Anti-sarcospan antibodies

Abstract

The present disclosure relates to antibodies or antigen-binding fragments thereof that bind to sarcospan. The present disclosure also relates to the use of anti-sarcospan antibodies or antigen-binding fragments in methods of detecting and / or measuring sarcospan expression.

Description

UCH-38625 ANTI-SARCOSPAN ANTIBODIES CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 626,856, filed on January 30, 2024, the content of which is hereby incorporated by reference in its entirety. BACKGROUND

[0002] Sarcospan (SSPN) is a transmembrane protein that is broadly expressed at the cell surface of many tissues. SSPN is a core component of the dystrophin-glycoprotein complex that links the intracellular actin cytoskeleton with the extracellular matrix. Loss of dystrophin at the muscle cell surface, and a concomitant loss of the entire dystrophin- glycoprotein complex, including SSPN, have been linked to the development of muscular disorders such as Duchene muscular dystrophy, Becker muscular dystrophy, and X-linked dilated cardiomyopathy.

[0003] Development of SSPN antibodies is challenged by the presence of its four transmembrane domains and limited antigenic epitopes. Highly specific and robust antibodies to SSPN are needed for basic research focused on molecular mechanisms of SSPN rescue, pre-clinical studies, and biomarker evaluation in human samples. SUMMARY

[0004] The present disclosure provides antibodies, and antigen-binding fragments thereof, that specifically target SSPN. In some embodiments, the present disclosure provides anti-SSPN antibodies capable of specific binding of SSPN at three epitopes of mouse and human SSPN protein. The antibodies of the present disclosure can be used to recognize, detect, and / or quantify SSPN in a variety of applications including different types of immunoassays (immunoblotting, ELISA, immunofluorescence imaging, and immunoprecipitation).

[0005] In a first aspect, the present disclosure provides an antibody, or antigen- binding fragment thereof, that specifically targets SSPN, wherein the antibody wherein the antibody or antigen-binding fragment comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein (i) the VL comprises a VL complementary determining region (CDR) 1 (LCDR1) having a sequence as set forth in SEQ ID NO: 1UCH-38625 (QSSETVYKNNYLSWF), a VL CDR 2 (LCDR2) having a sequence as set forth in SEQ ID NO: 2 (FLIYGASTLAS), and a VL CDR 3 (LCDR3) having a sequence as set forth in SEQ ID NO: 3 (GGGFSSSSDDT); and the VH comprises a VH CDR 1 (HCDR1) having a sequence as set forth in SEQ ID NO: 4 (GFSLSSYAMS), a VH CDR 2 (HCDR2) having a sequence as set forth in SEQ ID NO: 5 (YIGIINAAGSAYYARWVNG), and a VH CDR 3 (HCDR3) having a sequence as set forth in SEQ ID NO: 6 (VRGSIPYSGGVSL); (ii) the VL comprises a LCDR1 having a sequence as set forth in SEQ ID NO: 31 (VRGSIPYSGGVSL), a LCDR2 having a sequence as set forth in SEQ ID NO: 32 (LLIYGASTLAS), and a LCDR3 having a sequence as set forth in SEQ ID NO: 33 (GGGYDTSRDDV); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 34 (GFSLSTYAMS), a HCDR2 having a sequence as set forth in SEQ ID NO: 35 (YIGIINTGGSAYYASWAEG), and a HCDR3 having a sequence as set forth in SEQ ID NO: 36 (VRGSIAYTAGVTL); (iii) the VL comprises a LCDR1 having a sequence as set forth in SEQ ID NO: 61 (QASQSVYKNNYLSWF), a LCDR2 having a sequence as set forth in SEQ ID NO: 62 (LLIYGASTLAS), and a LCDR3 having a sequence as set forth in SEQ ID NO: 63 (GGGDASSSSDDA); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 64 (GFSLSNYAMS), a HCDR2 having a sequence as set forth in SEQ ID NO: 65 (YIGIINTGGSAYYASWAEG), and a HCDR3 having a sequence as set forth in SEQ ID NO: 66 (IINTAGSAYYARWVNG); (iv) the VL comprises a LCDR1 having a sequence as set forth in SEQ ID NO: 91 (QSSESVYKNNYLSWF), a LCDR2 having a sequence as set forth in SEQ ID NO: 92 (LLIYGASTLAS), and a LCDR3 having a sequence as set forth in SEQ ID NO: 93 (GGGYDTSRDDV); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 94 (GFSLSTYAMS), a HCDR2 having a sequence as set forth in SEQ ID NO: 95 (YIGIINTGGSAYYASWAKG), and a HCDR3 having a sequence as set forth in SEQ ID NO: 96 (VRGSIAYTAGVTL); (v) the VL comprises a LCDR1 having a sequence as set forth in SEQ ID NO: 121 (QSSESVYNNNYLSWF), a LCDR2 having a sequence as set forth in SEQ ID NO: 122 (LLIYGISTLAS), and a LCDR3 having a sequence as set forth in SEQ ID NO: 123 (GGGYSTSSDDT); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 124 (GFSLSSYAMS), a HCDR2 having a sequence as set forth in SEQ ID NO: 125 (YIGIINAGGIPYYANWVNG), and a HCDR3 having a sequence as set forth in SEQ ID NO: 126 (LWGPGTLVTVSS); (vi) the VL comprises aUCH-38625 LCDR1 having a sequence as set forth in SEQ ID NO: 151 (QSSETVYKNNYLSWF), a LCDR2 having a sequence as set forth in SEQ ID NO: 152 (FLIYGASTLAS), and a LCDR3 having a sequence as set forth in SEQ ID NO: 153 (GGGFSSSSDDT); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 154 (GFSLSSYAMS), a HCDR2 having a sequence as set forth in SEQ ID NO: 155 (YIGIINAAGSAYYARWVNG), and a HCDR3 having a sequence as set forth in SEQ ID NO: 156 (VRGSIPYSGGVSL); (vii) the VL comprises a LCDR1 having a sequence as set forth in SEQ ID NO: 181 (QSSESVYKNNYLSWF), a LCDR2 having a sequence as set forth in SEQ ID NO: 182 (LLIYGASTLAS), and a LCDR3 having a sequence as set forth in SEQ ID NO: 183 (CGGGYSTSSDDT); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 184 (GFSLSSYAVS), a HCDR2 having a sequence as set forth in SEQ ID NO: 185 (IVNTAGSAYYANWVNG), and a HCDR3 having a sequence as set forth in SEQ ID NO: 186 (VRGSIAYTAGVAL); (viii) the VL comprises a LCDR1 having a sequence as set forth in SEQ ID NO: 211 (RASQSLVHSNGNTYLHWY), a LCDR2 having a sequence as set forth in SEQ ID NO: 212 (LLIYKVSNRFF), and a LCDR3 having a sequence as set forth in SEQ ID NO: 213 (SQSEHVWT); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 214 (GYSITSDYAWN), a HCDR2 having a sequence as set forth in SEQ ID NO: 215 (WMAYITYTGRTLYNPSLES), and a HCDR3 having a sequence as set forth in SEQ ID NO: 216 (ARSFAY); (ix) the VL comprises a LCDR1 having a sequence as set forth in SEQ ID NO: 241 (RASQSLVHSNGNTYLH), a LCDR2 having a sequence as set forth in SEQ ID NO: 242 (LLIYKVSNRFF), and a LCDR3 having a sequence as set forth in SEQ ID NO: 243 (SQSEHVWT); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 244 (GYSITSDYAWN), a HCDR2 having a sequence as set forth in SEQ ID NO: 245 (WMAYITYTGRTLYNPSLES), and a HCDR3 having a sequence as set forth in SEQ ID NO: 246 (ARSFAY); (x) the VL comprises a LCDR1 having a sequence as set forth in SEQ ID NO: 271 (KSSQSLLKSRTRKNHLAWY), a LCDR2 having a sequence as set forth in SEQ ID NO: 272 (LLIYWASTRES), and a LCDR3 having a sequence as set forth in SEQ ID NO: 273 (KQSYDLYT); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 274 (GFTFSDYAMA), a HCDR2 having a sequence as set forth in SEQ ID NO: 275 (WIAFLSNLAKNVYYADTVAG), and a HCDR3 having a sequence as set forth in SEQ ID NO: 276 (ARGNGDYHAMDY); (xi) the VL comprises aUCH-38625 LCDR1 having a sequence as set forth in SEQ ID NO: 331 (KSSQSLLKSRTRRNYLA), a LCDR2 having a sequence as set forth in SEQ ID NO: 332 (LLIYWASTRES), and a LCDR3 having a sequence as set forth in SEQ ID NO: 333 (KQSYDLYT); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 334 (GFTFSDYGMA), a HCDR2 having a sequence as set forth in SEQ ID NO: 335 (WIAFISNLAYNIYYADTVTG), and a HCDR3 having a sequence as set forth in SEQ ID NO: 336 (ARGNGNYDAVDY); (xii) the VL comprises a LCDR1 having a sequence as set forth in SEQ ID NO: 361 (KSSQSLLKSRTRRNYLAWY), a LCDR2 having a sequence as set forth in SEQ ID NO: 362 (LLIYWASTRES), and a LCDR3 having a sequence as set forth in SEQ ID NO: 363 (KQSYDLYT); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 364 (GFTFSDYGMA), a HCDR2 having a sequence as set forth in SEQ ID NO: 365 (WIAFISNLAYNIYYADTVTG), and a HCDR3 having a sequence as set forth in SEQ ID NO: 366 (ARGNGNYDAVDY); or (xiii) the VL comprises a LCDR1 having a sequence as set forth in SEQ ID NO: 391 (KSSQSLLKSRTRKNYLAWY), a LCDR2 having a sequence as set forth in SEQ ID NO: 392 (LLIYWASTRES), and a LCDR3 having a sequence as set forth in SEQ ID NO: 393 (KQSYDLYT); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 394 (GFTFSDYAMA), a HCDR2 having a sequence as set forth in SEQ ID NO: 395 (WVTFISDLAYNIYYADTVT), and a HCDR3 having a sequence as set forth in SEQ ID NO: 396 (ARGNGDYDAMDY).

[0006] In some embodiments, the VL comprises a VL framework region 1 (FR1) having a sequence selected from SEQ ID NOs: 449, 457, 465, 473, 481, 489, 497, 505, 513, 521, 529, 537, 545, 553, 561, 569, 577, 585, 593, 601, 609, 617, 625, 633, 641, 649, 657, 665, 673, 681, 689, 697, 705, 713, 721, 729, 737, 745, 753, 761, 769, 777, 785, 793, 801, 809, 817, 825, 833, 841, 849, 857, 865, 873, 881, and 889, a VL framework region 2 (FR2) having a sequence selected from SEQ ID NOs: 450, 458, 466, 474, 482, 490, 498, 506, 514, 522, 530, 538, 546, 554, 562, 570, 578, 586, 594, 602, 610, 618, 626, 634, 642, 650, 658, 666, 674, 682, 690, 698, 706, 714, 722, 730, 738, 746, 754, 762, 770, 778, 786, 794, 802, 810, 818, 826, 834, 842, 850, 858, 866, 874, 882, and 890, a VL framework region 3 (FR3) having a sequence selected from SEQ ID NOs: 451, 459, 467, 475, 483, 491, 499, 507, 515, 523, 531, 539, 547, 555, 563, 571, 579, 587, 595, 603, 611, 619, 627, 635, 643, 651, 659, 667, 675, 683, 691, 699, 707, 715, 723, 731, 739, 747, 755, 763, 771, 779, 787, 795, 803,UCH-38625 811, 819, 827, 835, 843, 851, 859, 867, 875, 883, and 891, and a VL framework region 4 (FR4) having a sequence selected from SEQ ID NOs: 452, 460, 468, 476, 484, 492, 500, 508, 516, 524, 532, 540, 548, 556, 564, 572, 580, 588, 596, 604, 612, 620, 628, 636, 644, 652, 660, 668, 676, 684, 692, 700, 708, 716, 724, 732, 740, 748, 756, 764, 772, 780, 788, 796, 804, 812, 820, 828, 836, 844, 852, 860, 868, 876, 884, and 892.

[0007] In some embodiments, the VH comprises a VH framework region 1 (FR1) having a sequence selected from SEQ ID NOs: 453, 461, 469, 477, 485, 493, 501, 509, 517, 525, 533, 541, 549, 557, 565, 573, 581, 589, 597, 605, 613, 621, 629, 637, 645, 653, 661, 669, 677, 685, 693, 701, 709, 717, 725, 733, 741, 749, 757, 765, 773, 781, 789, 797, 805, 813, 821, 829, 837, 845, 853, 861, 869, 877, 885, and 893, a VH framework region 2 (FR2) having a sequence selected from SEQ ID NOs: 454, 462, 470, 478, 486, 494, 502, 510, 518, 526, 534, 542, 550, 558, 566, 574, 582, 590, 598, 606, 614, 622, 630, 638, 646, 654, 662, 670, 678, 686, 694, 702, 710, 718, 726, 734, 742, 750, 758, 766, 774, 782, 790, 798, 806, 814, 822, 830, 838, 846, 854, 862, 870, 878, 886, and 894, a VH framework region 3 (FR3) having a sequence selected from SEQ ID NOs: 455, 463, 471, 479, 487, 495, 503, 511, 519, 527, 535, 543, 551, 559, 567, 575, 583, 591, 599, 607, 615, 623, 631, 639, 647, 655, 663, 671, 679, 687, 695, 703, 711, 719, 727, 735, 743, 751, 759, 767, 775, 783, 791, 799, 807, 815, 823, 831, 839, 847, 855, 863, 871, 879, 887, and 895, and a VH framework region 4 (FR4) having a sequence selected from SEQ ID NOs: 456, 464, 472, 480, 488, 496, 504, 512, 520, 528, 536, 544, 552, 560, 568, 576, 584, 592, 600, 608, 616, 624, 632, 640, 648, 656, 664, 672, 680, 688, 696, 704, 712, 720, 728, 736, 744, 752, 760, 768, 776, 784, 792, 800, 808, 816, 824, 832, 840, 848, 856, 864, 872, 880, 888, and 896.

[0008] In some embodiments, the VH and the VL comprise amino acid sequences selected from: (a) SEQ ID NOs: 421 and 422, respectively; (b) SEQ ID NOs: 423 and 424, respectively; (c) SEQ ID NOs: 425 and 426, respectively; (d) SEQ ID NOs: 427 and 428, respectively; (e) SEQ ID NOs: 429 and 430, respectively; (f) SEQ ID NOs: 431 and 432, respectively; (g) SEQ ID NOs: 433 and 434, respectively; (h) SEQ ID NOs: 435 and 436, respectively; (i) SEQ ID NOs: 437 and 438, respectively; (j) SEQ ID NOs: 439 and 440, respectively; (k) SEQ ID NOs: 441 and 442, respectively; (l) SEQ ID NOs: 443 and 444, respectively; (m) SEQ ID NOs: 445 and 446, respectively; and (n) SEQ ID NOs: 447 and 448, respectively.UCH-38625

[0009] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region. In some embodiments, the heavy chain constant region comprises an amino acid sequence selected from: (a) SEQ ID NO: 900; (b) SEQ ID NO: 901; (c) SEQ ID NO: 902; and (d) SEQ ID NO: 903.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain constant region. In some embodiments, the light chain constant region comprises an amino acid sequence selected from: (a) SEQ ID NO: 897; (b) SEQ ID NO: 898; and (c) SEQ ID NO: 899.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region and a light chain constant region comprising the amino acid sequences selected from: (a) SEQ ID NOs: 900 and 897, respectively; (b) SEQ ID NOs: 900 and 898, respectively; (c) SEQ ID NOs: 901 and 899, respectively; (d) SEQ ID Nos: 902 and 899, respectively; and (e) SEQ ID NOs: 903 and 899, respectively.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain sequence and a light chain sequence comprising the amino acid sequences selected from: (a) SEQ ID NOs: 904 and 918, respectively; (b) SEQ ID NOs: 905 and 919, respectively; (c) SEQ ID NOs: 906 and 920, respectively; (d) SEQ ID NOs: 907 and 921, respectively; (e) SEQ ID NOs: 908 and 922, respectively; (f) SEQ ID NOs: 909 and 923, respectively; (g) SEQ ID NOs: 910 and 924, respectively; (h) SEQ ID NOs: 911 and 925, respectively; (i) SEQ ID NOs: 912 and 926, respectively; (j) SEQ ID NOs: 913 and 927, respectively; (k) SEQ ID NOs: 914 and 928, respectively; (l) SEQ ID NOs: 915 and 929, respectively; (m) SEQ ID NOs: 916 and 930, respectively; and (n) SEQ ID NOs: 917 and 931, respectively.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope, wherein the epitope is (i) an epitope of human SSPN, (ii) an epitope of mouse SSPN, or (iii) both an epitope of human SSPN and an epitope of mouse SSPN.

[0014] In some embodiments, the epitope is an intracellular epitope or an extracellular epitope. In some embodiments, the epitope is a C-terminal epitope or a large extracellular loop (LEL) epitope.

[0015] In some embodiments, the epitope is a C-terminal epitope within residues 219- 243 of human SSPN. In some embodiments, the epitope is a C-terminal epitope comprisingUCH-38625 an amino acid sequence within SEQ ID NO: 932. In some embodiments, the epitope is a C- terminal epitope within residues 219-243 of mouse SSPN. In some embodiments, the epitope is a C-terminal epitope comprising an amino acid sequence within SEQ ID NO: 936.

[0016] In some embodiments, the epitope is an LEL epitope within residues 167-186 of human SSPN. In some embodiments, the epitope is an LEL epitope comprising an amino acid sequence within SEQ ID NO: 933.

[0017] In some embodiments, the epitope is an N-terminal epitope. In some embodiments, the epitope is an N-terminal epitope within residues 1-25 of human SSPN. In some embodiments, the N-terminal epitope comprises an amino acid sequence within SEQ ID NO: 937.

[0018] In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope of mouse SSPN. In some embodiments, the epitope is an LEL epitope within residues 167-186 of mouse SSPN. In some embodiments, the epitope is an LEL epitope comprising an amino acid sequence within SEQ ID NO: 934.

[0019] In some embodiments, the epitope is an intracellular epitope. In some embodiments, the epitope is an N-terminal epitope. In some embodiments, the N-terminal epitope is within residues 1-25 of mouse SSPN. In some embodiments, the N-terminal epitope comprises an amino acid sequence within SEQ ID NO: 935.

[0020] In another aspect, the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding the heavy chain and the light chain of the antibody or antigen- binding fragment herein described. In another aspect, the present disclosure provides an expression vector comprising the nucleic acid encoding the heavy chain and the light chain of the antibody or antigen-binding fragment herein described. In another aspect, the present disclosure provides a cell comprising the expression vector herein described.

[0021] In another aspect, the present disclosure provides a method of detecting SSPN in a sample comprising contacting the sample with an antibody or antigen-binding fragment herein described and determining the presence or absence of SSPN in the sample. In some embodiments, the sample comprises a cell, a tissue, a biological fluid, an aqueous suspension, or a living organism such as a mammalian model of disease or a human. In some embodiments, the sample comprises skeletal muscle cells, smooth muscle cells,UCH-38625 cardiomyocytes, adipocytes, kidney epithelial cells, immune cells, chondrocytes, fibroblasts, plasma cells, tumor cells, neurons, or any SSPN expressing entity.

[0022] In some embodiments, determining the presence or absence of SSPN in the sample comprises the use of an immunoassay. In some embodiments, determining the presence or absence of SSPN in the sample comprises the use of an immunoblotting assay, an immunoprecipitation assay, an immunofluorescence assay, or an enzyme-linked immunosorbent assay (ELISA) assay. In some embodiments, SSPN antibodies could be used to detect the presence or absence of SSPN in a living organism. In some embodiments, use of SSPN antibodies may have a functional consequence such as affecting SSPN properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The Drawings included herein, which include the following Figures, are for illustration purposes only and not for limitation.

[0024] FIG.1A is a schematic diagram representing predicted membrane topology of human SSPN (hSSPN) protein and the corresponding epitope regions (i.e., a large extracellular loop (LEL) epitope region and a C-terminal epitope region) targeted by the anti- SSPN antibodies herein provided.

[0025] FIG.1B is a schematic diagram representing predicted membrane topology of mouse SSPN (mSPPN) protein and the corresponding epitope regions (i.e., an N-terminal epitope region) targeted by the anti-SSPN antibodies herein provided.

[0026] FIG.1C is a schematic diagram showing a comparison of the amino acid residues of hSSPN and mSSPN in the epitope regions targeted by the anti-SSPN antibodies herein provided.

[0027] FIG.2A is a series of graphs showing the immune response to the antigen consisting of amino acid residues 1-25 of the N-terminus of mouse SSPN.

[0028] FIG.2B is a series of images showing immunoblotting of total muscle lysates from mice overexpressing mouse SSPN (mSSPN) in lane 1, human SSPN (hSSPN) in lane 2, wild-type (WT) mouse muscle in lane 3, a mouse model of Duchenne muscular dystrophy (mdx) in lane 4, and SSPN null mice (SSPN- / -) in lane 5, with rabbit sera after 3rdand 4thimmunizations (bleed I and bleed II respectively).

[0029] FIG.3A is a graph showing validation of purified rabbit polyclonal antibodies by ELISA. Terminal bleeds from rabbits #8870, #8871, #8872 and #8873 were purified byUCH-38625 affinity chromatography. Titer of purified antibodies was estimated by ELISA with SUMO tagged SSPN or unrelated GST-tagged protein (0.5 µg / ml). Antibody dilution: original purified antibodies were first diluted at 1:1000, followed by 8-point 1:3 serial dilution. Detection: HRP conjugated goat anti‐rabbit IgG (Jackson ImmunoResearch, 111‐035‐045) (1:5000).

[0030] FIG.3B is an image showing immunoblotting of total muscle lysates from mice overexpressing mouse SSPN (mSSPN) in lane 1, human SSPN (hSSPN) in lane 2, wild- type (WT) mouse muscle in lane 3, a mouse model of Duchenne muscular dystrophy (mdx) in lane 4, and SSPN null mice (SSPN- / -) in lane 5.20 µg of total protein per line for all lines, except mSSPN where 2 µg of total protein per line was loaded) with purified rabbit polyclonal antibodies #8870. Arrow denotes mouse SSPN.

[0031] FIG.3C is a series of immunofluorescence images of transverse cross- sections of quadriceps muscles of WT SSPN- / -mice and human control (hamstring) stained with purified rabbit polyclonal antibodies #8870. Scale bar – 50µm.

[0032] FIG.4A is an image showing immunoblotting of total muscle lysates from mice overexpressing mouse SSPN (mSSPN) in lane 1, human SSPN (hSSPN) in lane 2, wild- type (WT) mouse muscle in lane 3, a mouse model of Duchenne muscular dystrophy (mdx) in lane 4, and SSPN null mice (SSPN- / -) with commercially available mouse monoclonal antibody E2 (Santa Cruz) and with rabbit monoclonal antibody 10B8.20 µg of total protein per lane for all lanes, except mSSPN where 2 µg of total protein per line was loaded. Film exposure time was 15 sec.

[0033] FIG.4B is a series of immunofluorescence images of transverse cross- sections of WT, SSPN- / -quadriceps, WT hearts and human hamstring (control) stained with rabbit monoclonal antibody 10B8 and commercially available mouse monoclonal antibody E2 for comparison. Acquisition time for E2 – 1sec, for 10B8 – 0.8 sec. Scale bar – 50 μm.

[0034] FIG.5A is a graph showing reactivity of mouse sera to C-terminus fragment of human SSPN in ELISA.

[0035] FIG.5B is a graph showing reactivity of mouse sera to LEL fragment of human SSPN in ELISA.

[0036] FIG.6A is a series of images showing immunoblotting of total muscle lysates from mice overexpressing mouse SSPN (mSSPN) in lane 1, human SSPN (hSSPN) in lane 2,UCH-38625 wild-type (WT) mouse muscle in lane 3, a mouse model of Duchenne muscular dystrophy (mdx) in lane 4, and SSPN null mice (SSPN- / -) with commercially available mouse monoclonal antibody E2 (Santa Cruz) and antibodies 289-F15 and 290-F25.20 µg of total protein per line was loaded, except for mSSPN where 2 µg of total protein per line was loaded. Film exposure time for all antibodies was 2 min.

[0037] FIG.6B is a series of immunofluorescence images of transverse cross- sections from quadriceps muscle tissue of WT mice, SSPN- / -mice, and human control (hamstring tissue) stained with commercially available mouse monoclonal antibody E2 and antibodies 289-F15 and 290-F25. Acquisition time for all antibodies was 1 sec. Scale bar – 50 µm.

[0038] FIG.7 is a series of immunofluorescence images showing transverse cross- sections of human healthy control muscle tissue, human Becker muscular dystrophy (BMD) hamstring tissue, mouse WT muscle tissue and mouse SSPN- / -quadricep tissue stained with mouse monoclonal antibodies (289-17, 289-18, 290-4, and 290-11) and commercially available mouse monoclonal antibody E2 for comparison. Acquisition time for all antibodies was 1 sec. Scale bar – 50 µm.

[0039] FIG.8 is a series of images showing affinity purification of SSPN by monoclonal antibodies 10B8, 290-04, 20E11, 290-17, 290-05, 290-06, 290-11, 289-18, and 289-17. Quadricep muscle lysates from wild-type (WT), mSSPN-TG (mTG), hSSPN-TG (hTG) and SSPN- / -were immunoprecipitated with the indicated rabbit (10B8, 20E11) or mouse (290-04, 290-05, 290-17, 290-11, 290-06, 289-17, 289-18, and E2) monoclonal antibodies. Successful pull-down of SSPN was assessed by immunoblotting equimolar amounts of lysates with E2 or 10B8 anti-SSPN antibodies. Lysates incubated with protein A / G PLUS-agarose beads in the absence of antibody was used as a negative control. Black arrow: human SSPN (hSSPN) monomer, gray arrow: mouse SSPN (mSSPN) monomer, black arrowhead: hSSPN dimer, gray arrowhead: mSSPN dimer, #: IgG light chain, *: IgG heavy chain, IP: antibody used for immunoprecipitation, IB: antibody used for immunoblotting. All blots were processed in parallel and developed with identical film exposure time.

[0040] FIG.9A is a graph showing B-cell linear epitope prediction for mouse SSPN by Bepipred linear epitope prediction 2.0 tool. Areas labeled with a (*) are predicted immunogenic epitopes and areas labeled with an (x) are non-immunogenic hydrophobicUCH-38625 regions of SSPN protein. Boxes denote fragments used for immunization to generate rabbit antibodies.

[0041] FIG.9B is a graph showing B-cell linear epitope prediction for human SSPN by Bepipred linear epitope prediction 2.0 tool. Areas labeled with a (*) are predicted immunogenic epitopes and areas labeled with an (x) are non-immunogenic hydrophobic regions of SSPN protein. Boxes denote fragments used for immunization to generate mouse antibodies.

[0042] FIG.10 is a series of immunofluorescence images showing transverse cross- sections of quadriceps from transgenic mice overexpressing mouse SSPN, WT, mdx and mouse SSPN- / -stained with non-purified rabbit immune sera from mouse 8870, 8871, 8872, 8873, and 8874 (diluted 1:50 in 3% BSA in PBS). Scale bar – 50 µm.

[0043] FIG.11 is a series of graphs showing the validation of rabbit immune sera (bleeds III, IV and V) by ELISA. Rabbit immune response to SSPN after 5th(bleed III), 6th(bleed IV) and 7th(bleed V) immunizations analyzed by indirect ELISA. Serial dilutions of antisera (original sera were first diluted at 1:1000, followed by 8-point 1:3 serial dilution) were analyzed by ELISA using SUMO-tagged SSPN protein (0.5µg / ml). Detection: HRP conjugated goat anti‐rabbit IgG was used for final detection (1:5000).

[0044] FIG.12 is a series of graphs showing reactivity of mouse sera #4 and #5 to C- terminus and LEL fragment of human SSPN in ELISA. Testing of mice immune response to hSSPN aa219-243 in ELISA (Internal serial #289 for anti-C-terminus sera) after 3rdboost (antisera #4) and 4thboost (antisera #5). Plate coating: Biotin-hSSPN aa219-243 (HRYQVFYVGVRICSLTASEGPQQKI) (SEQ ID NO: 932), Coating concentration: 1µg / ml. Secondary antibody: Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L). Secondary antibody dilution ratio: 1:10000. (B) Mouse antisera #4 and 5 response to hSSPN aa167-186 tested in ELISA (Internal serial #290 for anti-LEL sera). Plate coating: Biotin-hSSPN aa167-186 PSSEPLSRTFVYRDVTDCTS (SEQ ID NO: 933). Coating concentration: 1 µg / mL. Secondary antibody: Peroxidase-conjugated AffiniPure Goat Anti Mouse IgG (H+L).

[0045] FIG.13A is a series of images showing immunoblotting of total muscle lysates from mice overexpressing mouse SSPN (mSSPN), human SSPN (hSSPN), WT mice, mdx mice and SSPN- / -mice with crude mouse sera (diluted 1:100 in in Tris-buffer saline, pHUCH-38625 7.4 with Halt protease and phosphatase inhibitors) after rounds of immunization with hSSPN aa219-243.20 µg of total protein per line for all lines, except mSSPN where 2 µg of total protein per line was loaded.

[0046] FIG.13B is a series of images showing immunoblotting of total muscle lysates from mice overexpressing mouse SSPN (mSSPN), human SSPN (hSSPN), WT mice, mdx mice and SSPN- / -mice with crude mouse sera (diluted 1:100 in in Tris-buffer saline, pH 7.4 with Halt protease and phosphatase inhibitors) after rounds of immunization with hSSPN aa167-186.20 µg of total protein per line for all lines, except mSSPN where 2 µg of total protein per line was loaded. DETAILED DESCRIPTION

[0047] The present disclosure is based, at least in part, on the discovery of antibodies that bind sarcospan (SSPN). Highly specific and robust anti-SSPN antibodies are vital for evaluating of the efficiency of SSPN-modulating therapies, pre-clinical studies, and basic research focused on molecular mechanisms of SSPN rescue.

[0048] SSPN is a transmembrane protein that forms part of the dystrophin-associated protein complex and supports the linking between the extracellular matrix and the intracellular cytoskeleton. SSPN homo-oligomers create a scaffold for many cell surface receptors that interact with the extracellular matrix and play roles in cell adhesion, cell signaling, and mechanotransduction.

[0049] SSPN interacts tightly with many multimeric transmembrane protein complexes including the dystrophin-glycoprotein-complex (DGC), the utrophin-glycoprotein complex (UGC), and the α7β1-integrin complex. Within the DGC and UGC, SSPN mediates protein-protein interactions between the dystroglycans (α- and β-subunits) and the sarcoglycan (SG) subcomplex, and this interaction is critical for membrane targeting and stable interaction with laminin. In skeletal muscles, SSPN is enriched at the myotendinous junction (MTJ) and the neuromuscular junction (NMJ), where SSPN interacts with ECM receptors and mechanotransducers proteins. Many forms of muscular dystrophy result from a loss of muscle cell attachment to extracellular matrix. For instance, perturbations of sarcoglycan-SSPN subcomplex are associated with autosomal recessive Limb-girdle muscular dystrophy (LGMD). It is well established that stable interactions among the integral membrane proteins are critical for function of adhesion complexes and prevention ofUCH-38625 muscular dystrophy. Despite their importance, the factors that determine the structural integrity of the DGC, UGC, and α7β1-integrin complex are not well understood.

[0050] Exploration of the therapeutic benefit of SSPN-based sarcolemma stabilization of DMD skeletal and cardiac muscle has revealed that SSPN overexpression is effective at improving numerous aspects of Duchenne muscular dystrophy DMD pathology. Even low levels of SSPN overexpression can enhance mdx cardiac membrane stability, upregulate utrophin expression, and improve cardiac function, in addition to addressing skeletal muscle pathology and contraction-induced injury to myofibers. The availability of specific and robust anti-SSPN antibodies is vital for evaluating of the efficiency of SSPN-modulating therapies.

[0051] Certain terms are defined below. The publications and other reference materials referenced herein are hereby incorporated by reference.

[0052] In this application, unless otherwise clear from context, (i) the terms “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article; (ii) the term “or” may be understood to mean “and / or”; (iii) the terms “comprising” and "including" may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) where ranges are provided, endpoints are included.

[0053] As used herein, “about” will be understood by persons of ordinary skill and will vary to some extent depending on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill given the context in which it is used, "about" will mean up to plus or minus 10% of the particular value.

[0054] As used herein, the term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phospho serine. The term “amino acid analogs” refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurringUCH-38625 amino acid. The term “amino acid mimetics” refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.

[0055] Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, can be referred to by their commonly accepted single-letter codes.

[0056] As used herein, the term “antibody” refers to a whole antibody comprising two light chain polypeptides and two heavy chain polypeptides. Whole antibodies include different antibody isotypes including IgM, IgG, IgA, IgD, and IgE antibodies. The term “antibody” includes a polyclonal antibody, a monoclonal antibody, a chimerized or chimeric antibody, a humanized antibody, a primatized antibody, a deimmunized antibody, and a fully human antibody. The antibody can be a purified or a recombinant antibody.

[0057] As used herein, the terms “antibody fragment,” “antigen-binding fragment,” “antigen binding portion” or similar terms refer to a fragment of an antibody that retains the ability to bind to a target antigen (e.g., SSPN). Such fragments include, e.g., a single chain antibody, a single chain Fv fragment (scFv), an Fd fragment, a Fab fragment, a Fab' fragment, or an F(ab')2 fragment. An scFv fragment is a single polypeptide chain that includes both the heavy and light chain variable regions of the antibody from which the scFv is derived. In addition, intrabodies, minibodies, triabodies, and diabodies are also included in the definition of antibody and are compatible for use in the methods described herein. See, e.g., Todorovska et al., (2001) J. Immunol. Methods 248(l):47-66; Hudson and Kortt, (1999) J. Immunol. Methods 231(1): 177-189; Poljak, (1994) Structure 2(12): 1121-1123; Rondon and Marasco, (1997) Annu. Rev. Microbiol.51:257-283, the disclosures of each of which are incorporated herein by reference in their entirety.

[0058] As used herein, the term “antibody fragment” also includes, e.g., single domain antibodies such as camelized single domain antibodies. See, e.g., Muyldermans et al., (2001) Trends Biochem. Sci.26:230-235; Nuttall et al., (2000) Curr. Pharm. Biotech.1:253- 263; Reichmann et al., (1999) J. Immunol. Meth.231:25-38; PCT application publication nos. WO 94 / 04678 and WO 94 / 25591; and U.S. patent no.6,005,079, all of which are incorporated herein by reference in their entireties. In some embodiments, the disclosureUCH-38625 provides single domain antibodies comprising two VH domains with modifications such that single domain antibodies are formed.

[0059] In some embodiments, an antigen-binding fragment includes the variable region of a heavy chain polypeptide and the variable region of a light chain polypeptide. In some embodiments, an antigen-binding fragment described herein comprises the CDRs of the light chain and heavy chain polypeptide of an antibody.

[0060] The term “CDR” refers to a complementarity determining region within an immunoglobulin variable region sequence. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1, CDR2 and CDR3. The term “CDR set” refers to a group of three CDRs that are present in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al. (1987) Sequences of Proteins of Immunological Interest, Fourth Edition. US Govt. Printing Off. No.165-492; and Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition. NIH Publication No.91-3242) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody or binding protein, but also provides precise residue boundaries defining the three CDRs in each heavy or light chain sequence. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia and Lesk (1987) J. Mol. Biol.196:901-917; Chothia et al. (1989) Nature 342:877-883) found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as L1, L2 and L3 or H1, H2 and H3 where the “L” and the “H” designates the light chain and the heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described in Padlan (1995) FASEB J.9:133-139 and MacCallum (1996) J. Mol. Biol. 262(5):732-45). Still other CDR boundary definitions may not strictly follow one of the herein described systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding.UCH-38625

[0061] It will also be understood by one of ordinary skill in the art that the antibodies suitable for use in the methods disclosed herein may be altered such that they vary in sequence from the naturally occurring or native sequences from which they were derived, while retaining the desirable activity of the native sequences. For example, nucleotide or amino acid substitutions leading to conservative substitutions or changes at “non-essential” amino acid residues may be made. Mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0062] The antibodies suitable for use in the methods disclosed herein may comprise conservative amino acid substitutions at one or more amino acid residues, e.g., at essential or non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in a binding polypeptide is preferably replaced with another amino acid residue from the same side chain family. In certain embodiments, a string of amino acids can be replaced with a structurally similar string that differs in order and / or composition of side chain family members. Alternatively, in certain embodiments, mutations may be introduced randomly along all or part of a coding sequence, such as by saturation mutagenesis, and the resultant mutants can be incorporated into binding polypeptides provided herein and screened for their ability to bind to the desired target.

[0063] The term “epitope,” as used herein, refers to the region of or within SSPN which is bound by an antibody or antigen-binding fragment herein described. In some embodiments, the epitope is a linear epitope. “Linear epitope” refers to the region of or within SSPN which is bound by the antibody or antigen-binding fragment and which region is composed of contiguous amino acids of the amino acid sequence of SSPN. The amino acids of a linear epitope are adjacent to each other in the primary structure of SSPN. Accordingly, a linear epitope is a fragment or portion of the amino acid sequence of theUCH-38625 antigen, i.e., SSPN. In other various embodiments, the epitope is a conformational or structural epitope. The terms “conformational epitope” or “structural epitope” refer to an epitope which is composed of amino acids which are located in close proximity to one another only when SSPN is in its properly folded state. Unlike linear epitopes, the amino acids of a conformational or structural epitope are not adjacent to each other in the primary structure (i.e., amino acid sequence) of SSPN. A conformational or structural epitope is not made of contiguous amino acids of the amino acid sequence of the antigen (e.g., SSPN).

[0064] As used herein, the term “isolated antibody” is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to human SSPN is substantially free of antibodies that specifically bind antigens other than SSPN). An isolated antibody that specifically binds to an epitope may, however, have cross-reactivity to other SSPN proteins from different species. However, the antibody continues to display specific binding to human SSPN in a specific binding assay as described herein. In addition, an isolated antibody is typically substantially free of other cellular material and / or chemicals. In some embodiments, a combination of “isolated” antibodies having different SSPN specificities is combined in a well-defined composition.

[0065] As used herein, the term “isolated nucleic acid molecule” refers to nucleic acids encoding antibodies and / or antibody portions (e.g., VH, VL, CDR3) that bind to SSPN and is intended to refer to a nucleic acid molecule in which the nucleotide sequences encoding the antibody and / or antibody portion thereof are free of other nucleotide sequences encoding antibodies or antibody portions that bind antigens other than SSPN.

[0066] As used herein the term “KD” or “KD” refers to the equilibrium dissociation constant of a binding reaction between an antibody and an antigen. The value of KD is a numeric representation of the ratio of the antibody off-rate constant (kd) to the antibody on- rate constant (ka). The value of KD is inversely related to the binding affinity of an antibody to an antigen. The smaller the KD value the greater the affinity of the antibody for its antigen. Affinity is the strength of binding of a single molecule to its ligand and is typically measured and reported by the equilibrium dissociation constant (KD), which is used to evaluate and rank order strengths of bimolecular interactions.UCH-38625

[0067] As used herein, the term “kd” or “kd” (alternatively “koff” or “koff”) is intended to refer to the off-rate constant for the dissociation of an antibody from an antibody / antigen complex. The value of kd is a numeric representation of the fraction of complexes that decay or dissociate per second, and is expressed in units sec-1.

[0068] As used herein, the term “ka” or “ka” (alternatively “kon” or “kon”) is intended to refer to the on-rate constant for the association of an antibody with an antigen. The value of ka is a numeric representation of the number of antibody / antigen complexes formed per second in a 1 molar (1M) solution of antibody and antigen, and is expressed in units M-1sec-1.

[0069] As used herein, the term “monoclonal antibody” refers to an antibody which displays a single binding specificity and affinity for a particular epitope.

[0070] As used herein, the term “nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res.19:5081, 1991; Ohtsuka et al., Biol. Chem.260:2605-2608, 1985; and Cassol et al, 1992; Rossolini et al, Mol. Cell. Probes 8:91-98, 1994). For arginine and leucine, modifications at the second base can also be conservative. The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0071] The term “percent identity,” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the “percent identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or,UCH-38625 alternatively, exist over the full length of the two sequences to be compared. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0072] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math.2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat’l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra). One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol.215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website.

[0073] The term “polyclonal antibody” describes a composition of different (diverse) antibody molecules which is capable of binding to or reacting with a specific antigenic determinant on the same or on different antigens.

[0074] As used herein, the term “purified” or “isolated” as applied to any of the proteins (antibodies or fragments) described herein refers to a polypeptide that has been separated or purified from components (e.g., proteins or other naturally-occurring biological or organic molecules) which naturally accompany it, e.g., other proteins, lipids, and nucleic acid in a prokaryote expressing the proteins. Typically, a polypeptide is purified when it constitutes at least 60 (e.g., at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 92, at least 95, at least 97, or at least 99) %, by weight, of the total protein in a sample.

[0075] The term “treat,” as used herein, means decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease / disorder,UCH-38625 lessen the severity of the disease / disorder, or improve at least one symptom associated with the disease / disorder.

[0076] As used herein, the term “subject” is intended to include human and non- human animals. Exemplary human subjects include a human patient having a disease / disorder, e.g., a disease / disorder described herein or a normal subject. The term “non- human animals” of one aspect of the invention includes all vertebrates, e.g., non-mammals (such as chickens, amphibians, reptiles) and mammals, such as non-human primates, domesticated and / or agriculturally useful animals, e.g., sheep, dog, cat, cow, pig, etc.

[0077] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the presently disclosed methods and compositions. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Anti-SSPN Antibodies and Antigen-Binding Fragments Thereof

[0078] In some aspects, the present disclosure provides antibodies and antigen- binding fragments that specifically bind to SSPN. In some aspects, the disclosure provides anti-SSPN antibodies that are useful for detecting and / or measuring SSPN in a sample (e.g., expression level of SSPN in a tissue sample).

[0079] SSPN is a transmembrane protein that has intracellular and extracellular domains. The present disclosure provides antibodies with binding specificity to at least one of three domains or regions of SSPN: the N-terminus, the large extracellular loop (LEL; disposed between transmembrane domain 3 and 4), and the C-terminus (FIGs.1A and 1B).

[0080] In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, binds to an epitope. The epitope may be, for example (i) an epitope of human SSPN, (ii) an epitope of mouse SSPN, or (iii) both an epitope of human SSPN and an epitope of mouse SSPN. In some embodiments, the epitope is an intracellular epitope of SSPN. In some embodiments, the epitope is an extracellular epitope of SSPN. In some embodiments, the epitope comprises 4 or more (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acids. In some embodiments, the epitope comprises 4 or more (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,UCH-38625 14, or 15) contiguous amino acids. In some embodiments, the epitope comprises 4 or more (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) non-contiguous amino acids.

[0081] In some embodiments, the epitope is a C-terminal epitope. In some embodiments, the C-terminal epitope is within the C-terminal region. In some embodiments, the C-terminal epitope is within residues 219-243 (i.e., the C-terminal region) of human SSPN. In some embodiments, the C-terminal epitope comprises an amino acid sequence within SEQ ID NO: 932. In some embodiments, the C-terminal epitope is an amino acid sequence within SEQ ID NO: 932. In some embodiments, the C-terminal epitope comprises at least 4 (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acids within SEQ ID NO: 932.

[0082] In some embodiments, the C-terminal epitope is within residues 219-243 of mouse SSPN. In some embodiments, the C-terminal epitope comprises an amino acid sequence within SEQ ID NO: 936. In some embodiments, the C-terminal epitope is an amino acid sequence within SEQ ID NO: 936. In some embodiments, the C-terminal epitope comprises at least 4 (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acids within SEQ ID NO: 936.

[0083] As used herein, the term “LEL” refers to the large extracellular loop of SSPN. A “LEL epitope” is an epitope located at least partially or fully within the LEL. In some embodiments, the epitope is an LEL epitope of human SSPN. In some embodiments, the LEL epitope is within residues 167-186 of human SSPN. In some embodiments, the epitope is an LEL epitope comprising an amino acid sequence within SEQ ID NO: 933. In some embodiments, the LEL epitope is an amino acid sequence within SEQ ID NO: 933. In some embodiments, the LEL epitope comprises at least 4 (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acids within SEQ ID NO: 933.

[0084] In some embodiments, the epitope is an LEL epitope of mouse SSPN. In some embodiments, the LEL epitope is within residues 167-186 of human SSPN. In some embodiments, the epitope is an LEL epitope comprising an amino acid sequence within SEQ ID NO: 934. In some embodiments, the LEL epitope is an amino acid sequence within SEQ ID NO: 934. In some embodiments, the LEL epitope comprises at least 4 (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acids within SEQ ID NO: 934.

[0085] In some embodiments, the epitope is an N-terminal epitope of mouse SSPN. In some embodiments, the epitope is an N-terminal epitope within residues 1-25 of mouseUCH-38625 SSPN. In some embodiments, the N-terminal epitope comprises an amino acid sequence within SEQ ID NO: 935. In some embodiments, the N-terminal epitope is an amino acid sequence within SEQ ID NO: 935. In some embodiments, the N-terminal epitope comprises at least 4 (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acids within SEQ ID NO: 935.

[0086] In some embodiments, the epitope is an N-terminal epitope of human SSPN. In some embodiments, the epitope is an N-terminal epitope within residues 1-25 of human SSPN. In some embodiments, the N-terminal epitope comprises an amino acid sequence within SEQ ID NO: 937. In some embodiments, the N-terminal epitope is an amino acid sequence within SEQ ID NO: 937. In some embodiments, the N-terminal epitope comprises at least 4 (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acids within SEQ ID NO: 937.

[0087] In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a binding affinity against SSPN of from about 0.5 nM to about 50 nM (e.g., from about 0.5 nM to about 40 nM, from about 0.5 nM to about 30 nM, from about 0.5 nM to about 20 nM, from about 0.5 nM to about 10 nM, from about 0.5 nM to about 5 nM, from about 0.5 nM to about 2 nM, from about 0.5 nM to about 1 nM, from about 1 nM to about 50 nM, from about 10 nM to about 50 nM, from about 20 nM to about 50 nM, from about 30 nM to about 50 nM, or from about 40 nM to about 50 nM).

[0088] In some embodiments, the present disclosure provides a nucleic acid comprising a nucleotide encoding the VL, the VH, the heavy chain, the light chain, or any combination thereof, of an antibody or antigen-binding fragment described herein. In some embodiments, the nucleic acid is a DNA. In some embodiments, the nucleic acid is an RNA.

[0089] In some embodiments, the present disclosure provides an expression vector. The expression vector may comprise a nucleic acid encoding a VL, a VH, a heavy chain, a light chain, or any combination thereof of an antibody or antigen-binding fragment described herein. In some embodiments, the present disclosure provides a cell comprising the expression vector as herein disclosed. Variable regions

[0090] In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a light chain variable region (VL) and a heavy chain variable region (VH). In some embodiments, the VL comprises a VL complementary determining region (CDR) 1UCH-38625 (LCDR1), a CDR 2 (LCDR2), and a CDR 3 (LCDR3). In some embodiments, the VH comprises a VH CDR 1 (HCDR1), a VH CDR 2 (HCDR2), and a VH CDR 3 (HCDR3). In some embodiments, the VL comprises a VL complementary determining region (CDR) 1 (LCDR1), a CDR 2 (LCDR2), and a CDR 3 (LCDR3), and the VH comprises a VH CDR 1 (HCDR1), a VH CDR 2 (HCDR2), and a VH CDR 3 (HCDR3).

[0091] The positions of the CDRs and framework regions of the antibody or antigen- binding fragment thereof can be determined using various well known definitions in the art, e.g., Kabat, Chothia, international ImMunoGeneTics database (IMGT) (on the worldwide web at imgt.cines.fr / ), and Martin (Enhanced Chothia) (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication No.91-3242; Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Martin, AC. Proteins, 25(1):130-133 (1996), Lefranc et al., Dev. Comp. Immunol., 29(3):185-203 (2005); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997), each incorporated by reference in its entirety).

[0092] The CDRs of the anti-SSPN antibody or antigen-binding fragment thereof provided herein are provided using Kabat, Chothia, IMGT, and Martin definitions (see Table 2). In some embodiments, the CDRs of the anti-SSPN antibody or antigen-binding fragment thereof may be defined by combining the CDRs using Kabat, Chothia, IMGT, and Martin definitions (see CDRs labeled as “combined” in Table 2).

[0093] In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VL comprising a LCDR1 having a sequence as set forth in SEQ ID NO: 1 (QSSETVYKNNYLSWF), a VL CDR 2 (LCDR2) having a sequence as set forth in SEQ ID NO: 2 (FLIYGASTLAS), and a VL CDR 3 (LCDR3) having a sequence as set forth in SEQ ID NO: 3 (GGGFSSSSDDT); and the VH comprises a VH CDR 1 (HCDR1) having a sequence as set forth in SEQ ID NO: 4 (GFSLSSYAMS), a VH CDR 2 (HCDR2) having a sequence as set forth in SEQ ID NO: 5 (YIGIINAAGSAYYARWVNG), and a VH CDR 3 (HCDR3) having a sequence as set forth in SEQ ID NO: 6 (VRGSIPYSGGVSL).

[0094] In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VL comprising a LCDR1 having a sequence as set forth in SEQ ID NO: 31 (VRGSIPYSGGVSL), a LCDR2 having a sequence as set forth in SEQ ID NO: 32UCH-38625 (LLIYGASTLAS), and a LCDR3 having a sequence as set forth in SEQ ID NO: 33 (GGGYDTSRDDV); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 34 (GFSLSTYAMS), a HCDR2 having a sequence as set forth in SEQ ID NO: 35 (YIGIINTGGSAYYASWAEG), and a HCDR3 having a sequence as set forth in SEQ ID NO: 36 (VRGSIAYTAGVTL).

[0095] In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VL comprising a LCDR1 having a sequence as set forth in SEQ ID NO: 61 (QASQSVYKNNYLSWF), a LCDR2 having a sequence as set forth in SEQ ID NO: 62 (LLIYGASTLAS), and a LCDR3 having a sequence as set forth in SEQ ID NO: 63 (GGGDASSSSDDA); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 64 (GFSLSNYAMS), a HCDR2 having a sequence as set forth in SEQ ID NO: 65 (YIGIINTGGSAYYASWAEG), and a HCDR3 having a sequence as set forth in SEQ ID NO: 66 (IINTAGSAYYARWVNG).

[0096] In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VL comprising a LCDR1 having a sequence as set forth in SEQ ID NO: 91 (QSSESVYKNNYLSWF), a LCDR2 having a sequence as set forth in SEQ ID NO: 92 (LLIYGASTLAS), and a LCDR3 having a sequence as set forth in SEQ ID NO: 93 (GGGYDTSRDDV); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 94 (GFSLSTYAMS), a HCDR2 having a sequence as set forth in SEQ ID NO: 95 (YIGIINTGGSAYYASWAKG), and a HCDR3 having a sequence as set forth in SEQ ID NO: 96 (VRGSIAYTAGVTL).

[0097] In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VL comprising a LCDR1 having a sequence as set forth in SEQ ID NO: 121 (QSSESVYNNNYLSWF), a LCDR2 having a sequence as set forth in SEQ ID NO: 122 (LLIYGISTLAS), and a LCDR3 having a sequence as set forth in SEQ ID NO: 123 (GGGYSTSSDDT); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 124 (GFSLSSYAMS), a HCDR2 having a sequence as set forth in SEQ ID NO: 125 (YIGIINAGGIPYYANWVNG), and a HCDR3 having a sequence as set forth in SEQ ID NO: 126 (LWGPGTLVTVSS).

[0098] In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VL comprising a LCDR1 having a sequence as set forth in SEQ ID NO:UCH-38625 151 (QSSETVYKNNYLSWF), a LCDR2 having a sequence as set forth in SEQ ID NO: 152 (FLIYGASTLAS), and a LCDR3 having a sequence as set forth in SEQ ID NO: 153 (GGGFSSSSDDT); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 154 (GFSLSSYAMS), a HCDR2 having a sequence as set forth in SEQ ID NO: 155 (YIGIINAAGSAYYARWVNG), and a HCDR3 having a sequence as set forth in SEQ ID NO: 156 (VRGSIPYSGGVSL).

[0099] In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VL comprising LCDR1 having a sequence as set forth in SEQ ID NO: 181 (QSSESVYKNNYLSWF), a LCDR2 having a sequence as set forth in SEQ ID NO: 182 (LLIYGASTLAS), and a LCDR3 having a sequence as set forth in SEQ ID NO: 183 (CGGGYSTSSDDT); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 184 (GFSLSSYAVS), a HCDR2 having a sequence as set forth in SEQ ID NO: 185 (IVNTAGSAYYANWVNG), and a HCDR3 having a sequence as set forth in SEQ ID NO: 186 (VRGSIAYTAGVAL).

[0100] In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VL comprising a LCDR1 having a sequence as set forth in SEQ ID NO: 211 (RASQSLVHSNGNTYLHWY), a LCDR2 having a sequence as set forth in SEQ ID NO: 212 (LLIYKVSNRFF), and a LCDR3 having a sequence as set forth in SEQ ID NO: 213 (SQSEHVWT); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 214 (GYSITSDYAWN), a HCDR2 having a sequence as set forth in SEQ ID NO: 215 (WMAYITYTGRTLYNPSLES), and a HCDR3 having a sequence as set forth in SEQ ID NO: 216 (ARSFAY).

[0101] In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VL comprising a LCDR1 having a sequence as set forth in SEQ ID NO: 241 (RASQSLVHSNGNTYLH), a LCDR2 having a sequence as set forth in SEQ ID NO: 242 (LLIYKVSNRFF), and a LCDR3 having a sequence as set forth in SEQ ID NO: 243 (SQSEHVWT); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 244 (GYSITSDYAWN), a HCDR2 having a sequence as set forth in SEQ ID NO: 245 (WMAYITYTGRTLYNPSLES), and a HCDR3 having a sequence as set forth in SEQ ID NO: 246 (ARSFAY).UCH-38625

[0102] In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VL comprising a LCDR1 having a sequence as set forth in SEQ ID NO: 271 (KSSQSLLKSRTRKNHLAWY), a LCDR2 having a sequence as set forth in SEQ ID NO: 272 (LLIYWASTRES), and a LCDR3 having a sequence as set forth in SEQ ID NO: 273 (KQSYDLYT); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 274 (GFTFSDYAMA), a HCDR2 having a sequence as set forth in SEQ ID NO: 275 (WIAFLSNLAKNVYYADTVAG), and a HCDR3 having a sequence as set forth in SEQ ID NO: 276 (ARGNGDYHAMDY).

[0103] In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VL comprising a LCDR1 having a sequence as set forth in SEQ ID NO: 331 (KSSQSLLKSRTRRNYLA), a LCDR2 having a sequence as set forth in SEQ ID NO: 332 (LLIYWASTRES), and a LCDR3 having a sequence as set forth in SEQ ID NO: 333 (KQSYDLYT); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 334 (GFTFSDYGMA), a HCDR2 having a sequence as set forth in SEQ ID NO: 335 (WIAFISNLAYNIYYADTVTG), and a HCDR3 having a sequence as set forth in SEQ ID NO: 336 (ARGNGNYDAVDY).

[0104] In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VL comprising a LCDR1 having a sequence as set forth in SEQ ID NO: 361 (KSSQSLLKSRTRRNYLAWY), a LCDR2 having a sequence as set forth in SEQ ID NO: 362 (LLIYWASTRES), and a LCDR3 having a sequence as set forth in SEQ ID NO: 363 (KQSYDLYT); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 364 (GFTFSDYGMA), a HCDR2 having a sequence as set forth in SEQ ID NO: 365 (WIAFISNLAYNIYYADTVTG), and a HCDR3 having a sequence as set forth in SEQ ID NO: 366 (ARGNGNYDAVDY).

[0105] In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VL comprising a LCDR1 having a sequence as set forth in SEQ ID NO: 391 (KSSQSLLKSRTRKNYLAWY), a LCDR2 having a sequence as set forth in SEQ ID NO: 392 (LLIYWASTRES), and a LCDR3 having a sequence as set forth in SEQ ID NO: 393 (KQSYDLYT); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 394 (GFTFSDYAMA), a HCDR2 having a sequence as set forth in SEQ ID NO: 395UCH-38625 (WVTFISDLAYNIYYADTVT), and a HCDR3 having a sequence as set forth in SEQ ID NO: 396 (ARGNGDYDAMDY).

[0106] In some embodiments, the antibody, or antigen binding fragment thereof, comprises a VL comprising at least one framework region (VL FR) and a VH comprising at least one framework region (VH FR).

[0107] In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VL comprising a VL framework region 1 (FR1) having a sequence selected from SEQ ID NOs: 449, 457, 465, 473, 481, 489, 497, 505, 513, 521, 529, 537, 545, 553, 561, 569, 577, 585, 593, 601, 609, 617, 625, 633, 641, 649, 657, 665, 673, 681, 689, 697, 705, 713, 721, 729, 737, 745, 753, 761, 769, 777, 785, 793, 801, 809, 817, 825, 833, 841, 849, 857, 865, 873, 881, and 889, a VL framework region 2 (FR2) having a sequence selected from SEQ ID NOs: 450, 458, 466, 474, 482, 490, 498, 506, 514, 522, 530, 538, 546, 554, 562, 570, 578, 586, 594, 602, 610, 618, 626, 634, 642, 650, 658, 666, 674, 682, 690, 698, 706, 714, 722, 730, 738, 746, 754, 762, 770, 778, 786, 794, 802, 810, 818, 826, 834, 842, 850, 858, 866, 874, 882, and 890, a VL framework region 3 (FR3) having a sequence selected from SEQ ID NOs: 451, 459, 467, 475, 483, 491, 499, 507, 515, 523, 531, 539, 547, 555, 563, 571, 579, 587, 595, 603, 611, 619, 627, 635, 643, 651, 659, 667, 675, 683, 691, 699, 707, 715, 723, 731, 739, 747, 755, 763, 771, 779, 787, 795, 803, 811, 819, 827, 835, 843, 851, 859, 867, 875, 883, and 891, and a VL framework region 4 (FR4) having a sequence selected from SEQ ID NOs: 452, 460, 468, 476, 484, 492, 500, 508, 516, 524, 532, 540, 548, 556, 564, 572, 580, 588, 596, 604, 612, 620, 628, 636, 644, 652, 660, 668, 676, 684, 692, 700, 708, 716, 724, 732, 740, 748, 756, 764, 772, 780, 788, 796, 804, 812, 820, 828, 836, 844, 852, 860, 868, 876, 884, and 892 (as shown in Table 2).

[0108] In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VH comprising a VH framework region 1 (FR1) having a sequence selected from SEQ ID NOs: 453, 461, 469, 477, 485, 493, 501, 509, 517, 525, 533, 541, 549, 557, 565, 573, 581, 589, 597, 605, 613, 621, 629, 637, 645, 653, 661, 669, 677, 685, 693, 701, 709, 717, 725, 733, 741, 749, 757, 765, 773, 781, 789, 797, 805, 813, 821, 829, 837, 845, 853, 861, 869, 877, 885, and 893, a VH framework region 2 (FR2) having a sequence selected from SEQ ID NOs: 454, 462, 470, 478, 486, 494, 502, 510, 518, 526, 534, 542, 550, 558, 566, 574, 582, 590, 598, 606, 614, 622, 630, 638, 646, 654, 662, 670, 678, 686, 694,UCH-38625 702, 710, 718, 726, 734, 742, 750, 758, 766, 774, 782, 790, 798, 806, 814, 822, 830, 838, 846, 854, 862, 870, 878, 886, and 894, a VH framework region 3 (FR3) having a sequence selected from SEQ ID NOs: 455, 463, 471, 479, 487, 495, 503, 511, 519, 527, 535, 543, 551, 559, 567, 575, 583, 591, 599, 607, 615, 623, 631, 639, 647, 655, 663, 671, 679, 687, 695, 703, 711, 719, 727, 735, 743, 751, 759, 767, 775, 783, 791, 799, 807, 815, 823, 831, 839, 847, 855, 863, 871, 879, 887, and 895, and a VH framework region 4 (FR4) having a sequence selected from SEQ ID NOs: 456, 464, 472, 480, 488, 496, 504, 512, 520, 528, 536, 544, 552, 560, 568, 576, 584, 592, 600, 608, 616, 624, 632, 640, 648, 656, 664, 672, 680, 688, 696, 704, 712, 720, 728, 736, 744, 752, 760, 768, 776, 784, 792, 800, 808, 816, 824, 832, 840, 848, 856, 864, 872, 880, 888, and 896 (as shown in Table 2).

[0109] In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VH comprising an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, or 447 (Table 2). In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VH comprising the amino acid sequence of SEQ ID NO: 421. In some embodiments, the anti- SSPN antibody, or antigen-binding fragment thereof, comprises a VH comprising the amino acid sequence of SEQ ID NO: 423. In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VH comprising the amino acid sequence of SEQ ID NO: 425. In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VH comprising the amino acid sequence of SEQ ID NO: 427. In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VH comprising the amino acid sequence of SEQ ID NO: 429. In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VH comprising the amino acid sequence of SEQ ID NO: 431. In some embodiments, the anti- SSPN antibody, or antigen-binding fragment thereof, comprises a VH comprising the amino acid sequence of SEQ ID NO: 433. In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VH comprising the amino acid sequence of SEQ ID NO: 435. In some embodiments, the anti-SSPN antibody, or antigen-bindingUCH-38625 fragment thereof, comprises a VH comprising the amino acid sequence of SEQ ID NO: 437. In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VH comprising the amino acid sequence of SEQ ID NO: 439. In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VH comprising the amino acid sequence of SEQ ID NO: 441. In some embodiments, the anti- SSPN antibody, or antigen-binding fragment thereof, comprises a VH comprising the amino acid sequence of SEQ ID NO: 443. In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VH comprising the amino acid sequence of SEQ ID NO: 445. In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VH comprising the amino acid sequence of SEQ ID NO: 447.

[0110] In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VL comprising an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, or 448 (Table 2). In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VL comprising the amino acid sequence of SEQ ID NO: 422. In some embodiments, the anti- SSPN antibody, or antigen-binding fragment thereof, comprises a VL comprising the amino acid sequence of SEQ ID NO: 424. In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VL comprising the amino acid sequence of SEQ ID NO: 426. In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VL comprising the amino acid sequence of SEQ ID NO: 428. In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VL comprising the amino acid sequence of SEQ ID NO: 430. In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VL comprising the amino acid sequence of SEQ ID NO: 432. In some embodiments, the anti- SSPN antibody, or antigen-binding fragment thereof, comprises a VL comprising the amino acid sequence of SEQ ID NO: 434. In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VL comprising the amino acid sequence of SEQ ID NO: 436. In some embodiments, the anti-SSPN antibody, or antigen-bindingUCH-38625 fragment thereof, comprises a VL comprising the amino acid sequence of SEQ ID NO: 438. In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VL comprising the amino acid sequence of SEQ ID NO: 440. In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VL comprising the amino acid sequence of SEQ ID NO: 442. In some embodiments, the anti- SSPN antibody, or antigen-binding fragment thereof, comprises a VL comprising the amino acid sequence of SEQ ID NO: 444. In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VL comprising the amino acid sequence of SEQ ID NO: 446. In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a VL comprising the amino acid sequence of SEQ ID NO: 448.

[0111] In some embodiments, the VL and the VH comprise amino acid sequences having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequences selected from: SEQ ID NOs: 421 and 422, respectively; SEQ ID NOs: 423 and 424, respectively; SEQ ID NOs: 425 and 426, respectively; SEQ ID NOs: 427 and 428, respectively; SEQ ID NOs: 429 and 430, respectively; SEQ ID NOs: 431 and 432, respectively; SEQ ID NOs: 433 and 434, respectively; SEQ ID NOs: 435 and 436, respectively; SEQ ID NOs: 437 and 438, respectively; SEQ ID NOs: 439 and 440, respectively; SEQ ID NOs: 441 and 442, respectively; SEQ ID NOs: 443 and 444, respectively; SEQ ID NOs: 445 and 446, respectively; and SEQ ID NOs: 447 and 448, respectively.

[0112] In some embodiments, the VL and the VH comprise amino acid sequences selected from: SEQ ID NOs: 421 and 422, respectively; SEQ ID NOs: 423 and 424, respectively; SEQ ID NOs: 425 and 426, respectively; SEQ ID NOs: 427 and 428, respectively; SEQ ID NOs: 429 and 430, respectively; SEQ ID NOs: 431 and 432, respectively; SEQ ID NOs: 433 and 434, respectively; SEQ ID NOs: 435 and 436, respectively; SEQ ID NOs: 437 and 438, respectively; SEQ ID NOs: 439 and 440, respectively; SEQ ID NOs: 441 and 442, respectively; SEQ ID NOs: 443 and 444, respectively; SEQ ID NOs: 445 and 446, respectively; and SEQ ID NOs: 447 and 448, respectively.UCH-38625 Heavy chain and Light chain constant regions

[0113] In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a heavy chain constant region.

[0114] In some embodiments, the heavy chain constant region comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 900. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 900.

[0115] In some embodiments, the heavy chain constant region comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 901. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 901.

[0116] In some embodiments, the heavy chain constant region comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 902. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 902.

[0117] In some embodiments, the heavy chain constant region comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 903. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 903.

[0118] In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a light chain constant region.

[0119] In some embodiments, the light chain constant region comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 897. In some embodiments, the light chain constant region comprises the amino acid sequence of SEQ ID NO: 897.UCH-38625

[0120] In some embodiments, the light chain constant region comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 898. In some embodiments, the light chain constant region comprises the amino acid sequence of SEQ ID NO: 898.

[0121] In some embodiments, the light chain constant region comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 899. In some embodiments, the light chain constant region comprises the amino acid sequence of SEQ ID NO: 899.

[0122] In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a heavy chain constant region comprising the amino acid sequence as set forth in SEQ ID NO: 900 and a light chain constant region comprising the amino acid sequence as set forth in SEQ ID NO: 897. In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a heavy chain constant region comprising the amino acid sequence as set forth in SEQ ID NO: 900 and a light chain constant region comprising the amino acid sequence as set forth in SEQ ID NO: 898. In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a heavy chain constant region comprising the amino acid sequence as set forth in SEQ ID NO: 901 and a light chain constant region comprising the amino acid sequence as set forth in SEQ ID NO: 899. In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a heavy chain constant region comprising the amino acid sequence as set forth in SEQ ID NO: 902 and a light chain constant region comprising the amino acid sequence as set forth in SEQ ID NO: 899. In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a heavy chain constant region comprising the amino acid sequence as set forth in SEQ ID NO: 903 and a light chain constant region comprising the amino acid sequence as set forth in SEQ ID NO: 899. Heavy chain and light chain regions

[0123] In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, comprises a heavy chain sequence.UCH-38625

[0124] In some embodiments, the antibody, or antigen binding fragment thereof, comprises a heavy chain (HC) and a light chain (LC) comprising amino acid sequences having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequences selected from: SEQ ID NOs: 904 and 918, respectively; SEQ ID NOs: 905 and 919, respectively; SEQ ID NOs: 906 and 920, respectively; SEQ ID Nos: 907 and 921, respectively; SEQ ID NOs: 908 and 922, respectively; SEQ ID NOs: 909 and 923, respectively; SEQ ID NOs: 910 and 924, respectively; SEQ ID NOs: 911 and 925, respectively; SEQ ID NOs: 912 and 926, respectively; SEQ ID NOs: 913 and 927, respectively; SEQ ID NOs: 914 and 928, respectively; SEQ ID NOs: 915 and 929, respectively; SEQ ID NOs: 916 and 930, respectively; and SEQ ID NOs: 917 and 931, respectively.

[0125] In some embodiments, the antibody, or antigen binding fragment thereof, comprises an HC and an LC wherein the HC comprises SEQ ID NO: 904 and the LC comprises SEQ ID NO: 918, respectively. In some embodiments, the antibody, or antigen binding fragment thereof, comprises an HC and an LC wherein the HC comprises SEQ ID NO: 905 and the LC comprises SEQ ID NO: 919, respectively. In some embodiments, the antibody, or antigen binding fragment thereof, comprises an HC and an LC wherein the HC comprises SEQ ID NO: 906 and the LC comprises SEQ ID NO: 920, respectively. In some embodiments, the antibody, or antigen binding fragment thereof, comprises an HC and an LC wherein the HC comprises SEQ ID NO: 907 and the LC comprises SEQ ID NO: 921, respectively. In some embodiments, the antibody, or antigen binding fragment thereof, comprises an HC and an LC wherein the HC comprises SEQ ID NO: 908 and the LC comprises SEQ ID NO: 922, respectively. In some embodiments, the antibody, or antigen binding fragment thereof, comprises an HC and an LC wherein the HC comprises SEQ ID NO: 909 and the LC comprises SEQ ID NO: 923, respectively. In some embodiments, the antibody, or antigen binding fragment thereof, comprises an HC and an LC wherein the HC comprises SEQ ID NO: 910 and the LC comprises SEQ ID NO: 924, respectively. In some embodiments, the antibody, or antigen binding fragment thereof, comprises an HC and an LC wherein the HC comprises SEQ ID NO: 911 and the LC comprises SEQ ID NO: 925, respectively. In some embodiments, the antibody, or antigen binding fragment thereof,UCH-38625 comprises an HC and an LC wherein the HC comprises SEQ ID NO: 912 and the LC comprises SEQ ID NO: 926, respectively. In some embodiments, the antibody, or antigen binding fragment thereof, comprises an HC and an LC wherein the HC comprises SEQ ID NO: 913 and the LC comprises SEQ ID NO: 927, respectively. In some embodiments, the antibody, or antigen binding fragment thereof, comprises an HC and an LC wherein the HC comprises SEQ ID NO: 914 and the LC comprises SEQ ID NO: 928, respectively. In some embodiments, the antibody, or antigen binding fragment thereof, comprises an HC and an LC wherein the HC comprises SEQ ID NO: 915 and the LC comprises SEQ ID NO: 929, respectively. In some embodiments, the antibody, or antigen binding fragment thereof, comprises an HC and an LC wherein the HC comprises SEQ ID NO: 916 and the LC comprises SEQ ID NO: 930, respectively. In some embodiments, the antibody, or antigen binding fragment thereof, comprises an HC and an LC wherein the HC comprises SEQ ID NO: 917 and the LC comprises SEQ ID NO: 931, respectively. Anti-SSPN polyclonal antibodies

[0126] In some aspects, the present disclosure provides polyclonal antibodies that specifically bind to at least one epitope of SSPN. In some embodiments, the polyclonal antibodies bind to human SSPN, mouse SSPN, or both mouse and human SSPN (Table 1). In some embodiments, the polyclonal antibodies bind to a region within an N-terminal fragment (i.e., an N-terminal epitope) of SSPN. In some embodiments, the polyclonal antibodies bind to a region within an N-terminal fragment of mouse SSPN. In some embodiments, the polyclonal antibodies target a region within a C-terminal fragment (i.e., an C-terminal epitope) of SSPN. In some embodiments, the polyclonal antibodies target a region within a C-terminal fragment of mouse SSPN.

[0127] In some embodiments, the polyclonal antibodies are heterogenous populations of antibody molecules derived from the sera of immunized animals. In some embodiments, unfractionated immune serum can be used for the methods disclosed herein. Methods of producing anti-SSPN antibodies

[0128] Methods of producing monoclonal or polyclonal antibodies that react specifically with antigens can be prepared using standard methodologies. For example,UCH-38625 preparation of monoclonal antibodies by immunizing mice with an appropriate immunogen is described in, e.g., Coligan, Current Protocols in Immunology (1991): Harlow & Lane, ANTIBODIES, A LABORATORY MANUAL, Cold Spring harbor Publication, New York (1988); Goding, Monoclonal Antibodies: Principles and Practice (2d ed.1986); Kohler & Milstein, Nature 256:495497 (1975). Antibody preparation by selection of antibodies from libraries of nucleic acids encoding recombinant antibodies packaged in phage or similar vectors is described in, e.g., Huse, et al., Science 246:1275-1281 (1989) and Ward, et al., Nature 341:544-546 (1989). In addition, antibodies can be produced recombinantly using methods known in the art and described in e.g., Sambrook, et al., Molecular Cloning, A laboratory Manual (2nd ed.1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994).

[0129] The antibodies or antigen-binding fragments thereof described herein can be produced using a variety of techniques known in the art of molecular biology and protein chemistry. For example, a nucleic acid encoding one or both of the heavy and light chain polypeptides of an antibody can be inserted into an expression vector that contains transcriptional and translational regulatory sequences, which include, e.g., promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, transcription terminator signals, polyadenylation signals, and enhancer or activator sequences. The regulatory sequences include a promoter and transcriptional start and stop sequences. In addition, the expression vector can include more than one replication system such that it can be maintained in two different organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification.

[0130] Several possible vector systems are available for the expression of cloned heavy chain and light chain polypeptides from nucleic acids in mammalian cells. One class of vectors relies upon the integration of the desired gene sequences into the host cell genome. Cells which have stably integrated DNA can be selected by simultaneously introducing drug resistance genes such as E. coli gpt (Mulligan and Berg (1981) Proc Natl Acad Sci USA 78:2072) or Tn5 neo (Southern and Berg (1982) MolAppl Genet1:327). The selectable marker gene can be either linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection (Wigler et al. (1979) Cell 16:77). A second class of vectors utilizes DNA elements which confer autonomously replicating capabilities to anUCH-38625 extrachromosomal plasmid. These vectors can be derived from animal viruses, such as bovine papillomavirus (Sarver et al. (1982) Proc Natl Acad Sci USA, 79:7147), cytomegalovirus, polyoma virus (Deans et al. (1984) Proc Natl Acad Sci USA 81: 1292), or SV40 virus (Lusky and Botchan (1981) Nature 293:79).

[0131] The expression vectors can be introduced into cells in a manner suitable for subsequent expression of the nucleic acid. The method of introduction is largely dictated by the targeted cell type, discussed below. Exemplary methods include CaP04 precipitation, liposome fusion, cationic liposomes, electroporation, viral infection, dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct microinjection. Appropriate host cells for the expression of antibodies or antigen-binding fragments thereof include yeast, bacteria, insect, plant, and mammalian cells. Of particular interest are bacteria such as E. coli, fungi such as Saccharomyces cerevisiae and Pichia pastoris, insect cells such as SF9, mammalian cell lines (e.g., human cell lines), as well as primary cell lines.

[0132] Following expression, the antibodies and fragments thereof can be isolated. An antibody or fragment thereof can be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological, and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse- phase HPLC chromatography. For example, an antibody can be purified using a standard anti-antibody column (e.g., a protein-A or protein-G column). Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. See, e.g., Scopes (1994) "Protein Purification, 3rd edition," Springer- Verlag, New York City, New York. The degree of purification necessary will vary depending on the desired use. In some instances, no purification of the expressed antibody or fragments thereof will be necessary. Methods for determining the yield or purity of a purified antibody or fragment thereof are known in the art and include, e.g., Bradford assay, UV spectroscopy, Biuret protein assay, Lowry protein assay, amido black protein assay, high pressure liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoretic methods (e.g., using a protein stain such as Coomassie Blue or colloidal silver stain).

[0133] In some embodiments, the polyclonal antibodies, or antigen-binding fragment thereof, are produced by immunization of an animal using an antigen comprising an epitopeUCH-38625 of interest (e.g., an N-terminal epitope of SSPN, a LEL epitope of SSPN, or a C-terminus epitope). In some embodiments, the antigen used to immunize a host animal is a synthetic polypeptide comprising amino acid residues 219-243 of the C-terminus of human SSPN (FIG.1A). In some embodiments, the antigen used to immunize a host animal is a synthetic polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 932. In some embodiments, the antigen used to immunize a host animal is a synthetic polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 936.

[0134] In some embodiments, the antigen used to immunize a host animal is a synthetic polypeptide comprising amino acid residues 167-186 of the LEL of human SSPN (FIG.1B). In some embodiments, the antigen used to immunize a host animal is a synthetic polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 933. In some embodiments, the antigen used to immunize a host animal is a synthetic polypeptide comprising amino acid residues 167-186 of the LEL of mouse SSPN. In some embodiments, the antigen used to immunize a host animal is a synthetic polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 934.

[0135] In some embodiments, the antigen used to immunize a host animal comprises the N-terminal fragment of mouse SSPN. In some embodiments, the antigen comprises amino acids 1-25 of the N-terminus of mouse SSPN (FIG.1B). In some embodiments, the antigen comprises SEQ ID NO: 935.

[0136] In some embodiments, the antigen used to immunize a host animal comprises the N-terminal fragment of human SSPN. In some embodiments, the antigen comprises amino acids 1-25 of the N-terminus of human SSPN. In some embodiments, the antigen comprises SEQ ID NO: 937.

[0137] In some embodiments, the synthetic polypeptide is covalently conjugated to a tag that promotes immunogenicity. In some embodiments, the synthetic polypeptide is covalently conjugated to keyhole limpet hemocyanin (KLH). In some embodiments, the antigen is fused to a tag useful for purification of the antigen (e.g., a hexahistidine-tag (his- tag, polyhistidine-tag), a streptavidin tag (strep-tag), a SBP-tag (a streptavidin binding tag), or a GST(glutathione-S-transferase)-tag). In some embodiments, the tag is a protein purification tag. In some embodiments, the antigen is fused to a protein purification tag wherein the purification tag is glutathione S-transferase (GST).UCH-38625

[0138] In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, is produced in a mammalian animal. In some embodiments, animals of Rodentia, Lagomorpha, or Primates are used. Animals of Rodentia include, for example, mouse, rat, and hamster. Animals of Lagomorpha include, for example, rabbit. Animals of Primates include, for example, a monkey of Catarrhini (old world monkey) such as Macaca fascicularis, rhesus monkey, baboon, and chimpanzees. In some embodiments, the anti-SSPN antibodies are produced in immunized rabbits. In some embodiments, the anti-SSPN antibodies are produced in immunized mice.

[0139] Methods for immunizing animals with antigens are known in the art. In some embodiments, intramuscular, intraperitoneal, subcutaneous injection, or any combination thereof, of the antigen is used for immunization of the host animal. In some embodiments, the antigen may be administered with an appropriate amount of a standard adjuvant, such as Freund's complete adjuvant, using a protocol for inducing high titer antibodies specific to SSPN as described in Crosbie et al., J Cell Biol 1999, 145, 153-165; Crosbie, et al., Hum Mol Genet 2000, 9, 2019-2027; Peter, et al., J Cell Biol 2008, 183, 419-427; and Marshall, et al., J Cell Biol 2012, 197, herein incorporated by reference in their entireties. In some embodiments, booster compositions of the antigen may be administered to the animals until the anti-SSPN antibody titer reaches a plateau with Freund’s incomplete adjuvant.

[0140] In some embodiments, after immunization, serum is examined by a standard method for an increase in the amount of desired antibodies (e.g., enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, immunofluorescence, or any combination thereof). In some embodiments, antibody titers are analyzed using ELISA (as shown in FIG.11). In some embodiments, the immune response of the host animal to immunization with the antigen is evaluated using immunoblotting of non-purified sera blotted with total protein lysates one or more model organisms expressing varying levels of SSPN (e.g., total protein lysate of muscle tissue from wild-type, mdx, SSPN-TG and SSPN-null mice, FIG.2B). In some embodiments, anti-SSPN antibodies are affinity-purified for evaluating the binding specificity to SSPN using immunofluorescence analyses (IFA) (FIG. 3C).

[0141] In some embodiments, the animal is boosted with the same antigen of interest, but conjugated to a different protein and / or through a different cross-linking reagent.UCH-38625 Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.

[0142] In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, is generated using standard hybridoma generation methodologies as described in Kohler, et al., Nature 1975, 256, 495-497, doi:10.1038 / 256495a0, herein incorporated by reference in its entirety.

[0143] In some embodiments, the anti-SSPN antibody, or antigen-binding fragment thereof, exhibits robust specific signal with minimal background in immunoblotting and IFA (as shown in FIG.2A, FIG.2B, FIG.3B, and FIG.3C).

[0144] In some embodiments, single cell sorting technology (e.g., fluorescence- activated cell sorting (FACS) is used to screen antibody candidates collected from cells (e.g., B-cells) from an immunized animal. In some embodiments, antibody candidates that exhibit highest titers, measured using ELISA (as shown in FIGs.5A-5B, FIG.12, Table 6, Table 7, and Table 8), are selected for evaluation using immunoblotting with total muscle lysate samples from mice expressing varying levels of SSPN (Table 5 and FIGs.13A-13B). In some embodiments, antibody candidates exhibiting recognition of SSPN using immunoblotting are evaluated using IFA analysis (Table 7 and Table 12). In some embodiments, antibodies exhibiting robust signal-to-noise using IFA are selected for RNA extraction, RT-PCR, cloning of variable regions of IgG genes and construction of a linear expression module (LEM). In some embodiments, the LEM for each antibody candidate is expressed in HEK 293-F cells, purified, and evaluated for retention of anti-SSPN activity using ELISA, immunoblotting, IFA, or any combination thereof. In some embodiments, antibody candidates exhibiting robust signal-to-noise ratio using immunoblotting and IFA are selected for hybridoma generation for subcloning and expansion. Methods of detecting SSPN

[0145] In some aspects, the present disclosure provides a method of detecting SSPN in a sample. In some embodiments, the method of detecting SSPN comprises contacting the sample with an anti-SSPN antibody or antigen-binding fragment herein described and determining the presence or absence of SSPN in the sample. In some embodiments, the sample comprises a cell, a tissue, a biological fluid, an aqueous suspension, or a living organism such as a mammalian model of disease or a human. In some embodiments, theUCH-38625 sample comprises skeletal muscle cells, smooth muscle cells, cardiomyocytes, adipocytes, kidney epithelial cells, immune cells, chondrocytes, fibroblasts, plasma cells, tumor cells, neurons, or any SSPN expressing entity. In some embodiments, the sample comprises skeletal muscle cells, smooth muscle cells, cardiomyocytes, adipocytes, kidney epithelial cells, immune cells, chondrocytes, fibroblasts, plasma cells, tumor cells, or neurons. In some embodiments, the sample comprises any SSPN expressing entity.

[0146] In some embodiments, determining the presence or absence of SSPN in the sample comprises the use of an immunoassay. In some embodiments, determining the presence or absence of SSPN in the sample comprises the use of an immunoblotting assay, an immunoprecipitation assay, an immunofluorescence assay, or an enzyme-linked immunosorbent assay (ELISA) assay.

[0147] In some embodiments, contacting a sample with the anti-SSPN antibody, or antigen-binding fragment thereof, of the present disclosure results in increased signal-to- noise ratio, improved sensitivity, improved binding specificity, compared to anti-SSPN antibodies known in the art. In some embodiments, detecting SSPN in a sample comprises in vivo imaging. In some embodiments, the anti-SSPN antibody is used for treatment of a disease and / or a condition in a subject. Methods of Use

[0148] In some aspects, this disclosure provides methods for providing therapy to a subject and / or treating a disease or disorder comprising administering to a subject any of the herein provided anti-SSPN antibodies.

[0149] In some embodiments, the disclosure provides methods for preventing, treating, and / or ameliorating a disease or disorder associated with dysregulation of cellular matrix (e.g., extracellular matrix (ECM) dysregulation). Cellular matrix dysregulation can develop into serious pathology including, but not limited to fibrosis, insulin resistance, and / or cancer metastasis. For example, increased ECM deposition contributes to the pathogenesis of fibrosis and development of obesity-associated metabolic complications. Elevated levels of cell-ECM adhesion proteins have been associated with malignant diseases in a variety of tissues and organs, contributing to increased cell proliferation, resistance to apoptosis, elevated cell motility and invasion, and promotion of angiogenesis.UCH-38625

[0150] In some embodiments, the method comprises targeting SSPN with an anti- SSPN antibody or antigen-binding fragment thereof as an ECM component to prevent aberrant ECM remodeling. In some embodiments, the method comprises targeting SSPN with an anti-SSPN antibody or antigen-binding fragment thereof as an ECM component to prevent aberrant ECM remodeling in a subject. In some embodiments, the method comprises targeting SSPN with an anti-SSPN antibody or antigen-binding fragment thereof as an ECM component to treat aberrant ECM remodeling. In some embodiments, the method comprises targeting SSPN with an anti-SSPN antibody or antigen-binding fragment thereof as an ECM component to treat aberrant ECM remodeling in a subject. In some embodiments, the method comprises targeting SSPN with an anti-SSPN antibody or antigen-binding fragment thereof as an ECM component to ameliorate aberrant ECM remodeling. In some embodiments, the method comprises targeting SSPN with an anti-SSPN antibody or antigen-binding fragment thereof as an ECM component to ameliorate aberrant ECM remodeling in a subject.

[0151] In some embodiments, the method comprises targeting SSPN with an anti- SSPN antibody or antigen-binding fragment thereof as an ECM component to prevent tumor cell invasion. In some embodiments, the method comprises targeting SSPN with an anti- SSPN antibody or antigen-binding fragment thereof as an ECM component to prevent tumor cell invasion in a subject. In some embodiments, the method comprises targeting SSPN with an anti-SSPN antibody or antigen-binding fragment thereof as an ECM component to treat tumor cell invasion. In some embodiments, the method comprises targeting SSPN with an anti-SSPN antibody or antigen-binding fragment thereof as an ECM component to treat tumor cell invasion in a subject. In some embodiments, the method comprises targeting SSPN with an anti-SSPN antibody or antigen-binding fragment thereof as an ECM component to ameliorate tumor cell invasion. In some embodiments, the method comprises targeting SSPN with an anti-SSPN antibody or antigen-binding fragment thereof as an ECM component to ameliorate tumor cell invasion in a subject.

[0152] In some embodiments, the method comprises targeting SSPN with an anti- SSPN antibody or antigen-binding fragment thereof as an ECM component to prevent obesity-associated metabolic disorders. In some embodiments, the method comprises targeting SSPN with an anti-SSPN antibody or antigen-binding fragment thereof as an ECM component to prevent obesity-associated metabolic disorders in a subject. In someUCH-38625 embodiments, the method comprises targeting SSPN with an anti-SSPN antibody or antigen- binding fragment thereof as an ECM component to treat obesity-associated metabolic disorders. In some embodiments, the method comprises targeting SSPN with an anti-SSPN antibody or antigen-binding fragment thereof as an ECM component to treat obesity- associated metabolic disorders in a subject. In some embodiments, the method comprises targeting SSPN with an anti-SSPN antibody or antigen-binding fragment thereof as an ECM component to ameliorate obesity-associated metabolic disorders. In some embodiments, the method comprises targeting SSPN with an anti-SSPN antibody or antigen-binding fragment thereof as an ECM component to ameliorate obesity-associated metabolic disorders in a subject.

[0153] In some aspects, the present disclosure provides methods for providing therapy and / or treating a disease or disorder comprising administering to a subject a composition comprising the antibody or antigen binding fragment herein disclosed. In some embodiments, the composition comprises a pharmaceutically acceptable carrier.

[0154] The above-described antibodies, antigen binding fragments thereof, or antibody-conjugates are useful in, inter alia, methods for treating or preventing a variety of diseases or disorders in a subject. The anti-SSPN antibodies can be administered to a subject, e.g., a human subject, using a variety of methods that depend, in part, on the route of administration. The route can be, e.g., intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal (IP) injection, intramuscular injection (IM), or intrathecal injection (IT). The injection can be in a bolus or a continuous infusion.

[0155] Administration can be achieved by, e.g., local infusion, injection, or by means of an implant. The composition can be delivered to the subject by way of an implantable device based on, e.g., diffusive, erodible, or convective systems, e.g., osmotic pumps, biodegradable implants, electro diffusion systems, electroosmosis systems, vapor pressure pumps, electrolytic pumps, effervescent pumps, piezoelectric pumps, erosion-based systems, or electromechanical systems.

[0156] In some embodiments, an anti-SSPN antibody or antigen-binding fragment thereof is therapeutically delivered to a subject by way of local administration.

[0157] As described above, the compositions described herein (e.g., anti-SSPN compositions) can be used to treat a variety of diseases or disorders such as but not limited to:UCH-38625 muscular dystrophy, diseases associated with ECM abnormalities encompassing the immune, musculoskeletal, cardiovascular, pulmonary, and renal systems. In some embodiments, the anti-SSPN antibody or antigen-binding fragment thereof is used to treat muscular dystrophies and / or myopathies; skin, cartilage, and bone diseases (e.g., Ehlers-Danlos syndrome, osteogenesis imperfecta, Marfan syndrome), vascular system disorders (e.g., atherosclerosis, vessel aneurysm), inflammation, fibrosis, metabolic disorders such as obesity-associated insulin resistance, and broad cellular dysregulations found in malignancy and cancer cachexia. Kits

[0158] In some embodiments, the disclosure provides a kit comprising an anti-SSPN antibody described herein. In some embodiments, a kit includes an anti-SSPN antibody as disclosed herein, and instructions for use. The kits may comprise, in a suitable container, an anti-SSPN antibody, one or more controls, and various buffers, reagents, enzymes and other standard ingredients well known in the art.

[0159] The container can include at least one vial, well, test tube, flask, bottle, syringe, or other container means, into which an anti-SSPN antibody may be placed, and in some instances, suitably aliquoted. Where an additional component is provided, the kit can contain additional containers into which this component may be placed. The kits can also include a means for containing an anti-SSPN antibody and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow- molded plastic containers into which the desired vials are retained. Containers and / or kits can include labeling with instructions for use and / or warnings.UCH-38625 Table 1. Summary of anti-SSPN antibodiesTable 2. Antibody sequencesUCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625UCH-38625EXEMPLIFICATION Example 1: Generation and characterization of anti-SSPN rabbit antibodies

[0160] Anti-SSPN polyclonal antibodies with specificity to the N-terminus of mouse SSPN were generated. Rabbit polyclonal antibody specificity to the N-terminal epitope of mouse SSPN was tested using immunoblotting (IB), immunofluorescent analysis (IFA), immunoprecipitation (IP), and enzyme-linked immunosorbent assay (ELISA). Recombinant SSPN protein production

[0161] Two recombinant SSPN fusion proteins were generated for rabbit immunizations. N-terminal epitope was selected from the fragment of mouse SSPN protein (amino acids 1-25, having amino acid sequence MGRKPSPRAQELPEEEARTCCGCRF, SEQ ID NO: 935) and was fused with Glutathione S-transferase (GST). GST-fusion to the N- terminus of mouse SSPN (GST-NT) was engineered by subcloning a cDNA fragment encoding the first 25 amino acids of murine SSPN into BamHI and EcoRI sites of the pGEX4T1 vector (Amersham Biosciences). GST-SSPN fusion protein was expressed and purified from E. coli Rosetta (DE3) by ABclonal Technology (Massachusetts, IL). Bacterial cells were grown in LB medium at 37 ℃ until they reached a cellular optimal density (OD600) in the range of 0.5 to 0.6. At this point, recombinant protein expression was induced during exponential growth by treatment with 0.4 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) at 37 ℃ for 4 h. The cells were harvested and lysed by sonication in PBS plus 1% Triton X-100. Clarified lysates were affinity purified using glutathione-sepharose column chromatography according to the manufacturer’s instructions (ThermoFisher Scientific,UCH-38625 16109). On average, 10-12mg of GST-fused SSPN protein per 300 mL of growth medium was obtained after purification.

[0162] Small Ubiquitin-like Modifier (SUMO)-fused N-terminal fragment of mouse SSPN protein (aa 1-25) for antibody screening purposes was generated by ABclonal using pET-28a SUMO expression vector. Protein expression in E. coli Rosetta (DE3) strain was induced with 0.4 mM of IPTG for 4 h at 37°C. The cells were harvested and lysed by sonication in PBS plus 1% Triton X-100. Protein purification was performed by ABclonal using immobilized nickel ion-affinity column chromatography (ThermoFisher Scientific, 90098). On average, 8-10 mg of SUMO-fused SSPN protein per 300 mL of growth medium was obtained after purification. Rabbit Immunization

[0163] Rabbit antibodies were developed against N-terminal fragment of mouse SSPN. Five female adult New Zealand white rabbits (Rabbit #: 8870, 8871, 8872, 8873, and 8874) were immunized with the purified recombinant fusion proteins described above. Rabbits were injected intramuscularly (in both quadriceps) and subcutaneously with 1 mg (total) of purified N-terminal SSPN GST-fused recombinant protein in Freund’s Complete Adjuvant. Pre-immune sera (5 mL from each rabbit) were collected from the lateral ear vein prior to immunization. After initial immunization, each rabbit received three boosters with 500 μg of antigen in Freund’s Incomplete Adjuvant subcutaneously. Bleeds I and II were collected from the lateral ear vein after the second and third booster for testing immune response to SSPN protein (FIGs.2A and 2B). Rabbits selected for generation of monoclonal antibodies received a final injection with 500μg of antigen intravenously four days before spleen collection (see Table 3 below).UCH-38625 Table 3. Immunization schedule of rabbits

[0164] Rabbits selected for extended immunization and generation of polyclonal antibodies received four additional boosters with 250μg of antigen in Freund’s Incomplete Adjuvant via subcutaneous route of administration. Production bleeds III-V were collected from the lateral ear vein after each booster (see Table 4 below). Terminal bleeds for polyclonal antibody generation were performed via cardiac puncture under anesthesia. Animal housing, immunization, and blood collection procedures were performed by ABclonal, according to the approved protocols and guidelines of the Institutional Animal Care and Use Committee (IACUC). Table 4. Immunization schedule and test blood collection of rabbits selected or extended immunization to generate polyclonal antibodies.UCH-38625Methods of detecting recombinant SSPN using indirect ELISA assay

[0165] Side-by-side comparison of bleed I and II results suggested that rabbits #8871, #8872, #8873 and #8874 had enhanced response to recombinant SSPN after subsequent booster doses (FIG.2A), while rabbit #8870 developed robust immune response after second boost and this response maintained at the same level after the third boost (FIG.2A).

[0166] To confirm whether rabbits develop immune response to SSPN, sera from bleeds I and II were analyzed by ABclonal using indirect ELISA with SUMO-tagged SSPN protein to determine anti-SSPN antibody titer. Small Ubiquitin-like Modifier (SUMO)-fused N-terminal fragment of mouse SSPN protein (aa 1-25) was created using pET-28a SUMO expression vector. Briefly, serial dilutions of antisera from bleeds I and II (original sera were first diluted at 1:1000, followed by 8-point 1:3 serial dilutions were used for analysis). 96- well plates were precoated with 25 µL / well of SUMO-tagged SSPN (0.5 µg / mL) in carbonate-bicarbonate coating buffer, pH 9.66 (Sigma, SRE0102) overnight. After three rounds of washing with washing buffer (sodium phosphate-buffer pH 7.4 (ThermoFisher Scientific, J62152-K2) with 0.1% Tween-20), plates were blocked with 3% BSA in sodium phosphate-buffer, pH 7.4 for 2 h, and then probed with serial dilutions of antisera in carbonate-bicarbonate coating buffer, pH 9.66 and incubated for 1 h at 37 ℃. After three rounds of washing, a peroxidase-conjugated goat anti-rabbit IgG antibody (Jackson ImmunoResearch, 111-005-046) at 1:5,000 in dilution buffer (1% BSA in sodium phosphate- buffer pH 7.4 with 0.1% Tween-20) was added to the plate and incubated for 1h at RT. The plate was washed four times as described above. The enzymatic reaction was conducted with 100 µL / well in ready-to-use tetramethylbenzidine (TMB) solution (ThermoFisher Scientific, N301) at RT and stopped by 50 µL / well of 2M sulfuric acid. The optical density was measured at 450 nm using SpectraMax M2 microplate reader (Molecular Devices) (FIG.2A).UCH-38625 Methods used for immunoblotting for detection of SSPN in skeletal muscle protein lysate

[0167] To generate protein lysates, quadriceps muscles were harvested from 12-15 weeks old male mice of each genotype, snap-frozen in liquid nitrogen, pulverized under liquid nitrogen and homogenized on ice in 30x volume of RIPA buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% Na deoxycholate (DOC), 0.1% sodium dodecyl sulfate (SDS) with protease inhibitor cocktail (ThermoFisher, 1860932), and phosphatase inhibitor cocktail (ThermoFisher Scientific, 78428). Lysates were incubated for 1h at 4oC with rotation and clarified by centrifugation at 18,000xg for 15min. Protein concentrations of clarified supernatants were quantified using the DC Protein Assay (Bio- Rad, 5000114). Supernatants (20 µg of total protein) were separated by SDS-PAGE on 12 % Bolt Bis-Tris gels (ThermoFisher Scientific, NW00122) in MOPS SDS Running Buffer (Invitrogen, B000102). PageRuler Plus Prestained Protein Ladder (ThermoFisher Scientific, 26619) was used as a molecular weight marker. Immunoblotting was performed using the following procedure. nitrocellulose membranes (Li-Cor Biosciences, 926-31092) were blocked with blocking buffer (5% nonfat dry milk (Nestle Carnation) in Tris-buffer, pH 7.4 (ThermoFisher Scientific, 28379) with 0.1% Tween-20 (TBS-T)) followed by incubation with primary antibodies (rabbit bleeds, purified polyclonal antibodies or supernatants from rabbit B-cell clones) overnight at 4oC with gentle rotation. Rabbit bleeds I and II were diluted in the blocking buffer 1:150 and 1:600 respectively (FIG.2B), purified polyclonal and monoclonal antibodies were tested at 0.5-1µg / mL concentrations (FIG.3B and FIG.4A), supernatants from intermediate B-cell rabbit clones during monoclonal antibody generation were diluted 1:5 in blocking buffer. Membranes were washed three times 5 min each in TBS-T before applying secondary antibody, horseradish peroxidase-conjugated anti-mouse (Abcam, ab6708) or anti-rabbit IgG (Abcam, ab6721), 1:30,000 in blocking buffer. The signal was detected with SuperSignal West Pico Plus Chemiluminescence kit (ThermoFisher Scientific, 34580) on Blue Basic Autoradiography Film (GeneMate, F-9023-8X10). Indirect immunofluorescent analysis (IFA) methods for detection of SSPN in transverse cryosections of human and mouse skeletal muscle

[0168] Human muscle tissue was obtained from healthy 13-year-old male left hamstring tissue from an anterior cruciate ligament (ACL) reconstruction. Mouse quadricepsUCH-38625 muscle tissues were frozen in liquid nitrogen-cooled isopentane, mounted on Tissue Plus OCT (Fisher HealthCare, 4585) and stored at -80oC. Transverse cryosections (10 μm in thickness) were mounted on glass slides and were blocked with 3% BSA at room temperature (RT) for 1 h, followed by incubation with avidin / biotin blocking kit according to the manufacture’s protocol (Vector Laboratories, SP-2001). Sections were washed with PBS, pH 7.4 (Fisher BioReagents, BP399-4) and incubated with primary antibodies overnight at 4°C. Primary antibodies were detected with species-specific biotinylated secondary antibodies give the information on these antibodies (Vector Laboratories, BA-1000) by incubating for 1h at RT. Bound antibodies were visualized by incubation with fluorescein-conjugated avidin D (Vector Laboratories, A-2001). Slides were wet mounted in Vectashield with (Vector Laboratories, H-1200) or without DAPI (Vector Laboratories, H-1000) before analysis by microscopy on Zeiss Axio Observer 7 or Axio Imager M2 (Carl Zeiss). Images were captured with Hammatsu ORCA-Flash 4.0 V3 digital complementary metal oxide semiconductor camera and either EC Plan-Neofluar 10x / 0.30 Ph1 or Plan-Aprochromat 20x / 0.8 M27 objectives (FIG.3C, FIG.4B, FIG.6B, FIG.7, and FIG.10). SSPN B-Cell linear epitope prediction

[0169] Identification of possible B-cell epitopes within the SSPN protein (FIG.1A and 1B), was performed using Bepipred Linear Epitope Prediction 2.0 tool from IEDB Analysis Resource available on the world wide web at (http: / / tools.iedb.org / ). Mouse SSPN (GenBank accession number U02487) and human SSPN (GenBank accession number AF016028) amino-acid sequence by using this tool (FIG.9A and 9B). Isolation and sorting of rabbit B-cells

[0170] Isolation and sorting of rabbit immune cells were performed by ABclonal using said company’s proprietary technology. The antigen-binding cells from the rabbit spleen were isolated by fluorescent-activated cell sorting (FACS) using an anti-IgM stain to identify naive B cells. Concurrent staining was performed with antigen labelled with two spectrally-distinct fluorophores for identification of antigen-specific B cells. Antigen-specific IgG+ / IgM- B cells were sorted and a single cell was placed into individual wells of 96-well plates. A total of twenty 96-well plates of B-cells were isolated, providing a potential 1,920UCH-38625 clones. All 1,920 B-cell clones were expanded for 12 days with ABclonal Technology’s proprietary cell culture system. All B-cell supernatants were screened in the ELISA for reactivity to the SSPN recombinant SUMO-tagged antigen by ABClonal. B-cell supernatants from the top 96 positive clones with the highest reactivity in ELISA were sent to UCLA and subjected to validation by immunoblotting and IFA. Based on the results of three assays, the top ten clones were selected for IgG gene isolation and generation of the linear expression modules. Generation of linear expression module (LEM)

[0171] Total RNA was purified from each preserved B-cell pellet for selected clones by ABclonal. Isolation of mRNA and amplification of cDNA fragments of IgG were performed by reverse transcription polymerase chain reaction (RT-PCR). Variable regions of heavy and light chains were amplified using cDNA after reverse transcription by ABclonal and used for construction of LEMs using protocol described in Clargo AM, Hudson AR, Ndlovu W, Wootton RJ, Cremin LA, O'Dowd VL, et al. (2014) The rapid generation of recombinant functional monoclonal antibodies from individual, antigen-specific bone marrow-derived plasma cells isolated using a novel fluorescence-based method. MAbs 6: 143–159.27044; doi: 10.4161 / mabs.27044 PMID: 24423622, and modified by ABclonal. The LEM construction was performed by Abclonal according to Abclonal protocol and involved inserting the Ig heavy chain and light chain genes into a linear construct for transient transfection into mammalian cells. This allowed expression and screening of a greater number of clones (due to reduced subcloning relative to methods that rely on generating expression vectors). Characterization of rabbit polyclonal anti-SSPN antibodies

[0172] To evaluate specific response to native SSPN protein, immunoblotting of non- purified rabbit sera with total protein lysates generated from muscles of wild-type mice, mdx mice, SSPN-TG mice, and SSPN-null mice (FIG.2B). These mouse lines exhibit various levels of SSPN protein expression (as shown in Table 5 below), from no expression in SSPN- nulls, reduced expression (compared to wild-type levels) in mdx to high-level expression (~30-fold vs wild-type) in SSPN-TG.UCH-38625 Table 5. Murine models used for anti-SSPN antibody development

[0173] Native mouse SSPN in the SSPN-TG lysate was detected, where SSPN is the most highly-expressed, with sera from all rabbits (FIG.2B, lane 1 on all immunoblot images). Subsequent boosters (Bleed II) increased specific signal with mSSPN across all five rabbits (FIG.2B, lane 1 on lower IB images). Specific signal with native SSPN in WT and mdx samples was observed only after third boost (Bleed II) in rabbits #8870, #8871, #8872. Sera from rabbits # 8873 and 8874 did not recognize native SSPN in WT and mdx samples and generated high non-specific background including prominent IgG light chain bands running at very similar molecular weight with SSPN (25kD) in all tested samples including SSPN-null.

[0174] Bleeds II from all rabbits were also analyzed by indirect IFA (FIG.10). Crude non-purified sera (rabbits #8870, #8871, #8872, #8873) recognized native SSPN in mSSPN- overexpressing tissues (mSSPN-TG) and WT muscle tissue using indirect IFA (FIG.10). Positive signal in mouse tissues overexpressing SSPN by immunoblotting was detected with sera from all rabbits. As serum from rabbit #8874 did not recognize mouse SSPN in indirect IFA (FIG.10) and produced highest background in immunoblotting (FIG.2B), we decided to exclude this rabbit from further antibody development. Rabbit #8873 was also excluded from extended immunization because of background issues in immunoblots (FIG.2B). However,UCH-38625 using IFA serum from rabbit #8873 was observed to recognize native mouse SSPN in mSSPN-TG and wild-type muscle tissue without high background in SSPN-null tissues (FIG. 10). Based on these results, rabbit #8873 was selected for affinity-purification of its polyclonal anti-SSPN antibodies from the terminal bleed for future use in IFA.

[0175] After evaluating the results of three assays (ELISA, IB and IFA), rabbit #8871 was selected for monoclonal antibody production based on results of ELISA, IFA and immunoblotting. Serum from rabbit #8871 had high titer to SSPN protein in ELISA and exhibited robust specific signal with minimal background in immunoblotting and IFA (FIG. 2A, FIG.2B, and FIG.10). Serum from rabbits #8870 and #8872 demonstrated good signal- to-background ratio in immunoblotting (FIG.2B) and IFA (FIG.10), recognizing native SSPN protein in wild-type, SSPN-TG, and mdx tissue. Rabbits #8870 and #8872 were selected for extended immunization and generation of polyclonal antibodies to mouse SSPN (Table 4). Antibody titers were analyzed by ELISA (FIG.11) and revealed that both rabbits maintained good immune response against antigens during extended immunization.

[0176] Terminal bleeds from the rabbits selected for extended immunization (#8870, #8872) and from the rabbit #8873 excluded from extended immunization, as well as from rabbit #8871, selected for monoclonal antibody development, were affinity purified and evaluated by ELISA, IFA and immunoblotting. For ELISA analysis, specific SSPN signal was evaluated with SUMO-tagged SSPN (FIG.3A). Possible cross-reactivity with GST that was used as a fusion protein for immunization was assessed using an unrelated GST recombinant fusion protein (data not shown). Purified antibodies demonstrated high titers against SSPN antigens with similar OD450readings across all sera samples. All samples exhibited low cross-reactivity with GST protein. Purified antibodies from rabbits #8870, #8871 and #8872 recognized mouse SSPN with high affinity and specificity in immunoblotting and IFA. Antibodies from rabbit #8873 generated strong specific signals in IFA and immunoblotting; however, an extra low-molecular weight band was observed in SSPN-null sample by immunoblotting (data not shown), so use of this antibody was limited to IFA. Representative immunoblotting data and IFA images with purified antibody from rabbit #8870 serum are presented in FIGs.3B and 3C.UCH-38625 Characterization of rabbit monoclonal anti-SSPN antibodies

[0177] Rabbit #8871 was selected for monoclonal antibody development based on results of ELISA, IFA and immunoblotting. Over 900 single cell clones were screened by ELISA using SUMO-tagged SSPN protein to select SSPN-specific clones not cross-reacting with fusion protein (GST) used for immunization. Cell supernatants from 96 clones with highest titers in ELISA (as shown in Table 6) were selected for further evaluation by immunoblotting with total muscle lysates. Table 6. Validation of intermediate rabbit clones after primary screening by ELISA

[0178] Total lysates were prepared from wild-type (C57 BL / 6), mdx, mouse SSPN- TG (Ln28) and SSPN-null mice (Table 5). 39 clones were found that recognized mouse SSPN in immunoblotting and were then evaluated for SSPN detection using IFA on human and mouse muscle sections (Table 7). Of the 39 clones, 15 clones with robust signals in both immunoblotting and IFA were selected for the next step of monoclonal antibody developmentUCH-38625 that included performing RNA extraction from selected clones, RT-PCR, cloning of variable region of IgG genes, and construction of an LEM. Table 7. Validation of intermediate rabbit clones after primary screening by western blot and IFAUCH-38625

[0179] LEMs were transiently transfected into HEK 293-F cells and secondary ELISA screening of the supernatants from transiently transfected cells was performed (Table 8). Table 8. Selected rabbit clones LEM supernatant ELISA Results

[0180] 13 out of 15 selected clones retained their specific anti-SSPN activity in ELISA after construction of linear expression cassettes. The 13 clones were re-screened by immunoblot and IFA, and the 7 best performing clones were selected for expression and large-scale purification of monoclonal antibodies. All 7 clones retained their activity after large-scale antibody production, producing robust specific signal in ELISA, IFA, and immunoblotting (data not shown). Representative data from validation of antibody 10B8 by IFA and immunoblot are provided (FIG.4A and 4B) in comparison to the commercially available mouse monoclonal antibody E2 (Santa Cruz, sc393187). Antibody 10B8 outperformed E2 in terms of reactivity and specificity. Antibodies 8870 and 10B8 (FIG.3B and FIG.4A) exhibited a clean background signal without the non-specific bands detected by the E2 antibodies in immunoblotting (FIG.6A, lower panel labeled E2). The rabbit polyclonal and monoclonal antibodies exhibited strong specific signals with mouse SSPNUCH-38625 protein, but not with human SSPN in immunoblotting and IFA (FIGs.3A-3C and FIGs.4A- 4B) supporting that the developed rabbit antibodies were mouse SSPN-specific. Example 2: Generation and characterization of anti-SSPN mouse antibodies Methods for generation of synthetic SSPN polypeptides

[0181] Two peptides of human SSPN (GenBank accession number AF016028) from the C-terminus region (aa 219-243, HRYQVFYVGVRICSLTASEGPQQKI, SEQ ID NO: 932) and a fragment of large extracellular loop (LEL) (aa 167-186, PSSEPLSRTFVYRDVTDCTS, SEQ ID NO: 933) were synthetized by ABClonal using companies’ protocol. To ensure immunogenicity, synthetic peptides were chemically conjugated to keyhole limpet hemocyanin (KLH). Mouse immunizations

[0182] All of the housing and immunization procedures were performed by ABclonal according to the approved protocols and guidelines of IACUC. Briefly, two-months old female BALB / c mice (five mice per antigen) were injected subcutaneously with 100μg synthetic antigen peptide supplemented with Complete Freund’s Adjuvant for the primary injection (Day 1), and 100 μg antigen peptide supplemented with Incomplete Freund’s Adjuvant for subsequent four boosting injections (Day 22, 36, 50, 64) (See Table 9 below). Table 9. Immunization schedule of miceUCH-38625

[0183] The first immune bleed (Bleed I) was collected after two boosting injections on Day 43. Bleeds II and III (4thand 5thantisera) were collected on Days 57 and 70, respectively. All bleeds were analyzed by ELISA using biotin-tagged peptides to determine SSPN antibody titer. To choose the best mouse for the monoclonal antibody development, the 4thand 5thantisera were tested (diluted 1:250 in blocking buffer) by immunoblotting using the same workflow as for testing of the rabbit anti-sera (see Example 1 above).

[0184] For evaluation of immune response in mouse after immunization, mouse sera were tested by ELISA against SsPN peptides by ABclonal. To analyze immune response to human SSPN C-terminal fragment, 96-well plates were precoated with biotin-conjugated hSSPN (aa 219-243, HRYQVFYVGVRICSLTASEGPQQKI (SEQ ID NO: 932)) at 1 µg / mL in carbonate-bicarbonate coating buffer, pH 9.66 (Sigma, SRE0102) overnight. To analyze immune response to human SSPN LEL fragment, biotin-hSSPN (aa 167-186, PSSEPLSRTFVYRDVTDCTS (SEQ ID NO: 933)) at 1 µg / mL in carbonate-bicarbonate coating buffer was used as an antigen. Mouse sera from bleeds II and III were first diluted at 1:1000, followed by 8-point 1:3 serial dilutions in carbonate-bicarbonate coating buffer, pH 9.66 and incubated for 1 h at 37 ℃. Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch, 111-005-046), 1:10,000 in dilution buffer (1% BSA in sodium phosphate-buffer pH 7.4 with 0.1% Tween-20) was added to the plate and incubated for 1h at RT. All washing, blocking steps and enzymatic reaction were performed as described above. Methods used for mouse monoclonal antibody generation

[0185] Standard hybridoma generation approach was used for creation of mouse monoclonal antibodies by ABclonal. Briefly, for a mouse selected for monoclonal antibody generation, 150 μg antigen without adjuvant was administered through peritoneal and intravenous (tail vein) routes a day before splenectomy. Mouse spleen cells were fused with SP2 / 0 myeloma cells PEG1500 (Roche Diagnostics, Indianapolis, IN, USA). Hybridomas were grown in RPMI 1640 (Gibco, 11875), supplemented with 10% FBS (Sigma, F4135), 100 U / mL of penicillin (Sigma), 100 µg / mL streptomycin (Sigma), and 0.25 µg / mL amphotericin B (Sigma).UCH-38625

[0186] Over 900 clones were screened by ELISA to detect clones generating antibodies with the highest titer by ABclonal. Based on the ELISA results, 100 clones (50 for each antigen, see Table 10 and Table 11 below) were selected and sent to UCLA for testing by immunoblotting and IFA (methods of performing immunoblotting and IFA were used as in Example 1 above). Table 10. List of fusion clones selected for further characterization of AK289M03 (immunized with C-terminus of hSSPN, aa 219-243UCH-38625Table 11. List of fusion clones selected for further characterization of AK290M02 (immunized with LEL fragment of hSSPN, aa 167-186)UCH-38625

[0187] Based on the results of immunoblotting and IFA, we selected intermediate hybridoma clones recognizing native SSPN with high affinity and specificity (see Table 12 below), for subcloning and expansion. Table 12. Testing of intermediate mouse clones of 289 series (to hSSPN C-terminus) and 290 series (to hSSPN LEL) by WB and IFA.UCH-38625UCH-38625

[0188] Multiple rounds of subcloning were performed on selected clones until monoclonal hybridoma cell lines were established using limiting dilution method by ABclonal as described in Dale. O Starkie, Joanne E. Compson, Stephen Rapecki, Daniel J. Lightwood. Generation of Recombinant Monoclonal Antibodies from Immunised Mice and Rabbits via Flow Cytometry and Sorting of Antigen-Specific IgG+Memory B Cells.2016. After subcloning and stabilization, clones were evaluated using ELISA by ABClonal and 80 clones (40 per each antigen) (see Table 13 and Table 14 below) with highest titers to SSPN were re-screened by immunoblotting and IFA. Table 13. Results of ELISA testing of 40 best clones to hSSPN C-terminus after subcloning and stabilizationUCH-38625Table 14. Results of ELISA testing of 40 best clones to hSSPN LEL after subcloning and stabilizationUCH-38625

[0189] Based on results of re-screening, we selected seven clones recognizing native SSPN with highest affinity and specificity. These clones were re-tested by ELISA (see Table 16 and Table 17 below), mouse monoclonal antibodies were purified from selected clones by ABclonal. Table 16. Results of ELISA evaluation of the final selected clones to hSSPN C-terminusTable 17. Results of ELISA evaluation of the final selected clones to hSSPN LELUCH-38625Determining efficiency of immunoprecipitation for affinity purification of SSPN

[0190] Snap-frozen quadriceps muscles from of wild-type (C57 BL / 6), mdx, mouse SSPN-TG (Ln28) and SSPN-null mice were pulverized using a metal mortar and pestle. Ground tissue was homogenized in Dounce homogenizer in ice-cold IP lysis buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, pH 7.4) containing Halt protease (Thermo Scientific, 1860932) and phosphatase inhibitors (Thermo Scientific, 78428), transferred to the 1.5 mL Eppendorf tubes prechilled on ice and rotated at 4oC for 1 h. Lysates were clarified by centrifuging at 20,000 x g for 15 min at 4oC. Protein concentration was determined with the DC Protein Assay (Bio-Rad). To minimize non-specific binding, muscle lysates (250 mg) were pre-cleared using 50 μL protein A / G PLUS-agarose beads (SCBT, sc2003) for 1 h at 4oC with gentle rotation. Beads were sedimented by centrifugation at 1,000 x g for 5 min at 4oC min and pre-cleared lysates were transferred to a new prechilled Eppendorf tube containing 5 µg of antibodies followed by rotation for 1 h at 4oC. SSPN-antibody conjugates were captured with 50 μL protein A / G PLUS-agarose beads, pre-equilibrated with cold IP wash buffer overnight at 4oC with rotation before washing five times with IP wash buffer (50 mM Tris, 150 mM NaCl, 0.1% NP-40, pH 7.4) containing Halt protease and phosphatase inhibitors. Bound proteins were eluted by heating at 70oC for 10 min with 2x NuPAGE sample buffer (Thermo Scientific, NP0007). Centrifugation steps were performed at 1,000 x g for 5 min at 4oC. Equimolar amounts of eluates were resolved by SDS-PAGE on 12% Bolt Bis-Tris gels (Thermo Scientific, NW00122BOX). PageRuler Plus Prestained Protein Ladder (Thermo Scientific, PI26619) was used as a molecular weight marker. Immunoblotting was performed as described in Example 1 above. Characterization of mouse monoclonal antibodies for LEL and C-terminus epitopes of human SSPN

[0191] Mouse monoclonal antibodies were developed against two fragments of human SSPN. One targeted epitope was the C-terminus region including amino acid residues 219-243 (SEQ ID NO: 932) (FIG.1A and FIG.1C). Another targeted epitope was a fragmentUCH-38625 of the LEL of SSPN including amino acid residues 167-186 (SEQ ID NO: 933) (FIG.1A and FIG.1C).

[0192] Two of five mice immunized with the C-terminus fragment of hSSPN (mice #289-02 and #289-03) exhibited the highest titers of anti-SSPN antibodies in ELISA (FIG. 5A). For mouse #289-02, the OD450was 1.03 at 1:8,000 sera dilution after the first bleed (immune serum #3). For mouse #289-03, the highest OD450 was 0.84 after the second bleed (immune serum #4) (FIG.5A and FIG.12).

[0193] Of the mice immunized by LEL fragment of hSSPN, mouse #290-01 developed the best response to the immunogen with OD450of 1.3 at 1:16,000 dilution (3rdsera collection) and OD450 of 1.1 (5thsera collection). Mouse #290-02 also demonstrated stable immune response to the immunogen with highest reactivity at the 4thsera collection (OD450=0.71 at 1:16,000 dilution) and the 5thsear collection (OD450= 0.38 at 1:16,000 dilution) (FIG.5B and FIG.12).

[0194] To choose the best mouse for the monoclonal antibody development, immune sera #4 and #5 were tested by immunoblotting with total muscle lysates from wild-type, mdx, mSSPN-TG, hSSPN-TG, and SSPN-null mice (FIGs.13A-13B). Human SSPN was detected by immunoblotting immune sera from mouse #289-03 and mouse #289-01. Among mice immunized by LEL fragment of hSSPN, both mouse and human SSPN were detected in immune sera from mouse #290-02. Interestingly, immune sera from mouse #290-01, which demonstrated highest antibody titers using ELISA, did not generate specific signal with human or mouse SSPN using immunoblotting. Based on the results of immunoblotting and ELISA, mouse #289-03 was chosen from the cohort of mice immunized by C-terminus of hSSPN for monoclonal antibody development (mouse #289-01 was used as a backup). From the cohort on mice, immunized by LEL fragment, mouse #290-02 was selected for monoclonal antibody development.

[0195] After two additional boosts, splenocytes from the selected mice were harvested, fused with myeloma cells, and supernatants from the fused cells were tested by ELISA. One hundred clones exhibiting the highest titers in ELISA (50 for each antigen) (Table 10 and Table 11) were selected for further evaluation by immunoblotting and IFA with the goal of determining hybridomas for subcloning and expansion (Table 12). Based on results of immunoblotting and IFA, 23 clones for subcloning and stabilization were selected.UCH-38625 Fourteen clones from the group specific to C-terminus of human SSPN and 9 clones specific to LEL fragment (selected clones are marked with asterisk in Table 12). Results of immunoblotting and IFA evaluation of two representative intermediate clones, 289-F15 and 290-F5 are presented in FIG.6A and FIG.6B. Clone 289-F15 developed against C-terminus of human SSPN, recognized only human SSPN in immunoblotting and IFA (FIG.6B).289- F15 exceeded commercially available mouse monoclonal antibodies E2 in terms of specificity (no extra bands in immunoblot (FIG.6A)) and sensitivity (higher signal in IFA with human muscle tissue compared to E2 (FIG.6B). Clone 290-F5 recognized both mouse and human SSPN in immunoblotting and IFA, produced high specific signal with human SSPN in immunoblotting, however specific signal in IFA with human muscle tissue was relatively low (FIG.6A and FIG.6B).

[0196] After completion of subcloning and stabilization, forty hybridoma cell supernatants (20 clones per antigen) were selected based on ELISA data (Table 13 and Table 14) for re-screening in immunoblotting and IFA to choose the 2-3 best clones per antigen as final deliverables.

[0197] During evaluation of clones after subcloning and stabilization, it was observed that some of the clones recognized native SSPN in IFA, but neither of supernatants generated signal in immunoblotting (data not shown). This suggested that clones working in both applications were not stable and did not survive subcloning and stabilization steps. We selected five clones from the cohort generated against LEL fragment and two clones specific to C-terminus. C-terminus clones were human specific, clones to LEL fragment were either mouse-specific or recognized both mouse and human SSPN (Table 1). Monoclonal antibodies from all selected clones were purified and evaluated by indirect ELISA with biotinylated SSPN fragments (Table 15 and Table 16).

[0198] IFA evaluation of finally selected clones demonstrated that the mouse anti- SSPN antibodies generate more intense signal with native mouse SSPN in muscle sections from WT mice compared to commercially available mouse monoclonal antibody E2 (FIG.7). The herein developed human SSPN-specific antibodies significantly overperformed E2 in IFA and effectively recognized native SSPN in healthy human control muscle samples and in muscle samples from patients with Becker muscular dystrophy where SSPN is significantlyUCH-38625 reduced at sarcolemma (FIG.7). This made the herein provided human-specific antibodies a unique tool for SSPN detection for research and clinical purposes.

[0199] To interrogate whether herein developed anti-SSPN antibodies would affinity purify SSPN, immunoprecipitation on quadricep muscle lysates from WT, mSSPN-TG, hSSPN-TG, and SSPN-null mice was performed. Nine of the herein developed monoclonal anti-SSPN antibodies and the commercially available E2 were tested. The efficiency of SSPN pull-down was assessed by SDS-PAGE analysis of eluates followed by immunoblotting with either E2 antibody, which recognizes both human and mouse SSPN, or rabbit monoclonal antibody 10B8. All tested antibodies captured SSPN with variable affinity (FIG.8). SSPN was observed in monomer state, homo oligomers (dimers, closed arrowhead; trimers, open arrowhead), consistent with its function as a teteraspanin-like protein (FIG.8). Although the E2 antibody successfully captured SSPN, the newly developed antibodies resulted in higher specificity of affinity purified SSPN with markedly reduced background (FIG.8). When immunoblotting with E2, endogenous SSPN in WT eluates was not detected (FIG.8). However, when lysates were affinity purified with E2 and eluates immunoblotted, in parallel, with either E2 or 10B8 antibodies, endogenous SSPN in WT was detected by 10B8 (FIG.8). This suggested that endogenous SSPN, after affinity purification of WT lysates, was not detected due to the lower levels of endogenous SSPN found in WT tissue coupled with the reduced sensitivity of the commercially available E2 antibody relative to the herein developed rabbit monoclonal antibody 10B8. Antibody 10B8 showed it could capture mouse SSPN with greater affinity then human SSPN. In contrast, antibody 20E11 was able to capture only mouse SSPN (FIG.8), suggesting that both rabbit monoclonal antibodies bind distinct epitopes within the N-terminus. The N-terminus peptide of mSSPN shares 52% identity with hSSPN (FIG.1C). The binding of hSSPN by antibody 10B8 came as a surprise since it was not able to detect hSSPN by immunofluorescence or immunoblot analysis.

[0200] All herein developed antibodies targeting the LEL domain (290-04, 290-05, 290-17, 290-06, 290-11) generated using residues 167-186 of hSSPN (SEQ ID NO: 933), detected both mouse and human SSPN (FIG.8). These antibodies were unable to detect SSPN by immunoblotting suggesting that the epitope binds a fully or partially folded protein which was lost after denaturing with SDS. Interestingly, 290-04 and 290-05 recognize recombinant SSPN protein in sandwich ELISA, while 290-11 and 290-17 do not (data notUCH-38625 shown). However, all four of these clones captured hSSPN with greater affinity compared to mSSPN (FIG.8), consistent with use of human peptide which shares 95% identity to mSSPN (FIG.1C). Taken together with the performance of these antibodies in immunofluorescence analysis and sandwich ELISA, it is possible that antibodies 290-11, 290-17, and 290-04 / 290- 05 have distinct epitopes within the LEL region. Furthermore, the antibodies that do not work in sandwich ELISA however are able to capture SSPN by immunoprecipitation, suggesting that the epitope region may be disrupted by the small amounts of Triton X-100, SDS, and DOC present in the more stringent extraction buffer used for sandwich ELISA, yet remains intact when solubilized with a mild NP-40 detergent used for IP (Table 1).

[0201] Antibodies developed against residues 219-243 of the human C-terminus, 289- 17 and 289-18, despite sharing 64% identity with mSSPN (FIG.1C), captures only hSSPN, consistent with immunofluorescence analysis. The capability of the herein developed antibodies of binding to distinct epitopes within SSPN, in addition to selectively capturing either mouse or human SSPN or both mouse and human will be beneficial in downstream applications. INCORPORATION BY REFERENCE

[0202] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0203] Also incorporated by reference in their entirety are any polynucleotide and polypeptide sequences which reference an accession number correlating to an entry in a public database, such as those maintained by The Institute for Genomic Research (TIGR) on the world wide web at tigr.org and / or the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov. EQUIVALENTS

[0204] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

UCH-38625 CLAIMS 1. An antibody, or antigen-binding fragment thereof, that specifically binds sarcospan (SSPN), wherein the antibody or antigen-binding fragment comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein: (i) the VL comprises a VL complementary determining region (CDR) 1 (LCDR1) having a sequence as set forth in SEQ ID NO: 1 (QSSETVYKNNYLSWF), a VL CDR 2 (LCDR2) having a sequence as set forth in SEQ ID NO: 2 (FLIYGASTLAS), and a VL CDR 3 (LCDR3) having a sequence as set forth in SEQ ID NO: 3 (GGGFSSSSDDT); and the VH comprises a VH CDR 1 (HCDR1) having a sequence as set forth in SEQ ID NO: 4 (GFSLSSYAMS), a VH CDR 2 (HCDR2) having a sequence as set forth in SEQ ID NO: 5 (YIGIINAAGSAYYARWVNG), and a VH CDR 3 (HCDR3) having a sequence as set forth in SEQ ID NO: 6 (VRGSIPYSGGVSL); (ii) the VL comprises a LCDR1 having a sequence as set forth in SEQ ID NO: 31 (VRGSIPYSGGVSL), a LCDR2 having a sequence as set forth in SEQ ID NO: 32 (LLIYGASTLAS), and a LCDR3 having a sequence as set forth in SEQ ID NO: 33 (GGGYDTSRDDV); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 34 (GFSLSTYAMS), a HCDR2 having a sequence as set forth in SEQ ID NO: 35 (YIGIINTGGSAYYASWAEG), and a HCDR3 having a sequence as set forth in SEQ ID NO: 36 (VRGSIAYTAGVTL); (iii) the VL comprises a LCDR1 having a sequence as set forth in SEQ ID NO: 61 (QASQSVYKNNYLSWF), a LCDR2 having a sequence as set forth in SEQ ID NO: 62 (LLIYGASTLAS), and a LCDR3 having a sequence as set forth in SEQ ID NO: 63 (GGGDASSSSDDA); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 64 (GFSLSNYAMS), a HCDR2 having a sequence as set forth in SEQ ID NO: 65 (YIGIINTGGSAYYASWAEG), and a HCDR3 having a sequence as set forth in SEQ ID NO: 66 (IINTAGSAYYARWVNG); (iv) the VL comprises a LCDR1 having a sequence as set forth in SEQ ID NO: 91 (QSSESVYKNNYLSWF), a LCDR2 having a sequence as set forth in SEQ ID NO: 92 (LLIYGASTLAS), and a LCDR3 having a sequence as set forth in SEQ ID NO: 93 (GGGYDTSRDDV); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 94 (GFSLSTYAMS), a HCDR2 having a sequence as set forth in SEQ ID NO: 95UCH-38625 (YIGIINTGGSAYYASWAKG), and a HCDR3 having a sequence as set forth in SEQ ID NO: 96 (VRGSIAYTAGVTL); (v) the VL comprises a LCDR1 having a sequence as set forth in SEQ ID NO: 121 (QSSESVYNNNYLSWF), a LCDR2 having a sequence as set forth in SEQ ID NO: 122 (LLIYGISTLAS), and a LCDR3 having a sequence as set forth in SEQ ID NO: 123 (GGGYSTSSDDT); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 124 (GFSLSSYAMS), a HCDR2 having a sequence as set forth in SEQ ID NO: 125 (YIGIINAGGIPYYANWVNG), and a HCDR3 having a sequence as set forth in SEQ ID NO: 126 (LWGPGTLVTVSS); (vi) the VL comprises a LCDR1 having a sequence as set forth in SEQ ID NO: 151 (QSSETVYKNNYLSWF), a LCDR2 having a sequence as set forth in SEQ ID NO: 152 (FLIYGASTLAS), and a LCDR3 having a sequence as set forth in SEQ ID NO: 153 (GGGFSSSSDDT); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 154 (GFSLSSYAMS), a HCDR2 having a sequence as set forth in SEQ ID NO: 155 (YIGIINAAGSAYYARWVNG), and a HCDR3 having a sequence as set forth in SEQ ID NO: 156 (VRGSIPYSGGVSL); (vii) the VL comprises a LCDR1 having a sequence as set forth in SEQ ID NO: 181 (QSSESVYKNNYLSWF), a LCDR2 having a sequence as set forth in SEQ ID NO: 182 (LLIYGASTLAS), and a LCDR3 having a sequence as set forth in SEQ ID NO: 183 (CGGGYSTSSDDT); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 184 (GFSLSSYAVS), a HCDR2 having a sequence as set forth in SEQ ID NO: 185 (IVNTAGSAYYANWVNG), and a HCDR3 having a sequence as set forth in SEQ ID NO: 186 (VRGSIAYTAGVAL); (viii) the VL comprises a LCDR1 having a sequence as set forth in SEQ ID NO: 211 (RASQSLVHSNGNTYLHWY), a LCDR2 having a sequence as set forth in SEQ ID NO: 212 (LLIYKVSNRFF), and a LCDR3 having a sequence as set forth in SEQ ID NO: 213 (SQSEHVWT); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 214 (GYSITSDYAWN), a HCDR2 having a sequence as set forth in SEQ ID NO: 215 (WMAYITYTGRTLYNPSLES), and a HCDR3 having a sequence as set forth in SEQ ID NO: 216 (ARSFAY); (ix) the VL comprises a LCDR1 having a sequence as set forth in SEQ ID NO: 271 (KSSQSLLKSRTRKNHLAWY), a LCDR2 having a sequence as set forth in SEQ ID NO:UCH-38625 272 (LLIYWASTRES), and a LCDR3 having a sequence as set forth in SEQ ID NO: 273 (KQSYDLYT); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 274 (GFTFSDYAMA), a HCDR2 having a sequence as set forth in SEQ ID NO: 275 (WIAFLSNLAKNVYYADTVAG), and a HCDR3 having a sequence as set forth in SEQ ID NO: 276 (ARGNGDYHAMDY); (x) the VL comprises a LCDR1 having a sequence as set forth in SEQ ID NO: 331 (KSSQSLLKSRTRRNYLA), a LCDR2 having a sequence as set forth in SEQ ID NO: 332 (LLIYWASTRES), and a LCDR3 having a sequence as set forth in SEQ ID NO: 333 (KQSYDLYT); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 334 (GFTFSDYGMA), a HCDR2 having a sequence as set forth in SEQ ID NO: 335 (WIAFISNLAYNIYYADTVTG), and a HCDR3 having a sequence as set forth in SEQ ID NO: 336 (ARGNGNYDAVDY); (xi) the VL comprises a LCDR1 having a sequence as set forth in SEQ ID NO: 361 (KSSQSLLKSRTRRNYLAWY), a LCDR2 having a sequence as set forth in SEQ ID NO: 362 (LLIYWASTRES), and a LCDR3 having a sequence as set forth in SEQ ID NO: 363 (KQSYDLYT); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 364 (GFTFSDYGMA), a HCDR2 having a sequence as set forth in SEQ ID NO: 365 (WIAFISNLAYNIYYADTVTG), and a HCDR3 having a sequence as set forth in SEQ ID NO: 366 (ARGNGNYDAVDY); or (xii) the VL comprises a LCDR1 having a sequence as set forth in SEQ ID NO: 391 (KSSQSLLKSRTRKNYLAWY), a LCDR2 having a sequence as set forth in SEQ ID NO: 392 (LLIYWASTRES), and a LCDR3 having a sequence as set forth in SEQ ID NO: 393 (KQSYDLYT); and the VH comprises a HCDR1 having a sequence as set forth in SEQ ID NO: 394 (GFTFSDYAMA), a HCDR2 having a sequence as set forth in SEQ ID NO: 395 (WVTFISDLAYNIYYADTVT), and a HCDR3 having a sequence as set forth in SEQ ID NO: 396 (ARGNGDYDAMDY).

2. The antibody or antigen-binding fragment of claim 1, wherein the VL comprises a VL framework region 1 (FR1) having a sequence selected from SEQ ID NOs: 449, 457, 465, 473, 481, 489, 497, 505, 513, 521, 529, 537, 545, 553, 561, 569, 577, 585, 593, 601, 609, 617, 625, 633, 641, 649, 657, 665, 673, 681, 689, 697, 705, 713, 721, 729, 737, 745, 753, 761, 769, 777, 785, 793, 801, 809, 817, 825, 833, 841, 849, 857, 865, 873, 881, and 889, aUCH-38625 VL framework region 2 (FR2) having a sequence selected from SEQ ID NOs: 450, 458, 466, 474, 482, 490, 498, 506, 514, 522, 530, 538, 546, 554, 562, 570, 578, 586, 594, 602, 610, 618, 626, 634, 642, 650, 658, 666, 674, 682, 690, 698, 706, 714, 722, 730, 738, 746, 754, 762, 770, 778, 786, 794, 802, 810, 818, 826, 834, 842, 850, 858, 866, 874, 882, and 890, a VL framework region 3 (FR3) having a sequence selected from SEQ ID NOs: 451, 459, 467, 475, 483, 491, 499, 507, 515, 523, 531, 539, 547, 555, 563, 571, 579, 587, 595, 603, 611, 619, 627, 635, 643, 651, 659, 667, 675, 683, 691, 699, 707, 715, 723, 731, 739, 747, 755, 763, 771, 779, 787, 795, 803, 811, 819, 827, 835, 843, 851, 859, 867, 875, 883, and 891, and a VL framework region 4 (FR4) having a sequence selected from SEQ ID NOs: 452, 460, 468, 476, 484, 492, 500, 508, 516, 524, 532, 540, 548, 556, 564, 572, 580, 588, 596, 604, 612, 620, 628, 636, 644, 652, 660, 668, 676, 684, 692, 700, 708, 716, 724, 732, 740, 748, 756, 764, 772, 780, 788, 796, 804, 812, 820, 828, 836, 844, 852, 860, 868, 876, 884, and 892.

3. The antibody or antigen-binding fragment of claim 1 or 2, wherein the VH comprises a VH framework region 1 (FR1) having a sequence selected from SEQ ID NOs: 453, 461, 469, 477, 485, 493, 501, 509, 517, 525, 533, 541, 549, 557, 565, 573, 581, 589, 597, 605, 613, 621, 629, 637, 645, 653, 661, 669, 677, 685, 693, 701, 709, 717, 725, 733, 741, 749, 757, 765, 773, 781, 789, 797, 805, 813, 821, 829, 837, 845, 853, 861, 869, 877, 885, and 893, a VH framework region 2 (FR2) having a sequence selected from SEQ ID NOs: 454, 462, 470, 478, 486, 494, 502, 510, 518, 526, 534, 542, 550, 558, 566, 574, 582, 590, 598, 606, 614, 622, 630, 638, 646, 654, 662, 670, 678, 686, 694, 702, 710, 718, 726, 734, 742, 750, 758, 766, 774, 782, 790, 798, 806, 814, 822, 830, 838, 846, 854, 862, 870, 878, 886, and 894, a VH framework region 3 (FR3) having a sequence selected from SEQ ID NOs: 455, 463, 471, 479, 487, 495, 503, 511, 519, 527, 535, 543, 551, 559, 567, 575, 583, 591, 599, 607, 615, 623, 631, 639, 647, 655, 663, 671, 679, 687, 695, 703, 711, 719, 727, 735, 743, 751, 759, 767, 775, 783, 791, 799, 807, 815, 823, 831, 839, 847, 855, 863, 871, 879, 887, and 895, and a VH framework region 4 (FR4) having a sequence selected from SEQ ID NOs: 456, 464, 472, 480, 488, 496, 504, 512, 520, 528, 536, 544, 552, 560, 568, 576, 584, 592, 600, 608, 616, 624, 632, 640, 648, 656, 664, 672, 680, 688, 696, 704, 712, 720, 728, 736, 744, 752, 760, 768, 776, 784, 792, 800, 808, 816, 824, 832, 840, 848, 856, 864, 872, 880, 888, and 896.UCH-38625 4. The antibody or antigen-binding fragment of any one of claims 1-3, wherein the VH and the VL comprise amino acid sequences selected from: (a) SEQ ID NOs: 421 and 422, respectively; (b) SEQ ID NOs: 423 and 424, respectively; (c) SEQ ID NOs: 425 and 426, respectively; (d) SEQ ID NOs: 427 and 428, respectively; (e) SEQ ID NOs: 429 and 430, respectively; (f) SEQ ID NOs: 431 and 432, respectively; (g) SEQ ID NOs: 433 and 434, respectively; (h) SEQ ID NOs: 435 and 436, respectively; (i) SEQ ID NOs: 437 and 438, respectively; (j) SEQ ID NOs: 439 and 440, respectively; (k) SEQ ID NOs: 441 and 442, respectively; (l) SEQ ID NOs: 443 and 444, respectively; (m) SEQ ID NOs: 445 and 446, respectively; and (n) SEQ ID NOs: 447 and 448, respectively.

5. The antibody or antigen-binding fragment of any one of claims 1-4, comprising a heavy chain constant region.

6. The antibody or antigen-binding fragment of claim 5, wherein the heavy chain constant region comprises an amino acid sequence selected from: (a) SEQ ID NO: 900; (b) SEQ ID NO: 901; (c) SEQ ID NO: 902; and (d) SEQ ID NO:

903.

7. The antibody or antigen-binding fragment of any one of claims 1-6, comprising a light chain constant region.UCH-38625 8. The antibody or antigen-binding fragment of claim 7, wherein the light chain constant region comprises an amino acid sequence selected from: (a) SEQ ID NO: 897; (b) SEQ ID NO: 898; and (c) SEQ ID NO:

899.

9. The antibody or antigen binding fragment of any one of claims 1-8, comprising a heavy chain constant region and a light chain constant region comprising amino acid sequences selected from: (a) SEQ ID NOs: 900 and 897, respectively; (b) SEQ ID NOs: 900 and 898, respectively; (c) SEQ ID NOs: 901 and 899, respectively; (d) SEQ ID Nos: 902 and 899, respectively; and (e) SEQ ID NOs: 903 and 899, respectively.

10. The antibody or antigen binding fragment of any one of claims 1-9, comprising a heavy chain and a light chain comprising the amino acid sequences selected from: (a) SEQ ID NOs: 904 and 918, respectively; (b) SEQ ID NOs: 905 and 919, respectively; (c) SEQ ID NOs: 906 and 920, respectively; (d) SEQ ID NOs: 907 and 921, respectively; (e) SEQ ID NOs: 908 and 922, respectively; (f) SEQ ID NOs: 909 and 923, respectively; (g) SEQ ID NOs: 910 and 924, respectively; (h) SEQ ID NOs: 911 and 925, respectively; (i) SEQ ID NOs: 912 and 926, respectively; (j) SEQ ID NOs: 913 and 927, respectively; (k) SEQ ID NOs: 914 and 928, respectively; (l) SEQ ID NOs: 915 and 929, respectively; (m) SEQ ID NOs: 916 and 930, respectively; and (n) SEQ ID NOs: 917 and 931, respectively.UCH-38625 11. The antibody or antigen-binding fragment of any one of claims 1-10, wherein the antibody or antigen-binding fragment thereof binds to an epitope, wherein the epitope is (i) an epitope of human SSPN, (ii) an epitope of mouse SSPN, or (ii) both an epitope of human SSPN and an epitope of mouse SSPN.

12. The antibody or antigen-binding fragment of claim 11, wherein the epitope is an intracellular epitope or an extracellular epitope.

13. The antibody or antigen-binding fragment of claim 11, wherein the epitope is a C- terminal epitope or a large extracellular loop (LEL) epitope.

14. The antibody or antigen-binding fragment of claim 11, wherein the epitope is a C- terminal epitope within residues 219-243 of human SSPN.

15. The antibody or antigen-binding fragment of claim 11, wherein the epitope is a C- terminal epitope comprising an amino acid sequence within SEQ ID NO:

932.

16. The antibody or antigen-binding fragment of claim 11, wherein the epitope is a C- terminal epitope comprising an amino acid sequence within SEQ ID NO:

936.

17. The antibody or antigen-binding fragment of claim 11, wherein the epitope is an LEL epitope within residues 167-186 of human SSPN.

18. The antibody or antigen-binding fragment of claim 11, wherein the epitope is an LEL epitope comprising an amino acid sequence within SEQ ID NO:

933.

19. The antibody or antigen-binding fragment of claim 11, wherein the epitope is an N- terminal epitope.

20. The antibody or antigen-binding fragment of claim 11, wherein the epitope is an N- terminal epitope within residues 1-25 of human SSPN.UCH-38625 21. The antibody or antigen-binding fragment of claim 19, wherein the N-terminal epitope comprises an amino acid sequence within SEQ ID NO:

937.

22. The antibody or antigen-binding fragment of any one of claims 1-10, wherein the antibody or antigen-binding fragment thereof binds to an epitope of mouse SSPN.

23. The antibody or antigen-binding fragment of claim 22, wherein the epitope is an LEL epitope within residues 167-186 of mouse SSPN.

24. The antibody or antigen-binding fragment of claim 22, wherein the epitope is an LEL epitope comprising an amino acid sequence within SEQ ID NO:

934.

25. The antibody or antigen-binding fragment of claim 22, wherein the epitope is an intracellular epitope.

26. The antibody or antigen-binding fragment of claim 22, wherein the epitope is an N- terminal epitope.

27. The antibody or antigen-binding fragment of claim 26, wherein the N-terminal epitope is within residues 1-25 of mouse SSPN.

28. The antibody or antigen-binding fragment of claim 26, wherein the N-terminal epitope comprises an amino acid sequence within SEQ ID NO:

935.

29. A nucleic acid comprising a nucleotide sequence encoding the heavy chain and the light chain of the antibody or antigen-binding fragment of any one of claims 1-28.

30. An expression vector comprising the nucleic acid of claim 29.

31. A cell comprising the expression vector of claim 30.UCH-38625 32. A method of detecting SSPN in a sample comprising: (a) contacting the sample with an antibody or antigen-binding fragment of any one of claims 1-28; and (b) determining the presence or absence of SSPN in the sample.

33. The method of claim 32, wherein the sample comprises a cell, a tissue, a biological fluid, an aqueous suspension, or a living organism such as a mammalian model of disease or a human.

34. The method of claim 33, wherein the sample comprises skeletal muscle cells, smooth muscle cells, cardiomyocytes, adipocytes, kidney epithelial cells, neurons, immune cells, chondrocytes, fibroblasts, plasma cells, tumor cells, neurons, or any SSPN expressing entity.

35. The method of any one of claims 32-34, wherein step (b) comprises the use of an immunoassay.

36. The method of any one of claims 32-35, wherein step (b) comprises the use of an immunoblotting assay, an immunoprecipitation assay, an immunofluorescence assay, or an enzyme-linked immunosorbent assay (ELISA) assay.

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