Compositions and methods comprising SFRP2 antagonists

Abstract

The present disclosure provides humanized antibodies and antigen-binding fragments thereof that specifically bind to SFRP2, as well as compositions comprising such humanized antibodies or antigen-binding fragments. In some embodiments, the humanized antibodies or antigen-binding fragments can be used to treat diseases or conditions associated with increased SFRP2, such as cancer. In some embodiments, the antibodies or antigen-binding fragments can be used to treat osteosarcoma.

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 124,345, filed December 11, 2020, which is incorporated herein by reference in its entirety.

[0002] 2. Reference to Electronically Submitted Sequence Listings The contents of the electronically transmitted Sequence Listing in an ASCII text file filed with this application (Name: 421_497_PCT_ST25.txt; Size: 27 kilobytes, and Creation Date: December 8, 2021) are hereby incorporated by reference in their entirety.

[0003] 3.Technical Field The present disclosure relates generally to humanized anti-SFRP2 antibodies and antigen-binding fragments thereof, compositions comprising such antibodies and antigen-binding fragments thereof, methods of making and using humanized anti-SFRP2 antibodies and antigen-binding fragments thereof, and methods of treating diseases such as cancer, which methods comprise administering humanized anti-SFRP2 antibodies and antigen-binding fragments thereof, optionally as part of a combination therapy, to a subject in need thereof. [Background technology]

[0004] 4.Background technology Secreted frizzled-related protein-2 (SFRP2) has been shown to be a key regulator of breast cancer (Lee, JL, et al., Breast Cancer Res Treat 84(2):139-49(2004); Bhati R., et al., Am J Pathol 172(5):1381-90(2008); Fontenot, E., et al., Molecular Cancer Therapeutics 12(5):685-95(2013); Lee, JL, et al., Breast Cancer Res Treat. 100(1):49-58(2006)), angiosarcoma (Bhati, R., et al., Am J Pathol. 172(5):1381-90(2008); Fontenot, E., et al., Molecular Cancer Therapeutics 12(5):685-95(2013)), osteosarcoma (Techavichit, P., et al., BMC Cancer 16(1):869(2016)), rhabdomyosarcoma (Singh, S., et al., The American Journal of Pathology 177(4):2055-66(2010)), alveolar soft tissue sarcoma (Tanaka, M., et al., Cancer Res 77(4):897-907(2017)), malignant glioma (Roth, W., et al. al., Oncogene19(37):4210-20(2000)), multiple myeloma (Oshima, T., et al., Blood 106(9):3160-5(2005)), renal cell carcinoma (Yamamura, S., et al. Mol Cancer It has been shown to promote tumor growth in many cancers, including prostate cancer (Sun, Y., et al., Oncogene 35(33):4321-34(2016)), lung cancer (Xiao, X., et al., Oncol Rep 34(5):2259-66(2015)), and melanoma (Kaur, A., et al., Nature 532(7598):250-4(2016)).

[0005] Osteosarcoma (OS) is a common primary malignant bone tumor in the pediatric population. The current standard of care consists of chemotherapy and surgical resection of the tumor. However, even with chemotherapy, only two-thirds of patients with resectable disease are cured. Long-term survival occurs in only <30% of patients with metastatic or recurrent tumors (NC Institute, S. Database, Ed. (2017), vol. 2017). The lung is the most common site of metastasis, with approximately 80% of metastases occurring in this anatomical location (Lindsey, BA et al., Rheumatol Ther 4:25-43 (2017)).

[0006] Increased SFRP2 expression levels in OS patient samples correlate with decreased survival, and SFRP2 overexpression suppresses normal osteoblast differentiation, promotes OS characteristics, and facilitates angiogenesis (Kim, H., et al., Proc Natl Acad Sci USA 115:E11128-E11137 (2018)). Functional studies revealed that stable overexpression of SFRP2 in localized human and mouse OS cells significantly increased cell migration and invasive capacity in vitro and enhanced metastatic potential in vivo (Techavichit, P., et al., BMC Cancer 16:869 (2016)). Furthermore, knockdown of SFRP2 in metastatic OS cells demonstrated reduced cell migration and invasive capacity in vitro, thus confirming the crucial biological phenotype executed by SFRP2 (Techavichit, P., et al., BMC Cancer 16:869 (2016)). Thus, SFRP2 has emerged as a potential therapeutic target for osteosarcoma, but the lack of new active agents has hindered progress in increasing survival for over 30 years, and therefore novel therapeutic approaches are seriously needed (Wedekind, MF, et al., Pediatr Blood Cancer 65:e27227 (2018)).

[0007] Furthermore, PD-L1 expression in osteosarcoma correlates with immune cell infiltration and has been found to be significantly associated with poor 5-year event-free survival (EFS) (Koirala, P., et al., SciRep 6:30093 (2016)). Despite these findings, a phase II trial of pembrolizumab (SARC028) lacked efficacy in treating osteosarcoma, in which only 5% of patients with metastatic osteosarcoma had an objective response (Tawbi, HA, et al., Lancet Oncol 18:1493-1501 (2017)). These findings further demonstrate the urgent need for new therapies for osteosarcoma. Summary of the Invention

[0008] 5. Summary of the Invention The present disclosure generally relates to a humanized anti-SFRP2 antibody or antigen-binding fragment thereof that binds to secreted frizzled-related protein 2 (SFRP2), wherein the antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) polypeptide that is at least 90%, 95%, or 99% identical to a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4, and further wherein the antibody or antigen-binding fragment thereof comprises a variable light chain (VL) polypeptide that is at least 90%, 95%, or 99% identical to a polypeptide comprising the amino acid sequence of SEQ ID NO: 5, 6, 7, 8, or 9.

[0009] Furthermore, the present disclosure generally relates to a humanized anti-SFRP2 antibody or antigen-binding fragment thereof that binds to secreted frizzled-related protein 2 (SFRP2), wherein the antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) polypeptide and a variable light chain (VL) polypeptide comprising the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:6, respectively (Ab1).

[0010] Furthermore, the present disclosure generally relates to a humanized anti-SFRP2 antibody or antigen-binding fragment thereof that binds to secreted frizzled-related protein 2 (SFRP2), wherein the antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) polypeptide and a variable light chain (VL) polypeptide comprising the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:7, respectively (Ab2).

[0011] Furthermore, the present disclosure generally relates to a humanized anti-SFRP2 antibody or antigen-binding fragment thereof that binds to secreted frizzled-related protein 2 (SFRP2), wherein the antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) polypeptide and a variable light chain (VL) polypeptide comprising the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:8, respectively (Ab3).

[0012] Furthermore, the present disclosure generally relates to a humanized anti-SFRP2 antibody or antigen-expressing fragment thereof that binds to secreted frizzled-related protein 2 (SFRP2), wherein the antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) polypeptide and a variable light chain (VL) polypeptide comprising the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:9, respectively (Ab4).

[0013] Furthermore, the present disclosure generally relates to a humanized anti-SFRP2 antibody or antigen-binding fragment thereof that binds to secreted frizzled-related protein 2 (SFRP2), wherein the antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) polypeptide and a variable light chain (VL) polypeptide comprising the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:6, respectively (Ab5).

[0014] Furthermore, the present disclosure generally relates to a humanized anti-secreted frizzled-related protein 2 (SFRP2) antibody or antigen-binding fragment thereof that binds to SFRP2, the antibody or antigen-binding fragment thereof comprising a variable heavy chain (VH) polypeptide and a variable light chain (VL) polypeptide comprising the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:7, respectively (Ab6).

[0015] Furthermore, the present disclosure generally relates to a humanized anti-secreted frizzled-related protein 2 (SFRP2) antibody or antigen-binding fragment thereof that binds to SFRP2, the antibody or antigen-binding fragment thereof comprising a variable heavy chain (VH) polypeptide and a variable light chain (VL) polypeptide comprising the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:8, respectively (Ab7).

[0016] Furthermore, the present disclosure generally relates to a humanized anti-secreted frizzled-related protein 2 (SFRP2) antibody or antigen-binding fragment thereof that binds to SFRP2, the antibody or antigen-binding fragment thereof comprising a variable heavy chain (VH) polypeptide and a variable light chain (VL) polypeptide comprising the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:9, respectively (Ab8).

[0017] In some aspects, provided herein is a humanized anti-SFRP2 antibody or antigen-binding fragment thereof that binds to secreted frizzled-related protein 2 (SFRP2), wherein the antibody or antigen-binding fragment thereof comprises a complementarity-determining region (CDR) H1 comprising the amino acid sequence of SEQ ID NO: 19, a CDR H2 comprising the amino acid sequence of SEQ ID NO: 20, a CDR H3 comprising the amino acid sequence of SEQ ID NO: 21, a CDR L1 comprising the amino acid sequence of SEQ ID NO: 22, a CDR L2 comprising the amino acid sequence of SEQ ID NO: 23, and a CDR L3 (Ab8) comprising the amino acid sequence of SEQ ID NO: 24.

[0018] In some aspects, provided herein is a humanized anti-SFRP2 antibody or antigen-binding fragment thereof that binds to secreted frizzled related protein 2 (SFRP2), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain polypeptide and a light chain polypeptide comprising the amino acid sequences of SEQ ID NO: 15 and SEQ ID NO: 16, respectively (Ab8).

[0019] Furthermore, the present disclosure generally relates to a humanized anti-SFRP2 antibody or antigen-binding fragment thereof that binds to secreted frizzled-related protein 2 (SFRP2), wherein the antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) polypeptide and a variable light chain (VL) polypeptide comprising the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:6, respectively (Ab9).

[0020] Furthermore, the present disclosure generally relates to a humanized anti-SFRP2 antibody or antigen-binding fragment thereof that binds to secreted frizzled-related protein 2 (SFRP2), wherein the antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) polypeptide and a variable light chain (VL) polypeptide comprising the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:7, respectively (Ab10).

[0021] Furthermore, the present disclosure generally relates to a humanized anti-SFRP2 antibody or antigen-binding fragment thereof that binds to secreted frizzled-related protein 2 (SFRP2), wherein the antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) polypeptide and a variable light chain (VL) polypeptide comprising the amino acid sequences of SEQ ID NO:4 and SEQ ID NO:8, respectively (Ab11).

[0022] Furthermore, the present disclosure generally relates to a humanized anti-secreted frizzled-related protein 2 (SFRP2) antibody or antigen-binding fragment thereof that binds to SFRP2, the antibody or antigen-binding fragment thereof comprising a variable heavy chain (VH) polypeptide and a variable light chain (VL) polypeptide comprising the amino acid sequences of SEQ ID NO:4 and SEQ ID NO:9, respectively (Ab12).

[0023] Furthermore, the present disclosure generally relates to a humanized anti-SFRP2 antibody or antigen-binding fragment thereof that binds to secreted frizzled-related protein 2 (SFRP2), wherein the antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) polypeptide and a variable light chain (VL) polypeptide comprising the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:5, respectively (Ab13).

[0024] Furthermore, the present disclosure generally relates to a humanized anti-SFRP2 antibody or antigen-binding fragment thereof that binds to secreted frizzled-related protein 2 (SFRP2), wherein the antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) polypeptide and a variable light chain (VL) polypeptide comprising the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:9, respectively (Ab14).

[0025] Furthermore, the present disclosure generally relates to a humanized anti-SFRP2 antibody or antigen-binding fragment thereof that binds to secreted frizzled-related protein 2 (SFRP2), wherein the antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) polypeptide and a variable light chain (VL) polypeptide comprising the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:5, respectively (Ab15).

[0026] Furthermore, the present disclosure generally relates to a humanized anti-SFRP2 antibody or antigen-binding fragment thereof that binds to secreted frizzled-related protein 2 (SFRP2), wherein the antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) polypeptide and a variable light chain (VL) polypeptide comprising the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:5, respectively (Ab16).

[0027] Furthermore, the present disclosure generally relates to a humanized anti-secreted frizzled-related protein 2 (SFRP2) antibody or antigen-binding fragment thereof that binds to SFRP2, the antibody or antigen-binding fragment thereof comprising a variable heavy chain (VH) polypeptide and a variable light chain (VL) polypeptide comprising the amino acid sequences of SEQ ID NO:4 and SEQ ID NO:5, respectively (Ab17).

[0028] Furthermore, the present disclosure generally relates to a humanized anti-SFRP2 antibody or antigen-binding fragment thereof that binds to secreted frizzled-related protein 2 (SFRP2), wherein the antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) polypeptide and a variable light chain (VL) polypeptide comprising the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:8, respectively (Ab18).

[0029] Furthermore, the present disclosure generally relates to a humanized anti-SFRP2 antibody or antigen-binding fragment thereof that binds to secreted frizzled-related protein 2 (SFRP2), wherein the antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) polypeptide and a variable light chain (VL) polypeptide comprising the amino acid sequences of SEQ ID NO:4 and SEQ ID NO:6, respectively (Ab19).

[0030] Furthermore, the present disclosure generally relates to a humanized anti-secreted frizzled-related protein 2 (SFRP2) antibody or antigen-binding fragment thereof that binds to SFRP2, the antibody or antigen-binding fragment thereof comprising a variable heavy chain (VH) polypeptide and a variable light chain (VL) polypeptide comprising the amino acid sequences of SEQ ID NO:4 and SEQ ID NO:7, respectively (Ab20).

[0031] Furthermore, the present disclosure generally relates to a humanized anti-SFRP2 antibody or antigen-binding fragment thereof that binds to secreted frizzled-related protein 2 (SFRP2), the antibody or antigen-binding fragment thereof being selected from the group consisting of: i. SEQ ID NO:1 and SEQ ID NO:6, respectively (Ab1); ii. SEQ ID NO:1 and SEQ ID NO:7, respectively (Ab2); iii. SEQ ID NO:1 and SEQ ID NO:8, respectively (Ab3); iv. SEQ ID NO:1 and SEQ ID NO:9, respectively (Ab4); v. SEQ ID NO:2 and SEQ ID NO:6, respectively (Ab5); vi. SEQ ID NO:2 and SEQ ID NO:7, respectively (Ab6); vii. SEQ ID NO:2 and SEQ ID NO:8, respectively (Ab7); viii. SEQ ID NO:2 and SEQ ID NO:9, respectively (Ab8); ix. SEQ ID NO:3 and SEQ ID NO:6, respectively (Ab9); x. SEQ ID NO:1 and SEQ ID NO:2, respectively (Ab1); and SEQ ID NO:7 (Ab10), xi. SEQ ID NO:4 and SEQ ID NO:8 (Ab11), respectively; xii. SEQ ID NO:4 and SEQ ID NO:9 (Ab12), respectively; xiii. SEQ ID NO:3 and SEQ ID NO:5 (Ab13), respectively; xiv. SEQ ID NO:3 and SEQ ID NO:9 (Ab14), respectively; xv. SEQ ID NO:1 and SEQ ID NO:5 (Ab15), respectively; xvi. SEQ ID NO:2 and SEQ ID NO:5 (Ab16), respectively; xvii. SEQ ID NO:4 and SEQ ID NO:5 (Ab17), respectively; xviii. SEQ ID NO:3 and SEQ ID NO:8 (Ab18), respectively; xix. SEQ ID NO:4 and SEQ ID NO:6 (Ab19), respectively; or xx. VH chain polypeptides and VL chain polypeptides comprising the amino acid sequences of SEQ ID NO:4 and SEQ ID NO:7 (Ab20), respectively.

[0032] In some aspects, the antibody or antigen-binding fragment comprises a heavy chain constant region and a light chain constant region. In some aspects, the antibody or antigen-binding fragment is a monoclonal antibody. In some aspects, the antibody is a full-length antibody. In some aspects, the antibody or antigen-binding fragment is an antigen-binding fragment.

[0033] In some aspects, the antibody or antigen-binding fragment thereof comprises an IC50 value of 0.7 or less, 0.6 or less, or 0.5 or less relative to the IC50 value of an antibody or antigen-binding fragment thereof comprising the VH chain polypeptide and VL chain polypeptide of SEQ ID NOs: 10 and 11, respectively (Mo.1), wherein these IC50 values ​​are measured by ELISA assay.

[0034] Additionally, the present disclosure generally relates to isolated polynucleotides comprising nucleic acid molecules encoding the heavy chain variable region or heavy chain of an antibody or antigen-binding fragment thereof discussed herein. In some embodiments, the nucleic acid molecule encodes the VH of SEQ ID NO: 1, 2, 3, or 4.

[0035] Additionally, the present disclosure generally relates to isolated polynucleotides comprising nucleic acid molecules encoding the light chain variable region or light chain of an antibody or antigen-binding fragment thereof discussed herein. In some embodiments, the nucleic acid molecule encodes the VL of SEQ ID NO: 5, 6, 7, 8, or 9.

[0036] Additionally, the present disclosure generally relates to isolated polynucleotides comprising nucleic acid molecules encoding the heavy chain variable region or heavy chain of an antibody or antigen-binding fragment thereof discussed herein, and the light chain variable region or light chain of an antibody or antigen-binding fragment thereof discussed herein.

[0037] Additionally, the present disclosure generally relates to an isolated polynucleotide comprising a nucleic acid molecule encoding a polynucleotide discussed herein, e.g., a heavy chain variable region or heavy chain of an antibody or antigen-binding fragment thereof, e.g., of an antibody or antigen-binding fragment thereof, discussed herein, and an isolated vector comprising a light chain variable region or light chain of an antibody or antigen-binding fragment thereof, e.g., of an antibody or antigen-binding fragment thereof, discussed herein.

[0038] Furthermore, the present disclosure generally relates to a host cell comprising (a) a polynucleotide discussed herein, (b) a vector discussed herein, or (c) a first vector comprising a nucleic acid molecule encoding a heavy chain variable region or heavy chain of an antibody or antigen-binding fragment thereof of an antibody or antigen-binding fragment thereof discussed herein, and a second vector comprising a nucleic acid molecule encoding a light chain variable region or light chain of an antibody or antigen-binding fragment thereof of an antibody or antigen-binding fragment thereof discussed herein. In some aspects, the host cell is selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, YB / 20, NS0, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, HepG2, SP2 / 0, R1.1, BW, LM, COS1, COS7, BSC1, BSC40, BMT10 cells, plant cells in tissue culture, insect cells, and human cells. In some embodiments, the host cell is a CHO cell.

[0039] Furthermore, the present disclosure generally relates to methods for producing an antibody or antigen-binding fragment thereof that binds to SFRP2, the method comprising culturing a host cell as discussed herein such that a nucleic acid molecule is expressed and an antibody or antigen-binding fragment thereof is produced, and optionally, the method further comprises isolating the antibody or antigen-binding fragment thereof from the culture. In some aspects, the isolated antibody or antigen-binding fragment thereof is substantially free of precipitate. In some aspects, the isolated antibody or antigen-binding fragment thereof comprises a humanized antibody.

[0040] Additionally, the present disclosure generally relates to an isolated antibody or antigen-binding fragment thereof that specifically binds to secreted frizzled-related protein 2 (SFRP2) and that is encoded by a polynucleotide described herein or produced by a method described herein.

[0041] Additionally, the present disclosure generally relates to pharmaceutical compositions comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof discussed herein and a pharmaceutically acceptable excipient.

[0042] Furthermore, the present disclosure generally relates to a method of treating cancer in a patient, the method comprising administering to the patient a pharmaceutical composition discussed herein. In some aspects, the cancer is breast cancer, malignant glioma, multiple myeloma, renal cell carcinoma, kidney cancer, prostate cancer, lung cancer, melanoma, non-small cell lung cancer, pancreatic cancer, colorectal cancer, bladder cancer, hepatocellular carcinoma, gastrointestinal cancer, and sarcoma, including, but not limited to, angiosarcoma, osteosarcoma, rhabdomyosarcoma, and alveolar soft part sarcoma. In some aspects, the cancer is osteosarcoma. In some aspects, the method of treating cancer in a patient further comprises administering an antagonist of an inhibitory immune checkpoint molecule, optionally, the immune checkpoint molecule is PD-1. In some aspects, the PD-1 antagonist is an anti-PD-1 antibody or antigen-binding fragment thereof, optionally wherein the anti-PD-1 antibody or antigen-binding fragment thereof is selected from the group consisting of nivolumab, pembrolizumab, MEDI-0680 (AMP-514), camrelizumab (SHR-1210), tislelizumab (BGB-A317), and spartalizumab (NPVPDR001, NVS240118, PDR001). [Brief explanation of the drawings]

[0043] 6. Brief description of the drawings [Figure 1A] 1 presents an image of a Coomassie blue stained SDS-PAGE gel of various different purified humanized anti-SFRP2 antibodies according to Example 1. Lanes of the SDS-PAGE gel were loaded as follows: 1 = MW marker, 2 = Ab1, 3 = Ab2, 4 = Ab3, 5 = Ab4, 6 = Ab5, 7 = Ab6, 8 = Ab7, 9 = Ab8, 10 = Ab9, 11 = MW marker, 12 = MW marker, 13 = Ab10, 14 = Ab11, 15 = Ab12, 16 = MW marker, 17 = Ab13, and lane 18 = Ab14.

[0044] [Figure 1B]1 presents an image of a Coomassie blue stained SDS-PAGE gel of various different purified chimeric antibodies according to Example 1. Lanes of the SDS-PAGE gel were loaded as follows: 1 = MW marker, 2 = Chi.1 purified from HEK cells, 3 = Chi.2 purified from HEK cells, 4 = MW marker, 5 = Chi.1 purified from NS0 cells, 6 = Chi.2 purified from NS0 cells.

[0045] [Figure 2] A table of relative IC50 values ​​for various different humanized anti-SFRP2 antibodies is presented according to Example 2. The relative IC50 value for each antibody was calculated by dividing the IC50 value of the test antibody by the IC50 value of antibody Mo.1 assayed on the same ELISA plate.

[0046] [Figure 3] Presented are images of an endothelial tube formation assay taken according to Example 2. In Figure 3, 1 = control, 2 = treatment with Ab8, and 3 = treatment with Ab11.

[0047] [Figure 4] 1A-1C present graphical representations of cell-based assays performed according to Example 3. A presents a graphical representation of an assay in which an osteosarcoma model cell line (RF577 cell line) was treated with either IgG1 (control) or Ab8. B presents a graphical representation of a T cell apoptosis assay in which T cells were treated with Ab8, and also presents positive and negative controls.

[0048] [Figure 5] 1 presents a graphical representation of SFRP2 serum levels in various different mice administered various different treatments according to Example 4. SFRP2 levels are presented for tumor-free mice and for mice bearing metastatic osteosarcoma treated with IgG1, PD-1 mAb alone, Ab8 alone, or a combination of Ab8 and PD-1 mAb. The X-axis represents the treatment administered, and the Y-axis represents the SFRP2 serum concentration (ng / ml).

[0049] [Figure 6] 10 presents an image of a Western blot measuring CD38 levels according to Example 5. Splenocytes from mice bearing metastatic osteosarcoma were treated with either IgG1 or Ab8, followed by Western blot analysis according to Example 5. Actin was used for normalization.

[0050] [Figure 7] 1 presents a graphical representation of data relating to the treatment of metastatic osteosarcoma growth according to Example 6. A presents a graphical representation of an in vivo metastatic RF577 osteosarcoma experiment performed according to Example 6. The X-axis represents the treatment used and the Y-axis represents the number of lung surface metastases observed. B presents a graphical representation of a toxicity study performed according to Example 6. The X-axis represents the week of treatment of the mice and the Y-axis represents the weight of the mice. Filled circles represent IgG1, filled squares represent PD1 mAb, triangles represent Ab8, and inverted triangles represent the combination treatment of Ab8 and PD1 mAb.

[0051] [Figure 8] 1 presents a graphical representation of data relating to the treatment of metastatic osteosarcoma growth according to Example 6. The X-axis represents the treatment used, and the Y-axis represents the percentage of lung area occupied by the observed tumor. The percentage of lung occupied by tumor was calculated by dividing the tumor area by the normal lung area and multiplying by 100. Treatments were normalized to the control.

[0052] [Figure 9A] An image (FIG. 9A) of an endothelial tube formation assay according to Example 2 and a graphical representation of the results of the endothelial tube formation assay (FIG. 9B) are presented. In FIG. 9A, 1 = control, 2 = Ab11, and 3 = Ab8. FIG. 9B presents a graphical representation of tube branch points relative to control (# branch points treated / # branch points in control) as exhibited by each antibody in the tube formation assay according to Example 2. A lower percentage indicates stronger inhibition of endothelial tube formation. [Figure 9B]An image (FIG. 9A) of an endothelial tube formation assay according to Example 2 and a graphical representation of the results of the endothelial tube formation assay (FIG. 9B) are presented. In FIG. 9A, 1 = control, 2 = Ab11, and 3 = Ab8. FIG. 9B presents a graphical representation of tube branch points relative to control (# branch points treated / # branch points in control) as exhibited by each antibody in the tube formation assay according to Example 2. A lower percentage indicates stronger inhibition of endothelial tube formation.

[0053] [Figure 10] Figure 1 shows images of the biodistribution of IgG1 and hSFRP2 mAbs in control and breast cancer model mice after tail vein injection of fluorophore-tagged nanoparticles. Arrows point to tumors.

[0054] [Figure 11A] 1 presents a graphical representation of an in vivo tumor inhibition study using hSFRP2 mAb. Tumor volume (mm3) over a 97-day period is presented in response to treatment with hSFRP2 mAb or IgG1 starting on day 19. [Figure 11B] 1 presents a graphical representation of an in vivo tumor inhibition study using hSFRP2 mAb. The percentage of animals in this study that had distant metastases after a 97-day period in response to treatment with hSFRP2 mAb or IgG1 is presented. [Figure 11C] 1 presents a graphical representation of an in vivo tumor inhibition study using hSFRP2 mAb. Weights of hSFRP2 mAb- and IgG1-treated animals measured weekly over a 97-day period are presented. DETAILED DESCRIPTION OF THE INVENTION

[0055] 7. MODE FOR CARRYING OUT THE INVENTION Provided herein are humanized antibodies and antigen-binding fragments thereof that bind to SFRP2. In some embodiments, the humanized anti-SFRP2 antibodies and antigen-binding fragments thereof provided herein are used to treat diseases associated with increased levels of SFRP2 expression, such as various different cancers, e.g., osteosarcoma. The humanized anti-SFRP2 antibodies and antigen-binding fragments thereof can, for example, selectively induce apoptosis of SFRP2-expressing osteosarcoma cells and / or other tumor cells; for example, the humanized anti-SFRP2 antibodies do not induce apoptosis in T cells, but induce apoptosis of osteosarcoma cells and / or other tumor cells, reduce the amount of intracellular SFRP2, reduce serum levels of SFRP2 in treated subjects, reduce metastatic tumor growth of osteosarcoma and / or other tumor types in subjects, reduce the amount of osteosarcoma lung metastases in patients, and / or reduce the amount of CD38 protein in patients. These activities of humanized anti-SFRP2 antibodies and antigen-binding fragments thereof may facilitate effective treatment of diseases in which SFRP2 is overexpressed and / or in which SFRP2 expression or overexpression is associated with a variety of different disease states, such as cancers, e.g., osteosarcoma.

[0056] Also provided herein are isolated nucleic acids (polynucleotides), such as complementary DNA (cDNA), encoding such humanized antibodies and antigen-binding fragments thereof. Further provided are vectors (e.g., expression vectors) and cells (e.g., host cells) containing nucleic acids (polynucleotides) encoding such humanized antibodies and antigen-binding fragments thereof. Methods for producing such humanized antibodies and antigen-binding fragments thereof are also provided.

[0057] In other aspects, provided herein are methods for, for example, modulating SFRP2 activity using such humanized antibodies and antigen-binding fragments thereof. SFRP2 activity can be modulated, for example, by binding of the humanized antibodies and antigen-binding fragments described herein to SFRP2. Modulation of SFRP2 activity by the antibodies and antigen-binding fragments described herein can result in a reduction in the amount of SFRP2 in cells, a reduction in serum levels of SFRP2 in a treated subject, a reduction in the growth of metastatic osteosarcoma in a subject, a reduction in the amount of osteosarcoma lung metastases in a patient, or the selective induction of apoptosis of osteosarcoma cells, e.g., a humanized anti-SFRP2 antibody does not induce apoptosis in T cells, but does induce apoptosis of osteosarcoma cells, and / or induces a reduction in the amount of CD38 protein in a subject.

[0058] In further aspects, the humanized anti-SFRP2 antibodies and antigen-binding fragments thereof provided herein are used to treat diseases such as cancer, e.g., osteosarcoma. In some aspects, treatment comprises administering an effective amount of one or more humanized anti-SFRP2 antibodies and antigen-binding fragments thereof described herein to a subject in need thereof. In some aspects, such diseases include, but are not limited to, cancers such as breast cancer, malignant glioma, multiple myeloma, renal cell carcinoma, kidney cancer, prostate cancer, lung cancer, melanoma, non-small cell lung cancer, pancreatic cancer, colorectal cancer, bladder cancer, hepatocellular carcinoma, gastrointestinal cancer, and sarcomas, including, but not limited to, angiosarcoma, osteosarcoma, rhabdomyosarcoma, and alveolar soft part sarcoma. In some aspects, such diseases include diseases in which increased expression of SFRP2, overexpression of SFRP2, and / or expression levels of SFRP2 associated with a disease state occur, such as breast cancer, malignant glioma, multiple myeloma, renal cell carcinoma, kidney cancer, prostate cancer, lung cancer, melanoma, non-small cell lung cancer, pancreatic cancer, colorectal cancer, bladder cancer, hepatocellular carcinoma, gastrointestinal cancer, and sarcomas, including but not limited to angiosarcoma, osteosarcoma, rhabdomyosarcoma, and alveolar soft part sarcoma.

[0059] 6.1 Terminology As used herein, the term "SFRP2" refers to a secreted frizzled-related protein 2 polypeptide, including, but not limited to, a native SFRP2 polypeptide and any naturally occurring variants thereof. As used herein, the term "human SFRP2" refers to a polypeptide comprising the amino acid sequence of SEQ ID NO: 12. "SFRP2 polynucleotide," "SFRP2 nucleotide," or "SFRP2 nucleic acid" refers to any polynucleotide encoding SFRP2, including those described above.

[0060] The term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing (e.g., glycoprotein), via at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, and any other immunoglobulin molecule so long as the antibody exhibits the desired biological activity. Antibodies may be of any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), designated alpha, delta, epsilon, gamma, and mu, respectively, based on the identity of their heavy chain constant domains. The different classes of immunoglobulins have distinct and well-known subunit structures and three-dimensional configurations. Antibodies can be naked, part of a fusion protein, or conjugated to other molecules such as toxins, radioisotopes, etc.

[0061] The term "antibody fragment" refers to a portion of an antibody. An "antigen-binding fragment," "antigen-binding domain," or "antigen-binding region" refers to the portion of an antibody that binds to an antigen. An antigen-binding fragment may contain an antigen-determining region of an antibody (e.g., a complementarity-determining region (CDR)). An antigen-binding fragment may contain part or all of the VH and / or VL chain polypeptides of an antibody. Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single-chain antibodies. Antigen-binding fragments of antibodies can be derived from any animal species, such as rodents (e.g., mice, rats, or hamsters) and humans, or can be artificially produced.

[0062] The terms "anti-SFRP2 antibody," "SFRP2 antibody," and "antibody that binds to SFRP2" refer to an antibody that can bind to SFRP2 with sufficient affinity such that the antibody is useful as a diagnostic, therapeutic, and / or modulator of SFRP2 activity.

[0063] A "monoclonal" antibody or antigen-binding fragment thereof refers to a homogeneous population of antibodies or antigen-binding fragments involved in highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically include different antibodies directed against different antigenic determinants. The term "monoclonal" antibody or antigen-binding fragment thereof encompasses both intact and full-length monoclonal antibodies, as well as antibody fragments (Fab, Fab', F(ab')2, Fv, etc.), single-chain (scFv) variants, fusion proteins containing an antibody portion, and any other modified immunoglobulin molecule containing an antigen recognition site. Furthermore, a "monoclonal" antibody or antigen-binding fragment thereof refers to such antibodies and antigen-binding fragments thereof produced in any number of ways, including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals.

[0064] As used herein, the terms "variable region" and "variable domain" are used interchangeably and are common in the art. The variable region typically refers to a portion of an antibody, generally a portion of either the light or heavy chain, typically the amino-terminal 110-120 or 110-125 amino acids in the mature heavy chain and about 90-115 amino acids in the mature light chain, which differ extensively in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen.

[0065] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

[0066] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

[0067] The "hypervariable regions" of each chain are held together in close proximity by FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 1992; Chothia et al., Conformations of immunoglobulin hypervariable regions. Nature (1989) 342:877-83). As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody responsible for antigen binding. Hypervariable regions generally comprise amino acid residues from the "complementarity-determining regions" or "CDRs," the latter of which are responsible for the greatest sequence variability and / or antigen recognition.

[0068] The term "Kabat numbering" and similar terms are art-recognized and refer to a system for numbering amino acid residues within the heavy and light chain variable regions of an antibody or antigen-binding fragment thereof (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242).

[0069] As used herein, the terms "constant region" or "constant domain" are interchangeable and have their common meaning in the art. The constant region is the portion of an antibody, e.g., the carboxyl-terminal portion of the light and / or heavy chain, that is not directly involved in binding the antibody to an antigen but can exhibit various effector functions, such as interaction with Fc receptors. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence compared to the immunoglobulin variable domain. In certain embodiments, the antibody or antigen-binding fragment comprises a constant region or portion thereof sufficient for antibody-dependent cell-mediated cytotoxicity (ADCC).

[0070] As used herein, the term "heavy chain," when used in reference to an antibody, can refer to any distinct type, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant domain, which gives rise to the IgA, IgD, IgE, IgG, and IgM classes, including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4, respectively. Heavy chain amino acid sequences are well known in the art. In some aspects, the heavy chain is a human heavy chain.

[0071] As used herein, the term "light chain," when used in reference to an antibody, can refer to any distinct type, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In some embodiments, the light chain is a human light chain.

[0072] The term "chimeric" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof whose amino acid sequences are derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of an antibody or antigen-binding fragment thereof from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capacity, while the constant regions are homologous to the sequences of an antibody or antigen-binding fragment thereof from another (usually human) species to avoid eliciting an immune response from that species.

[0073] The term "humanized" antibody or antigen-binding fragment thereof refers to forms of non-human (e.g., murine) antibodies or antigen-binding fragments that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences. Typically, humanized antibodies or antigen-binding fragments thereof are human immunoglobulins in which residues from the complementarity-determining regions (CDRs) are replaced by residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, hamster) having the desired specificity, affinity, and capacity (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)). Humanized antibodies or antigen-binding fragments thereof can be further modified by substitution of additional residues within the Fv framework regions and / or within the substituted non-human residues to refine and optimize the specificity, affinity, and / or potency of the antibody or antigen-binding fragment. Generally, a humanized antibody or antigen-binding fragment thereof comprises a VH and VL domain that contain substantially all of at least one, typically two or three, of the CDR regions corresponding to a non-human immunoglobulin, while all or substantially all of the FR regions are of human immunoglobulin consensus sequences. A humanized antibody or antigen-binding fragment thereof can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Patent No. 5,225,539, Roguska et al., Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994), and Roguska et al., Protein Eng. 9(10):895-904 (1996). In some embodiments, a "humanized antibody" is a resurfaced antibody.

[0074] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or antigen-binding fragment thereof) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody or antigen-binding fragment thereof and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D Affinity can be expressed as the equilibrium dissociation constant (K D ), and the equilibrium association constant (K A ) can be measured and / or expressed in a number of ways known in the art, including, but not limited to, K D is k off / k on It is calculated from the quotient of K A is k on / k off It is calculated from the quotient of k on refers to, for example, the association rate constant of an antibody or its antigen-binding fragment to an antigen, and k off k refers to, for example, the dissociation rate constant of an antibody or antigen-binding fragment thereof from an antigen. on and k off can be determined by techniques known to those skilled in the art, such as BIAcore® or KinExA.

[0075] As used herein, the terms "immunospecifically bind," "immunospecifically recognize," "specifically bind," and "specifically recognize" are analogous terms in the context of an antibody or antigen-binding fragment thereof. These terms indicate that the antibody or antigen-binding fragment thereof binds to an epitope via its antigen-binding domain, and that the binding involves complementarity between the antigen-binding domain and the epitope. Thus, an antibody that "specifically binds" to human SFRP2 (e.g., SEQ ID NO: 12) may also bind to SFRP2 from other species and / or SFRP2 proteins produced from other human alleles.

[0076] A "blocking" or "blocking" or "inhibitory" or "inhibiting" antibody is one that, when bound to a target protein, reduces or inhibits (partially or completely) binding of that target protein to one or more ligands and / or reduces or inhibits (partially or completely) one or more activities or functions of the target protein when bound to the target protein.

[0077] As used herein, "epitope" is a term used in the art and refers to a localized region of an antigen to which an antibody or antigen-binding fragment thereof can specifically bind. An epitope can be, for example, consecutive amino acids of a polypeptide (a linear or continuous epitope), or an epitope can be joined together from, for example, two or more non-contiguous regions of a polypeptide(s) (a conformational, non-linear, discontinuous, or discontinuous epitope). In some embodiments, the epitope to which an antibody or antigen-binding fragment thereof binds can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., alanine scanning or other site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be achieved using any of the methods known in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen NE (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251:6300-6303).Crystals of an antibody or antigen-binding fragment thereof and its antigen can be studied using well-known X-ray diffraction techniques and refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds. Wyckoff HW et al., US2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, eds. Carter CW, Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323). Mutagenesis mapping studies can be accomplished using any method known to those of skill in the art. See, for example, Champe M et al., (1995) J Biol Chem 270:1388-1394 and Cunningham BC & Wells JA (1989) Science 244:1081-1085 for a description of mutagenesis techniques, including alanine scanning mutagenesis techniques.

[0078] An antibody is said to "competitively inhibit" the binding of a reference antibody to a given epitope if it preferentially binds to that epitope or an overlapping epitope, blocking to some extent the binding of the reference antibody to the epitope. Competitive inhibition can be determined by any method known in the art, for example, a competitive ELISA assay.

[0079] An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition is a polypeptide, antibody, polynucleotide, vector, cell, or composition in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to the extent that they are no longer in the form in which they are found in nature. In some aspects, an isolated antibody, polynucleotide, vector, cell, or composition is substantially pure.

[0080] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. A polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The term also encompasses amino acid polymers that are modified naturally or by intervention, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as polypeptides containing other modifications known in the art. Because the polypeptides of the present disclosure are based on antibodies, it is understood that in some aspects the polypeptides can occur as single chains or associated chains.

[0081] As used herein, the term "host cell" can be any type of cell, e.g., a primary cell, a cell in culture, or a cell from a cell line. In some aspects, the term "host cell" refers to a cell that has been transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due, for example, to mutations or environmental influences that may occur in subsequent generations, or to integration of the nucleic acid molecule into the host cell genome.

[0082] As used herein, the term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. The formulation can be sterile.

[0083] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the composition is contemplated. Supplementary active compounds can also be incorporated into the composition.

[0084] The terms "administer," "administering," "administration," and the like, as used herein, refer to methods that can be used to deliver a drug, e.g., a humanized anti-SFRP2 antibody or antigen-binding fragment thereof, to a desired site of biological action. Administration techniques that can be used in the agents and methods described herein can be found, for example, in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon, and Remington's, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, Pa.

[0085] As used herein, the terms "subject" and "patient" are used interchangeably. A subject can be a mammal, such as a non-human animal (e.g., a cow, pig, horse, cat, dog, rat, mouse, monkey, or other primate, etc.). In some embodiments, the subject is a mouse. In some embodiments, the subject is a cynomolgus monkey. In some embodiments, the subject is a human.

[0086] The term "therapeutically effective amount" refers to an amount of a drug, e.g., a humanized anti-SFRP2 antibody or antigen-binding fragment thereof, effective to treat a disease or condition of interest. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells, reduce tumor size or burden, inhibit (i.e., slow to some extent, and in some embodiments, stop) cancer cell invasion into peripheral organs, inhibit (i.e., slow to some extent, and in some embodiments stop) tumor metastasis, inhibit tumor growth to some extent, inhibit angiogenesis to some extent, alleviate to some extent one or more symptoms associated with cancer, and / or result in a favorable response such as progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), complete response (CR), partial response (PR), or in some cases stable disease (SD), reduction in progressive disease (PD), reduction in time to progression (TTP), or any combination thereof. To the extent the drug can prevent growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic.

[0087] The terms "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" and the like refer to a therapeutic measure that cures, slows, relieves the symptoms of, and / or halts the progression of, a diagnosed pathological condition or disorder. Thus, a person in need of treatment includes a person already diagnosed with or suspected of having the disorder. In some aspects, a subject is successfully "treated" for cancer according to the methods provided herein if the patient exhibits one or more of the following: a reduction in the number or complete absence of cancer cells, a reduction in tumor size, an inhibition or absence of cancer cell invasion to peripheral organs, including, for example, spread of cancer to soft tissue and bone, an inhibition or absence of tumor metastasis, an inhibition or absence of non-tumor growth, an alleviation of one or more symptoms associated with the particular cancer, a reduction in morbidity and mortality, an improvement in quality of life, a reduction in the tumorigenicity, tumorigenic frequency, or tumorigenic volume of the tumor, a reduction in the number or frequency of cancer stem cells in the tumor, differentiation of tumorigenic cells to a non-tumorous state, an improvement in progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), including a CR (complete response), a partial response (PR), an increase in stable disease (SD), a decrease in progressive disease (PD), a reduction in time to progression (TTP), or any combination thereof.In some embodiments, the treatment comprises reducing the amount of SFRP2 in a cell by contacting the cell with a humanized anti-SFRP2 antibody, or antigen-binding fragment thereof, described herein; reducing serum SFRP2 levels in the patient by administering to the patient a therapeutically effective amount of a humanized anti-SFRP2 antibody, or antigen-binding fragment thereof, described herein; selectively inducing apoptosis in osteosarcoma cells in the patient, e.g., the humanized anti-SFRP2 antibody does not induce apoptosis in T cells but induces apoptosis in osteosarcoma cells; and administering a therapeutically effective amount of a humanized anti-SFRP2 antibody, or antigen-binding fragment thereof, described herein. or antigen-binding fragment thereof to the patient, reducing the amount of osteosarcoma lung metastases in the patient by administering to the patient a therapeutically effective amount of a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein, reducing the amount of CD38 protein in the patient by administering to the patient a therapeutically effective amount of a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein, and treating the patient diagnosed with osteosarcoma by administering to the patient a therapeutically effective amount of a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein. In some aspects, the treatment is a monotherapy treatment comprising treatment with a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein. In some aspects, the treatment comprises a combination therapy comprising treatment with a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein and an antagonist of an inhibitory immune checkpoint molecule, optionally the immune checkpoint molecule is PD-1. In some aspects, the PD-1 antagonist is an anti-PD-1 antibody or antigen-binding fragment thereof, optionally wherein the anti-PD-1 antibody or antigen-binding fragment thereof is selected from the group consisting of nivolumab, pembrolizumab, MEDI-0680 (AMP-514), camrelizumab (SHR-1210), tislelizumab (BGB-A317), and spartalizumab (NPVPDR001, NVS240118, PDR001).

[0088] The terms "cancer" and "cancerous" refer to or describe the physiological condition in a mammal in which a population of cells is characterized by uncontrolled cell growth. In some embodiments, cancer includes increased expression of SFRP2 and / or SFRP2 expression levels indicative of a disease state. Examples include breast cancer, malignant glioma, multiple myeloma, renal cell carcinoma, kidney cancer, prostate cancer, lung cancer, melanoma, non-small cell lung cancer, pancreatic cancer, colorectal cancer, bladder cancer, hepatocellular carcinoma, gastrointestinal cancer, and sarcomas, including, but not limited to, angiosarcoma, osteosarcoma, rhabdomyosarcoma, and alveolar soft part sarcoma.

[0089] As used in this disclosure and the claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0090] Whenever an embodiment is described herein using the word "comprising," it is understood that other similar embodiments are also provided that are described in terms of "consisting of" and / or "consisting essentially of." In this disclosure, "comprises," "comprising," "containing," and "having" and the like can mean "includes," "including," and the terms "consisting essentially of" or "consisting essentially of" are open-ended, allowing for the presence of more than what is recited, so long as it excludes prior art aspects, while the basic or novel characteristics of what is recited are not changed by the presence of more than what is recited.

[0091] As used herein, unless otherwise specified or clear from the context, the term "or" is understood to be inclusive. When used in a phrase such as "A and / or B" herein, the term "and / or" is intended to include both "A and B," "A or B," "A," and "B." Similarly, when used in a phrase such as "A, B, and / or C," the term "and / or" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0092] As used herein, the terms "about" and "approximately," when used to modify a numerical value or numerical range, indicate that deviations of up to 10% above and below that value or range remain within the intended meaning of the recited value or range. It is understood that any embodiment described herein using the language "about" or "approximately" a numerical value or range also provides otherwise similar embodiments that refer to the specific numerical value or range.

[0093] "Sequence identity" refers to the degree of identity between two sequences (e.g., amino acid sequences or nucleic acid sequences). Sequence identity can be determined by aligning two sequences and introducing gaps to maximize the identity between the sequences. Alignment can be generated using programs known in the art. For the purposes of this specification, alignment of nucleotide sequences can be performed using the blastn program set to default parameters, and alignment of amino acid sequences can be performed using the blastp program set to default parameters (see the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov).

[0094] Any composition or method provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0095] 6.2 Antibodies In one aspect, provided herein are humanized antibodies and antigen-binding fragments thereof that bind to SFRP2, such as human SFRP2. In particular aspects, provided herein are humanized antibodies and antigen-binding fragments thereof that specifically bind to human SFRP2. The amino acid sequence of human SFRP2 is known in the art and is provided herein as represented in SEQ ID NO: 12. MLQGPGSLLLLFLASHCCLGSARGLFLFGQPDFSYKRSNCKPIPVNLQLCHGIEYQNMRLPNLLGHETMKEVLEQAGAWIPLVMKQCHPDTKKFLCSLFAPVCLDDLDETIQPCHSLCVQVKDRCAPVMSAFGFPWPDMLECDRFPQ DNDLCIPLASSDHLLPATEEAPKVCEACKNKNDDDNDIMETLCKNDFALKIKVKEITYINRDTKIILETKSKTIYKLNGVSERDLKKSVLWLKDSLQCTCEEMNDINAPYLVMGQKQGGELVITSVKRWQKGQREFKRISRSIRKLQC

[0096] In some embodiments, the humanized anti-SFRP2 antibodies or antigen-binding fragments thereof described herein bind to SFRP2, e.g., human SFRP2, and comprise a VH chain having at least 90% sequence identity, at least 95% sequence identity, at least 99% sequence identity, or 100% sequence identity to a VH chain selected from Table 1. [Table 1]

[0097] In some embodiments, the humanized anti-SFRP2 antibodies or antigen-binding fragments thereof described herein bind to SFRP2, e.g., human SFRP2, and comprise a VL chain having at least 90% sequence identity, at least 95% sequence identity, at least 99% sequence identity, or 100% sequence identity to a VL chain selected from Table 2. [Table 2]

[0098] In some aspects, the humanized anti-SFRP2 antibodies or antigen-binding fragments thereof described herein bind to SFRP2, e.g., human SFRP2, and comprise a VH chain of Table 1 and a VL chain of Table 2.

[0099] In some aspects, the humanized anti-SFRP2 antibodies or antigen-binding fragments thereof described herein bind to SFRP2, e.g., human SFRP2, and comprise a VH chain and a VL chain listed in Table 3. [Table 3]

[0100] In some embodiments, provided herein are humanized anti-SFRP2 antibodies comprising a heavy chain and a light chain. With respect to the heavy chain, in some embodiments, the heavy chain of a humanized anti-SFRP2 antibody described herein can be an alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments, the heavy chain of a humanized anti-SFRP2 antibody described herein can comprise a human alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments, the humanized anti-SFRP2 antibody described herein comprises a heavy chain, wherein the amino acid sequence of the VH domain comprises the amino acid sequence set forth in Table 1, and the constant region of the heavy chain comprises the amino acid sequence of a human gamma (γ) heavy chain constant region. In some embodiments, a humanized anti-SFRP2 antibody described herein, e.g., an antibody that binds human SFRP2, comprises a heavy chain in which the amino acid sequence of the VH domain comprises the amino acid sequence set forth in Table 1, and the heavy chain constant region comprises the amino acid sequence of an IgG1 heavy chain constant region. In some embodiments, a humanized anti-SFRP2 antibody described herein, e.g., an antibody that binds human SFRP2, comprises a heavy chain in which the amino acid sequence of the VH domain comprises the sequence set forth in Table 1, and the heavy chain constant region comprises the amino acids of a human heavy chain described herein or known in the art. Non-limiting examples of human constant region sequences are described in the art, see, e.g., U.S. Pat. No. 5,693,780 and Kabat EA et al., (1991), supra. In some embodiments, the heavy chain comprises a VH domain corresponding to SEQ ID NO: 2. In some embodiments, the heavy chain comprises the sequence of SEQ ID NO: 15. In some embodiments, the heavy chain is encoded by the polynucleotide sequence of SEQ ID NO: 17.

[0101] With respect to the light chain, in some embodiments, the light chain of a humanized anti-SFRP2 antibody described herein is a kappa light chain. In some embodiments, the light chain of a humanized anti-SFRP2 antibody described herein is a lambda light chain. In some embodiments, the light chain of a humanized anti-SFRP2 antibody described herein is a human kappa light chain or a human lambda light chain. In some embodiments, the light chain of a humanized anti-SFRP2 antibody described herein is a human kappa light chain. In some embodiments, the light chain comprises a VL domain corresponding to SEQ ID NO: 9. In some embodiments, the light chain comprises the sequence of SEQ ID NO: 16. In some embodiments, the light chain is encoded by the polynucleotide sequence of SEQ ID NO: 18.

[0102] In some embodiments, a humanized anti-SFRP2 antibody described herein, e.g., an antibody that binds human SFRP2, comprises a light chain in which the amino acid sequence of its VL domain comprises a sequence set forth in Table 2, and in which the constant region of the light chain comprises the amino acid sequence of a human kappa light chain constant region. In some embodiments, a humanized anti-SFRP2 antibody described herein, e.g., an antibody that binds human SFRP2, comprises a light chain in which the amino acid sequence of its VL domain comprises a sequence set forth in Table 2, and in which the constant region of the light chain comprises the amino acid sequence of a human lambda light chain constant region. In some embodiments, a humanized anti-SFRP2 antibody described herein, e.g., an antibody that binds human SFRP2, comprises a light chain in which the amino acid sequence of its VL domain comprises a sequence set forth in Table 2, and in which the constant region of the light chain comprises the amino acid sequence of a human kappa or lambda light chain constant region. Non-limiting examples of human constant region sequences are described in the art, see, e.g., U.S. Pat. No. 5,693,780 and Kabat EA et al., (1991), supra.

[0103] In some aspects, the humanized anti-SFRP2 antibodies described herein, e.g., antibodies that bind to human SFRP2, comprise a VH domain and a VL domain comprising the amino acid sequence of any of the humanized anti-SFRP2 antibodies described herein, and the constant region comprises the amino acid sequence of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, or the constant region of a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule. In some embodiments, the humanized anti-SFRP2 antibodies described herein, e.g., antibodies that bind to human SFRP2, comprise a VH domain and a VL domain comprising the amino acid sequence of any of the humanized anti-SFRP2 antibodies described herein, wherein the constant region comprises the amino acid sequence of a constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, of any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecules. In some embodiments, the constant region comprises the amino acid sequence of a constant region of a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, of any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecules.

[0104] In some aspects, the humanized anti-SFRP2 antibodies described herein, e.g., antibodies that bind to human SFRP2, comprise a heavy chain whose VH domain amino acid sequence comprises an amino acid sequence set forth in Table 1 and whose heavy chain constant region comprises the amino acid sequence of an IgG1 heavy chain constant region, and / or a light chain whose VL domain amino acid sequence comprises a sequence set forth in Table 2 and whose light chain constant region comprises the amino acid sequence of a human kappa light chain constant region.

[0105] In some aspects, a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein, e.g., an antibody that binds to human SFRP2, comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises a VH domain comprising the amino acid sequence of an antibody listed in Table 1 (e.g., SEQ ID NO: 1, 2, 3, or 4), (ii) the light chain comprises a VL domain comprising the amino acid sequence of the same antibody listed in Table 2 (e.g., SEQ ID NO: 5, 6, 7, 8, or 9), (iii) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG1 heavy chain, and (iv) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa light chain. In some aspects, a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein, e.g., an antibody that binds to human SFRP2, comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 15 and / or the light chain comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the heavy chain comprises an amino acid sequence having at least 90% sequence identity, at least 95% sequence identity, at least 99% sequence identity, or 100% sequence identity to SEQ ID NO: 15. In some embodiments, the light chain comprises an amino acid sequence having at least 90% sequence identity, at least 95% sequence identity, at least 99% sequence identity, or 100% sequence identity to SEQ ID NO: 16. In some embodiments, the heavy and / or light chains comprise the amino acid sequences set forth in Table 4. [Table 4]

[0106] In some aspects, the humanized anti-SFRP2 antibodies described herein, e.g., antibodies that bind to human SFRP2, comprise a heavy chain and a light chain, wherein the heavy chain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 2, and / or the light chain comprises a VL domain comprising the amino acid sequence of SEQ ID NO: 9.

[0107] In some embodiments, a humanized anti-SFRP2 antibody described herein, e.g., an antibody that binds to human SFRP2, comprises a complementarity determining region (CDR) H1 comprising the amino acid sequence of SEQ ID NO: 19, a CDR H2 comprising the amino acid sequence of SEQ ID NO: 20, a CDR H3 comprising the amino acid sequence of SEQ ID NO: 21, a CDR L1 comprising the amino acid sequence of SEQ ID NO: 22, a CDR L2 comprising the amino acid sequence of SEQ ID NO: 23, and a CDR L3 comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the humanized anti-SFRP2 antibody comprises CDR sequences comprising the amino acid sequences shown in Table 5. [Table 5]

[0108] Non-limiting examples of human constant regions are described in the art, see, eg, Kabat EA et al., (1991) supra.

[0109] In some embodiments, the anti-SFRP2 antibody or antigen-binding fragment thereof comprises a chimeric antibody. In some embodiments, the chimeric anti-SFRP2 antibody comprises a VH chain polypeptide of SEQ ID NO: 10 and a VL chain polypeptide of SEQ ID NO: 11 (referred to as Chi.1). In some embodiments, the chimeric anti-SFRP2 antibody comprises a VH chain polypeptide of SEQ ID NO: 10 and a VL chain polypeptide of SEQ ID NO: 13 (referred to as Chi.2). In some embodiments, the chimeric anti-SFRP2 antibody comprises a human constant region, such as any of those discussed herein. In some examples, the chimeric anti-SFRP2 antibody comprises an IgG1 constant region.

[0110] 6.3 Antibody activity In some embodiments, the humanized anti-SFRP2 antibodies described herein, e.g., antibodies that bind to human SFRP2, comprise an IC50 value of 0.7 or less, 0.6 or less, or 0.5 or less relative to the IC50 value of an antibody or antigen-binding fragment thereof comprising the VH chain polypeptide and VL chain polypeptide of SEQ ID NOs: 10 and 11, respectively. Relative IC50 values ​​can be calculated, for example, using the method described in Example 2. For example, a humanized anti-SFRP2 antibody or antigen-binding fragment thereof can be prepared as a dilution series and then premixed with a fixed concentration of biotinylated antibody comprising SEQ ID NOs: 10 and 11 before incubating at room temperature on a microtiter plate pre-coated with peptide B (SEQ ID NO: 14), the peptide comprising amino acids 202-220 of human SFRP2 (SEQ ID NO: 12). Results from competitive ELISA analysis can be used to calculate the IC50 of each antibody. 50 The IC values ​​can then be calculated as the IC values ​​of the antibodies containing SEQ ID NOs: 10 and 11 included on each ELISA plate. 50 can be normalized to a value.

[0111] In some embodiments, the humanized anti-SFRP2 antibodies described herein, e.g., antibodies that bind to human SFRP2, inhibit endothelial tube formation. Inhibition of endothelial tube formation can be measured as described in Example 2. For example, an endothelial tube formation assay using 2H11 cells can be performed, in which 2H11 mouse endothelial cells treated with SFRP2, which promotes tube formation, are further treated with a humanized anti-SFRP2 antibody or a control antibody, e.g., IgG1 treatment, and the number of branch points is measured and compared with positive and negative controls. Alternatively, an endothelial tube formation assay using SVR angiosarcoma cells can be performed, in which the SVR angiosarcoma cells are treated with a humanized anti-SFRP2 antibody or a control antibody, e.g., IgG1 treatment, and the number of branch points is counted and / or the percentage of inhibition is calculated. As shown by the results of such a tube formation assay, the humanized anti-SFRP2 antibody Ab8 was the only antibody that statistically significantly inhibited tube formation compared to the control treatment (*p=0.03). This result was surprising because, for example, the heavy chain of Ab8 differs from the heavy chains of Ab2 and Ab3 by a single amino acid, while the light chain of Ab8 differs from the light chain of Ab11 by a single amino acid. However, Ab2, Ab3, and Ab11 did not statistically significantly inhibit tube formation. Thus, the results of this endothelial tube formation assay demonstrate the unique and surprising properties of a humanized anti-SFRP2 antibody comprising the variable heavy chain amino acid sequence of SEQ ID NO:2 and the variable light chain amino acid sequence of SEQ ID NO:9, i.e., Ab8.

[0112] In some embodiments, the humanized anti-SFRP2 antibodies described herein, e.g., antibodies that bind to human SFRP2, induce apoptosis of SFRP2-expressing osteosarcoma cells. In some embodiments, such induction of apoptosis can be measured as described in Example 3. For example, RF577 cell lines, which endogenously express SFRP2, can be plated and then treated with various concentrations of humanized anti-SFRP2 antibodies or an IgG1 control antibody, and apoptosis can be measured using techniques known in the art. In some embodiments, apoptosis of osteosarcoma cells is increased by 10% or more, 20% or more, 30% or more, or 40% or more compared to a control treatment, e.g., treatment with an IgG antibody. In some embodiments, the humanized anti-SFRP2 antibody-induced induction of apoptosis of SFRP2-expressing osteosarcoma cells is selective; e.g., the humanized anti-SFRP2 antibody does not induce apoptosis in T cells. In some examples, such measurements can be performed as described in Example 3. For example, CD4+ and CD8+ cells can be isolated from a source, e.g., mouse splenocytes, according to methods known in the art, and then treated with a humanized anti-SFRP2 antibody or a control, e.g., an IgG1 antibody. After treatment, the cells can be stained and analyzed by flow cytometry to detect measured apoptosis of the cells. In some embodiments, the percentage of apoptotic cells is unchanged compared to a control sample and / or the percentage of apoptotic cells is less than the number of cells in a positive control in which apoptosis occurs.

[0113] In some embodiments, the humanized anti-SFRP2 antibodies described herein, e.g., antibodies that bind human SFRP2, inhibit osteosarcoma-associated lung metastases, either as monotherapy or when administered in combination with a PD-1 inhibitor, e.g., a PD-1 mAb. Such inhibition can be measured as generally described in Example 6. For example, osteosarcoma lung metastases can be generated in C57 / B16 mice by tail vein injection with RF577 tumor cells. Treatment can begin a predetermined length of time after tumor cell injection, e.g., 12 days, and include an IgG1 control antibody, a humanized anti-SFRP2 antibody, a PD-1 inhibitor, or a combination of a humanized anti-SFRP2 antibody and a PD-1 inhibitor. After a predetermined length of time, the lungs of the mice can be analyzed using high-resolution photographs, and the number of metastatic surface lung nodules in each treatment group can be quantified. In some embodiments, treatment with a humanized anti-SFRP2 antibody or antigen-binding fragment thereof, either when administered as monotherapy or in combination with an antagonist of an inhibitory immune checkpoint molecule, reduces the number of lung surface metastases compared to an IgG1 control, and optionally the immune checkpoint molecule is PD-1. In some embodiments, the SFRP2 antibody or antigen-binding fragment thereof, e.g., when administered as monotherapy, e.g., in combination with an antagonist of an inhibitory immune checkpoint molecule, reduces lung metastatic tumor volume by 50% or more, 60% or more, 70% or more, or 80% or more compared to a control.

[0114] In some embodiments, a humanized anti-SFRP2 antibody described herein, e.g., an antibody that binds to human SFRP2, reduces SFRP2 serum levels, either as monotherapy or when administered in combination with an antagonist of an inhibitory immune checkpoint molecule, optionally the immune checkpoint molecule being PD-1. Such reduction can be measured as generally described in Example 4. For example, blood can be collected from control mice or RF577-bearing mice treated with an IgG1 control antibody, a humanized anti-SFRP2 antibody, an antagonist of an inhibitory immune checkpoint molecule, e.g., a PD-1 inhibitor, or a combination of both antibodies, and serum isolation can be performed. Serum samples can then be processed using a RayBiotech Mouse SFRP2 ELISA kit (ELM-SFRP-2; Peachtree Corners, GA, USA) according to the manufacturer's protocol. In some embodiments, the humanized anti-SFRP2 antibodies described herein, e.g., antibodies that bind to human SFRP2, either as monotherapy or when administered in combination with an antagonist of an inhibitory immune checkpoint molecule, reduce SFRP2 serum levels compared to treatment with a control, e.g., IgG1.

[0115] In some embodiments, the humanized anti-SFRP2 antibodies described herein, e.g., antibodies that bind to human SFRP2, reduce CD38 levels. Such reductions can be measured as generally described in Example 5. For example, mice bearing RF577 OS lung metastases can be prepared as known in the art, and splenocytes can be harvested from these mice at predetermined times. After harvesting, the splenocytes can be treated with an IgG1 control antibody or a humanized anti-SFRP2 antibody. After treatment, the splenocytes can be lysed and prepared for Western blot analysis to examine CD38 levels using standard protocols. In some embodiments, the humanized anti-SFRP2 antibodies described herein reduce CD38 levels by 50% or more, 60% or more, 70% or more, or 80% or more compared to treatment with a control, e.g., IgG1.

[0116] 6.4 Antigen-binding fragments In some embodiments, antigen-binding fragments of the humanized anti-SFRP2 antibodies described herein, e.g., humanized anti-SFRP2 antibodies that bind to human SFRP2, are provided. Exemplary antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and scFv, where the Fab, Fab', F(ab')2, or scFv comprises the heavy chain variable region sequence and light chain variable region sequence of the humanized anti-SFRP2 antibody described herein. The Fab, Fab', F(ab')2, or scFv can be generated by any technique known to those of skill in the art, including but not limited to those discussed below. In some embodiments, an antigen-binding fragment, such as a Fab, Fab', F(ab')2, or scFv, further comprises a moiety that extends the half-life of the antibody in vivo. This moiety is also referred to as a "half-life extending moiety." Any moiety known to those of skill in the art to extend the half-life of an antigen-binding fragment, such as a Fab, Fab', F(ab')2, or scFv, in vivo can be used. For example, the half-life extending moiety can comprise an Fc region, a polymer, albumin, or an albumin-binding protein or compound. The polymer can comprise a natural or synthetic, optionally substituted, linear or branched polyalkylene, polyalkenylene, polyoxylalkylene, polysaccharide, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, methoxypolyethylene glycol, lactose, amylose, dextran, glycogen, or a derivative thereof. The substituent can comprise one or more hydroxyl, methyl, or methoxy groups. In some embodiments, an antigen-binding fragment such as a Fab, Fab', F(ab')2, or scFv can be modified by the addition of one or more C-terminal amino acids for attachment of the half-life extending moiety. In some embodiments, the half-life extending moiety is polyethylene glycol or human serum albumin. In some embodiments, an antigen-binding fragment such as a Fab, Fab', F(ab')2, or scFv is fused to an Fc region.

[0117] The humanized anti-SFRP2 antibody or antigen-binding fragment thereof may be fused or conjugated (e.g., covalently or non-covalently bound) to a detectable label or substance. Examples of detectable labels or substances include enzyme labels such as glucose oxidase, radioisotopes such as iodine (I, I), carbon (C), sulfur (S), tritium (H), indium (In), and technetium (Tc), luminescent labels such as luminol, and fluorescent labels such as fluorescein and rhodamine, and biotin.

[0118] 6.5 Antibody production Humanized anti-SFRP2 antibodies and antigen-binding fragments thereof can be produced by any method known in the art for the synthesis of humanized antibodies and antigen-binding fragments. The methods described herein utilize, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields that are within the skill of the art. These techniques are described, for example, in the references cited herein and are explained more fully therein. See, for example, Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annually updated); Current Protocols in Immunology, John Wiley & Sons (1987 and annually updated); Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al., (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

[0119] In certain aspects, provided herein are methods for making a humanized anti-SFRP2 antibody or antigen-binding fragment thereof, comprising culturing a cell or host cell described herein (e.g., a cell or host cell comprising a polynucleotide encoding an antibody or antigen-binding fragment thereof described herein). In certain aspects, provided herein are methods for making a humanized anti-SFRP2 antibody or antigen-binding fragment thereof, comprising expressing (e.g., recombinantly expressing) the antibody or antigen-binding fragment thereof using a cell or host cell described herein (e.g., a cell or host cell comprising a polynucleotide encoding an antibody or antigen-binding fragment thereof described herein). In some aspects, the cell is an isolated cell. In some aspects, the encoding polynucleotide has been introduced into the cell. In some aspects, the method further comprises purifying the antibody or antigen-binding fragment obtained from the cell or host cell.

[0120] Monoclonal antibody or its antigen-binding fragment can be prepared using a variety of techniques known in the art, including the use of hybridoma, recombinant, phage display technology, yeast-based display technology, or a combination thereof.For example, monoclonal antibody or its antigen-binding fragment can be produced using hybridoma technology, which is known in the art and includes, for example, the technique taught in Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed., 1988), Hammerling GJ et al., Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, NY, 1981), or the technique described in Kohler G & Milstein C (1975) Nature 256:495. Examples of yeast-based display methods that can be utilized to select and generate the antibodies described herein include those disclosed in, for example, WO2009 / 036379A2, WO2010 / 105256, and WO2012 / 009568, each of which is incorporated by reference herein in its entirety.

[0121] In some embodiments, the monoclonal antibody or antigen-binding fragment is an antibody or antigen-binding fragment produced by a clonal cell (e.g., a hybridoma or a host cell producing a recombinant antibody or antigen-binding fragment), and the antibody or antigen-binding fragment comprises a humanized anti-SFRP2 antibody or antigen-binding fragment thereof. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof can be a Fab fragment or a F(ab')2 fragment. The monoclonal antibodies or antigen-binding fragments thereof described herein can be produced by hybridoma methods, e.g., as described in Kohler G & Milstein C (1975) Nature 256:495, or can be isolated from phage libraries, e.g., using the techniques described herein. Other methods for the preparation of clonal cell lines and the monoclonal antibodies and antigen-binding fragments thereof expressed thereby are well known in the art (see, e.g., Chapter 11 in Short Protocols in Molecular Biology (2002) 5th Ed., Ausubel FM et al., supra).

[0122] Antigen-binding fragments of the antibodies described herein can be produced by any technique known to those skilled in the art. For example, the Fab and F(ab')2 fragments described herein can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). Fab fragments correspond to one of the two identical arms of a tetrameric antibody molecule and contain an intact light chain paired with the VH and CH1 domains of the heavy chain. F(ab')2 fragments contain the two antigen-binding arms of a tetrameric antibody molecule linked by disulfide bonds in the hinge region.

[0123] In some aspects, the humanized anti-SFRP2 antibodies or antigen-binding fragments thereof described herein are produced by culturing host cells, such as CHO cells, to express nucleic acid molecule(s) encoding the antibodies or antigen-binding fragments thereof, thereby producing the humanized anti-SFRP2 antibodies or antigen-binding fragments thereof, and optionally isolating the antibodies or antigen-binding fragments thereof from the culture. In some aspects, the host cells are CHO cells. In some aspects, the isolated antibodies or antigen-binding fragments thereof are substantially free of precipitates, i.e., free of precipitates when examined, for example, by eye, A280, size exclusion chromatography, and / or dynamic light scattering. In some aspects, the humanized anti-SFRP2 antibodies or antigen-binding fragments thereof described herein are produced by CHO cells transduced with a vector, for example, a vector harvested from 293 GP cells transduced with a retrovector (GPEX vector) made from a genetic construct developed to express the humanized antibodies or antigen-binding fragments described herein.

[0124] 6.6 Polynucleotides In certain aspects, provided herein are polynucleotides comprising nucleotide sequences encoding humanized anti-SFRP2 antibodies or antigen-binding fragments thereof, as well as vectors, e.g., vectors comprising such polynucleotides for recombinant expression in host cells (e.g., E. coli and mammalian cells).

[0125] In certain aspects, provided herein is a polynucleotide comprising a nucleotide sequence encoding a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein, including a polypeptide comprising an amino acid sequence described herein.

[0126] Also provided herein is a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 1-9 and 15-16.

[0127] Also provided herein is a kit, vector, or host cell comprising (i) a first polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4, and (ii) a second polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 5, 6, 7, 8, or 9. In a kit comprising such first and second polynucleotides, the first and second polynucleotides can be in the same vector or in different vectors. In a host cell comprising such first and second polynucleotides, the first and second polynucleotides can be in the same vector or in different vectors.

[0128] In some aspects, provided herein is a polynucleotide comprising a nucleotide sequence encoding a humanized anti-SFRP2 antibody or antigen-binding fragment thereof, or a fragment thereof comprising a VH domain comprising an amino acid sequence described herein (see, e.g., Table 1). In some aspects, provided herein is a polynucleotide comprising a nucleotide sequence encoding a humanized anti-SFRP2 antibody or antigen-binding fragment thereof comprising a VL domain comprising an amino acid sequence described herein (see, e.g., Table 2).

[0129] In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a heavy chain variable region (e.g., a VH comprising the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4) and a heavy chain constant region, e.g., a human gamma (γ) heavy chain constant region, e.g., a human IgG1 constant region.

[0130] In some aspects, the polynucleotide comprises a nucleic acid sequence encoding a light chain variable region (e.g., a VL comprising the amino acid sequence of SEQ ID NO: 5, 6, 7, 8, or 9) and a light chain constant region, e.g., a human lambda or kappa light chain constant region, e.g., a human kappa light chain constant region.

[0131] Also provided herein are polynucleotides encoding the humanized anti-SFRP2 antibodies, or antigen-binding fragments thereof, or domains thereof described herein, optimized, for example, by codon / RNA optimization, substitution with a heterologous signal sequence, and removal of mRNA instability elements. Methods for generating optimized nucleic acids encoding humanized anti-SFRP2 antibodies, or antigen-binding fragments thereof, or domains thereof (e.g., heavy chain, light chain, VH domain, or VL domain) for recombinant expression by introducing codon changes (e.g., codon changes that encode the same amino acid due to the degeneracy of the genetic code; see Table 6 herein below) and / or removing inhibitory regions of the mRNA can be carried out accordingly, for example, by adapting the optimization methods described in U.S. Patent Nos. 5,965,726, 6,174,666, 6,291,664, 6,414,132, and 6,794,498.

[0132] Polynucleotides encoding the antibodies, or antigen-binding fragments thereof, or domains thereof described herein can be generated from nucleic acid from a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the antibody of interest. Using such PCR amplification methods, nucleic acids containing sequences encoding the light and / or heavy chains of an antibody or antigen-binding fragment thereof can be obtained. Using such PCR amplification methods, nucleic acids containing sequences encoding the variable light chain region and / or variable heavy chain region of an antibody or antigen-binding fragment thereof can be obtained. The amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning, e.g., to generate humanized antibodies or antigen-binding fragments thereof.

[0133] The polynucleotides provided herein may be, for example, in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and the DNA may be double-stranded or single-stranded. If single-stranded, the DNA may be the coding strand or the non-coding (antisense) strand. In some embodiments, the polynucleotide is a cDNA or DNA lacking one or more endogenous introns. In some embodiments, the polynucleotide is a non-naturally occurring polynucleotide. In some embodiments, the polynucleotide is recombinantly produced. In some embodiments, the polynucleotide is isolated. In some embodiments, the polynucleotide is substantially pure. In some embodiments, the polynucleotide is purified from natural components.

[0134] 6.7 Cells and Vectors In certain aspects, provided herein are polynucleotides comprising nucleotide sequences encoding humanized anti-SFRP2 antibodies and antigen-binding fragments thereof, or vectors (e.g., expression vectors) comprising such domains for recombinant expression in a host cell, preferably a mammalian cell. Also provided herein are cells, e.g., host cells, comprising such vectors for recombinantly expressing a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein, e.g., Ab1 through Ab20, e.g., an antibody comprising a VH chain polypeptide of SEQ ID NO: 1, 2, 3, or 4, and a VL chain polypeptide of SEQ ID NO: 5, 6, 7, 8, or 9. In some aspects, provided herein are methods for producing an antibody or antigen-binding fragment thereof described herein, comprising expressing such an antibody or antigen-binding fragment thereof in a host cell.

[0135] In some aspects, recombinant expression of a humanized anti-SFRP2 antibody or antigen-binding fragment thereof or domain thereof (e.g., a heavy or light chain described herein) involves construction of an expression vector containing a polynucleotide encoding the antibody or antigen-binding fragment thereof or domain thereof. Once a polynucleotide encoding an antibody or antigen-binding fragment thereof or domain thereof (e.g., a heavy or light chain variable domain) described herein is obtained, a vector for production of the antibody or antigen-binding fragment thereof can be generated by recombinant DNA technology using techniques well known in the art. Thus, described herein are methods for preparing proteins by expressing polynucleotides containing nucleotide sequences encoding an antibody or antigen-binding fragment thereof or domain thereof (e.g., a light or heavy chain). Methods well known to those skilled in the art can be used to construct expression vectors containing an antibody or antigen-binding fragment thereof or domain thereof (e.g., a light or heavy chain) coding sequence and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors containing a nucleotide sequence encoding an antibody or antigen-binding fragment thereof, heavy or light chain, heavy or light chain variable domain, or heavy or light chain CDR described herein, operably linked to a promoter. Such vectors may contain, for example, a nucleotide sequence encoding the constant region of an antibody or antigen-binding fragment thereof (see, e.g., International Publication Nos. WO 86 / 05807 and WO 89 / 01036 and U.S. Patent No. 5,122,464), and the variable domain of an antibody or antigen-binding fragment thereof may be cloned into such a vector for expression of the entire heavy chain, the entire light chain, or both the heavy and light chains.

[0136] The expression vector can be transferred into cells (e.g., host cells) by conventional techniques, and the resulting cells can then be cultured by conventional techniques to produce an antibody or antigen-binding fragment thereof described herein, e.g., Ab1-Ab20, e.g., an antibody comprising a VH chain polypeptide of SEQ ID NO: 1, 2, 3, or 4, and a VL chain polypeptide of SEQ ID NO: 5, 6, 7, 8, or 9. Accordingly, provided herein are host cells containing a polynucleotide encoding an antibody or antigen-binding fragment thereof described herein, e.g., Ab1-Ab20, e.g., comprising a VH chain polypeptide of SEQ ID NO: 1, 2, 3, or 4, and a VL chain polypeptide of SEQ ID NO: 5, 6, 7, 8, or 9, or a domain thereof, operably linked to a promoter for expression of such sequences in the host cell. In some embodiments, for expression of a double-chain antibody or antigen-binding fragment thereof, vectors encoding both the heavy and light chains separately can be co-expressed in the host cell for expression of the entire immunoglobulin, as described in more detail below. In some embodiments, the host cell contains a vector comprising polynucleotides encoding both the heavy and light chains of an antibody described herein, e.g., Ab1 through Ab20, e.g., an antibody comprising a VH chain polypeptide of SEQ ID NO: 1, 2, 3, or 4 and a VL chain polypeptide of SEQ ID NO: 5, 6, 7, 8, or 9, or a domain thereof. In some embodiments, the host cell contains two different vectors: a first vector comprising polynucleotides encoding the heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof described herein, and a second vector comprising polynucleotides encoding the light chain or light chain variable region of an antibody or antigen-binding fragment thereof described herein. In some embodiments, a first host cell contains a first vector comprising polynucleotides encoding the heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof described herein, and a second host cell contains a second vector comprising polynucleotides encoding the light chain or light chain variable region of an antibody or antigen-binding fragment thereof described herein.In some embodiments, the heavy chain / heavy chain variable region is expressed by a first cell in association with a light chain / light chain variable region of a second cell to form a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein. In some embodiments, provided herein is a population of host cells comprising such a first host cell and such a second host cell.

[0137] In some embodiments, provided herein are populations of vectors comprising a first vector comprising a polynucleotide encoding the light chain / light chain variable region of a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein, and a second vector comprising a polynucleotide encoding the heavy chain / heavy chain variable region of a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein. Alternatively, a single vector capable of encoding and expressing both heavy and light chain polypeptides can be used.

[0138] A variety of host-expression vector systems can be utilized to express the antibodies and antigen-binding fragments thereof described herein (e.g., antibodies or antigen-binding fragments thereof comprising a VH chain polypeptide of SEQ ID NO: 1, 2, 3, or 4 and comprising a VL chain polypeptide of SEQ ID NO: 5, 6, 7, 8, or 9) (see, e.g., U.S. Pat. No. 5,807,715). Such host-expression systems represent vehicles in which a coding sequence of interest can be produced and subsequently purified, but also represent cells which, when transformed or transfected with the appropriate nucleotide coding sequence, are capable of expressing the antibodies or antigen-binding fragments thereof described herein in situ. These include bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody coding sequences, recombinant yeast (e.g., Saccharomyces Pichia) containing the antibody coding sequences, insect cell lines transformed with recombinant yeast expression vectors containing the antibody coding sequences, insect cell lines infected with recombinant viral expression vectors (e.g., baculovirus) containing the antibody coding sequences, and plant cell lines (e.g., Chlamydomonas spp.) infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the antibody coding sequences. reinhardtii), or mammalian cell lines (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 cells) harboring a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter, a vaccinia virus 7.5K promoter).In some embodiments, cells for expressing the antibodies and antigen-binding fragments thereof described herein (e.g., antibodies or antigen-binding fragments thereof comprising a VH chain polypeptide of SEQ ID NO: 1, 2, 3, or 4 and comprising a VL chain polypeptide of SEQ ID NO: 5, 6, 7, 8, or 9) are CHO cells, e.g., the GPEx® Chinese Hamster Ovary (GCHO) cell line. In some embodiments, cells for expressing the antibodies described herein are human cells, e.g., a human cell line. In some embodiments, the mammalian expression vector is pOptiVEC™ or pcDNA3.3. In some embodiments, bacterial cells such as E. coli, or eukaryotic cells (e.g., mammalian cells), particularly for the expression of whole recombinant antibody molecules, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary (CHO) cells, in conjunction with a vector such as the major intermediate-early gene promoter element from human cytomegalovirus, are an effective expression system for antibodies (Foecking MK & Hofstetter H (1986) Gene 45:101-105, and Cockett MI et al., (1990) Biotechnology 8:662-667). In some embodiments, the antibodies or antigen-binding fragments thereof described herein are produced by CHO cells or NS0 cells. In some embodiments, the antibodies or antigen-binding fragments thereof described herein are produced by CHO cells transduced with a vector.

[0139] In addition, a host cell strain can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can contribute to the function of the protein. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK293, NIH3T3, W138, BT483, Hs578T, HTB2, BT2O, and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10, and HsS78Bst cells. In some embodiments, the humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein (an antibody or antigen-binding fragment thereof comprising a VH chain polypeptide of SEQ ID NO: 1, 2, 3 or 4 and a VL chain polypeptide of SEQ ID NO: 5, 6, 7, 8 or 9) is produced in mammalian cells, such as CHO cells.

[0140] Once an antibody or antigen-binding fragment thereof described herein is produced by recombinant expression, it can be purified by any method known in the art for the purification of immunoglobulin molecules, for example, by chromatography (e.g., ion exchange, affinity, particularly for a particular antigen following protein A, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins. Additionally, the antibodies or antigen-binding fragments thereof described herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0141] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are isolated or purified. Generally, an isolated antibody or antigen-binding fragment thereof is substantially free of other antibodies or antigen-binding fragments thereof having antigen specificities different from those of the isolated antibody or antigen-binding fragment thereof. For example, in some embodiments, preparations of the antibodies or antigen-binding fragments thereof described herein are substantially free of cellular material and / or chemical precursors. Furthermore, in some embodiments, the antibodies or antigen-binding fragments thereof described herein are isolated or purified, e.g., partially purified, and the isolated or purified antibodies or antigen-binding fragments thereof are substantially free of precipitates, i.e., free of precipitates when examined, for example, by eye, A280, size exclusion chromatography, and / or dynamic light scattering.

[0142] 6.8 Pharmaceutical Compositions Provided herein are compositions comprising a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein, and in some cases, further comprising an antagonist of an inhibitory immune checkpoint molecule. In some embodiments, the humanized anti-SFRP2 antibody or antigen-binding fragment thereof having the desired purity is present in a pharmaceutical composition containing, for example, a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed. Suitable formulations for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives.

[0143] In some embodiments, the pharmaceutical composition comprises phosphate buffered saline (PBS), e.g., PBS at about pH 7.4. In some embodiments, the pharmaceutical formulation comprises sodium acetate and / or sodium chloride, e.g., about 10 mM sodium acetate and about 100 mM sodium chloride, at a pH of about 5.5. In some embodiments, the pharmaceutical formulation comprises sodium acetate and / or sodium chloride, e.g., about 20 mM sodium acetate and about 100 mM sodium chloride, at a pH of about 5.5. In some embodiments, the pharmaceutical formulation comprises citrate and / or sodium chloride, e.g., about 10 mM citrate and about 150 mM sodium chloride, at a pH of about 5. In some embodiments, the pharmaceutical formulation comprises citrate and / or sodium chloride, e.g., about 20 mM citrate and about 150 mM sodium chloride, at a pH of about 5. In some embodiments, the pharmaceutical formulation comprises histidine and / or sodium chloride, e.g., about 10 mM histidine and about 150 mM sodium chloride, at a pH of about 6. In some embodiments, the pharmaceutical formulation comprises histidine and / or sodium chloride, e.g., about 20 mM citrate and about 150 mM sodium chloride, at a pH of about 6. In some embodiments, the pharmaceutical formulation comprises phosphate and / or sodium chloride, e.g., about 10 mM phosphate and about 150 mM sodium chloride, at a pH of about 7. In some embodiments, the pharmaceutical formulation comprises phosphate and / or sodium chloride, e.g., about 20 mM phosphate and about 150 mM sodium chloride, at a pH of about 7. In some embodiments, the pharmaceutical formulation comprises phosphate and / or sodium chloride, e.g., about 10 mM phosphate and about 150 mM sodium chloride, at a pH of about 8. In some embodiments, the pharmaceutical formulation comprises phosphate and / or sodium chloride, e.g., about 20 mM phosphate and about 150 mM sodium chloride, at a pH of about 8. In some embodiments, the pharmaceutical formulation comprises tris and / or sodium chloride, e.g., about 10 mM tris and about 150 mM sodium chloride, at a pH of about 9. In some embodiments, the pharmaceutical formulation comprises tris and / or sodium chloride, e.g., about 20 mM phosphate and about 150 mM sodium chloride, at a pH of about 9.In some embodiments, the pharmaceutical composition comprises one or more excipients. In some embodiments, the one or more excipients include sodium chloride, a polysorbate, e.g., polysorbate 80, sucrose, and / or arginine. In some embodiments, the pharmaceutical composition comprises a buffer at a desired pH and further comprises sodium chloride, e.g., about 100 mM sodium chloride, e.g., about 120 mM sodium chloride. In some embodiments, the pharmaceutical composition comprises a buffer at a desired pH and further comprises sodium chloride and a polysorbate, e.g., about 120 mM sodium chloride and 0.01% polysorbate 80, e.g., 150 mM NaCl and 0.05% polysorbate 20, e.g., 75 mM NaCl and 0.05% polysorbate 20. In some embodiments, the pharmaceutical composition comprises a buffer at a desired pH and further comprises sucrose, e.g., about 6% sucrose. In some embodiments, the pharmaceutical composition comprises a buffer at a desired pH and further comprises sucrose and a polysorbate, e.g., about 6% sucrose and about 0.01% polysorbate 80, e.g., about 10% sucrose and about 0.05% polysorbate 20. In some embodiments, the pharmaceutical composition comprises a buffer at a desired pH and further comprises sucrose, arginine, and a polysorbate, e.g., about 10% sucrose, about 50 mM arginine, and about 0.05% polysorbate 20. In some embodiments, the pharmaceutical composition comprises a buffer at a desired pH and further comprises arginine and a polysorbate, e.g., about 50 mM arginine and about 0.05% polysorbate 20. In some embodiments, the pharmaceutical composition comprises a buffer at a desired pH and further comprises sodium chloride, sucrose, arginine, and a polysorbate, e.g., about 75 mM NaCl, about 5% sucrose, about 50 mM arginine, and about 0.05% polysorbate 20.

[0144] In some embodiments, the pharmaceutical composition comprises a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein and a pharmaceutically acceptable carrier (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd ed., Pharmaceutical Press (2000)). In some embodiments, the pharmaceutical compositions described herein are for use as medicaments. Compositions to be used for in vivo administration can be sterilized. This is readily accomplished, for example, by filtration through sterile filtration membranes.

[0145] The pharmaceutical compositions described herein can be used to exert biological effect(s) in vivo or in vitro. For example, the pharmaceutical compositions described herein can be used to selectively induce apoptosis of SFRP2-expressing osteosarcoma cells. For example, a humanized anti-SFRP2 antibody does not induce apoptosis in T cells, but does induce apoptosis of osteosarcoma cells, reduces the amount of intracellular SFRP2, reduces serum levels of SFRP2 in a treated subject, reduces the growth of metastatic osteosarcoma in a subject, reduces the amount of osteosarcoma lung metastases in a patient, reduces the amount of CD38 protein in a patient, and / or treats diseases or conditions in which SFRP2 is overexpressed and / or in which SFRP2 expression or overexpression is associated with disease states such as various cancers, e.g., osteosarcoma.

[0146] In some aspects, the pharmaceutical compositions provided herein are used to treat diseases or conditions such as cancer. Examples of cancers that can be treated as provided herein include, but are not limited to, breast cancer, angiosarcoma, osteosarcoma, rhabdomyosarcoma, alveolar soft part sarcoma, malignant glioma, multiple myeloma, renal cell carcinoma, prostate cancer, lung cancer, and melanoma. In some aspects, the cancer can be an early-stage cancer or a late-stage cancer. In some aspects, the cancer is a primary tumor.

[0147] 6.9 Usage and Methods In various aspects, provided herein are in vitro and in vivo methods using the humanized anti-SFRP2 antibodies or antigen-binding fragments thereof described herein, or the pharmaceutical compositions described herein. Such methods include inducing apoptosis of SFRP2-expressing osteosarcoma cells (e.g., a humanized anti-SFRP2 antibody does not induce apoptosis in T cells but does induce apoptosis in osteosarcoma cells), reducing the amount of intracellular SFRP2, reducing serum levels of SFRP2 in a treated subject, reducing the growth of metastatic osteosarcoma in a subject, reducing the amount of osteosarcoma lung metastases in a patient, reducing the amount of CD38 protein in a patient, and / or treating diseases or conditions in which SFRP2 is overexpressed and / or in which SFRP2 expression or overexpression is associated with a variety of different disease states, such as osteosarcoma.

[0148] In some embodiments, methods for treating cancer are provided herein. In some embodiments, treating cancer includes reducing the amount of SFRP2 in cells by contacting the cells with a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein, or a pharmaceutical composition thereof; reducing serum SFRP2 levels in a patient by administering to the patient a therapeutically effective amount of a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein, or a pharmaceutical composition thereof; selectively inducing apoptosis in osteosarcoma cells in the patient, for example, the humanized anti-SFRP2 antibody does not induce apoptosis in T cells, but inducing apoptosis in osteosarcoma cells by administering a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein, or a pharmaceutical composition thereof; reducing the amount of osteosarcoma lung metastases in the patient by therapeutically administering to the patient a therapeutically effective amount of a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein or a pharmaceutical composition thereof; reducing the amount of CD38 protein in the patient by administering to the patient a therapeutically effective amount of a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein or a pharmaceutical composition thereof; or treating a patient diagnosed with osteosarcoma by administering to the patient a therapeutically effective amount of a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein or a pharmaceutical composition thereof. In some embodiments, the treatment is a monotherapy treatment comprising treatment with a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein or a pharmaceutical composition thereof. In some embodiments, the treatment comprises a combination therapy comprising treatment with a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein and an antagonist of an inhibitory immune checkpoint molecule, optionally PD-1, or a pharmaceutical composition thereof. In some embodiments, the cancer treated by the methods of the present invention is a cancer with increased SFRP2 (eg, a cancer with increased SFRP2 mRNA and / or increased SFRP2 protein).In some embodiments, the cancer is osteosarcoma.

[0149] Additionally, methods of treating cancer are provided herein, in some aspects, where the cancers treated by the methods described herein include breast cancer, malignant glioma, multiple myeloma, renal cell carcinoma, kidney cancer, prostate cancer, lung cancer, melanoma, non-small cell lung cancer, pancreatic cancer, colorectal cancer, bladder cancer, hepatocellular carcinoma, gastrointestinal cancer, and sarcomas, including, but not limited to, angiosarcoma, osteosarcoma, rhabdomyosarcoma, and alveolar soft part sarcoma. In some aspects, the cancer can be an early-stage cancer or a late-stage cancer. In some aspects, the cancer is a primary tumor. In some aspects, the cancer is one in which SFRP2 expression levels compared to normal, e.g., SFRP2 overexpression, are indicative of a disease state and / or cancerous state. In some embodiments, treating cancer includes reducing the amount of SFRP2 in a cell by contacting the cell with a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein or a pharmaceutical composition thereof; reducing serum SFRP2 levels in the patient by administering to the patient a therapeutically effective amount of a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein or a pharmaceutical composition thereof; selectively inducing apoptosis in cancer cells in the patient, for example, by administering a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein or a pharmaceutical composition thereof, the humanized anti-SFRP2 antibody does not induce apoptosis in T cells but does induce apoptosis in cancerous cells; reducing the amount of metastases, for example lung metastases, in a patient by administering to the patient a therapeutically effective amount of a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein or a pharmaceutical composition thereof; reducing the amount of CD38 protein in a patient by administering to the patient a therapeutically effective amount of a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein or a pharmaceutical composition thereof; treating a patient diagnosed with cancer by administering to the patient a therapeutically effective amount of a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein or a pharmaceutical composition thereof;These methods include reducing the amount of proliferating cancer cells in a patient by administering a therapeutically effective amount of a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein, or a pharmaceutical composition thereof; increasing the amount of necrotic cancer cells in a patient by administering a therapeutically effective amount of a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein, or a pharmaceutical composition thereof; reducing the volume of a tumor(s) in a patient by administering a therapeutically effective amount of a humanized anti-SFRP2 antibody or antigen-binding fragment thereof, or a pharmaceutical composition described herein; or selectively reducing PD-1 levels in T cells of a patient by administering a therapeutically effective amount of a humanized anti-SFRP2 antibody or antigen-binding fragment thereof, or a pharmaceutical composition described herein to a patient in need thereof. In some embodiments, the treatment is a monotherapy treatment comprising treatment with a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein, or a pharmaceutical composition thereof. In some embodiments, the treatment comprises a combination therapy comprising treatment with a humanized anti-SFRP2 antibody or antigen-binding fragment thereof described herein and, optionally, an antagonist of an inhibitory immune checkpoint molecule, which is PD-1, or a pharmaceutical composition thereof. In some embodiments, the cancer treated by the methods of the invention is a cancer with increased SFRP2 (e.g., a cancer with increased SFRP2 mRNA and / or increased SFRP2 protein).

[0150] In some embodiments, methods of treating cancer, e.g., breast cancer, angiosarcoma, osteosarcoma, rhabdomyosarcoma, alveolar soft part sarcoma, malignant glioma, multiple myeloma, renal cell carcinoma, prostate cancer, lung cancer, or melanoma, are provided, the methods comprising administering a humanized anti-SFRP2 antibody or antigen-binding fragment thereof, or a pharmaceutical composition thereof, the method further comprising administering an antagonist of an inhibitory immune checkpoint molecule. In some embodiments, the inhibitory checkpoint molecule is PD-1 (programmed cell death protein-1). In some embodiments, the PD-1 antagonist is an antibody against PD-1. PD-1 antibodies include, for example, OPDIVO (nivolumab), KEYTRUDA (pembrolizumab), MEDI-0680 (AMP-514; WO2012 / 145493), camrelizumab (SHR-1210), tislelizumab (BGB-A317), or spartalizumab (NPVPDR001, NVS240118, PDR001). A recombinant protein composed of the extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1, called AMP-224, can also be used to antagonize the PD-1 receptor. In some embodiments, a humanized anti-SFRP2 antibody or antigen-binding fragment thereof, or a pharmaceutical composition thereof, is administered in combination with radiation therapy and / or a chemotherapeutic agent.

[0151] 6.10 Administration and Dosing The humanized SFRP2 antibody or antigen-binding fragment thereof provided herein, or pharmaceutical composition thereof provided herein, can be administered by any suitable means, including parenteral, intrapulmonary, intranasal, intratumoral, intralesional, intracerebrospinal, intracranial, intraspinal, intrasynovial, intrathecal, oral, topical, or inhalation routes. Parenteral infusions include intramuscular, intravenous, intraarterial, intraarticular, intraperitoneal, or subcutaneous administration, either as a bolus or by continuous infusion over a period of time. In some embodiments, administration is intravenous. In some embodiments, administration is subcutaneous.

[0152] The appropriate dosage and administration regimen of the humanized anti-SFRP2 antibody or antigen-binding fragment thereof provided herein, or the pharmaceutical composition provided herein, when used alone or in combination with one or more other additional therapeutic agents, e.g., one or more antagonists of inhibitory immune checkpoint molecules, will depend on the disease being treated, the severity and course of the disease, the route of administration, and other factors.

[0153] In some embodiments, the humanized anti-SFRP2 antibody or antigen-binding fragment thereof is administered to a patient in need thereof, for example, in an amount of 0.1 mg / kg body weight to 200 mg / kg body weight, e.g., 0.1 mg / kg body weight to about 100 mg / kg body weight, e.g., about 1 to 30 mg / kg body weight, e.g., about 5 to 15 mg / kg body weight. In some embodiments, the humanized anti-SFRP2 antibody or antigen-binding fragment thereof is administered at 0.1 mg / kg body weight or less, 0.25 mg / kg body weight or less, 0.50 mg / kg body weight or less, 0.75 mg / kg body weight or less, 1 mg / kg body weight or less, 2 mg / kg body weight or less, 3 mg / kg body weight or less, 4 mg / kg body weight or less, 5 mg / kg body weight or less, 6 mg / kg body weight or less, 7 mg / kg body weight or less, 8 mg / kg body weight or less, 9 mg / kg body weight or less, 10 mg / kg body weight or less, 15 mg / kg body weight or less, 20 mg / kg body weight or less, 25 mg / kg body weight or less, 30 mg / kg body weight or less, 35 mg / kg body weight or less, 40 mg / kg body weight or less, 45 mg / kg body weight or less, 50 mg / kg body weight or less, or 60 mg / kg body weight or less, 70 mg / kg body weight or less, 80 mg / kg body weight or less, 90 mg / kg body weight or less, 100 mg / kg body weight or less, or 200 mg / kg body weight or less. In some embodiments, the humanized anti-SFRP2 antibody or antigen-binding fragment thereof is administered at a concentration of about 0.1 mg / kg body weight to about 100 mg / kg body weight, about 0.5 mg / kg body weight to about 50 mg / kg body weight, about 1.0 mg / kg body weight to about 40 mg / kg body weight, about 1.0 to about 30.0 mg / kg body weight, about 1.0 mg / kg body weight to about 25 mg / kg body weight, about 1.0 mg / kg body weight to about 20 mg / kg body weight, about 1.0 mg / kg body weight to about 15 mg / kg body weight, about 2.0 to about 30 mg / kg body weight, about 2.0 to about 25 mg / kg body weight, about 2.0 to about 25 mg / kg body weight, about 2.0 to about 30 ... It is administered at about 20 mg / kg body weight, about 2.0 to about 15 mg / kg body weight, about 3.0 to about 30 mg / kg body weight, about 3.0 to about 25 mg / kg body weight, about 3.0 to about 20 mg / kg body weight, about 3.0 to about 15 mg / kg body weight, about 4.0 to about 30 mg / kg body weight, about 4.0 to about 25 mg / kg body weight, about 4.0 to about 20 mg / kg body weight, about 4.0 to about 15 mg / kg body weight, about 5.0 to about 30 mg / kg body weight, about 5.0 to about 25 mg / kg body weight, about 5.0 to about 20 mg / kg body weight, or about 5.0 to about 15 mg / kg body weight.

[0154] In some embodiments, the humanized anti-SFRP2 antibody or antigen-binding fragment thereof is administered, e.g., to a patient in need thereof, as part of a combination therapy in the amounts described herein. In some embodiments, the combination therapy includes one or more antagonists of inhibitory immune checkpoint molecules, e.g., one or more PD-1 inhibitors. In some embodiments, the combination therapy includes one or more PD-1 inhibitors, which are administered in an amount of 0.1 mg / kg body weight to 200 mg / kg body weight, e.g., 0.1 mg / kg body weight to about 100 mg / kg body weight. In some embodiments, the one or more PD-1 inhibitors are administered in an amount of 0.1 mg / kg body weight or less, 0.25 mg / kg body weight or less, 0.50 mg / kg body weight or less, 0.75 mg / kg body weight or less, 1 mg / kg body weight or less, 2 mg / kg body weight or less, 3 mg / kg body weight or less, 4 mg / kg body weight or less, 5 mg / kg body weight or less, 6 mg / kg body weight or less, 7 mg / kg body weight or less, 8 mg / kg body weight or less, 9 mg / kg body weight or less, 10 mg / kg body weight or less, 15 mg / kg body weight or less, 20 mg / kg body weight or less, 25 mg / kg body weight or less, 30 mg / kg body weight or less, 35 mg / kg body weight or less, 40 mg / kg body weight or less, 45 mg / kg body weight or less, 50 mg / kg body weight or less, 60 mg / kg body weight or less, 70 mg / kg body weight or less, 80 mg / kg body weight or less, 90 mg / kg body weight or less, 100 mg / kg body weight or less, or 200 mg / kg body weight or less. In some embodiments, the one or more PD-1 inhibitors are administered in an amount of about 0.1 mg / kg body weight to about 20 mg / kg body weight, about 0.5 mg / kg body weight to about 15 mg / kg body weight, about 1.0 mg / kg body weight to about 10 mg / kg body weight, about 2.0 mg / kg body weight to about 10.0 mg / kg body weight, about 3.0 mg / kg body weight to about 10.0 mg / kg body weight, about 4.0 mg / kg body weight to about 10.0 mg / kg body weight, about 5.0 mg / kg body weight to about 10.0 mg / kg body weight, about 6.0 mg / kg body weight to about 10.0 mg / kg body weight, about 7.0 mg / kg body weight to about 10.0 mg / kg body weight, about 7.5 mg / kg body weight to about 10 mg / kg body weight, or about 8.0 mg / kg body weight to about 10 mg / kg body weight.

[0155] In some embodiments, the PD-1 antagonist is administered daily, once every three days, once every week, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every eight weeks, or once every 12 weeks.

[0156] In some embodiments, the humanized anti-SFRP2 antibody or antigen-binding fragment thereof and the one or more PD-1 inhibitors are administered for at least 3 days, at least 30 days, at least 42 days, at least 8 weeks, at least 12 weeks, at least 24 weeks, at least 6 months, or at least 12 months, or until the cancer recurs.

[0157] In some embodiments, the humanized anti-SFRP2 antibody or antigen-binding fragment thereof is administered two or more times with an interval between the two administrations, hi some embodiments, the interval is at least about 1 hour, at least about 12 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 1 month, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 2 months, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 3 months, at least about 6 months, or at least about 12 months. In some embodiments, the interval is about 1 hour, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 2 months, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 3 months, about 6 months, or about 12 months. In some embodiments, the interval is the same across doses. In some embodiments, the interval is different across doses.

[0158] In some embodiments, the humanized anti-SFRP2 antibody or antigen-binding fragment thereof is administered as a combination therapy with one or more antagonists of inhibitory immune checkpoint molecules, e.g., one or more PD-1 inhibitors. In some embodiments, the humanized anti-SFRP2 antibody or antigen-binding fragment thereof and the one or more antagonists of inhibitory immune checkpoint molecules are administered simultaneously, separately, or sequentially. In some embodiments, the humanized anti-SFRP2 antibody or antigen-binding fragment thereof is administered first, and the one or more antagonists of inhibitory immune checkpoint molecules are administered after a time interval. In some embodiments, the one or more antagonists of inhibitory immune checkpoint molecules are administered first, and the humanized anti-SFRP2 antibody or antigen-binding fragment thereof is administered after a time interval. In some embodiments, the interval is at least about 1 hour, at least about 12 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 1 month, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 2 months, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 3 months, at least about 6 months, or at least about 12 months. In some embodiments, the interval is about 1 hour, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 2 months, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 3 months, about 6 months, or about 12 months. In some embodiments, the interval is the same across doses. In some embodiments, the interval is different across doses.

[0159] In some embodiments, the humanized anti-SFRP2 antibody or antigen-binding fragment thereof is administered, e.g., to a patient in need thereof, as part of a combination therapy that optionally includes administration of one or more antagonists of inhibitory immune checkpoint molecules, which combination therapy further includes radiation therapy, chemotherapy, cytokine therapy, and / or gene therapy.

[0160] In some aspects, provided herein is a humanized anti-SFRP2 antibody or antigen-binding fragment thereof for use as a medicament, or a pharmaceutical composition provided herein.

[0161] In some embodiments, provided herein is a humanized anti-SFRP2 antibody, or antigen-binding fragment thereof, or pharmaceutical composition provided herein for use in a method for treating cancer, e.g., osteosarcoma. In some embodiments, provided herein is a humanized anti-SFRP2 antibody, or antigen-binding fragment thereof, or pharmaceutical composition provided herein for use in a method for treating cancer in a subject, comprising administering to the subject an effective amount of a humanized anti-SFRP2 antibody, or antigen-binding fragment thereof, or pharmaceutical composition provided herein. [Example]

[0162] 8. Working Example Example 1: Preparation of humanized anti-SFRP2 antibodies In this example, humanized anti-SFRP2 antibodies were prepared as follows. First, sequencing of the variable heavy and light chain regions was performed on the murine anti-SFRP2 antibody variable region genes obtained from the anti-SFRP2 hybridoma of the Mo.1 antibody (VH chain of SEQ ID NO: 10, VL chain of SEQ ID NO: 11). After sequencing, the sequence information was used to design a series of humanized antibody variants. Composite V region genes were generated using synthetic oligonucleotides encoding combinations of selected human sequence segments. The VH and VL chain constructs were then cloned into vectors containing either an IgG1 heavy chain or a kappa light chain. The amino acid sequences and sequence identifiers of the variable heavy and light chain regions cloned into the vectors are presented in Tables 1 and 2 below. TIFF0007747350000006.tif94164 TIFF0007747350000007.tif116164

[0163] Each possible combination of VH and VL chains, i.e., a total of 20 pairings, was stably transfected into NS0 cells via electroporation for expression testing. These combinations are presented in Table 3 below. TIFF0007747350000008.tif150164

[0164] Although successful transfection and stable clone selection were achieved for the majority of VH and VL chain combinations evaluated, some chain combinations, such as some containing VK1, did not yield clones (e.g., VH1 / VK1, VH2 / VK1, and VH4 / VK1).

[0165] Various different antibodies expressed from NS0 cells were purified from cell culture supernatants on a Protein A Sepharose column (GE Healthcare), buffer exchanged into PBS pH 7.4, and analyzed by reducing SDS-PAGE (see Figure 1A). Bands corresponding to the predicted sizes of the VH and VL chain polypeptides were observed in each case (see Figure 1A). Lanes on the SDS-PAGE gel were loaded as follows: 1 = MW marker, 2 = Ab1, 3 = Ab2, 4 = Ab3, 5 = Ab4, 6 = Ab5, 7 = Ab6, 8 = Ab7, 9 = Ab8, 10 = Ab9, 11 = MW marker, 12 = MW marker, 13 = Ab10, 14 = Ab11, 15 = Ab12, 16 = MW marker, 17 = Ab13, and lane 18 = Ab14 (see Figure 1A).

[0166] In addition to the humanized antibodies Ab1-Ab20 discussed above, two chimeric antibodies, Chi.1 and Chi.2, were prepared as follows. The Mo.1 VH and VL chains (SEQ ID NOs: 10 and 11, respectively) were cloned into IgG1 VH and VK chain expression vectors. The Chi.1 antibody was first generated and sequenced. During sequencing, it was noted that Chi.1 contains a lysine at Kabat position 106a, while Mo.1 contains a glutamine at Kabat position 106a. Therefore, a second chimeric antibody, Chi.2, was prepared, which contains a glutamine at Kabat position 106a. The single residue difference at Kabat position 106a was not found to affect the binding of the chimeric antibodies to SFRP2 (data not shown). The VH and VL chains of Chi.1 contained the amino acid sequences of SEQ ID NOs: 10 and 11, respectively. The VH and VL chains of Chi.2 contained the amino acid sequences of SEQ ID NO: 10 and SEQ ID NO: 13, respectively.

[0167] Chi.1 and Chi.2, independently expressed in HEK and NS0 cells, were purified from cell culture supernatants on a Protein A Sepharose column (GE Healthcare), buffer exchanged into PBS pH 7.4, and analyzed by reducing SDS-PAGE (see Figure 1B). Bands corresponding to the predicted sizes of the VH and VL chain polypeptides were observed in each case (see Figure 1B). Lanes on the SDS-PAGE gel were loaded as follows: 1 = MW marker; 2 = Chi.1 purified from HEK cells; 3 = Chi.2 purified from HEK cells; 4 = MW marker; 5 = Chi.1 purified from NS0 cells; 6 = Chi.2 purified from NS0 cells. No significant differences in expression or purity of Chi.1 were observed compared to Chi.2.

[0168] Example 2: Comparative analysis of anti-SFRP2 antibodies In this example, antibodies Ab1-Ab14 prepared as in Example 1 were analyzed for comparison. Ab1-Ab14 were also compared to the parent murine antibody, Mo.1, which binds to SFRP2 (SEQ ID NO: 12). Murine antibody Mo.1 contained a VH chain polypeptide sequence of SEQ ID NO: 10 and a VL chain polypeptide sequence of SEQ ID NO: 11.

[0169] To compare the performance of each of the different antibody constructs with each other and with Mo.1, Ab1-Ab14 and Mo.1 were evaluated in a competitive binding assay (ELISA). Briefly, a 3-fold dilution series of Ab1-Ab14, ranging from 10 μg / ml to 0.0046 μg / ml, was premixed with a fixed concentration of biotinylated Mo.1 antibody (0.16 μg / ml, final concentration) and then incubated for 1 hour at room temperature on a NUNC IMMUNO MAXISORP™ 96-well flat-bottom microtiter plate pre-coated with a 1 / 5000 dilution of peptide B (SEQ ID NO: 14) in carbonate buffer. Binding of the biotinylated antibody was detected using streptavidin-HRP and TMB substrate.

[0170] Using the results obtained from the competitive ELISA analysis, the IC of each antibody was calculated. 50 The IC value was calculated and used as the IC value of the mouse Mo.1 antibody contained in each ELISA plate. 50 The relative IC values ​​obtained were normalized to 50 The values ​​are shown in Figure 2. The obtained IC 50 As can be observed from the values, all antibodies tested, ie, Ab1-Ab14, showed improved binding to SFRP2 / peptide B when compared to the murine antibody Mo.1.

[0171] To further evaluate antibody performance, each of antibodies Ab1-Ab14 was evaluated in an endothelial tube formation assay. Briefly, 2H11 mouse endothelial cells (#CRL-2163, ATCC®, Manassas, VA, USA) were cultured in Opti-MEM (#22600134, Thermo Fisher Scientific, Waltham, MA, USA) containing 5% heat-inactivated fetal bovine serum (FBS, #FB-12, Omega Scientific, Biel / Bienne, Switzerland) and 1% penicillin / streptomycin (v / v). Cells were cultured at 37°C in a humidified 5% CO2-95% room air atmosphere. Cultured 2H11 endothelial cells were then placed in Opti-MEM with 5% FBS and allowed to settle for 24 hours. Quiescence was induced by maintaining cells in Opti-MEM overnight with 2.5% FBS. Matrigel™ (#ECM625, Millipore, Bedford, MA, USA) was polymerized in wells of a 96-well plate according to the In Vitro Angiogenesis Assay protocol (#ECM625 Millipore). In this assay, an IgG1 control (5 μM) was compared to samples independently treated with one of Ab1-Ab14 at increasing concentrations of 0.5, 1, 5, 10, or 20 μM for each antibody. Each sample, except for the negative control treated with IgG1 alone, was treated with 30 nM SFRP2, which promotes tube formation. The positive control was treated with both IgG1 and SFRP2. Treatments resuspended in Opti-MEM with 2.5% FBS were preincubated for 90 minutes on a rocker at 37°C and 5% CO2 before adding to the cells. 1.9 x 10 4The cells were resuspended in 150 ml of pre-incubation medium and then incubated for an additional 30 minutes on a rocker at 37°C and 5% CO2. Finally, the cell suspension was added to each well pre-coated with polymerized Matrigel™. Control cells received fresh Opti-MEM with 2.5% FBS and 5 mM IgG1. For each treatment condition, after 4 hours of incubation at 37°C and 5% CO2, images were acquired using a 4x objective on an EVOS FL digital imaging system (Thermo Fisher Scientific, Waltham, MA, USA).

[0172] Referring now to Figure 3, Figure 3 shows representative results of a control assay (see 1 in Figure 3) and treatment with Ab8 (see 2 in Figure 3) or Ab11 (see 3 in Figure 3). As can be seen in the images in Figure 3, both Ab8 and Ab11 significantly reduced the number of endothelial branch points compared to the control.

[0173] Antibody performance was further evaluated in a tube formation assay using SVR angiosarcoma cells. Briefly, SVR angiosarcoma cells were plated in Matrigel at 12,000 cells / well and treated with either control, Ab2, Ab3, Ab8, Ab11, or Ab12 at 100 ng / μl (n=4 per group). After 4 hours, a photograph of the tubes in the wells was taken under a microscope, and branch points were counted using Image J. This was done by clicking each branch point on the image with the mouse. With each click, a color number corresponding to the type counted was displayed on the image, and the corresponding counter was updated to count all branch points on the image.

[0174] Referring now to Figure 9A, Figure 9A presents representative results of assays for control treatment (see 1 in Figure 9A), as well as treatment with Ab11 (see 2 in Figure 9A) or Ab8 (see 3 in Figure 9A). As can be observed in the images in Figure 9A, Ab8 significantly reduced the number of endothelial branch points compared to both Ab11 and the control.

[0175] Referring now to Figure 9B, data obtained from the above tube formation assay are presented as percent inhibition (treated / control) x 100. A two-tailed T-test was performed and significance was found at P<0.05. The results of the formation assay were quantified as percent inhibition (treated / control) x 100. As can be observed in Figure 9B, treatment with Ab8 was the only treatment that significantly inhibited tube formation compared to the control (*p=0.03).

[0176] Example 3: Humanized anti-SFRP2 antibodies selectively induce apoptosis in osteosarcoma cells but not in T cells In this example, we measured the effect of humanized anti-SFRP2 antibody, Ab8, on the treatment of RF577 osteosarcoma cells. Furthermore, we measured the effect of humanized anti-SFRP2 antibody, Ab8, on T cell apoptosis.

[0177] For the RF577 cell line assay, the RF577 cell line, which endogenously expresses SFRP2, was plated in 96-well plates (#0030730119; Eppendorf, Hamburg, Germany) at 1.0 × 10 4Cells were plated at 1000 cells / well. The next day, cells were treated with 10 μM Ab8 or 10 μM IgG1 control for 1 hour at 37°C, 5% CO2. Apoptosis was measured according to the protocol of the Apoptosis Detection Kit (#PK-CA707-30017; PromoCell, GmbH, Heidelberg, Germany). Apoptotic cells were FITC-positive, and necrotic cells were Texas Red-positive. Images were acquired using a 10x objective lens on an EVOS FLc digital imaging system (Thermo Fisher Scientific). Cells were counted using ImageJ cell counting software. Each data point represents the result of three independent experiments, each containing four separate wells (n = 12).

[0178] Referring now to Figure 4A, there was a significant increase in the percentage of apoptotic cells after treatment with Ab8. In RF577 cells, Ab8 increased apoptosis from 11.8±0.3% in IgG-treated cells to 52.4±0.08% (n=12, **p<0.0001) (see Figure 4A).

[0179] For the T cell apoptosis assay, splenocytes isolated from C57BL6 mice were stimulated with TCR and 6000 U / mL IL-2 for 48 hours. After stimulation, splenocytes were removed from the wells, washed twice with PBS, and CD4+ / CD8+ T cells were selected by negative subtraction using a mixture of the following biotinylated antibodies diluted 1:200: TER119 (#116204), CD25 (#102004), GR-1 (#108404), NK1.1 (#108704), CD11C (#117304), CD11B (#101204), and CD19 (#101504), all from BioLegend (San Diego, CA, USA), followed by incubation on ice for 15 minutes. The cells were then incubated for 20 min at RT on a magnetic tube holder containing 200 μL of streptavidin-coupled bead solution (#557812, BD Biosciences, Franklin Lakes, NJ, USA). CD4+ and CD8+ cells were isolated from the supernatant, and other cells bound to the beads were discarded.

[0180] To measure the effect of Ab8 on T cell apoptosis, T cells (CD4 + and CD8 + ) were isolated as described above, treated with IgG1 (10 μM) or Ab8 (10 μM) for 24 hours, stained for Hoechst and Annexin V, and then analyzed by flow cytometry. Positive controls for apoptosis were obtained from T cells that had been subjected to various freeze / thaw cycles in the DMSO-containing medium used for the experiment. The percentage of apoptotic cells in the IgG1 control-treated samples was significantly lower than that in the positive control group (n = 3, *p < 0.001) (see Figure 4B). Compared to the IgG1-treated samples, the percentage of apoptotic cells remained unchanged in the Ab8-treated samples (n = 3, p = NS) (see Figure 4B).

[0181] Example 4: Monotherapy and combination therapy including humanized anti-SFRP2 antibodies reduce SFRP2 serum levels In this example, the effects of monotherapy and combination therapy, including the use of the humanized anti-SFRP2 antibody Ab8, on reducing SFRP2 serum levels were evaluated.

[0182] Blood was collected from C57BL6 control mice (n = 3) or RF577-bearing mice treated with IgG1 (n = 9), Ab8 (n = 12), PD-1 mAb (n = 8) (anti-mouse PD-1 / CD279 monoclonal antibody purchased from Bioxcell, Lebanon, NH, USA (#BE0273)), or a combination of both antibodies (n = 12) from the inferior vena cava immediately after euthanasia and laparotomy. Serum isolation was performed using BD Vacutainer EDTA SST tubes (#367981; Becton Dickinson and Company, Franklin Lakes, NJ, USA) according to the manufacturer's protocol. Serum samples were then processed using the RayBiotech Mouse SFRP2 ELISA kit (ELM-SFRP-2; Peachtree Corners, GA, USA) according to the manufacturer's protocol. Finally, absorbance was read at 450 nm using a Synergy2 plate reader with Gen5 2.06 software (BioTek Instruments, Winooski, VT, USA).

[0183] Referring now to Figure 5, ELISA was used to compare serum levels of SFRP2 in all treatment groups of C57 / BL6 mice bearing metastatic RF577 OS and tumor-free C57 / BL6 mice. SFRP2 protein levels in the serum of control tumor-bearing mice (n = 9) were increased compared to non-tumor-bearing mice (n = 8) (32.6 ± 2.64 ng / ml vs. 9.30 ± 2.52 ng / mL, respectively, p < 0.01, Figure 5). In addition, SFRP2 levels were compared among treatment groups: control (n = 9), PD-1 mAb (n = 8), Ab8 (n = 12), and combination therapy (n = 12). All treatment groups had significantly lower SFRP2 levels compared to IgG control-treated mice (32.6 ± 2.64 ng / ml for IgG control; 11.7 ± 3.12 ng / ml for PD-1 mAb; 9.14 ± 2.02 ng / ml for Ab8; 10.5 ± 2.30 ng / ml for combination therapy; p < 0.01; Figure 5).

[0184] Example 5: Treatment of metastatic osteosarcoma with a humanized anti-SFRP2 antibody reduces CD38 levels In this example, the effect of treating metastatic osteosarcoma with the humanized anti-SFRP2 antibody, Ab8, on CD38 levels was evaluated.

[0185] Mice bearing RF577 OS lung metastases were prepared as generally described in Example 4 above. Splenocytes were harvested from these mice on day 49 and treated with IgG1 control (n=5) or Ab8 (n=5). After treatment, splenocytes were lysed and prepared for Western blot analysis probing for CD38 using standard protocols. Densitometry was performed on ImageJ comparing the loading control with the protein of interest. Density was calculated by multiplying the average intensity by the surface area of ​​each band. The loading control was used to eliminate sample-to-sample variation. Final results were obtained by normalizing each value to the untreated control.

[0186] Referring now to Figure 6, mean relative CD38 protein levels normalized to actin were reduced by 82% in T cells from Ab8-treated mice compared to the IgG1-treated control group (p=0.004).

[0187] Example 6: Monotherapy and combination therapy containing humanized anti-SFRP2 antibodies inhibit osteosarcoma lung metastasis In this example, monotherapy and combination therapies, each involving the use of the humanized anti-SFRP2 antibody Ab8, were evaluated for their efficacy in inhibiting osteosarcoma lung metastasis in an in vivo model.

[0188] 5 x 10 pre-filtered and resuspended in PBS 5 Osteosarcoma lung metastases were generated in C57 / B16 mice by tail vein injection using RF577 tumor cells / 100 μl. A total of 62 mice were injected. 12 days after tumor cell injection, treatment was initiated with either an IgG1 control antibody (4 mg / kg, iv, weekly; n = 13), a PD-1 mAb (200 μg / 100 μL ip, q3 days; n = 14), an Ab8 (4 mg / kg iv, q3 days; n = 15), or a combination of both treatments (n = 14) and continued for 49 days. After 49 days, mice were euthanized and lungs were excised. High-resolution photographs were taken and used to quantify metastatic lung surface nodules in each treatment group. The PD-1 mAb used was an anti-mouse PD-1 / CD279 monoclonal antibody purchased from Bioxcell, Lebanon, NH USA (#BE0273).

[0189] Quantification of this data is shown in Figure 7A. Referring to Figure 7A, the number of surface metastases in 56 mice analyzed on day 49 of treatment was 11.5 ± 2.5 in the IgG1-treated group, 6.7 ± 3 in the PD-1 mAb-treated group, 7.8 ± 1.3 in the Ab8-treated group, and 4.2 ± 1.1 for the combination therapy (p = 0.018, comparing IgG1 vs. the combination; see Figure 7A). As can be observed from the data presented in Figure 7A, the combination therapy significantly reduced lung surface metastases compared to the IgG1 control (*p = 0.018). As can be observed from the data presented in Figure 7A, monotherapy with Ab8 also reduced lung surface metastases compared to the IgG1 control.

[0190] In parallel with the above study, animal weights were measured starting on the first day of treatment and then weekly until the final week of treatment. The collected data is presented in Figure 7B. As shown in Figure 7B, no significant reduction in body weight was observed in any of the treatment groups.

[0191] A second study similar to the one described above was conducted to evaluate the effects of monotherapy and combination therapy, including treatment of osteosarcoma lung metastases with the humanized anti-SFRP2 antibody Ab8. This study was generally performed as described above, except that the percentage of mouse lung occupied by tumor was measured after lung harvest. This measurement was calculated by dividing the tumor area by the normal lung area and multiplying by 100, with treatment normalized to control. The results from this study are shown in Figure 8. As can be seen in Figure 8, treatment with Ab8 reduced lung metastatic tumor volume by 71% compared to control, and combination therapy reduced tumor volume by 82% compared to control. [Table 6]

[0192] Example 7: Efficacy and biodistribution of hSFRP2 mAb in MDA-231 human triple-negative breast cancer method Clinical Trial Design: An orthotopic model was used in nude mice bearing human triple-negative breast cancer MDA-MB-231 cells to evaluate the in vivo kinetics of hSFRP2 mAb through treatment with NIR-fluorophore-conjugated hSFRP2 mAb. Cellular association of the mAb was monitored over 72 hours by measuring Dylight 755 release using an in vivo Maestro imaging system. NIR-tagged IgG1 control was used as a treatment control, and tumor-free mice served as healthy controls. The efficacy of hSFRP2 mAb treatment was investigated in a separate in vivo experiment using the same orthotopic model.

[0193] Cell Culture. MDA-MB-231 cells (ATCC, Manassas, VA, USA) were cultured in DMEM (#30-202, ATCC®) containing 10% heat-inactivated FBS (#BT201-500-D, BioFluid, Fleming Island, FL, USA) and 1% penicillin / streptomycin (#MT30009C, Thermo Fisher Scientific, Waltham, MA, USA) at 37°C, 5% CO2, and 95% humidity. Cells were certified by ATCC® and tested for rodent pathogens by Charles River Research Animal (Wilmington, MA, USA) prior to in vivo injection.

[0194] Antibody and protein control IgG1 was obtained from Novartis (Basel, Switzerland) as omalizumab (#NDC50242-040-62). It was reconstituted per packaging and diluted with PBS to a dose of 4 mg / kg for in vivo treatment. Humanized SFRP2 mAb (Ab8) was constructed as described above in Example 1 and purified to remove endotoxin. For in vivo treatment, it was diluted with PBS to a dose of 4 mg / kg.

[0195] mouse. In vivo biodistribution using imaging Female nude mice were purchased from Charles River Laboratories. Half of the mice (n = 6) were injected with 5 million MDA-MB-231 cells in 100 μL of a suspension of 50% HBSS and 50% Matrigel (#354234, Corning, Corning, NY, USA). Mice were imaged when they had palpable tumors, and non-tumor-bearing mice were imaged as controls. hSFRP2 mAb or IgG1 control treatment was administered via tail vein injection at a concentration of 4 mg / kg.

[0196] In vivo study of hSFRP2 mAb treatment in an orthotopic triple-negative breast cancer model Female nude mice were purchased from Envigo at 8 weeks of age. Mice were injected into the right mammary fat pad with 5 million MDA-MB-231 cells in a 100 μL suspension of 50% HBSS and 50% basement membrane HC Matrigel phenol red-free (#354262, Corning, Corning, NY, USA). Tumor size was measured every 3 days using a caliper, and the volume was calculated using the formula (L × W²) / ². Once tumors reached a volume of 50 mm² on day 19, tumors were cultured. 3 Treatment began when tumor size approached 100 μg / day. Mice were randomly assigned to either an IgG1 control treatment group (n=11) or an hSFRP2 mAb treatment group (n=11). Treatment was delivered intravenously via tail vein injection at 4 mg / kg every 3 days until control tumors reached 2 cm, at which time the experiment was terminated. IgG1 was administered weekly, and hSFRP2 mAb was administered every 3 days according to previous MTD and PK studies. Tumor volume was measured every 3 days during treatment, and body weights were recorded weekly. Treatment ended 78 days after control tumors reached a maximum dimension of 2 cm.

[0197] Imaging. The Maestro in vivo imaging system was used to assess the biodistribution of IgG1 and hSFRP2 mAb in mice via fluorescent labeling of each treatment. Imaging was performed before injection, immediately after injection, and 24, 48, 72, and 96 hours after injection.

[0198] Statistics. Tumor volumes of the IgG1 control and hSFRP2 mAb-treated groups were compared by ANOVA with interaction. Adjustments for multiple comparisons were made using the Sidak technique. A p-value of <0.05 was considered statistically significant in all analyses. Statistical analyses were completed using the STATA statistical software package (version 15.0).

[0199] result Humanized SFRP2 mAb preferentially localizes to tumors in vivo. To determine the biodistribution of hSFRP2 mAb treatment in an orthotopic model of triple-negative breast cancer, NIR-tagged hSFRP2 mAb was administered to tumor-bearing mice via tail vein injection, and fluorescence was measured over 96 hours using a Maestro in vivo imaging system (Figure 10). Fluorescence was compared with three non-tumor-bearing mice that received hSFRP2 mAb as a control. At 24 hours, fluorescence was visualized in the liver, bladder, and tumor of tumor-bearing mice. For 72 hours, fluorescence persisted within the tumor according to the previously determined half-life. During this time, fluorescence dissipated from the liver, and the bladder maintained fluorescence as expected due to urinary excretion. Non-tumor-bearing mice showed no fluorescence in the mammary fat pad or any other specific region other than the bladder. Tumor-bearing mice (n = 3) and non-tumor-bearing mice (n = 3) were administered NIR-tagged IgG1 as a control. The hSFRP2 mAb NIR tag showed distribution of fluorescence in all three mice, specifically localizing to the tumor, in contrast to the IgG1 NIR tagged control, which did not express fluorescence within the tumor.

[0200] Humanized SFRP2 mAb inhibits tumor growth in vivo To evaluate whether hSFRP2 mAb inhibits tumor growth in vivo, we used an orthotopic triple-negative breast cancer model of MDA-MB-231 cells in the mammary fat pad of nude mice. Treatment was initiated 19 days after tumor cell inoculation until tumor volume reached 50 mm. 3Treatment began when the tumor reached a mass of 1000 mg / kg. Mice were injected via the tail vein with either hSFRP2 mAb (4 mg / kg every 3 days, n=11) or IgG1 control (4 mg / kg weekly, n=11) for 11 weeks. Tumors were measured every 3 days with a caliper, and volume was calculated as (L x W^2) / 2. During the course of treatment, one IgG1 control-treated mouse expired early due to ascites. This mouse had no primary tumor at necropsy and was removed from the study. At the end of the study, one mouse from each treatment group was also removed due to the absence of tumor at necropsy. The mean tumor volume at the end of the experiment was 2998 mm in the IgG1 control group. 3 (n=9, 95%CI), 1159mm in the hSFRP2 group 3 (n=10, 95%CI 800-1519mm 3 ). The HSFRP2 mAb-treated group experienced a 61% reduction in tumor volume, which was a significant reduction in tumor volume compared to the IgG1 control group (p<0.001, FIG. 11A). At necropsy, four IgG1 control-treated mice (40%) had distant metastasis, and one hSFRP2-treated mouse (10%) had distant metastasis (FIG. 11B). There was no significant weight loss, hair loss, or lethargy in any of the mice over the course of treatment (FIG. 11C).

[0201] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

[0202] All references (e.g., publications or patents or patent applications) cited in this specification are incorporated herein in their entirety for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0203] Other embodiments are within the scope of the following claims. Further aspects of the present invention are described below: [Section 1] A humanized anti-SFRP2 antibody or antigen-binding fragment thereof that binds to secreted frizzled-related protein 2 (SFRP2), wherein the humanized antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) polypeptide and a variable light chain (VL) polypeptide comprising the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 9, respectively. [Section 2] A humanized anti-SFRP2 antibody or antigen-binding fragment thereof that binds to secreted frizzled-related protein 2 (SFRP2), wherein the humanized antibody or antigen-binding fragment thereof comprises a complementarity-determining region (CDR) H1 having the amino acid sequence of SEQ ID NO: 19, a CDR H2 having the amino acid sequence of SEQ ID NO: 20, a CDR H3 having the amino acid sequence of SEQ ID NO: 21, a CDR L1 having the amino acid sequence of SEQ ID NO: 22, a CDR L2 having the amino acid sequence of SEQ ID NO: 23, and a CDR L3 having the amino acid sequence of SEQ ID NO: 24. [Section 3] A humanized anti-SFRP2 antibody or antigen-binding fragment thereof that binds to secreted frizzled-related protein 2 (SFRP2), wherein the humanized antibody or antigen-binding fragment thereof comprises a heavy chain polypeptide and a light chain polypeptide having the amino acid sequences of SEQ ID NO: 15 and SEQ ID NO: 16, respectively. [Section 4] Item 3. The humanized antibody or antigen-binding fragment thereof according to Item 1 or 2, wherein the humanized antibody or antigen-binding fragment comprises a heavy chain constant region and a light chain constant region. [Section 5] Item 10. The humanized antibody or antigen-binding fragment thereof of any one of the preceding items, wherein the humanized antibody or antigen-binding fragment is a monoclonal antibody. [Section 6] Item 10. The humanized antibody or antigen-binding fragment thereof of any one of the preceding items, which is a full-length antibody. [Section 7] Item 10. The humanized antibody or antigen-binding fragment thereof of any one of the preceding items, which is a humanized antigen-binding fragment. [Section 8] 5. The humanized antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the humanized antibody or antigen-binding fragment thereof comprises an IC50 value of 0.7 or less relative to the IC50 value of an antibody or antigen-binding fragment thereof comprising the VH chain polypeptide and VL chain polypeptide of SEQ ID NOs: 10 and 11, respectively, wherein the IC50 value is measured by ELISA assay. [Section 9] An isolated polynucleotide comprising a nucleic acid molecule encoding the heavy chain variable region or heavy chain of the humanized antibody or antigen-binding fragment thereof according to any one of Items 1 to 8. [Section 10] The isolated polynucleotide of paragraph 9, wherein the nucleic acid molecule encodes the VH of SEQ ID NO:2. [Section 11] An isolated polynucleotide comprising a nucleic acid molecule encoding the light chain variable region or the light chain of the humanized antibody or antigen-binding fragment thereof according to any one of Items 1 to 8. [Section 12] 12. The isolated polynucleotide of paragraph 11, wherein the nucleic acid molecule encodes the VL of SEQ ID NO:9. [Section 13] An isolated polynucleotide comprising a nucleic acid molecule encoding the heavy chain variable region or heavy chain of the humanized antibody or antigen-binding fragment thereof according to any one of Items 1 to 8, and the light chain variable region or light chain of the humanized antibody or antigen-binding fragment thereof according to any one of Items 1 to 8. [Section 14] Item 14. An isolated vector comprising the polynucleotide according to any one of Items 9 to 13. [Section 15] A host cell comprising: (a) the polynucleotide according to any one of Items 9 to 13; (b) the vector according to Item 14; or (c) a first vector comprising the polynucleotide according to Item 9 or 10; and a second vector comprising the polynucleotide according to Item 11 or 12. [Section 16] 16. The host cell of paragraph 15, which is selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, YB / 20, NS0, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, HepG2, SP2 / 0, R1.1, BW, LM, COS1, COS7, BSC1, BSC40, BMT10 cells, plant cells in tissue culture, insect cells, and human cells. [Section 17] The host cell of paragraph 15 or paragraph 16, wherein the host cell is a CHO cell. [Section 18] A method for producing a humanized antibody or antigen-binding fragment thereof that binds to SFRP2, comprising culturing the host cell of any one of claims 14 to 17 so that a nucleic acid molecule is expressed and the humanized antibody or antigen-binding fragment thereof is produced, and optionally, the method further comprises isolating the humanized antibody or antigen-binding fragment thereof from the culture. [Section 19] 19. The method of claim 18, wherein the isolated humanized antibody or antigen-binding fragment thereof is substantially free of precipitates. [Section 20] An isolated humanized antibody or antigen-binding fragment thereof that specifically binds to secreted frizzled-related protein 2 (SFRP2) and is encoded by the polynucleotide of any one of items 9 to 13 or produced by the method of item 18. [Section 21] A pharmaceutical composition comprising a therapeutically effective amount of the humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 8 and 20, and a pharmaceutically acceptable excipient. [Section 22] 22. A method for treating cancer in a patient, comprising administering to the patient the pharmaceutical composition of claim 21. [Section 23] Item 23. The method according to Item 22, wherein the cancer is breast cancer, angiosarcoma, osteosarcoma, rhabdomyosarcoma, alveolar soft part sarcoma, malignant glioma, multiple myeloma, renal cell carcinoma, kidney cancer, prostate cancer, lung cancer, melanoma, non-small cell lung cancer, pancreatic cancer, colorectal cancer, bladder cancer, hepatocellular carcinoma, sarcoma, or gastrointestinal cancer. [Section 24] 24. The method of any one of items 22 to 23, further comprising administering an antagonist of an inhibitory immune checkpoint molecule, optionally wherein the immune checkpoint molecule is PD-1. [Section 25] 25. The method of claim 24, wherein the PD-1 antagonist is an anti-PD-1 antibody or antigen-binding fragment thereof, and optionally the anti-PD-1 antibody or antigen-binding fragment thereof is selected from the group consisting of nivolumab, pembrolizumab, MEDI-0680 (AMP-514), camrelizumab (SHR-1210), tislelizumab (BGB-A317), and spartalizumab (NPVPDR001, NVS240118, PDR001).

Claims

1. A humanized anti-SFRP2 antibody or antigen-binding fragment thereof that binds to secreted frizzled-related protein 2 (SFRP2), wherein the humanized anti-SFRP2 antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) polypeptide and a variable light chain (VL) polypeptide comprising the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:9, respectively.

2. A humanized anti-SFRP2 antibody or antigen-binding fragment thereof that binds to secreted frizzled-related protein 2 (SFRP2), wherein the humanized anti-SFRP2 antibody or antigen-binding fragment thereof comprises a heavy chain polypeptide and a light chain polypeptide comprising the amino acid sequences of SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

3. The humanized anti-SFRP2 antibody or antigen-binding fragment thereof according to claim 1 , wherein the humanized anti-SFRP2 antibody or antigen-binding fragment thereof comprises a heavy chain constant region and a light chain constant region.

4. The humanized anti-SFRP2 antibody or antigen-binding fragment thereof according to claim 1, wherein the humanized anti-SFRP2 antibody or antigen-binding fragment thereof is a monoclonal antibody.

5. The humanized anti-SFRP2 antibody or antigen-binding fragment thereof according to claim 1, which is a full-length antibody.

6. The humanized anti-SFRP2 antibody or antigen-binding fragment thereof according to claim 1, which is a humanized antigen-binding fragment.

7. 2. The humanized anti-SFRP2 antibody or antigen-binding fragment thereof according to claim 1, wherein the humanized anti-SFRP2 antibody or antigen-binding fragment thereof comprises an IC50 value of 0.7 or less relative to the IC50 value of an antibody or antigen-binding fragment thereof comprising the VH chain polypeptide and VL chain polypeptide of SEQ ID NOs: 10 and 11, respectively, wherein the IC50 value is measured by ELISA assay.

8. An isolated polynucleotide comprising a nucleic acid molecule encoding the heavy chain variable region or heavy chain of the humanized anti-SFRP2 antibody or antigen-binding fragment thereof of claim 1 and the light chain variable region or light chain of the humanized anti-SFRP2 antibody or antigen-binding fragment thereof of claim 1.

9. An isolated vector comprising the polynucleotide of claim 8.

10. A host cell comprising the polynucleotide of claim 8.

11. 11. A method for producing a humanized antibody or antigen-binding fragment thereof that binds to secreted frizzled-related protein 2 (SFRP2), comprising culturing the host cell of claim 10 so that a nucleic acid molecule is expressed and the humanized antibody or antigen-binding fragment thereof is produced.

12. 12. The method of claim 11, wherein the method further comprises isolating the humanized antibody or antigen-binding fragment thereof from the culture.

13. 9. An isolated humanized antibody or antigen-binding fragment thereof that specifically binds to secreted frizzled-related protein 2 (SFRP2) and is encoded by the polynucleotide of claim 8.

14. A pharmaceutical composition comprising a therapeutically effective amount of the humanized anti-SFRP2 antibody or antigen-binding fragment thereof according to claim 1 and a pharmaceutically acceptable excipient.

Images