6192a3b1de659ef33b92c0fe629fe164 biomolecules-15-01602.pdf 40ffdb4fe7e88f187faf8caedca79852bfa05033 biomolecules-15-01602.pdf 4fcaaefd75a5d682a61ed9d9eff946b6a1f00c39dd34c64850aafe062d026bc5 biomolecules-15-01602.pdf Title: ScFv-h3D6 Prevents Bapineuzumab-Induced Hemorrhagic Events in the APP23 Mouse Model of Alzheimer’s Disease Subject: The occurrence of amyloid-related imaging abnormalities (ARIAs), found in clinical trials for A-immunotherapy, has been related to the antibody’s effector function on glial activation by the Fc portion of the antibody. The use of single-chain variable fragments (scFv) has been proposed as a safer therapeutic strategy. Here, the effects of the mice format of bapineuzumap (mAb-m3D6) and its scFv (scFv-h3D6) on the occurrence of ARIAs in the APP23 mouse model of Alzheimer’s disease (AD) and cerebral amyloid angiopathy (CAA) have been addressed by magnetic resonance imaging (MRI). Results are supported by histological and/or biochemical determinations. Aged APP23 mice showed a significantly higher number of microhemorrhages than non-transgenic mice. mAb-m3D6 produced an increase in the number of new hemorrhagic events, mainly in the cortex, whereas scFv-h3D6 did not. Both mAb-m3D6 and scFv-h3D6 reduced A levels by the same extent. Axonal/myelin damage was found in the frontal corpus callosum of APP23 mice, which did not recover after treatment. In conclusion, the scFv-h3D6 format appears safer than the full-length mAb in the APP23 model of AD and CAA. This finding is highly relevant in light of the new FDA- and EMA-approved mAbs, which exclude APOE4 allele carriers due to the occurrence of hemorrhages. Keywords: Alzheimer’s disease; amyloid-related imaging abnormalities; cerebral amyloid angiopathy; immunotherapy; magnetic resonance imaging; single-chain variable fragment Author: Silvia Lope-Piedrafita, Gabriel Serra-Mir, Paula Melón, Anna Bonaterra, Mar Hernández-Guillamon and Sandra Villegas Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Sat Nov 15 10:15:09 2025 CET ModDate: Sat Nov 15 10:17:58 2025 CET Custom Metadata: yes Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 24 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2337253 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- AZOXTQ+PazoMath-Italic Type 1 Builtin yes yes yes 144 0 VKTEFL+URWPalladioL-Bold Type 1 Custom yes yes yes 145 0 AIRHKL+VnURWPalladioL Type 1 Custom yes yes yes 146 0 WGTJKN+URWPalladioL-Roma Type 1 Custom yes yes yes 147 0 JPUAMM+EURM10 Type 1 Builtin yes yes yes 148 0 RPRLGB+URWPalladioL-Ital Type 1 Custom yes yes yes 149 0 YLLRKM+CMSY10 Type 1 Builtin yes yes yes 260 0 JOELGB+CMEX10 Type 1 Builtin yes yes yes 297 0 GIGFZE+CMR10 Type 1 Builtin yes yes yes 298 0 URYLRZ+URWPalladioL-BoldItal Type 1 Custom yes yes yes 340 0 ESSRQF+VnURWPalladioL-Italic Type 1 Custom yes yes yes 520 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-12-18 03:13:47 CET RepresentationInformation: biomolecules-15-01602.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-12-17 10:43:29 CET Size: 2337253 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 681 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Item: Title: Materials and Methods Children: Item: Title: ScFv-h3D6 Production Item: Title: mAb-m3D6 Production Item: Title: Animals Item: Title: Experimental Design and Statistics Item: Title: Magnetic Resonance Imaging (MRI) Children: Item: Title: Volume Measurements Item: Title: Imaging ARIA-H Item: Title: Imaging ARIA-E Item: Title: Axonal and Myelin Damage Item: Title: Sample Collection and Processing Item: Title: Immunofluorescence Item: Title: Image Capture and Processing Item: Title: Enzyme-Linked Immunosorbent Assays (ELISAs) Item: Title: Results Children: Item: Title: Hemorrhagic Events Induced by mAb-m3D6 Are Prevented by scFv-h3D6 Item: Title: Both, mAb-m3D6 and scFv-h3D6 Do Not Trigger a General Inflammatory Response Item: Title: Both mAb-m3D6 and scFv-h3D6 Reduce A Levels Item: Title: Reduction in A Is Not Enough to Recover from Neurodegeneration in Late Stages of AD Item: Title: Discussion Item: Title: Conclusions Item: Title: References Info: Title: ScFv-h3D6 Prevents Bapineuzumab-Induced Hemorrhagic Events in the APP23 Mouse Model of Alzheimer’s Disease Author: Silvia Lope-Piedrafita, Gabriel Serra-Mir, Paula Melón, Anna Bonaterra, Mar Hernández-Guillamon and Sandra Villegas Subject: The occurrence of amyloid-related imaging abnormalities (ARIAs), found in clinical trials for A-immunotherapy, has been related to the antibody’s effector function on glial activation by the Fc portion of the antibody. The use of single-chain variable fragments (scFv) has been proposed as a safer therapeutic strategy. Here, the effects of the mice format of bapineuzumap (mAb-m3D6) and its scFv (scFv-h3D6) on the occurrence of ARIAs in the APP23 mouse model of Alzheimer’s disease (AD) and cerebral amyloid angiopathy (CAA) have been addressed by magnetic resonance imaging (MRI). Results are supported by histological and/or biochemical determinations. Aged APP23 mice showed a significantly higher number of microhemorrhages than non-transgenic mice. mAb-m3D6 produced an increase in the number of new hemorrhagic events, mainly in the cortex, whereas scFv-h3D6 did not. Both mAb-m3D6 and scFv-h3D6 reduced A levels by the same extent. Axonal/myelin damage was found in the frontal corpus callosum of APP23 mice, which did not recover after treatment. In conclusion, the scFv-h3D6 format appears safer than the full-length mAb in the APP23 model of AD and CAA. This finding is highly relevant in light of the new FDA- and EMA-approved mAbs, which exclude APOE4 allele carriers due to the occurrence of hemorrhages. Keywords: Alzheimer’s disease; amyloid-related imaging abnormalities; cerebral amyloid angiopathy; immunotherapy; magnetic resonance imaging; single-chain variable fragment Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Sat Nov 15 10:15:09 CET 2025 ModDate: Sat Nov 15 10:17:58 CET 2025 ID: 0xc477912c84360b3a6d55d03c16015fd0, 0x9a1853ae4aa656e5f2f618844045fb41 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: AZOXTQ+PazoMath-Italic FontSubset: true FirstChar: 35 LastChar: 108 FontDescriptor: FontName: AZOXTQ+PazoMath-Italic Flags: Symbolic FontBBox: -70, -277, 902, 733 FontFile: true ToUnicode: true Font: BaseFont: VKTEFL+URWPalladioL-Bold FontSubset: true FirstChar: 2 LastChar: 243 FontDescriptor: FontName: VKTEFL+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: AIRHKL+VnURWPalladioL FontSubset: true FirstChar: 160 LastChar: 225 FontDescriptor: FontName: AIRHKL+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: WGTJKN+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: WGTJKN+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: JPUAMM+EURM10 FontSubset: true FirstChar: 11 LastChar: 34 FontDescriptor: FontName: JPUAMM+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true ToUnicode: true Font: BaseFont: YLLRKM+CMSY10 FontSubset: true FirstChar: 0 LastChar: 106 FontDescriptor: FontName: YLLRKM+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true ToUnicode: true Font: BaseFont: URYLRZ+URWPalladioL-BoldItal FontSubset: true FirstChar: 110 LastChar: 110 FontDescriptor: FontName: URYLRZ+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: RPRLGB+URWPalladioL-Ital FontSubset: true FirstChar: 3 LastChar: 237 FontDescriptor: FontName: RPRLGB+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: ESSRQF+VnURWPalladioL-Italic FontSubset: true FirstChar: 225 LastChar: 225 FontDescriptor: FontName: ESSRQF+VnURWPalladioL-Italic Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: JOELGB+CMEX10 FontSubset: true FirstChar: 16 LastChar: 118 FontDescriptor: FontName: JOELGB+CMEX10 Flags: Symbolic FontBBox: -24, -2960, 1454, 772 FontFile: true ToUnicode: true Font: BaseFont: GIGFZE+CMR10 FontSubset: true FirstChar: 40 LastChar: 61 FontDescriptor: FontName: GIGFZE+CMR10 Flags: Symbolic FontBBox: -40, -250, 1009, 750 FontFile: true ToUnicode: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 34, 177, 152, 186 Annotation: Subtype: Link Rect: 34, 167, 80, 174 Annotation: Subtype: Link Rect: 36, 75, 152, 84 Annotation: Subtype: Link Rect: 34, 63, 89, 72 Annotation: Subtype: Link Rect: 34, 773, 170, 809 Annotation: Subtype: Link Rect: 515, 773, 560, 809 Annotation: Subtype: Link Rect: 167, 663, 178, 677 Annotation: Subtype: Link Rect: 429, 663, 440, 677 Annotation: Subtype: Link Rect: 166, 650, 177, 663 Annotation: Subtype: Link Rect: 282, 650, 293, 663 Annotation: Subtype: Link Rect: 417, 122, 424, 131 ActionDest: 109 Annotation: Subtype: Link Rect: 188, 80, 195, 89 ActionDest: 109 Annotation: Subtype: Link Rect: 417, 27, 560, 37 Annotation: Subtype: Link Rect: 34, 344, 87, 363 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 416, 730, 423, 740 ActionDest: 109 Annotation: Subtype: Link Rect: 426, 730, 432, 740 ActionDest: 109 Annotation: Subtype: Link Rect: 345, 661, 352, 670 ActionDest: 109 Annotation: Subtype: Link Rect: 333, 633, 340, 642 ActionDest: 109 Annotation: Subtype: Link Rect: 440, 591, 447, 600 ActionDest: 109 Annotation: Subtype: Link Rect: 410, 508, 417, 517 ActionDest: 109 Annotation: Subtype: Link Rect: 417, 508, 429, 517 ActionDest: 109 Annotation: Subtype: Link Rect: 243, 480, 250, 489 ActionDest: 109 Annotation: Subtype: Link Rect: 544, 396, 556, 405 ActionDest: 109 Annotation: Subtype: Link Rect: 226, 355, 238, 364 ActionDest: 109 Annotation: Subtype: Link Rect: 297, 243, 309, 252 ActionDest: 109 Annotation: Subtype: Link Rect: 530, 215, 542, 224 ActionDest: 109 Annotation: Subtype: Link Rect: 265, 160, 276, 169 ActionDest: 109 Annotation: Subtype: Link Rect: 376, 132, 388, 141 ActionDest: 109 Annotation: Subtype: Link Rect: 362, 76, 369, 85 ActionDest: 109 Annotation: Subtype: Link Rect: 369, 76, 381, 85 ActionDest: 109 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 341, 744, 353, 754 ActionDest: 109 Annotation: Subtype: Link Rect: 319, 689, 331, 698 ActionDest: 109 Annotation: Subtype: Link Rect: 332, 689, 344, 698 ActionDest: 109 Annotation: Subtype: Link Rect: 417, 661, 429, 670 ActionDest: 109 Annotation: Subtype: Link Rect: 438, 591, 450, 600 ActionDest: 109 Annotation: Subtype: Link Rect: 357, 536, 369, 545 ActionDest: 109 Annotation: Subtype: Link Rect: 544, 466, 556, 475 ActionDest: 244 Annotation: Subtype: Link Rect: 214, 424, 226, 433 ActionDest: 244 Annotation: Subtype: Link Rect: 438, 396, 450, 405 ActionDest: 244 Annotation: Subtype: Link Rect: 453, 396, 465, 405 ActionDest: 244 Annotation: Subtype: Link Rect: 360, 368, 371, 377 ActionDest: 244 Annotation: Subtype: Link Rect: 234, 341, 246, 350 ActionDest: 244 Annotation: Subtype: Link Rect: 390, 341, 402, 350 ActionDest: 244 Annotation: Subtype: Link Rect: 403, 341, 415, 350 ActionDest: 244 Annotation: Subtype: Link Rect: 277, 327, 289, 336 ActionDest: 244 Annotation: Subtype: Link Rect: 289, 327, 301, 336 ActionDest: 244 Annotation: Subtype: Link Rect: 302, 327, 314, 336 ActionDest: 244 Annotation: Subtype: Link Rect: 289, 299, 301, 308 ActionDest: 244 Annotation: Subtype: Link Rect: 544, 285, 556, 294 ActionDest: 244 Annotation: Subtype: Link Rect: 468, 229, 480, 238 ActionDest: 244 Annotation: Subtype: Link Rect: 217, 187, 229, 196 ActionDest: 244 Annotation: Subtype: Link Rect: 230, 187, 242, 196 ActionDest: 244 Annotation: Subtype: Link Rect: 275, 159, 286, 169 ActionDest: 244 Annotation: Subtype: Link Rect: 287, 159, 299, 169 ActionDest: 244 Annotation: Subtype: Link Rect: 400, 90, 412, 99 ActionDest: 244 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 407, 657, 419, 667 ActionDest: 244 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 217, 701, 223, 713 ActionDest: 33 Annotation: Subtype: Link Rect: 498, 565, 510, 574 ActionDest: 244 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 511, 758, 523, 767 ActionDest: 244 Annotation: Subtype: Link Rect: 237, 730, 249, 740 ActionDest: 244 Annotation: Subtype: Link Rect: 414, 654, 426, 663 ActionDest: 244 Annotation: Subtype: Link Rect: 403, 225, 415, 234 ActionDest: 244 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 519, 730, 530, 740 ActionDest: 244 Annotation: Subtype: Link Rect: 478, 398, 490, 407 ActionDest: 244 Annotation: Subtype: Link Rect: 345, 342, 357, 351 ActionDest: 244 Annotation: Subtype: Link Rect: 414, 232, 426, 241 ActionDest: 244 Annotation: Subtype: Link Rect: 432, 162, 444, 171 ActionDest: 244 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 350, 703, 362, 712 ActionDest: 244 Annotation: Subtype: Link Rect: 467, 329, 479, 338 ActionDest: 244 Annotation: Subtype: Link Rect: 383, 177, 395, 186 ActionDest: 244 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 450, 674, 462, 683 ActionDest: 244 Annotation: Subtype: Link Rect: 462, 674, 474, 683 ActionDest: 244 Annotation: Subtype: Link Rect: 211, 603, 218, 615 ActionDest: 310 Annotation: Subtype: Link Rect: 531, 592, 543, 601 ActionDest: 244 Annotation: Subtype: Link Rect: 544, 592, 556, 601 ActionDest: 338 Annotation: Subtype: Link Rect: 358, 561, 365, 573 ActionDest: 75 Annotation: Subtype: Link Rect: 473, 547, 480, 559 ActionDest: 75 Annotation: Subtype: Link Rect: 330, 491, 337, 503 ActionDest: 75 Page: Sequence: 10 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 282, 731, 294, 740 ActionDest: 109 Annotation: Subtype: Link Rect: 294, 730, 306, 740 ActionDest: 109 Annotation: Subtype: Link Rect: 296, 672, 303, 684 ActionDest: 311 Annotation: Subtype: Link Rect: 288, 630, 295, 642 ActionDest: 311 Annotation: Subtype: Link Rect: 199, 589, 206, 601 ActionDest: 311 Annotation: Subtype: Link Rect: 234, 533, 241, 545 ActionDest: 311 Annotation: Subtype: Link Rect: 204, 453, 216, 462 ActionDest: 338 Annotation: Subtype: Link Rect: 403, 440, 415, 448 ActionDest: 244 Annotation: Subtype: Link Rect: 416, 439, 428, 448 ActionDest: 244 Annotation: Subtype: Link Rect: 428, 439, 440, 448 ActionDest: 244 Annotation: Subtype: Link Rect: 441, 439, 453, 448 ActionDest: 244 Annotation: Subtype: Link Rect: 251, 325, 258, 337 ActionDest: 91 Annotation: Subtype: Link Rect: 372, 325, 379, 337 ActionDest: 91 Annotation: Subtype: Link Rect: 297, 286, 309, 295 ActionDest: 338 Annotation: Subtype: Link Rect: 449, 175, 461, 184 ActionDest: 244 Page: Sequence: 12 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 471, 337, 483, 347 ActionDest: 338 Annotation: Subtype: Link Rect: 375, 296, 386, 305 ActionDest: 244 Annotation: Subtype: Link Rect: 387, 296, 399, 305 ActionDest: 244 Annotation: Subtype: Link Rect: 399, 296, 411, 305 ActionDest: 244 Annotation: Subtype: Link Rect: 412, 296, 424, 305 ActionDest: 244 Annotation: Subtype: Link Rect: 491, 254, 503, 263 ActionDest: 338 Annotation: Subtype: Link Rect: 338, 212, 350, 221 ActionDest: 244 Annotation: Subtype: Link Rect: 217, 182, 224, 194 ActionDest: 396 Annotation: Subtype: Link Rect: 327, 182, 334, 194 ActionDest: 396 Annotation: Subtype: Link Rect: 291, 98, 298, 110 ActionDest: 396 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 535, 744, 542, 754 ActionDest: 109 Annotation: Subtype: Link Rect: 324, 717, 336, 726 ActionDest: 109 Annotation: Subtype: Link Rect: 417, 619, 429, 628 ActionDest: 244 Annotation: Subtype: Link Rect: 333, 589, 340, 601 ActionDest: 396 Annotation: Subtype: Link Rect: 440, 509, 452, 518 ActionDest: 244 Annotation: Subtype: Link Rect: 452, 509, 464, 518 ActionDest: 244 Annotation: Subtype: Link Rect: 299, 465, 306, 477 ActionDest: 397 Annotation: Subtype: Link Rect: 205, 453, 217, 462 ActionDest: 338 Annotation: Subtype: Link Rect: 543, 437, 550, 449 ActionDest: 397 Annotation: Subtype: Link Rect: 435, 411, 447, 421 ActionDest: 338 Annotation: Subtype: Link Rect: 544, 398, 556, 407 ActionDest: 244 Annotation: Subtype: Link Rect: 477, 370, 489, 379 ActionDest: 338 Annotation: Subtype: Link Rect: 490, 370, 502, 379 ActionDest: 338 Annotation: Subtype: Link Rect: 544, 342, 556, 351 ActionDest: 338 Annotation: Subtype: Link Rect: 485, 314, 497, 323 ActionDest: 338 Annotation: Subtype: Link Rect: 302, 272, 314, 281 ActionDest: 338 Annotation: Subtype: Link Rect: 469, 216, 481, 226 ActionDest: 338 Annotation: Subtype: Link Rect: 481, 216, 493, 226 ActionDest: 338 Annotation: Subtype: Link Rect: 394, 144, 401, 156 ActionDest: 397 Annotation: Subtype: Link Rect: 200, 116, 207, 128 ActionDest: 397 Annotation: Subtype: Link Rect: 532, 116, 539, 128 ActionDest: 397 Annotation: Subtype: Link Rect: 332, 75, 339, 87 ActionDest: 99 Page: Sequence: 15 Page: Sequence: 16 Page: Sequence: 17 Page: Sequence: 18 Annotations: Annotation: Subtype: Link Rect: 428, 744, 440, 754 ActionDest: 338 Annotation: Subtype: Link Rect: 441, 744, 453, 754 ActionDest: 338 Annotation: Subtype: Link Rect: 346, 605, 358, 614 ActionDest: 338 Annotation: Subtype: Link Rect: 358, 605, 370, 614 ActionDest: 338 Annotation: Subtype: Link Rect: 207, 452, 219, 461 ActionDest: 244 Annotation: Subtype: Link Rect: 288, 438, 300, 447 ActionDest: 244 Annotation: Subtype: Link Rect: 404, 271, 411, 280 ActionDest: 109 Annotation: Subtype: Link Rect: 254, 118, 266, 127 ActionDest: 338 Annotation: Subtype: Link Rect: 266, 118, 278, 127 ActionDest: 338 Annotation: Subtype: Link Rect: 498, 104, 510, 113 ActionDest: 338 Annotation: Subtype: Link Rect: 513, 104, 525, 113 ActionDest: 338 Annotation: Subtype: Link Rect: 477, 76, 488, 85 ActionDest: 338 Page: Sequence: 19 Annotations: Annotation: Subtype: Link Rect: 287, 717, 299, 726 ActionDest: 338 Annotation: Subtype: Link Rect: 264, 661, 276, 670 ActionDest: 509 Annotation: Subtype: Link Rect: 196, 633, 208, 642 ActionDest: 509 Annotation: Subtype: Link Rect: 415, 563, 427, 572 ActionDest: 509 Annotation: Subtype: Link Rect: 473, 549, 485, 559 ActionDest: 509 Annotation: Subtype: Link Rect: 185, 466, 197, 475 ActionDest: 509 Annotation: Subtype: Link Rect: 197, 466, 209, 475 ActionDest: 509 Annotation: Subtype: Link Rect: 273, 452, 284, 461 ActionDest: 509 Annotation: Subtype: Link Rect: 390, 438, 402, 447 ActionDest: 509 Annotation: Subtype: Link Rect: 536, 103, 560, 114 Annotation: Subtype: Link Rect: 165, 90, 385, 101 Page: Sequence: 20 Page: Sequence: 21 Annotations: Annotation: Subtype: Link Rect: 169, 637, 427, 648 Annotation: Subtype: Link Rect: 519, 624, 557, 635 Annotation: Subtype: Link Rect: 109, 599, 146, 609 Annotation: Subtype: Link Rect: 293, 573, 331, 584 Annotation: Subtype: Link Rect: 337, 573, 373, 584 Annotation: Subtype: Link Rect: 274, 534, 312, 545 Annotation: Subtype: Link Rect: 519, 509, 557, 520 Annotation: Subtype: Link Rect: 59, 497, 95, 507 Annotation: Subtype: Link Rect: 184, 459, 221, 469 Annotation: Subtype: Link Rect: 383, 432, 420, 443 Annotation: Subtype: Link Rect: 427, 432, 463, 443 Annotation: Subtype: Link Rect: 221, 408, 258, 418 Annotation: Subtype: Link Rect: 484, 381, 522, 392 Annotation: Subtype: Link Rect: 449, 356, 486, 367 Annotation: Subtype: Link Rect: 140, 318, 177, 328 Annotation: Subtype: Link Rect: 184, 318, 220, 328 Annotation: Subtype: Link Rect: 442, 279, 479, 290 Annotation: Subtype: Link Rect: 486, 279, 522, 290 Annotation: Subtype: Link Rect: 59, 242, 96, 252 Annotation: Subtype: Link Rect: 102, 242, 138, 252 Annotation: Subtype: Link Rect: 59, 216, 96, 226 Annotation: Subtype: Link Rect: 404, 190, 442, 201 Annotation: Subtype: Link Rect: 480, 164, 518, 175 Annotation: Subtype: Link Rect: 437, 139, 474, 150 Annotation: Subtype: Link Rect: 399, 100, 436, 111 Page: Sequence: 22 Annotations: Annotation: Subtype: Link Rect: 90, 731, 127, 742 Annotation: Subtype: Link Rect: 133, 731, 169, 742 Annotation: Subtype: Link Rect: 176, 731, 243, 742 Annotation: Subtype: Link Rect: 155, 706, 193, 716 Annotation: Subtype: Link Rect: 199, 706, 235, 716 Annotation: Subtype: Link Rect: 241, 706, 309, 716 Annotation: Subtype: Link Rect: 350, 679, 387, 690 Annotation: Subtype: Link Rect: 393, 679, 429, 690 Annotation: Subtype: Link Rect: 441, 654, 478, 665 Annotation: Subtype: Link Rect: 485, 654, 521, 665 Annotation: Subtype: Link Rect: 287, 616, 324, 627 Annotation: Subtype: Link Rect: 245, 578, 283, 588 Annotation: Subtype: Link Rect: 413, 552, 450, 563 Annotation: Subtype: Link Rect: 435, 526, 472, 537 Annotation: Subtype: Link Rect: 517, 501, 555, 512 Annotation: Subtype: Link Rect: 307, 475, 345, 486 Annotation: Subtype: Link Rect: 179, 438, 216, 448 Annotation: Subtype: Link Rect: 446, 411, 483, 422 Annotation: Subtype: Link Rect: 323, 373, 361, 384 Annotation: Subtype: Link Rect: 367, 373, 403, 384 Annotation: Subtype: Link Rect: 172, 335, 410, 346 Annotation: Subtype: Link Rect: 360, 309, 397, 320 Annotation: Subtype: Link Rect: 403, 309, 439, 320 Annotation: Subtype: Link Rect: 446, 309, 513, 320 Annotation: Subtype: Link Rect: 275, 271, 312, 282 Annotation: Subtype: Link Rect: 319, 271, 355, 282 Annotation: Subtype: Link Rect: 519, 258, 557, 269 Annotation: Subtype: Link Rect: 59, 246, 95, 256 Annotation: Subtype: Link Rect: 247, 220, 285, 231 Annotation: Subtype: Link Rect: 243, 194, 560, 205 Annotation: Subtype: Link Rect: 56, 182, 298, 193 Annotation: Subtype: Link Rect: 82, 144, 119, 154 Annotation: Subtype: Link Rect: 213, 118, 251, 129 Annotation: Subtype: Link Rect: 95, 93, 133, 103 Page: Sequence: 23 Annotations: Annotation: Subtype: Link Rect: 515, 731, 552, 742 Annotation: Subtype: Link Rect: 467, 718, 504, 729 Annotation: Subtype: Link Rect: 510, 718, 546, 729 Annotation: Subtype: Link Rect: 59, 680, 96, 690 Annotation: Subtype: Link Rect: 102, 680, 138, 690 Annotation: Subtype: Link Rect: 476, 654, 513, 665 Annotation: Subtype: Link Rect: 519, 654, 555, 665 Annotation: Subtype: Link Rect: 109, 616, 146, 627 Annotation: Subtype: Link Rect: 417, 590, 454, 601 Annotation: Subtype: Link Rect: 59, 553, 96, 563 Annotation: Subtype: Link Rect: 59, 502, 96, 512 Annotation: Subtype: Link Rect: 155, 462, 192, 473 Annotation: Subtype: Link Rect: 418, 437, 456, 448 Annotation: Subtype: Link Rect: 346, 399, 384, 410 Annotation: Subtype: Link Rect: 470, 373, 507, 384 Annotation: Subtype: Link Rect: 332, 347, 370, 358 Annotation: Subtype: Link Rect: 376, 347, 412, 358 Annotation: Subtype: Link Rect: 391, 322, 428, 333 Annotation: Subtype: Link Rect: 435, 322, 471, 333 Annotation: Subtype: Link Rect: 59, 285, 96, 295 Annotation: Subtype: Link Rect: 102, 285, 138, 295 Annotation: Subtype: Link Rect: 438, 271, 476, 282 Annotation: Subtype: Link Rect: 395, 233, 432, 244 Annotation: Subtype: Link Rect: 292, 195, 329, 205 Annotation: Subtype: Link Rect: 423, 169, 460, 180 Annotation: Subtype: Link Rect: 82, 131, 119, 141 Annotation: Subtype: Link Rect: 271, 93, 309, 103 Annotation: Subtype: Link Rect: 464, 79, 502, 90 Page: Sequence: 24 Annotations: Annotation: Subtype: Link Rect: 210, 731, 247, 742 Annotation: Subtype: Link Rect: 372, 718, 560, 729 Annotation: Subtype: Link Rect: 56, 706, 162, 716 Annotation: Subtype: Link Rect: 372, 679, 409, 690 Annotation: Subtype: Link Rect: 416, 679, 452, 690 Annotation: Subtype: Link Rect: 352, 654, 390, 665 Annotation: Subtype: Link Rect: 396, 654, 432, 665 Annotation: Subtype: Link Rect: 254, 628, 292, 639 Annotation: Subtype: Link Rect: 298, 628, 334, 639 Annotation: Subtype: Link Rect: 198, 616, 560, 627 Annotation: Subtype: Link Rect: 56, 603, 133, 614 Annotation: Subtype: Link Rect: 95, 565, 133, 576 Annotation: Subtype: Link Rect: 445, 539, 483, 550 Checksum: bb863da1 Type: CRC32 Checksum: 6192a3b1de659ef33b92c0fe629fe164 Type: MD5 Checksum: 40ffdb4fe7e88f187faf8caedca79852bfa05033 Type: SHA-1 Checksum: 4fcaaefd75a5d682a61ed9d9eff946b6a1f00c39dd34c64850aafe062d026bc5 Type: SHA-256