Methods of treating cancer using fusion proteins specific for CD137 and CD228

Abstract

The disclosure provides methods for treating cancer using fusion proteins that bind both CD137 and CD228.

Description

Attorney Docket No.01218-0032-00PCT Methods of Treating Cancer Using Fusion Proteins Specific for CD137 and CD228 I. CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority of US Provisional Application No. 63 / 554,297, filed February 16, 2024, the content of which is incorporated by reference herein in its entirety. II. SEQUENCE LISTING

[0002] The present 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 January 29, 2025, is named “01218-0032-00PCT-ST26.xml” and is 308,864 bytes in size. III. INTRODUCTION AND SUMMARY

[0003] Cluster of differentiation 228 or CD228 (also known as melanotransferrin, MELTF, p97 and MFI2) is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein that belongs to the transferrin family of iron-binding proteins and was first described as an oncofetal protein highly expressed on malignant melanoma cells (Rose et al., Proc Natl Acad Sci U S A, 1986).

[0004] CD228 is expressed in a variety of cancers, including melanoma, mesothelioma, thyroid cancer, lung cancer, liver cancer, pancreatic cancer, head and neck cancer, stomach cancer, colorectal cancer, urothelial cancer, breast cancer, and cervical cancer. Melanoma, also known as malignant melanoma, is a type of cancer that develops from melanocytes, which are pigment-containing cells. Melanoma is the most dangerous type of skin cancer. In 2015, there were 3.1 million people with active disease and melanoma resulted in 59,800 deaths. Surgery can be effective for early-stage melanoma but may not be a treatment option for disease that has metastasized to distant organs. Melanomas that spread often do so to the lymph nodes in the area before spreading elsewhere. Attempts to improve survival by removing lymph nodes surgically were associated with many complications but no overall survival benefit. Immunotherapy, chemotherapy, and radiation therapy have all been used, but are often not curative, particularly for late-stage melanoma. When there is distant metastasis, the cancer is generally considered incurable. The five-year survival rate of stage IV disease is 15-20%.

[0005] CD137 (also known as 4-1BB and TNFRSF9) is a co-stimulatory immune receptor and a member of the tumor necrosis factor receptor (TNFR) superfamily. It is primarily expressed on activated CD4+ and CD8+ T cells, activated B cells, and natural killer (NK) cells but can also be found on resting monocytes and dendritic cells (Li and Liu, Clin Pharmacol, 2013), orAttorney Docket No.01218-0032-00PCT endothelial cells (Snell et al., Immunol Rev, 2011). CD137 plays an important role in regulation of the immune response and thus is a target for cancer immunotherapy. CD137 ligand (CD137L) is the only known natural ligand of CD137, is constitutively expressed on several types of antigen presenting cells, such as activated B cells, monocytes, and splenic dendritic cells, and can be induced on T lymphocytes.

[0006] CD137L is a trimeric protein that exists as a membrane-bound form and as a soluble variant. The ability of soluble CD137L to activate CD137, e.g., on CD137-expressing lymphocytes is limited, however, and large concentrations are required to elicit an effect (Wyzgol et al., J Immunol, 2009). The natural way of activation of CD137 is via the engagement of a CD137-positive cell with a CD137L-positive cell. CD137 activation is then thought to be induced by clustering through CD137L on the opposing cell, leading to signaling via TRAF1, 2 and 3 (Yao et al., Nat Rev Drug Discov, 2013; Snell et al., Immunol Rev, 2011) and further concomitant downstream effects in the CD137-positive T cell. In the case of T cells activated by recognition of their respective cognate targets, the effects elicited by co-stimulation of CD137 are a further enhanced activation, enhanced survival and proliferation, the production of pro-inflammatory cytokines and an improved capacity to kill.

[0007] The present disclosure provides, among other things, methods for treating cancer using fusion proteins that bind both CD137 and CD228.

[0008] The present disclosure provides, among other things, the following numbered embodiments.

[0009] Embodiment 1 is a method for treating a cancer in a human subject, comprising administering to the subject a fusion protein that binds both CD137 and CD228, or is a fusion protein that binds both CD137 and CD228 for use in treating a cancer in a human subject, or is use of a fusion protein that binds both CD137 and CD228 in the manufacture of a medicament for treating a cancer in a human subject, wherein the fusion protein comprises at least two subunits in any order, wherein a first subunit comprises an antibody or an antigen-binding domain thereof specific for CD228, wherein a second subunit comprises a lipocalin mutein specific for CD137, and wherein the fusion protein is administered to the subject at a dose of 0.15 to 22.5 mg / kg.

[0010] Embodiment 2 is the method, fusion protein for use, or use of embodiment 1, wherein the fusion protein is administered to the subject at a dose of 0.15, 0.3, 0.45, 0.5, 1.0, 1.5, 2.5, 3.0, 4.5, 5.0, 7.5, 10.0, 15.0, or 22.5 mg / kg.

[0011] Embodiment 3 is the method, fusion protein for use, or use of embodiment 1, wherein the fusion protein is administered to the subject every week (Q1W).Attorney Docket No.01218-0032-00PCT

[0012] Embodiment 4 is the method, fusion protein for use, or use of embodiment 3, wherein the fusion protein is administered to the subject for at least one 21-day cycle.

[0013] Embodiment 5 is the method, fusion protein for use, or use of embodiment 3 or 4, wherein the fusion protein is administered to the subject for one, two, three, four, or five 21-day cycles.

[0014] Embodiment 6 is the method, fusion protein for use, or use of embodiment 4 or 5, wherein the fusion protein is administered to the subject on days 1, 8, and 15 of the cycle or each of the cycles.

[0015] Embodiment 7 is the method, fusion protein for use, or use of any one of embodiments 3 to 6, wherein the fusion protein is administered to the subject at a dose of 0.15 to 7.5 mg / kg.

[0016] Embodiment 8 is the method, fusion protein for use, or use of any one of embodiments 3 to 7, wherein the fusion protein is administered to the subject at a dose of 0.15, 0.5, 1.5, 2.5, 5.0, or 7.5 mg / kg.

[0017] Embodiment 9 is the method, fusion protein for use, or use of embodiment 1, wherein the fusion protein is administered to the subject every two weeks (Q2W).

[0018] Embodiment 10 is the method, fusion protein for use, or use of embodiment 9, wherein the fusion protein is administered to the subject for at least one 28-day cycle.

[0019] Embodiment 11 is the method, fusion protein for use, or use of embodiment 9 or 10, wherein the fusion protein is administered to the subject for one, two, three, four, or five 28- day cycles.

[0020] Embodiment 12 is the method, fusion protein for use, or use of embodiment 10 or 11, wherein the fusion protein is administered to the subject on days 1 and 15 of the cycle or each of the cycles.

[0021] Embodiment 13 is the method, fusion protein for use, or use of any one of embodiments 9 to 12, wherein the fusion protein is administered to the subject at a dose of 0.3 to 15.0 mg / kg.

[0022] Embodiment 14 is the method, fusion protein for use, or use of any one of embodiments 9 to 13wherein the fusion protein is administered to the subject at a dose of 0.3, 1.0, 3.0, 5.0, 10.0, or 15.0 mg / kg.

[0023] Embodiment 15 is the method, fusion protein for use, or use of embodiment 1, wherein the fusion protein is administered to the subject every three weeks (Q3W).

[0024] Embodiment 16 is the method, fusion protein for use, or use of embodiment 15, wherein the fusion protein is administered to the subject for at least one 21-day cycle.

[0025] Embodiment 17 is the method, fusion protein for use, or use of embodiment 15 or 16, wherein the fusion protein is administered to the subject for one, two, three, four, or five 21- day cycles.Attorney Docket No.01218-0032-00PCT

[0026] Embodiment 18 is the method, fusion protein for use, or use of embodiment 16 or 17, wherein the fusion protein is administered to the subject on day 1 of the cycle or each of the cycles.

[0027] Embodiment 19 is the method, fusion protein for use, or use of any one of embodiments 15 to 18, wherein the fusion protein is administered to the subject at a dose of 0.45 to 22.5 mg / kg.

[0028] Embodiment 20 is the method, fusion protein for use, or use of any one of embodiments 15 to 19, wherein the fusion protein is administered to the subject at a dose of 0.45, 1.5, 4.5, 7.5, 15.0, or 22.5 mg / kg.

[0029] Embodiment 21 is the method, fusion protein for use, or use of any one of embodiments 1 to 20, wherein the fusion protein is administered to the subject intravenously.

[0030] Embodiment 22 is the method, fusion protein for use, or use of any one of embodiments 1 to 21, wherein the fusion protein is administered to the subject as a monotherapy.

[0031] Embodiment 23 is the method, fusion protein for use, or use of any one of embodiments 1 to 22, wherein the cancer has relapsed or become refractory to a prior therapy for the cancer.

[0032] Embodiment 24 is the method, fusion protein for use, or use of any one of embodiments 1 to 23, wherein the cancer is a metastatic cancer or an unresectable cancer.

[0033] Embodiment 25 is the method, fusion protein for use, or use of any one of embodiments 1 to 24, wherein the cancer is a solid cancer.

[0034] Embodiment 26 is the method, fusion protein for use, or use of any one of embodiments 1 to 25, wherein the cancer is lung cancer, melanoma, pancreatic cancer, mesothelioma, colorectal cancer (CRC), thyroid cancer, breast cancer, cholangiocarcinoma, esophageal cancer, head and neck cancer, liver cancer, stomach cancer, urothelial cancer, or cervical cancer.

[0035] Embodiment 27 is the method, fusion protein for use, or use of any one of embodiments 1 to 26, wherein the cancer is cutaneous melanoma, non-small cell lung cancer (NSCLC), colorectal cancer (CRC), pancreatic cancer, or mesothelioma.

[0036] Embodiment 28 is the method, fusion protein for use, or use of any one of embodiments 1 to 27, wherein the cancer is cutaneous melanoma.

[0037] Embodiment 29 is the method, fusion protein for use, or use of embodiment 28, wherein the subject has received a PD-1 inhibitor or a PD-L1 inhibitor.

[0038] Embodiment 30 is the method, fusion protein for use, or use of embodiment 29, wherein the subject has received the PD-1 inhibitor or the PD-L1 inhibitor in combination with another checkpoint inhibitor.Attorney Docket No.01218-0032-00PCT

[0039] Embodiment 31 is the method, fusion protein for use, or use of embodiment 30, wherein the other checkpoint inhibitor is an inhibitor of the CTLA-4 pathway, the LAG-3 pathway, or the TIM-3 pathway.

[0040] Embodiment 32 is the method, fusion protein for use, or use of any one of embodiments 29 to 31, wherein the cutaneous melanoma is resistant or has become refractory to the PD-1 inhibitor or the PD-L1 inhibitor.

[0041] Embodiment 33 is the method, fusion protein for use, or use of any one of embodiments 29 to 32, wherein the PD-1 inhibitor is an anti-PD-1 antibody, or wherein the PD- L1 inhibitor is an anti-PD-L1 antibody.

[0042] Embodiment 34 is the method, fusion protein for use, or use of embodiment 33, wherein the anti-PD-1 antibody is nivolumab, pembrolizumab, cemiplimab, dostarlimab, or retifanlimab, or wherein the anti-PD-L1 antibody is atezolizumab, avelumab, or durvalumab.

[0043] Embodiment 35 is the method, fusion protein for use, or use of any one of embodiments 1 to 34, wherein the cancer is cutaneous melanoma, and the subject has received a BRAF / MEK targeted therapy.

[0044] Embodiment 36 is the method, fusion protein for use, or use of embodiment 35, wherein the BRAF / MEK targeted therapy is a BRAF inhibitor or a MEK inhibitor.

[0045] Embodiment 37 is the method, fusion protein for use, or use of embodiment 36, wherein the BRAF inhibitor is vemurafenib, dabrafenib, or encorafenib, or wherein the MEK inhibitor is trametinib, cobimetinib, or binimetinib.

[0046] Embodiment 38 is the method, fusion protein for use, or use of any one of embodiments 1 to 37, wherein the amino acid sequence of the lipocalin mutein has at least 85% sequence identity to an amino acid sequence selected from SEQ ID NOs: 32-38.

[0047] Embodiment 39 is the method, fusion protein for use, or use of any one of embodiments 1 to 38, wherein the amino acid sequence of the lipocalin mutein comprises an amino acid sequence selected from SEQ ID NOs: 32-38 or a fragment or variant thereof.

[0048] Embodiment 40 is the method, fusion protein for use, or use of any one of embodiments 1 to 37, wherein the amino acid sequence of the lipocalin mutein has at least 85% sequence identity to an amino acid sequence selected from SEQ ID NOs: 39-57.

[0049] Embodiment 41 is the method, fusion protein for use, or use of any one of embodiments 1 to 37 and 40, wherein the amino acid sequence of the lipocalin mutein comprises an amino acid sequence selected from SEQ ID NOs: 39-57 or a fragment or variant thereof.

[0050] Embodiment 42 is the method, fusion protein for use, or use of any one of embodiments 1 to 37, 40, and 41, wherein the amino acid sequence of the lipocalin mutein comprises the amino acid sequence of SEQ ID NO: 40.

[0051] Embodiment 43 is the method, fusion protein for use, or use of any one of embodiments 1 to 42, wherein one subunit is linked to another subunit via a linker.Attorney Docket No.01218-0032-00PCT

[0052] Embodiment 44 is the method, fusion protein for use, or use of any one of embodiments 1 to 43, wherein the second subunit is linked at the N-terminus via a linker to the N- or C-terminus of each heavy chain constant region (CH) of the first subunit or the N- or C- terminus of each light chain constant region (CL) of the first subunit.

[0053] Embodiment 45 is the method, fusion protein for use, or use of embodiment 43 or 44, wherein the linker is an unstructured glycine-serine linker, a polyproline linker, a proline- alanine-serine polymer, or a linker selected from SEQ ID NOs: 13-23.

[0054] Embodiment 46 is the method, fusion protein for use, or use of any one of embodiments 43 to 45, wherein the linker is an unstructured (Gly-Gly-Gly-Gly-Ser)3 linker (SEQ ID NO: 13).

[0055] Embodiment 47 is the method, fusion protein for use, or use of any one of embodiments 1 to 46, wherein the antibody or the antigen-binding domain thereof comprises: a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 110, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 111, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 112, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 116, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 118; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 113, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 114, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 115, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 119, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 120, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 121; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 130, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 131, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 132, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 136, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 137, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 138; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 133, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 134, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 135, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 139, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 140, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 141; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 150, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 151, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 152, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 156, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 157, and (f) CDR-L3 comprising the aminoAttorney Docket No.01218-0032-00PCT acid sequence of SEQ ID NO: 158; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 153, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 154, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 155, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 159, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 160, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 161; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 170, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 171, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 172, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 173, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 174, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 175, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 179, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 180, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 181; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 190, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 191, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 192, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 196, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 197, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 198; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 193, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 194, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 195, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 199, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 200, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 201; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 210, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 211, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 212, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 216, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 217, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 218; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 213, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 214, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 215, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 219, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 220, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 221; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 230, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:Attorney Docket No.01218-0032-00PCT 231, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 232, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 236, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 237, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 238; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 233, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 234, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 235, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 239, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 240, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 241; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 252, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 256, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 257, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 258; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 253, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 254, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 255, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 259, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 260, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 261; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 270, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 271, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 272, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 276, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 277, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 278; or a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 273, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 274, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 275, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 279, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 280, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 281.

[0056] Embodiment 48 is the method, fusion protein for use, or use of embodiment 47, wherein the antibody or the antigen-binding domain thereof comprises: a VH comprising (a) CDR- H1 comprising the amino acid sequence of SEQ ID NO: 210, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 211, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 212, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 216, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 217, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 218; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 213, (b) CDR-H2 comprising the amino acidAttorney Docket No.01218-0032-00PCT sequence of SEQ ID NO: 214, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 215, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 219, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 220, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 221; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 252, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 256, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 257, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 258; or a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 253, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 254, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 255, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 259, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 260, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 261.

[0057] Embodiment 49 is the method, fusion protein for use, or use of embodiment 47 or 48, wherein the antibody or the antigen-binding domain thereof comprises: a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 210, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 211, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 212, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 216, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 217, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 218; or a VH comprising (a) CDR- H1 comprising the amino acid sequence of SEQ ID NO: 213, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 214, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 215, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 219, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 220, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 221.

[0058] Embodiment 50 is the method, fusion protein for use, or use of embodiment 47, wherein the antibody or the antigen-binding domain thereof comprises: a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 122, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 124; a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 142, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 144; a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 162, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 164; a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 182, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 184; a VH comprising an amino acid sequence havingAttorney Docket No.01218-0032-00PCT at least 95% sequence identity to SEQ ID NO: 202, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 204; a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 222, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 224; a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 242, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 244; a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 262, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 264; or a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 282, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 284.

[0059] Embodiment 51 is the method, fusion protein for use, or use of any one of embodiments 47 to 50, wherein the antibody or the antigen-binding domain thereof comprises: a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 222, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 224; or a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 262, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 264.

[0060] Embodiment 52 is the method, fusion protein for use, or use of any one of embodiments 47 to 51, wherein the antibody or the antigen-binding domain thereof comprises: a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 222, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 224.

[0061] Embodiment 53 is the method, fusion protein for use, or use of embodiment 47 to 50, wherein the antibody or the antigen-binding domain thereof comprises: a VH comprising the amino acid sequence of SEQ ID NO: 122, and a VL comprising the amino acid sequence of SEQ ID NO: 124; a VH comprising the amino acid sequence of SEQ ID NO: 142, and a VL comprising the amino acid sequence of SEQ ID NO: 144; a VH comprising the amino acid sequence of SEQ ID NO: 162, and a VL comprising the amino acid sequence of SEQ ID NO: 164; a VH comprising the amino acid sequence of SEQ ID NO: 182, and a VL comprising the amino acid sequence of SEQ ID NO: 184; a VH comprising the amino acid sequence of SEQ ID NO: 202, and a VL comprising the amino acid sequence of SEQ ID NO: 204; a VH comprising the amino acid sequence of SEQ ID NO: 222, and a VL comprising the amino acid sequence of SEQ ID NO: 224; a VH comprising the amino acid sequence of SEQ ID NO: 242, and a VL comprising the amino acid sequence of SEQ ID NO: 244; a VH comprising the amino acid sequence of SEQ ID NO: 262, and a VL comprising the amino acid sequence of SEQ ID NO: 264; or a VH comprising theAttorney Docket No.01218-0032-00PCT amino acid sequence of SEQ ID NO: 282, and a VL comprising the amino acid sequence of SEQ ID NO: 284.

[0062] Embodiment 54 is the method, fusion protein for use, or use of any one of embodiments 47, 48, 50, 51, and 53, wherein the antibody or the antigen-binding domain thereof comprises: a VH comprising the amino acid sequence of SEQ ID NO: 222, and a VL comprising the amino acid sequence of SEQ ID NO: 224; or a VH comprising the amino acid sequence of SEQ ID NO: 262, and a VL comprising the amino acid sequence of SEQ ID NO: 264.

[0063] Embodiment 55 is the method, fusion protein for use, or use of any one of embodiments 47 to 54, wherein the antibody or the antigen-binding domain thereof comprises: a VH comprising the amino acid sequence of SEQ ID NO: 222, and a VL comprising the amino acid sequence of SEQ ID NO: 224.

[0064] Embodiment 56 is the method, fusion protein for use, or use of any one of embodiments 47, 50, and 53, wherein the antibody or the antigen-binding domain thereof comprises: a heavy chain (HC) comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 126, and a light chain (LC) comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 128; a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 146, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 148; a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 166, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 168; a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 186, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 188; a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 206, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 208; a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 226, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 228; a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 246, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 248; a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 266, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 268; or a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 286, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 288.

[0065] Embodiment 57 is the method, fusion protein for use, or use of any one of embodiments 47, 48, 50, 51, 53, 54, and 56, wherein the antibody or the antigen-binding domain thereof comprises: a HC comprising an amino acid sequence having at least 95% sequenceAttorney Docket No.01218-0032-00PCT identity to SEQ ID NO: 226, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 228; or a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 266, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 268.

[0066] Embodiment 58 is the method, fusion protein for use, or use of any one of embodiments 47 to 57, wherein the antibody or the antigen-binding domain thereof comprises: a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 226, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 228.

[0067] Embodiment 59 is the method, fusion protein for use, or use of any one of embodiments 47, 50, 53, and 56, wherein the antibody or the antigen-binding domain thereof comprises: a HC comprising the amino acid sequence of SEQ ID NO: 126, and a LC comprising the amino acid sequence of SEQ ID NO: 128; a HC comprising the amino acid sequence of SEQ ID NO: 146, and a LC comprising the amino acid sequence of SEQ ID NO: 148; a HC comprising the amino acid sequence of SEQ ID NO: 166, and a LC comprising the amino acid sequence of SEQ ID NO: 168; a HC comprising the amino acid sequence of SEQ ID NO: 186, and a LC comprising the amino acid sequence of SEQ ID NO: 188; a HC comprising the amino acid sequence of SEQ ID NO: 206, and a LC comprising the amino acid sequence of SEQ ID NO: 208; a HC comprising the amino acid sequence of SEQ ID NO: 226, and a LC comprising the amino acid sequence of SEQ ID NO: 228; a HC comprising the amino acid sequence of SEQ ID NO: 246, and a LC comprising the amino acid sequence of SEQ ID NO: 248; a HC comprising the amino acid sequence of SEQ ID NO: 266, and a LC comprising the amino acid sequence of SEQ ID NO: 268; or a HC comprising the amino acid sequence of SEQ ID NO: 286, and a LC comprising the amino acid sequence of SEQ ID NO: 288.

[0068] Embodiment 60 is the method, fusion protein for use, or use of any one of embodiments 47, 48, 50, 51, 53, 54, 56, 57, and 59, wherein the antibody or the antigen-binding domain thereof comprises: a HC comprising the amino acid sequence of SEQ ID NO: 226, and a LC comprising the amino acid sequence of SEQ ID NO: 228; or a HC comprising the amino acid sequence of SEQ ID NO: 266, and a LC comprising the amino acid sequence of SEQ ID NO: 268.

[0069] Embodiment 61 is the method, fusion protein for use, or use of any one of embodiments 47 to 60, wherein the antibody or the antigen-binding domain thereof comprises: a HC comprising the amino acid sequence of SEQ ID NO: 226, and a LC comprising the amino acid sequence of SEQ ID NO: 228.

[0070] Embodiment 62 is the method, fusion protein for use, or use of any one of embodiments 1 to 61, wherein the antibody is a monoclonal antibody.Attorney Docket No.01218-0032-00PCT

[0071] Embodiment 63 is the method, fusion protein for use, or use of any one of embodiments 1 to 62, wherein the antibody is a humanized or chimeric antibody.

[0072] Embodiment 64 is the method, fusion protein for use, or use of any one of embodiments 1 to 63, wherein the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody.

[0073] Embodiment 65 is the method, fusion protein for use, or use of any one of embodiments 1 to 64, wherein the antibody is an IgG4 antibody.

[0074] Embodiment 66 is the method, fusion protein for use, or use of embodiment 65, wherein the antibody comprises one or more of the following mutations: S228P, N297A, F234A, L235A, M428L, N434S, M252Y, S254T, and T256E.

[0075] Embodiment 67 is the method, fusion protein for use, or use of any one of embodiments 1 to 48, 50, 51, 53, 54, 56, 57, 59, 60, and 62 to 66, wherein the fusion protein comprises an amino acid sequence shown in any one of SEQ ID NOs: 75, 76 and 78-83, or wherein the fusion protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or higher sequence identity to an amino acid sequence shown in any one of SEQ ID NOs: 75, 76 and 78-83.

[0076] Embodiment 68 is the method, fusion protein for use, or use of any one of embodiments 1 to 48, 50, 51, 53, 54, 56, 57, 59, 60, and 62 to 67, wherein the fusion protein comprises amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or higher sequence identity to the amino acid sequences shown in SEQ ID NOs: 80 and 76, SEQ ID NOs: 82 and 79, SEQ ID NOs: 75 and 81, or SEQ ID NOs: 78 and 83.

[0077] Embodiment 69 is the method, fusion protein for use, or use of any one of embodiments 1 to 48, 50, 51, 53, 54, 56, 57, 59, 60, and 62 to 68, wherein the fusion protein comprises the amino acid sequences shown in SEQ ID NOs: 80 and 76, SEQ ID NOs: 82 and 79, SEQ ID NOs: 75 and 81, or SEQ ID NOs: 78 and 83.

[0078] Embodiment 70 is the method, fusion protein for use, or use of any one of embodiments 1 to 69, wherein the fusion protein comprises the amino acid sequences shown in SEQ ID NOs: 80 and 76. IV. DEFINITIONS

[0079] The following list defines terms, phrases, and abbreviations used throughout the instant specification. All terms listed and defined herein are intended to encompass all grammatical forms.

[0080] As used herein, unless otherwise specified, “CD137” means human CD137 (huCD137). Human CD137 means a full-length protein defined by UniProt Q07011, a fragment thereof, or a variant thereof. CD137 is also known as 4-1BB, tumor necrosis factor receptorAttorney Docket No.01218-0032-00PCT superfamily member 9 (TNFRSF9), and induced by lymphocyte activation (ILA). In some particular embodiments, CD137 of non-human species, e.g., cynomolgus CD137 or mouse CD137, is used.

[0081] As used herein, unless otherwise specified, “CD228” means human CD228. Human CD228 means a full-length protein defined by UniProt P08582, a mature form thereof, an isoform thereof, a fragment thereof, or a variant thereof. Human CD228 is encoded by the MELTF gene. CD228 is also known as melanotransferrin, MELTF, p97 and MFI2, which terms may be used interchangeably herein. In some particular embodiments, CD228 of non-human species, e.g., cynomolgus CD228 or mouse CD228, is used.

[0082] As used herein, “binding affinity” describes the ability of a biomolecule (e.g., a polypeptide or a protein) of the disclosure (e.g., a lipocalin mutein, an antibody, an antigen-binding domain thereof, a fusion protein, or any other peptide or protein) to bind a selected target (and form a complex). Binding affinity is measured by a number of methods known to those skilled in the art including, but not limited to, fluorescence titration, enzyme-linked immunosorbent assay (ELISA)-based assays, including direct and competitive ELISA, calorimetric methods, such as isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). These methods are well-established in the art and some examples of such methods are further described herein. Binding affinity is thereby reported as a value of the dissociation constant (KD), half maximal effective concentration (EC50), or half maximal inhibitory concentration (IC50) measured using such methods. A lower KD, EC50, or IC50value reflects better (higher) binding ability (affinity). Accordingly, the binding affinities of two biomolecules toward a selected target can be measured and compared. When comparing the binding affinities of two biomolecules toward the selected target, the term “comparable to”, “about the same,” “substantially the same” or “substantially similar” means one biomolecule has a binding affinity reported as a KD, an EC50, or an IC50value that is identical or similar to that of another molecule within the experimental variability of the binding affinity measurement. Preferably, “comparable to”, “about the same,” “substantially the same” or “substantially similar” relate to a value that is within 50% deviation to a given reference value, more preferably within 20% deviation, most preferably within 10% deviation. The experimental variability of the binding affinity measurement is dependent upon the specific method used and is known to those skilled in the art.

[0083] As used herein, the term “substantially” may also refer to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.Attorney Docket No.01218-0032-00PCT

[0084] As used herein, the term “detect”, “detection”, “detectable”, or “detecting” is understood both on a quantitative and a qualitative level, as well as a combination thereof. It thus includes quantitative, semi-quantitative, and qualitative measurements performed on a biomolecule of the disclosure.

[0085] As used herein, “detectable affinity” generally means the binding ability between a biomolecule and its target, reported by a KD, EC50, or IC50value, is at most about 10-5M or lower. A binding affinity, reported by a KD, EC50, or IC50value, higher than 10-5M is generally no longer measurable with common methods such as ELISA and SPR and is therefore of secondary importance. Thus, “detectable affinity” may refer to a KDvalue of about 10-5M or lower as determined by ELISA or SPR, preferably SPR.

[0086] As used herein, “specific for”, “specific binding”, “specifically bind”, or “binding specificity” relates to the ability of a biomolecule to discriminate between the desired target (for example, CD137 and CD228) and one or more reference targets (for example, cellular receptor for neutrophil gelatinase-associated lipocalin). It is understood that such specificity is not an absolute but a relative property and can be determined, for example, by means of SPR, western blots, ELISA, fluorescence activated cell sorting (FACS), radioimmunoassay (RIA), electrochemiluminescence (ECL), immunoradiometric assay (IRMA), ImmunoHistoChemistry (IHC), and peptide scans.

[0087] When used herein in the context of a biomolecule, such as an antibody, an antigen- binding domain thereof, or a fusion protein, of the present disclosure that binds to CD137 and / or CD228, the term “specific for”, “specific binding”, “specifically bind”, or “binding specificity” means that the biomolecule binds to, reacts with, or is directed against CD137 and / or CD228, as described herein, but does not substantially bind another protein. The term “another protein” includes any proteins that are not CD137 or CD228 nor proteins closely related to or being homologous to CD137 or CD228. However, CD137 or CD228 from species other than human and fragments and / or variants of CD137 or CD228 are not excluded by the term “another protein.” The term “does not substantially bind” means that a biomolecule of the present disclosure binds another protein with lower binding affinity than CD137 and / or CD228, i.e., shows a cross-reactivity of less than 30%, preferably 20%, more preferably 10%, particularly preferably less than 9, 8, 7, 6, or 5%. Whether the biomolecule specifically reacts as defined herein above can easily be tested, inter alia, by comparing the reaction of a biomolecule of the present disclosure with CD137 and / or CD228 and the reaction of said biomolecule with (an)other protein(s).

[0088] As used herein, the term “lipocalin” refers to a monomeric protein of approximately 18-20 kDa in weight, having a cylindrical β-pleated sheet supersecondary structural region comprising a plurality of β-strands (preferably eight β-strands designated A to H) connected pair-Attorney Docket No.01218-0032-00PCT wise by a plurality of (preferably four) loops at one end to thereby comprise a ligand-binding pocket and define the entrance to the ligand-binding pocket. Preferably, the loops comprising the ligand-binding pocket used in the present disclosure are loops connecting the open ends of β- strands A and B, C and D, E and F, and G and H, and are designated loops AB, CD, EF, and GH. It is well-established that the diversity of said loops in the otherwise rigid lipocalin scaffold gives rise to a variety of different binding modes among the lipocalin family members, each capable of accommodating targets of different sizes, shape, and chemical character (reviewed, e.g., in Skerra, Biochim Biophys Acta, 2000; Flower et al., Biochim Biophys Acta, 2000; Flower, Biochem J, 1996). It is understood that the lipocalin family of proteins has naturally evolved to bind a wide spectrum of ligands, sharing unusually low levels of overall sequence conservation (often with sequence identities of less than 20%) yet retaining a highly conserved overall folding pattern. The correspondence between positions in various lipocalins is also well-known to one of skill in the art (see, e.g., U.S. Patent No.7,250,297). Proteins falling in the definition of “lipocalin” as used herein include, but are not limited to, human lipocalins including tear lipocalin (Tlc, Lcn1), Lipocalin-2 (Lcn2) or neutrophil gelatinase-associated lipocalin (NGAL), apolipoprotein D (ApoD), apolipoprotein M, α1-acid glycoprotein 1, α1-acid glycoprotein 2, α1-microglobulin, complement component 8γ, retinol-binding protein (RBP), the epididymal retinoic acid-binding protein, glycodelin, odorant-binding protein IIa, odorant-binding protein IIb, lipocalin-15 (Lcn15), and prostaglandin D synthase.

[0089] As used herein, unless otherwise specified, “tear lipocalin” refers to human tear lipocalin (hTlc) and further refers to mature human tear lipocalin. The term “mature” when used to characterize a protein means a protein essentially free from the signal peptide. A “mature hTlc” of the instant disclosure refers to the mature form of human tear lipocalin, which is free from the signal peptide. Mature hTlc is described by residues 19-176 of the sequence deposited with the SWISS-PROT Data Bank under Accession Number P31025, and its amino acid sequence is shown in SEQ ID NO: 1.

[0090] As used herein, “Lipocalin-2” or “neutrophil gelatinase-associated lipocalin” refers to human Lipocalin-2 (hLcn2) or human neutrophil gelatinase-associated lipocalin (hNGAL) and further refers to the mature human Lipocalin-2 or mature human neutrophil gelatinase-associated lipocalin. The term “mature” when used to characterize a protein means a protein essentially free from the signal peptide. A “mature hNGAL” of the instant disclosure refers to the mature form of human neutrophil gelatinase-associated lipocalin, which is free from the signal peptide. Mature hNGAL is described by residues 21-198 of the sequence deposited with the SWISS-PROT Data Bank under Accession Number P80188, and its amino acid sequence is shown in SEQ ID NO: 2.

[0091] As used herein, a “native sequence” refers to a protein or a polypeptide having a sequence that occurs in nature or having a wild-type sequence, regardless of its mode ofAttorney Docket No.01218-0032-00PCT preparation. Such native sequence protein or polypeptide can be isolated from nature or can be produced by other means, such as by recombinant or synthetic methods.

[0092] The “native sequence lipocalin” refers to a lipocalin having the same amino acid sequence as the corresponding polypeptide derived from nature. Thus, a native sequence lipocalin can have the amino acid sequence of the respective naturally occurring (wild-type) lipocalin from any organism, in particular, a mammal. The term “native sequence”, when used in the context of a lipocalin, specifically encompasses naturally occurring truncated or secreted forms of the lipocalin, naturally occurring variant forms such as alternatively spliced forms and naturally occurring allelic variants of the lipocalin. The terms “native sequence lipocalin” and “wild- type lipocalin” are used interchangeably herein.

[0093] As used herein, a “mutein”, a “mutated” entity (whether protein or nucleic acid), or “mutant” refers to the exchange, deletion, or insertion of one or more amino acids or nucleotides, compared to the naturally occurring (wild-type) protein or nucleic acid. Said term also includes fragments of a mutein as described herein. The present disclosure explicitly encompasses lipocalin muteins (also referred to as Anticalin® proteins), as described herein, having a cylindrical β-pleated sheet supersecondary structural region comprising eight β-strands connected pair-wise by four loops at one end to thereby comprise a ligand-binding pocket and define the entrance of the ligand-binding pocket, wherein at least one amino acid located within said four loops has been mutated as compared to the native sequence lipocalin. Lipocalin muteins of the present disclosure preferably have the function of binding CD137 as described herein.

[0094] As used herein, the term “fragment”, in connection with the lipocalin muteins of the disclosure, refers to proteins or polypeptides derived from full-length mature hTlc or hNGAL or lipocalin muteins that are N-terminally and / or C-terminally truncated, i.e., lacking at least one of the N-terminal and / or C-terminal amino acids. Such fragments may include at least 10 or more, such as 20 or 30 or more consecutive amino acids of the primary sequence of mature hTlc or hNGAL or the lipocalin mutein it is derived from and are usually detectable in an immunoassay of mature hTlc or hNGAL. Such a fragment may lack up to 2, up to 3, up to 4, up to 5, up to 10, up to 15, up to 20, up to 25, or up to 30 (including all numbers in between) of the N-terminal and / or C-terminal amino acids. As an illustrative example, such a fragment may lack the one, two, three, or four N-terminal (His-His-Leu-Leu) (SEQ ID NO: 5) and / or one or two C-terminal amino acids (Ser-Asp) of mature hTlc. It is understood that the fragment is preferably a functional fragment of mature hTlc or hNGAL or the lipocalin mutein from which it is derived, which means that it preferably retains the binding specificity, preferably to CD137, of mature hTlc / hNGAL or lipocalin mutein it is derived from. As an illustrative example, such a functional fragment may comprise at least amino acids at positions 5-153, 5-150, 9-148, 12-140, 20-135, or 26-133 corresponding to the linear polypeptide sequence of mature hTlc. As another illustrative example, such a functionalAttorney Docket No.01218-0032-00PCT fragment may comprise at least amino acids at positions 13–157, 15-150, 18-141, 20-134, 25- 134, or 28-134 corresponding to the linear polypeptide sequence of mature hNGAL.

[0095] A “fragment” with respect to the corresponding target CD137 or CD228 of an antibody, an antigen-binding domain thereof, or a fusion protein of the disclosure, refers to N- terminally and / or C-terminally truncated CD137 or CD228 or protein domains of CD137 or CD228. Fragments of CD137 or fragments of CD228 as described herein retain the capability of the full- length CD137 or CD228 to be recognized and / or bound by an antibody, an antigen-binding domain thereof, or a fusion protein of the disclosure. As an illustrative example, the fragment may be an extracellular domain of CD137. For example, such an extracellular domain of CD137 may comprise amino acids of the extracellular subdomains of CD137, such as the individual or combined amino acid sequences of domain 1 (residues 24-45 of UniProt Q07011), domain 2 (residues 46-86), domain 3 (87-118) and domain 4 (residues 119-159).

[0096] As used herein, the term “variant” relates to derivatives of a protein or polypeptide that include mutations, for example by substitutions, deletions, insertions, and / or chemical modifications of an amino acid sequence or nucleotide sequence. In some embodiments, such mutations and / or chemical modifications do not reduce the functionality of the protein or peptide. Such substitutions may be conservative, i.e., an amino acid residue is replaced with a chemically similar amino acid residue. Examples of conservative substitutions are the replacements among the members of the following groups: 1) alanine, serine, threonine, and valine; 2) aspartic acid, glutamic acid, glutamine, asparagine, and histidine; 3) arginine, lysine, glutamine, asparagine, and histidine; 4) isoleucine, leucine, methionine, valine, alanine, phenylalanine, threonine, and proline; and 5) isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Such variants include proteins or polypeptides, wherein one or more amino acids have been substituted by their respective D-stereoisomers or by amino acids other than the naturally occurring 20 amino acids, such as, for example, ornithine, hydroxyproline, citrulline, homoserine, hydroxylysine, norvaline. Such variants also include, for instance, proteins or polypeptides in which one or more amino acid residues are added or deleted at the N- and / or C-terminus. Generally, a variant has at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95% or at least about 98% amino acid sequence identity with the native sequence protein or polypeptide. A variant preferably retains the biological activity, e.g., binding the same target, of the protein or polypeptide it is derived from.

[0097] The term “variant”, as used herein with respect to CD137 or CD228 relates to CD137 or CD228 or a fragment thereof, respectively, that has one or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 40, 50, 60, 70, 80 or more amino acid substitutions, deletions and / or insertions in comparison to the native sequence of CD137 or CD228 (wild-type CD137 or CD228), such as CD137 as deposited with UniProt Q07011 or CD228Attorney Docket No.01218-0032-00PCT as deposited with UniProt P08582 as described herein. A CD137 variant or a CD228 variant, respectively, has preferably an amino acid identity of at least 50%, 60%, 70%, 80%, 85%, 90% or 95% with a wild-type CD137 or CD228. A CD137 variant or a CD228 variant as described herein retains the ability to bind antibodies, antigen-binding domains thereof, lipocalin muteins, or fusion proteins specific to CD137 and / or CD228 disclosed herein.

[0098] The term “variant”, as used herein with respect to a lipocalin mutein, relates to a lipocalin mutein or fragment thereof of the disclosure, wherein the sequence has mutations, including substitutions, deletions, insertions, and / or chemical modifications. A variant of a lipocalin mutein as described herein retains the biological activity, e.g., binding to CD137, of the lipocalin mutein from which it is derived. Generally, a lipocalin mutein variant has at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, or 98% amino acid sequence identity with the lipocalin mutein from which it is derived.

[0099] The term “variant”, as used herein with respect to an antibody or an antigen- binding domain thereof relates to an antibody or an antigen-binding domain thereof of the disclosure, wherein the sequence has mutations, including substitutions, deletions, insertions, and / or chemical modifications. A variant of an antibody or an antigen-binding domain thereof as described herein retains the biological activity, e.g., binding to CD228, of the antibody or antigen- binding domain thereof from which it is derived. Generally, a variant of an antibody or an antigen- binding domain thereof has at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, or 98% amino acid sequence identity with the antibody or antigen-binding domain thereof from which it is derived.

[0100] As used herein, the term “mutagenesis” refers to the introduction of mutations into a polynucleotide or amino acid sequence. Mutations are preferably introduced under experimental conditions such that the amino acid naturally occurring at a given position of the protein or polypeptide sequence can be altered, for example substituted by at least one amino acid. The term “mutagenesis” also includes the (additional) modification of the length of sequence segments by deletion or insertion of one or more amino acids. Thus, it is within the scope of the disclosure that, for example, one amino acid at a chosen sequence position is replaced by a stretch of three amino acids, leading to an addition of two amino acid residues compared to the length of the respective segment of the native protein or polypeptide amino acid sequence. Such an insertion or deletion may be introduced independently from each other in any of the sequence segments that can be subjected to mutagenesis in the disclosure. In one exemplary embodiment of the disclosure, an insertion may be introduced into an amino acid sequence segment corresponding to the loop AB of the native sequence lipocalin (cf. International Patent Publication No. WO 2005 / 019256, which is incorporated herein by reference in its entirety).Attorney Docket No.01218-0032-00PCT

[0101] As used herein, the term “random mutagenesis” means that no predetermined mutation (alteration of an amino acid) is present at a certain sequence position but that at least two amino acids can be incorporated with a certain probability at a predefined sequence position during mutagenesis.

[0102] As used herein, the term “sequence identity” or “identity” denotes a property of sequences that measures their similarity or relationship. The term “sequence identity” or “identity” as used in the present disclosure means the percentage of pair-wise identical residues – following (homologous) alignment of a sequence of a protein or polypeptide of the disclosure with a sequence in question – with respect to the number of residues in the longer of these two sequences. Sequence identity is measured by dividing the number of identical amino acid residues by the total number of residues and multiplying the product by 100.

[0103] As used herein, the term “sequence homology” or “homology” has its usual meaning, and a homologous amino acid includes identical amino acids as well as amino acids which are regarded to be conservative substitutions at equivalent positions in the linear amino acid sequence of a protein or polypeptide of the disclosure (e.g., any fusion proteins or lipocalin muteins of the disclosure).

[0104] A skilled artisan will recognize available computer programs, for example BLAST (Altschul et al., Nucleic Acids Res, 1997), BLAST2 (Altschul et al., J Mol Biol, 1990), and Smith- Waterman (Smith and Waterman, J Mol Biol, 1981), for determining sequence homology or sequence identity using standard parameters. The percentage of sequence homology or sequence identity can, for example, be determined herein using the program BLASTP, version 2.2.5 (November 16, 2002; (Altschul et al., Nucleic Acids Res, 1997). In some embodiments, the percentage of homology is based on the alignment of the entire protein or polypeptide sequences (matrix: BLOSUM 62; gap costs: 11.1; cutoff value set to 10-3) including the propeptide sequences, preferably using the wild-type protein scaffold as reference in a pairwise comparison. It is calculated as the percentage of numbers of “positives” (homologous amino acids) indicated as result in the BLASTP program output divided by the total number of amino acids selected by the program for the alignment.

[0105] Specifically, in order to determine whether the amino acid sequence of a lipocalin (mutein) is different from that of a reference (wild-type) lipocalin with regard to a certain position in the amino acid sequence of the reference (wild-type) lipocalin, a skilled artisan can use means and methods well-known in the art, e.g., alignments, either manually or by using computer programs such as BLAST 2.0, which stands for Basic Local Alignment Search Tool, or ClustalW, or any other suitable program which is suitable to generate sequence alignments. Accordingly, the amino acid sequence of a reference (wild-type) lipocalin can serve as “subject sequence” orAttorney Docket No.01218-0032-00PCT “reference sequence”, while the amino acid sequence of a lipocalin mutein serves as “query sequence”. The terms “wild-type sequence”, “reference sequence” and “subject sequence” are used interchangeably herein. A preferred wild-type sequence of a lipocalin is the sequence of hTLc as shown in SEQ ID NO: 1 or hNGAL as shown in SEQ ID NO: 2.

[0106] “Gaps” are spaces in an alignment that are the result of additions or deletions of amino acids. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved, and have deletions, additions, or replacements, may have a lower degree of sequence identity.

[0107] As used herein, the term “position” means the position of either an amino acid within an amino acid sequence disclosed herein or the position of a nucleotide within a nucleic acid sequence disclosed herein. It is to be understood that when the term “correspond” or “corresponding” is used herein in the context of the amino acid sequence positions of one or more lipocalin muteins, a corresponding position is not only determined by the number of the preceding nucleotides or amino acids. Accordingly, the absolute position of a given amino acid in accordance with the disclosure may vary from the corresponding position due to deletion or addition of amino acids elsewhere in a (mutant or wild-type) lipocalin. Similarly, the absolute position of a given nucleotide in accordance with the present disclosure may vary from the corresponding position due to deletions or additional nucleotides elsewhere in a mutein or wild- type lipocalin 5’-untranslated region (UTR) including the promoter and / or any other regulatory sequences or gene regions (including exons and introns).

[0108] A “corresponding position” in accordance with the disclosure may be the sequence position that aligns to the sequence position it corresponds to in a pairwise or multiple sequence alignment according to the present disclosure. It is preferably to be understood that for a “corresponding position” in accordance with the disclosure, the absolute positions of nucleotides or amino acids may differ from adjacent nucleotides or amino acids but said adjacent nucleotides or amino acids which may have been exchanged, deleted, or added may be comprised by the same one or more “corresponding positions”.

[0109] In addition, for a corresponding position in a lipocalin mutein based on a reference sequence in accordance with the disclosure, it is preferably to be understood that the positions of nucleotides or amino acids of a lipocalin mutein can structurally correspond to the positions elsewhere in a reference lipocalin (wild-type lipocalin) or another lipocalin mutein, even if they may differ in the absolute position numbers, as appreciated by the skilled person in light of the highly-conserved overall folding pattern among lipocalins.

[0110] As used interchangeably herein, the terms “conjugate”, “conjugation”, “fuse”, “fusion”, or “linked” refer to the joining together of two or more subunits, through all forms ofAttorney Docket No.01218-0032-00PCT covalent or non-covalent linkage, by means including, but not limited to, genetic fusion, chemical conjugation, coupling through a linker or a cross-linking agent, and non-covalent association.

[0111] The term “fusion polypeptide” or “fusion protein” as used herein refers to a polypeptide or protein comprising two or more subunits. In some embodiments, a fusion protein as described herein comprises two or more subunits, at least one of these subunits being capable of specifically binding to CD137, and a further subunit capable of specifically binding to CD228. Within the fusion protein, these subunits may be linked by covalent or non-covalent linkage. Preferably, the fusion protein is a translational fusion between the two or more subunits. The translational fusion may be generated by genetically engineering the coding sequence for one subunit in a reading frame with the coding sequence of a further subunit. Both subunits may be interspersed by a nucleotide sequence encoding a linker. However, the subunits of a fusion protein of the present disclosure may also be linked through chemical conjugation. The subunits forming the fusion protein are typically linked to each other as follows: C-terminus of one subunit to N-terminus of another subunit, or C-terminus of one subunit to C-terminus of another subunit, or N-terminus of one subunit to N-terminus of another subunit, or N-terminus of one subunit to C- terminus of another subunit. The subunits of the fusion protein can be linked in any order and may include more than one of any of the constituent subunits. If one or more of the subunits are part of a protein (complex) that consists of more than one polypeptide chain, the term “fusion protein” may also refer to the protein comprising the fused sequences and all other polypeptide chain(s) of the protein (complex). As an illustrative example, where a full-length immunoglobulin / antibody is fused to a lipocalin mutein via a heavy or light chain of the immunoglobulin / antibody, the term “fusion protein” may refer to the single polypeptide chain comprising the lipocalin mutein and the heavy or light chain of the immunoglobulin / antibody. The term “fusion protein” may also refer to the entire immunoglobulin / antibody (both light and heavy chains) and the lipocalin mutein fused to one or both of its heavy and / or light chains.

[0112] As used herein, the term “subunit” of a fusion protein disclosed herein refers to a single protein or a separate polypeptide chain, which can form a stable folded structure by itself and may define a unique function of providing a binding motif towards a target. In some embodiments, a preferred subunit of the disclosure is a lipocalin mutein. In some other embodiments, a preferred subunit of the disclosure is an antibody, such as a full-length antibody, or an antigen-binding domain / fragment thereof.

[0113] A “linker” that may be comprised by a fusion protein of the present disclosure joins together two or more subunits of a fusion protein as described herein. The linkage can be covalent or non-covalent. A preferred covalent linkage is via a peptide bond, such as a peptide bond between amino acids. A preferred linker is a peptide linker. Accordingly, in a preferred embodiment, said linker comprises one or more amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,Attorney Docket No.01218-0032-00PCT 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids. Preferred peptide linkers are described herein, including glycine-serine (GS) linkers, glycosylated GS linkers, and proline-alanine-serine polymer (PAS) linkers. In some preferred embodiments, a GS linker, such as a (G4S)3linker as described in SEQ ID NO: 13, is used to join together the subunits of a fusion protein. Other preferred linkers include chemical linkers.

[0114] As used herein, the term “albumin” includes all mammalian albumins, such as human serum albumin or bovine serum albumin or rat serum albumin.

[0115] As used herein, the term “organic molecule” or “small organic molecule” denotes an organic molecule comprising at least two carbon atoms, but preferably not more than 7 or 12 rotatable carbon bonds, having a molecular weight in the range between 100 and 2,000 daltons, preferably between 100 and 1,000 daltons, and optionally including one or two metal atoms.

[0116] A “sample” is defined as a biological sample taken from any subject. Biological samples include, but are not limited to, blood, serum, urine, feces, semen, or tissue, including tumor tissue.

[0117] A “subject” is a vertebrate, preferably a mammal, more preferably a human. The term “mammal” is used herein to refer to any animal classified as a mammal, including, without limitation, humans, domestic and farm animals, and zoo, sports, or pet animals, such as sheep, dogs, horses, cats, cows, rats, pigs, apes such as cynomolgus monkeys, to name only a few illustrative examples. Preferably, the “mammal” used herein is human. The term does not necessarily indicate that the subject has been diagnosed with a particular disease, but typically refers to an individual under medical supervision.

[0118] The terms “therapy,” “treating,” “treatment,” and “amelioration” refer to any reduction in the severity of symptoms. In the case of treating cancer, treatment can refer to reducing, e.g., tumor size, number of cancer cells, growth rate, metastatic activity, cell death of non-cancer cells, etc. As used herein, the terms “treat” and “prevent” are not intended to be absolute terms. Treatment and prevention can refer to any delay in onset, amelioration of symptoms, improvement in patient survival, increase in survival time or rate, etc. Treatment and prevention can be complete (no detectable symptoms remaining) or partial, such that symptoms are less frequent or severe than in a patient without the treatment described herein. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment. In some aspects, the severity of disease is reduced by at least 10%, as compared, e.g., to the individual before administration or to a control individual not undergoing treatment. In some aspects, the severity of disease is reduced by at least 25%, 50%, 75%, 80%, or 90%, or in some cases, no longer detectable using standard diagnostic techniques.Attorney Docket No.01218-0032-00PCT

[0119] As used herein, a “therapeutic amount,” “effective amount,” or “therapeutically effective amount” of an agent (e.g., a fusion protein as described herein) is an amount of the agent that prevents, alleviates, abates, ameliorates, or reduces the severity of symptoms of a disease (e.g., a cancer) in a subject.

[0120] As used herein, “resistant” or “refractory” refers to a cancer that responds poorly or does not respond to a therapy for the cancer. In some embodiments, the therapy is indicated for treating the cancer. In some embodiments, the cancer is resistant or refractory to the therapy before or at the beginning of the therapy. In other embodiments, the cancer becomes resistant or refractory to the therapy during the course of the therapy.

[0121] As used herein, “relapse” refers to progression of a cancer after an initial period of responsiveness (e.g., complete response or partial response) to a therapy. The initial period of responsiveness may comprise, for example, a reduction in cancer size, a reduction in the number of cancer cells, no increase in cancer size, no increase in the number of cancer cells, slowing of cancer growth, or slowing of the increase in the number of cancer cells. The progression may comprise, for example, an increase in cancer size, an increase in the number of cancer cells, acceleration of cancer growth, or acceleration of the increase in the number of cancer cells. The initial period of responsiveness may involve the number of cancer cells, or the cancer size, falling below a clinical threshold. The progression may involve the number of cancer cells, or the cancer size, rising above a clinical threshold.

[0122] The terms “administer,” “administered,” or “administering” refer to methods of delivering agents, compounds, or compositions to the desired site of biological action. These methods include, but are not limited to, topical delivery, parenteral delivery, intravenous delivery, intradermal delivery, intramuscular delivery, colonic delivery, rectal delivery, or intraperitoneal delivery. Administration techniques that are optionally employed with the agents and methods described herein, include e.g., as discussed in Goodman and Gilman, THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, current ed.; Pergamon; and Remington's, PHARMACEUTICAL SCIENCES (current edition), Mack Publishing Co., Easton, PA.

[0123] As used herein, “antibody” includes whole antibodies or any antigen-binding fragment (i.e., “antigen-binding portion” or “antigen-binding domain”) or single chain thereof. The terms “antibody” and “immunoglobulin” can be and are used interchangeably herein. A whole antibody refers to a glycoprotein comprising at least two heavy chains (HCs) and two light chains (LCs) inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable domain / region (VHor HCVR) and a heavy chain constant region (CH). The heavy chain constant region is comprised of three domains, CH1, CH2and CH3. Each light chain is comprised of a light chain variable domain / region (VLor LCVR) and a light chain constant region (CL). TheAttorney Docket No.01218-0032-00PCT light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VHand VLis composed of three CDRs and four FRs, arranged in the following order from the amino- terminus to the carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen (for example, CD228). The constant regions of the antibodies may optionally mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0124] As used herein, “antigen-binding fragment” (also referred to as “antigen-binding domain”) of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., CD228). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding fragment” of an antibody include (i) a Fab fragment consisting of the VH, VL, CLand CH1domains; (ii) a F(ab′)2fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab′ fragment consisting of the VH, VL, CLand CH1domains and the region between the CH1and CH2domains; (iv) an Fd fragment consisting of the VHand CH1domains; (v) a single-chain Fv fragment consisting of the VHand VLdomains of a single arm of an antibody, (vi) a dAb fragment (Ward et al., Nature, 1989) consisting of a VHdomain; (vii) an isolated complementarity determining region (CDR) or a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker; (viii) a “diabody” comprising the VHand VLconnected in the same polypeptide chain using a short linker (see, e.g., patent documents EP 404,097; WO 93 / 11161; and Holliger et al., Proc Natl Acad Sci U S A, 1993); and (ix) a “domain antibody fragment” containing only the VHor VL, where in some instances two or more VHregions are covalently joined.

[0125] Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof (e.g., humanized, chimeric, or multispecific). Antibodies may also be fully human.

[0126] As used herein, “framework” or “FR” refers to the variable domain residues other than the hypervariable region (CDR) residues.

[0127] “Fragment crystallizable region” or “Fc region” refers to the C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof numbering according to EUAttorney Docket No.01218-0032-00PCT index of Kabat (Johnson and Wu, Nucleic Acids Res, 2000). The C-terminal lysine (residue 447 according to EU index of Kabat) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue. Suitable native-sequence Fc regions for use in the antibodies of the disclosure include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0128] “Fc receptor” or “FcR” refers to a receptor that binds to the Fc region of an antibody.

[0129] As used herein, “isolated antibody” refers to an antibody that is substantially free of its natural environment. For instance, an isolated antibody is substantially free of cellular material and other proteins from the cell or tissue source from which it is derived. An “isolated antibody” further refers to an antibody that is substantially free of other antibodies having different antigenic specificities. In the present case, an isolated antibody that binds specifically CD228 is substantially free of antibodies that specifically bind antigens other than CD228. However, an isolated antibody that specifically binds CD228 may have cross-reactivity to other antigens, such as CD228 molecules from other species.

[0130] As used herein, “monoclonal antibody” refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0131] As used herein, “humanized antibody” refers to an antibody that consists of the CDRs of antibodies derived from mammals other than human, and the FR region and the constant region of a human antibody or derived from a human antibody. A humanized antibody may comprise a variable domain that has a variable region amino acid sequence which, analyzed as a whole, is closer to human than to other species as assessed using the Immunogenetics Information System (IMGT) DomainGapAlign tool, as described by Ehrenmann et al. (2010). A humanized antibody may be useful as an effective component in a therapeutic agent due to the reduced antigenicity. The term “therapeutic agent” or “therapeutically active agent”, as used herein, refers to an agent which is therapeutically useful. A therapeutic agent may be any agent for the prevention, amelioration, or treatment of a disease, a physiological condition, a symptom, or for the evaluation or diagnosis thereof.

[0132] As used herein, “human antibody” includes antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of theAttorney Docket No.01218-0032-00PCT disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0133] As used herein, “antibody clone X” may also be referred to as “OMTX.” For example, “antibody clone 30” may also be referred to as “OMT30.” Antibody clone 8 may also be referred to as “EE03”; antibody clone 24 may also be referred to as “EB03”; antibody clone 30 may also be referred to as “OC04”; antibody clone 35 may also be referred to as “OB02”; and antibody clone 36 may also be referred to as “OE12.”

[0134] As used herein, “AAFX” refers to a fusion protein containing antibody clone X and the lipocalin mutein having the amino acid sequence of SEQ ID NO: 40 (Lipocalin Mutein I). For example, “AAF30” refers to a fusion protein containing antibody clone 30 and Lipocalin Mutein I. The notation “HC” in the name of a fusion protein refers to the lipocalin mutein therein being conjugated to the antibody via the antibody’s heavy chain, and the notation “LC” in the name of a fusion protein refers to the lipocalin mutein therein being conjugated to the antibody via the antibody’s light chain. A fusion protein name that contains a clone number followed by “HC” or “LC” refers to a fusion protein containing that antibody clone and Lipocalin Mutein I, conjugated via the antibody’s heavy chain or light chain, respectively. For example, “30HC” refers to a fusion protein containing antibody clone 30 and Lipocalin Mutein I conjugated thereto via the antibody’s heavy chain.

[0135] An antibody or fusion protein may also be denoted by its heavy and light chain sequences (e.g., the fusion protein having the sequences of SEQ ID NOs: 80 and 76), one of which in the case of a fusion protein (e.g., SEQ ID NO: 80) contains the sequence of a lipocalin mutein. V. DETAILED DESCRIPTION OF THE DISCLOSURE

[0136] The fusion proteins that target both CD137 and CD228 as provided herein are based on the recognition that a bivalent CD137-binder, such as an antibody, may not be sufficient by itself to cluster CD137 on T cells or NK cells and lead to efficient activation, similar to the lack of activity of the trivalent soluble CD137L. A second problem observed with bivalent anti-CD137 antibodies is that active doses can in some instances elicit on-target liver toxicity, indicating a need for a more specific, tumor-targeted mode of action.

[0137] Based in part upon these various insights, the present disclosure provides, among other things, novel approaches for simultaneously engaging CD137 and CD228 via one or moreAttorney Docket No.01218-0032-00PCT fusion proteins having binding specificity for CD137 and binding specificity for CD228. The binding specificity for CD228 may be provided by an antibody or antigen-binding domain thereof that binds CD228 as provided herein. Provided fusion proteins are designed to promote CD137 clustering by bridging CD137-positive T cells with CD228-expressing tumor cells located in the tumor microenvironment. Hence, the fusion proteins are designed to allow the localized induction of antigen-specific T cells in the tumor microenvironment, potentially reducing peripheral toxicity. This is in contrast to, for example, urelumab, a 4-1BB agonist that is active in solid tumors but that elicits on-target liver toxicity at active doses.

[0138] Based in part upon these various insights, the present disclosure also provides methods for treating cancer using fusion proteins that bind both CD137 and CD228. The disclosure also provides methods of making fusion proteins described herein as well as compositions comprising such proteins. A. Methods for treating cancer using fusion proteins of the disclosure.

[0139] In some embodiments, provided herein is a method for treating a cancer in a human subject, comprising administering to the subject any one of the fusion proteins disclosed herein, wherein the fusion protein is administered to the subject at a dose of 0.15 to 22.5 mg / kg. In some embodiments, provided herein is method for treating a cancer in a human subject, comprising administering to the subject a fusion protein that binds both CD137 and CD228, wherein the fusion protein comprises at least two subunits in any order, wherein a first subunit comprises an antibody or an antigen-binding domain thereof specific for CD228, wherein a second subunit comprises a lipocalin mutein specific for CD137, and wherein the fusion protein is administered to the subject at a dose of 0.15 to 22.5 mg / kg.

[0140] In some embodiments, the fusion protein is administered to the subject at a dose of 0.15, 0.3, 0.45, 0.5, 1.0, 1.5, 2.5, 3.0, 4.5, 5.0, 7.5, 10.0, 15.0, or 22.5 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 0.15 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 0.3 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 0.45 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 0.5 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 1.0 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 1.5 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 2.5 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 3.0 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 4.5 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 5.0 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 7.5 mg / kg. In someAttorney Docket No.01218-0032-00PCT embodiments, the fusion protein is administered to the subject at a dose of 10.0 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 15.0 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 22.5 mg / kg.

[0141] In some embodiments, the fusion protein is administered to the subject every week (Q1W). In some embodiments, the fusion protein is administered to the subject for at least one 21-day cycle. In some embodiments, the fusion protein is administered to the subject for one, two, three, four, or five 21-day cycles. In some embodiments, the fusion protein is administered to the subject for one 21-day cycle. In some embodiments, the fusion protein is administered to the subject for two 21-day cycles. In some embodiments, the fusion protein is administered to the subject for three 21-day cycles. In some embodiments, the fusion protein is administered to the subject for four 21-day cycles. In some embodiments, the fusion protein is administered to the subject for five 21-day cycles. In some embodiments, the fusion protein is administered to the subject on days 1, 8, and 15 of the cycle or each of the cycles. In some embodiments, the fusion protein is administered to the subject at a dose of 0.15 to 7.5 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 0.15, 0.5, 1.5, 2.5, 5.0, or 7.5 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 0.15 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 0.5 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 1.5 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 2.5 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 5.0 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 7.5 mg / kg.

[0142] In some embodiments, the fusion protein is administered to the subject every two weeks (Q2W). In some embodiments, the fusion protein is administered to the subject for at least one 28-day cycle. In some embodiments, the fusion protein is administered to the subject for one, two, three, four, or five 28-day cycles. In some embodiments, the fusion protein is administered to the subject for one 28-day cycle. In some embodiments, the fusion protein is administered to the subject for two 28-day cycles. In some embodiments, the fusion protein is administered to the subject for three 28-day cycles. In some embodiments, the fusion protein is administered to the subject for four 28-day cycles. In some embodiments, the fusion protein is administered to the subject for five 28-day cycles. In some embodiments, the fusion protein is administered to the subject on days 1 and 15 of the cycle or each of the cycles. In some embodiments, the fusion protein is administered to the subject at a dose of 0.3 to 15.0 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 0.3, 1.0, 3.0, 5.0, 10.0, or 15.0 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 0.3 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 1.0 mg / kg. In some embodiments, the fusion protein is administered to the subject at aAttorney Docket No.01218-0032-00PCT dose of 3.0 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 5.0 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 10.0 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 15.0 mg / kg.

[0143] In some embodiments, the fusion protein is administered to the subject every three weeks (Q3W). In some embodiments, the fusion protein is administered to the subject for at least one 21-day cycle. In some embodiments, the fusion protein is administered to the subject for one, two, three, four, or five 21-day cycles. In some embodiments, the fusion protein is administered to the subject for one 21-day cycle. In some embodiments, the fusion protein is administered to the subject for two 21-day cycles. In some embodiments, the fusion protein is administered to the subject for three 21-day cycles. In some embodiments, the fusion protein is administered to the subject for four 21-day cycles. In some embodiments, the fusion protein is administered to the subject for five 21-day cycles. In some embodiments, the fusion protein is administered to the subject on day 1 of the cycle or each of the cycles. In some embodiments, the fusion protein is administered to the subject at a dose of 0.45 to 22.5 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 0.45, 1.5, 4.5, 7.5, 15.0, or 22.5 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 0.45 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 1.5 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 4.5 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 7.5 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 15.0 mg / kg. In some embodiments, the fusion protein is administered to the subject at a dose of 22.5 mg / kg.

[0144] In some embodiments, the fusion protein is administered to the subject intravenously. In some embodiments, the fusion protein is administered to the subject as a monotherapy.

[0145] In some embodiments, the cancer has relapsed or become refractory to a prior therapy for the cancer. In some embodiments, the cancer is a metastatic cancer or an unresectable cancer. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is lung cancer, melanoma, pancreatic cancer, mesothelioma, colorectal cancer (CRC), thyroid cancer, breast cancer, cholangiocarcinoma, esophageal cancer, head and neck cancer, liver cancer, stomach cancer, urothelial cancer, or cervical cancer. In some embodiments, the cancer is cutaneous melanoma, non-small cell lung cancer (NSCLC), CRC, pancreatic cancer, or mesothelioma. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is NSCLC. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is cutaneous melanoma. In some embodiments, the cancer is pancreatic cancer. In someAttorney Docket No.01218-0032-00PCT embodiments, the cancer is mesothelioma. In some embodiments, the cancer is CRC. In some embodiments, the cancer is thyroid cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is cholangiocarcinoma. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is stomach cancer. In some embodiments, the cancer is urothelial cancer. In some embodiments, the cancer is cervical cancer.

[0146] In some embodiments, the cancer is cutaneous melanoma. In some embodiments, the subject has received a PD-1 inhibitor or a PD-L1 inhibitor. In some embodiments, the subject has received a PD-1 inhibitor. In some embodiments, the subject has received a PD-L1 inhibitor. In some embodiments, the subject has received the PD-1 inhibitor or the PD-L1 inhibitor in combination with another checkpoint inhibitor. In some embodiments, the subject has received the PD-1 inhibitor in combination with another checkpoint inhibitor. In some embodiments, the subject has received the PD-L1 inhibitor in combination with another checkpoint inhibitor. In some embodiments, the other checkpoint inhibitor is an inhibitor of the CTLA-4 pathway, the LAG-3 pathway, or the TIM-3 pathway. In some embodiments, the other checkpoint inhibitor is an inhibitor of the CTLA-4 pathway. In some embodiments, the other checkpoint inhibitor is an inhibitor of the LAG-3 pathway. In some embodiments, the other checkpoint inhibitor is an inhibitor of the TIM-3 pathway. In some embodiments, the cutaneous melanoma is resistant or has become refractory to the PD-1 inhibitor or the PD-L1 inhibitor. In some embodiments, the cutaneous melanoma is resistant or has become refractory to the PD-1 inhibitor. In some embodiments, the cutaneous melanoma is resistant or has become refractory to the PD-L1 inhibitor. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody, or the PD-L1 inhibitor is an anti-PD-L1 antibody. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody. In some embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody. In some embodiments, the anti-PD-1 antibody is nivolumab, pembrolizumab, cemiplimab, dostarlimab, or retifanlimab, or the anti-PD-L1 antibody is atezolizumab, avelumab, or durvalumab. In some embodiments, the anti-PD-1 antibody is nivolumab. In some embodiments, the anti-PD-1 antibody is pembrolizumab. In some embodiments, the anti-PD-1 antibody is cemiplimab. In some embodiments, the anti-PD-1 antibody is dostarlimab. In some embodiments, the anti-PD-1 antibody is retifanlimab. In some embodiments, the anti-PD-L1 antibody is atezolizumab. In some embodiments, the anti-PD-L1 antibody is avelumab. In some embodiments, the anti-PD-L1 antibody is durvalumab.

[0147] In some embodiments, the cancer is cutaneous melanoma, and the subject has received a BRAF / MEK targeted therapy. In some embodiments, the BRAF / MEK targeted therapy is a BRAF inhibitor or a MEK inhibitor. In some embodiments, the BRAF / MEK targeted therapy is a BRAF inhibitor. In some embodiments, the BRAF / MEK targeted therapy is a MEK inhibitor. InAttorney Docket No.01218-0032-00PCT some embodiments, the BRAF inhibitor is vemurafenib, dabrafenib, or encorafenib, or the MEK inhibitor is trametinib, cobimetinib, or binimetinib. In some embodiments, the BRAF inhibitor is vemurafenib. In some embodiments, the BRAF inhibitor is dabrafenib. In some embodiments, the BRAF inhibitor is encorafenib. In some embodiments, the MEK inhibitor is trametinib. In some embodiments, the MEK inhibitor is cobimetinib. In some embodiments, the MEK inhibitor is binimetinib.

[0148] In some embodiments, prior to administering the fusion protein to the subject, a long-acting oral antihistamine is administered to the subject. In some embodiments, the evening prior to administering the fusion protein to the subject, a long-acting oral antihistamine is administered to the subject.

[0149] In some embodiments, prior to administering the fusion protein to the subject, an antipyretic, an antihistamine, a corticosteroid, and an oral long-acting antihistamine are administered to the subject. In some embodiments, 45 to 90 minutes prior to administering the fusion protein to the subject, an antipyretic, an antihistamine, a corticosteroid, and an oral long- acting antihistamine are administered to the subject. In some embodiments, prior to administering the fusion protein to the subject, an antipyretic, an antihistamine, and an oral long-acting antihistamine are administered to the subject. In some embodiments, 45 to 90 minutes prior to administering the fusion protein to the subject, an antipyretic, an antihistamine, and an oral long- acting antihistamine are administered to the subject. In some embodiments, after the start of administering the fusion protein to the subject, the antipyretic and the antihistamine are further administered to the subject. In some embodiments, 4 to 6 hours after the start of administering the fusion protein to the subject, the antipyretic and the antihistamine are further administered to the subject.

[0150] In some embodiments, the antipyretic is 500 to 1000 mg acetaminophen (paracetamol). In some embodiments, the antipyretic is administered to the subject intravenously or orally. In some embodiments, the antihistamine is an H1 blocker. In some embodiments, the antihistamine is 25 to 50 mg diphenhydramine. In some embodiments, the antihistamine is administered to the subject intravenously or orally. In some embodiments, the corticosteroid is 100 mg hydrocortisone. In some embodiments, the corticosteroid is 200 mg hydrocortisone, and the fusion protein is administered to the subject every week (Q1W) at a dose of 2.5 mg / kg or higher, is administered to the subject every two weeks (Q2W) at a dose of 5.0 mg / kg or higher, or is administered to the subject every three weeks (Q3W) at a dose of 7.5 mg / kg or higher. In some embodiments, the corticosteroid is administered to the subject intravenously. In some embodiments, the long-acting oral antihistamine is 10 mg cetirizine, administered orally.

[0151] In some embodiments, prior to administering and / or after the start of administeringAttorney Docket No.01218-0032-00PCT the fusion protein to the subject, an H2 blocker and / or a leukotriene inhibitor is administered to the subject. In some embodiments, prior to administering and / or after the start of administering the fusion protein to the subject, greater than 100 mg or greater than 200 mg hydrocortisone and / or a longer acting steroid than hydrocortisone is administered to the subject.

[0152] In some embodiments, prior to administering the fusion protein to the subject, the subject has an absolute neutrophil count (ANC) ≥ 1500 / μL (1.5 ×10^9 / L). In some embodiments, prior to administering the fusion protein to the subject, the subject has hemoglobin (Hgb) ≥ 9 g / dL. In some embodiments, prior to administering the fusion protein to the subject, the subject has platelet count ≥ 100,000 / μL. In some embodiments, prior to administering the fusion protein to the subject, the subject has serum bilirubin ≤ 1.5 × upper limit of normal (ULN), or ≤ 3 × ULN if the subject has documented hepatic tumor involvement, or direct bilirubin ≤ 1.5 × ULN if the subject has Gilbert’s disease. In some embodiments, prior to administering the fusion protein to the subject, the subject has individual estimated glomerular filtration rate (GFR) ≥ 45 mL / min / 1.73 m^2, measured using, for example the Modification of Diet in Renal Disease (MDRD) study equation. In some embodiments, prior to administering the fusion protein to the subject, the subject has alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ≤ 3 × ULN, or ALT and AST ≤ 5 × ULN if there is evidence of hepatic involvement by malignant disease in the subject. B. Exemplary antibodies or antigen-binding domains thereof specific for CD228.

[0153] In some embodiments, an antibody or an antigen-binding domain thereof that binds CD228 (e.g., an independent antibody or antigen-binding domain thereof, or an antibody or antigen- binding domain thereof that is comprised in any one of the fusion proteins disclosed herein) is provided, wherein the antibody or the antigen-binding domain thereof comprises: i) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 110, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 111, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 112, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 116, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 118; ii) a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 113, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 114, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 115, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 119, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 120, and (f) CDR-Attorney Docket No.01218-0032-00PCT L3 comprising the amino acid sequence of SEQ ID NO: 121; iii) a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 130, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 131, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 132, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 136, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 137, and (f) CDR- L3 comprising the amino acid sequence of SEQ ID NO: 138; iv) a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 133, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 134, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 135, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 139, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 140, and (f) CDR- L3 comprising the amino acid sequence of SEQ ID NO: 141; v) a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 150, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 151, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 152, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 156, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 157, and (f) CDR- L3 comprising the amino acid sequence of SEQ ID NO: 158; vi) a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 153, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 154, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 155, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 159, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 160, and (f) CDR- L3 comprising the amino acid sequence of SEQ ID NO: 161; vii) a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 170, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 171, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 172, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and (f) CDR- L3 comprising the amino acid sequence of SEQ ID NO: 178; viii) a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 173, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 174, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 175, and a VLAttorney Docket No.01218-0032-00PCT comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 179, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 180, and (f) CDR- L3 comprising the amino acid sequence of SEQ ID NO: 181; ix) a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 190, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 191, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 192, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 196, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 197, and (f) CDR- L3 comprising the amino acid sequence of SEQ ID NO: 198; x) a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 193, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 194, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 195, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 199, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 200, and (f) CDR- L3 comprising the amino acid sequence of SEQ ID NO: 201; xi) a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 210, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 211, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 212, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 216, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 217, and (f) CDR- L3 comprising the amino acid sequence of SEQ ID NO: 218; xii) a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 213, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 214, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 215, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 219, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 220, and (f) CDR- L3 comprising the amino acid sequence of SEQ ID NO: 221; xiii) a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 230, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 231, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 232, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 236, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 237, and (f) CDR- L3 comprising the amino acid sequence of SEQ ID NO: 238; xiv) a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:Attorney Docket No.01218-0032-00PCT 233, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 234, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 235, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 239, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 240, and (f) CDR- L3 comprising the amino acid sequence of SEQ ID NO: 241; xv) a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 252, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 256, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 257, and (f) CDR- L3 comprising the amino acid sequence of SEQ ID NO: 258; xvi) a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 253, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 254, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 255, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 259, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 260, and (f) CDR- L3 comprising the amino acid sequence of SEQ ID NO: 261; xvii) a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 270, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 271, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 272, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 276, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 277, and (f) CDR- L3 comprising the amino acid sequence of SEQ ID NO: 278; or xviii) a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 273, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 274, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 275, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 279, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 280, and (f) CDR- L3 comprising the amino acid sequence of SEQ ID NO: 281.

[0154] In some embodiments, the antibody or the antigen-binding domain thereof that binds CD228 provided herein (e.g., an independent antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof that is comprised in any one of the fusion proteins disclosed herein) comprises: i) a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:Attorney Docket No.01218-0032-00PCT 210, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 211, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 212, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 216, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 217, and (f) CDR- L3 comprising the amino acid sequence of SEQ ID NO: 218; ii) a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 213, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 214, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 215, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 219, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 220, and (f) CDR- L3 comprising the amino acid sequence of SEQ ID NO: 221; iii) a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 252, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 256, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 257, and (f) CDR- L3 comprising the amino acid sequence of SEQ ID NO: 258; or iv) a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 253, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 254, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 255, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 259, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 260, and (f) CDR- L3 comprising the amino acid sequence of SEQ ID NO: 261.

[0155] In some embodiments, the antibody or the antigen-binding domain thereof that binds CD228 provided herein (e.g., an independent antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof that is comprised in any one of the fusion proteins disclosed herein) comprises: a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 210, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 211, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 212, and a VL comprising (d) CDR- L1 comprising the amino acid sequence of SEQ ID NO: 216, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 217, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 218; or a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 213, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 214, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 215, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 219, (e) CDR-L2 comprising the amino acid sequence of SEQ IDAttorney Docket No.01218-0032-00PCT NO: 220, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 221.

[0156] In some embodiments, the antibody or the antigen-binding domain thereof that binds CD228 provided herein (e.g., an independent antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof that is comprised in any one of the fusion proteins disclosed herein) comprises: i) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 122, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 124; ii) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 142, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 144; iii) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 162, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 164; iv) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 182, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 184; v) a VH comprising an amino acid sequence having at least 80%, at least 85%, at leastAttorney Docket No.01218-0032-00PCT 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 202, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 204; vi) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 222, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 224; vii) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 242, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 244; viii) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 262, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 264; or ix) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 282, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 284.

[0157] In some embodiments, the antibody or the antigen-binding domain thereof that bindsAttorney Docket No.01218-0032-00PCT CD228 provided herein (e.g., an independent antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof that is comprised in any one of the fusion proteins disclosed herein) comprises: a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 222, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 224; or a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 262, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 264.

[0158] In some embodiments, the antibody or the antigen-binding domain thereof that binds CD228 provided herein (e.g., an independent antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof that is comprised in any one of the fusion proteins disclosed herein) comprises: a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 222, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 224.

[0159] In some embodiments, the antibody or the antigen-binding domain thereof that binds CD228 provided herein (e.g., an independent antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof that is comprised in any one of the fusion proteins disclosed herein) comprises: i) a VH comprising the amino acid sequence of SEQ ID NO: 122, and a VL comprising the amino acid sequence of SEQ ID NO: 124; ii) a VH comprising the amino acid sequence of SEQ ID NO: 142, and a VL comprising the amino acid sequence of SEQ ID NO: 144; iii) a VH comprising the amino acid sequence of SEQ ID NO: 162, and a VL comprising the amino acid sequence of SEQ ID NO: 164; iv) a VH comprising the amino acid sequence of SEQ ID NO: 182, and a VL comprising the amino acid sequence of SEQ ID NO: 184;Attorney Docket No.01218-0032-00PCT v) a VH comprising the amino acid sequence of SEQ ID NO: 202, and a VL comprising the amino acid sequence of SEQ ID NO: 204; vi) a VH comprising the amino acid sequence of SEQ ID NO: 222, and a VL comprising the amino acid sequence of SEQ ID NO: 224; vii) a VH comprising the amino acid sequence of SEQ ID NO: 242, and a VL comprising the amino acid sequence of SEQ ID NO: 244; viii) a VH comprising the amino acid sequence of SEQ ID NO: 262, and a VL comprising the amino acid sequence of SEQ ID NO: 264; or ix) a VH comprising the amino acid sequence of SEQ ID NO: 282, and a VL comprising the amino acid sequence of SEQ ID NO: 284.

[0160] In some embodiments, the antibody or the antigen-binding domain thereof that binds CD228 provided herein (e.g., an independent antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof that is comprised in any one of the fusion proteins disclosed herein) comprises: a VH comprising the amino acid sequence of SEQ ID NO: 222, and a VL comprising the amino acid sequence of SEQ ID NO: 224; or a VH comprising the amino acid sequence of SEQ ID NO: 262, and a VL comprising the amino acid sequence of SEQ ID NO: 264.

[0161] In some embodiments, the antibody or the antigen-binding domain thereof that binds CD228 provided herein (e.g., an independent antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof that is comprised in any one of the fusion proteins disclosed herein) comprises: a VH comprising the amino acid sequence of SEQ ID NO: 222, and a VL comprising the amino acid sequence of SEQ ID NO: 224.

[0162] In some embodiments, the antibody or the antigen-binding domain thereof that binds CD228 provided herein (e.g., an independent antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof that is comprised in any one of the fusion proteins disclosed herein) comprises: i) a heavy chain (HC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 126, and a light chain (LC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 128; ii) a HC comprising an amino acid sequence having at least 80%, at least 85%, at leastAttorney Docket No.01218-0032-00PCT 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 146, and a LC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 148; iii) a HC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 166, and a LC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 168; iv) a HC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 186, and a LC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 188; v) a HC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 206, and a LC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 208; vi) a HC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 226, and a LC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 228; vii) a HC comprising an amino acid sequence having at least 80%, at least 85%, at leastAttorney Docket No.01218-0032-00PCT 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 246, and a LC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 248; viii) a HC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 266, and a LC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 268; or ix) a HC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 286, and a LC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 288.

[0163] In some embodiments, the antibody or the antigen-binding domain thereof that binds CD228 provided herein (e.g., an independent antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof that is comprised in any one of the fusion proteins disclosed herein) comprises: a HC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 226, and a LC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 228; or a HC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 266, and a LC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 268.

[0164] In some embodiments, the antibody or the antigen-binding domain thereof that bindsAttorney Docket No.01218-0032-00PCT CD228 provided herein (e.g., an independent antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof that is comprised in any one of the fusion proteins disclosed herein) comprises: a HC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 226, and a LC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 228.

[0165] In some embodiments, the antibody or the antigen-binding domain thereof that binds CD228 provided herein (e.g., an independent antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof that is comprised in any one of the fusion proteins disclosed herein) comprises: i) a HC comprising the amino acid sequence of SEQ ID NO: 126, and a LC comprising the amino acid sequence of SEQ ID NO: 128; ii) a HC comprising the amino acid sequence of SEQ ID NO: 146, and a LC comprising the amino acid sequence of SEQ ID NO: 148; iii) a HC comprising the amino acid sequence of SEQ ID NO: 166, and a LC comprising the amino acid sequence of SEQ ID NO: 168; iv) a HC comprising the amino acid sequence of SEQ ID NO: 186, and a LC comprising the amino acid sequence of SEQ ID NO: 188; v) a HC comprising the amino acid sequence of SEQ ID NO: 206, and a LC comprising the amino acid sequence of SEQ ID NO: 208; vi) a HC comprising the amino acid sequence of SEQ ID NO: 226, and a LC comprising the amino acid sequence of SEQ ID NO: 228; vii) a HC comprising the amino acid sequence of SEQ ID NO: 246, and a LC comprising the amino acid sequence of SEQ ID NO: 248; viii) a HC comprising the amino acid sequence of SEQ ID NO: 266, and a LC comprising the amino acid sequence of SEQ ID NO: 268; or ix) a HC comprising the amino acid sequence of SEQ ID NO: 286, and a LC comprising the amino acid sequence of SEQ ID NO: 288.

[0166] In some embodiments, the antibody or the antigen-binding domain thereof that binds CD228 provided herein (e.g., an independent antibody or antigen-binding domain thereof, or anAttorney Docket No.01218-0032-00PCT antibody or antigen-binding domain thereof that is comprised in any one of the fusion proteins disclosed herein) comprises: a HC comprising the amino acid sequence of SEQ ID NO: 226, and a LC comprising the amino acid sequence of SEQ ID NO: 228; or a HC comprising the amino acid sequence of SEQ ID NO: 266, and a LC comprising the amino acid sequence of SEQ ID NO: 268.

[0167] In some embodiments, the antibody or the antigen-binding domain thereof that binds CD228 provided herein (e.g., an independent antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof that is comprised in any one of the fusion proteins disclosed herein) comprises: a HC comprising the amino acid sequence of SEQ ID NO: 226, and a LC comprising the amino acid sequence of SEQ ID NO: 228.

[0168] In some embodiments, the antibody that binds CD228 provided herein (e.g., an independent antibody or an antibody that is comprised in any one of the fusion proteins disclosed herein) is a monoclonal antibody. In some embodiments, the antibody that binds CD228 provided herein is a humanized or chimeric antibody. In some embodiments, the antibody that binds CD228 provided herein is an IgG1, IgG2, IgG3, or IgG4 antibody.

[0169] In some embodiments, CDR sequences disclosed herein are defined according to the Kabat numbering scheme as described in Kabat et al. (1991),“Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. In some embodiments, CDR sequences disclosed herein are defined according to the IMGT method as described in Lefranc, M.-P., The Immunologist, 7, 132-136 (1999).

[0170] Antibodies specifically binding to CD228 as included in fusion proteins of the disclosure may comprise an Fc part which allows for extending the in vivo half-life of the bispecific binding molecule of the disclosure. In some embodiments, such Fc part is preferably from human origin, more preferably a human Fc part of an IgG1 or lgG4 antibody, even more preferably an engineered human Fc part of an IgG1 or lgG4 with activating or silencing effector functions. In some embodiments, silencing effector functions may be preferred over activating effector functions. In some embodiments, such an Fc part is an engineered to silence effector functions with mutation(s) at positions 234 and / or 235, numbering according to EU index of Kabat (Johnson and Wu, Nucleic Acids Res, 2000). In some embodiments, mutations in positions F234 and L235 of a provided anti-CD228 antibody may be introduced to silence effector functions. In other embodiments, mutations in positions D265 and P329 of a provided anti-CD228 antibody may be introduced to silence effector function. Numbering for both sets of these potential mutations is according to the EU index of Kabat (Shields et al., J Biol Chem, 2001). In some embodiments, the provided CD228 antibody has an engineered IgG4 backbone with the mutations S228P, F234A and L235A.

[0171] Various techniques for the production of antibodies and antigen-binding domainsAttorney Docket No.01218-0032-00PCT thereof are well known in the art and described, e.g., in Altshuler et al. (2010). Thus, for example, polyclonal antibodies can be obtained from the blood of an animal following immunization with an antigen in mixture with additives and adjuvants and monoclonal antibodies can be produced by any technique which provides antibodies produced by continuous cell line cultures. Examples of such techniques are described, e.g., Harlow and Lane (1999), (1988), and include the hybridoma technique originally described by Köhler and Milstein, 1975, the trioma technique, the human B cell hybridoma technique (see e.g., Li et al., Proc Natl Acad Sci U S A, 2006; Kozbor and Roder, Immunol Today, 1983) and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., Cancer Res, 1984). Furthermore, recombinant antibodies may be obtained from monoclonal antibodies or can be prepared de novo using various display methods such as phage, ribosomal, mRNA, or cell display. In some embodiments, a suitable system for the expression of the recombinant (humanized) antibodies or fragments thereof may be selected from, for example, bacteria, yeast, insects, mammalian cell lines or transgenic animals or plants (see, e.g., US Patent No.6,080,560; Holliger and Hudson, Nat Biotechnol, 2005). Further, techniques described for the production of single chain antibodies (see, inter alia, US Patent No.4,946,778) can be adapted to produce single chain antibodies specific for the target of this invention. Surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies. C. Exemplary fusion proteins specific for CD137 and CD228 of the disclosure.

[0172] In some embodiments, a provided fusion protein contains at least two subunits in any order: (1) a first subunit that comprises a full-length antibody or an antigen-binding domain thereof specific for CD228, and (2) a second subunit that comprises a lipocalin mutein specific for CD137. In some embodiments, in the fusion protein provided herein, the antibody or the antigen- binding domain thereof is any one of the antibodies or the antigen-binding domains thereof disclosed herein, in particular any one of the antibodies or the antigen-binding domains thereof disclosed in Section III.B above.

[0173] In some embodiments, a provided fusion protein also may contain at least one additional subunit, for example, a third subunit. For instance, a fusion protein may contain a third subunit specific for CD137. In some embodiments, a third subunit may be or comprise a lipocalin mutein specific for CD137. For example, two lipocalin muteins may be fused to a first antibody subunit, one at the C-terminus and one at the N-terminus of the antibody. In some embodiments, lipocalin muteins may be fused to the heavy chain or light chain of an antibody.

[0174] In some embodiments, provided fusion proteins may comprise one or more additional subunits (e.g., a fourth, fifth, or sixth subunit).Attorney Docket No.01218-0032-00PCT

[0175] In some embodiments, at least one subunit may be fused at its N-terminus and / or its C-terminus to another subunit.

[0176] In some embodiments, at least one subunit can be linked to another subunit via a linker. In some further embodiments, a linker is a peptide linker, for example, a glycine-serine (GS) linker, such as an unstructured GS linker or a glycosylated GS linker, or a proline-alanine- serine polymer (PAS) linker. In some embodiments, the GS linker is a (Gly4Ser)3linker ((G4S)3) as shown in SEQ ID NO: 13. Other exemplary linkers are shown in SEQ ID NOs: 14-23. In some embodiments, a peptide linker may have from 1 to 50 amino acids, such as 1, 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 1718, 19, 20, 25, 30, 35, 40, 45 or 50 amino acids. For example, when a first subunit comprises an antibody, such as a full-length antibody, a second subunit may be linked via a peptide linker between the N-terminus of the second subunit and the C-terminus of a heavy chain constant region (CH) of said antibody. In some further embodiments, a third subunit may be linked via a peptide linker between the N-terminus of the third subunit and the C-terminus of a light chain constant region (CL) of said antibody.

[0177] Generally, one subunit may be fused at its N-terminus and / or its C-terminus to another subunit. For example, in some embodiments, a lipocalin mutein subunit can be fused at its N-terminus and / or its C-terminus to an antibody subunit. For further example, one lipocalin mutein can be linked, preferably via a peptide linker, to the C-terminus of the antibody heavy chain (HC), the N-terminus of the HC, the C-terminus of the antibody light chain (LC), and / or the N-terminus of the LC.

[0178] In some embodiments, a lipocalin mutein subunit can be fused at its N-terminus and / or its C-terminus to an antibody fragment. For example, in some embodiments, a lipocalin mutein may be linked, preferably via a peptide linker, to the C-terminus of a heavy chain constant region (CH) or the C-terminus of a light chain constant region (CL) of the antibody.

[0179] In some embodiments, when one subunit comprises an antibody, such as a full- length antibody, the antibody and a second subunit may be linked between the N-terminus of the second subunit and the C-terminus of a heavy chain constant region (CH) of said antibody.

[0180] In some embodiments, a third subunit may be linked via the N-terminus of the third subunit and the C-terminus of a light chain constant region (CL) of said antibody.

[0181] In some embodiments, the fusion protein comprises an antibody, such as a full- length antibody, specific for CD228 and two lipocalin muteins specific for CD137, wherein each of the lipocalin muteins is fused at its N-terminus via a peptide linker to the C-terminus of a heavy chain of the antibody.Attorney Docket No.01218-0032-00PCT

[0182] In some embodiments, the fusion protein comprises an antibody, such as a full- length antibody, specific for CD228 and two lipocalin muteins specific for CD137, wherein each of the lipocalin muteins is fused at its N-terminus via a peptide linker to the C-terminus of a light chain of the antibody.

[0183] In some embodiments, with respect to a fusion protein of the disclosure, wherein at least one subunit may be or comprise an antibody, such as a full-length antibody, the Fc function of the Fc region of the antibody to Fc receptor-positive cell may be preserved at the same time while the fusion protein is simultaneously engaging CD137 and CD228.

[0184] In some embodiments, wherein at least one subunit of a provided fusion protein may be or comprise an antibody, such as a full-length antibody, the Fc function of the Fc region of the antibody to Fc receptor-positive cell may be reduced or fully suppressed by protein engineering while the fusion protein is simultaneously engaging CD137 and CD228. In some embodiments, this may be achieved, for example, by switching from the IgG1 backbone to IgG4, as IgG4 is known to display reduced Fc-gamma receptor interactions compared to IgG1. In some embodiments, to further reduce the residual binding to Fc-gamma receptors, mutations may be introduced into the IgG4 backbone such as F234A and L235A. In some embodiments, an S228P mutation may also be introduced into the IgG4 backbone to minimize the exchange of IgG4 half- antibody (Silva et al., J Biol Chem, 2015). In some embodiments, F234A and L235A mutations may be introduced for decreased ADCC and ADCP (Glaesner et al., Diabetes Metab Res Rev, 2010) and / or M428L and N434S mutations or M252Y, S254T, and T256E mutations for extended serum half-life (Dall'Acqua et al., J Biol Chem, 2006; Zalevsky et al., Nat Biotechnol, 2010). In some embodiments, an additional N297A mutation may be present in the antibody heavy chain of the fusion protein in order to remove the natural glycosylation motif.

[0185] In some embodiments, the Fc portion of an antibody included in a fusion protein of the disclosure may contribute to maintaining the serum levels of the fusion protein. For example, when the Fc portion binds to Fc receptors on endothelial cells and phagocytes, the fusion protein may become internalized and recycled back to the bloodstream, enhancing its half-life within the body.

[0186] In one aspect, fusion proteins of the disclosure bind CD137 with high affinity. In another aspect, provided fusion proteins bind CD228 with high affinity. In some preferred embodiments, provided fusion proteins simultaneously bind CD137 and CD228. In some embodiments, the simultaneous binding to CD137 and CD228 allows provided fusion proteins to exhibit a durable anti-tumor or anti-infection response.

[0187] In some embodiments, a provided fusion protein comprises an amino acid sequence shown in any one of SEQ ID NOs: 75, 76 and 78-83.Attorney Docket No.01218-0032-00PCT

[0188] In some embodiments, a provided fusion protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or even higher sequence identity to an amino acid sequence shown in any one of SEQ ID NOs: 75, 76 and 78-83.

[0189] In some embodiments, a provided fusion protein comprises amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or even higher sequence identity to the amino acid sequences shown in SEQ ID NOs: 80 and 76, SEQ ID NOs: 82 and 79, SEQ ID NOs: 75 and 81, SEQ ID NOs: 78 and 83, SEQ ID NOs: 96 and 97, SEQ ID NOs: 98 and 99, SEQ ID NOs: 100 and 101, SEQ ID NOs: 102 and 103, SEQ ID NOs: 104 and 105, or SEQ ID NOs: 106 and 107.

[0190] In some embodiments, a provided fusion protein comprises the amino acid sequences shown in SEQ ID NOs: 80 and 76, SEQ ID NOs: 82 and 79, SEQ ID NOs: 75 and 81, SEQ ID NOs: 78 and 83, SEQ ID NOs: 96 and 97, SEQ ID NOs: 98 and 99, SEQ ID NOs: 100 and 101, SEQ ID NOs: 102 and 103, SEQ ID NOs: 104 and 105, or SEQ ID NOs: 106 and 107. In one embodiment, a provided fusion protein comprises the amino acid sequences shown in SEQ ID NOs: 80 and 76. In one embodiment, a provided fusion protein comprises the amino acid sequences shown in SEQ ID NOs: 82 and 79. In one embodiment, a provided fusion protein comprises the amino acid sequences shown in SEQ ID NOs: 75 and 81. In one embodiment, a provided fusion protein comprises the amino acid sequences shown in SEQ ID NOs: 78 and 83. In one embodiment, a provided fusion protein comprises the amino acid sequences shown in SEQ ID NOs: 96 and 97. In one embodiment, a provided fusion protein comprises the amino acid sequences shown in SEQ ID NOs: 98 and 99. In one embodiment, a provided fusion protein comprises the amino acid sequences shown in SEQ ID NOs: 100 and 101. In one embodiment, a provided fusion protein comprises the amino acid sequences shown in SEQ ID NOs: 102 and 103. In one embodiment, a provided fusion protein comprises the amino acid sequences shown in SEQ ID NOs: 104 and 105. In one embodiment, a provided fusion protein comprises the amino acid sequences shown in SEQ ID NOs: 106 and 107. D. Exemplary lipocalin muteins of the disclosure.

[0191] Lipocalins are proteinaceous binding molecules that have naturally evolved to bind ligands. Lipocalins occur in many organisms, including vertebrates, insects, plants, and bacteria. The members of the lipocalin protein family (Pervaiz and Brew, FASEB J, 1987) are typically small, secreted proteins and have a single polypeptide chain. They are characterized by a range of different molecular-recognition properties: their binding to various, principally hydrophobic small molecules (such as retinoids, fatty acids, cholesterols, prostaglandins, biliverdins, pheromones, tastants, and odorants), and their binding to specific cell-surface receptors and theirAttorney Docket No.01218-0032-00PCT formation of macromolecular complexes. Although they have, in the past, been classified primarily as transport proteins, it is now clear that the lipocalins fulfill a variety of physiological functions. These include roles in retinol transport, olfaction, pheromone signaling, and the synthesis of prostaglandins. Lipocalins have also been implicated in the regulation of the immune response and the mediation of cell homeostasis (reviewed, e.g., in Flower et al., Biochim Biophys Acta, 2000; Flower, Biochem J, 1996).

[0192] Lipocalins share unusually low levels of overall sequence conservation, often with sequence identities of less than 20%. In strong contrast, their overall folding pattern is highly conserved. The central part of the lipocalin structure consists of a single eight-stranded anti- parallel β-sheet closed back on itself to form a continuously hydrogen-bonded β-barrel. This β- barrel forms a central cavity. One end of the barrel is sterically blocked by the N-terminal peptide segment that runs across its bottom as well as three peptide loops connecting the β-strands. The other end of the β-barrel is open to the solvent and encompasses a target-binding site, which is formed by four flexible peptide loops (AB, CD, EF, and GH). It is the diversity of the loops in the otherwise rigid lipocalin scaffold that gives rise to a variety of different binding modes each capable of accommodating targets of different size, shape, and chemical character (reviewed, e.g., in Skerra, Biochim Biophys Acta, 2000; Flower et al., Biochim Biophys Acta, 2000; Flower, Biochem J, 1996).

[0193] A lipocalin mutein according to the present disclosure may be a mutein of any lipocalin. Examples of suitable lipocalins (also sometimes designated as “reference lipocalin,” “wild-type lipocalin,” “reference protein scaffolds,” or simply “scaffolds”) of which a mutein may be used include, but are not limited to, tear lipocalin (lipocalin-1, Tlc, or von Ebner’s gland protein), retinol binding protein, neutrophil lipocalin-type prostaglandin D-synthase, β-lactoglobulin, bilin- binding protein (BBP), apolipoprotein D (APOD), neutrophil gelatinase-associated lipocalin (NGAL), α2-microglobulin-related protein (A2m), 24p3 / uterocalin (24p3), von Ebner’s gland protein 1 (VEGP 1), von Ebner’s gland protein 2 (VEGP 2), and Major allergen Can f 1 (ALL-1). In related embodiments, a lipocalin mutein is derived from the lipocalin group consisting of human tear lipocalin (hTlc), human neutrophil gelatinase-associated lipocalin (hNGAL), human apolipoprotein D (hAPOD) and the bilin-binding protein of Pieris brassicae.

[0194] The amino acid sequence of a lipocalin mutein according to the disclosure may have a high sequence identity as compared to the reference (or wild-type) lipocalin from which it is derived, for example, hTlc or hNGAL, when compared to sequence identities with another lipocalin (see also above). In this general context, the amino acid sequence of a lipocalin mutein according to the disclosure is at least substantially similar to the amino acid sequence of the corresponding reference (wild-type) lipocalin, with the proviso that there may be gaps (as defined herein) in an alignment that are the result of additions or deletions of amino acids. A respectiveAttorney Docket No.01218-0032-00PCT sequence of a lipocalin mutein of the disclosure, being substantially similar to the sequences of the corresponding reference (wild-type) lipocalin, has, in some embodiments, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 87%, or at least 90% identity, including at least 95% identity to the sequence of the corresponding lipocalin. In this regard, a lipocalin mutein of the disclosure of course may contain substitutions as described herein which renders the lipocalin mutein capable of binding to CD137.

[0195] Typically, a lipocalin mutein contains one or more mutated amino acid residues – relative to the amino acid sequence of the wild-type or reference lipocalin, for example, hTlc and hNGAL – in the four loops at the open end that comprise a ligand-binding pocket and define the entrance of the ligand-binding pocket (cf. above). As explained above, these regions are essential in determining the binding specificity of a lipocalin mutein for the desired target. In some embodiments, a lipocalin mutein of the disclosure may also contain mutated amino acid residues regions outside of the four loops. In some embodiments, a lipocalin mutein of the disclosure may contain one or more mutated amino acid residues in one or more of the three peptide loops (designated BC, DE, and FG) connecting the β-strands at the closed end of the lipocalin. In some embodiments, a mutein derived from tear lipocalin, NGAL or a homologue thereof, may have 1, 2, 3, 4, or more mutated amino acid residues at any sequence position in the N-terminal region and / or in the three peptide loops BC, DE, and FG arranged at the end of the β-barrel structure that is located opposite to the natural lipocalin binding pocket. In some embodiments, a mutein derived from tear lipocalin, NGAL or a homologue thereof, may have no mutated amino acid residues in peptide loop DE arranged at the end of the β-barrel structure, compared to wild-type sequence of tear lipocalin, NGAL or a homologue thereof.

[0196] In some embodiments, a lipocalin mutein according to the disclosure may include one or more, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or even more mutated amino acid residues in comparison to the amino acid sequence of a corresponding reference (wild-type) lipocalin, provided that such a lipocalin mutein is capable of binding to CD137. In some embodiments, a lipocalin mutein of the disclosure includes at least two, including 2, 3, 4, 5, or even more, mutated amino acid residues, where a native amino acid residue of the corresponding reference (wild-type) lipocalin is substituted by an arginine residue.

[0197] Any types and numbers of mutations, including substitutions, deletions, and insertions, are envisaged as long as a provided lipocalin mutein retains its capability to bind CD137, and / or it has a sequence identity of at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or higher identity to the amino acid sequence of the reference (wild-type) lipocalin, for example, mature hTlc or mature hNGAL.

[0198] In some embodiments, a substitution is a conservative substitution. In someAttorney Docket No.01218-0032-00PCT embodiments, a substitution is a non-conservative substitution or one or more from the exemplary substitutions below.

[0199] Specifically, in order to determine whether the amino acid sequence of a lipocalin (mutein) is different from that of a reference (wild-type) lipocalin with regard to a certain position in the amino acid sequence of the reference (wild-type) lipocalin, a skilled artisan can use means and methods well-known in the art, e.g., alignments, either manually or by using computer programs such as BLAST2.0, which stands for Basic Local Alignment Search Tool or ClustalW or any other suitable program which is suitable to generate sequence alignments. Accordingly, the amino acid sequence of a reference (wild-type) lipocalin can serve as “subject sequence” or “reference sequence”, while the amino acid sequence of a lipocalin mutein serves as “query sequence” (see also above).

[0200] Conservative substitutions are generally the following substitutions, listed according to the amino acid to be mutated, each followed by one or more replacement(s) that can be taken to be conservative: AlaSer, Thr, or Val; ArgLys, Gln, Asn, or His; Asn → Gln, Glu, Asp, or His; AspGlnGlu → Asp, Asn, Gln, or His; His → Arg, Lys, Asn, Gln, Asp, or Glu; IleLeu Ile, Val, Met, Ala, Phe, Pro, Tyr, or Trp; Lys → Arg, His, Gln, or Asn; Met → Thr, Leu, Tyr, Ile, Phe, Val, Ala, Pro, or Trp; Phe → Thr, Met, Leu, Tyr, Ile, Pro, Trp, Val, or Ala; SerAla, or Val; ThrIle, Met, Val, Phe, Pro, or Leu; Trp → Tyr, Phe, Met, Ile, or Leu; Tyr → Trp, Phe, Ile, Leu, or Met; Val → Thr, Ile, Leu, Met, Phe, Ala, Ser, or Pro. Other substitutions are also permissible and can be determined empirically or in accordance with other known conservative or non-conservative substitutions. As a further orientation, the following groups each contain amino acids that can typically be taken to define conservative substitutions for one another: i) Alanine (Ala), Serine (Ser), Threonine (Thr), Valine (Val); ii) Aspartic acid (Asp), Glutamic acid (Glu), Glutamine (Gln), Asparagine (Asn), Histidine (His); iii) Arginine (Arg), Lysine (Lys), Glutamine (Gln), Asparagine (Asn), Histidine (His); iv) Isoleucine (Ile), Leucine (Leu), Methionine (Met), Valine (Val), Alanine (Ala), Phenylalanine (Phe), Threonine (Thr), Proline (Pro); v) Isoleucine (Ile), Leucine (Leu), Methionine (Met), Phenylalanine (Phe), Tyrosine (Tyr), Tryptophan (Trp).

[0201] If such conservative substitutions result in a change in biological activity, then more substantial changes, such as the following, or as further described below in reference to aminoAttorney Docket No.01218-0032-00PCT acid classes, may be introduced and the products be screened for a desired characteristic. Examples of such more substantial changes are: Ala → Leu or Phe; Arg → Glu; Asn → Ile, Val, or Trp; Asp → Met; CysPro; Gln → Phe; Glu → Arg; His → Gly; IleLys, Glu, or Gln; Leu → Lys or Ser; Lys → Tyr; MetGlu; PheGlu, Gln, or Asp; Trp → Cys; Tyr → Glu or Asp; Val → Lys, Arg, His.

[0202] In some embodiments, substantial modifications in the physical and biological properties of the lipocalin (mutein) are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

[0203] Naturally occurring residues are divided into groups based on common side-chain properties: (1) hydrophobic: methionine, alanine, valine, leucine, iso-leucine; (2) neutral hydrophilic: cysteine, serine, threonine, asparagine, glutamine; (3) acidic: aspartic acid, glutamic acid; (4) basic: histidine, lysine, arginine; (5) residues that influence chain orientation: glycine, proline; and (6) aromatic: tryptophan, tyrosine, phenylalanine. In some embodiments, substitutions may entail exchanging a member of one of these classes for a member of another class.

[0204] Any cysteine residue not involved in maintaining the proper conformation of the respective lipocalin also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the lipocalin to improve its stability. E. Exemplary CD137-specific lipocalin muteins of the disclosure.

[0205] As noted above, a lipocalin is a polypeptide defined by its supersecondary structure, namely a cylindrical β-pleated sheet supersecondary structural region comprising eight β-strands connected pair-wise by four loops at one end to define thereby a binding pocket. The present disclosure is not limited to lipocalin muteins specifically disclosed herein. In this regard, the disclosure relates to a lipocalin mutein having a cylindrical β-pleated sheet supersecondary structural region comprising eight β-strands connected pair-wise by four loops at one end to define thereby a binding pocket, wherein at least one amino acid of each of at least three of said four loops has been mutated and wherein said lipocalin is effective to bind CD137 with detectable affinity.

[0206] In some embodiments, lipocalin muteins disclosed herein may be or comprise a mutein of mature human tear lipocalin (hTlc). A mutein of mature hTlc may be designated herein as an “hTlc mutein”. In some other embodiments, a lipocalin mutein disclosed herein is a muteinAttorney Docket No.01218-0032-00PCT of mature human neutrophil gelatinase-associated lipocalin (hNGAL). A mutein of mature hNGAL may be designated herein as an “hNGAL mutein”.

[0207] In one aspect, the present disclosure includes any number of lipocalin muteins derived from a reference (wild-type) lipocalin, preferably derived from mature hTlc or mature hNGAL, that bind CD137 with detectable affinity. In a related aspect, the disclosure includes various lipocalin muteins that are capable of activating the downstream signaling pathways of CD137 by binding to CD137. In this sense, CD137 can be regarded as a non-natural target of the reference (wild-type) lipocalin, preferably hTlc or hNGAL, where “non-natural target” refers to a substance that does not bind to the reference (wild-type) lipocalins under physiological conditions. By engineering reference (wild-type) lipocalins with one or more mutations at certain sequence positions, the present inventors have demonstrated that high affinity and high specificity for the non-natural target, CD137, is possible. In some embodiments, at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or even more nucleotide triplet(s) encoding certain sequence positions on wild-type lipocalins, a random mutagenesis may be carried out through substitution at these positions by a subset of nucleotide triplets, with the aim of generating a lipocalin mutein which is capable of binding CD137.

[0208] In some embodiments, lipocalin muteins of the disclosure may have mutated, including substituted, deleted and inserted, amino acid residue(s) at one or more sequence positions corresponding to sequence positions in the linear polypeptide sequence of a reference lipocalin, preferably hTlc or hNGAL. In some embodiments, the number of amino acid residues of a lipocalin mutein of the disclosure that are mutated in comparison with the amino acid sequence of the reference lipocalin, preferably hTlc or hNGAL, is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more such as 25, 30, 35, 40, 45 or 50, with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 being preferred and 9, 10 or 11 being even more preferred. However, it is preferred that a lipocalin mutein of the disclosure is still capable of binding CD137.

[0209] In some embodiments, a lipocalin mutein of the present disclosure may lack 1, 2, 3, 4 or more amino acids at its N-terminal end and / or 1, 2 or more amino acids at its C-terminal end, in comparison to the respective reference (wild-type) lipocalin; for example, SEQ ID NOs: 32-38. In some embodiments, the present disclosure encompasses hTlc muteins as defined above, in which the first one, two, three, or four N-terminal amino acid residues of the sequence of mature hTlc (His-His-Leu-Leu; positions 1-4) (SEQ ID NO: 5) and / or the last one or two C- terminal amino acid residues (Ser-Asp; positions 157-158) of the linear polypeptide sequence of the mature hTlc have been deleted (e.g., SEQ ID NOs: 32-38). In some embodiments, the present disclosure encompasses hNGAL muteins as defined above, in which amino acid residues (Lys- Asp-Pro, positions 46-48) of the linear polypeptide sequence of the mature hNGAL have been deleted (SEQ ID NO: 43). Further, a lipocalin mutein of the disclosure may include the wild-typeAttorney Docket No.01218-0032-00PCT (natural) amino acid sequence of the reference (wild-type) lipocalin, preferably hTlc or hNGAL, outside the mutated amino acid sequence positions.

[0210] In some embodiments, one or more mutated amino acid residues incorporated into a lipocalin mutein of the disclosure do not substantially hamper or interfere with the binding activity to the designated target and the folding of the mutein. Such mutations, including substitutions, deletions and insertions, can be accomplished at the DNA level using established standard methods (Sambrook and Russell, 2001, Molecular cloning: a laboratory manual). In some embodiments, (a) mutated amino acid residue(s) at one or more sequence positions corresponding to the linear polypeptide sequence of the reference (wild-type) lipocalin, preferably hTlc or hNGAL, is / are introduced through random mutagenesis by substituting the nucleotide triplet(s) encoding the corresponding sequence positions of the reference lipocalin with a subset of nucleotide triplets.

[0211] In some embodiments, a provided lipocalin mutein that binds CD137 with detectable affinity may include at least one amino acid substitution of a native cysteine residue by another amino acid, for example, a serine residue. In some embodiments, a lipocalin mutein that binds CD137 with detectable affinity may include one or more non-native cysteine residues substituting one or more amino acids of a reference (wild-type) lipocalin, preferably hTlc or hNGAL. In some embodiments, a lipocalin mutein according to the disclosure includes at least two amino acid substitutions of a native amino acid by a cysteine residue, hereby to form one or more cysteine bridges. In some embodiments, said cysteine bridge may connect at least two loop regions. The definition of these regions is used herein in accordance with Skerra, Biochim Biophys Acta (2000), Flower (1996) and Breustedt et al. (2005).

[0212] Generally, a lipocalin mutein of the disclosure may have at least about 70%, including at least about 80%, such as at least about 85%, amino acid sequence identity with the amino acid sequence of the mature hTlc (SEQ ID NO: 1) or mature hNGAL (SEQ ID NO: 2).

[0213] In some aspects, the present disclosure provides CD137-binding hTlc muteins. In this regard, the disclosure provides one or more hTlc muteins that are capable of binding CD137 with an affinity measured by a KDof about 300 nM, 200 nM, 150 nM, 100 nM, or lower. In some embodiments, provided hTlc muteins are capable of binding CD137 with an EC50value of about 250 nM, 150 nM, 100 nM, 50 nM, 20 nM, or even lower. In some other embodiments, the CD137- binding hTlc muteins may be cross-reactive with cynomolgus CD137 (cyCD137).

[0214] In some embodiments, an hTlc mutein of the disclosure may interfere with the binding of CD137L to CD137.Attorney Docket No.01218-0032-00PCT

[0215] In some embodiments, provided hTlc muteins may comprise a mutated amino acid residue at one or more positions corresponding to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150, and 153 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1).

[0216] In some embodiments, provided hTlc muteins may comprise a mutated amino acid residue at one or more positions corresponding to positions 26-34, 55-58, 60-61, 65, 104-106, and 108 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1).

[0217] In some embodiments, provided hTlc muteins may further comprise a mutated amino acid residue at one or more positions corresponding to positions 101, 111, 114 and 153 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1).

[0218] In some embodiments, provided hTlc muteins may comprise a mutated amino acid residue at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or even more positions corresponding to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150 and 153 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1). In some preferred embodiments, the provided hTlc muteins are capable of binding CD137, in particular human CD137.

[0219] In some embodiments, provided hTlc muteins may comprise a mutated amino acid residue at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or even more positions corresponding to positions 26-34, 55-58, 60-61, 65, 104-106 and 108 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1). In some preferred embodiments, the provided hTlc muteins are capable of binding CD137, in particular human CD137.

[0220] In some embodiments, a lipocalin mutein according to the disclosure may include at least one amino acid substitution of a native cysteine residue by, e.g., a serine residue. In some embodiments, an hTlc mutein according to the disclosure includes an amino acid substitution of a native cysteine residue at positions corresponding to positions 61 and / or 153 of the linear polypeptide sequence of mature hTlc (SEQ ID NO:1) by another amino acid, such as a serine residue. In this context it is noted that it has been found that removal of the structural disulfide bond (on the level of a respective naïve nucleic acid library) of wild-type hTlc that is formed by the cysteine residues 61 and 153 (cf. Breustedt et al., J Biol Chem, 2005) may provide hTlc muteins that are not only stably folded but are also able to bind a given non-natural target with high affinity. In some embodiments, the elimination of the structural disulfide bond may provide the further advantage of allowing for the generation or deliberate introduction of non-natural disulfide bonds into muteins of the disclosure, thereby, increasing the stability of the muteins. However, hTlc muteins that bind CD137 and that have the disulfide bridge formed between Cys 61 and Cys 153 are also part of the present disclosure.Attorney Docket No.01218-0032-00PCT

[0221] In some particular embodiments, an hTlc mutein of the disclosure may include the amino acid substitutions Cys 61 → Ala, Phe, Lys, Arg, Thr, Asn, Gly, Gln, Asp, Asn, Leu, Tyr, Met, Ser, Pro or Trp and / or Cys 153 → Ser or Ala, at positions corresponding to positions 61 and / or 153 of the linear polypeptide sequence of mature hTlc (SEQ ID NO:1).

[0222] In some embodiments, either two or all three of the cysteine codons at positions corresponding to positions 61, 101 and 153 of the linear polypeptide sequence of mature hTlc (SEQ ID NO:1) are replaced by a codon of another amino acid. Further, in some embodiments, an hTlc mutein according to the disclosure includes an amino acid substitution of a native cysteine residue at the position corresponding to position 101 of the linear polypeptide sequence of mature hTlc (SEQ ID NO:1) by a serine residue or a histidine residue.

[0223] In some embodiments, a mutein according to the disclosure comprises an amino acid substitution of a native amino acid by a cysteine residue at positions corresponding to positions 28 or 105 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1). Further, in some embodiments, a mutein according to the disclosure comprises an amino acid substitution of a native arginine residue at the position corresponding to position 111 of the linear polypeptide sequence of mature hTlc (SEQ ID NO:1) by a proline residue. Further, in some embodiments, a mutein according to the disclosure comprises an amino acid substitution of a native lysine residue at the position corresponding to position 114 of the linear polypeptide sequence of mature hTlc (SEQ ID NO:1) by a tryptophan residue or a glutamic acid.

[0224] In some embodiments, provided CD137-binding hTlc muteins may comprise, at one or more positions corresponding to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150, and 153 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1), one or more of the following mutated amino acid residues: Ala 5 → Val or Thr; Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Thr 42 → Ser; Gly 46 → Asp; Lys 52 → Glu; Leu 56 → Ala; Ser 58 → Asp; Arg 60 → Pro; Cys 61 → Ala; Lys 65 → Arg or Asn; Thr 71 → Ala; Val 85 → Asp; Lys 94 → Arg or Glu; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp; Lys 121 → Glu; Ala 133 → Thr; Arg 148 → Ser; Ser 150 → Ile; and Cys 153 → Ser. In some embodiments, an hTlc mutein of the disclosure comprises two or more, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more, or even all mutated amino acid residues at these sequence positions of mature hTlc (SEQ ID NO: 1).

[0225] In some embodiments, provided CD137-binding hTlc muteins may comprise one of the following sets of mutated amino acid residues in comparison with the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1):Attorney Docket No.01218-0032-00PCT (a) Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Leu 56 → Ala; Ser 58 → Asp; Arg 60 → Pro; Cys 61 → Ala; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp; and Cys 153 → Ser; (b) Ala 5 → Thr; Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Leu 56 → Ala; Ser 58 → Asp; Arg 60 → Pro; Cys 61 → Ala; Lys 65 → Arg; Val 85 → Asp; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp; Lys 121 → Glu; Ala 133 → Thr; and Cys 153 → Ser; (c) Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Leu 56 → Ala; Ser 58 → Asp; Arg 60 → Pro; Cys 61 → Ala; Lys 65 → Asn; Lys 94 → Arg; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp; Lys 121 → Glu; Ala 133 → Thr; and Cys 153 → Ser; (d) Ala 5 → Val; Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Leu 56 → Ala; Ser 58 → Asp; Arg 60 → Pro; Cys 61 → Ala; Lys 65 → Arg; Lys 94 → Glu; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp; Lys 121 → Glu; Ala 133 → Thr; and Cys 153 → Ser; (e) Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Thr 42 → Ser; Leu 56 → Ala; Ser 58 → Asp; Arg 60 → Pro; Cys 61 → Ala; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp; Ser 150 → Ile; and Cys 153 → Ser; (f) Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Lys 52 → Glu; Leu 56 → Ala; Ser 58 → Asp; Arg 60 → Pro; Cys 61 → Ala; Thr 71 → Ala; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp; Ala 133 → Thr; Arg 148 → Ser; Ser 150 → Ile; and Cys 153 → Ser; and (g) Ala 5 → Thr; Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Gly 46 → Asp; Leu 56 → Ala; Ser 58 → Asp; Arg 60 → Pro; Cys 61 → Ala; Thr 71 → Ala; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp; Ser 150 → Ile; and Cys 153 → Ser.

[0226] In some embodiments, the residual region, i.e., the region differing from positionsAttorney Docket No.01218-0032-00PCT corresponding to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104- 106, 108, 111, 114, 121, 133, 148, 150, and 153 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1), of an hTlc mutein of the disclosure may comprise the wild-type (natural) amino acid sequence of the linear polypeptide sequence of mature hTlc outside the mutated amino acid sequence positions.

[0227] In some embodiments, an hTlc mutein of the disclosure has at least 70% sequence identity or at least 70% sequence homology to the sequence of mature hTlc (SEQ ID NO: 1). As an illustrative example, the mutein of SEQ ID NO: 32 has an amino acid sequence identity or a sequence homology of approximately 84% with the amino acid sequence of the mature hTlc.

[0228] In some embodiments, an hTlc mutein of the disclosure comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 32-38 or a fragment or variant thereof.

[0229] In some embodiments, an hTlc mutein of the disclosure has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or higher sequence identity to an amino acid sequence selected from SEQ ID NOs: 32-38.

[0230] The present disclosure also includes structural homologues of an hTlc mutein having an amino acid sequence selected from SEQ ID NOs: 32-38, which structural homologues have an amino acid sequence homology or sequence identity of more than about 60%, preferably more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 92% and most preferably more than 95% in relation to said hTlc mutein.

[0231] In some embodiments, provided hNGAL muteins may comprise a mutated amino acid residue at one or more positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132 and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

[0232] In some embodiments, provided hNGAL muteins may comprise a mutated amino acid residue at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or even more positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2). In some preferred embodiments, the provided hNGAL muteins are capable of binding CD137, in particular human CD137.

[0233] In some embodiments, provided hNGAL muteins may comprise a mutated amino acid residue at one or more positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 87, 96, 100, 103, 106, 125, 127, 132 and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2) In some preferred embodiments, the provided hNGAL muteins are capable of binding CD137, in particular human CD137.Attorney Docket No.01218-0032-00PCT

[0234] In some embodiments, provided hNGAL muteins may comprise a mutated amino acid residue at one or more positions corresponding to positions 36, 87, and 96 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2) and at one or more positions corresponding to positions 28, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 94, 100, 103, 106, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

[0235] In other some embodiments, provided hNGAL muteins may comprise a mutated amino acid residue at one or more positions corresponding to positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

[0236] In other embodiments, provided hNGAL muteins may comprise a mutated amino acid residue at one or more positions corresponding to positions 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 96, 100, 103, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2) and at one or more positions corresponding to positions 20, 25, 33, 44, 59, 71, 78, 80, 82, 87, 92, 98, 101, and 122 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

[0237] In some embodiments, a lipocalin mutein according to the disclosure may comprise at least one amino acid substitution of a native cysteine residue by, e.g., a serine residue. In some embodiments, an hNGAL mutein according to the disclosure may comprise an amino acid substitution of a native cysteine residue at positions corresponding to positions 76 and / or 175 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2) by another amino acid, such as a serine residue. In this context, it is noted that it has been found that removal of the structural disulfide bond (on the level of a respective naïve nucleic acid library) of wild-type hNGAL that is formed by the cysteine residues 76 and 175 (cf. Breustedt et al., J Biol Chem, 2005) may provide hNGAL muteins that are not only stably folded but are also able to bind a given non-natural target with high affinity. In some embodiments, the elimination of the structural disulfide bond may provide the further advantage of allowing for the generation or deliberate introduction of non-natural disulfide bonds into muteins of the disclosure, thereby, increasing the stability of the muteins. However, hNGAL muteins that bind CD137 and that have the disulfide bridge formed between Cys 76 and Cys 175 are also part of the present disclosure.

[0238] In some embodiments, provided CD137-binding hNGAL muteins may comprise, at one or more positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132 and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), one or more of the following mutated amino acid residues: Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Arg or Lys; Gln 49 → Val, Ile, His, Ser or Asn; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Met, Ala or Gly; Leu 70 → Ala, Lys, Ser or Thr; Arg 72 →Attorney Docket No.01218-0032-00PCT Asp; Lys 73 → Asp; Asp 77 → Met, Arg, Thr or Asn; Trp 79 → Ala or Asp; Arg 81 → Met, Trp or Ser; Phe 83 → Leu; Cys 87 → Ser; Leu 94 → Phe; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu and Lys 134 → Tyr. In some embodiments, an hNGAL mutein of the disclosure comprises two or more, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, even more such as 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or all mutated amino acid residues at these sequence positions of mature hNGAL (SEQ ID NO: 2).

[0239] In some embodiments, provided CD137-binding hNGAL muteins may comprise, at one or more positions corresponding to positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), one or more of the following mutated amino acid residues: Gln 20 → Arg; Asn 25 → Tyr or Asp; Gln 28 → His; Val 33 → Ile; Leu 36 →Met; Ala 40 → Asn; Ile 41 → Leu; GluVal or Asp; Gln 49 → His; Tyr 52 →Ser or Gly; Lys 59 → Asn; Ser 68 → Asp; Leu 70 → Met; Phe 71 → Leu; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln or His; Tyr 78 → His; Trp 79 → Ile; Ile 80 → Asn; Arg 81 → Trp or Gln; Thr 82 → Pro; Cys 87 → Ser; Phe 92 → Leu or Ser; Asn 96 → Phe; Lys 98 → Arg; Tyr 100 → Asp; Pro 101 → Leu; Leu 103 → His or Pro; Phe 122 → Tyr; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp; and Lys 134 → Gly.

[0240] In some embodiments, provided CD137-binding hNGAL muteins may comprise, at one or more positions corresponding to positions 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 96, 100, 103, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), one or more of the following mutated amino acid residues: Leu 36 →Met; Ala 40 → Asn; Ile Gln 49 → His; Tyr 52 →Ser or Gly; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; AspGln or His; Trp 79 → Ile; Arg 81Trp or Gln; AsnPhe; Tyr 100 → Asp; Leu 103 → His or Pro; Lys 125 → Ser; Ser 127Ile; Tyr 132 → Trp; and Lys 134 → Gly. In some embodiments, provided CD137-binding hNGAL muteins may further comprise, at one or more positions corresponding to positions 20, 25, 33, 44, 59, 71, 78, 80, 82, 92, 98, 101, and 122 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), one or more of the following mutated amino acid residues: Gln 20 → Arg; Asn 25 → Tyr or Asp; Val 33 → Ile; Glu 44 → Val or Asp; Lys 59 → Asn; Phe 71 → Leu; Tyr 78 → His; Ile 80 → Asn; Thr 82 → Pro; Phe 92 → Leu or Ser; Lys 98 → Arg; Pro 101 → Leu; and Phe 122 → Tyr.

[0241] In some embodiments, provided CD137-binding hNGAL muteins may comprise one of the following sets of mutated amino acid residues in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2):Attorney Docket No.01218-0032-00PCT (a) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Lys; Gln 49 → Asn; Tyr 52 → Met; Ser 68 → Gly; Leu 70 → Thr; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → Ala; Arg 81 → Ser; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu; and Lys 134 → Tyr; (b) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Arg; Gln 49 → Ile; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Met; Leu 70 → Lys; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Met; Trp 79 → Asp; Arg 81 → Trp; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu; and Lys 134 → Tyr; (c) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Arg; Gln 49 → Asn; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Ala; Leu 70 → Ala; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → Asp; Arg 81 → Trp; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu; and Lys 134 → Tyr; (d) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Lys; Gln 49 → Asn; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Ala; Leu 70 → Ala; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → Asp; Arg 81 → Trp; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu; and Lys 134 → Tyr; (e) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Lys; Gln 49 → Ser; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Gly; Leu 70 → Ser; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → Ala; Arg 81 → Met; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu; and Lys 134 → Tyr; (f) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Lys; Gln 49 → Val; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Gly; Leu 70 → Thr; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Arg; Trp 79 → Asp; Arg 81 → Ser; Cys 87 → Ser; Leu 94 → Phe; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu; and Lys 134 → Tyr; (g) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Arg; Gln 49 → His; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Gly; Leu 70 → Thr; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → Ala; Arg 81 → Ser; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu; and Lys 134 → Tyr; (h) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Lys; Gln 49 → Asn; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Gly; Leu 70 → Thr; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → Ala; Arg 81 → Ser; Phe 83 → Leu; Cys 87 → Ser; Leu 94 → Phe; Asn 96 →Attorney Docket No.01218-0032-00PCT Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu; and Lys 134 → Tyr; or (i) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Arg; Gln 49 → Ser; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Ala; Leu 70 → Thr; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Asn; Trp 79 → Ala; Arg 81 → Ser; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu; and Lys 134 → Tyr.

[0242] In some further embodiments, in the residual region, i.e., the region differing from positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132 and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), an hNGAL mutein of the disclosure may include the wild-type (natural) amino acid sequence of mature hNGAL outside the mutated amino acid sequence positions.

[0243] In some other embodiments, provided CD137-binding hNGAL muteins may comprise one of the following sets of mutated amino acid residues in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2): (a) Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Ser; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Trp 79 → Ile; Arg 81 → Trp; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp; and Lys 134 → Gly; (b) Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Ser; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Trp 79 → Ile; Arg 81 → Trp; Phe 92 → Leu; Asn 96 → Phe; Lys 98 → Arg; Tyr 100 → Asp; Pro 101 → Leu; Leu 103 → His; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp; and Lys 134 → Gly; (c) Asn 25 → Tyr; Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Gly; Ser 68 → Asp; Leu 70 → Met; Phe 71 → Leu; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Trp 79 → Ile; Arg 81 → Gln; Phe 92 → Ser; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp; and Lys 134 → Gly; (d) Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Gly; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Tyr 78 → His; Trp 79 → Ile; Arg 81 → Trp; Phe 92 → Leu; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp; and Lys 134 → Gly; (e) Asn 25 → Asp; Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Gly; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Trp 79 → Ile;Attorney Docket No.01218-0032-00PCT Arg 81 → Trp; Phe 92 → Leu; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp; and Lys 134 → Gly; (f) Val 33 → Ile; Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Gly; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Trp 79 → Ile; Arg 81 → Trp; Phe 92 → Leu; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp; and Lys 134 → Gly; (g) Gln 20 → Arg; Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Glu 44 → Val; Gln 49 → His; Tyr 52 → Gly; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Trp 79 → Ile; Arg 81 → Trp; Phe 92 → Leu; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Phe 122 → Tyr; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp; and Lys 134 → Gly; (h) Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Ser; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Trp 79 → Ile; Ile 80 → Asn; Arg 81 → Trp; Thr 82 → Pro; Asn 96 → Phe; Tyr 100 → Asp; Pro 101 → Leu; Leu 103 → Pro; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp; and Lys 134 → Gly; (i) Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Gly; Lys 59 → Asn; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Trp 79 → Ile; Arg 81 → Trp; Phe 92 → Leu; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp; and Lys 134 → Gly; and (j) Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Glu 44 → Asp; Gln 49 → His; Tyr 52 → Ser; Ser 68 → Asp; Leu 70 → Met; Phe 71 → Leu; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → His; Trp 79 → Ile; Arg 81 → Trp; Phe 92 → Leu; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp; and Lys 134 → Gly.

[0244] In some embodiments, in the residual region, i.e., the region differing from positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), of an hNGAL mutein of the disclosure may include the wild-type (natural) amino acid sequence of mature hNGAL outside the mutated amino acid sequence positions.

[0245] In some embodiments, an hNGAL mutein of the disclosure has at least 70% sequence identity or at least 70% sequence homology to the sequence of mature hNGAL (SEQ ID NO: 2). As an illustrative example, the mutein of the SEQ ID NO: 40 has an amino acid sequence identity or a sequence homology of approximately 87% with the amino acid sequence of the mature hNGAL.

[0246] In some embodiments, an hNGAL mutein of the disclosure comprises an aminoAttorney Docket No.01218-0032-00PCT acid sequence as set forth in any one of SEQ ID NOs: 39-57 or a fragment or variant thereof.

[0247] In some embodiments, an hNGAL mutein of the disclosure has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or higher sequence identity to an amino acid sequence selected from SEQ ID NOs: 39-57.

[0248] The present disclosure also includes structural homologues of an hNGAL mutein having an amino acid sequence selected from SEQ ID NOs: 39-57, which structural homologues have an amino acid sequence homology or sequence identity of more than about 60%, preferably more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 92% and most preferably more than 95% in relation to said hNGAL mutein.

[0249] In some embodiments, the present disclosure provides a lipocalin mutein that binds CD137 with an affinity measured by a KDof about 5 nM or lower, wherein the lipocalin mutein has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or higher sequence identity to the amino acid sequence of SEQ ID NO: 40.

[0250] Suitable lipocalin muteins specific for CD137 are also described in WO 2016 / 177762, which is incorporated herein by reference in its entirety.

[0251] In some embodiments, a lipocalin mutein of the present disclosure can comprise a heterologous amino acid sequence at its N-or C-terminus, preferably C-terminus, such as a Strep II tag (SEQ ID NO: 12) or a cleavage site sequence for certain restriction enzymes, without affecting the biological activity (binding to its target, e.g., CD137) of the lipocalin mutein.

[0252] In some embodiments, further modifications of a lipocalin mutein may be introduced in order to modulate certain characteristics of the mutein, such as to improve folding stability, serum stability, protein resistance or water solubility or to reduce aggregation tendency, or to introduce new characteristics to the mutein. In some embodiments, modification(s) may result in two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) characteristics of a provided mutein being modulated.

[0253] For example, it is possible to mutate one or more amino acid sequence positions of a lipocalin mutein to introduce new reactive groups, for example, for the conjugation to other compounds, such as polyethylene glycol (PEG), hydroxyethyl starch (HES), biotin, peptides or proteins, or for the formation of non-naturally occurring disulphide linkages. The conjugated compound, for example, PEG and HES, can in some cases increase the serum half-life of the corresponding lipocalin mutein.

[0254] In some embodiments, a reactive group of a lipocalin mutein may occur naturally in its amino acid sequence, such as naturally occurring cysteine residues in said amino acidAttorney Docket No.01218-0032-00PCT sequence. In some other embodiments, such reactive group may be introduced via mutagenesis. In case a reactive group is introduced via mutagenesis, one possibility is the mutation of an amino acid at the appropriate position by a cysteine residue. Exemplary possibilities of such a mutation to introduce a cysteine residue into the amino acid sequence of an hTlc mutein include the substitutions Thr 40→ Cys, Glu 73→ Cys, Arg 90→ Cys, Asp 95→ Cys, and Glu 131→ Cys of the wild-type sequence of hTlc (SEQ ID NO: 1). Exemplary possibilities of such a mutation to introduce a cysteine residue into the amino acid sequence of an hNGAL mutein include the introduction of a cysteine residue at one or more of the sequence positions that correspond to sequence positions 14, 21, 60, 84, 88, 116, 141, 145, 143, 146 or 158 of the wild-type sequence of hNGAL (SEQ ID NO: 2).The generated thiol moiety may be used to PEGylate or HESylate the mutein, for example, in order to increase the serum half-life of a respective lipocalin mutein.

[0255] In some embodiments, in order to provide suitable amino acid side chains as new reactive groups for conjugating one of the above compounds to a lipocalin mutein, artificial amino acids may be introduced to the amino acid sequence of a lipocalin mutein. Generally, such artificial amino acids are designed to be more reactive and thus to facilitate the conjugation to the desired compound. Such artificial amino acids may be introduced by mutagenesis, for example, using an artificial tRNA, such as para-acetyl-phenylalanine.

[0256] In some embodiments, a lipocalin mutein of the disclosure is fused at its N- terminus or its C-terminus to a protein, a protein domain or a peptide, for instance, an antibody, a signal sequence and / or an affinity tag. In some other embodiments, a lipocalin mutein of the disclosure is conjugated at its N-terminus or its C-terminus to a partner, which is a protein, a protein domain or a peptide; for instance, an antibody, a signal sequence and / or an affinity tag.

[0257] Affinity tags such as the Strep-tag or Strep-tag II (Schmidt et al., J Mol Biol, 1996), the c-myc-tag, the FLAG-tag, the His-tag or the HA-tag or proteins such as glutathione-S- transferase, which allow easy detection and / or purification of recombinant proteins, are examples of suitable fusion partners. Proteins with chromogenic or fluorescent properties such as the green fluorescent protein (GFP) or the yellow fluorescent protein (YFP) are suitable fusion partners for lipocalin muteins of the disclosure as well. In general, it is possible to label the lipocalin muteins of the disclosure with any appropriate chemical substance or enzyme, which directly or indirectly generates a detectable compound or signal in a chemical, physical, optical, or enzymatic reaction. For example, a fluorescent or radioactive label can be conjugated to a lipocalin mutein to generate fluorescence or x-rays as detectable signal. Alkaline phosphatase, horseradish peroxidase and β-galactosidase are examples of enzyme labels (and at the same time optical labels) which catalyze the formation of chromogenic reaction products. In general, all labels commonly used for antibodies (except those exclusively used with the sugar moiety in the Fc part of immunoglobulins) can also be used for conjugation to the lipocalin muteins of the disclosure.Attorney Docket No.01218-0032-00PCT

[0258] In some embodiments, a lipocalin mutein of the disclosure may be fused or conjugated to a moiety that extends the serum half-life of the mutein (in this regard see also International Patent Publication No. WO 2006 / 056464, where such strategies are described with reference to muteins of human neutrophil gelatinase-associated lipocalin (hNGAL) with binding affinity for CTLA-4). The moiety that extends the serum half-life may be a PEG molecule, a HES molecule, a fatty acid molecule, such as palmitic acid (Vajo and Duckworth, Pharmacol Rev, 2000), an Fc part of an immunoglobulin, a CH3 domain of an immunoglobulin, a CH4 domain of an immunoglobulin, an albumin binding peptide, an albumin binding protein, or a transferrin, to name only a few.

[0259] In some embodiments, if PEG is used as a conjugation partner, the PEG molecule can be substituted, unsubstituted, linear, or branched. It can also be an activated polyethylene derivative. Examples of suitable compounds are PEG molecules as described in International Patent Publication No. WO 1999 / 64016, in U.S. Patent No. 6,177,074, or in U.S. Patent No. 6,403,564 in relation to interferon, or as described for other proteins such as PEG- modified asparaginase, PEG-adenosine deaminase (PEG-ADA) or PEG-superoxide dismutase (Fuertges and Abuchowski, Journal of Controlled Release, 1990). The molecular weight of such a polymer, such as polyethylene glycol, may range from about 300 to about 70,000 daltons, including, for example, polyethylene glycol with a molecular weight of about 10,000, of about 20,000, of about 30,000 or of about 40,000 daltons. Moreover, as, e.g., described in U.S. Patent No. 6,500,930 or 6,620,413, carbohydrate oligomers and polymers such as HES can be conjugated to a mutein of the disclosure for the purpose of serum half-life extension.

[0260] In some embodiments, if an Fc part of an immunoglobulin is used for the purpose of prolonging the serum half-life of the lipocalin muteins of the disclosure, the SynFusion™ technology, commercially available from Syntonix Pharmaceuticals, Inc. (MA, USA), may be used. The use of this Fc-fusion technology allows the creation of longer-acting biopharmaceuticals and may, for example, consist of two copies of the mutein linked to the Fc region of an antibody to improve pharmacokinetics, solubility, and production efficiency.

[0261] Examples of albumin binding peptides that can be used to extend the serum half- life of a lipocalin mutein are, for instance, those having a Cys-Xaa1-Xaa2-Xaa3-Xaa4-Cys consensus sequence, wherein Xaa1is Asp, Asn, Ser, Thr, or Trp; Xaa2is Asn, Gln, His, Ile, Leu, or Lys; Xaa3is Ala, Asp, Phe, Trp, or Tyr; and Xaa4is Asp, Gly, Leu, Phe, Ser, or Thr as described in U.S. Patent Publication No.2003 / 0069395 or Dennis et al. (2002). The albumin binding protein fused or conjugated to a lipocalin mutein to extend serum half-life may be a bacterial albumin binding protein, an antibody, an antibody fragment including domain antibodies (see U.S. Patent No.6,696,245, for example), or a lipocalin mutein with binding activity for albumin. Examples of bacterial albumin binding proteins include streptococcal protein G (Konig and Skerra, J ImmunolAttorney Docket No.01218-0032-00PCT Methods, 1998).

[0262] In some embodiments, if the albumin-binding protein is an antibody fragment it may be a domain antibody. Domain Antibodies (dAbs) are engineered to allow precise control over biophysical properties and in vivo half-life to create the optimal safety and efficacy product profile. Domain Antibodies are for example commercially available from Domantis Ltd. (Cambridge, UK, and MA, USA).

[0263] In some embodiments, albumin itself (Osborn et al., J Pharmacol Exp Ther, 2002), or a biologically active fragment of albumin can be used as a partner of a lipocalin mutein of the disclosure to extend serum half-life. The term “albumin” includes all mammal albumins such as human serum albumin or bovine serum albumin or rat albumin. The albumin or fragment thereof can be recombinantly produced as described in U.S. Patent No.5,728,553 or European Patent Publication Nos. EP0330451 and EP0361991. Accordingly, recombinant human albumin (e.g., Recombumin® from Novozymes Delta Ltd., Nottingham, UK) can be conjugated or fused to a lipocalin mutein of the disclosure.

[0264] In some embodiments, if a transferrin is used as a partner to extend the serum half-life of the lipocalin muteins of the disclosure, the muteins can be genetically fused to the N- or C-terminus, or both, of non-glycosylated transferrin. Non-glycosylated transferrin has a half-life of 14-17 days, and a transferrin fusion protein will similarly have an extended half-life. The transferrin carrier also provides high bioavailability, biodistribution and circulating stability. This technology is commercially available from BioRexis (BioRexis Pharmaceutical Corporation, PA, USA). Recombinant human transferrin (DeltaFerrin™) for use as a protein stabilizer / half-life extension partner is also commercially available from Novozymes Delta Ltd. (Nottingham, UK).

[0265] Yet another alternative to prolong the half-life of the lipocalin muteins of the disclosure is to fuse to the N- or C-terminus of a mutein a long, unstructured, flexible glycine-rich sequence (for example poly-glycine with about 20 to 80 consecutive glycine residues). This approach disclosed in International Patent Publication No. WO 2007 / 038619, for example, has also been term “rPEG” (recombinant PEG). F. Production of exemplary provided antibodies or antigen-binding domains thereof specific for CD228 and fusion proteins specific for CD137 and CD228.

[0266] In some embodiments, the present disclosure provides nucleic acid molecules (e.g., DNA or RNA) that include nucleotide sequences encoding provided antibodies, antigen-binding domains thereof, or fusion proteins. In some embodiments, the disclosure encompasses a vector containing a provided nucleic acid molecule. In some embodiments, the disclosure encompasses a host cell containing a provided nucleic acid molecule or vector. Since the degeneracy of theAttorney Docket No.01218-0032-00PCT genetic code permits substitutions of certain codons by other codons specifying the same amino acid, the disclosure is not limited to a specific nucleic acid molecule encoding an antibody, antigen- binding domain thereof, or fusion protein as described herein, rather, encompassing all nucleic acid molecules that include nucleotide sequences encoding a functional antibody, antigen-binding domain thereof, or fusion protein. In this regard, the present disclosure also relates to nucleotide sequences encoding provided antibodies, antigen-binding domains thereof, or fusion proteins.

[0267] A nucleic acid molecule, such as DNA, is referred to as “capable of expressing a nucleic acid molecule” or “able to allow expression of a nucleotide sequence” if it includes sequence elements that contain information regarding to transcriptional and / or translational regulation, and such sequences are “operably linked” to the nucleotide sequence encoding the protein. An operable linkage is a linkage in which the regulatory sequence elements and the sequence to be expressed are connected in a way that enables gene expression. The precise nature of the regulatory regions necessary for gene expression may vary among species, but in general these regions include a promoter, which, in prokaryotes, contains both the promoter per se, i.e., DNA elements directing the initiation of transcription, as well as DNA elements which, when transcribed into RNA, will signal the initiation of translation. Such promoter regions normally include 5’ non-coding sequences involved in initiation of transcription and translation, such as the -35 / -10 boxes and the Shine-Dalgarno element in prokaryotes or the TATA box, CAAT sequences, and 5’-capping elements in eukaryotes. These regions can also include enhancer or repressor elements as well as translated signal and leader sequences for targeting the native protein to a specific compartment of a host cell.

[0268] In addition, 3’ non-coding sequences may contain regulatory elements involved in transcriptional termination, polyadenylation or the like. If, however, these termination sequences are not satisfactorily functional in a particular host cell, then they may be substituted with signals functional in that cell.

[0269] Therefore, a nucleic acid molecule of the disclosure may be “operably linked” to one or more regulatory sequences, such as a promoter sequence, to allow expression of this nucleic acid molecule. In some embodiments, a nucleic acid molecule of the disclosure includes a promoter sequence and a transcriptional termination sequence. Suitable prokaryotic promoters are, for example, the tet promoter, the lacUV5 promoter or the T7 promoter. Examples of promoters useful for expression in eukaryotic cells are the SV40 promoter or the CMV promoter.

[0270] In some embodiments, a nucleic acid molecule encoding a lipocalin mutein disclosed in this application may be “operably linked” to another nucleic acid molecule encoding an antibody of the disclosure to allow expression of a fusion protein disclosed herein.

[0271] In some embodiments, provided methods may include subjecting at least oneAttorney Docket No.01218-0032-00PCT nucleic acid molecule encoding mature hTlc to mutagenesis at nucleotide triplets coding for one or more positions corresponding to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150 and 153 of the linear polypeptide sequence of hTlc (SEQ ID NO: 1), to obtain lipocalin muteins as included in provided fusion proteins. In some embodiments, provided methods may include subjecting at least one nucleic acid molecule encoding mature hNGAL to mutagenesis at nucleotide triplets coding for one or more positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132 and 134 of the linear polypeptide sequence of hNGAL (SEQ ID NO: 2), to obtain lipocalin muteins as included in provided fusion proteins. In some embodiments, a provided method may include subjecting at least one nucleic acid molecule encoding mature hNGAL to mutagenesis at nucleotide triplets coding for one or more positions corresponding to positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132, and 134 of the linear polypeptide sequence of hNGAL (SEQ ID NO: 2), to obtain lipocalin muteins as included in provided fusion proteins.

[0272] In addition, with respect to hTlc muteins or hNGAL muteins of the disclosure as included in the fusion proteins, in some embodiments, the naturally occurring disulfide bond between Cys 61 and Cys 153 or Cys 76 and Cys 175, respectively, may be removed. Accordingly, such muteins can be produced in a cell compartment having a reducing redox milieu, for example, in the cytoplasm of Gram-negative bacteria.

[0273] With further respect to provided hTlc muteins or hNGAL muteins of the disclosure as included in the fusion proteins, the disclosure also includes nucleic acid molecules encoding such muteins which, in some embodiments, may include one or more additional mutations outside the indicated sequence positions of experimental mutagenesis. Such mutations are often tolerated or can even prove to be advantageous, for example, if they contribute to an improved folding efficiency, serum stability, thermal stability or ligand binding affinity of the lipocalin muteins and / or the fusion proteins.

[0274] In some embodiments, provided nucleic acid molecules can also be part of a vector or any other kind of cloning vehicle, such as a plasmid, a phagemid, a phage, a baculovirus, a cosmid or an artificial chromosome.

[0275] In some embodiments, a provided nucleic acid molecule may be included in a phagemid. As used in this context, a phagemid vector denotes a vector encoding the intergenic region of a temperate phage, such as M13 or f1, or a functional part thereof fused to the cDNA of interest. For example, in some embodiments, after superinfection of bacterial host cells with such a provided phagemid vector and an appropriate helper phage (e.g., M13K07, VCS-M13 or R408) intact phage particles are produced, thereby enabling physical coupling of the encodedAttorney Docket No.01218-0032-00PCT heterologous cDNA to its corresponding polypeptide displayed on the phage surface (Lowman, Annu Rev Biophys Biomol Struct, 1997, Rodi and Makowski, Curr Opin Biotechnol, 1999).

[0276] In accordance with various embodiments, cloning vehicles can include, aside from the regulatory sequences described above and a nucleic acid sequence encoding an antibody, antigen-binding domain thereof, or fusion protein as described herein, replication and control sequences derived from a species compatible with the host cell that is used for expression as well as selection markers conferring a selectable phenotype on transformed or transfected cells. Large numbers of suitable cloning vectors are known in the art and are commercially available.

[0277] The disclosure also relates, in some embodiments, to methods for the production of antibodies, antigen-binding domains thereof, or fusion proteins of the disclosure starting from a nucleic acid coding for an antibody, antigen-binding domain thereof, or fusion protein or any subunit(s) therein using genetic engineering methods. In some embodiments, a provided method can be carried out in vivo, wherein a provided antibody, antigen-binding domain thereof, or fusion protein can, for example, be produced in a bacterial or eukaryotic host organism, and then isolated from this host organism or its culture. It is also possible to produce an antibody, antigen-binding domain thereof, or fusion protein of the disclosure in vitro, for example, using an in vitro translation system.

[0278] When producing an antibody, antigen-binding domain thereof, or fusion protein in vivo, a nucleic acid encoding such antibody, antigen-binding domain thereof, or fusion protein may be introduced into a suitable bacterial or eukaryotic host organism using recombinant DNA technology well known in the art. In some embodiments, a DNA molecule encoding an antibody, antigen-binding domain thereof, or fusion protein as described herein (for example, SEQ ID NOs: 91-94 and the sequence pairs of SEQ ID NOs: 91 and 88, 93 and 90, 87 and 92, or 89 and 94), and in particular a cloning vector containing the coding sequence of such an antibody, antigen- binding domain thereof, or fusion protein can be transformed into a host cell capable of expressing the gene. Transformation can be performed using standard techniques. Thus, the disclosure is also directed to host cells containing a nucleic acid molecule as disclosed herein.

[0279] In some embodiments, transformed host cells may be cultured under conditions suitable for expression of the nucleotide sequence encoding an antibody, antigen-binding domain thereof, or fusion protein of the disclosure. In some embodiments, host cells can be prokaryotic, such as Escherichia coli (E. coli) or Bacillus subtilis, or eukaryotic, such as Saccharomyces cerevisiae, Pichia pastoris, SF9 or High5 insect cells, immortalized mammalian cell lines (e.g., HeLa cells or CHO cells) or primary mammalian cells.

[0280] In some embodiments, where a lipocalin mutein of the disclosure, including as comprised in a fusion protein disclosed herein, includes intramolecular disulfide bonds, it may beAttorney Docket No.01218-0032-00PCT preferred to direct the nascent protein to a cell compartment having an oxidizing redox milieu using an appropriate signal sequence. Such an oxidizing environment may be provided by the periplasm of Gram-negative bacteria such as E. coli, in the extracellular milieu of Gram-positive bacteria or the lumen of the endoplasmic reticulum of eukaryotic cells and usually favors the formation of structural disulfide bonds.

[0281] In some embodiments, it is also possible to produce an antibody, antigen-binding domain thereof, or fusion protein of the disclosure in the cytosol of a host cell, preferably E. coli. In this case, a provided antibody, antigen-binding domain thereof, or fusion protein can either be directly obtained in a soluble and folded state or recovered in the form of inclusion bodies, followed by renaturation in vitro. A further option is the use of specific host strains having an oxidizing intracellular milieu, which may thus allow the formation of disulfide bonds in the cytosol (Venturi et al., J Mol Biol, 2002).

[0282] In some embodiments, an antibody, antigen-binding domain thereof, or fusion protein of the disclosure as described herein may be not necessarily generated or produced, in whole or in part, via use of genetic engineering. Rather, such protein can also be obtained by any of the many conventional and well-known techniques such as plain organic synthesis strategies, solid phase-assisted synthesis techniques, commercially available automated synthesizers, or by in vitro transcription and translation. It is, for example, possible that promising antibodies, antigen- binding domains thereof, or fusion proteins or lipocalin muteins included in such fusion proteins are identified using molecular modeling, synthesized in vitro, and investigated for the binding activity for the target(s) of interest. Methods for the solid phase and / or solution phase synthesis of proteins are well known in the art (see, e.g., Bruckdorfer et al., Curr Pharm Biotechnol, 2004).

[0283] In some embodiments, an antibody, antigen-binding domain thereof, or fusion protein of the disclosure may be produced by in vitro transcription / translation employing well- established methods known to those skilled in the art.

[0284] In some further embodiments, antibodies, antigen-binding domains thereof, or fusion proteins as described herein may also be prepared by conventional recombinant techniques alone or in combination with conventional synthetic techniques.

[0285] Moreover, in some embodiments, a fusion protein according to the present disclosure may be obtained by conjugating together individual subunits, e.g., antibodies and muteins as included in the fusion protein. Such conjugation can be, for example, achieved through all forms of covalent or non-covalent linkage using conventional methods.

[0286] The skilled worker will appreciate methods useful to prepare antibodies, antigen- binding domains thereof, or fusion proteins contemplated by the present disclosure but whoseAttorney Docket No.01218-0032-00PCT protein or nucleic acid sequences are not explicitly disclosed herein. As an overview, such modifications of the amino acid sequence include, e.g., directed mutagenesis of single amino acid positions to simplify sub-cloning of a protein gene or its parts by incorporating cleavage sites for certain restriction enzymes. Also, these mutations can be incorporated to further improve the affinity of an antibody, antigen-binding domain thereof, or fusion protein for its target(s) (e.g., CD137 and / or CD228). Furthermore, mutations can be introduced to modulate one or more characteristics of the protein such as to improve folding stability, serum stability, protein resistance or water solubility or to reduce aggregation tendency, if necessary.

[0287] Additional objects, advantages, and features of this disclosure will become apparent to those skilled in the art upon examination of the following Examples and the attached Figures, which are not intended to be limiting. Thus, it should be understood that although the present disclosure is specifically disclosed by exemplary embodiments and optional features, modification and variation of the disclosures embodied therein herein disclosed may be resorted to by those skilled in the art and that such modifications and variations are considered to be within the scope of this disclosure. VI. EXAMPLES Example 1. Clinical Study of a Fusion Protein That Binds Both CD137 and CD228

[0288] A phase 1, open-label, multicenter, dose-escalation, dose-optimization, and dose- expansion study designed to evaluate the safety, tolerability, pharmacokinetics (PK), and antitumor activity of PF-08046049 (also known as SGN-BB228, a fusion protein that binds both CD137 and CD228 and comprising the amino acid sequences shown in SEQ ID NOs: 80 and 76) in adults with metastatic or unresectable melanoma or other advanced solid tumors (e.g., non- small cell lung cancer (NSCLC), colorectal cancer (CRC), pancreatic cancer, and mesothelioma) is undertaken.

[0289] There is no requirement for subject selection based on CD228 expression. The study includes dose-escalation (Part A), followed by cohorts for an optional dose and schedule optimization (Part B), and disease-specific cohorts in dose-expansion (Part C). The study may enroll approximately 300 subjects with melanoma and other advanced solid tumors, including approximately 70 subjects to be evaluated in dose-escalation (including approximately 15 subjects in a biology backfill cohort), approximately 30 subjects in the optional dose-optimization cohorts (with approximately 15 subjects per dose / schedule for 2 doses / schedules), and approximately 200 subjects across 5 disease-specific expansion cohorts (up to 40 subjects per disease cohort).

[0290] During Part A (dose-escalation) of the study, approximately 70 subjects withAttorney Docket No.01218-0032-00PCT cutaneous melanoma are treated with PF-08046049 as an intravenous (IV) infusion on Day 1 and Day 15 of a 28-day cycle (Q2W). The initial dose level is 0.3 mg / kg and at least 6 dose levels (0.3, 1.0, 3.0, 5.0, 10.0, or 15.0 mg / kg) are planned to evaluate safety and tolerability and to identify the maximum tolerated dose (MTD) and / or recommended dose of PF-08046049. Alternate dosing schedule(s) (e.g., Q1W or Q3W) may also be evaluated. Under the Q1W schedule, the subjects are treated with PF-08046049 as an IV infusion on Day 1, Day 8, and Day 15 of a 21-day cycle, and at least 6 dose levels (0.15, 0.5, 1.5, 2.5, 5.0, or 7.5 mg / kg) are planned; under the Q3W schedule, the subjects are treated with PF-08046049 as an IV infusion on Day 1 of a 21-day cycle, and at least 6 dose levels (0.45, 1.5, 4.5, 7.5, 15.0, or 22.5 mg / kg) are planned. Dose-escalation is conducted using the mTPI method (Ji et al., Clin Trials. 2010; 7(6): 653-63) with the dose-escalation rules determined based on a target dose limiting toxicity (DLT) rate of 25% with a 5% margin. The MTD is defined as the highest dose that does not cause unacceptable toxicity. A dose is defined as having unacceptable toxicity if there is a more than 95% posterior probability that the DLT rate is greater than the target level. A minimum of 6 subjects are evaluated at the estimated MTD or recommended dose before these are determined. Safety, PK, pharmacodynamics, and biomarker analyses, as well as preliminary antitumor activity, are used to determine a recommended dose.

[0291] Also, for biology backfill cohorts, additional subjects are enrolled therein at tested dose levels that are deemed safe (that is, “backfilled”). For subjects who consent to a biology backfill cohort, fresh tumor biopsies at screening and during Cycle 2 are required. These biopsies are used to investigate the mechanism of action (MOA), support evidence of intratumoral pharmacodynamic effects of the drug, correlate with serum pharmacodynamic markers like soluble 4-1BB, and resistance mechanisms of PF-08046049. The dose escalation continues uninterrupted during biology backfill cohort enrollment.

[0292] During Part B (dose optimization) of the study, approximately 30 subjects with advanced cutaneous melanoma are enrolled and randomly allocated to a recommended dosing schedule from Part A or an alternate dosing schedule (e.g., Q1W, 21-day cycle; Q2W, 28-day cycle; or Q3W, 21-day cycle) with equal probability. Up to 2 dosing schedules are evaluated in parallel cohorts with up to approximately 15 subjects per schedule. This part allows optimization of the dose and schedule that becomes recommended for expansion.

[0293] During Part C (dose expansion) of the study, based on a recommended dose and schedule identified in Part A and / or Part B, dose-expansion cohorts open in melanoma, NSCLC, CRC, pancreatic cancer, and / or mesothelioma. Each disease cohort may enroll up to 40 subjects, with an interim analysis performed after treatment of approximately 15 subjects in that disease cohort to determine whether to continue enrollment of the remaining 25 subjects in that disease cohort.Attorney Docket No.01218-0032-00PCT

[0294] The initial and alternate dosing schedules and levels are summarized in Tables 1 and 2 below.

[0295] Table 1: Initial and alternate treatment schedules

[0296] Table 2: Planned dose levels for initial and alternate schedules

[0297] To prevent occurrence of infusion-related reactions (IRRs), subjects may also receive premedication and postmedication as follows: • Long-acting oral antihistamine: administer the evening before PF-08046049 infusion, such as: o Cetirizine, oral, 10 mg • Antipyretic + antihistamine + corticosteroid + oral long-acting antihistamine: administer 45 to 90 minutes prior to PF-08046049 infusion, such as: o Acetaminophen (paracetamol), oral, 500 to 1000 mg. o Diphenhydramine, IV, 25 to 50 mg (or equivalent H1 blocker). o Hydrocortisone, IV, 100 mg (200 mg at DL4 or higher). o Cetirizine, oral, 10 mg • Repeat acetaminophen (paracetamol) and antihistamine 1 time, 4 to 6 hours after the start of infusion of the initial and subsequent doses. Dosing may be IV or oral. • Additional premedications and / or additional repeat-doses of premedications may be administered if warranted in the judgement of the treating physician, based on the subject’s individual clinical status, risk for developing IRRs, and / or institutional practice.

[0298] If no Grade 2 or higher IRR is experienced during the first 2 cycles, then corticosteroid medication may be decreased or omitted beginning with Cycle 3 Day 1 dose onwards. If IRRs occur despite premedications:Attorney Docket No.01218-0032-00PCT • Treat with supportive care based on symptoms. • Higher dose of diphenhydramine (e.g., 50 mg IV) or additional premedications (e.g., H2 blockers or leukotriene inhibitors) may be considered. • Consider rate reduction. • Consider higher dose of corticosteroid and / or longer acting steroid with subsequent cycles.

[0299] Subjects receive study treatment until progressive disease, unacceptable toxicity, withdrawal of consent, initiation of subsequent therapy, or study termination, whichever occurs first. The determination of antitumor activity is based on objective response rate (ORR) assessments as defined by Response Evaluation Criteria in Solid Tumors Version 1.1 (RECIST v.1.1) and by Modified RECIST v.1.1 for immune-based therapeutics (iRECIST).

[0300] The inclusion criteria for the study are as follows: 1. One of the following tumor types: a. Part A and Part B: Subjects must have histologically or cytologically confirmed metastatic or unresectable cutaneous melanoma. Subjects must have disease that is relapsed, refractory, or intolerant to standard of care therapies and in the judgement of the investigator must not have other therapeutic options known to provide clinical benefit. b. Part C: Subjects must have disease that is relapsed, refractory, or intolerant to standard of care therapies and in the judgement of the investigator must not have other therapeutic options known to provide clinical benefit. Subjects must have histologically or cytologically confirmed metastatic or unresectable solid malignancy from one of the following tumor types: - Cutaneous melanoma - NSCLC - CRC - Pancreatic cancer - Mesothelioma 2. A pre-treatment biopsy or submission of archival tissue is required. Subjects enrolled in biology backfill cohorts must agree to a pre-treatment biopsy and an on-treatment biopsy during Cycle 2 which are required. 3. For subjects with cutaneous melanoma in Parts A, B, and CAttorney Docket No.01218-0032-00PCT a. Must have been previously treated with an anti-programmed death-1 (anti-PD- 1) or anti-programmed death ligand-1 (anti-PD-L1) agent administered either as monotherapy, or in combination with other checkpoint inhibitors or other therapies. b. Subjects with a targetable BRAF mutation must have been treated with, been intolerant of, or been deemed ineligible to receive treatment with BRAF / MEK targeted therapy prior to study entry. Subjects with a targetable BRAF mutation who are rapidly progressing should be treated with BRAF / MEK targeted therapy to stabilize disease prior to enrollment, unless contraindicated. 4. Age 18 years or older. 5. An Eastern Cooperative Oncology Group (ECOG) Performance Status score of 0 or 1. 6. Measurable disease per Response Evaluation Criteria in Solid Tumors Version 1.1 (RECIST v.1.1) at baseline. 7. The following baseline laboratory data: • absolute neutrophil count (ANC) ≥ 1500 / μL (1.5 ×10^9 / L) • hemoglobin (Hgb) ≥ 9 g / dL • platelet count ≥ 100,000 / μL • serum bilirubin ≤ 1.5 × upper limit of normal (ULN) or ≤ 3 × ULN for subjects with documented hepatic tumor involvement or direct bilirubin ≤ 1.5 × ULN for subjects with Gilbert’s disease • individual estimated glomerular filtration rate (GFR) ≥ 45 mL / min / 1.73 m^2 using the Modification of Diet in Renal Disease (MDRD) study equation as applicable • Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ≤ 3 × ULN (≤ 5 × ULN if there is evidence of hepatic involvement by malignant disease) 8. Subjects of childbearing potential, under the following conditions: a. Must have a negative serum or urine pregnancy test (minimum sensitivity 25 mIU / mL or equivalent units of beta human chorionic gonadotropin (β-hCG)) result within 7 days prior to the first dose of PF-08046049. Subjects with false positive results and documented verification that the subject is not pregnant are eligible for participation.Attorney Docket No.01218-0032-00PCT b. Must agree not to try to become pregnant during the study and for at least 4 months after the final dose of study drug c. Must agree not to breastfeed or donate ova, starting at time of informed consent and continuing through 4 months after the final dose of study drug d. If sexually active in a way that could lead to pregnancy, must consistently use the acceptable combinations of contraceptive methods starting at time of informed consent and continuing throughout the study and for at least 4 months after the final dose of study drug 9. Subjects born male, under the following conditions: a. Must agree not to donate sperm starting at time of informed consent and continuing throughout the study period and for at least 4 months after the final dose of study drug. b. If sexually active with a person of childbearing potential in a way that could lead to pregnancy, must consistently use the acceptable combinations of contraceptive methods starting at time of informed consent and continuing throughout the study and for at least 4 months after the final dose of study drug. c. If sexually active with a person who is pregnant, breastfeeding, or of childbearing potential, must consistently use a male condom starting at time of informed consent and continuing throughout the study and for at least 4 months after the final dose of study drug. 10. The subject must provide written informed consent.

[0301] Subjects are excluded from the study if any of the following criteria apply: 1. History of another malignancy within 3 years before the first dose of study drug, or any evidence of residual disease from a previously diagnosed malignancy. Exceptions are malignancies with a negligible risk of metastasis or death (e.g., 5-year overall survival (OS) ≥ 90%), such as adequately treated carcinoma in situ of the cervix, non-melanoma skin carcinoma, localized prostate cancer, ductal carcinoma in situ, or Stage I uterine cancer. 2. Known active central nervous system metastases or leptomeningeal disease. Subjects with previously treated brain metastases may participate provided they are clinically stable for at least 4 weeks prior to study entry after brain metastasis treatment, they have no new or enlarging brain metastases, and are off of corticosteroids prescribed for symptoms associated with brain metastases for at least 7 days prior to the first dose of study drug.Attorney Docket No.01218-0032-00PCT 3. Any active viral, bacterial, or fungal infection (per the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE), Version 5.0) within 2 weeks prior to the first dose of study drug, unless deemed not clinically significant by the investigator (e.g., onychomycosis). Routine antimicrobial prophylaxis is permitted. 4. Known to be positive for hepatitis B by surface antigen expression. Known to have active hepatitis C infection (positive by polymerase chain reaction (PCR) or on antiviral therapy for hepatitis C within the last 6 months). However, subjects who have been treated for hepatitis C infection are permitted if they have documented sustained virologic response of 12 weeks. 5. Known to be positive for human immunodeficiency virus (HIV). 6. Documented history of a cerebral vascular event (stroke or transient ischemic attack), unstable angina, myocardial infarction, congestive heart failure or cardiac symptoms consistent with New York Heart Association Class III-IV within 6 months prior to their first dose of study drug. 7. Prior therapies cannot include any drugs targeting CD228 (e.g., SGN-CD228A) or 4- 1BB (e.g., urelumab, utomilumab) for all subjects. 8. Immunotherapy, biologics, and / or other approved or investigational antitumor treatment that is not completed 4 weeks prior to first dose of study drug, or within 2 weeks prior to the first dose of study drug if the underlying disease has progressed on treatment. 9. Subjects who are breastfeeding, pregnant, or planning to become pregnant from time of informed consent until at least 24 months after final dose of study drug administration. 10. Known hypersensitivity to any excipient contained in the drug formulation of PF- 08046049. 11. Grade 3 or higher pulmonary disease unrelated to underlying malignancy. 12. Estimated life expectancy <12 weeks, in the opinion of the investigator. 13. Other serious underlying medical condition that, in the opinion of the investigator, would impair the subject’s ability to receive or tolerate the planned treatment and follow- up. 14. Any toxicity associated with prior therapy that has not returned to baseline or that is ≥ Grade 2, with the exception of alopecia and other adverse events (AEs) that are not deemed clinically significant in the opinion of the investigator, and AEs that are sequelae of prior immune-related-AEs which are adequately treated and stable.Attorney Docket No.01218-0032-00PCT 15. Live or live-attenuated vaccine(s) received within 30 days prior to Cycle 1 Day 1. 16. Melanoma subtypes including acral, uveal, and mucosal are excluded. 17. Unstable cardiovascular function within 6 months prior to first dose of study drug.

[0302] Specific objectives and corresponding endpoints for the study are summarized in Table 3 below.

[0303] Table 3: Objectives and corresponding endpointsVII. TABLE OF SEQUENCESAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCTAttorney Docket No.01218-0032-00PCT

Claims

Attorney Docket No.01218-0032-00PCT CLAIMS 1. A method for treating a cancer in a human subject, comprising administering to the subject a fusion protein that binds both CD137 and CD228, wherein the fusion protein comprises at least two subunits in any order, wherein a first subunit comprises an antibody or an antigen- binding domain thereof specific for CD228, wherein a second subunit comprises a lipocalin mutein specific for CD137, and wherein the fusion protein is administered to the subject at a dose of 0.15 to 22.5 mg / kg.

2. The method of claim 1, wherein the fusion protein is administered to the subject at a dose of 0.15, 0.3, 0.45, 0.5, 1.0, 1.5, 2.5, 3.0, 4.5, 5.0, 7.5, 10.0, 15.0, or 22.5 mg / kg.

3. The method of claim 1, wherein the fusion protein is administered to the subject every week (Q1W).

4. The method of claim 3, wherein the fusion protein is administered to the subject for at least one 21-day cycle.

5. The method of claim 3 or 4, wherein the fusion protein is administered to the subject for one, two, three, four, or five 21-day cycles.

6. The method of claim 4 or 5, wherein the fusion protein is administered to the subject on days 1, 8, and 15 of the cycle or each of the cycles.

7. The method of any one of claims 3 to 6, wherein the fusion protein is administered to the subject at a dose of 0.15 to 7.5 mg / kg.

8. The method of any one of claims 3 to 7, wherein the fusion protein is administered to the subject at a dose of 0.15, 0.5, 1.5, 2.5, 5.0, or 7.5 mg / kg.

9. The method of claim 1, wherein the fusion protein is administered to the subject every two weeks (Q2W).

10. The method of claim 9, wherein the fusion protein is administered to the subject for at least one 28-day cycle.

11. The method of claim 9 or 10, wherein the fusion protein is administered to the subject for one, two, three, four, or five 28-day cycles.Attorney Docket No.01218-0032-00PCT 12. The method of claim 10 or 11, wherein the fusion protein is administered to the subject on days 1 and 15 of the cycle or each of the cycles.

13. The method of any one of claims 9 to 12, wherein the fusion protein is administered to the subject at a dose of 0.3 to 15.0 mg / kg.

14. The method of any one of claims 9 to 13wherein the fusion protein is administered to the subject at a dose of 0.3, 1.0, 3.0, 5.0, 10.0, or 15.0 mg / kg.

15. The method of claim 1, wherein the fusion protein is administered to the subject every three weeks (Q3W).

16. The method of claim 15, wherein the fusion protein is administered to the subject for at least one 21-day cycle.

17. The method of claim 15 or 16, wherein the fusion protein is administered to the subject for one, two, three, four, or five 21-day cycles.

18. The method of claim 16 or 17, wherein the fusion protein is administered to the subject on day 1 of the cycle or each of the cycles.

19. The method of any one of claims 15 to 18, wherein the fusion protein is administered to the subject at a dose of 0.45 to 22.5 mg / kg.

20. The method of any one of claims 15 to 19, wherein the fusion protein is administered to the subject at a dose of 0.45, 1.5, 4.5, 7.5, 15.0, or 22.5 mg / kg.

21. The method of any one of claims 1 to 20, wherein the fusion protein is administered to the subject intravenously.

22. The method of any one of claims 1 to 21, wherein the fusion protein is administered to the subject as a monotherapy.

23. The method of any one of claims 1 to 22, wherein the cancer has relapsed or become refractory to a prior therapy for the cancer.Attorney Docket No.01218-0032-00PCT 24. The method of any one of claims 1 to 23, wherein the cancer is a metastatic cancer or an unresectable cancer.

25. The method of any one of claims 1 to 24, wherein the cancer is a solid cancer.

26. The method of any one of claims 1 to 25, wherein the cancer is lung cancer, melanoma, pancreatic cancer, mesothelioma, colorectal cancer (CRC), thyroid cancer, breast cancer, cholangiocarcinoma, esophageal cancer, head and neck cancer, liver cancer, stomach cancer, urothelial cancer, or cervical cancer.

27. The method of any one of claims 1 to 26, wherein the cancer is cutaneous melanoma, non- small cell lung cancer (NSCLC), colorectal cancer (CRC), pancreatic cancer, or mesothelioma.

28. The method of any one of claims 1 to 27, wherein the cancer is cutaneous melanoma.

29. The method of claim 28, wherein the subject has received a PD-1 inhibitor or a PD-L1 inhibitor.

30. The method of claim 29, wherein the subject has received the PD-1 inhibitor or the PD-L1 inhibitor in combination with another checkpoint inhibitor.

31. The method of claim 30, wherein the other checkpoint inhibitor is an inhibitor of the CTLA-4 pathway, the LAG-3 pathway, or the TIM-3 pathway.

32. The method of any one of claims 29 to 31, wherein the cutaneous melanoma is resistant or has become refractory to the PD-1 inhibitor or the PD-L1 inhibitor.

33. The method of any one of claims 29 to 32, wherein the PD-1 inhibitor is an anti-PD-1 antibody, or wherein the PD-L1 inhibitor is an anti-PD-L1 antibody.

34. The method of claim 33, wherein the anti-PD-1 antibody is nivolumab, pembrolizumab, cemiplimab, dostarlimab, or retifanlimab, or wherein the anti-PD-L1 antibody is atezolizumab, avelumab, or durvalumab.

35. The method of any one of claims 1 to 34, wherein the cancer is cutaneous melanoma, and the subject has received a BRAF / MEK targeted therapy.Attorney Docket No.01218-0032-00PCT 36. The method of claim 35, wherein the BRAF / MEK targeted therapy is a BRAF inhibitor or a MEK inhibitor.

37. The method of claim 36, wherein the BRAF inhibitor is vemurafenib, dabrafenib, or encorafenib, or wherein the MEK inhibitor is trametinib, cobimetinib, or binimetinib.

38. The method of any one of claims 1 to 37, wherein the amino acid sequence of the lipocalin mutein has at least 85% sequence identity to an amino acid sequence selected from SEQ ID NOs: 32-38.

39. The method of any one of claims 1 to 38, wherein the amino acid sequence of the lipocalin mutein comprises an amino acid sequence selected from SEQ ID NOs: 32-38 or a fragment or variant thereof.

40. The method of any one of claims 1 to 37, wherein the amino acid sequence of the lipocalin mutein has at least 85% sequence identity to an amino acid sequence selected from SEQ ID NOs: 39-57.

41. The method of any one of claims 1 to 37 and 40, wherein the amino acid sequence of the lipocalin mutein comprises an amino acid sequence selected from SEQ ID NOs: 39-57 or a fragment or variant thereof.

42. The method of any one of claims 1 to 37, 40, and 41, wherein the amino acid sequence of the lipocalin mutein comprises the amino acid sequence of SEQ ID NO:

40.

43. The method of any one of claims 1 to 42, wherein one subunit is linked to another subunit via a linker.

44. The method of any one of claims 1 to 43, wherein the second subunit is linked at the N- terminus via a linker to the N- or C-terminus of each heavy chain constant region (CH) of the first subunit or the N- or C-terminus of each light chain constant region (CL) of the first subunit.

45. The method of claim 43 or 44, wherein the linker is an unstructured glycine-serine linker, a polyproline linker, a proline-alanine-serine polymer, or a linker selected from SEQ ID NOs: 13-23.Attorney Docket No.01218-0032-00PCT 46. The method of any one of claims 43 to 45, wherein the linker is an unstructured (Gly-Gly-Gly- Gly-Ser)3linker (SEQ ID NO: 13).

47. The method of any one of claims 1 to 46, wherein the antibody or the antigen-binding domain thereof comprises: a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 110, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 111, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 112, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 116, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 118; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 113, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 114, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 115, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 119, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 120, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 121; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 130, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 131, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 132, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 136, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 137, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 138; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 133, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 134, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 135, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 139, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 140, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 141; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 150, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 151, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 152, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 156, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 157, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 158; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 153, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 154, and (c) CDR-H3Attorney Docket No.01218-0032-00PCT comprising the amino acid sequence of SEQ ID NO: 155, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 159, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 160, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 161; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 170, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 171, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 172, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 173, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 174, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 175, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 179, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 180, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 181; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 190, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 191, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 192, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 196, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 197, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 198; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 193, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 194, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 195, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 199, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 200, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 201; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 210, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 211, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 212, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 216, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 217, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 218; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 213, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 214, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 215, and a VL comprising (d) CDR-L1Attorney Docket No.01218-0032-00PCT comprising the amino acid sequence of SEQ ID NO: 219, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 220, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 221; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 230, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 231, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 232, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 236, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 237, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 238; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 233, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 234, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 235, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 239, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 240, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 241; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 252, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 256, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 257, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 258; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 253, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 254, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 255, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 259, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 260, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 261; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 270, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 271, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 272, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 276, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 277, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 278; or a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 273, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 274, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 275, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 279, (e) CDR-L2 comprising the aminoAttorney Docket No.01218-0032-00PCT acid sequence of SEQ ID NO: 280, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:

281.

48. The method of claim 47, wherein the antibody or the antigen-binding domain thereof comprises: a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 210, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 211, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 212, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 216, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 217, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 218; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 213, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 214, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 215, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 219, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 220, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 221; a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 252, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 256, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 257, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 258; or a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 253, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 254, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 255, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 259, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 260, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:

261.

49. The method of claim 47 or 48, wherein the antibody or the antigen-binding domain thereof comprises: a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 210, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 211, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 212, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 216, (e) CDR-L2 comprising the amino acidAttorney Docket No.01218-0032-00PCT sequence of SEQ ID NO: 217, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 218; or a VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 213, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 214, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 215, and a VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 219, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 220, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:

221.

50. The method of claim 47, wherein the antibody or the antigen-binding domain thereof comprises: a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 122, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 124; a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 142, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 144; a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 162, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 164; a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 182, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 184; a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 202, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 204; a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 222, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 224; a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 242, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 244; a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 262, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 264; orAttorney Docket No.01218-0032-00PCT a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 282, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

284.

51. The method of any one of claims 47 to 50, wherein the antibody or the antigen-binding domain thereof comprises: a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 222, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 224; or a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 262, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

264.

52. The method of any one of claims 47 to 51, wherein the antibody or the antigen-binding domain thereof comprises: a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 222, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

224.

53. The method of claim 47 to 50, wherein the antibody or the antigen-binding domain thereof comprises: a VH comprising the amino acid sequence of SEQ ID NO: 122, and a VL comprising the amino acid sequence of SEQ ID NO: 124; a VH comprising the amino acid sequence of SEQ ID NO: 142, and a VL comprising the amino acid sequence of SEQ ID NO: 144; a VH comprising the amino acid sequence of SEQ ID NO: 162, and a VL comprising the amino acid sequence of SEQ ID NO: 164; a VH comprising the amino acid sequence of SEQ ID NO: 182, and a VL comprising the amino acid sequence of SEQ ID NO: 184; a VH comprising the amino acid sequence of SEQ ID NO: 202, and a VL comprising the amino acid sequence of SEQ ID NO: 204; a VH comprising the amino acid sequence of SEQ ID NO: 222, and a VL comprising the amino acid sequence of SEQ ID NO: 224; a VH comprising the amino acid sequence of SEQ ID NO: 242, and a VL comprising the amino acid sequence of SEQ ID NO: 244; a VH comprising the amino acid sequence of SEQ ID NO: 262, and a VL comprising the amino acid sequence of SEQ ID NO: 264; orAttorney Docket No.01218-0032-00PCT a VH comprising the amino acid sequence of SEQ ID NO: 282, and a VL comprising the amino acid sequence of SEQ ID NO:

284.

54. The method of any one of claims 47, 48, 50, 51, and 53, wherein the antibody or the antigen- binding domain thereof comprises: a VH comprising the amino acid sequence of SEQ ID NO: 222, and a VL comprising the amino acid sequence of SEQ ID NO: 224; or a VH comprising the amino acid sequence of SEQ ID NO: 262, and a VL comprising the amino acid sequence of SEQ ID NO:

264.

55. The method of any one of claims 47 to 54, wherein the antibody or the antigen-binding domain thereof comprises: a VH comprising the amino acid sequence of SEQ ID NO: 222, and a VL comprising the amino acid sequence of SEQ ID NO:

224.

56. The method of any one of claims 47, 50, and 53, wherein the antibody or the antigen-binding domain thereof comprises: a heavy chain (HC) comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 126, and a light chain (LC) comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 128; a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 146, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 148; a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 166, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 168; a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 186, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 188; a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 206, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 208; a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 226, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 228;Attorney Docket No.01218-0032-00PCT a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 246, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 248; a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 266, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 268; or a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 286, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

288.

57. The method of any one of claims 47, 48, 50, 51, 53, 54, and 56, wherein the antibody or the antigen-binding domain thereof comprises: a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 226, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 228; or a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 266, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

268.

58. The method of any one of claims 47 to 57, wherein the antibody or the antigen-binding domain thereof comprises: a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 226, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

228.

59. The method of any one of claims 47, 50, 53, and 56, wherein the antibody or the antigen- binding domain thereof comprises: a HC comprising the amino acid sequence of SEQ ID NO: 126, and a LC comprising the amino acid sequence of SEQ ID NO: 128; a HC comprising the amino acid sequence of SEQ ID NO: 146, and a LC comprising the amino acid sequence of SEQ ID NO: 148; a HC comprising the amino acid sequence of SEQ ID NO: 166, and a LC comprising the amino acid sequence of SEQ ID NO: 168; a HC comprising the amino acid sequence of SEQ ID NO: 186, and a LC comprising the amino acid sequence of SEQ ID NO: 188; a HC comprising the amino acid sequence of SEQ ID NO: 206, and a LC comprising the amino acid sequence of SEQ ID NO: 208;Attorney Docket No.01218-0032-00PCT a HC comprising the amino acid sequence of SEQ ID NO: 226, and a LC comprising the amino acid sequence of SEQ ID NO: 228; a HC comprising the amino acid sequence of SEQ ID NO: 246, and a LC comprising the amino acid sequence of SEQ ID NO: 248; a HC comprising the amino acid sequence of SEQ ID NO: 266, and a LC comprising the amino acid sequence of SEQ ID NO: 268; or a HC comprising the amino acid sequence of SEQ ID NO: 286, and a LC comprising the amino acid sequence of SEQ ID NO:

288.

60. The method of any one of claims 47, 48, 50, 51, 53, 54, 56, 57, and 59, wherein the antibody or the antigen-binding domain thereof comprises: a HC comprising the amino acid sequence of SEQ ID NO: 226, and a LC comprising the amino acid sequence of SEQ ID NO: 228; or a HC comprising the amino acid sequence of SEQ ID NO: 266, and a LC comprising the amino acid sequence of SEQ ID NO:

268.

61. The method of any one of claims 47 to 60, wherein the antibody or the antigen-binding domain thereof comprises: a HC comprising the amino acid sequence of SEQ ID NO: 226, and a LC comprising the amino acid sequence of SEQ ID NO:

228.

62. The method of any one of claims 1 to 61, wherein the antibody is a monoclonal antibody.

63. The method of any one of claims 1 to 62, wherein the antibody is a humanized or chimeric antibody.

64. The method of any one of claims 1 to 63, wherein the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody.

65. The method of any one of claims 1 to 64, wherein the antibody is an IgG4 antibody.

66. The method of claim 65, wherein the antibody comprises one or more of the following mutations: S228P, N297A, F234A, L235A, M428L, N434S, M252Y, S254T, and T256E.

67. The method of any one of claims 1 to 48, 50, 51, 53, 54, 56, 57, 59, 60, and 62 to 66, wherein the fusion protein comprises an amino acid sequence shown in any one of SEQ ID NOs: 75, 76 and 78-83, or wherein the fusion protein comprises an amino acid sequence having atAttorney Docket No.01218-0032-00PCT least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or higher sequence identity to an amino acid sequence shown in any one of SEQ ID NOs: 75, 76 and 78-83.

68. The method of any one of claims 1 to 48, 50, 51, 53, 54, 56, 57, 59, 60, and 62 to 67, wherein the fusion protein comprises amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or higher sequence identity to the amino acid sequences shown in SEQ ID NOs: 80 and 76, SEQ ID NOs: 82 and 79, SEQ ID NOs: 75 and 81, or SEQ ID NOs: 78 and 83.

69. The method of any one of claims 1 to 48, 50, 51, 53, 54, 56, 57, 59, 60, and 62 to 68, wherein the fusion protein comprises the amino acid sequences shown in SEQ ID NOs: 80 and 76, SEQ ID NOs: 82 and 79, SEQ ID NOs: 75 and 81, or SEQ ID NOs: 78 and 83.

70. The method of any one of claims 1 to 69, wherein the fusion protein comprises the amino acid sequences shown in SEQ ID NOs: 80 and 76.

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