expression vector that is suitable for expression in a selected host cell.

[0237] Vectors comprising nucleic acids that encode the 41BB-binding molecules described herein are provided. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In some embodiments, a vector is selected that is optimized for expression of polypeptides in a desired cell type, such as CHO or CHO-derived cells, or in NSO cells. Exemplary such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

[0238] In particular, a DNA vector that encodes a desired 41BB-binding molecule, such as a fusion protein, can be used to facilitate the methods of preparing the 41BB-binding molecules described herein and to obtain significant quantities. The DNA sequence can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of theAtorney Docket No. 01202-0066-00PCT inserted protein-coding sequence. A variety of host-vector systems may be utilized to express the proteincoding sequence. These include mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage DNA, plasmid DNA or cosmid DNA. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.

[0239] The disclosure also provides methods of producing a 41BB-binding molecule by culturing a cell under conditions that lead to expression of the molecules, wherein the cell comprises an isolated nucleic acid molecule encoding a 41BB-binding molecule described herein, and / or vectors that include these isolated nucleic acid sequences.

[0240] In some embodiments, a 41BB-binding molecule may be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression may be carried out, for example, according to procedures known in the art. Exemplary eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44. Lecl3 CHO cells, and FUT8 CHO cells; PER.C6® cells (Crucell); and NSO cells. In some embodiments, the 41BB-binding molecules may be expressed in yeast. See, e.g., U.S. Publication No. US 2006 / 0270045. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the polypeptide. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.

[0241] Introduction of one or more nucleic acids (such as vectors) into a desired host cell may be accomplished by any method, including but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, etc. Nonlimiting exemplary methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rded. Cold Spring Harbor Laboratory Press (2001). Nucleic acids may be transiently or stably transfected in the desired host cells, according to any suitable method.

[0242] Host cells comprising any of the nucleic acids or vectors described herein are also provided. In some embodiments, a host cell that expresses a 41BB-binding molecule described herein is provided. The 41BB-binding molecules expressed in host cells can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include the 4 IBB ECD and agents that bind Fc regions. For example, a Protein A, Protein G, Protein A / G, or an antibody affinity column may be used to bind the Fc region and to purify a 41BB-binding molecule that comprises an Fc region. Hydrophobic interactive chromatography, for example, a butyl or phenyl column, may also suitable for purifying some polypeptides such as antibodies. Ion exchange chromatography (for example anion exchangeAttorney Docket No. 01202-0066-00PCT chromatography and / or cation exchange chromatography) may also suitable for purifying some polypeptides such as antibodies. Mixed-mode chromatography (for example reversed phase / anion exchange, reversed phase / cation exchange, hydrophilic interaction / anion exchange, hydrophilic interaction / cation exchange, etc.) may also suitable for purifying some polypeptides such as antibodies. Many methods of purifying polypeptides are known in the art.

[0243] In some embodiments, the 41BB-binding molecule is produced in a cell-free system.Nonlimiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al., Biotechnol. Adv. 21: 695-713 (2003).

[0244] In some embodiments, 41BB-binding molecules prepared by the methods described above are provided. In some embodiments, the 41BB-binding molecule is prepared in a host cell. In some embodiments, the 41BB-binding molecule is prepared in a cell-free system. In some embodiments, the 41BB-binding molecule is purified. In some embodiments, a cell culture media comprising a 41BB-binding molecule is provided.

[0245] In some embodiments, compositions comprising 41BB-binding molecules prepared by the methods described above are provided. In some embodiments, the composition comprises a 41BB-binding molecule prepared in a host cell. In some embodiments, the composition comprises a 41BB-binding molecule prepared in a cell-free system. In some embodiments, the composition comprises a purified 41BB-binding molecule.V. PHARMACEUTICAL COMPOSITIONS AND FORMULATIONS

[0246] Provided herein are pharmaceutical compositions containing any of the 41BB-binding molecules provided herein or engineered cells expressing the same. In some embodiments, 41BB-binding molecules, such as fusion proteins of the disclosure (also referred to herein as “active compounds”), and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. In some embodiments, engineered cells expressing a chimeric receptor, such as a chimeric antigen receptor, containing a 41BB-binding molecule provided herein can be incorporated into pharmaceutical compositions suitable for administration.

[0247] In some embodiments, compositions comprising 41BB-binding molecules are provided in formulations with a wide variety of pharmaceutically acceptable carriers (see, for example, Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7thed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rded., Pharmaceutical Press (2000)). Various pharmaceutically acceptable carriers, which include vehicles, adjuvants, and diluents, are available. Moreover, various pharmaceutically acceptable auxiliaryAttorney Docket No. 01202-0066-00PCT substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are also available.

[0248] The pharmaceutical compositions can be included in a kit, container, pack, or dispenser together with instructions for administration. These pharmaceutical compositions can be included in diagnostic kits with instructions for use.VI. METHODS OF TREATMENT AND USES

[0249] The 41BB-binding molecules or engineered cells expressing the same described herein are useful in a variety of therapeutic, diagnostic and prophylactic indications. For example, the 41BB-binding molecules or engineered cells are useful in treating a variety of diseases and disorders in a subject. Such methods and uses include therapeutic methods and uses, for example, involving administration of the molecules or engineered cells, or compositions containing the same, to a subject having a disease, condition, or disorder, such as a tumor or cancer, an inflammatory or immune (e.g., autoimmunity) disorder. In some embodiments, the molecule ore engineered cell is administered in an effective amount to effect treatment of the disease or disorder. Uses include uses of molecules containing the 41BB-binding molecules or engineered cells in such methods and treatments, and in the preparation of a medicament in order to carry out such therapeutic methods. In some embodiments, the methods are carried out by administering the 41BB-binding molecules or engineered cells, or compositions comprising the same, to the subject having or suspected of having the disease or condition. In some embodiments, the methods thereby treat the disease or condition or disorder in the subject.

[0250] In some embodiments, a provided 41BB-binding molecule is capable of modulating, e.g. increasing, an immune response when administered to a subject, such as by costimulation of 4 IBB and engagement of a T cell activating receptor signal in a cell. In some embodiments, a provided 41BB-binding molecule is capable of modulating, e.g. increasing, an immune response when administered to a subject, such as by costimulation of 4 IBB and engagement of an NK cell activating receptor signal in a cell. In some embodiments, provided herein is a method of modulating an immune response in a subject by administering a therapeutically effective amount of a provided 41BB-binding molecule, or pharmaceutical compositions thereof. In some embodiments, the method of modulating an immune response increases or enhances an immune response in a subject. For example, the increase or enhanced response may be an increase in cell-mediated immunity. In some examples, the method increases T-cell activity, such as cytolytic T-cell (CTL) activity. In some examples, the method increases NK-cell activity, such as ADCC and NK-mediated cytotoxicity. In some embodiments, the modulated (e.g., increased) immune response is against a tumor or cancer. In some embodiments, the modulated (e.g., increased) immune response is against a pathogenic B cell and / or an auto-reactive T cell.Attorney Docket No. 01202-0066-00PCT

[0251] In some embodiments, administration of a 41BB-binding molecule, such as an 41BB-Fc fusion protein or a multispecific 41BB-binding molecule containing an Fc region, may activate innate immune cells via engagement of FcyRs through the Fc-region of the 41BB-binding molecule.Administration of such 41BB-binding molecules may agonize, stimulate, activate, and / or augment innate immune cell effector functions, including ADCC, cytokine release, degranulation and / or ADCP.

[0252] In some embodiments, the provided methods are for treating a disease or condition in a subject by administering a therapeutically effective amount of any of the provided 41 BB-binding molecules or engineered cells or pharmaceutical compositions thereof. In some embodiments, the disease or condition is a tumor or a cancer. In other embodiments, the disease or condition is an immune disease. In other embodiments, the disease or condition is an inflammatory condition. Generally, alleviation or treatment of a disease or disorder involves the lessening of one or more symptoms or medical problems associated with the disease or disorder. For example, in the case of cancer, the therapeutically effective amount of the drug can accomplish one or a combination of the following: reduce the number of cancer cells; reduce the tumor size; inhibit ( / .<?.. to decrease to some extent and / or stop) cancer cell infiltration into peripheral organs; inhibit tumor metastasis; inhibit, to some extent, tumor growth; and / or relieve to some extent one or more of the symptoms associated with the cancer. In some embodiments, a composition of this disclosure can be used to prevent the onset or reoccurrence of the disease or disorder in a subject, e.g. , a human or other mammal, such as a non-human primate, companion animal (e.g., cat, dog, horse), farm animal, work animal, or zoo animal. The terms subject and patient are used interchangeably herein.

[0253] In some embodiments, the 41BB-binding molecules or engineered cells, or pharmaceutical compositions thereof, can be used to inhibit growth of mammalian cancer cells (such as human cancer cells). A method of treating cancer can include administering an effective amount of any of the pharmaceutical compositions described herein to a subject with cancer. The effective amount of the pharmaceutical composition can be administered to inhibit, halt, or reverse progression of cancers.Human cancer cells can be treated in vivo, or ex vivo. In ex vivo treatment of a human patient, tissue or fluids containing cancer cells are treated outside the body and then the tissue or fluids are reintroduced back into the patient. In some embodiments, the cancer is treated in a human patient in vivo by administration of the therapeutic composition into the patient.

[0254] In some embodiments the 41BB-binding molecules or engineered cells, or pharmaceutical compositions thereof, are administered as a neoadjuvant therapy prior to resection. In some embodiments, neoadjuvant therapy refers to therapy to shrink and / or downgrade the tumor prior to any surgery. In some embodiments, neoadjuvant therapy means chemotherapy administered to cancer patients prior to surgery. In some embodiments, neoadjuvant therapy means an antibody is administered to cancer patients prior to surgery. Types of cancers for which neoadjuvant chemotherapy is commonly considered include, for example, breast, colorectal, ovarian, cervical, bladder, and lung. In someAttorney Docket No. 01202-0066-00PCT embodiments, the antibodies are used as a neoadjuvant therapy in the treatment of a cancer. In some embodiments, the use is prior to resection.

[0255] Non-liming examples of disease include: all types of cancers (breast, lung, colorectal, prostate, melanomas, head and neck, pancreatic, etc.), rheumatoid arthritis, Crohn’s disuse, SLE, cardiovascular damage, ischemia, etc. For example, indications would include leukemias, including T-cell acute lymphoblastic leukemia (T-ALL), lymphoblastic diseases including multiple myeloma, and solid tumors, including lung, colorectal, prostate, pancreatic, and breast, including triple negative breast cancer. For example, indications include bone disease or metastasis in cancer, regardless of primary tumor origin; breast cancer, including by way of non-limiting example, ER / PR+ breast cancer, Her2+ breast cancer, triple -negative breast cancer; colorectal cancer; endometrial cancer; gastric cancer; glioblastoma; head and neck cancer, such as esophageal cancer; lung cancer, such as by way of nonlimiting example, non-small cell lung cancer; multiple myeloma ovarian cancer; pancreatic cancer; prostate cancer; sarcoma, such as osteosarcoma; renal cancer, such as by way of nonlimiting example, renal cell carcinoma; and / or skin cancer, such as by way of nonlimiting example, squamous cell cancer, basal cell carcinoma, or melanoma. In some embodiments, the cancer is a squamous cell cancer. In some embodiments, the cancer is a skin squamous cell carcinoma. In some embodiments, the cancer is an esophageal squamous cell carcinoma. In some embodiments, the cancer is a head and neck squamous cell carcinoma. In some embodiments, the cancer is a lung squamous cell carcinoma.

[0256] In certain embodiments, the cancer is a metastatic cancer, refractory cancer, or recurrent cancer.

[0257] In some embodiments, a therapeutically effective amount of a 41BB-binding molecule of the disclosure relates generally to the amount needed to achieve a therapeutic objective. Typically, precise amount of the compositions of the present disclosure to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, type, extent and symptoms of disease, e.g., inflammation, infection or metastasis, and condition of the patient (subject).

[0258] In some embodiments, administration of the provided 41BB-binding molecules modulates immunological activity. In some embodiments, the 41BB-binding molecules modulate immunological activity by eliminating, sequestering, or inactivating immune cells mediating or capable of mediating an undesired immune response (e.g., pathogenic B cells, or auto-reactive T cells); inducing, generating, or turning on immune cells that mediate or are capable of mediating a protective immune response; changing the physical or functional properties of immune cells; or a combination of these effects.

[0259] Non-liming examples of immune and inflammatory diseases include: Systemic Lupus Erythematosus (SLE), rheumatoid arthritis (RA), Graves disease, Myasthenia Gravis (gMG), Type 1 Diabetes, Hashimoto's Thyroiditis, multiple sclerosis (MS), psoriasis & inflammatory bowel disease (IBD), autoimmune hepatitis, Sjogren’s syndrome, antiphospholipid syndrome, and pemphigus vulgaris.Attorney Docket No. 01202-0066-00PCT Combination Therapy

[0260] 41BB-binding molecules or engineered cells of the present disclosure can be administered alone or in combination with other modes of treatment, such as other anti -cancer agents, antiinflammatory agents, agents that modulate autoimmunity. They can be provided before, substantially contemporaneous with, or after other modes of treatment (i.e., concurrently or sequentially). In some embodiments, the method of treatment described herein can further include administering: radiation therapy, chemotherapy, vaccination, targeted tumor therapy, CAR-T therapy, oncolytic virus therapy, cancer immunotherapy, cytokine therapy, surgical resection, chromatin modification, ablation, cryotherapy, an antisense agent against, a siRNA agent, a microRNA agent,, or a biologic, such as an antibody, cytokine, or receptor extracellular domain-Fc fusion.VII. EXAMPLES

[0261] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. The examples are not intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (for example, amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.Example 1: Generation of Optimized 41BB sdAb

[0262] A bivalent 4 IBB VHH-Fc fusion protein comprising the previously described humanized 41BB VHH designated “hzRH3v5-l” and referred to herein as “RH3p” (SEQ ID NO: 42) was generated (designated cx7999; SEQ ID NO: 100) and the in vivo pharmacokinetics (PK) was evaluated rats. 8-10 week old female Sprague Dawley rats from Charles River Laboratories (3 per group) were injected once i.v. with cx7999 at either 30 mg / kg or 0.3 mg / kg. Serum samples were collected at 0.5, 4, 24, 96, 168, 240, 336, and 504 h post-injection. The samples were analyzed by ELISA to determine the concentration of cx7999 over time. The ELISA was performed essentially as follows: 96-well MaxiSorp plates were coated with the capture antibody (goat anti-human IgG Fc; Jackson ImmunoResearch 109-005-098 at 1 pg / mL,100 uL / well) and plates were incubated overnight at 4 degrees C. Plates were washed with PBS / 0.05% Tween-20 (300 pL / wash, 3 washes). All subsequent washes were performed similarly. Plates were blocked with PBS / 1% BSA (200 uL / well) for 1 h at RT with shaking and then washed. Cx7999 was diluted with 2% Sprague Dawley serum (BioIVT; diluted with PBS) 0.5 ug / mL, added to dilution plates (180 uL / well), and then serially diluted 1:3 in 2% serum to cover a concentration range of 0.5 ug / mL to 640 fg / mL. Serum samples were thawed at RT, mixed gently, diluted 1:50 using PBS and added to dilution plates. Serum samples were then serially diluted 1: 10 in 2% serum, respectively to generate final dilutions of 1: 100 and 1: 1000 or 1:500 and 1:5000, respectively. Test article titrations and serum sampleAtorney Docket No. 01202-0066-00PCT dilutions were added to the assay plates (100 uL / well) and plates were incubated at RT with shaking for 1 h. Detection antibody (HRP-conjugated goat anti-human IgG Fc; Jackson ImmunoResearch 709-035-098; stock at 0.8 mg / mL) was diluted 1:20000 with PBS / 0.05% Tween. Plates were washed and detection antibody was added (100 uL / well). Plates were incubated at RT for 1 h, washed, and then peroxidase substrate was added (100 uL / well; SureBlue 5120-0077). Plates were incubated at RT for 8 minutes and then absorbance at 650 nm was read using an Emax microplate reader. Matrix only replicate values were averaged and subtracted from respective standard and sample values. Standard curves were generated using a four-parameter fit ([Agonist] vs. response - Variable slope) with the following model equation: Y = Botom + (XAHillslope) * (Top-Botom) / (XAHillSlope + EC50AHillSlope). The results are shown in FIGS. 1A-1B.

[0263] These results show that the 4 IBB VHH-Fc fusion protein cx7999 comprising the RH3p VHH has a poor PK profile, evident at both 30 and 0.3 mg / kg dose levels (FIGS, 1A and IB, respectively). At 168 hours the average drug concentration in the serum in the 30 mg / kg group was just 0.8 pg / mL and the drug concentration dropped below the limit of detection in the 0.3 mg / kg group.

[0264] The amino acid sequence of RH3p was further modified by introducing a variety of substitutions to rehumanize to enhance the PK profile (i.e., increase the serum half-life) while maintaining the binding affinity for 41BB. Numerous clones were generated and evaluated for monovalent binding, and two variants, designated “RH3v23” and “RH3v24” were identified for further testing. The sequences of RH3v23 and RH3v24 are presented in SEQ ID NOs: 44 and 45, respectively. Variants of RH3v23 and RHv24 (designated “RH3v23.1” and “RHv24.1”) comprising an R at position 112 (EU numbering) are presented in SEQ ID NOs: 98 and 99, respectively. Figure 2 shows an alignment of the parental clone (RH5p) and the optimized variants. The CDRs of the optimized variants according to the AbM, Kabat, IMGT, Chothia, and Contact definitions are provided in Table 3 above. As shown in FIG. 2 the optimized variants comprise numerous amino acid changes across the VHH including in the CDRs and framework regions.

[0265] RH3p, RH3v23 and RHv24 were cloned and expressed as bivalent constructs with an IgGl Fc xELL (designated cxl04, cxl 1318 and cxl 1317, respectively; SEQ ID NOs: 101-103, respectively) and tested for binding to HEK-293 freestyle cells transfected with full length human or cynomolgus monkey 4 IBB by FACS. The FACS studies were performed essentially as follows: 4 IBB transfected, and untransfected HEK-293 freestyle cells were plated at 50K cells per well in a microtiter plate and spun down to remove the culture media, 100 pL of titrated test articles, cxl04, cxl 1317 and cxl 1318 (7 point 6 fold dilutions starting at 1000 nM in FACS buffer) was added to the cells and incubated at 4C for 1 hour. The plates were spun down and washed 2x with FACS buffer, anti-hFc-647 secondary from Jackson Immunoresearch was at 1 / 500 dilution and plates were incubated for 1 hour at 4C, plates were spun down and washed lx with FACS buffer before resuspending in 30ul FACS buffer and binding was determined by flow cytometry quantitating anti-human A647 mean fluorescent intensity using anAttorney Docket No. 01202-0066-00PCT Intellicyt iQue Plus. The data was plotted and analyzed using GraphPad Prism analysis software. The apparent affinity of the observed binding interaction (Kd in nM) was determined using the One Site -total analysis formula. The results are shown in FIGS. 3A-3C.

[0266] As shown in FIGS. 3A-3B all the 41BB VHH Fc fusions bind both human 41BB (FIG. 3A) and cynomolgus monkey 4 IBB (FIG. 3B), with the constructs comprising the optimized 4 IBB VHHs, RH2v23 and RHv24 (cxl 1318 and cxl 1317) exhibiting higher binding affinity. All of test articles exhibited minimal binding to untrasfected cells (FIG. 3C).

[0267] The parental and optimized 4 IBB VHHs were evaluated in silico for their immunogenicity potential. A commercial vendor (EpiVax®, Providence, Rhode Island, USA) performed in silico immunogenicity assessments on the sequences of RH3p, RHv24. In brief, EpiVax evaluated the amino acid sequences of the 4 IBB VHHs for predicted T-cell epitopes using their proprietary EpiMatrix™ algorithm. The amino acid sequences were scored for total predicted T-cell epitope content and ranked against the EpiMatrix Protein Immunogenicity Scale where the molecule could be compared directly to proteins of known immunogenicity. The parental RH3p VHH was found to have a score of 12.18 putting it above the average score of 10 antibodies know to induce anti -therapeutic responses in more than 5% of patients while the optimized RH3v24 VHH was found to have a significantly reduced score of -6.50 which is below the average score of 10 antibodies known to induce anti -therapeutic responses in more than 5% of patients. RH3v23 differs from RH3v24 by just one amino acid and is expected to have a similarly reduced score.

[0268] The in vivo pharmacokinetics (PK) of the 4 IBB VHH Fc fusion proteins comprising the optimized 41BB VHHs RH3v23 (cxl 1318) and RHv24 (cxl 1317) was evaluated in a mouse PK study. 9 week old female Balb / c mice (3 per group) were injected once i.v. with the test article at either 50 mg / kg or 0.5 mg / kg. Serum samples were collected at 0.5, 6, 24, 96, andl68 h post-injection. The samples were analyzed by ELISA to determine the concentration of the test articles over time. The results are shown in FIGS. 4A-4B.

[0269] These results show that the 41BB VHH-Fc fusion proteins cxl 1318 and cxl 1317 comprising the RH3v23 (FIG. 4A) and RH3v24 (FIG. 4B), respectively have greatly improved PK profiles, evident at both 50 and 0.5 mg / kg dose levels. At 168 hours the average drug concentration in the serum in the 50 mg / kg group was 78 pg / mL for cxl 1318 and 92 pg / mL for cxl 1317, and for the 0.5 mg / mk group was 14.9 pg / mL for cxl 1318 and 13.6 pg / mL for cxl 1317.

[0270] These data demonstrate that the optimized VHHs are readily incorporated into fusion proteins (e.g., a fusion protein comprising an Fc region) where they exhibit enhanced affinity, reduced immunogenicity potential, and improved in vivo PK profiles as compared to the parental VHH.

[0271] The disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting of the disclosure. Scope of the disclosure is thus indicated by theAtorney Docket No. 01202-0066-00PCT appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced herein.Table of Certain SequencesSEQ Description SequenceID NO1 human IgGl Fc region (no PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEV hinge) KFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK2 human IgG2 Fc region (no PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQ hinge) FNWYVDGVEVHNAKTKPREEQFNSTFRWSVLTWHQDWLNGKE YKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK3 human IgG3 Fc region (no PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEV hinge) QFKWYVDGVEVHNAKTKPREEQYNSTFRWSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFF LYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK4 human IgG4 Fc region (no PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEV hinge) QFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK5 human IgGl hinge EPKSCDKTHTCPPC6 modified human IgGl EPKSSDKTHTCPPChinge7 lower human IgGl hinge DKTHTCPPC8 human IgG2 hinge ERKCCVECPPC9 human IgG3 hinge KTPLDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP EPKSCDTPPPCPRC10 human IgG4 hinge ESKYGPPCPSC11 modified human IgG4 ESKYGPPCPPChinge12 human IgGl Fc xELL DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVWDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK13 IgGl Fc region xELL DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVWDVS H435R HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSL SPGK14 IgGl Fc region xELL DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVWDVS M252Y and M428V (YV) HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSWHEALHNHYTQKSLSLSPGKAtorney Docket No. 01202-0066-00PCT IgGl Fc region xELL DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVWDVS M252Y and M428L (YL) HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSL SPGKIgGl 18 Fc region xELL DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVWDVS M252Y19, M428L, H435R HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ (YLR) DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNRYTQKSLSL SPGKIgGl Fc region xELL DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVWDVS M252Y, M22428V, H435R HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ (YVR) DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSWHEALHNRYTQKSLSL SPGKIgGl Fc region xELL DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVWDVS S354C T366Wknob HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCR DELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKIgGl Fc region xELL DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVWDVS H435R S354C T366W HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ knob DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCR DELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSL SPGKIgGl Fc region xELL DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVWDVS M252Y and M428V (YV) HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ S354C T366Wknob DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCR DELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSWHEALHNHYTQKSLSL SPGKIgGl Fc region xELL DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVWDVS M252Y and M428L (YL) HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ S354C T366Wknob DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCR DELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSL SPGKIgGl Fc region xELL DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVWDVS M252Y, M428L, H435R HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ (YLR) S354C T366W DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCR DELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDknobSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNRYTQKSLSL SPGKIgGl Fc region xELL DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVWDVS M252Y, M428V, H435R HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ (YVR) S354C T366W DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCR DELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDknobSDGSFFLYSKLTVDKSRWQQGNVFSCSWHEALHNRYTQKSLSL SPGKIgGl Fc region xELL DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVWDVS T366S, L368A, Y407V HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ hole DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDAtorney Docket No. 01202-0066-00PCT SDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKIgGl Fc region xELL DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVWDVS H435R, T366S, L368A, HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ Y407V hole DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSR DELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSL SPGKIgGl Fc region xELL DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVWDVS M252Y and M428V (YV) HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ T366S, L368A, Y407V DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSR DELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDholeSDGSFFLVSKLTVDKSRWQQGNVFSCSWHEALHNHYTQKSLSL SPGKIgGl Fc region xELL DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVWDVS M252Y and M428L (YL) HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ T366S, L368A, Y407V DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSR DELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDholeSDGSFFLVSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSL SPGKIgGl Fc region xELL DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVWDVS M252Y, M428L, H435R HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ (YLR) T366S, L368A, DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSR DELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDY407V holeSDGSFFLVSKLTVDKSRWQQGNVFSCSVLHEALHNRYTQKSLSL SPGKIgGl Fc region xELL DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVWDVS M252Y, M428V, H435R HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ (YVR) T366S, L368A, DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSR DELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDY407V holeSDGSFFLVSKLTVDKSRWQQGNVFSCSWHEALHNRYTQKSLSL SPGKHuman IgG4 Fc region ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCV WD VS Q E D P E VQ FNW YVD GVE VHNAKT KPREEQFNS T YRWS VL TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGKIgG4 Fc region S228P (P) ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCV WD VS Q E D P E VQ FNW YVD GVE VHNAKT KPREEQFNS T YRWS VL TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGKIgG4 Fc region S228P, ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLYISRTPEVTCV M252Y and M428V (PYV) WD VS Q E D P E VQ FNW YVD GVE VHNAKT KPREEQFNS T YRWS VL TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSWHEALHNHYTQ KSLSLSLGKLinker 1 GGGGLinker 2 GSGGGSLinker 3 (GGS)2 GGSGGSLinker 4 (GGS)3 GGGSGGGSGGGSLinker 5 (GGS)4 GGSGGSGGSGGSLinker 6 (GGS)5 GGSGGSGGSGGSGGSLinker 7 GSGGSLinker 8 GGSGGGSGGGSAtorney Docket No. 01202-0066-00PCT 41BB MGNSCYNIVATLLLVLNFERTRSLQD P C S N C P AGT FCDNNRNQI CSPCPPNSFSSAGGQRTCDICRQCKG VFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTK KGCKDCCFGTFNDQKRGICRPWTNCS L D GK S VLVN GT KE RD WC GPS PAD L S P GAS S VT P PAP ARE P GH S PQIISFFLALTSTALLFLLFFLTLRF SWKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL signal sequence is underlinedRH3p EVQLLESGGGEVQPGGSLRLSCAASGFSFSINAMGWYRQAPGKR RE FVAAI E S GRNT VYAE S VKGRFT I S RDNAKNT VYLQMS S LRAE DTAVYYCGLLKGNRWSPSVAYWGQGTLVTVKP RH3con EVQLLESGGGEVQPGGSLRLSCAASGFTFDINAMGWYRQAPGKE REFVAAIESGGXiTVYAESVKGRFTISRDNAKNTLYLQMSSLRDE DTAVYYCALLKGNRWSPSVAYWGQGTLVTVXzP wherein Xi is N or S ; and Xz is K or R RH3v23 EVQLLESGGGEVQPGGSLRLSCAASGFTFDINAMGWYRQAPGKE REFVAAIESGGNTVYAESVKGRFTISRDNAKNTLYLQMSSLRDE DTAVYYCALLKGNRWSPSVAYWGQGTLVTVKP RH3v24 EVQLLESGGGEVQPGGSLRLSCAASGFTFDINAMGWYRQAPGKE REFVAAIESGGSTVYAESVKGRFTISRDNAKNTLYLQMSSLRDE DTAVYYCALLKGNRWSPSVAYWGQGTLVTVKPAbM CDR1: RH3p GFSFSINAMGAbM CDR1: RH3v23, GFTFDINAMGRH3v24AbM CDR2: RH3p AIESGRNTVAbM CDR2: RH3v23 AIESGGNTVAbM CDR2: RH3v24 AIESGGSTVAbm CDR2: con AIESGGXiTVwherein Xi is N or SAbM CDR3: RH3p, LKGNRWSPSVAYRH3v23, RH3v24KabatCDRl: RH3p, INAMGRH3v23, RH3v24Kabat CDR2: RH3p Al ES GRNT VYAE SVKGKabat CDR2: RH3v23 AIESGGNTVYAESVKGKabat CDR2: RH3v24 AIESGGSTVYAESVKGKabat CDR2: CON AIESGGXiTVYAESVKGwherein Xi is N or SKabat CDR3: RH3p, LKGNRWSPSVAYRH3v23, RH3v24IMGT CDR1: RH3p GFSFSINAIMGTCDR1: RH3v23, GFTFDINARH3v24IMGT CDR2: RH3p IESGRNTIMGT CDR2: RH3v23 IESGGNTIMGT CDR2: RH3v24 IESGGSTIMGT CDR2: con IESGGXITwherein Xi is N or SIMGT CDR3: RH3p GLLKGNRWSPSVAYIMGTCDR3: RH3v23, ALLKGNRWSPSVAYRH3v24Chothia CDR1: RH3p GFSFSINChothia CDR1: RH3v23, GFTFDINRH3v24Atorney Docket No. 01202-0066-00PCT Chothia CDR2: RH3p ESGRNChothia CDR2: RH3v23 ESGGNChothia CDR2: RH3v24 ESGGSChothia CDR2: con ESGGXiwherein Xi is N or SChothia CDR3: RH3p, LKGNRWSPSVAYRH3v23, RH3v24Contact CDR1 : RH3p SINAMGContact CDR1: RH3v23, DINAMGRH3v24Contact CDR2: RH3p FVAAIESGRNTVChothia CDR2: RH3v23 FVAAIESGGNTVChothia CDR2: RH3v24 FVAAIESGGSTVChothia CDR2: con FVAAIESGGXiTVwherein Xi is N or SContact CDR3 : RH3p GLLKGNRWSPSVAContact CDR3: RH3v23, ALLKGNRWSPSVARH3v24Linker 9 (GGGGS ) n, wherein n is 1 to 10Linker 10 (GGGGGS ) n, wherein n is 1 to 10Linker 11 PGGGGVH (SP34) EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKG LEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNL KT EDTAMYYCVRHGN FGN S YVS WFAYWGQGT LVT VS A VL (SP34) QAWTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDH LFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIY FCALWYSNLWVFGGGTKLTVL VH13 (44Cys) EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKC LEWVARI RS KYNN YAT YYAD S VKGRFT I S RDDAKNT L YLQMS S L RAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVKP VL10 (lOOcys) QAWTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQ AFRGLIGGTNKRAPGVPARFSGSLLGGKAALTISGAQPEDEADY YCALWYSNHWVFGCGTKLEIKAbMHCDRl: SP34, GFTFNTYAMNVH13AbM HCDR2: SP34, RIRSKYNNYATYVH13AbMHCDR3: SP34, HGNFGNSYVSWFAYVH13AbMLCDRl: SP34 RSSTGAVTTSNYANAbM LCDR1: VL10 GSSTGAVTTSNYANAbM LCDR2: SP34, VL10 GTNKRAPAbMLCDR3: SP34 ALWYSNLWVAbM LCDR3: VL10 ALWYSNHWVRH3v23.1 EVQLLESGGGEVQPGGSLRLSCAASGFTFDINAMGWYRQAPGKE REFVAAIESGGNTVYAESVKGRFTISRDNAKNTLYLQMSSLRDE DTAVYYCALLKGNRWSPSVAYWGQGTLVTVRP RH3v24.1 EVQLLESGGGEVQPGGSLRLSCAASGFTFDINAMGWYRQAPGKE REFVAAIESGGSTVYAESVKGRFTISRDNAKNTLYLQMSSLRDE DTAVYYCALLKGNRWSPSVAYWGQGTLVTVRPcx7999 EVQLVESGGGEVQPGGSLRLSCAASGFSFSINAMGWYRQAPGKR RE FVAAI E S GRNTVYAE S VKGRFT I S RDNAKNT VYLQMS S LRAEDTAVYYCGLLKGNRWSPSVAYWGQGTLVTVKPGGGGDKTHTCPAtorney Docket No. 01202-0066-00PCT PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKex 104 (RH3p Fc xELL EVQLLESGGGEVQPGGSLRLSCAASGFSFSINAMGWYRQAPGKR fusion) RE FVAAI E S GRNT VYAE S VKGRFT I S RDNAKNT VYLQMS S LRAE DTAVYYCGLLKGNRWSPSVAYWGQGTLVTVKPGGGGDKTHTCP PCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK cxll318 (RH3v23 Fc EVQLLESGGGEVQPGGSLRLSCAASGFTFDINAMGWYRQAPGKE xELL fusion) REFVAAIESGGNTVYAESVKGRFTISRDNAKNTLYLQMSSLRDE DTAVYYCALLKGNRWSPSVAYWGQGTLVTVKPGGGGDKTHTCP PCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK cxll317 (RH3v24 Fc EVQLLESGGGEVQPGGSLRLSCAASGFTFDINAMGWYRQAPGKE xELL fusion) REFVAAIESGGSTVYAESVKGRFTISRDNAKNTLYLQMSSLRDE DTAVYYCALLKGNRWSPSVAYWGQGTLVTVKPGGGGDKTHTCP PCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

Attorney Docket No. 01202-0066-00PCT What is Claimed1. A 4 IBB-binding molecule, comprising at least one heavy chain only variable domain that specifically binds 4 IBB (4 IBB VHH), wherein at least one 4 IBB VHH comprises a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 47, 53, 60, 68, and 75; a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 51, 57, 64, 72, and 79; and a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:52, 58, 66, 73, and 81.

2. The 41BB-binding molecule of claim 1, wherein at least one 41BB VHH comprises CDR1, CDR2, and CDR3 sequences selected from: SEQ ID NOs: 47, 51 and 52; SEQ ID NOs: 53, 57, and 58; SEQ ID NOs: 60, 64, and 66; SEQ ID NOs: 68, 72, and 73; and SEQ ID NOs: 75, 79, and 81.

3. The 41BB-binding molecule of claim 1, wherein at least one 41BB VHH comprises CDR1, CDR2, and CDR3 sequences selected from: SEQ ID NOs: 47, 49, and 52; SEQ ID NOs: 47, 50, and 52; SEQ ID NOs: 53, 55, 58; SEQ ID NOs: 53, 56, 58; SEQ ID NOs: 60, 62, and 66; SEQ ID NOs: 60, 63, and 66; SEQ ID NOs: 68, 70, and 73; SEQ ID NOs: 68, 71, and 73; SEQ ID NOs: 75, 77 and 81; and SEQ ID NOs: 75, 78, and 81.

4. The 41BB-binding molecule of any one of claims 1-3, wherein at least one 41BB VHH comprises the sequence of any of SEQ ID NO: 44, 45, 98, and 99, or an amino acid sequence that exhibits at least an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 44, 45, 98, and 99.

5. A 4 IBB-binding molecule, comprising at least one heavy chain only variable domain that specifically binds 4 IBB (4 IBB VHH), wherein at least one 4 IBB VHH comprises the sequence of any of SEQ ID NO: 44, 45, 98, and 99, or an amino acid sequence that exhibits at least an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 44, 45, 98, and 99.

6. The 41BB-binding molecule of any one of claims 1-5, wherein the 41BB is a human 41BB.

7. The 41BB-binding molecule of any one of claims 1-6, comprising two 41BB VHHs.

8. The 41BB-binding molecule of any one of claims 1-7, comprising three 41BB VHHs.

9. The 41BB-binding molecule of any one of claims 1-8, wherein the molecule comprises one or more additional binding domains that bind to a target other than 4 IBB.

10. The 41BB-binding molecule of claim 9, wherein the additional binding domain, or independently each of the additional binding domains is a domain that binds to an antigen selected from the group consisting of: 1-92-LFA-3, 5T4, ADAM-9, Alpha-4 integrin, Alpha-V integrin, alpha4betal integrin, alpha4beta7 integrin, AGR2, Anti-Lewis-Y, Apelin J receptor, APRIL, B7-H3, B7-H4, B7-H6, BAFF, BCMA, BTLA, C5 complement, C-242, CA9, CAI 9-9, (Lewis a), Carbonic anhydrase 9, CD2,Atorney Docket No. 01202-0066-00PCT CD3, CD6, CD9, CDlla, CD16, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD138, CD166, CD172A, CD248, CDH6, CDH17, CEACAM5 (CEA), CEACAM6 (NCA-90), CLAUDIN-3, CLAUDIN-4, CLAUDIN-18.2, cMet, Collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, Endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, Folate receptor alpha (FRa), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GP Ilb / IIIa receptors, GPC3, Gpl30, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, Hyaluronidase, ICOS, IFNalpha, IFNbeta, IFNgamma, IgE, IgE Receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rbetal, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R (wsxl), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, Insulin Receptor, Jagged Ligands, Jagged 1, Jagged 2, KISSl-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPDl, MCSP, Mesothelin, MRP4, MUC1, Mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, NKp46, Nicastrin, Notch Receptors, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, 0X40, PAR2, PDGF-AA, PDGF-BB, PDGFRalpha, PDGFRbeta, PD-1, PD-L1, PD-L2, Phosphatidyl-serine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, R0R1, SIGLEC-6, SLC44A4, Sphingosine 1 Phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFbeta, TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFalpha, TNFR, TNFR2, NFRS12A, TRAIL-R1, TRAIL-R2, Transferrin, Transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, and WISP-3.

11. The 4 IBB-binding molecule of claim 9, wherein at least one of the one or more additional binding domains binds to an activating receptor on an immune cell.

12. The 41BB-binding molecule of claim 11, wherein the immune cell is a T cell.

13. The 41BB-binding molecule of claim 11 or 12, wherein the activating receptor is CD3, optionally wherein the activating receptor is CD3e.

14. The 4 IBB-binding molecule of claim 11 or 12, wherein the activating receptor is a T cell receptor (“TCR”) comprising an alpha, beta, delta, or gamma chain, optionally wherein the activating receptor is a gamma / delta (y5) TCR.

15. The 41BB-binding molecule of claim 11, wherein the immune cell is an NK cell.

16. The 41BB-binding molecule of claim 11 or 15, wherein the activating receptor is CD16a.

17. The 41BB-binding molecule of claim 11 or 15, wherein the activating receptor is NKp46.Attorney Docket No. 01202-0066-00PCT 18. The 41BB-binding molecule of any one of claims 11-17, wherein the molecule further comprises at least one additional binding domain for an antigen on the surface of a target cell (TCSA-binding domain).

19. The 41BB-binding molecule of claim 18, wherein the TCSA-binding domain, or independently each of the TCSA-binding domains, is a domain that binds antigen on the surface of a target cell selected from the group consisting of: 1-92-LFA-3, 5T4, ADAM-9, Alpha-4 integrin, Alpha-V integrin, alpha4betal integrin, alpha4beta7 integrin, AGR2, Anti-Lewis-Y, Apelin J receptor, APRIL, B7-H3, B7-H4, B7-H6, BAFF, BCMA, BTLA, C5 complement, C-242, CA9, CA19-9, (Lewis a), Carbonic anhydrase 9, CD2, CD3, CD6, CD9, CDlla, CD16, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD138, CD166, CD172A, CD248, CDH6, CDH17, CEACAM5 (CEA), CEACAM6 (NCA-90), CLAUDIN-3, CLAUDIN-4, CLAUDIN-18.2, cMet, Collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, Endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, Folate receptor alpha (FRa), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GP Ilb / IIIa receptors, GPC3, Gpl30, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, Hyaluronidase, ICOS, IFNalpha, IFNbeta, IFNgamma, IgE, IgE Receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rbetal, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R (wsxl), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, Insulin Receptor, lagged Ligands, lagged 1, lagged 2, KISSl-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPDl, MCSP, Mesothelin, MRP4, MUC1, Mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, NKp46, Nicastrin, Notch Receptors, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, 0X40, PAR2, PDGF-AA, PDGF-BB, PDGFRalpha, PDGFRbeta, PD-1, PD-L1, PD-L2, Phosphatidyl-serine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, R0R1, SIGLEC-6, SLC44A4, Sphingosine 1 Phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFbeta, TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFalpha, TNFR, TNFR2, NFRS12A, TRAIL-Rl, TRAIL-R2, Transferrin, Transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, and WISP-3.

20. The 41BB-binding molecule of any one of claims 9-19, wherein at least one of the additional binding domains comprises an antibody or antigen-binding fragment thereof.

21. The 41BB-binding molecule of claim 20, wherein each of the additional binding domains comprises an antibody or antigen-binding fragment thereof.Atorney Docket No. 01202-0066-00PCT 22. The 41BB-binding molecule of claim 20 or 21, wherein the antibody or antigen-binding fragment thereof, or independently each antibody or antigen-binding fragment thereof, is an Fv, a disulfide-stabilized Fv (dsFv), scFv, a Fab, a F(ab')2, a single domain antibody (sdAb).

23. The 41BB-binding molecule of any one of claims 11-22, wherein the molecule comprises one binding domain that binds to an activating receptor on an immune cell.

24. The 41BB-binding molecule of any one of claims 11-22, wherein the molecule comprises two binding domains that bind to an activating receptor on an immune cell.

25. The 41BB-binding molecule of claim 24, wherein the domains that binds to an activating receptor on an immune cell bind the same epitope of an activating receptor.

26. The 41BB-binding molecule of claim 24, wherein the domains that binds to an activating receptor on an immune cell bind to different activating receptors, or different epitopes of the same activating receptor.

27. The 41BB-binding molecule of any one of claims 18-26, wherein the molecule comprises one TCSA-binding domain.

28. The 41BB-binding molecule of any one of claims 18-26, wherein the molecule comprises two or more TCSA-binding domains.

29. The 41BB-binding molecule of claim 28, wherein at least two of the two or more TCSA-binding domains bind to the same epitope of a TCSA.

30. The 41BB-binding molecule of claim 28, wherein at least two of the two or more TCSA-binding domains bind to different TCSA, or different epitopes of the same TCSA.

31. The 41BB-binding molecule of any one of claims 1-30, wherein the molecule comprises an Fc region.

32. The 41BB-binding molecule of any one of claims 9-31, wherein the molecule comprises an immunoglobulin Fc region that links the at least one 4 IBB VHH and the one or more additional binding domain.

33. The 41BB-binding molecule of any one of claims 1-32, wherein the molecule comprises at least two polypeptide chains each comprising an Fc region polypeptide.

34. The 41BB-binding molecule of any one of claims 31-33, wherein the Fc region is a homodimeric Fc region.

35. The 41BB-binding molecule of any one of claims 31-34, wherein the Fc region comprises the sequence of any of SEQ ID NO: 1-4, 12-17, or 30-32, or an amino acid that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOS: 1-4, 12-17, or 30-32.

36. The 41BB-binding molecule of any one of claims 31-33, wherein the Fc region is a heterodimeric Fc region.Attorney Docket No. 01202-0066-00PCT 37. The 41BB-binding molecule of claim 36, where the heterodimeric Fc region comprises paired or complementary amino acid modifications.

38. The 41BB-binding molecule of claim 36 or 37, wherein the heterodimeric Fc region comprises “knob” and “hole” modifications.

39. The 41BB-binding molecule of any one of claims 36—38, wherein the heterodimeric Fc region comprises a first Fc polypeptide and a second Fc polypeptide, wherein the first Fc polypeptide comprises any of SEQ ID NOs: 18-23, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 18-23, and the second Fc polypeptide comprises any of SEQ ID NOs: 24-29 or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs:24-29.

40. The 41BB-binding molecule of any one of claims 1-14 and 18-39, wherein the molecule comprises at least one TCSA-binding domain, and at least one binding domain capable of binding to a and activating receptor expressed on a T cell.

41. The 41BB-binding molecule of claim 40, wherein the activating receptor is CD3.

42. The 41BB-binding molecule of claim 41, wherein the CD3 is CD3e.

43. The 41BB-binding molecule of claim 40, wherein the activating receptor is a TCR comprising an alpha, beta, delta, or gamma chain.

44. The 41BB-binding molecule of claim 40, wherein the activating receptor is a gamma / delta (y5) TCR.

45. The 41BB-binding molecule of any one of claims 1-11, and 15-39, wherein the molecule comprises least one TCSA-binding domain, and at least one binding domain capable of binding to a activating receptor expressed on an NK cell.

46. The 41BB-binding molecule of any one of claims 1-14, and 18-44, wherein the molecule comprises a first component containing an immunoglobulin Fc region and a second component comprising a CD3 -binding region, wherein the first and second components are coupled by a linker, the Fc region is positioned N-terminal to the CD3-binding region; and one or both of the first and second components comprises (1) a TCSA-binding domain and (2) a 41BB-binding domain.

47. The 41BB-binding molecule of any one of claims 1-46, wherein at least one 41BB VHH comprises the sequence of SEQ ID NO: 44.

48. The 41BB-binding molecule of any one of claims 1-46, wherein at least one 41BB VHH comprises the sequence of SEQ ID NO: 45.

49. The 41BB-binding molecule of any one of claims 1-46, wherein at least one 41BB VHH comprises the sequence of SEQ ID NO: 98.

50. The 41BB-binding molecule of any one of claims 1-46, wherein at least one 41BB VHH comprises the sequence of SEQ ID NO: 99.Attorney Docket No. 01202-0066-00PCT 51. A polynucleotide(s) encoding the 41BB-binding molecule of any one of claims 1-50.

52. A vector(s) comprising the polynucleotide(s) of claim 51.

53. A cell or host cell comprising polynucleotide (s) of claim 51 or the vector(s) of claim 50.

54. A method of producing a 41BB-binding molecule, the method comprising introducing into a cell or host cell a polynucleotide or polynucleotides of claim 51 or a vector or vectors of claim 52, and culturing the cell or host cell under conditions to produce the 41BB-binding molecule.

55. The method of claim 54, further comprising isolating or purifying the 41BB-binding molecule from the cell or host cell.

56. A 41BB-binding molecule produced by the method of claim 54 or 55.

57. A pharmaceutical composition comprising the 41BB-binding molecule of any of claims 1-50 and 56.

58. The pharmaceutical composition of claim 57, comprising a pharmaceutically acceptable carrier.

59. A method of increasing T cell proliferation or activation comprising contacting T cells with the 41BB-binding molecule of any of claims 1-14, 18-44, 47-50 and 56, or the pharmaceutical composition of claim 57 or 58.

60. The method of claim 59, wherein the T cells are in vitro.

61. The method of claim 59, wherein the T cells are in vivo.

62. Use of the 41BB-binding molecule of any of claims 1-14, 18-50 and 56, or the pharmaceutical composition of claim 57 or 58 in the manufacture of a medicament for increasing T cell proliferation or activation, wherein the medicament is provided for contact with the T cells.

63. A method of increasing NK cell proliferation or activation comprising contacting NK cells with the 41BB-binding molecule of any of claims 1-11, 15-39, 45, 47-50 and 56, or the pharmaceutical composition of claim 57 or 58.

64. The method of claim 63, wherein the NK cells are in vitro.

65. The method of claim 63, wherein the NK cells are in vivo.

66. Use of the 41BB-binding molecule of any of claims 1-11, 15-39, 45, 47-50 and 56, or the pharmaceutical composition of claim 57 or 58 in the manufacture of a medicament for increasing NK cell proliferation or activation, wherein the medicament is provided for contact with the NK cells.

67. A method of stimulating or inducing an immune response in a subject, the method comprising administering, to a subject in need thereof, the 41BB-binding molecule of any of claims 1-50 and 56, or the pharmaceutical composition of claim 57 or 58.

68. The method of claim 67, wherein the immune response is increased against a target cell.

69. The method of claim 67 or 68, wherein the method treats a disease or condition in the subject.Attorney Docket No. 01202-0066-00PCT 70. The method of claim 69, wherein the disease or condition is a tumor or a cancer, an immune disease or an inflammatory disease.

71. The method of any one of claims 62-70, wherein the subject is a human.

72. Use of the 41BB-binding molecule of any of claims 1-50 and 56, or the pharmaceutical composition of claim 57 or 58, in the manufacture of a medicament for stimulating or inducing an immune response.

73. The use of claim 72, wherein the immune response is increased against a target cell.

74. The use of claim 72 or 73, wherein the use treats a disease or condition in the subject.

75. The use of claim 74, wherein the disease or condition is a tumor or a cancer, an immune disorder or an inflammatory disease.

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