0 9 Agonistic agonistic JJ 10 18 activity activity NN 19 21 of of IN 22 23 a a DT 24 37 CD40-specific cd40-specific JJ 38 50 single-chain single-chain JJ 51 53 Fv Fv NNP 54 65 constructed construct VBN 66 70 from from IN 71 74 the the DT 75 83 variable variable JJ 84 91 regions region NNS 92 94 of of IN 95 98 mAb mab NN 99 104 G28-5 g28-5 NN 104 105 . . . 107 108 A a DT 109 121 single-chain single-chain JJ 122 124 Fv Fv NNP 125 126 ( ( ( 126 129 sFv sFv NNP 129 130 ) ) ) 131 134 was be VBD 135 144 expressed express VBN 145 149 from from IN 150 153 the the DT 154 162 variable variable JJ 163 170 regions region NNS 171 173 of of IN 174 177 the the DT 178 191 CD40-specific cd40-specific JJ 192 195 mAb mab NN 196 201 G28-5 g28-5 NN 201 202 . . . 203 206 The the DT 207 215 molecule molecule NN 216 221 bound bind VBD 222 226 CD40 cd40 NN 227 231 with with IN 232 233 a a DT 234 238 high high JJ 239 247 affinity affinity NN 248 249 ( ( ( 249 252 2.2 2.2 CD 253 255 nM nM NNP 255 256 ) ) ) 257 260 and and CC 261 264 was be VBD 265 266 a a DT 267 274 monomer monomer NN 275 277 in in IN 278 286 solution solution NN 286 287 . . . 288 300 Surprisingly surprisingly RB 300 301 , , , 302 307 G28-5 G28-5 NNP 308 311 sFv sFv NNP 312 315 was be VBD 316 317 a a DT 318 324 potent potent JJ 325 329 CD40 cd40 NN 330 337 agonist agonist NN 338 342 that that WDT 343 350 rapidly rapidly RB 351 362 crosslinked crosslinke VBD 363 367 CD40 cd40 NN 368 370 on on IN 371 374 the the DT 375 379 cell cell NN 380 387 surface surface NN 388 391 but but CC 392 395 did do VBD 396 399 not not RB 400 409 crosslink crosslink VB 410 417 CD40-Ig cd40-ig NN 418 420 in in IN 421 429 solution solution NN 429 430 . . . 431 436 G28-5 G28-5 NNP 437 440 sFv sFv NNP 441 444 was be VBD 445 446 a a DT 447 451 more more RBR 452 458 potent potent JJ 459 466 agonist agonist NN 467 471 than than IN 472 477 G28-5 G28-5 NNP 478 481 IgG IgG NNP 482 485 and and CC 486 489 was be VBD 490 494 able able JJ 495 497 to to TO 498 507 stimulate stimulate VB 508 512 CD40 cd40 NN 513 522 responses response NNS 523 525 by by IN 526 527 B B NNP 528 533 cells cell NNS 534 537 and and CC 538 547 monocytes monocyte NNS 547 548 . . . 549 554 G28-5 G28-5 NNP 555 558 IgG IgG NNP 559 568 partially partially RB 569 576 blocked block VBD 576 577 , , , 578 585 whereas whereas IN 586 591 G28-5 G28-5 NNP 592 595 sFv sFv NNP 596 605 augmented augment VBD 606 610 CD40 cd40 NN 611 620 responses response NNS 621 627 during during IN 628 639 stimulation stimulation NN 640 644 with with IN 645 652 natural natural JJ 653 659 ligand ligand NN 660 661 ( ( ( 661 669 gp39-CD8 gp39-cd8 NN 670 676 fusion fusion NN 677 684 protein protein NN 684 685 ) ) ) 685 686 . . . 687 692 These these DT 693 700 results result NNS 701 709 indicate indicate VBP 710 714 that that IN 715 718 the the DT 719 729 functional functional JJ 730 738 activity activity NN 739 741 of of IN 742 749 ligands ligand NNS 750 755 built build VBN 756 760 from from IN 761 764 the the DT 765 772 binding bind VBG 773 777 site site NN 778 780 of of IN 781 786 G28-5 G28-5 NNP 787 789 is be VBZ 790 796 highly highly RB 797 806 dependent dependent JJ 807 811 upon upon IN 812 815 the the DT 816 820 size size NN 821 824 and and CC 825 833 physical physical JJ 834 844 properties property NNS 845 847 of of IN 848 851 the the DT 852 860 molecule molecule NN 861 865 both both CC 866 868 in in IN 869 877 solution solution NN 878 881 and and CC 882 884 on on IN 885 888 the the DT 889 893 cell cell NN 894 902 surfaces surface NNS 902 903 . . .