Dual binding compositions targeting vegfa and PD-1

Abstract

The present disclosure relates to dual binding compositions specific for PD-1 and VEGFA.

Description

Docket No. 56146-753.601DUAL BINDING COMPOSITIONS TARGETING VEGFA AND PD-1CROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 739,333 filed December 27, 2024, which application is incorporated herein by reference in its entirety.BRIEF SUMMARY

[0002] Described herein are dual binding compositions which are specific for vascular endothelial growth factor A (VEGFA) and programmed cell death protein 1 (PD-1). Such compositions are potentially useful in the treatment of cancer.

[0003] Further provided herein are methods of treating cancer and other disease with the aforementioned composition, as well as pharmaceutical compositions comprising the same.

[0004] In one aspect provided herein is a dual binding composition, comprising: a) a first binding domain targeting programmed cell death protein 1 (PD-1); and b) a second binding domain targeting vascular endothelial growth factor A (VEGFA). In some embodiments, wherein the first binding domain comprises a VHH having a VH CDR1, VH CDR2, and VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 each have sequences respectively selected from 510, 511, and 512; SEQ ID NOs: 502, 503, and 504; SEQ ID NOs: 506, 507, and 508; SEQ ID NOs: SEQ ID NOs: 514, 515, and 516; SEQ ID NOs: 518, 511, and 520; SEQ ID NOs: 522, 523, and 524; SEQ ID NOs: 526, 527, and 528; SEQ ID NOs: 530, 531, and 532; SEQ ID NOs: 534, 535, and 536; SEQ ID NOs: 538, 539, and 540; SEQ ID NOs: 542, 543, and 544; SEQ ID NOs: 546, 547, and 548; SEQ ID NOs: 550, 511, and 552; SEQ ID NOs: 554, 555, and 556; SEQ ID NOs: 558, 543, and 560; SEQ ID NOs: 562, 563, and 564; SEQ ID NOs: 566, 567, and 568; SEQ ID NOs: 570, 511, and 572; SEQ ID NOs: 574, 567, and 576; SEQ ID NOs: 578, 579, and 580; SEQ ID NOs: 582, 555, and 584; SEQ ID NOs: 586, 587, and 588; SEQ ID NOs: 590, 591, and 592; SEQ ID NOs: 594, 555, and 596; SEQ ID NOs: 578, 599, and 600; SEQ ID NOs: 602, 511, and 604; SEQ ID NOs: 606, 567, and 608; SEQ ID NOs: 610, 591, and 612; SEQ ID NOs: 614, 615, and 616; SEQ ID NOs: 618, 567, and 620; SEQ ID NOs: 622, 567, and 624; SEQ IDNOs: 626, 511, and 628; SEQ ID NOs: 630, 563, and 632; SEQ ID NOs: 634, 599, and 636; SEQ ID NOs: 638, 555, and 640; SEQ ID NOs: 642, 567, and 644; SEQ ID NOs: 646, 511, and 648; SEQ ID NOs: 650, 515, and 652; SEQ ID NOs: 654, 547, and 656; SEQ ID NOs: 658, 659, and 660; SEQ ID NOs: 662, 663, and 664; SEQ ID NOs: 421, 591, and 668; SEQ ID NOs: 670, 591, and 672; SEQ ID NOs: 674, 675, and 676; SEQ ID NOs: 678, 679, and 680; SEQ ID NOs: 682, 599, and 684;Docket No. 56146-753.601SEQ ID NOs: 686, 547, and 688; SEQ ID NOs: 690, 691, and 692; and SEQ ID NOs: 510, 699, and 512.

[0005] In some embodiments, the VHH comprises the amino acid sequence set forth in any one of SEQ ID NOs: 509, 501, 505, 513, 517, 521, 525, 529, 533, 537, 541, 545, 549, 553, 557, 561, 565, 569, 573, 577, 581, 585, 589, 593, 597, 601, 605, 609, 613, 617, 621, 625, 629, 633, 637, 641, 645, 649, 653, 657, 661, 665, 669, 673, 677, 681, 685, 689, 693, 694, 695, 696, 697, and 698.

[0006] In some embodiments, the VH CDR1, VH CDR2, and VH CDR3 each have sequences respectively selected from: 510, 511, and 512; 570, 511, and 572; 602, 511, and 604; 626, 511, and 628; 658, 659, and 660; and 510, 699, and 512. In some embodiments, the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 509, 569, 601, 625, 657, 693, 694, 695, 696, 697, and 698. In some embodiments, the VHH comprises the amino acid sequence set forth in any one of SEQ ID NOs: 509, 569, 601, 625, 657, 693, 694, 695, 696, 697, and 698.

[0007] In some embodiments, the VH CDR1 has a sequence of SEQ ID NO: 510, the VH CDR2 has a sequence of SEQ ID NO: 511, and the VH CDR3 has a sequence of SEQ ID NO: 512. In some embodiments, the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 509, 693, 694, 695, 696, and 698. In some embodiments, the VHH comprises the amino acid sequence set forth in any one of SEQ ID NOs: 509, 693, 694, 695, 696, and 698. In some embodiments, the VHH comprises the amino acid sequence of SEQ ID NO: 509. In some embodiments, the VHH comprises the amino acid sequence of SEQ ID NO: 693. In some embodiments, the VHH comprises the amino acid sequence of SEQ ID NO: 694. In some embodiments, the VHH comprises the amino acid sequence of SEQ ID NO: 695. In some embodiments, the VHH comprises the amino acid sequence of SEQ ID NO: 696. In some embodiments, the VHH comprises the amino acid sequence of SEQ ID NO: 698.

[0008] In some embodiments, the VH CDR1 has a sequence of SEQ ID NO: 570, the VH CDR2 has a sequence of SEQ ID NO: 511, and the VH CDR3 has a sequence of SEQ ID NO: 572. In some embodiments, the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 569. In some embodiments, the VHH comprises the amino acid sequence set forth in SEQ ID NO: 569.

[0009] In some embodiments, the VH CDR1 has a sequence of SEQ ID NO: 602, the VH CDR2 has a sequence of SEQ ID NO: 511, and the VH CDR3 has a sequence of SEQ ID NO: 604. In some embodiments, the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%,Docket No. 56146-753.60196%, 97%, 98%, or 99% identical to SEQ ID NO: 601. In some embodiments, the VHH comprises the amino acid sequence set forth in SEQ ID NO: 601.

[0010] In some embodiments, wherein the VH CDR1 has a sequence of SEQ ID NO: 626, the VH CDR2 has a sequence of SEQ ID NO: 511, and the VH CDR3 has a sequence of SEQ ID NO: 628. In some embodiments, the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 625. In some embodiments, the VHH comprises the amino acid sequence set forth in SEQ ID NO: 625.

[0011] In some embodiments, the VH CDR1 has a sequence of SEQ ID NO: 658, the VH CDR2 has a sequence of SEQ ID NO: 659, and the VH CDR3 has a sequence of SEQ ID NO: 660. In some embodiments, the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 657. In some embodiments, the VHH comprises the amino acid sequence set forth in SEQ ID NO: 657.

[0012] In some embodiments, the VH CDR1 has a sequence of SEQ ID NO: 510, the VH CDR2 has a sequence of SEQ ID NO: 699, and the VH CDR3 has a sequence of SEQ ID NO: 512. In some embodiments, the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 569. In some embodiments, the VHH comprises the amino acid sequence set forth in SEQ ID NO: 569.

[0013] In some embodiments, the first binding domain targeting programmed cell death protein 1 (PD-1) is capable of disrupting the interaction of PD-1 with programmed cell death ligand 1 (PD-L1).

[0014] In some embodiments, the second binding domain targeting VEGFA is capable of disrupting the interaction of VEGFA with one or more of its receptors.

[0015] In some embodiments, the second binding domain is comprised in an antigen binding fragment derived from an antibody.

[0016] In some embodiments, the second binding domain comprises a heavy chain variable domain (VH) comprising a heavy chain first complementary determining region (VH CDR1), a heavy chain second complementary determining region (VH CDR2), and a heavy chain third complementary determining region (VH CDR3). In some embodiments, the VH is comprised in a Fab, a Fab’, F(ab')2, a bispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), a bispecific scFv, disulfide stabilized Fv (dsFv), a minibody, a diabody, a bispecific diabody, triabody, a tetrabody, a maxibody, a camelid, a VHH, a single domain light chain antibody, a Fab-Fc, a scFv-Fc, or a bispecific antibody. In some embodiments, the VH comprises a set of VH CDR1, VH CDR2, and VH CDR3 derived from an antibody in Table 2ADocket No. 56146-753.601or Table 2B. In some embodiments, the VH comprise an amino acid sequence of a VH set forth in Table 2A or Table 2B.

[0017] In some embodiments, the VH comprises a VH CDR1 having a sequence GYTFTNYGMN (SEQ ID NO: 123), a VH CDR2 having a sequence WINTYTGEPTYAADFK (SEQ ID NO: 124), and a VH CDR3 having a sequence YPHYYGSSHWYFDV (SEQ ID NO: 125). In some embodiments, the VH comprises a VH having a sequence EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTG EPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVW GQGTLVTV (SEQ ID NO: 122).

[0018] In some embodiments, wherein the second binding domain comprises a light chain variable domain (VL) comprising a light chain first complementary determining region (VL CDR1), a light chain second complementary determining region (VL CDR2), and a light chain third complementary determining region (VL CDR3). In some embodiments, the VL is comprised in the Fab, Fab’, F(ab')2, bispecific F(ab')2, variable fragment (Fv), single chain variable fragment (scFv), bispecific scFv, disulfide stabilized Fv (dsFv), minibody, diabody, bispecific diabody, triabody, tetrabody, maxibody, Fab-Fc, scFv-Fc, or bispecific antibody in which the VH of the second binding domain is comprised. In some embodiments, the VL comprises a set of VL CDR1, VL CDR2, and VL CDR3 derived from an antibody in Table 2A. In some embodiments, the VL comprises a VL CDR1 having a sequence SASQDISNYLN (SEQ ID NO: 128), a VL CDR2 having a sequence FTSSLHS (SEQ ID NO: 129), and a VL CDR3 having a sequence QQYSTVPWT (SEQ ID NO: 130). In some embodiments, the VL comprises a sequence DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK (SEQ ID NO: 127).

[0019] In some embodiments, the second binding domain comprises a VH having a VH CDR1 having a sequence GYTFTNYGMN (SEQ ID NO: 123), a VH CDR2 having a sequence WINTYTGEPTYAADFK (SEQ ID NO: 124), and a VH CDR3 having a sequence YPHYYGSSHWYFDV (SEQ ID NO: 125), and a VL having a VL CDR1 having a sequence SASQDISNYLN (SEQ ID NO: 128), a VL CDR2 having a sequence FTSSLHS (SEQ ID NO: 129), and a VL CDR3 having a sequence QQYSTVPWT (SEQ ID NO: 130). In some embodiments, the second binding domain comprises a VH having a sequence EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTG EPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVW GQGTLVTV (SEQ ID NO: 122) and a VL having a sequenceDocket No. 56146-753.601DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK (SEQ ID NO: 127).

[0020] In some embodiments, the second binding domain is an scFv. In some embodiments, the second binding domain is a Fab.

[0021] In some embodiments, the second binding domain is a single domain antibody. In some embodiments, the second binding domain is a single domain heavy chain antibody (VHH). In some embodiments, the second binding domain comprises: a) a VH CDR1 sequence of AYPMM (SEQ ID NO: 202), a VH CDR2 sequence of EISPSGSYTYYADSVRG (SEQ ID NO: 203), and a VH CDR3 sequence of DPRKLDY (SEQ ID NO: 204); b) a VH CDR1 sequence of LYDMM (SEQ ID NO: 206), a VH CDR2 sequence of FIGGDGLNTYYADSVKG (SEQ ID NO: 207), and a VH CDR3 sequence of AGTQFDY (SEQ ID NO: 208); c) a VH CDR1 sequence of WYPMW (SEQ ID NO: 210), a VH CDR2 sequence of LIEGQGDRTYYADSVKG (SEQ ID NO: 211), and a VH CDR3 sequence of AGDRTAGSRGNSFD Y (SEQ ID NO: 212); d) a VH CDR1 sequence of AYPMM (SEQ ID NO: 202), a VH CDR2 sequence of EISPSGSYTYYADSVKG (SEQ ID NO: 215), and a VH CDR3 sequence of DPRKFDY (SEQ ID NO: 216); or e) a VH CDR1 having the sequence SYSMG (SEQ ID NO: 218), a VH CDR2 having the sequence AISKGGYKYDAVSLEG (SEQ ID NO: 219) or the sequence of SEQ ID NO: 279, and a VH CDR3 having the sequence SRAYGSSRLRLADTYEY (SEQ ID NO: 220). In some embodiments, the second binding domain comprises a sequence selected from: a) EVQLLVSGGGLVDPGGSLRLSCAASGFTFKAYPMMWVRQAPGKGLEWVSEISPSGSYT YYADSVRGRFFISRDNSKNTLYLQMNSLRAEDTAVYYCAKDPRKLDYWGQGTLWTVSS (SEQ ID NO: 201); b) EVQLLESGGGLVQPGGSLRLSCAASGFTFHLYDMMWVRQAPGKGLEWVSFIGGDGLN TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAGTQFDYWGQGTLVTV SS (SEQ ID NO: 205); c) EVQLLESGGGLVQPGGSLRLSCAASGFTFQWYPMWWVRQAPGKGLEWVSLIEGQGDR TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAGDRTAGSRGNSFDYW GQGTLVTVSS (SEQ ID NO: 209); d) EVQLLESGGGLVQPGGSLRLSCAASGFTFGAYPMMWVRQAPGKGLEWVSEISPSGSYT YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDPRKFDYWGQGTLVTVSS (SEQ ID NO: 213); e) D VQLVESGGGL VQPGGSLRLSC AASGRTF S S YSMGWFRQ APGKEREF VVAISKGGYKY DAVSLEGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGDocket No. 56146-753.601QGTLVTVSS (SEQ ID NO: 217); f) D VQLVESGGGL VQPGGSLRLSC AASGRTF S S YSMGWFRQAPGKEREF VVAISKGGYKY D AVSLEGRFTISRDNAKNT VYLQINSLRPEDT AVYYC AS SRAYGS SRLRLADTYEYWG QGTLVTVSS (SEQ ID NO: 221); h) D VQLVESGGGL VQPGGSLRLSC AASGRTF S S YSMGWFRQAPGKEREF VVAISKGGYKY D AVSLEGRFTISRDNAKNT VYLQINSLRPEDT AVYYC AS SRAYGS SRLRLADTYEYWG QGTLVTVSSPP (SEQ ID NO: 200); or g) any one of SEQ ID NOs: 280-284.

[0022] In some embodiments, the second binding domain comprises a light chain single domain antibody. In some embodiments, the second binding domain comprises a VL CDR1 having the sequence RASQWIGPELS (SEQ ID NO: 223), a VL CDR2 having the sequence HTSILQS (SEQ ID NO: 224), and a VL CDR3 having the sequence QQYMFQPRT (SEQ ID NO: 225). In some embodiments, the second binding domain comprises the sequence DIQMTQSPSSLSASVGDRVTITCRASQWIGPELSWYQQKPGKAPKLLIYHTSILQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQYMFQPRTFGQGTKVEIRR (SEQ ID NO: 222). In some embodiments, the second binding domain is an anti-VEGFA anticalin. In some embodiments, the second binding domain comprises the sequence DGGGIRRSMSGTWYLKAMTVDREFPEMNLESVTPMTLTLLKGHNLEAKVTMLISGRC QEVKAVLGRTKERKKYTADGGKHVAYIIPSAVRDHVIFYSEGQLHGKPVRGVKLVGRD PKNNLEALEDFEKAAGRLSTESILIPRQSETCSPG (SEQ ID NO: 226).

[0023] In another aspect provided herein is dual binding composition, comprising: a) a first binding domain targeting programmed cell death protein 1 (PD-1); b) a second binding domain targeting vascular endothelial growth factor A (VEGFA);wherein the second binding domain comprises a VHH having a VH CDR1, VH CDR2, and VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 each have sequences respectively selected from: SEQ ID NOs: 301, 302, and 303; SEQ ID NOs: 305, 306, and 307; SEQ ID NOs: 309, 310, and 311; SEQ ID NOs: 313, 314, and 315; SEQ ID NOs: 317, 318, and 319; SEQ ID NOs: 321, 322, and 323; SEQ ID NOs: 325, 326, and 327; SEQ ID NOs: 329, 330, and 331; SEQ ID NOs: 333, 334, and 335; SEQ ID NOs: 337, 338, and 339; SEQ ID NOs: 341, 342, and 343; SEQ ID NOs: 345, 346, and 347; SEQ ID NOs: 349, 350, and 351; SEQ ID NOs: 353, 354, and 355; SEQ ID NOs: 357, 358, and 359; SEQ ID NOs: 361, 362, and 363; SEQ ID NOs: 365, 366, and 367; SEQ ID NOs: 369, 370, and 371; SEQ ID NOs: 373, 374, and 375; SEQ ID NOs: 377, 378, and 379; SEQ ID NOs: 381, 382, and 383; SEQ ID NOs: 385, 386, and 387; SEQ ID NOs: 389, 390, and 391; SEQ ID NOs: 393, 394, and 395; SEQ ID NOs: 397, 398, and 399; SEQ ID NOs: 401, 402, and 403; SEQ ID NOs:Docket No. 56146-753.601405, 406, and 407; SEQ ID NOs: 409, 410, and 411; SEQ ID NOs: 413, 414, and 415; SEQ ID NOs: 417, 418, and 419; SEQ ID NOs: 421, 422, and 423; SEQ ID NOs: 425, 426, and 427; SEQ ID NOs: 429, 430, and 431; SEQ ID NOs: 433, 434, and 435; SEQ ID NOs: 437, 438, and 439; SEQ ID NOs: 441, 442, and 443; SEQ ID NOs: 445, 398, and 447; SEQ ID NOs: 449, 426, and 451; SEQ ID NOs: 453, 454, and 455; SEQ ID NOs: 457, 458, and 459; SEQ ID NOs: 461, 462, and 463; SEQ ID NOs: 465, 466, and 467; SEQ ID NOs: 469, 470, and 471; SEQ ID NOs: 473, 474, and 475; SEQ ID NOs: 477, 478, and 479; SEQ ID NOs: 481, 482, and 483; SEQ ID NOs: 485, 486, and 487; and SEQ ID NOs: 489, 490, and 491. In some embodiments, the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 300, 304, 308, 312, 316, 320, 324, 328, 332, 336, 340, 344, 348, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388, 392, 396, 400, 404, 408, 412, 416, 420, 424, 428, 432, 436, 440, 444, 448, 452, 456, 460, 464, 468, 472, 476, 480, 484, and 488. In some embodiments, the VHH comprises the amino acid sequence set forth in any one of SEQ ID NOs: 300, 304, 308, 312, 316, 320, 324, 328, 332, 336, 340, 344, 348, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388, 392, 396, 400, 404, 408, 412, 416, 420, 424, 428, 432, 436, 440, 444, 448, 452, 456, 460, 464, 468, 472, 476, 480, 484, and 488.

[0024] In some embodiments, the second binding domain targeting VEGFA is capable of disrupting the interaction of VEGFA with one or more of its receptors.

[0025] In some embodiments, the first binding domain is comprised in an antigen binding fragment derived from an antibody. In some embodiments, the first binding domain comprises a heavy chain variable domain (VH) comprising a heavy chain first complementary determining region (VH CDR1), a heavy chain second complementary determining region (VH CDR2), and a heavy chain third complementary determining region (VH CDR3). In some embodiments, the VH is comprised in a Fab, a Fab’, F(ab')2, a bispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), a bispecific scFv, disulfide stabilized Fv (dsFv), a minibody, a diabody, a bispecific diabody, triabody, a tetrabody, a maxibody, a camelid, a VHH, a Fab-Fc, a scFv-Fc, or a bispecific antibody.

[0026] In some embodiments, the VH comprises a set of VH CDR1, VH CDR2, and VH CDR3 derived from an antibody in Table 1A or Table IB. In some embodiments, the VH comprise an amino acid sequence of a VH set forth in Table 1 A or Table IB.

[0027] In some embodiments, the first binding domain is a VHH. In some embodiments, the first binding domain comprises: a) a VH CDR1 sequence of SEQ ID NO: 2; a VH CDR2 sequence of SEQ ID NO: 3; and a VH CDR3 sequence of SEQ ID NO: 4; b) a VH CDR1 sequence of SEQ IDDocket No. 56146-753.601NO: 6; a VH CDR2 sequence of SEQ ID NO: 7; and a VH CDR3 sequence of SEQ ID NO: 8; c) a VH CDR1 sequence of SEQ ID NO: 14; a VH CDR2 sequence of SEQ ID NO: 15; and a VH CDR3 sequence of CDR3 SEQ ID NO: 16; d) a VH CDR1 sequence of SEQ ID NO: 18; a VH CDR2 sequence of SEQ ID NO: 19; and a VH CDR3 sequence of CDR 3 of SEQ ID NO: 20; or e) a VH CDR1 sequence of SEQ ID NO: 288, a VH CDR2 sequence of SEQ ID NO: 289, and a VH CDR3 sequence of SEQ ID NO: 290. In some embodiments, the VHH comprises an amino acid sequence selected from: a) SEQ ID NO: 1; b) SEQ ID NO: 5; c) SEQ ID NO: 13; d) SEQ ID NO: 17; and e) SEQ ID NO: 287.

[0028] In some embodiments, the VH comprises: a) a VH CDR1 sequence of NYYMY (SEQ ID NO: 80), a VH CDR2 sequence of GINPSNGGTNFNEKFKN (SEQ ID NO: 81), and a VH CDR3 sequence of RDYRFDMGFD Y (SEQ ID NO: 82); b) a VH CDR1 sequence of NSGMH (SEQ ID NO:86), a VH CDR2 sequence of VIWYDGSKRYYADSVKG (SEQ ID NO: 87), and a VH CDR3 sequence of NDDY (SEQ ID NO: 88); or c) a VH CDR1 sequence of GYTFTSYYMY (SEQ ID NO: 113), a VH CDR2 sequence of GVNPSNGGTNFNEKFKS (SEQ ID NO: 114), and a VH CDR3 sequence of RDYRYDMGFDY (SEQ ID NO: 115).

[0029] In some embodiments, the VH comprises: a) a VH CDR1 sequence of NYYMY (SEQ ID NO: 80), a VH CDR2 sequence of GINPSNGGTNFNEKFKN (SEQ ID NO: 81), and a VH CDR3 sequence of RDYRFDMGFDY (SEQ ID NO: 82); b) a VH CDR1 sequence of NSGMH (SEQ ID NO:86), a VH CDR2 sequence of VIWYDGSKRYYADSVKG (SEQ ID NO: 87), and a VH CDR3 sequence of NDDY (SEQ ID NO: 88); or c) a VH CDR1 sequence of GYTFTSYYMY (SEQ ID NO: 113), a VH CDR2 sequence of GVNPSNGGTNFNEKFKS (SEQ ID NO: 114), and a VH CDR3 sequence of RDYRYDMGFDY (SEQ ID NO: 115). In some embodiments, the VH comprises: a) a VH having a sequence QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNG GTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQG TTVTV (SEQ ID NO: 48); b) a VH having a sequence QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSK RYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS(SEQ ID NO: 50); or c) a VH having a sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMYWVRQAPGQGLEWMGGVNPSNG GTNFNEKFKSRVTITADKSTSTAYMELSSLRSEDTAVYYCARRDYRYDMGFDYWGQG TTVTVSS (SEQ ID NO: 76).Docket No. 56146-753.601

[0030] In some embodiments, the first binding domain comprises a light chain variable domain (VL) comprising a light chain first complementary determining region (VL CDR1), a light chain second complementary determining region (VL CDR2), and a light chain third complementary determining region (VL CDR3). In some embodiments, the VL comprises a set of VL CDR1, VL CDR2, and VL CDR3 derived from an antibody in Table 1 A.

[0031] In some embodiments, the VL comprises a) a VL CDR1 sequence of RASKGVSTSGYSYLH (SEQ ID NO: 83), a VL CDR2 sequence of LASYLES (SEQ ID NO: 84), and a VL CDR3 sequence of QHSRDLPLT (SEQ ID NO: 85); b) a VL CDR1 sequence of RASQSVSSYLA (SEQ ID NO: 89), a VL CDR2 sequence of DASNRAT (SEQ ID NO: 90), and a VL CDR3 sequence of QQSSNWPRT (SEQ ID NO: 91); or c) a VL CDR1 sequence of RASKGVSTSGYSYLH (SEQ ID NO: 83), a VL CDR2 sequence of LASYLE (SEQ ID NO: 117), and a VL CDR3 sequence of QHSRELPLT (SEQ ID NO: 118).

[0032] In some embodiments, the VL comprises: a) a VL having a sequence EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLES GVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK (SEQ ID NO: 49); b) a VL having a sequence EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK (SEQ ID NO: 51); or c) a VL having a sequence EIVLTQSPATLSLSPGERATISCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLES GVPARFSGSGSGTDFTLTISSLEPEDFATYYCQHSRELPLTFGTGTKVEIK (SEQ ID NO: 77).In some embodiments, the first binding domain comprises: a) a VH having a VH CDR1 sequence of NYYMY (SEQ ID NO: 80), a VH CDR2 sequence of GINPSNGGTNFNEKFKN (SEQ ID NO: 81), and a VH CDR3 sequence of RDYRFDMGFDY (SEQ ID NO: 82), and a VL having a VL CDR1 sequence of RASKGVSTSGYSYLH (SEQ ID NO: 83), a VL CDR2 sequence of LASYLES (SEQ ID NO: 84), and a VL CDR3 sequence of QHSRDLPLT (SEQ ID NO: 85); or b) a VH having a VH CDR1 sequence of NSGMH (SEQ ID NO: 86), a VH CDR2 sequence of VIWYDGSKRYYADSVKG (SEQ ID NO: 87), and a VH CDR3 sequence of NDDY (SEQ ID NO: 88), and a VL having a VL CDR1 sequence of RASQSVSSYLA (SEQ ID NO: 89), a VL CDR2 sequence of DASNRAT (SEQ ID NO: 90), and a VL CDR3 sequence of QQSSNWPRT (SEQ ID NO: 91; or c) a VH having a VH CDR1 sequence of GYTFTSYYMY (SEQ ID NO: 113), a VH CDR2 sequence of GVNPSNGGTNFNEKFKSDocketNo. 56146-753.601(SEQ ID NO: 114), and a VH CDR3 sequence of RDYRYDMGFD Y (SEQ ID NO: 115), and a VL having a VL CDR1 sequence of RASKGVSTSGYSYLH (SEQ ID NO: 83), a VL CDR2 sequence of LASYLE (SEQ ID NO: 117), and a VL CDR3 sequence of QHSRELPLT (SEQ ID NO: 118).

[0033] In some embodiments, the first binding domain comprises: a) a VH having a sequence QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNG GTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQG TTVTV (SEQ ID NO: 48) and a VL having a sequence EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLES GVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK (SEQ ID NO: 49); b) a VH having a sequence QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSK RYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS(SEQ ID NO: 50) and a VL having a sequence EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK (SEQ ID NO: 51); or c) a VH having a sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMYWVRQAPGQGLEWMGGVNPSNG GTNFNEKFKSRVTITADKSTSTAYMELSSLRSEDTAVYYCARRDYRYDMGFDYWGQG TTVTVSS (SEQ ID NO: 76) and a VL having a sequence EIVLTQSPATLSLSPGERATISCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLES GVPARFSGSGSGTDFTLTISSLEPEDFATYYCQHSRELPLTFGTGTKVEIK (SEQ ID NO: 77)

[0034] In some embodiments, the first binding domain is an scFv. In some embodiments, the first binding domain is a Fab. In some embodiments, the composition comprises an Fc domain comprising first CH2 and CH3 domains on a first polypeptide chain and second CH2 and CH3 domains on a second polypeptide chain. In some embodiments, the Fc domain is derived from an IgG. In some embodiments, the Fc domain is derived from an IgGl or IgG4.

[0035] In some embodiments, the composition comprises a structure of the formula:X— Y~ ZX, w "7»T Z.Docket No. 56146-753.601wherein: Y is the first CH2 and CH3 domains; Y’ is the second CH2 and CH3 domains; X and X’ are each independently the first binding domain, the second binding domain, a copy of the first binding domain, a copy of the second binding domain, or absent; Z and Z’ are each independently the first binding domain, the second binding domain, a copy of the first binding domain, a copy of the second binding domain, a third binding domain targeting PD-1, or a fourth binding domain targeting VEGFA, or absent; wherein X, Y, and Z and X’, Y’ and Z’ are depicted in an N-terminal to C-terminal direction; and wherein each of X, X’, Z, and Z’ are independently and optionally connected to Y or Y’ via a peptide linker.

[0036] In some embodiments, X is the first binding domain; X’ is a copy of the first binding domain or absent; and one of Z or Z’ is the second binding domain and the other is absent or a copy of the second binding domain. In some embodiments, one of Z or Z’ is the second binding domain and the other is a copy of the second binding domain. In some embodiments, X is the second binding domain; X’ is a copy of the second binding domain or absent; and one of Z or Z’ is the first binding domain and the other is absent or a copy of the first binding domain. In some embodiments, one of Z or Z’ is the first binding domain and the other is a copy of the first binding domain.

[0037] In some embodiments, X is the first binding domain and is a Fab, X’ is a copy of the first binding domain, Z is the second binding domain and is an scFv, and Z’ is a copy of the second binding domain; Xis the first binding domain and is a Fab, X’ is a copy of the first binding domain; Z is the second binding domain and is a single domain antibody, and Z’ is a copy of the second binding domain; X is the first binding domain and is a Fab, X’ is absent, Z is the second binding domain and is an scFv, and Z’ is a copy of the second binding domain; X is the first binding domain and is a Fab, X’ is absent, Z is the second binding domain and is a single domain antibody, and Z’ is a copy of the second binding domain; X is the first binding domain and is a Fab, X’ is the second biding domain and is a Fab, and Z and Z’ are absent; X is the first binding domain and is a Fab, X’ is the second binding domain and is an scFv, and Z and Z’ are absent; X is the first binding domain and is an scFv, X’ is the second binding domain and is a Fab, and Z and Z’ are absent; X is the second binding domain and is a Fab, X’ is a copy of the second binding domain, Z is the first binding domain and is an scFv, and Z’ is a copy of the first binding domain; X is the second binding domain and is a Fab, X’ is a copy of the second binding domain; Z is the first binding domain and is a single domain antibody, and Z’ is a copy of the first binding domain; X is the second binding domain and is a Fab, X’ is absent, Z is the first binding domain and is an scFv, and Z’ is a copy of the first binding domain; X is the second binding domain and is a Fab,Docket No. 56146-753.601X’ is absent, Z is the first binding domain and is a single domain antibody, and Z’ is a copy of the first binding domain; X is the second binding domain and is a Fab, X’ is the first binding domain and is a Fab, Z is a fourth binding domain targeting VEGFA and is an scFv, and Z’ is absent; or X is the second binding domain and is a Fab, X’ is the first binding domain and is a Fab, Z is a fourth binding domain targeting VEGFA and is a single domain antibody, and Z’ is absent.

[0038] In some embodiments, the Fc domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in any one of SEQ ID NOs: 229-234 or 236-241.

[0039] In another aspect provided herein is a composition comprising a binding domain targeting programmed cell death protein 1 (PD-1); wherein the first binding domain comprises a VHH having a VH CDR1, VH CDR2, and VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 each have sequences respectively selected from wherein the first binding domain comprises a VHH having a VH CDR1, VH CDR2, and VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 each have sequences respectively selected from 510, 511, and 512; SEQ ID NOs: 502, 503, and 504; SEQ ID NOs: 506, 507, and 508; SEQ ID NOs: SEQ ID NOs: 514, 515, and 516; SEQ ID NOs: 518, 511, and 520; SEQ ID NOs: 522, 523, and 524; SEQ ID NOs: 526, 527, and 528; SEQ ID NOs: 530, 531, and 532; SEQ ID NOs: 534, 535, and 536; SEQ ID NOs: 538, 539, and 540; SEQ ID NOs: 542, 543, and 544; SEQ ID NOs: 546, 547, and 548; SEQ ID NOs: 550, 511, and 552; SEQ ID NOs: 554, 555, and 556; SEQ ID NOs: 558, 543, and 560; SEQ ID NOs: 562, 563, and 564; SEQ ID NOs: 566, 567, and 568; SEQ ID NOs: 570, 511, and 572; SEQ ID NOs: 574, 567, and 576; SEQ ID NOs: 578, 579, and 580; SEQ ID NOs: 582, 555, and 584; SEQ ID NOs: 586, 587, and 588; SEQ ID NOs: 590, 591, and 592; SEQ ID NOs: 594, 555, and 596; SEQ ID NOs: 578, 599, and 600; SEQ ID NOs: 602, 511, and 604; SEQ ID NOs: 606, 567, and 608; SEQ ID NOs: 610, 591, and 612; SEQ ID NOs: 614, 615, and 616; SEQ ID NOs: 618, 567, and 620; SEQ ID NOs: 622, 567, and 624; SEQ ID NOs: 626, 511, and 628; SEQ ID NOs: 630, 563, and 632; SEQ ID NOs: 634, 599, and 636; SEQ ID NOs: 638, 555, and 640; SEQ ID NOs: 642, 567, and 644; SEQ ID NOs: 646, 511, and 648; SEQ ID NOs: 650, 515, and 652; SEQ ID NOs: 654, 547, and 656; SEQ ID NOs: 658, 659, and 660; SEQ ID NOs: 662, 663, and 664; SEQ ID NOs: 421, 591, and 668; SEQ ID NOs: 670, 591, and 672; SEQ ID NOs: 674, 675, and 676; SEQ ID NOs: 678, 679, and 680; SEQ ID NOs: 682, 599, and 684; SEQ ID NOs: 686, 547, and 688; SEQ ID NOs: 690, 691, and 692; and SEQ ID NOs: 510, 699, and 512.

[0040] In some embodiments, the VHH comprises the amino acid sequence set forth in any one of SEQ ID NOs: 509, 501, 505, 513, 517, 521, 525, 529, 533, 537, 541, 545, 549, 553, 557, 561,Docket No. 56146-753.601565, 569, 573, 577, 581, 585, 589, 593, 597, 601, 605, 609, 613, 617, 621, 625, 629, 633, 637, 641, 645, 649, 653, 657, 661, 665, 669, 673, 677, 681, 685, 689, 693, 694, 695, 696, 697, and 698.

[0041] In some embodiments, the VH CDR1, VH CDR2, and VH CDR3 each have sequences respectively selected from: 510, 511, and 512; 570, 511, and 572; 602, 511, and 604; 626, 511, and 628; 658, 659, and 660; and 510, 699, and 512. In some embodiments, the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 509, 569, 601, 625, 657, 693, 694, 695, 696, 697, and 698. In some embodiments, the VHH comprises the amino acid sequence set forth in any one of SEQ ID NOs: 509, 569, 601, 625, 657, 693, 694, 695, 696, 697, and 698.

[0042] In some embodiments, the VH CDR1 has a sequence of SEQ ID NO: 510, the VH CDR2 has a sequence of SEQ ID NO: 511, and the VH CDR3 has a sequence of SEQ ID NO: 512. In some embodiments, the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 509, 693, 694, 695, 696, and 698. In some embodiments, the VHH comprises the amino acid sequence set forth in any one of SEQ ID NOs: 509, 693, 694, 695, 696, and 698. In some embodiments, the VHH comprises the amino acid sequence of SEQ ID NO: 509. In some embodiments, the VHH comprises the amino acid sequence of SEQ ID NO: 693. In some embodiments, the VHH comprises the amino acid sequence of SEQ ID NO: 694. In some embodiments, the VHH comprises the amino acid sequence of SEQ ID NO: 695. In some embodiments, the VHH comprises the amino acid sequence of SEQ ID NO: 696. In some embodiments, the VHH comprises the amino acid sequence of SEQ ID NO: 698.

[0043] In some embodiments, the VH CDR1 has a sequence of SEQ ID NO: 570, the VH CDR2 has a sequence of SEQ ID NO: 511, and the VH CDR3 has a sequence of SEQ ID NO: 572. In some embodiments, the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 569. In some embodiments, the VHH comprises the amino acid sequence set forth in SEQ ID NO: 569.

[0044] In some embodiments, the VH CDR1 has a sequence of SEQ ID NO: 602, the VH CDR2 has a sequence of SEQ ID NO: 511, and the VH CDR3 has a sequence of SEQ ID NO: 604. In some embodiments, the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 601. In some embodiments, the VHH comprises the amino acid sequence set forth in SEQ ID NO: 601.

[0045] In some embodiments, wherein the VH CDR1 has a sequence of SEQ ID NO: 626, the VH CDR2 has a sequence of SEQ ID NO: 511, and the VH CDR3 has a sequence of SEQ ID NO:Docket No. 56146-753.601628. In some embodiments, the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 625. In some embodiments, the VHH comprises the amino acid sequence set forth in SEQ ID NO: 625.

[0046] In some embodiments, the VH CDR1 has a sequence of SEQ ID NO: 658, the VH CDR2 has a sequence of SEQ ID NO: 659, and the VH CDR3 has a sequence of SEQ ID NO: 660. In some embodiments, the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 657. In some embodiments, the VHH comprises the amino acid sequence set forth in SEQ ID NO: 657.

[0047] In some embodiments, the VH CDR1 has a sequence of SEQ ID NO: 510, the VH CDR2 has a sequence of SEQ ID NO: 699, and the VH CDR3 has a sequence of SEQ ID NO: 512. In some embodiments, the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 569. In some embodiments, the VHH comprises the amino acid sequence set forth in SEQ ID NO: 569.

[0048] In some embodiments, the first binding domain targeting programmed cell death protein 1 (PD-1) is capable of disrupting the interaction of PD-1 with programmed cell death ligand 1 (PD-L1).

[0049] In another aspect herein is a comprising a binding domain targeting vascular endothelial growth factor A (VEGFA); wherein the VH CDR1, VH CDR2, and VH CDR3 each have sequences respectively selected from: SEQ ID NOs: 301, 302, and 303; SEQ ID NOs: 305, 306, and 307; SEQ ID NOs: 309, 310, and 311; SEQ ID NOs: 313, 314, and 315; SEQ ID NOs: 317, 318, and 319; SEQ ID NOs: 321, 322, and 323; SEQ ID NOs: 325, 326, and 327; SEQ ID NOs: 329, 330, and 331; SEQ ID NOs: 333, 334, and 335; SEQ ID NOs: 337, 338, and 339; SEQ ID NOs: 341, 342, and 343; SEQ ID NOs: 345, 346, and 347; SEQ ID NOs: 349, 350, and 351; SEQ ID NOs: 353, 354, and 355; SEQ ID NOs: 357, 358, and 359; SEQ ID NOs: 361, 362, and 363; SEQ ID NOs: 365, 366, and 367; SEQ ID NOs: 369, 370, and 371; SEQ ID NOs: 373, 374, and 375; SEQ ID NOs: 377, 378, and 379; SEQ ID NOs: 381, 382, and 383; SEQ ID NOs: 385, 386, and 387; SEQ ID NOs: 389, 390, and 391; SEQ ID NOs: 393, 394, and 395; SEQ ID NOs: 397, 398, and 399; SEQ ID NOs: 401, 402, and 403; SEQ ID NOs: 405, 406, and 407; SEQ ID NOs: 409, 410, and 411; SEQ ID NOs: 413, 414, and 415; SEQ ID NOs: 417, 418, and 419; SEQ ID NOs: 421, 422, and 423; SEQ ID NOs: 425, 426, and 427; SEQ ID NOs: 429, 430, and 431; SEQ ID NOs: 433, 434, and 435; SEQ ID NOs: 437, 438, and 439; SEQ ID NOs: 441, 442, and 443; SEQ ID NOs: 445, 398, and 447; SEQ ID NOs: 449, 426, and 451; SEQ ID NOs: 453, 454, and 455; SEQ ID NOs: 457, 458, and 459; SEQ ID NOs: 461, 462, and 463; SEQ ID NOs: 465, 466,Docket No. 56146-753.601and 467; SEQ ID NOs: 469, 470, and 471; SEQ ID NOs: 473, 474, and 475; SEQ ID NOs: 477, 478, and 479; SEQ ID NOs: 481, 482, and 483; SEQ ID NOs: 485, 486, and 487; and SEQ ID NOs: 489, 490, and 491. In some embodiments, the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 300, 304, 308, 312, 316, 320, 324, 328, 332, 336, 340, 344, 348, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388, 392, 396, 400, 404, 408, 412, 416, 420, 424, 428, 432, 436, 440, 444, 448, 452, 456, 460, 464, 468, 472, 476, 480, 484, and 488. In some embodiments, the VHH comprises the amino acid sequence set forth in any one of SEQ ID NOs: 300, 304, 308, 312, 316, 320, 324, 328, 332, 336, 340, 344, 348, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388, 392, 396, 400, 404, 408, 412, 416, 420, 424, 428, 432, 436, 440, 444, 448, 452, 456, 460, 464, 468, 472, 476, 480, 484, and 488. In some embodiments, the second binding domain targeting VEGFA is capable of disrupting the interaction of VEGFA with one or more of its receptors.

[0050] Also provided herein are fusion proteins of the binding domains described herein.

[0051] Further provided herein are nucleic acids encoding compositions described herein.

[0052] Also provided herein are host cells comprising the nucleic acids or compositions described herein.

[0053] Further provided herein are pharmaceutical compositions comprising the compositions herein.

[0054] In another aspect described herein is a method of treating cancer in a subject in need there comprising administering to the subject a composition described herein.

[0055] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.INCORPORATION BY REFERENCE

[0056] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.DocketNo. 56146-753.601BRIEF DESCRIPTION OF THE DRAWINGS

[0057] FIGURES 1A-1H depict formats of dual-binding compositions which contain anti-VEGFA and anti-PD-1 binding domains. In some embodiments, the anti-VEGFA binding domain comprises the VH and VL of Bevacizumab, or derivatives thereof. In some embodiments, the anti-PD-1 binding domains comprise the VH and VL of LZM-009, Pembrolizumab, or Nivolumab, or derivatives thereof. FIGs. 1 A and IB show formats which utilize Fabs as one set of binding domains and scFvs as the other set. In some embodiments, such as those depicted in FIGs. 1C-1H, a dual-binding composition comprises a single domain antibody (e.g., a VHH or light chain single domain antibody) as one of the binding domains (e.g., the anti-PD-1 or anti-VEGFA binding domain).

[0058] FIGURES 2A-2D depict formats of dual-binding compositions which contain anti-VEGFA and anti-PD-1 binding domains. These constructs contain a K248A substitution which can facilitate the conjugation of a single group to the construct, such as by using AJICAP™ technology. FIG. 2A and FIG. 2B depict embodiments in which a set of Fabs are used as binding domains and a set of scFvs are used as binding domains to the other target. In some embodiments, the anti-VEGFA binding domain comprises the VH and VL of Bevacizumab. In some embodiments, the anti-PD-1 binding domains comprise the VH and VL of LZM-009, Pembrolizumab, or Nivolumab. In some embodiments, such as those depicted in FIG. 2C and FIG.2D, a dual-binding composition comprises a single domain antibody (e.g., a VHH or light chain single domain antibody) as one of the binding domains (e.g., the anti-PD-1 or anti-VEGFA binding domain). In some embodiments, the anti-VEGFA single domain antibody binding domain is one of those described herein (e.g., a single domain antibody of any one of SEQ ID NOs:200, 201, 205, 209, 213, 217, 221, or 222, or a variant single domain antibody which comprises the CDRs set forth in one of those sequences). In some embodiments, the anti-PD-1 single domain antibody binding domain is one of those described herein (e.g., a single domain antibody of any one of SEQ ID NOs: 1, 5, 9, 17, 287 or one provided in Table IB (e.g., VHH47) or a variant single domain antibody which comprises the CDRs set forth in one of those sequences).

[0059] FIGURES 3A-3C depict formats of asymmetric dual-binding compositions in bispecific antibody formats containing anti-VEGFA and anti-PD-1 binding domains. FIG. 3 A depicts a format which uses two single Fabs as the binding domains in a typical bispecific antibody format. In the formats depicted, light chain pairing technology is utilized during manufacture to ensure proper pairing of VH and VL domains in the construct. In some embodiments, the anti-VEGFA binding domain comprises the VH and VL of Bevacizumab. In some embodiments, the anti-PD-Docket No. 56146-753.6011 binding domains comprise the VH and VL of LZM-009, Pembrolizumab, or Nivolumab. In some embodiments, such as that depicted in FIG. 3B, a second anti-VEGFA binding domain is incorporated via fusion to the C-terminus (optionally through a peptide linker). The second anti-VEGFA binding domain depicted in FIG. 3B is an scFv. In some embodiments, the second anti-VEGFA binding domain is derived from the same antibody as the first anti-VEGFA binding domain (e.g., the second anti-VEGFA binding domain contains the same VH and VL as the first anti-VEGFA binding domain). A similar embodiment is also depicted in FIG. 3C, which depicts a construct in which the second VEGFA binding domain is a single domain antibody (e.g., a VHH). In some embodiments, the single domain antibody is one of those described herein (e.g., a single domain antibody of any one of SEQ ID NOs: 200, 201, 205, 209, 213, 217, 221, or 222, or a variant single domain antibody which comprises the CDRs set forth in one of those sequences).

[0060] FIGURES 4A and 4B depict formats of asymmetric Fc domain containing dual-binding constructs in bispecific antibody formats containing anti-VEGFA and anti-PD-1 binding domains. This bispecific antibody format utilizes an scFv as one of the binding domains and eliminates the need for light chain pairing technology to be used in their manufacture. In some embodiments, the anti-VEGFA binding domain comprises the VH and VL of Bevacizumab, or derivatives thereof. In some embodiments, the anti-PD-1 binding domains comprise the VH and VL of LZM-009, Pembrolizumab, or Nivolumab, or derivatives thereof. FIG. 4A shows an embodiment with an anti-VEGF Fab and an anti-PD-1 scFv. FIG. 4B shows an embodiment with an anti-PD-1 Fab and an anti-VEGF scFv.

[0061] FIGURES 5A-5E show resulting traces of analytical heparin chromatography performed on Compositions 59, 74, 82, 88, and 96, respectively, as well as the relative retention times of control antibodies, from left to right, of Daratumumab, Obinutuzumab, Ustekinumab, Briakinumab, and Gantenerumab. Each of Compositions 59, 74, 82, 88, and 96 exhibited a retention time between that of Obinutuzumab and Ustekinumab.

[0062] FIGURE 6 depicts results of an accelerated stability experiment conducted on Compositions 10 and 46. Composition 10 showed faster degradation of percent monomer detected over time, with 68% of Composition 10 in monomeric form after 24 hours and 17% of Composition 10 in monomeric form after 48 hours. Composition 46 exhibited substantially slower degradation, retaining 96% monomeric form after 24 hours and 83% monomeric form after 48 hours.

[0063] FIGURES 7A-7E show the results of Dynamic Light Scattering (DLS) analysis (left panels) and analytical size exclusion chromatograph (aSEC) (right panels) for Compositions 59,Docket No. 56146-753.60174, 82, 88, and 96, respectively, in the presence or absence of VEGF. The notable shifts in both experiments supports cooperative effects (e.g., non-covalent multimerization) of these Compositions in the presence of VEGF.

[0064] FIGURE 8 shows the results of an anti-PD-1 ELISA assay using the indicated Compositions. Binding to human (left panel) and cynomolgus (right panel) PD-1 was measured.

[0065] FIGURE 9 shows the results of an PD-1 / PD-L1 blockade assay using the indicated Compositions.

[0066] FIGURE 10 shows the results of an anti-VEGF ELISA assay using the indicated Compositions.

[0067] FIGURE 11 shows the results of VEGF / VEGFR blocking assay using the indicated Compositions.

[0068] FIGURE 12 shows the results of a pharmacokinetic (PK) study of the indicated Compositions in C57BL / 6 mice transgenic for human FcRn.

[0069] FIGURE 13 shows results of a tumor growth inhibition study using the indicated Composition in an C57BL / 6 mouse model transgenic for human PD-1 / PD-L1 using an Mc38 tumor model overexpressing human VEGF A. Compositions 59 and 74 showed similar results (bottom lines in the chart), with Composition 74 (middle line in the chart) also showing significant tumor growth reduction comparted to vehicle (top line in the chart).

[0070] FIGURE 14 shows results of a tumor growth inhibition study using Composition 59 in a Balb / C nude mouse model using a SW620 CDX tumor model. The left panel shows average tumor size over time, and the right panel shows a survival curve.DETAILED DESCRIPTION

[0071] The following description and examples illustrate embodiments of the present disclosure in detail. It is to be understood that this present disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are numerous variations and modifications of this present disclosure, which are encompassed within its scope.

[0072] Although various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may be described herein in the context of separate embodiments for clarity, the present disclosure may also be implemented in a single embodiment.Docket No. 56146-753.601

[0073] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.Dual Binding Compositions

[0074] Provided herein in an aspect are dual binding compositions. In some embodiments, the dual binding compositions comprise at least one PD-1 binding domain and at least one VEGFA binding domain. It is expressly contemplated that additional features can be added to the dual binding compositions described herein, such as the conjugation or fusion of cytokines to the dual binding composition, or the addition of a further targeting group to the composition. Drug conjugates to the dual binding compositions described herein are also contemplated as being within the scope of the instant disclosure.Binding Domains

[0075] A dual binding composition described herein comprises one or more binding domains which specifically target one of PD-1 or VEGFA. In some preferred embodiments, a dual binding composition comprises both PD-1 and VEGFA binding domains.

[0076] A binding domain selectively binds or preferentially binds to a target if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances. As such, “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to specific binding means preferential binding where the affinity of the binding domain is at least at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater than the affinity of the binding domain for unrelated amino acid sequences.

[0077] In some embodiments, a binding domain of the disclosure can inhibit the action / activity of the substance to which it binds (e.g., by disruption an interaction of PD-1 with its receptor (e.g., PD-L1) and / or by disruption of VEGFA with one or more of its receptors (e.g., VEGFR1, VEGFR2, VEGFR3).

[0078] The binding domains of the instant disclosure can be of any desired format which binds specifically to the intended target. As such, the nature of the binding domains is not necessarily limited and can encompass, for example, polypeptides (e.g., antigen binding fragments derived from antibodies or other peptides which bind specifically for the target, such as variants ofDocket No. 56146-753.601receptors of the targets or other polypeptides), aptamers (e.g., nucleic acid aptamers), small molecules, and the like. In some embodiments, a binding domain of the instant disclosure is a polypeptide.

[0079] In some embodiments, a binding domain of the instant disclosure comprises an antigen binding fragment derived from an antibody, or a variant thereof. Antigen binding fragments of antibodies, including any of the antibodies herein (e.g., the anti-PD-1 antibodies or anti-VEGFA antibodies described below), are contemplated as being used as binding domains in dual binding compositions described herein. The terms “antigen binding portion of an antibody,” “antigen binding domain,” “antibody fragment,” or a “functional fragment of an antibody” are used interchangeably herein to refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. In some embodiments, such regions refer to the VH and / or the VL of the antibody, or a derivative or portion thereof. Representative antigen binding fragments include, but are not limited to, a Fab, a Fab', a F(ab')2, a bispecific F(ab')2, a trispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), a dsFv, a bispecific scFv, a variable heavy domain, a variable light domain, a variable NAR domain, bispecific scFv, an AVIMER®, a minibody, a diabody, a bispecific diabody, triabody, a tetrabody, a minibody, a maxibody, a camelid, a VHH, a minibody, an intrabody, fusion proteins comprising an antibody portion (e.g., a domain antibody), a single chain binding polypeptide, a scFv-Fc, a Fab-Fc, a bispecific T cell engager (BiTE; two scFvs produced as a single polypeptide chain, where each scFv comprises an amino acid sequences a combination of CDRs or a combination of VL / VL described herein), a tetravalent tandem diabody (TandAb; an antibody fragment that is produced as a non-covalent homodimer folder in a head-to-tail arrangement, e.g., a TandAb comprising an scFv, where the scFv comprises an amino acid sequences a combination of CDRs or a combination of VL / VL described herein), a Dual-Affinity Re-targeting Antibody (DART; different scFvs joined by a stabilizing interchain disulphide bond), a bispecific antibody (bscAb; two single-chain Fv fragments joined via a glycine-serine linker), a single domain antibody (sdAb), a fusion protein, a bispecific disulfide-stabilized Fv antibody fragment (dsFv-dsFv'; two different disulfide-stabilized Fv antibody fragments connected by flexible linker peptides).

[0080] In some embodiments, a binding domain of the instant disclosure is a Fab, a Fab’, F(ab')2, a bispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), a bispecific scFv, disulfide stabilized Fv (dsFv), a minibody, a diabody, a bispecific diabody, triabody, a tetrabody, a maxibody, a camelid, a single domain antibody (e.g., a VHH), a Fab-Fc, a scFv-Fc, or a bispecific antibody. In some embodiments, a binding domain of the instant disclosure is Fab,Docket No. 56146-753.601a Fab’, F(ab')2, a bispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), a bispecific scFv, disulfide stabilized Fv (dsFv), a minibody, a diabody, a bispecific diabody, triabody, a tetrabody, a maxibody, a cam elid, a single domain antibody (e.g., a VHH), a Fab-Fc, or a scFv-Fc. In some embodiments, a binding domain of the instant disclosure is Fab, a Fab’, F(ab')2, abispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), disulfide stabilized Fv (dsFv), a camelid, or a single domain antibody (e.g., VHH). In some embodiments, a binding domain of the instant disclosure is Fab, a Fab’, F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), disulfide stabilized Fv (dsFv), a camelid, or a single domain antibody (e.g., VHH). In some embodiments, a binding domain of the instant disclosure is Fab, a Fab’, a variable fragment (Fv), a single chain variable fragment (scFv), disulfide stabilized Fv (dsFv), or a single domain antibody (e.g., VHH). In some embodiments, a binding domain of the instant disclosure is Fab, a single chain variable fragment (scFv), or a single domain antibody such as a VHH. In some embodiments, each binding domain of a dual binding composition is independently one of those described above. In some embodiments, each binding domain of a dual binding composition described herein is independently a Fab, an scFv, or a single domain antibody, such as a VHH.

[0081] In some embodiments, a binding domain of the instant disclosure comprises a VH derived from an antibody, or a derivative thereof. In some embodiments, the VH retains the CDRs of the VH of the antibody from which the VH of the dual binding composition is derived (e.g., a heavy chain first complementary determining region (VH CDR1), a heavy chain second complementary determining region (VH CDR2), and a heavy chain third complementary determining region (VH CDR3)). In some embodiments, the VH retains the CDRS of the VH of the antibody from which it is derived and comprises one or more mutations in the framework region. In some embodiments, the VH retains the CDRs of the VH of the antibody from which it is derived and comprises up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutations in the framework region. In some embodiments, each mutation is a conservative mutation. In some embodiments, the binding domain comprises a VH of a corresponding antibody (e.g., with no mutations to the framework region). In some embodiments, the VH is comprised in a Fab, a Fab’, F(ab')2, a bispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), a bispecific scFv, disulfide stabilized Fv (dsFv), a minibody, a diabody, a bispecific diabody, triabody, a tetrabody, a maxibody, a camelid, a VHH, a Fab-Fc, a scFv-Fc, or a bispecific antibody. In some embodiments, the VH is comprised in a Fab, a Fab’, an scFv, or a VHH. In some embodiments, the VH is comprised in a Fab, anDocket No. 56146-753.601scFv, or a VHH. In some embodiments, the VH is comprised in a Fab or an scFv. In some embodiments, the VH is a VHH.

[0082] In some embodiments, a VHH of the instant disclosure can comprise one or more modifications which improve immunogenicity or reduce binding of pre-existing antibodies to the VHH. Examples of such modifications are described in, for example, U.S. Patent Publication Nos. US20180009888A9 (e.g., extension peptides of 1 to 5 extending beyond the C-terminal “SS” of the VHH, such as those consisting of the amino acids Ala and Gly), US20160207981A1 (e.g., substitutions of the C-terminal “SS” of the VHH, such as with an amino acid or peptide of a sequence E, SE, EG, SEG, EP, EPG, DP, DPG, K, SK, KP, KPG, RP, or RPG and / or substitutions of Leu 11 of the VHH, such as an LI IK, LI 1R, LI ID, or LI IE substitution), US20140161796A1 (e.g., deletions of certain sequences from the VHH), US20170121399A1 (e.g., substitutions of Leu 11 of the VHH (e.g., LI IK or LI IV) and / or Leu 89 (e.g., L89T), and Lin et al., “A structurebased engineering approach to abrogate pre-existing antibody binding to biotherapeutics,” PLoS ONE 16(7): e0254944. doi.org / 10.1371 / joumal.pone.0254944 (e.g., the addition of 1, 2, or 3 prolines beyond the C-terminal “SS” of the VHH, such as a two-proline peptide). In some embodiments, a VHH described herein comprises a C-terminal modification of the addition of 1, 2 or 3 prolines to the C-terminus of the VHH (e.g., to the C-terminus of any of the VHHs described herein). In some embodiments, a VHH described herein comprises a C-terminal modification of the addition of 2 prolines to the C-terminus of the VHH. In embodiments where two VHHs are linked in series (e.g., by a flexible peptide linker), in some instance only the C-terminal VHH will comprise the modification (e.g., only the C-terminal VHH comprises the sequence “PP” after the C-terminal “SS” of the VHH). In some instances, both VHHs linked in series will contain the modification (e.g., both will comprise the sequence “PP” after the C-terminal “SS” of each VHH).

[0083] In some embodiments, a binding domain of the instant disclosure comprises a VL derived from an antibody, or a derivative thereof. In some embodiments, the VL retains the CDRs of the VL of the antibody from which the VL of the dual binding composition is derived (e.g., a light chain first complementary determining region (VL CDR1), a light chain second complementary determining region (VL CDR2), and a light chain third complementary determining region (VL CDR3)). In some embodiments, the VL retains the CDRS of the VL of the antibody from which it is derived and comprises one or more mutations in the framework region. In some embodiments, the VL retains the CDRs of the VL of the antibody from which it is derived and comprises up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutations in the framework region. In some embodiments, each mutation is a conservative mutation. In some embodiments, the binding domain comprises a VLDocket No. 56146-753.601of a corresponding antibody (e.g., with no mutations to the framework region). In some embodiments, the VL is comprised in a Fab, a Fab’, F(ab')2, a bispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), a bispecific scFv, disulfide stabilized Fv (dsFv), a minibody, a diabody, a bispecific diabody, triabody, a tetrabody, a maxibody, a Fab-Fc, a scFv-Fc, or a bispecific antibody. In some embodiments, the VL is comprised in a Fab, a Fab’, or an scFv. In some embodiments, the VL is comprised in a Fab or an scFv. In some embodiments, the VL is comprised in the same Fab, Fab’, F(ab’)2, bispecific F(ab’)2, scFv, bispecific scFv, dsFv, minibody, diabody, bispecific diabody, triabody, tetrabody, maxibody, Fab-Fc, scFv-Fc, or bispecific antibody as the corresponding VH. In some embodiments, the VL is comprised in the same Fab, Fab’, or scFv as the corresponding VH. In some embodiments, the VL is comprised in the same Fab or scFv as the corresponding VH.

[0084] In some embodiments, a binding domain of the instant disclosure is a light chain single domain antibody.

[0085] In some embodiments, a binding domain specifically binds to one or more epitopes on one or more target antigens. In one instance, a binding domain selectively binds to an epitope on a single antigen.PD-1 Targeting Binding Domains

[0086] In some embodiments, a dual binding composition of the instant disclosure comprise one or more binding domains which target programmed cell death protein 1 (PD-1). In some embodiments, a binding domain incorporated into a dual binding composition of the disclosure specifically binds to PD-1. In some embodiments, the anti-PD-1 binding domain is capable of disrupting and / or preventing the interaction of PD-1 with programmed cell death ligand 1 (PD-Ll).

[0087] Programmed cell death protein 1 (also known as PD-1 and CD279), is a cell surface receptor that plays a role in down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. PD-1 is an immune cell inhibitory molecule that is expressed on activated B cells, T cells, and myeloid cells. PD-1 represents an immune checkpoint and guards against autoimmunity via a dual mechanism of promoting apoptosis (programmed cell death) in antigen-specific T-cells in lymph nodes while reducing apoptosis in regulatory T cells. PD-1 is a member of the CD28 / CTLA-4 / ICOS costimulatory receptor family that delivers negative signals that affect T and B cell immunity. PD-1 is monomeric both in solution as well as on cell surface, in contrast to CTLA-4 and other family members that are all disulfide-linked homodimers. Signaling through the PD-1 inhibitory receptor upon binding its ligand, PD-L1,Docket No. 56146-753.601suppresses immune responses against autoantigens and tumors and plays a role in the maintenance of peripheral immune tolerance. The interaction between PD-1 and PD-L1 results in a decrease in tumor infiltrating lymphocytes, a decrease in T cell receptor mediated proliferation, and immune evasion by the cancerous cells. A non-limiting, exemplary, human PD-1 amino acid sequence is MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLWTEGDNATFTCSFSN TSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRN DSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTP EPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 31).

[0088] In some embodiments, the anti -PD-1 binding domain is comprised in an antigen binding fragment derived from an antibody. For example, the anti-PD-1 binding domain can be derived from any anti-PD-1 antibody known in the art or which can be made according to well understood methods. In some embodiments, the antibody or antigen binding fragment thereof of a dual binding composition described herein is derived from an anti-PD-1 antibody or antigen binding fragment.

[0089] In one embodiment, an anti-PD-1 binding domain of a dual binding composition comprises and antigen binding fragment. In some embodiments, the anti-PD-1 binding domain of the disclosure comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) described herein, or of other anti-PD-1 antibodies or antigen binding fragments known in the art. In another embodiment, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment of the disclosure comprises a combination of complementarity determining regions (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3) described herein or of other anti-PD-1 antibodies or antigen binding fragments known in the art.

[0090] In one embodiment, an anti-PD-1 binding domain of the disclosure comprises the CDRs of an antibody selected from Tislelizumab, Baizean, 0KVO411B3N, BGB-A317, hu317-l / IgG4mt2, Sintilimab, Tyvyt, IBI-308, Toripalimab, TeRuiPuLi, Terepril, Tuoyi, JS-001, TAB-001, Camrelizumab, HR-301210, INCSHR-01210, SHR-1210, Cemiplimab, Cemiplimab-rwlc, LIBTAYO®, 6QVL057INT, H4H7798N, REGN-2810, SAR-439684, Avelumab, BAVENCIO®, 451238, KXG2PJ551I, MSB-0010682, MSB-0010718C, PF-06834635, Durvalumab, IMFINZI®, 28X28X9OKV, MEDI-4736, Lambrolizumab, Pembrolizumab, KEYTRUDA®, MK-3475, SCH-900475, h409All, Nivolumab, Nivolumab BMS, OPDIVO®, BMS-936558, MDX-1106, ONO-4538, Prolgolimab, Forteca, BCD-100, Penpulimab, AK-105, Zimberelimab,Docket No. 56146-753.601AB-122, GLS-010, WBP-3055, Balstilimab, 1Q2QT5M7EO, AGEN-2034, AGEN-2034w, Genolimzumab, Geptanolimab, APL-501, CBT-501, GB-226, Dostarlimab, ANB-011, GSK-4057190A, P0GVQ9A4S5, TSR-042, WBP-285, Serplulimab, HLX-10, CS-1003, Retifanlimab, 2Y3T5IF01Z, INCMGA-00012, INCMGA-0012, MGA-012, Sasanlimab, LZZ0IC2EWP, PF-06801591, RN-888, Spartalizumab, NVP-LZV-184, PDR-001, QOG25L6Z8Z, Relatlimab / nivolumab, BMS-986213, Cetrelimab, JNJ-3283, JNJ-63723283, LYK98WP91F, Tebotelimab, MGD-013, BCD-217, BAT-1306, HX-008, MEDI-5752, JTX-4014, Cadonilimab, AK-104, BI-754091, Pidilizumab, CT-011, MDV-9300, YBL-006, AMG-256, RG-6279, RO-7284755, BH-2950, IBI-315, RG-6139, RO-7247669, ONO-4685, AK-112, 609-A, LY-3434172, T-3011, MAX-10181, AMG-404, IBI-318, MGD-019, INCB-086550, ONCR-177, LY-3462817, RG-7769, RO-7121661, F-520, XmAb-23104, Pd-l-pik, SG-001, S-95016, Sym-021, LZM-009 (a.k.a., Lipustobart), Budigalimab, 6VDO4TY3OO, ABBV-181, PR-1648817, CC-90006, XmAb-20717, 2661380, AMP-224, B7-DCIg, EMB-02, ANB-030, PRS-332, [89Zr]Deferoxamide-pembrolizumab, 89Zr-Df-Pembrolizumab, [89Zr]Df-Pembrolizumab, STI-1110, STI-All 10, CX-188, mPD-1 Pb-Tx, MCLA-134, 244C8, ENUM 224C8, ENUM C8, 388D4, ENUM 388D4, ENUM D4, MEDI0680, or AMP-514 incorporated into a VH and VL. In some embodiments, the anti-PD-1 binding domain comprises the VH and VL of any one of these antibodies. In some embodiments, the anti-PD-1 binding domain comprises the VH and VL of any one of these antibodies or the VH and VL which include the CDRs of these antibodies in one of the following formats: a Fab, a Fab’, F(ab')2, a bispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), a bispecific scFv, disulfide stabilized Fv (dsFv), a minibody, a diabody, a bispecific diabody, triabody, a tetrabody, a maxibody, a Fab-Fc, a scFv-Fc, or a bispecific antibody. In some embodiments, the VH and VL of any one of these antibodies or the VH and VL which include the CDRs of these antibodies is comprised in a Fab, a Fab’, or an scFv. In some embodiments, the VH and VL any one of these antibodies or of the VH and VL which include the CDRs of these antibodies is comprised in a Fab or an scFv. In some embodiments, the VH and VL of any of these antibodies or the VH and VL which include the CDRs of these antibodies is comprised in a Fab. In some embodiments, the VH and VL of any of these antibodies or the VH and VL which include the CDRs of these antibodies is comprised in an scFv.

[0091] In some embodiments, an anti-PD-1 binding domain of the disclosure comprises the CDRs (e.g., VH and VL CDRs) of Tislelizumab, Sintilimab, Toripalimab, Terepril, Camrelizumab, Cemiplimab, Pembrolizumab Nivolumab, Prolgolimab, Penpulimab, Zimberelimab, Balstilimab,Docket No. 56146-753.601Genolimzumab, Geptanolimab, Dostarlimab, Serplulimab, Retifanlimab, Sasanlimab, Spartalizumab, Cetrelimab, Tebotelimab, Cadonilimab, Pidilizumab, LZM-009 (a.k.a. Lipustobart), or Budigalimab incorporated into a VH and VL. In one embodiment, an anti-PD-1 binding domain of the disclosure comprises the VH and VL of Tislelizumab, Sintilimab, Toripalimab, Terepril, Camrelizumab, Cemiplimab, Pembrolizumab Nivolumab, Prolgolimab, Penpulimab, Zimberelimab, Balstilimab, Genolimzumab, Geptanolimab, Dostarlimab, Serplulimab, Retifanlimab, Sasanlimab, Spartalizumab, Cetrelimab, Tebotelimab, Cadonilimab, Pidilizumab, LZM-009 (a.k.a. Lipustobart), or Budigalimab. In some embodiments, the anti-PD-1 binding domain comprises the VH and VL of any one of these antibodies or the VH and VL which include the CDRs of these antibodies in one of the following formats: a Fab, a Fab’, F(ab')2, a bispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), a bispecific scFv, disulfide stabilized Fv (dsFv), a minibody, a diabody, a bispecific diabody, triabody, a tetrabody, a maxibody, a Fab-Fc, a scFv-Fc, or a bispecific antibody. In some embodiments, the VH and VL of any one of these antibodies or the VH and VL which include the CDRs of these antibodies is comprised in a Fab, a Fab’, or an scFv. In some embodiments, the VH and VL any one of these antibodies or of the VH and VL which include the CDRs of these antibodies is comprised in a Fab or an scFv. In some embodiments, the VH and VL of any of these antibodies or the VH and VL which include the CDRs of these antibodies is comprised in a Fab. In some embodiments, the VH and VL of any of these antibodies or the VH and VL which include the CDRs of these antibodies is comprised in an scFv.

[0092] An anti-PD-1 binding domain can comprise a heavy chain, a VH, or a VH-CH1 domain (e.g., as in a Fab) having an amino acid sequence of any one of those set forth in Table 1 A, or a portion corresponding to a VH thereof (e.g., the portion depicted in bold). An anti-PD-1 binding domain can comprise (or further comprise) a light chain (e.g., as in a Fab) or VL having an amino acid sequence of any one of those described in Table 1A, or a portion corresponding to a VL thereof. In preferred embodiments, the heavy chain, VH, or VH-CH1 domain and VL or light chain are from the same antibody or antigen binding fragment described in Table 1 A.

[0093] In some embodiments, the anti-PD-1 binding domain comprises the VH and VL of Nivolumab, Pembrolizumab, LZM-009, Dostarlimab, Sintilimab, Spartalizumab, Tislelizumab, or Cemiplimab. In some embodiment, the anti-PD-1 binding domain comprises the VH and VL of Dostarlimab, Sintilimab, Spartalizumab, or Tislelizumab. In some embodiments, the anti-PD-1 binding domain comprises the VH and VL of Nivolumab, Pembrolizumab, LZM-009 (a.k.a. Lipustobart), or Cemiplimab. In some embodiments, the anti-PD-1 binding domain comprises theDocket No. 56146-753.601VH and VL of Nivolumab. In some embodiments, the anti-PD-1 binding domain comprises the VH and VL of Nivolumab in a Fab or scFv format. In some embodiments, the anti-PD-1 binding domain comprises the VH and VL of LZM-009 (a.k.a. Lipustobart). In some embodiments, the anti-PD-1 binding domain comprises the VH and VL of LZM-009 (a.k.a. Lipustobart) in a Fab or scFv format.

[0094] TABLE 1A provides the sequences of exemplary anti-PD-1 antibodies and anti-PD-1 antigen binding fragments which contain sets of CDRs which can be incorporated into VHs and / or VLs and used as anti-PD-1 binding domains as described herein. In some embodiments, the VHs and VLs of the antibodies in Table 1 A are incorporated into anti-PD-1 binding domains (e.g., in a Fab or scFv format). In some embodiments, CDRs of a VH described in Table 1 A is incorporated into a binding domain as a VHH. In some embodiments, a VH as described in Table 1A is incorporated into a binding domain as a VHH.

[0095] In some instances, the SEQ ID NOs listed in Table 1 A contain full-length heavy or light chains of the indicated antibodies with the VH or VL respectively indicated in bold. Where there is a reference herein to a VH or VL of a SEQ ID NO in Table 1A which contains a full-length heavy or light chain, it is intended to reference the bolded portion of the sequence. For example, reference to “a VH having an amino acid sequence shown in SEQ ID NO: 32” refers to the bolded portion of SEQ ID NO: 32 in Table 1 A.

[0096] An anti-PD-1 binding domain can comprise a VH having an amino acid sequence of any one of SEQ ID NOS: 32, 34, 36, 38, 40, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78. An anti-PD-1 binding domain can comprise (or further comprise) a VL having an amino acid sequence of any one of SEQ ID NOS: 33, 35, 37, 39, 41, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, and 79.

[0097] An anti-PD-1 binding domain can comprise a heavy chain or VH having an amino acid sequence of any one of SEQ ID NOS: 32, 34, 36, 38, 40, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78, or a portion corresponding to a VH thereof (e.g., the portion depicted in bold). An anti-PD-1 binding domain can comprise (or further comprise) a light chain or VL having an amino acid sequence of any one of SEQ ID NOS: 33, 35, 37, 39, 41, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, and 79, or a portion corresponding to a VL thereof.

[0098] In one instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 32, and a VL having an amino acid sequence shown in SEQ ID NO: 33. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acidDocket No. 56146-753.601sequence shown in SEQ ID NO: 34, and a VL having an amino acid sequence shown in SEQ ID NO: 35. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 36, and a VL having an amino acid sequence shown in SEQ ID NO: 37. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 38, and a VL having an amino acid sequence shown in SEQ ID NO: 39. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 40, and a VL having an amino acid sequence shown in SEQ ID NO: 41. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 46, and a VL having an amino acid sequence shown in SEQ ID NO: 47. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 48, and a VL having an amino acid sequence shown in SEQ ID NO: 49. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 50, and a VL having an amino acid sequence shown in SEQ ID NO: 51. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 52, and a VL having an amino acid sequence shown in SEQ ID NO: 53. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 54, and a VL having an amino acid sequence shown in SEQ ID NO: 55. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 56, and a VL having an amino acid sequence shown in SEQ ID NO: 57. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 58, and a VL having an amino acid sequence shown in SEQ ID NO: 59. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 60, and a VL having an amino acid sequence shown in SEQ ID NO: 61. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 62, and a VL having an amino acid sequence shown in SEQ ID NO: 63. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 64, and a VL having an amino acid sequence shown in SEQ ID NO: 65. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 66, and a VL having an amino acid sequence shown in SEQ ID NO: 67. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 68, and a VL having an amino acid sequence shown in SEQ ID NO: 69. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 70, and a VL having an amino acid sequence shown in SEQ IDDocket No. 56146-753.601NO: 71. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 72, and a VL having an amino acid sequence shown in SEQ ID NO: 73. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 74, and a VL having an amino acid sequence shown in SEQ ID NO: 75. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 76, and a VL having an amino acid sequence shown in SEQ ID NO: 77. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 78, and a VL having an amino acid sequence shown in SEQ ID NO: 79. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 9, and a VL having an amino acid sequence shown in SEQ ID NO: 10. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 11, and a VL having an amino acid sequence shown in SEQ ID NO: 12.

[0099] In one instance, an anti-PD-1 binding domain comprises a VH CDR1 having an amino acid sequence of SEQ ID NO: 80, a VH CDR2 having an amino acid sequence of SEQ ID NO: 81, a VH CDR3 having an amino acid sequence of SEQ ID NO: 82, VL CDR1 having an amino acid sequence of SEQ ID NO: 83, a VL CDR2 having an amino acid sequence of SEQ ID NO: 84, and a VL CDR3 having an amino acid sequence of SEQ ID NO: 85. In some embodiments, the anti-PD-1 binding domain comprises a corresponding VH and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the VH and VL of the parent antibody (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0100] In one instance, an anti-PD-1 binding domain comprises a VH CDR1 having an amino acid sequence of SEQ ID NO: 86, a VH CDR2 having an amino acid sequence of SEQ ID NO: 87, a VH CDR3 having an amino acid sequence of SEQ ID NO: 88, VL CDR1 having an amino acid sequence of SEQ ID NO: 89, a VL CDR2 having an amino acid sequence of SEQ ID NO: 90, and a VL CDR3 having an amino acid sequence of SEQ ID NO: 91. In some embodiments, the anti-PD-1 binding domain comprises a corresponding VH and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the VH and VL of the parent antibody (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0101] In one instance, an anti-PD-1 binding domain comprises a VH CDR1 having an amino acid sequence of SEQ ID NO: 92, a VH CDR2 having an amino acid sequence of SEQ ID NO:Docket No. 56146-753.60193, a VH CDR3 having an amino acid sequence of SEQ ID NO: 94, VL CDR1 having an amino acid sequence of SEQ ID NO: 95, a VL CDR2 having an amino acid sequence of SEQ ID NO: 96, and a VL CDR3 having an amino acid sequence of SEQ ID NO: 97. In some embodiments, the anti-PD-1 binding domain comprises a corresponding VH and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the VH and VL of the parent antibody (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0102] In one instance, an anti-PD-1 binding domain comprises a VH CDR1 having an amino acid sequence of SEQ ID NO: 98, a VH CDR2 having an amino acid sequence of SEQ ID NO: 99, a VH CDR3 having an amino acid sequence of SEQ ID NO: 100, VL CDR1 having an amino acid sequence of SEQ ID NO: 89, a VL CDR2 having an amino acid sequence of SEQ ID NO: 102, and a VL CDR3 having an amino acid sequence of SEQ ID NO: 103. In some embodiments, the anti-PD-1 binding domain comprises a corresponding VH and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the VH and VL of the parent antibody (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0103] In one instance, an anti-PD-1 binding domain comprises a VH CDR1 having an amino acid sequence of SEQ ID NO: 113, a VH CDR2 having an amino acid sequence of SEQ ID NO: 114, a VH CDR3 having an amino acid sequence of SEQ ID NO: 115, VL CDR1 having an amino acid sequence of SEQ ID NO: 83, a VL CDR2 having an amino acid sequence of SEQ ID NO: 117, and a VL CDR3 having an amino acid sequence of SEQ ID NO: 118. In some embodiments, the anti-PD-1 binding domain comprises a corresponding VH and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the VH and VL of the parent antibody (i.e., SEQ ID NOs: 76 and 77, respectively) (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0104] In some embodiments, a dual binding composition comprises one of the antibodies described in Table 1 A below (optionally comprising one or more modifications to the Fc domain, hinge region, or other modification described herein) fused to a binding domain specific for VEGFA (e.g., any of the VEGFA binding domains described herein). In some embodiments, the binding domain specific for VEGFA is fused to the C-terminus of the heavy chain of the antibody described in Table 1 A. In some embodiments, the binding domain specific for VEGFA fused to the antibody of Table 1A is an anti-VEGFA single-domain antibody as described herein.Docket No. 56146-753.601

[0105] In some embodiments, an anti-PD-1 binding domain comprises a single domain antibody. In some embodiments, the anti-PD-1 binding domain of a dual binding composition is a single domain antibody described in Table 1 A, or a variant thereof.

[0106] In some embodiments, the anti-PD-1 binding domain is a single domain antibody comprising a CDR1 as set forth in SEQ ID NO: 2, a CDR2 as set forth in SEQ ID NO: 3, and a CDR3 as set forth in SEQ ID NO: 4. In some embodiments, the CDRs are comprised in a VHH. In some embodiments, the anti-PD-1 binding domain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to the sequence set forth in SEQ ID NO: 1 (e.g., the anti-PD-1 binding domain comprises the indicated sequence identity to SEQ ID NO: 1 and retains the CDRs).

[0107] In some embodiments, the anti-PD-1 binding domain is a single domain antibody comprising a CDR1 as set forth in SEQ ID NO: 6, a CDR2 as set forth in SEQ ID NO: 7, and a CDR3 as set forth in SEQ ID NO: 8. In some embodiments, the CDRs are comprised in a VHH. In some embodiments, the anti-PD-1 binding domain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to the sequence set forth in SEQ ID NO: 5 (e.g., the anti-PD-1 binding domain comprises the indicated sequence identity to SEQ ID NO: 5 and retains the CDRs).

[0108] In some embodiments, the anti-PD-1 binding domain is a single domain antibody comprising a CDR1 as set forth in SEQ ID NO: 14, a CDR2 as set forth in SEQ ID NO: 15, and a CDR3 as set forth in SEQ ID NO: 16. In some embodiments, the CDRs are comprised in a VHH. In some embodiments, the anti-PD-1 binding domain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to the sequence set forth in SEQ ID NO: 13 (e.g., the anti-PD-1 binding domain comprises the indicated sequence identity to SEQ ID NO: 13 and retains the CDRs).

[0109] In some embodiments, the anti-PD-1 binding domain is a single domain antibody comprising a CDR1 as set forth in SEQ ID NO: 18, a CDR2 as set forth in SEQ ID NO: 19, and a CDR3 as set forth in SEQ ID NO: 20. In some embodiments, the CDRs are comprised in a VHH. In some embodiments, the anti-PD-1 binding domain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to the sequence set forth in SEQ ID NO: 17 (e.g., the anti-PD-1 binding domain comprises the indicated sequence identity to SEQ ID NO: 17 and retains the CDRs).

[0110] In some embodiments, the anti-PD-1 binding domain is a single domain antibody comprising a CDR1 as set forth in SEQ ID NO: 288, a CDR2 as set forth in SEQ ID NO: 289, andDocket No. 56146-753.601a CDR3 as set forth in SEQ ID NO: 290. In some embodiments, the CDRs are comprised in a VHH. In some embodiments, the anti-PD-1 binding domain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to the sequence set forth in SEQ ID NO: 287 (e.g., the anti-PD-1 binding domain comprises the indicated sequence identity to SEQ ID NO: 287 and retains the CDRs).

[0111] In some embodiments, a dual binding composition comprises one or two anti-PD-1 binding domains. In some embodiments, the dual binding composition comprises one anti-PD-1 binding domain. In some embodiments, the dual binding composition comprises two anti-PD-1 binding domains. In some embodiments, each anti-PD-1 binding domain is a Fab or scFv. In some embodiments, the dual binding composition comprises two copies of the same anti-PD-1 binding domain. In some embodiments, the dual binding composition comprises two anti-PD-1 Fabs. In some embodiments, the dual binding composition comprises one anti-PD-1 Fab and one anti-Pd-1 scFv. In some embodiments, the anti-PD-1 Fab and the anti-PD-1 scFv comprise the same VH and VL. In some embodiments, the dual binding composition comprises one anti-PD-1 Fab and one anti-PD-1 single domain antibody. In some embodiments, the dual binding composition comprises two anti-PD-1 single domain antibodies. In some embodiments, the dual binding composition comprises two anti-PD-1 VHHs.TABLE 1A - Exemplary Antibodies Targeting PD-1 From Which anti-PD-1 binding domains can be derivedDocket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601

[0112] In some embodiments, the anti-PD-1 binding domain is one provided in Table IB, or a derivative thereof. In some embodiments, the anti-PD-1 binding domain is one which comprises the CDRs of a VHH provided in Table IB (e.g., those of VHH 47, 62, 70, 76, 84, or 178). In some embodiments, the anti-PD-1 binding domain is one which comprises the CDRs of a VHH provided in Table IB and comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the corresponding full-length VHH sequence (e.g., VHH 47,Docket No. 56146-753.60162, 70, 76, 84, or 178). In some embodiments, the anti-PD-1 binding domain is one of the VHHs provided in Table IB (e.g., VHH 47, 62, 70, 76, 84, or 178).

[0113] In some embodiments, certain VHH domains of the instant disclosure provide certain advantages over other VHH domains which target PD-1. For example, in some embodiments an anti-PD-1 VHH (e.g., one described in table IB) exhibits enhanced binding to PD-1 than other anti-PD-1 VHH domains, improved ability to reduce interaction of PD-1 and PD-L1 than other anti-PD-1 VHH domains, enhanced selectivity for PD-1 compared to other anti-PD-1 VHH domains, improved stability compared to other anti-PD-1 VHH domains, improved manufacturability compared to other anti-PD-1 VHH domains (e.g., enhanced culture yield, enhanced purification yield, or enhanced expression yield), reduced immunogenicity compared to other anti-PD-1 VHH domains, reduced binding to pre-existing antibodies compared to other anti-PD-1 VHH domains, and / or other advantages. In some embodiments, such advantageous properties are particularly evident when the anti-PD-1 VHH domain of the instant disclosure is incorporated into a dual binding composition of a particular format (e.g., in the format depicted in, for example, FIG. 1C).Table IB - Exemplary anti-PD-1 VHH binding domainsDocket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601

[0114] In some embodiments, VHH 47 of the instant disclosure provide certain advantages over other VHH domains which target PD-1. For example, in some embodiments VHH47 exhibits one or more advantageous property selected from enhanced binding to PD-1 than other anti -PD-1 VHH domains, improved ability to inhibit the interaction of PD-1 and PD-L1 than other anti-PD-1 VHH domains, enhanced selectivity for PD-1 compared to other anti -PD-1 VHH domains, improved stability compared to other anti -PD-1 VHH domains, improved manufacturability compared to other anti-PD-1 VHH domains (e.g., enhanced culture yield, enhanced purification yield, or enhanced expression yield), reduced immunogenicity compared to other anti-PD-1 VHH domains, reduced binding to pre-existing antibodies compared to other anti-PD-1 VHH domains, and / or other advantages. In some embodiments, such advantageous properties are particularly evident when VHH47 of the instant disclosure is incorporated into a dual binding composition of the format depicted in FIG. 1C.

[0115] In some embodiments, an anti-PD-1 VHH of the dual binding composition comprises one or more modifications which imparts the VHH with reduced immunogenicity or reduced binding of pre-existing antibodies to the VHH. Examples of such modifications are described in, for example, U.S. Patent Publication Nos. US20180009888A9 (e.g., extension peptides of 1 to 5 extending beyond the C-terminal “SS” of the VHH, such as those consisting of the amino acids Ala and Gly), US20160207981A1 (e.g., substitutions of the C-terminal “SS” of the VHH, such as with an amino acid or peptide of a sequence E, SE, EG, SEG, EP, EPG, DP, DPG, K, SK, KP, KPG, RP, or RPG and / or substitutions of Leu 11 of the VHH, such as an LI IK, LI 1R, LI ID, or LI IE substitution), US20140161796A1 (e.g., deletions of certain sequences from the VHH), US20170121399A1 (e.g., substitutions of Leu 11 of the VHH (e.g., L11K or L11V) and / or Leu 89 (e.g., L89T)), and / . / / / etal., “A structure-based engineering approach to abrogate pre-existing antibody binding to biotherapeutics,” PLoS ONE 16(7): e0254944. doi.org / 10.1371 / journal.pone.0254944 (e.g., the addition of 1, 2, or 3 prolines beyond the C-Docket No. 56146-753.601terminal “SS” of the VHH, such as a two-proline peptide). In some embodiments, the anti-PD-1 VHH (e.g., any of those described in the tables above) comprises a C-terminal modification of the addition of 1, 2 or 3 prolines to the C-terminus of the VHH (e.g., to the C-terminus of any of the VHHs described herein). In some embodiments, the anti-PD-1 VHH comprises a C-terminal modification of the addition of 2 prolines to the C-terminus of the VHH. In embodiments where two anti-PD-1 VHHs are linked in series (e.g., by a flexible peptide linker), in some instance only the C-terminal VHH will comprise the modification (e.g., only the C-terminal VHH comprises the sequence “PP” after the C-terminal “SS” of the VHH). In some instances, both VHHs linked in series will contain the modification (e.g., both will comprise the sequence “PP” after the C-terminal “SS” of each VHH).

[0116] In some embodiments, the anti-PD-1 binding domain of the dual binding composition comprises a light chain constant region. In some embodiments, the light chain constant region is one which contains one or more modifications which enhance the stability and / or manufacturability of the binding domain (or the dual binding composition as a whole). Such modifications are described in, for example, U.S. Patent No. 9,777,067 and U.S. Patent Publication No. US20150239977A1 and include, for example, modifications of residue L154 of the light chain constant region (EU numbering), such as an L154K substitution and / or modifications of residue L201 (EU numbering), such as an L201K substitution. In some embodiments, the light chain constant region of the anti-PD-1 binding domain (e.g., a Fab) comprises an L154K substitution compared to the consensus sequence (e.g., as in SEQ ID NO: 277). In some embodiments, the light chain constant region of the anti-PD-1 binding domain (e.g., a Fab) comprises an L201K substitution compared to the consensus sequence. In some embodiments, the light chain constant region of the anti-PD-1 binding domain (e.g., a Fab) comprises L154K and L201K substitutions compared to the consensus sequence (e.g., as in SEQ ID NO: 278).VEGFA Targeting Binding Domains

[0117] In some embodiments, a dual binding composition of the instant disclosure comprise one or more binding domains which target vascular endothelial growth factor A (VEGFA or VEGF). In some embodiments, a binding domain incorporated into a dual binding composition of the disclosure specifically binds to VEGFA. In some embodiments, the anti-VEGFA binding domain is capable of disrupting and / or preventing the interaction of VEGFA with one or more of its receptors (e.g., VEGFR1, VEGFR2, and / or VEGFR3). In some embodiments, the anti- VEGFA binding domain is capable of disrupting and / or preventing the interaction of VEGFA withDocket No. 56146-753.601VEGFR1. In some embodiments, the anti- VEGFA binding domain is capable of disrupting and / or preventing the interaction of VEGFA with VEGFR2. In some embodiments, the anti- VEGFA binding domain is capable of disrupting and / or preventing the interaction of VEGFA with VEGFR3. In some embodiments, the anti- VEGFA binding domain is capable of disrupting and / or preventing the interaction of VEGFA with each of VEGFR1, VEGFR2, and VEGFR3.

[0118] A non-limiting, exemplary, human VEGFA amino acid sequence is LTDRQTDTAPSPSYHLLPGRRRTVDAAASRGQGPEPAPGGGVEGVGARGVALKLFVQL LGCSRFGGAVVRAGEAEPSGAARSASSGREEPQPEEGEEEEEKEEERGPQWRLGARKPG SWTGEAAVCADSAPAARAPQALARASGRGGRVARRGAEESGPPHSPSRRGSASRAGPG RASETMNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRS YCHPIETLVD1FQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMR1KP HQGQHIGEMSFLQHNKCECRCDKPRR (SEQ ID NO: 120) (UniProt ID A0A0A0MR43). VEGFA is also referred to simply as VEGF. The terms VEGF and VEGFA are used interchangeably herein.

[0119] In some embodiments, the anti-VEGFA binding domain is comprised in an antigen binding fragment derived from an antibody. For example, the anti- VEGFA binding domain can be derived from any anti- VEGFA antibody known in the art or which can be made according to well understood methods. In some embodiments, the binding domain of a dual binding composition described herein is derived from an anti-VEGFA antibody or antigen binding fragment.

[0120] In one embodiment, an anti-VEGFA binding domain of a dual binding composition comprises and antigen binding fragment. In some embodiments, an anti-VEGFA binding domain of the disclosure comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) described herein, or those of an antibody or antigen binding fragment otherwise known in the art. In another embodiment, an anti- VEGFA binding domain of the disclosure comprises a combination of complementarity determining regions (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3) described herein, orthose of an antibody or antigen binding fragment otherwise known in the art.

[0121] In one embodiment, an anti- VEGFA binding domain of the disclosure comprises the CDRs of an antibody selected from Bevacizumab, Brolucizumab, Faricimab, Ranibizumab, Ivonescimab, AI-081, HLX-04, or IBB 05 incorporated into a VH and VL. In some embodiments, the anti- VEGFA binding domain comprises the VH and VL of any one of these antibodies. In some embodiments, the anti-VEGFA binding domain comprises the VH and VL of any one ofDocket No. 56146-753.601these antibodies or the VH and VL which include the CDRs of these antibodies in one of the following formats: a Fab, a Fab’, F(ab')2, a bispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), a bispecific scFv, disulfide stabilized Fv (dsFv), a minibody, a diabody, a bispecific diabody, triabody, a tetrabody, a maxibody, a Fab-Fc, a scFv-Fc, or a bispecific antibody. In some embodiments, the VH and VL of any one of these antibodies or the VH and VL which include the CDRs of these antibodies is comprised in a Fab, a Fab’, or an scFv. In some embodiments, the VH and VL any one of these antibodies or of the VH and VL which include the CDRs of these antibodies is comprised in a Fab or an scFv. In some embodiments, the VH and VL of any of these antibodies or the VH and VL which include the CDRs of these antibodies is comprised in a Fab. In some embodiments, the VH and VL of any of these antibodies or the VH and VL which include the CDRs of these antibodies is comprised in an scFv.

[0122] In some embodiments, an anti-VEGFA binding domain of the disclosure comprises the CDRs (e.g., VH and VL CDRs) of Bevacizumab, Brolucizumab, Faricimab, Ranibizumab, Ivonescimab, AI-081, HLX-04, or IBI305 incorporated into a VH and VL. In one embodiment, an anti-PD-1 binding domain of the disclosure comprises the VH and VL of Bevacizumab, Brolucizumab, Faricimab, Ranibizumab, Ivonescimab, AI-081, HLX-04, or IBI305. In some embodiments, the anti-VEGFA binding domain comprises the VH and VL of any one of these antibodies or the VH and VL which include the CDRs of these antibodies in one of the following formats: a Fab, a Fab’, F(ab')2, a bispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), a bispecific scFv, disulfide stabilized Fv (dsFv), a minibody, a diabody, a bispecific diabody, triabody, a tetrabody, a maxibody, a Fab-Fc, a scFv-Fc, or a bispecific antibody. In some embodiments, the VH and VL of any one of these antibodies or the VH and VL which include the CDRs of these antibodies is comprised in a Fab, a Fab’, or an scFv. In some embodiments, the VH and VL any one of these antibodies or of the VH and VL which include the CDRs of these antibodies is comprised in a Fab or an scFv. In some embodiments, the VH and VL of any of these antibodies or the VH and VL which include the CDRs of these antibodies is comprised in a Fab. In some embodiments, the VH and VL of any of these antibodies or the VH and VL which include the CDRs of these antibodies is comprised in an scFv.

[0123] In some embodiments, the anti-VEGFA binding domain comprises the VH and VL of Bevacizumab, Brolucizumab, Faricimab, or Ranibizumab. In some embodiment, the anti-VEGFA binding domain comprises the VH and VL of Bevacizumab or Brolucizumab. In some embodiments, the anti-VEGFA binding domain comprises the VH and VL of Bevacizumab. In some embodiments, the anti-VEGFA binding domain comprises the VH and VL of Brolucizumab.Docket No. 56146-753.601In some embodiments, the anti- VEGFA binding domain comprises the VH and VL of Bevacizumab in a Fab or scFv format.

[0124] TABLE 2A provides the sequences of exemplary anti-VEGFA antibodies and anti-VEGFA antigen binding fragments which contain sets of CDRs which can be incorporated into VHs and / or VLs and used as anti-VEGFA binding domains as described herein. In some embodiments, the VHs and VLs of the antibodies in Table 2A are incorporated into anti-VEGFA binding domains (e.g., in a Fab or scFv format). In some embodiments, CDRs of a VH described in Table 2A is incorporated into a binding domain as a VHH. In some embodiments, a VH as described in Table 2A is incorporated into a binding domain as a VHH.

[0125] In some instances, the SEQ ID NOs listed in Table 2A contain full-length heavy or light chains of the indicated antibodies with the VH or VL respectively indicated in bold. Where there is a reference herein to a VH or VL of a SEQ ID NO in Table 2A which contains a full-length heavy or light chain, it is intended to reference the bolded portion of the sequence.

[0126] An anti-VEGFA binding domain can comprise a VH having an amino acid sequence of any one of those described in Table 2A. The anti- VEGFA binding domain can comprise (or further comprise) a VL having an amino acid sequence of any one of those described in Table 2A. In preferred embodiments, the VH and VL are from the same antibody or antigen binding fragment described in Table 2A.

[0127] An anti- VEGFA binding domain can comprise a heavy chain, a VH, or a VH-CH1 domain (e.g., as in a Fab) having an amino acid sequence of any one of those set forth in Table 2A, or a portion corresponding to a VH thereof (e.g., the portion depicted in bold). An anti-VEGFA binding domain can comprise (or further comprise) a light chain (e.g., as in a Fab) or VL having an amino acid sequence of any one of those described in Table 2 A, or a portion corresponding to a VL thereof. In preferred embodiments, the heavy chain, VH, or VH-CH1 domain and VL or light chain are from the same antibody or antigen binding fragment described in Table 2 A.

[0128] In some embodiments, an anti-VEGFA binding domain comprises a VH having an amino acid sequence shown in Table 2A, and a VL having an amino acid sequence shown in Table 2A. In some embodiments, an anti-VEGFA binding domain comprises a VH and VL of Bevacizumab as shown in Table 2A. In some embodiments, an anti-VEGFA binding domain comprises a VH and VL of Brolucizumab as shown in Table 2A. In some embodiments, an anti-VEGFA binding domain comprises a VH and VL of Faricimab as shown in Table 2A. In some embodiments, an anti-VEGFA binding domain comprises a VH and VL of Ranibizumab as shown in Table 2A. InDocket No. 56146-753.601some embodiments, an anti-VEGFA binding domain comprises a VH and VL of Ivonescimab as shown in Table 2A. In some embodiments, an anti-VEGFA binding domain comprises a VH and VL of AI-081 as shown in Table 2A. In some embodiments, an anti-VEGFA binding domain comprises a VH and VL of HLX-04 as shown in Table 2A. In some embodiments, an anti-VEGFA binding domain comprises a VH and VL of IBI305as shown in Table 2A.

[0129] In some embodiments, an anti-VEGFA binding domain comprises a VH CDR1 a VH CDR2 and a VH CDR3 of an antibody or antigen binding fragment as shown in Table 2A comprised in a VH and a VL CDR1, a VL CDR2, and a VL CDR3 of the same antibody or antigen binding fragment as shown in Table 2A comprised in a VL. In some embodiments, the anti-VEGFA binding domain comprises a corresponding VH and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the VH and VL of the parent antibody (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0130] In some embodiments, an anti-VEGFA binding domain comprises a VH CDR1 a VH CDR2 and a VH CDR3 of Bevacizumab as shown in Table 2A comprising in a VH and a VL CDR1, a VL CDR2, and a VL CDR3 of Bevacizumab as shown in Table 2A comprised in a VL. In some embodiments, the anti- VEGFA binding domain comprises a VH and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the VH and VL of Bevacizumab as shown in Table 2A (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0131] In some embodiments, an anti-VEGFA binding domain comprises a VH CDR1 a VH CDR2 and a VH CDR3 of Brolucizumab as shown in Table 2A comprised in a VH and a VL CDR1, a VL CDR2, and a VL CDR3 of Brolucizumab as shown in Table 2A comprised in a VL. In some embodiments, the anti-VEGFA binding domain comprises a VH and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the VH and VL of Brolucizumab as shown in Table 2 A (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0132] In some embodiments, an anti-VEGFA binding domain comprises a VH CDR1 a VH CDR2 and a VH CDR3 of Faricimab as shown in Table 2A comprised in a VH and a VL CDR1, a VL CDR2, and a VL CDR3 of Faricimab as shown in Table 2A comprised in a VL. In some embodiments, the anti-VEGFA binding domain comprises a VH and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the VH and VL of Faricimab asDocket No. 56146-753.601shown in Table 2A (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0133] In some embodiments, an anti-VEGFA binding domain comprises a VH CDR1 a VH CDR2 and a VH CDR3 of Ranibizumab as shown in Table 2A comprised in a VH and a VL CDR1, a VL CDR2, and a VL CDR3 of Ranibizumab as shown in Table 2A comprised in a VL. In some embodiments, the anti-VEGFA binding domain comprises a VH and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the VH and VL of Ranibizumab as shown in Table 2A (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0134] In some embodiments, an anti-VEGFA binding domain comprises a VH CDR1 a VH CDR2 and a VH CDR3 of Ivonescimab as shown in Table 2A comprised in a VH and a VL CDR1, a VL CDR2, and a VL CDR3 of Ivonescimab as shown in Table 2A comprised in a VL. In some embodiments, the anti-VEGFA binding domain comprises a VH and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the VH and VL of Ivonescimab as shown in Table 2A (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0135] In some embodiments, an anti-VEGFA binding domain comprises a VH CDR1 a VH CDR2 and a VH CDR3 of AI-081 as shown in Table 2A comprised in a VH and a VL CDR1, a VL CDR2, and a VL CDR3 of AI-081 as shown in Table 2A comprised in a VL. In some embodiments, the anti-VEGFA binding domain comprises a VH and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the VH and VL of AI-081 as shown in Table 2A (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0136] In some embodiments, an anti-VEGFA binding domain comprises a VH CDR1 a VH CDR2 and a VH CDR3 of HLX-04 as shown in Table 2A comprised in a VH and a VL CDR1, a VL CDR2, and a VL CDR3 of HLX-04 as shown in Table 2A comprised in a VL. In some embodiments, the anti-VEGFA binding domain comprises a VH and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the VH and VL of HLX-04 as shown in Table 2A (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0137] In some embodiments, an anti-VEGFA binding domain comprises a VH CDR1 a VH CDR2 and a VH CDR3 of IB 1305 as shown in Table 2A comprised in a VH and a VL CDR1, a VL CDR2, and a VL CDR3 of IBI305 as shown in Table 2A comprised in a VL. In someDocket No. 56146-753.601embodiments, the anti-VEGFA binding domain comprises a VH and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the VH and VL of IBI305 as shown in Table 2A (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0138] In some embodiments, the anti-VEGFA binding domain is a single domain antibody comprising a CDR1 as set forth in SEQ ID NO: 202, a CDR2 as set forth in SEQ ID NO: 203, and a CDR3 as set forth in SEQ ID NO: 204. In some embodiments, the CDRs are comprised in a VHH. In some embodiments, the anti-VEGFA binding domain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to the sequence set forth in SEQ ID NO: 201 (e.g., the anti-VEGFA binding domain comprises the indicated sequence identity to SEQ ID NO: 201 and retains the CDRs).

[0139] In some embodiments, the anti-VEGFA binding domain is a single domain antibody comprising a CDR1 as set forth in SEQ ID NO: 206, a CDR2 as set forth in SEQ ID NO: 207, and a CDR3 as set forth in SEQ ID NO: 208. In some embodiments, the CDRs are comprised in a VHH. In some embodiments, the anti-VEGFA binding domain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to the sequence set forth in SEQ ID NO: 205 (e.g., the anti-VEGFA binding domain comprises the indicated sequence identity to SEQ ID NO: 205 and retains the CDRs).

[0140] In some embodiments, the anti-VEGFA binding domain is a single domain antibody comprising a CDR1 as set forth in SEQ ID NO: 210, a CDR2 as set forth in SEQ ID NO: 211, and a CDR3 as set forth in SEQ ID NO: 212. In some embodiments, the CDRs are comprised in a VHH. In some embodiments, the anti-VEGFA binding domain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to the sequence set forth in SEQ ID NO: 209 (e.g., the anti-VEGFA binding domain comprises the indicated sequence identity to SEQ ID NO: 209 and retains the CDRs).

[0141] In some embodiments, the anti-VEGFA binding domain is a single domain antibody comprising a CDR1 as set forth in SEQ ID NO: 202, a CDR2 as set forth in SEQ ID NO: 215, and a CDR3 as set forth in SEQ ID NO: 216. In some embodiments, the CDRs are comprised in a VHH. In some embodiments, the anti-VEGFA binding domain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to the sequence set forth in SEQ ID NO: 213 (e.g., the anti-VEGFA binding domain comprises the indicated sequence identity to SEQ ID NO: 213 and retains the CDRs).Docket No. 56146-753.601

[0142] In some embodiments, the anti-VEGFA binding domain is a single domain antibody comprising a CDR1 as set forth in SEQ ID NO: 218, a CDR2 as set forth in SEQ ID NO: 219, and a CDR3 as set forth in SEQ ID NO: 220. In some embodiments, the CDRs are comprised in a VHH. In some embodiments, the anti-VEGFA binding domain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to the sequence set forth in SEQ ID NO: 200, 217, 221, 280, or 281 (e.g., the anti-VEGFA binding domain comprises the indicated sequence identity to SEQ ID NO: 200, 217, 221, 280, or 281 and retains the CDRs).

[0143] In some embodiments, the anti-VEGFA binding domain is a single domain antibody comprising a CDR1 as set forth in SEQ ID NO: 218, a CDR2 as set forth in SEQ ID NO: 279, and a CDR3 as set forth in SEQ ID NO: 220. In some embodiments, the CDRs are comprised in a VHH. In some embodiments, the anti-VEGFA binding domain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to the sequence set forth in SEQ ID NO: 282-284 (e.g., the anti-VEGFA binding domain comprises the indicated sequence identity to SEQ ID NO: 282-284 and retains the CDRs).

[0144] In some embodiments, the anti-VEGFA binding domain is a single domain antibody comprising a CDR1 as set forth in SEQ ID NO: 223, a CDR2 as set forth in SEQ ID NO: 224, and a CDR3 as set forth in SEQ ID NO: 225. In some embodiments, the CDRs are comprised in a light chain single domain antibody. In some embodiments, the anti-VEGFA binding domain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to the sequence set forth in SEQ ID NO: 222 (e.g., the anti-VEGFA binding domain comprises the indicated sequence identity to SEQ ID NO: 222 and retains the CDRs).

[0145] In some embodiments, a dual binding composition comprises one of the antibodies described in Table 2A below (optionally comprising one or more modifications to the Fc domain, hinge region, or other modification described herein) fused to a binding domain specific for PD-1 (e.g., any of the PD-1 binding domains described herein). In some embodiments, the binding domain specific for PD-1 is fused to the C-terminus of the heavy chain of the antibody described in Table 2 A. In some embodiments, the binding domain specific for PD-1 fused to the antibody of Table 2A is an anti-PD-1 single-domain antibody as described herein.

[0146] In some embodiments, a dual binding composition comprises one or two anti-VEGFA binding domains. In some embodiments, the dual binding composition comprises one anti-VEGFA binding domain. In some embodiments, the dual binding composition comprises two anti-VEGFA binding domains. In some embodiments, each anti-VEGFA binding domain is a Fab or scFv. In some embodiments, the dual binding composition comprises two copies of the sameDocket No. 56146-753.601anti-VEGFA binding domain. In some embodiments, the dual binding composition comprises two anti-VEGFA Fabs. In some embodiments, the dual binding composition comprises one anti-VEGFA Fab and one anti-VEGFA scFv. In some embodiments, the anti-VEGFA Fab and the anti-VEGFA scFv comprise the same VH and VL. In some embodiments, the dual binding composition comprises one anti-VEGFA Fab and one anti-VEGFA single domain antibody. In some embodiments, the dual binding composition comprises two anti-VEGFA single domain antibodies. In some embodiments, the dual binding composition comprises two anti-VEGFA VHHs.TABLE 2A - Exemplary Antibodies Targeting VEGFA From Which anti-VEGFA Binding Domains Can be DerivedDocket No. 56146-753.601VL:DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTS SLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKV EIK (SEQ ID NO: 127)VL CDR1: SASQDISNYLN (SEQ ID NO: 128)VL CDR2: FTSSLHS (SEQ ID NO: 129)VL CDR3: QQYSTVPWT (SEQ ID NO: 130)Docket No. 56146-753.601NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC (SEQ ID NO: 146)VL:DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSS LHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE IK (SEQ ID NO: 147)VL CDR1: SASQDISNYLN (SEQ ID NO: 128)VL CDR2: FTSSLHS (SEQ ID NO: 129)VL CDR3: QQYSTVPWT (SEQ ID NO: 130)Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601

[0147] In some embodiments, the anti-VEGFA binding domain is one provided in Table 2B, or a derivative thereof. In some embodiments, the anti-VEGFA binding domain is one which comprises the CDRs of a VHH provided in Table 2B. In some embodiments, the anti-VEGFA binding domain is one which comprises the CDRs of a VHH provided in Table 2B and comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the corresponding full-length VHH sequence. In some embodiments, the anti-VEGFA binding domain is one of the VHHs provided in Table 2B. In some embodiments, the C-terminal “PP” of the VHH in Table 2B can be omitted.Table 2B - Exemplary anti-VEGFA VHH binding domainsDocket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601

[0148] In some embodiments, the anti-VEGFA VHH of the dual binding composition comprises one or more modifications which imparts the VHH with reduced immunogenicity or reduced binding of pre-existing antibodies to the VHH. Examples of such modifications are described in, for example, U.S. Patent Publication Nos. US20180009888A9 (e.g., extension peptides of 1 to 5 extending beyond the C-terminal “SS” of the VHH, such as those consisting of the amino acidsDocket No. 56146-753.601Ala and Gly), US20160207981A1 (e.g., substitutions of the C-terminal “SS” of the VHH, such as with an amino acid or peptide of a sequence E, SE, EG, SEG, EP, EPG, DP, DPG, K, SK, KP, KPG, RP, or RPG and / or substitutions of Leu 11 of the VHH, such as an LI IK, LI 1R, LI ID, or LI IE substitution), US20140161796A1 (e.g., deletions of certain sequences from the VHH), US20170121399A1 (e.g., substitutions of Leu 11 of the VHH (e.g., L11K or L11V) and / or Leu 89 (e.g., L89T)), and / . / / / etal., “A structure-based engineering approach to abrogate pre-existing antibody binding to biotherapeutics,” PLoS ONE 16(7): e0254944. doi.org / 10.1371 / journal.pone.0254944 (e.g., the addition of 1, 2, or 3 prolines beyond the C-terminal “SS” of the VHH, such as a two-proline peptide). In some embodiments, the anti-VEGFA VHH (e.g., any of those described in the tables above) comprises a C-terminal modification of the addition of 1, 2 or 3 prolines to the C-terminus of the VHH (e.g., to the C-terminus of any of the VHHs described herein). In some embodiments, the anti-VEGFA VHH comprises a C-terminal modification of the addition of 2 prolines to the C-terminus of the VHH. In embodiments where two anti-VEGFA VHHs are linked in series (e.g., by a flexible peptide linker), in some instances only the C-terminal VHH will comprise the modification (e.g., only the C-terminal VHH comprises the sequence “PP” after the C-terminal “SS” of the VHH). In some instances, both VHHs linked in series will contain the modification (e.g., both will comprise the sequence “PP” after the C-terminal “SS” of each VHH).

[0149] In some embodiments, the anti-VEGFA binding domain of the dual binding composition comprises a light chain constant region. In some embodiments, the light chain constant region is one which contains one or more modifications which enhance the stability and / or manufacturability of the binding domain (or the dual binding composition as a whole). Such modifications are described in, for example, U.S. Patent No. 9,777,067 and U.S. Patent Publication No. US20150239977A1 and include, for example, modifications of residue L154 of the light chain constant region (EU numbering), such as an L154K substitution and / or modifications of residue L201 (EU numbering), such as an L201K substitution. In some embodiments, the light chain constant region of the anti-VEGFA binding domain (e.g., a Fab) comprises an L154K substitution compared to the consensus sequence (e.g., as in SEQ ID NO: 277). In some embodiments, the light chain constant region of the anti-VEGFA binding domain (e.g., a Fab) comprises an L201K substitution compared to the consensus sequence. In some embodiments, the light chain constant region of the anti-VEGFA binding domain (e.g., a Fab) comprises L154K and L201K substitutions compared to the consensus sequence (e.g., as in SEQ ID NO: 278).Docket No. 56146-753.601

[0150] In some embodiments, an anti-VEGFA binding domain according to the instant disclosure can be a different peptide which bind to VEGFA. In some embodiments, the anti-VEGFA binding domain comprises an anticalin which binds to VEGFA. In some embodiments, the anticalin comprises an amino acid sequence having at least 80%, 85%, 90% 95%, 96% 97%, 98%, 99%, or 100% identity to the sequence DGGGIRRSMSGTWYLKAMTVDREFPEMNLESVTPMTLTLLKGHNLEAKVTMLISGRC QEVKAVLGRTKERKKYTADGGKHVAYIIPSAVRDHVIFYSEGQLHGKPVRGVKLVGRD PKNNLEALEDFEKAAGRLSTESILIPRQSETCSPG (SEQ ID NO: 226). In some embodiments, the anti-VEGFA binding domain comprises the sequence of SEQ ID NO: 226.Scaffolds for Dual Binding Compositions

[0151] In some embodiments, the components of the dual binding composition (e.g., the anti-PD-1 binding domain and the anti-VEGFA binding domain) are all in covalent association. In some embodiments, each component is linked to the other portions of the dual binding composition via covalent bonds. In some embodiments, the portions of the dual binding composition are all linked to a scaffold group. Non-limiting examples of suitable scaffold groups include, for example, polypeptides (e.g., immunoglobulins or other biocompatible polypeptides), polymers (e.g., biocompatible polymers), particles (e.g., nanoparticles, microparticles, etc., such as those made from biocompatible polymers or metals), or any other such groups. In some preferred embodiments, all of the components are linked to a scaffold polypeptide, such as an immunoglobulin polypeptide. In some embodiments, one or more of the portions of the dual binding composition can be linked to the scaffold indirectly, such as through linkers (e.g., any of the linkers described herein, including peptide linkers for fusion proteins) or through other portions of the dual binding composition (e.g., the two binding domains are linked such that only one of the binding domains is attached to the scaffold (optionally through a suitable linker) and the other binding domain is linked to the binding domain which is linked to the scaffold, independently and optionally though another linker). In some embodiments, all of the components are linked as a fusion protein of one or more polypeptides which combine to form the dual binding composition (e.g., each component is fused, directly or indirectly, to a polypeptide scaffold, such as an Fc domain).Fc Domains as Scaffolds

[0152] In some embodiments, the dual binding composition comprises an Fc domain which acts as a scaffold. In some embodiments, the Fc domain is fused to one, both, or each of the binding domains (or, for binding domains which comprise a plurality of polypeptide chains, such as a Fab,Docket No. 56146-753.601one of the polypeptide chains of the binding domain). In some embodiments, the Fc domain is also fused to an additional group (e.g., a cytokine). In some embodiments, the Fc domain is attached (e.g., fused or conjugated) to an additional group (e.g., a cytokine) via a linker. In some embodiments, such a linker is attached to a side chain of an amino acid residue of the Fc domain (e.g., a K246, K248, K288, K290, or K317 residue of the Fc domain (EU numbering), such as by AJICAP™ technology). Any of the Fc domains described herein (e.g., any one of SEQ ID NOs: 229-234 or 236-241, or a variant thereof) can comprise such a linker attached. In some embodiments, one arm of an Fc domain comprises a modification which prevents attachment of a linker at a specific residue, thereby facilitating conjugation of only a single group to the dual binding composition (e.g., a substitution at one of residues K246, K248, K288, K290, or K317 on one arm of the Fc domain, thereby preventing attachment of the linker to that arm). In some embodiments, the Fc domain comprises a K246A, K248A, K288A, K290A, or K317A substitution. In some embodiments, the Fc domain comprises a K248A substitution. Any of the Fc domains described herein (e.g., any one of SEQ ID NOs: 229-234 or 236-241, or a variant thereof) can comprise such a modification.

[0153] In some embodiments, a dual binding composition comprises an Fc domain comprising first CH2 and CH3 domains on a first polypeptide chain and second CH2 and CH3 domains on a second polypeptide chain.

[0154] In some embodiments, the dual binding composition comprises an Fc domain. In some embodiments, the Fc domain is an IgG Fc domain, an IgA Fc domain, an IgD Fc domain, an IgM Fc domain, or an IgE Fc domain, or a derivative thereof. In some embodiments, the Fc domain is an IgG Fc domain, an IgA Fc domain, or an IgD Fc domain, or a derivative thereof. In some embodiments, the Fc domain is a human Fc domain, or a derivative thereof. In some embodiments, the Fc domain is a humanized. Fc domain, or a derivative thereof. In some embodiments, the Fc domain is an IgG Fc domain, or a derivative thereof. In some instances, an IgG Fc domain is an IgGl Fc domain, an IgG2a Fc domain, or an IgG4 Fc domain, or a derivative thereof. In some embodiments, the Fc domain is an IgGl Fc domain, or a derivative thereof. In some embodiments, the Fc domain is an IgG4 Fc domain, or a derivative thereof. In some embodiments, a derivative of an Fc domain is one which contains at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a corresponding natural Fc domain (e.g., each arm has the indicated sequence identity).

[0155] In some embodiments, the Fc domain of a dual binding composition is an IgGl Fc domain, or a derivative thereof. In some embodiments, the Fc domain comprises an amino acid sequenceDocket No. 56146-753.601having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a human IgGl Fc domain (i.e., the CH2 and CH3 domains of human IgGl) (e.g., each arm of the Fc domain has the indicated sequence identity). In some embodiments, the Fc domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in Table 3A below. In some embodiments, the Fc domain which comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in Table 3A below can further comprise residues corresponding to residues 231-238 of an IgGl (EU numbering) appended to the N-terminal residue of the sequences set forth in Table 3A below (e.g., a peptide sequence of APELLGGP (SEQ ID NO: 227) (WT IgGl “lower hinge”) or APEAAGGP (SEQ ID NO: 228) (LALA substituted “lower hinge”)). Such a sequence can also be considered to be co-extensive with the hinge region. Thus, when a dual binding composition described herein is described as having a hinge region described herein and a Fc domain as described herein, it is contemplated that the sequence of the hinge region described herein and the Fc domain described herein can contain overlap. In some embodiments, the Fc domain with the indicated sequence identity to the sequence set forth in Table 3A below retains the noted substitutions (all of which are numbered with EU numbering). In some embodiments, the Fc domain comprises one or more additional substitutions described herein (e.g., K248A on one arm, etc.). In some embodiments, the Fc domain comprises one or more additional substitutions described herein (e.g., K248A on one arm, etc.) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or more additional substitutions).Table 3A. Fc domains<<<Docket No. 56146-753.601

[0156] In some embodiments, the Fc domain of a dual binding composition is an IgG4 Fc domain, or a derivative thereof. In some embodiments, the Fc domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a human IgG4 Fc domain (i.e., the CH2 and CH3 domains of human IgG4). In some embodiments, the Fc domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in Table 3B below. In some embodiments,Docket No. 56146-753.601the Fc domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in Table 3B below in which the first 8 residues of the sequences set forth below are omitted (e.g., the sequence APEFLGGP (SEQ ID NO: 235) is omitted). Such a sequence can also be considered to be co-extensive with the hinge region. Thus, when a dual binding composition described herein is described as having a hinge region described herein and a Fc domain as described herein, it is contemplated that the sequence of the hinge region described herein and the Fc domain described herein can contain overlap (i.e., when a dual binding composition is described as having an Fc domain described herein and a hinge region described herein, any overlapping portions of such sequences can be attributed to both regions) In some embodiments, the Fc domain with the indicated sequence identity to the sequence set forth in Table 3B below retains the noted substitutions (all of which are numbered with EU numbering). In some embodiments, the Fc domain comprises one or more additional substitutions described herein (e.g., K248A on one arm, etc.) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or more additional substitutions).Table 3B. FC domains""""Docket No. 56146-753.601""

[0157] One or more mutations may be introduced in an Fc domain to reduce Fc-mediated effector functions of dual binding composition, such as, for example, antibody-dependent cellular cytotoxicity (ADCC) and / or complement function. In some instances, a modified Fc comprises a humanized IgG4 kappa isotype that contains a S229P Fc mutation. In some instances, a modified Fc comprises a human IgGl kappa where the heavy chain CH2 domain is engineered with a triple mutation such as, for example: (a) L238P, L239E, and P335S; or (2) K248; K288; and K317.

[0158] In some embodiments, the Fc domain comprises one or more modifications which favors heterodimerization of the two polypeptide which dimerize to form the Fc domains. Many such modifications are known in art for generating bispecific antibodies which can be applied to the instant disclosure. Such modifications are described in, for example, “Fc Engineering for Developing Therapeutic Bispecific Antibodies and Novel Scaffolds,” Liu et al., Front. Immunol., 26 January 2017 (doi.org / 10.3389 / fimmu.2017.00038) and include, for example, knob-into-hole technology (see, e.g., U.S. Patent No. 8,216,805) and modification introduced into one Fc domain to abrogate binding to protein A to facilitate purification of desired heterodimeric formats e.g., RF mutations, as described in, e.g., U.S. Patent No. 11,168,111). In some embodiments, the Fc domain of the dual binding compositions provided herein utilize knob-into-hole technology, forDocket No. 56146-753.601example the “hole” modifications of Y349C, T366S, L368A, and Y407V and the “knob” modifications of S354C and T366W (EU numbering). In some embodiments, the dual binding compositions provided herein utilize the RF mutations, e.g., H435R and Y436F mutations (EU numbering). In some embodiments, the dual binding compositions utilize both of these modifications together (e.g., one arm of the Fc domain of the dual binding composition having the hole and RF modifications, and one arm of the Fc domain of the dual binding composition having the knob modifications). In some embodiments, on arm of the Fc domain comprises T336W, H435R, and Y436F substitutions and the other arm of the Fc domain comprises T366S, L368A, Y407V substitutions. In some embodiments, one arm of the Fc domain comprises Y349C, T366S, L368A, and Y407V “hole” mutations and the other arm of the Fc domain comprises S354C and T366W “knob” mutations (EU numbering). Other combinations of such knob and hole modifications are well known in the art and compatible with the instant disclosure.

[0159] In some embodiments, one arm of the Fc domain of the dual binding composition comprises a mutation at a residue which eliminates the ability to conjugate an additional group (such as a cytokine) to that site, thereby allowing for easier preparation of a dual binding composition which contains only a single conjugated group attached (e.g., a single cytokine). For example, in instances where AJICAP™ technology is intended to be used to conjugate an additional group to one of residues K246, K248, K288, K290, or K317 (EU numbering), one arm of the Fc domain can comprise a mutation at the lysine residue to be targeted for conjugation to render it unavailable for reaction with the affinity peptide of the AJICAP™ technology. In one particular example, where it is intended to use an AJICAP™ affinity peptide to add a sulfide group to K248 for subsequent conjugation to the additional group (e.g., the cytokine, through an intermediate reaction with a heterobifunctional linking reagent described herein), the K248 residue of one arm of the Fc domain is mutated to a suitable residue which is incapable of reaction with the affinity peptide, such as a K248A substitution. Thus, in some embodiments, one arm of the Fc domain contains a substitution at one or more of residue K246, K248, K288, K290, or K317. In some embodiments, the substitution is one or more of a K246A, K248A, K288A, K290A, or K317A substitution. In some embodiments, one arm of the Fc domain contains a K248A substitution. In some embodiments, one arm of the Fc domain contains a K248A substitution and the other arm is conjugated to the additional group (e.g., the cytokine). Analogously, mutations at the other residues on one arm of the Fc domain can similarly be paired with conjugation at the corresponding unsubstituted residue on the other arm of the Fc domain (e.g., one arm with a K246A substitution and the other arm conjugated to the additional group).Docket No. 56146-753.601Such substitutions to facilitate conjugation to only one arm of the Fc domain are favorably paired with substitutions which help to facilitate the heterodimerization of the arms of the Fc domain (e.g., knob-into-hole modifications discussed above or other similar modifications known in the art).

[0160] In some embodiments, the constant domains (e.g., the Fc domain) of a dual binding composition described herein can comprise further modifications (either in place of or in addition to the other modifications described herein, such as those which favor heterodimerization of two different arms of the fusion dual binding). Such modifications to Fc domains are known in the art and include, for example, modifications which alter antibody effector functions (e.g., enhance or decease Fc receptor binding or activity, thereby altering antibody-dependent cellular cytotoxicity, complement dependent cytotoxicity, or other effects), improve half-life circulation, or otherwise alter the performance of the molecule. Such modifications are well known in the art and are described in, for example, “Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life,” Saunders et al., Fron. Immunol., 07 June 2019 (doi.org / 10.3389 / fimmu.2019.01296). Such modifications can be at any relevant portion of the fusion dual binding, including without limitation an Fc domain (e.g., either the CH2 or CH3 domain, or both), a hinge region, a CHI domain, a light chain constant region, and / or a framework region of an antigen binding domain (e.g., a VH or VL domain).Exemplary Dual Binder Compositions

[0161] In some embodiments, the dual binding compositions described herein comprise an Fc domain. In some embodiments, the dual binding composition comprises one or both of the anti-PD-1 and / or the anti-VEGF binding domains fused to an Fc scaffold. Non-limiting examples of Fc domain containing scaffolds fused to anti-PD-1 and / or anti-VEGF binding domains are depicted in FIGs. 1A-D, 2A-D, 3A-C, and 4A-B. Non-limiting examples of sequences of such dual binding compositions which comprise Fc domains can be found in the table below.Table 4A. Dual Binder CompositionsDocket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Additional Exemplary Dual Binder Compositions

[0162] Additional exemplary dual binder compositions according to the instant disclosure are described in Table 4B below. The composition numbers provided in the table are those referenced in the Examples provided herein. In the table below where multiple SEQ ID NOs are separated by semicolons, the full-length sequence can be arrived at by appending the indicated sequences together in an N-terminal to C-terminal direction. For example, the heavy chain of composition 59 has a sequence of EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTG EPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFP AVLQS SGLYSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGG GGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTVSDYDMAWYRQAPGKGRELVA GINDEGTTTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCARVVSGQQLVF PLDYWGQGSLVTVSS (SEQ ID NO: 176).Table 4B. Additional Exemplary Dual Binder CompositionsDocket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601Docket No. 56146-753.601

[0163] Additional Sequences

[0164] SEQ ID NO: 161 is EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTG EPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFP AVLQS SGLYSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQDocket No. 56146-753.601PREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0165] SEQ ID NO: 162 is QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNG GTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0166] SEQ ID NO: 163 is QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNG GTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGS GGGGSDVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISK GGYKYD AVSLEGRFTISRDNAKNT VYLQINSLRPEDT AVYYC AS SRAYGS SRLRLADT Y EYWGQGTLVTVSSGGGGSGGGGSDVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSM GWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVY YC AS SRAYGS SRLRL ADTYEYWGQGTL VT VS S .

[0167] SEQ ID NO: 164 is QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNG GTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFDocket No. 56146-753.601LVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGS GGGGSD VQLVESGGGLVQPGGSLRLSC AASGRTF S S YSMGWFRQAPGKEREF VVAISK GGYKYD AVSLEGRFTISRDNAKNT VYLQINSLRPEDT AVYYC AS SRAYGS SRLRLADT Y EYWGQGTLVTVSS.

[0168] SEQ ID NO: 165 is QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNG GTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGS GGGGSDVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISK GGYK YD AVSLEGRFTISRDNAKNT VYLQINSLRPEDT AVYYC AS SRAYGS SRLRLADT Y EYWGQGTLVTVSS.

[0169] SEQ ID NO: 166 is EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTG EPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFP AVLQS SGLYSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSG GGGSGGGGSDVQLVESGGGLVQPGGSLRLSCAASGLPFSDYSMGWFRQAPGKEREFV AGISGSSITTYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCATSGYSYVAGG MDVWGQGTTVTVSSGGGGSGGGGSGGGGSDVQLVESGGGLVQPGGSLRLSCAASGLP FSDYSMGWFRQAPGKEREFVAGISGSSITTYYADSVKGRFTISRDNSKNTLYLQMNSLR PEDT AVYYC AT SGYS YVAGGMD VWGQGTT VT VS S .

[0170] SEQ ID NO: 167 is QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNG GTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGDocket No. 56146-753.601TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

[0171] SEQ ID NO: 168 is EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTG EPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFP AVLQS SGLYSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0172] SEQ ID NO: 169 is QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNG GTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGS GGGGSDVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISK GGYKYD AVSLEGRFTISRDNAKNT VYLQINSLRPEDT AVYYC AS SRAYGS SRLRLADT Y EYWGQGTLVTVSSPPGGGGSGGGGSDVQLVESGGGLVQPGGSLRLSCAASGRTFSSYS MGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTA VYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSPP.

[0173] SEQ ID NO: 170 is EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTG EPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGDocket No. 56146-753.601VHTFP AVLQS SGLYSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP.

[0174] SEQ ID NO: 171 is QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNG GTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGS GGGGSDVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISK GGYKYD AVSLEGRFTISRDNAKNT VYLQINSLRPEDT AVYYC AS SRAYGS SRLRLADT Y EYWGQGTLVTVSSPP.

[0175] SEQ ID NO: 172 is QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNG GTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGS GGGGSDVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISK GGYK YD AVSLEGRFTISRDNAKNT VYLQINSLRPEDT AVYYC AS SRAYGS SRLRLADT Y EYWGQGTLVTVSSGGGGSGGGGSDVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSM GWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVY YCASSRAYGSSRLRLADTYEYWGQGTLVTVSSPP.

[0176] SEQ ID NO: 173 is QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGDocket No. 56146-753.601GTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0177] SEQ ID NO: 174 is QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNG GTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSDVQLVESGGG LVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTI SRDNAKNT VYLQINSLRPEDT AVYYC AS SRA YGS SRLRLADT YEYWGQGTL VT VS S .

[0178] SEQ ID NO: 175 is QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNG GTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGS GGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKGLEFVVAISK GGYKYD AVS VKGRFTISRDNAKNS VYLQMNSLRAEDT AVYYC AS SRAYGS SRLRL AD TYEYWGQGTL VT VS S .

[0179] In some embodiments, a dual binding composition is of the format shown in FIG. 1 A. In some embodiments, a dual binding composition is of the format shown in FIG. IB. In some embodiments, a dual binding composition is of the format shown in FIG. 1C. In someDocket No. 56146-753.601embodiments, a dual binding composition is of the format shown in FIG. ID. In some embodiments, a dual binding composition is of the format shown in FIG. IE. In some embodiments, a dual binding composition is of the format shown in FIG. IF. In some embodiments, a dual binding composition is of the format shown in FIG. 1G. In some embodiments, a dual binding composition is of the format shown in FIG. 1H. In some embodiments, a dual binding composition is of the format shown in FIG. 2A. In some embodiments, a dual binding composition is of the format shown in FIG. 2B. In some embodiments, a dual binding composition is of the format shown in FIG. 2C. In some embodiments, a dual binding composition is of the format shown in FIG. 2D. In some embodiments, a dual binding composition is of the format shown in FIG. 3A. In some embodiments, a dual binding composition is of the format shown in FIG. 3B. In some embodiments, a dual binding composition is of the format shown in FIG. 3C. In some embodiments, a dual binding composition is of the format shown in FIG. 4A. In some embodiments, a dual binding composition is of the format shown in FIG. 4B.Peptide Linkers

[0180] In some embodiments, the Fc domain is linked to the other components of the dual binding composition (e.g., the binding domains). In some embodiments, the Fc domain is linked to both binding domains as a fusion protein. In such instances, the linker (if present) comprises one or more peptide bonds between the Fc domain and the other group (e.g., the binding domain). In some embodiments, the linker between the Fc domain and the binding domain is a bond. In some embodiments, the linker between the Fc domain and the binding domain is a linking peptide. Nonlimiting examples of linking peptides include, but are not limited to (GS)n(SEQ ID NO: 23), (GGS)n (SEQ ID NO: 24), (GGGS)n(SEQ ID NO: 25), (GGSG)n(SEQ ID NO: 26), or (GGSGG)n(SEQ ID NO: 27), (GGGGS)n(SEQ ID NO: 28), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, a linking peptide can be GGGGS (SEQ ID NO: 21), (GGGGS)2 (SEQ ID NO: 22), (GGGGS)3 (SEQ ID NO: 29), or (GGGGS)4 (SEQ ID NO: 30). In some embodiments, a binding domain is fused to the C-terminal end of the Fc domain (optionally through a linking peptide). In some embodiments, the binding domain is fused to the N-terminal end of the Fc domain (optionally through a linking peptide). In embodiments wherein multiple portions of the dual binding composition are fused to the Fc domain, each peptide linker is independently selected and can be the same or different.

[0181] In embodiments in which a binding domain is fused to the N-terminus of the Fc domain, the linking peptide is desirably a hinge region of an antibody. For example, wherein the Fc domainDocket No. 56146-753.601is derived from an IgGl, an IgGl hinge region or variant thereof can desirably be used to link the binding domain to the Fc domain (e.g., a hinge region having a sequence EPKSCDKTHTCPPCPAPELLGGP (SEQ ID NO: 267) or a variant thereof (e.g., having a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto), such as a so-called “LALA” variant which comprises L234A and L235A substitutions (EU numbering) (i.e., having a sequence EPKSCDKTHTCPPCPAPEAAGGP (SEQ ID NO: 268)). In some embodiments, the hinge region comprises a sequence having at least 80%, 85%, 90%, 95%, or 100% identity to the sequence EPKSCDKTHTCPPCP (SEQ ID NO: 269). Similarly, when the Fc domain is derived from an IgG4, an IgG4 hinge region or variant thereof can serve as the linker between the binding domain (e.g., the Fab or scFv) and the Fc domain (e.g., an IgG4 hinge region of the sequence ESKYGPPCPSCPAPEFLGGP (SEQ ID NO: 270) or a variant thereof (e.g., having a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto)). In some embodiments, the hinge region comprises a sequence having at least 80%, 85%, 90%, 95%, or 100% identity to the sequence ESKYGPPCPSCP (SEQ ID NO: 271). In some embodiments, the linker peptide comprises an antibody hinge region, or an amino acid sequence having 1, 2, 3, 4, or 5 substitutions thereto (e.g., conservative substitutions). In some embodiments, a hinge region acts as the linker between a Fab binding domain as described herein and an Fc domain. In some embodiments, the linking peptide comprises a portion of a hinge region. In some embodiments, the linking peptide comprises a hinge region portion having the amino acid sequence DKTHTCPPCPAPELLGGP (SEQ ID NO: 265), or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the linking peptide comprises a hinge region portion having the amino acid sequence DKTHTCPPCPAPEAAGGP (SEQ ID NO: 266), or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.Domain Architecture of Dual binding Compositions

[0182] In the following section, the “first binding domain” refers to the anti-PD-1 binding domain of a dual binding composition and the “second binding domain” refers to the anti-VEGFA binding domain of the dual binding composition. Such binding domains can be any of the binding domains described herein.

[0183] In some embodiments, a dual binding composition comprises a structure of the formula:Docket No. 56146-753.601wherein:Y is the first CH2 and CH3 domains;Y’ is the second CH2 and CH3 domains;X and X’ are each independently the first binding domain, the second binding domain, a copy of the first binding domain, a copy of the second binding domain, or absent;Z and Z’ are each independently the first binding domain, the second binding domain, a copy of the first binding domain, a copy of the second binding domain, a third binding domain targeting PD-1, or a fourth binding domain targeting VEGFA, or absent;wherein X, Y, and Z and X’, Y’ and Z’ are depicted in an N-terminal to C-terminal direction; andwherein each of X, X’, Z, and Z’ are independently and optionally connected to Y or Y’ via a peptide linker.

[0184] In some embodiments, X is the first binding domain; X’ is a copy of the first binding domain or absent; and one of Z or Z’ is the second binding domain and the other is absent or a copy of the second binding domain. In some embodiments, one of Z or Z’ is the second binding domain and the other is a copy of the second binding domain.

[0185] In some embodiments, X is the second binding domain; X’ is a copy of the second binding domain or absent; and one of Z or Z’ is the first binding domain and the other is absent or a copy of the first binding domain. In some embodiments, one of Z or Z’ is the first binding domain and the other is a copy of the first binding domain.

[0186] In some embodiments, X is the first binding domain and is a Fab, X’ is a copy of the first binding domain, Z is the second binding domain and is an scFv, and Z’ is a copy of the second binding domain.

[0187] In some embodiments, X is the first binding domain and is a Fab, X’ is a copy of the first binding domain; Z is the second binding domain and is a single domain antibody, and Z’ is a copy of the second binding domain.

[0188] In some embodiments, X is the first binding domain and is a Fab, X’ is absent, Z is the second binding domain and is an scFv, and Z’ is a copy of the second binding domain. In some embodiments, X is the first binding domain and is a Fab, X’ is absent, Z is the second binding domain and is a single domain antibody, and Z’ is a copy of the second binding domain.Docket No. 56146-753.601

[0189] In some embodiments, X is the first binding domain and is a Fab, X’ is the second biding domain and is a Fab, and Z and Z’ are absent.

[0190] In some embodiments, X is the first binding domain and is a Fab, X’ is the second binding domain and is an scFv, and Z and Z’ are absent.

[0191] In some embodiments, X is the first binding domain and is an scFv, X’ is the second binding domain and is a Fab, and Z and Z’ are absent.

[0192] In some embodiments, X is the second binding domain and is a Fab, X’ is a copy of the second binding domain, Z is the first binding domain and is an scFv, and Z’ is a copy of the first binding domain.

[0193] In some embodiments, X is the second binding domain and is a Fab, X’ is a copy of the second binding domain; Z is the first binding domain and is a single domain antibody, and Z’ is a copy of the first binding domain.

[0194] In some embodiments, X is the second binding domain and is a Fab, X’ is absent, Z is the first binding domain and is an scFv, and Z’ is a copy of the first binding domain.

[0195] In some embodiments, X is the second binding domain and is a Fab, X’ is absent, Z is the first binding domain and is a single domain antibody, and Z’ is a copy of the first binding domain.

[0196] In some embodiments, X is the second binding domain and is a Fab, X’ is the first binding domain and is a Fab, Z is a fourth binding domain targeting VEGFA and is an scFv, and Z’ is absent.

[0197] In some embodiments, X is the second binding domain and is a Fab, X’ is the first binding domain and is a Fab, Z is a fourth binding domain targeting VEGFA and is a single domain antibody, and Z’ is absent.

[0198] In some embodiments wherein X, X’, Z, and / or Z’ are one of the binding domains, it is expressly contemplated that the binding domain can be linked to another binding domain in addition to the Fc domain. For example, in cases where Z is a VHH, the VHH of Z can be further linked to another binding domain (e.g., another VHH), either directly or, in preferred instances, by a peptide linker. For example, in some embodiments, Z is a VHH linked to another (i.e., a second) VHH via a peptide linker. An example of such a dual binding composition is depicted in FIG. 1G. In preferred embodiments, the two VHHs will share the same CDR sequences. In some embodiments, the two VHHs will comprise an identical amino acid sequence. In some embodiments, the second VHH will contain additional amino acids added to the C-terminus of the VHH (e.g., a series of one or more prolines attached to the C-terminus compared to the first VHH). In some embodiments, the two VHHs will have an identical amino acid sequence. In someDocket No. 56146-753.601embodiments wherein X, X’, Z, and / or Z’ are one of the binding domains, the binding domain is not further linked to an additional binding domain (i.e., the binding domain of X, X’, Z, and / or Z’ is the only binding domain at the position).

[0199] In some embodiments, a dual binding composition comprises:1) a heavy chain polypeptide comprising, in an N-terminal to C-terminal direction:a) a VH of an anti-VEGFA antibody (e.g., any of those as described herein (e.g., as described in Table 2 A));b) a CHI domain (e.g., any of those as described herein);c) a hinge region (e.g., any of those as described herein);d) an Fc domain (e.g., any of those as described herein);e) an optional peptide linker (e.g., any of those as described herein); f) one of:i) an anti-PD-1 single domain antibody (e.g., any of those as described herein); orii) an anti-PD-1 scFv comprising a VH and VL of an anti-PD-1 antibody (e.g., any of those as described herein), optionally connected by a second peptide linker (e.g., any of those as described herein). 2) a light chain polypeptide comprising, in an N-terminal to C-terminal direction:a) a VL of an anti-VEGFA antibody (e.g., the same antibody as the corresponding VH); andb) a light chain constant domain (e.g., any of those as described herein).

[0200] In some embodiments, a dual binding composition comprises1) a heavy chain polypeptide comprising, in an N-terminal to C-terminal direction:a) a VH of an anti-PD-1 antibody (e.g., any of those as described herein (e.g., as described in Table 1 A));b) a CHI domain (e.g., any of those as described herein);c) a hinge region (e.g., any of those as described herein);d) an Fc domain (e.g., any of those as described herein);e) an optional peptide linker (e.g., any of those as described herein); f) one of:i) an anti-VEGFA single domain antibody (e.g., any of those as described herein); orDocket No. 56146-753.601ii) an anti-VEGFA scFv comprising a VH and VL of an anti-VEGFA antibody (e.g., any of those as described herein), optionally connected by a second peptide linker (e.g., any of those as described herein).2) a light chain polypeptide comprising, in an N-terminal to C-terminal direction:a) a VL of an anti-PD-1 antibody (e.g., the same antibody as the corresponding VH); andb) a light chain constant domain (e.g., any of those as described herein).Pharmaceutical Compositions

[0201] In one aspect, provided herein is a pharmaceutical composition comprising a dual binding composition described herein; and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition further comprises one or more excipients, wherein the one or more excipients include, but are not limited to, a carbohydrate, an inorganic salt, an antioxidant, a surfactant, a buffer, or any combination thereof. In some embodiments the pharmaceutical composition further comprises one, two, three, four, five, six, seven, eight, nine, ten, or more excipients, wherein the one or more excipients include, but are not limited to, a carbohydrate, an inorganic salt, an antioxidant, a surfactant, a buffer, or any combination thereof.

[0202] In some embodiments, the pharmaceutical composition further comprises a carbohydrate. In certain embodiments, the carbohydrate is selected from the group consisting of fructose, maltose, galactose, glucose, D-mannose, sorbose, lactose, sucrose, trehalose, cellobiose raffinose, melezitose, maltodextrins, dextrans, starches, mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, cyclodextrins, and combinations thereof.

[0203] Alternately, or in addition, the pharmaceutical composition further comprises an inorganic salt. In certain embodiments, the inorganic salt is selected from the group consisting of sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium phosphate, potassium phosphate, sodium sulfate, or combinations thereof.

[0204] Alternately, or in addition, the pharmaceutical composition further comprises an antioxidant. In certain embodiments, the antioxidant is selected from the group consisting of ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, potassium metabisulfite, propyl gallate, sodium metabisulfite, sodium thiosulfate, vitamin E, 3,4-dihydroxybenzoic acid, and combinations thereof.

[0205] Alternately, or in addition, the pharmaceutical composition further comprises a surfactant. In certain embodiments, the surfactant is selected from the group consisting of polysorbates,Docket No. 56146-753.601sorbitan esters, lipids, phospholipids, phosphatidylethanolamines, fatty acids, fatty acid esters, steroids, EDTA, zinc, and combinations thereof.

[0206] Alternately, or in addition, the pharmaceutical composition further comprises a buffer. In certain embodiments, the buffer is selected from the group consisting of citric acid, sodium phosphate, potassium phosphate, acetic acid, ethanolamine, histidine, amino acids, tartaric acid, succinic acid, fumaric acid, lactic acid, tris, HEPES, or combinations thereof.

[0207] In some embodiments, the pharmaceutical composition is formulated for parenteral or enteral administration. In some embodiments, the pharmaceutical composition is formulated for intravenous (IV) or subcutaneous (SQ) administration. In some embodiments, the pharmaceutical composition is in a lyophilized form.Dosage Forms

[0208] The dual binding compositions described herein can be in a variety of dosage forms. In some embodiments, the dual binding composition is dosed as a solution. In some embodiments, the dual binding composition is dosed as an injectable solution. In some embodiments, the dual binding composition is dosed as an IV solution.Methods of Treatment

[0209] In one aspect, described herein, is a method of treating cancer in a human subject in need thereof, comprising: administering to the subject an effective amount of a dual binding composition described herein or a pharmaceutical composition as described herein.

[0210] In some embodiments, the cancer is a solid cancer. A cancer or tumor can be, for example, a primary cancer or tumor or a metastatic cancer or tumor. In some embodiments, the cancer is a solid cancer. In some embodiments, the solid cancer is adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, carcinoid cancer, cervical cancer, colorectal cancer, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal stromal tumor, germ cell cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine cancer, oral cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, pediatric cancer, penile cancer, pituitary cancer, prostate cancer, skin cancer, soft tissue cancer, spinal cord cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, ureteral cancer, uterine cancer, vaginal cancer, or vulvar cancer.

[0211] Combination therapies with one or more additional active agents are contemplated herein.

[0212] An effective response is achieved when the subject experiences partial or total alleviation or reduction of signs or symptoms of illness, and specifically includes, without limitation,Docket No. 56146-753.601prolongation of survival. The expected progression -free survival times may be measured in months to years, depending on prognostic factors including the number of relapses, stage of disease, and other factors. Prolonging survival includes without limitation times of at least 1 month (mo), about at least 2 mos., about at least 3 mos., about at least 4 mos., about at least 6 mos., about at least 1 year, about at least 2 years, about at least 3 years, about at least 4 years, about at least 5 years, etc. Overall or progression-free survival can be also measured in months to years. Alternatively, an effective response may be that a subject’s symptoms or cancer burden remain static and do not worsen. Further treatment of indications are described in more detail elsewhere herein. In some instances, a cancer or tumor is reduced by at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0213] In some embodiments, the dual binding composition is administered in a single dose of the effective amount of dual binding composition, including further embodiments in which (i) the dual binding composition is administered once a day. In some embodiments, the dual binding composition is administered periodically (e.g., multiple doses are administered at different time points).Definitions

[0214] All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0215] The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0216] The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. In this application, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0217] In this application, the use of “or” means “and / or” unless stated otherwise. The terms “and / or” and “any combination thereof’ and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated.Docket No. 56146-753.601Solely for illustrative purposes, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof’ can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C ” The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

[0218] The term “about” or “approximately” can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, z.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5 -fold, or within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0219] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

[0220] Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures. To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.

[0221] The term “Fc region” or “Fc domain” is used to define the amino acids of a C-terminal region of an immunoglobulin heavy chain. The “Fc region” or “Fc domain” may be a native sequence Fc region or a variant Fc region. As used herein, “Fc region” or “Fc domain” will frequently refer to such a region by itself (e.g., reference to an “Fc region” or “Fc domain” will refer to such amino acids without requiring the presence of any other portion of an antibody) and / or attached to binding domains described herein. Although the boundaries of the Fc region or Fc domain of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc regionDocket No. 56146-753.601is generally defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof (e.g., to K447 (EU numbering)). The numbering of the residues in the Fc region is that of the EU index as in Kabat. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3. In certain instances herein, an “Fc region” or “Fc domain” as used herein can also stretch from position Ser239 (EU numbering) or from Ala231 (EU numbering).

[0222] The term “hinge region” of an antibody or antigen binding fragment herein refers to the flexible part of an antibody molecule that connects a Fab to the Fc region of an antibody (e.g., as in a full-length antibody). In some embodiments, a “hinge region” can be used as a peptide linker to fuse any of the binding domains described herein to an Fc domain (e.g., a hinge region can be used to fuse an scFv binding domain to the N-terminal end of an Fc domain). The “hinge region” of an antibody generally refers residues Glu216 to Pro230 or Glu216 to Pro238 (EU numbering). The “hinge region” can be divided into an “upper portion” corresponding to residues Glu216 to Thr225 (IgGl) or Pro225(IgG4), a “core portion” corresponding to residues Cys226 to Pro230, and a “lower portion” corresponding to residues Ala231 to Pro238. In some instances, the “lower portion” and / or “core portion” of the hinge region can also be considered part of the Fc domain (i.e., the hinge region and the Fc domain can be co-extensive over certain residues). Thus, when a “hinge region” of the instant disclosure is described as a linker between an Fc domain and a binding domain herein, it is expressly contemplated that such a hinge region can include residues which overlap with the Fc domain.

[0223] As used herein, the term “antibody” refers to an immunoglobulin (Ig), polypeptide, or a protein having a binding domain which is, or is homologous to, an antigen binding domain. The term further includes “antigen binding fragments” and other interchangeable terms for similar binding fragments as described below. Native antibodies and native immunoglobulins (Igs) are generally heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light chains and two identical heavy chains. Each light chain is typically linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (“VH”) followed by a number of constant domains (“CH”). Each light chain has a variable domain at one end (“VL”) and a constant domain (“CL”) at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is alignedDocket No. 56146-753.601with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains.

[0224] “Antibodies” from which binding domains of the instant disclosure can be derived encompass, but are not limited to, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, bispecific antibodies, grafted antibodies, multispecific antibodies, heteroconjugate antibodies, humanized antibodies, human antibodies, deimmunized antibodies, mutants thereof, fusions thereof, immunoconjugates thereof, antigen binding fragments thereof, and / or any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

[0225] In some instances, an antibody is a humanized antibody. As used herein, “humanized” antibodies refer to forms of non -human (e.g., murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or fragments thereof that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and biological activity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences but are included to further refine and optimize antibody performance. In general, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Antibodies may have Fc regions modified as described in, for example, WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, or six) which are altered with respect to the original antibody, which are also termed one or more CDRs “derived from” one or more CDRs from the original antibody.

[0226] If needed, an antigen binding fragment described herein derived from an antibody used as a binding domain as described herein can be assessed for immunogenicity and, as needed, be deimmunized (z.e., the antibody is made less immunoreactive by altering one or more T cellDocket No. 56146-753.601epitopes). As used herein, a “deimmunized antibody” means that one or more T cell epitopes in an antibody sequence have been modified such that a T cell response after administration of the antibody to a subject is reduced compared to an antibody that has not been deimmunized. Analysis of immunogenicity and T-cell epitopes present in the antibodies and antigen binding fragments described herein can be carried out via the use of software and specific databases. Exemplary software and databases include iTope™ developed by Antitope of Cambridge, England. iTope™, is an in silico technology for analysis of peptide binding to human MHC class II alleles. The iTope™ software predicts peptide binding to human MHC class II alleles and thereby provides an initial screen for the location of such “potential T cell epitopes.” iTope™ software predicts favorable interactions between amino acid side chains of a peptide and specific binding pockets within the binding grooves of 34 human MHC class II alleles. The location of key binding residues is achieved by the in silico generation of 9mer peptides that overlap by one amino acid spanning the test antibody variable region sequence. Each 9mer peptide can be tested against each of the 34 MHC class II allotypes and scored based on their potential “fit” and interactions with the MHC class II binding groove. Peptides that produce a high mean binding score (>0.55 in the iTope™ scoring function) against >50% of the MHC class II alleles are considered as potential T cell epitopes. In such regions, the core 9 amino acid sequence for peptide binding within the MHC class II groove is analyzed to determine the MHC class II pocket residues (Pl, P4, P6, P7, and P9) and the possible T cell receptor (TCR) contact residues (P-1, P2, P3, P5, P8). After identification of any T-cell epitopes, amino acid residue changes, substitutions, additions, and / or deletions can be introduced to remove the identified T-cell epitope. Such changes can be made so as to preserve antibody structure and function while still removing the identified epitope. Exemplary changes can include, but are not limited to, conservative amino acid changes.

[0227] The “light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (“K” or “K”) or lambda (“X”), based on the amino acid sequences of their constant domains.

[0228] A “variable region” of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequenceDocket No. 56146-753.601variability (Kabat el al.. Sequences of Proteins of Immunological Interest, 5th Ed., 1991, National Institutes of Health, Bethesda Md., pages 647-669; hereafter “Kabat”); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-Iazikani et al. (1997) J. Molec. Biol. 273:927-948)). As used herein, a CDR may refer to CDRs defined by either approach or by a combination of both approaches. In some embodiments, a binding domain includes such a variable region derived from an antibody, or a derivative thereof.

[0229] With respect to antibodies and / or binding domains which are derived from antibodies, the term “variable domain” refers to the variable domains of antibodies that are used in the binding and specificity of an antibody for its particular antigen (or such a domain separated from the antibody as a binding domain as provided herein). However, the variability is not evenly distributed throughout the variable domains of antibodies. Rather, it is concentrated in three segments called hypervariable regions (also known as CDRs) in both the light chain and the heavy chain variable domains. More highly conserved portions of variable domains are called the “framework regions” or “FRs.” The variable domains of unmodified heavy and light chains each contain four FRs (FR1, FR2, FR3, and FR4), largely adopting a P-sheet configuration interspersed with three CDRs which form loops connecting and, in some cases, part of the P-sheet structure. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see, Kabat).

[0230] The terms “hypervariable region” and “CDR” when used herein, refer to the amino acid residues of an antibody or a binding domain derived from an antibody (i.e., in a binding domain as described herein) which are responsible for antigen binding. The CDRs comprise amino acid residues from three sequence regions which bind in a complementary manner to an antigen and are known as CDR1, CDR2, and CDR3 for each of the VH and VL chains. In the light chain variable domain, the CDRs typically correspond to approximately residues 24-34 (CDRL1), SO-56 (CDRL2), and 89-97 (CDRL3), and in the heavy chain variable domain the CDRs typically correspond to approximately residues 31-35 (CDRH1), 50-65 (CDRH2), and 95-102 (CDRH3) according to Kabat et al., Id. It is understood that the CDRs of different antibodies may contain insertions, thus the amino acid numbering may differ. The Kabat numbering system accounts for such insertions with a numbering scheme that utilizes letters attached to specific residues (e.g., 1 K, 27B, 27C, 27D, 27E, and 27F of CDRL1 in the light chain) to reflect any insertions in the numberings between different antibodies. Alternatively, in the light chain variable domain, the CDRs typically correspond to approximately residues 26-32 (CDRL1), 50-52 (CDRL2), and 91-96 (CDRL3), and in the heavy chain variable domain, the CDRs typically correspond toDocket No. 56146-753.601approximately residues 26-32 (CDRH1), 53-55 (CDRH2), and 96-101 (CDRH3) according to Chothia and Lesk (J Mol. Biol., 196: 901-917 (1987)).

[0231] As used herein, “framework region,” “FW,” or “FR” refers to framework amino acid residues that form a part of the antigen binding pocket or groove. In some embodiments, the framework residues form a loop that is a part of the antigen binding pocket or groove and the amino acids residues in the loop may or may not contact the antigen. Framework regions generally comprise the regions between the CDRs. In the light chain variable domain, the FRs typically correspond to approximately residues 0-23 (FRL1), 35-49 (FRL2), 57-88 (FRL3), and 98-109 and in the heavy chain variable domain the FRs typically correspond to approximately residues 0-30 (FRH1), 36-49 (FRH2), 66-94 (FRH3), and 103-133 according to Kabat et al., Id. As discussed above with the Kabat numbering for the light chain, the heavy chain too accounts for insertions in a similar manner (e.g., 35 A, 35B of CDRH1 in the heavy chain). Alternatively, in the light chain variable domain, the FRs typically correspond to approximately residues 0-25 (FRL1), 33-49 (FRL2) 53-90 (FRL3), and 97-109 (FRL4), and in the heavy chain variable domain, the FRs typically correspond to approximately residues 0-25 (FRH1), 33-52 (FRH2), 56-95 (FRH3), and 102-113 (FRH4) according to Chothia and Lesk, Id. The loop amino acids of a FR can be assessed and determined by inspection of the three-dimensional structure of an antibody heavy chain and / or antibody light chain. The three-dimensional structure can be analyzed for solvent accessible amino acid positions as such positions are likely to form a loop and / or provide antigen contact in an antibody variable domain. Some of the solvent accessible positions can tolerate amino acid sequence diversity and others (e.g., structural positions) are, generally, less diversified. The three-dimensional structure of the antibody variable domain can be derived from a crystal structure or protein modeling.

[0232] In the present disclosure, the following abbreviations (in the parentheses) are used in accordance with the customs, as necessary: heavy chain (H chain), light chain (L chain), heavy chain variable region (VH), light chain variable region (VL), complementarity determining region (CDR), first complementarity determining region (CDR1), second complementarity determining region (CDR2), third complementarity determining region (CDR3), heavy chain first complementarity determining region (VH CDR1), heavy chain second complementarity determining region (VH CDR2), heavy chain third complementarity determining region (VH CDR3), light chain first complementarity determining region (VL CDR1), light chain second complementarity determining region (VL CDR2), and light chain third complementarity determining region (VL CDR3).Docket No. 56146-753.601

[0233] As used herein, the term “avidity” refers to the resistance of a complex of two or more agents to dissociation after dilution. Apparent affinities can be determined by methods such as an enzyme-linked immunosorbent assay (ELISA) or any other suitable technique. Avidities can be determined by methods such as a Scatchard analysis or any other suitable technique.

[0234] As used herein, the term “affinity” refers to the equilibrium constant for the reversible binding of two agents and is expressed as KD. The binding affinity (KD) of an antibody or antigen binding fragment herein can be less than 500 nM, 475 nM, 450 nM, 425 nM, 400 nM, 375 nM, 350 nM, 325 nM, 300 nM, 275 nM, 250 nM, 225 nM, 200 nM, 175 nM, 150 nM, 125 nM, 100 nM, 90 nM, 80 nM, 70 nM, 50 nM, 50 nM, 49 nM, 48 nM, 47 nM, 46 nM, 45 nM, 44 nM, 43 nM, 42 nM, 41 nM, 40 nM, 39 nM, 38 nM, 37 nM, 36 nM, 35 nM, 34 nM, 33 nM, 32 nM, 31 nM, 30 nM, 29 nM, 28 nM, 27 nM, 26 nM, 25 nM, 24 nM, 23 nM, 22 nM, 21 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 990 pM, 980 pM, 970 pM, 960 pM, 950 pM, 940 pM, 930 pM, 920 pM, 910 pM, 900 pM, 890 pM, 880 pM, 870 pM, 860 pM, 850 pM, 840 pM, 830 pM, 820 pM, 810 pM, 800 pM, 790 pM, 780 pM, 770 pM, 760 pM, 750 pM, 740 pM, 730 pM, 720 pM, 710 pM, 700 pM, 690 pM, 680 pM, 670 pM, 660 pM, 650 pM, 640 pM, 630 pM, 620 pM, 610 pM, 600 pM, 590 pM, 580 pM, 570 pM, 560 pM, 550 pM, 540 pM, 530 pM, 520 pM, 510 pM, 500 pM, 490 pM, 480 pM, 470 pM, 460 pM, 450 pM, 440 pM, 430 pM, 420 pM, 410 pM, 400 pM, 390 pM, 380 pM, 370 pM, 360 pM, 350 pM, 340 pM, 330 pM, 320 pM, 310 pM, 300 pM, 290 pM, 280 pM, 270 pM, 260 pM, 250 pM, 240 pM, 230 pM, 220 pM, 210 pM, 200 pM, 190 pM, 180 pM, 170 pM, or any integer therebetween. Binding affinity may be determined using surface plasmon resonance (SPR), KINEXA® Biosensor, scintillation proximity assays, enzyme linked immunosorbent assay (ELISA), ORIGEN immunoassay (IGEN), fluorescence quenching, fluorescence transfer, yeast display, or any combination thereof. Binding affinity may also be screened using a suitable bioassay.

[0235] As used herein, a “Fab” refers to an antigen binding fragment which comprises one heavy chain variable domain (VH) linked to one constant domain (e.g., a CHI domain) derived from an antibody and one light chain variable domain (VL) linked to another constant domain (e.g., a light chain constant domain) derived from an antibody. In some embodiments, the VH is linked to a CHI domain (e.g., is fused to the CHI domain). In some embodiments, the VL is linked to the light chain constant domain. However, in other embodiments, alternative formats can be used, such as one in which the VH is linked to the light chain constant domain and the VL is linked to a CHI domain. Such alternative formats can be useful when such Fabs are intended to be used inDocket No. 56146-753.601bispecific antibody formats where light chain pairing can become complicated owing to the present of Fabs which bind to different antigens. Examples of technology where such approaches are used are described by, for example, Klein et al., “The use of CrossMAb technology for the generation of bi- and multi specific antibodies.” Mabs. 2016 Aug-Sep; 8(6): 1010-1020.

[0236] A CHI domain according to the instant disclosure (e.g., a CHI domain of a Fab as provided herein) can be one of those of any of the antibodies described herein, or a variant thereof. For example, a CHI domain can be one which has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the following sequences: a) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKRV (SEQ ID NO: 272); b) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTKTYTCNVDHKPSNTKVDKRV (SEQ ID NO: 273); c) ASKYGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTKTYTCNVDHKPSNTKVDKRV (SEQ ID NO: 274); or d) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 275).

[0237] A light chain constant region according to the instant disclosure can that of a lambda or kappa light chain, or a variant thereof. For example, a light chain constant region can be one which has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 276), RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAKQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 277), or RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAKQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGKSSPVTKSFNRGEC (SEQ ID NO: 278).

[0238] As used herein, an “scFv” refers to a single chain variable fragment which comprises a VH and VL linked via a peptide linker. In some embodiments, the linker is a flexible peptide linker, such as the GS linkers described herein.

[0239] As used herein, a “VHH” refers to a single-domain heavy chain antibody that binds to an antigen. Such single-domain antibodies are also referred to as “nanobodies” in the art. Such VHHsDocket No. 56146-753.601can be derived from the heavy-chain antibodies of camelids. A VHH lacks a corresponding VL domain.

[0240] Referred to herein are groups which are “attached” or “covalently attached” to residues of IL-18 polypeptides or other polypeptides. As used herein, “attached” or “covalently attached” means that the group is tethered to the indicated reside, and such tethering can include a linking group (i.e., a linker). Thus, for a group “attached” or “covalently attached” to a residue, it is expressly contemplated that such linking groups are also encompassed.

[0241] Binding affinity refers to the strength of a binding interaction between a single molecule and its ligand / binding partner. A higher binding affinity refers to a higher strength bond than a lower binding affinity. In some instances, binding affinity is measured by the dissociation constant (KD) between the two relevant molecules. When comparing KD values, a binding interaction with a lower value will have a higher binding affinity than a binding interaction with a higher value. For a protein-ligand interaction, KD is calculated according to the following formula:where [L] is the concentration of the ligand, [P] is the concentration of the protein, and [LP] is the concentration of the ligand / protein complex.

[0242] Referred to herein are certain amino acid sequences (e.g., polypeptide sequences) which have a certain percent sequence identity to a reference sequence or refer to a residue at a position corresponding to a position of a reference sequence. Sequence identity is measured by proteinprotein BLAST algorithm using parameters of Matrix BLOSUM62, Gap Costs Existence: 11, Extension:!, and Compositional Adjustments Conditional Compositional Score Matrix Adjustment. This alignment algorithm is also used to assess if a residue is at a “corresponding” position through an analysis of the alignment of the two sequences being compared.

[0243] The term “pharmaceutically acceptable” refers to approved or approvable by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia (U.S.P.) or other generally recognized pharmacopeia for use in animals, including humans.

[0244] A “pharmaceutically acceptable excipient, carrier, or diluent” refers to an excipient, carrier, or diluent that can be administered to a subject, together with an agent, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent.

[0245] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination ofDocket No. 56146-753.601numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, “nested sub-ranges” that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0246] Throughout the instant description, certain numerical or other similar values may be described as, for example, “at least” or “at most” a set of values indicated in a list form (e.g., “at least 2, 3, 4, 5, or 6”). In such cases, unless context clearly indicates otherwise, it is intended that the phrase “at least,” “at most,” or other similar term is applied individually to each value in the list. For example, the phrase “at least 2, 3, 4, 5, or 6” is to be interpreted as “at least 2, at least 3, at least 4, at least 5, or at least 6.”

[0247] Certain formulas and other illustrations provided herein depict triazole reaction products resulting from azide-alkyne cycloaddition reactions. While such formulas generally depict only a single regioisomer of the resulting triazole formed in the reaction, it is intended that the formulas encompass both resulting regioisomers. Thus, while the formulas depict only a single regioisomer (e.g.), it is intended that the other regioisomer (e.g.also encompassed.

[0248] The term “subject” refers to an animal which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, bovine, equine, canine, ovine, or feline.

[0249] The term “optional” or “optionally” denotes that a subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not.

[0250] The term “moiety” refers to a specific segment or functional group of a molecule. Chemical moieties are often recognized chemical entities embedded in or appended to a molecule.

[0251] As used herein, “conjugation handle” refers to a reactive group capable of forming a bond upon contacting a complementary reactive group. In some instances, a conjugation handle preferably does not have a substantial reactivity with other molecules which do not comprise theDocket No. 56146-753.601intended complementary reactive group. Non-limiting examples of conjugation handles, their respective complementary conjugation handles, and corresponding reaction products can be found in the table below. While table headings place certain reactive groups under the title “conjugation handle” or “complementary conjugation handle,” it is intended that any reference to a conjugation handle can instead encompass the complementary conjugation handles listed in the table (e.g., a trans-cyclooctene can be a conjugation handle, in which case tetrazine would be the complementary conjugation handle). In some instances, amine conjugation handles and conjugation handles complementary to amines are less preferable for use in biological systems owing to the ubiquitous presence of amines in biological systems and the increased likelihood for off-target conjugation.Table 5. Conjugation Handles

[0252] Throughout the instant application, prefixes are used before the term “conjugation handle” to denote the functionality to which the conjugation handle is linked. For example, a “protein conjugation handle” is a conjugation handle attached to a protein (either directly or through a linker), an “antibody conjugation handle” is a conjugation handle attached to an antibody (either directly or through a linker), and a “linker conjugation handle” is a conjugation handle attached to a linker group (e.g., a bifunctional linker used to link a synthetic protein and an antibody).

[0253] As used herein, “AJICAP™ technology,” “AJICAP™ methods,” and similar terms refer to systems and methods (currently produced by Ajinomoto Bio-Pharma Services (“Ajinomoto”)) for the site specific functionalization of antibodies and related molecules using affinity peptidesDocket No. 56146-753.601to deliver the desired functionalization to the desired site. General protocols for the AJICAP™ methodology are found at least in PCT Publication No. WO2018199337A1, PCT Publication No. WO2019240288 Al, PCT Publication No. WO2019240287A1, PCT Publication No. W02020090979A1, Matsuda et al., Mol. Pharmaceutics 2021, 18, 4058-4066, and Yamada et al., AJICAP: Affinity Peptide Mediated Regiodivergent Functionalization of Native Antibodies. Angew. Chem., Int. Ed. 2019, 58, 5592-5597, and in particular Examples 2-4 of US Patent Publication No. US20200190165A1. In some embodiments, such methodologies site specifically incorporate the desired functionalization at lysine residues at a position selected from position 246, position 248, position 288, position 290, and position 317 of an Fc region (e.g., an IgGl Fc region) (EU numbering). In some embodiments, the desired functionalization is incorporated at residue position 248 of an Fc region (EU numbering). In some embodiments, position 248 corresponds to the 18thresidue in a human IgG CH2 region (EU numbering).

[0254] SEQ ID NOs: 31 and 120 herein refer to human protein sequences. The remaining sequences are synthetic constructs.

[0255] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the disclosure as defined in the appended claims.

[0256] The present disclosure is further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the disclosure in any way.EXAMPLESExample 1A - Expression of Dual Binding Compositions

[0257] Expression: The molecules were expressed using ExpiCHO-S cell line (Thermofisher Scientific) and BalanCD transfectory CHO (Fujifilm) as culture media. The day of the transfection, the cells were centrifugated at low speed (500g / 15min), the media was carefully removed, and the cells were resuspended at a density of 6.0 million cells per ml with fresh BalanCD transfectory CHO media supplemented with 4mM L-Glutamine (Gibco).

[0258] The cells were transfected using FectoPro transfection reagent (Sartorius) as described by the manufacturer. Briefly, for a IL transfection reaction, a total of 0.8mg of plasmid DNA mix containing two plasmids encoding the different chains was diluted into 100ml of nonsupplemented BalanCD transfectory CHO media. The mixture was then poured into a container with 1.6ml of the FectoPro reagent, mixed vigorously and incubated for 10 minutes at roomDocket No. 56146-753.601temperature before the solution was added to the cells. After 4h of incubation at 37°C and 8% CO2, the cells were incubated at 32°C and 5% CO2. At Day 4, 6, 8 and 11, the cells were supplemented with balanCD Feed 4 (Fujifilm) and Glucose (Gibco) to maintain high viability before harvesting on Day 12.Example IB - Purification of Dual Binding Compositions

[0259] After expression, the culture media was centrifugated at 2000g during 15min to remove cells. The clarified media was then filtered on 0.22um filter with Diatomaceous earth as filter aid. The molecules were purified on an AKTA Pure coupled with a HiTrap MabSelect Sure column (Cytiva). After elution with 0.1M of glycine pH3.0, the fractions were pooled and neutralized by addition of 10% of the pool volume of IM acetate pH5.5 and the molecules were polished using a HiTrap SP HP with 50mM acetate Ph5.0 as mobile phase. The peak corresponding to the molecule of interest was collected, sterile filtered and the purity was assessed using SEC-HPLC, SDS-PAGE and LC-MS analysis.Example 2 - Biophysical CharacterizationExample 2A - Analytical Size Exclusion chromatography (aSEC)Protocol for aSEC

[0260] Sample preparation: Samples are diluted to a concentration between 0.5 and l.Omg / mL with their respective formulation buffer before centrifugation at 20000xg, 4°C for lOmin. The following Material was used 10X PBS pH7.2 (Phosphate-Buffered Saline, Ref. 70013-032, Gibco). The column was XBridge Premier Protein SEC Column (7.8 x 300 mm, 2.5 pm, 250A pore size, Ref. 186009962, Waters). The following parameters were used: System Suitability Test (SST) BEH200 SEC Protein Standard mix (Ref. 186006518, Waters); Flow rate 0.75 mL / min; Elution buffer IX PBS pH 7.2 (Filtered on 0.22um and sonicated); Temperature 25°C; Sample loading lOuL; Elution 100% of IX PBS pH7.2; Data acquisition 24 min; Wavelengths 220nm and 280nm.Results of aSEC

[0261] Table 6 Summarizes the results of aSEC.Table 6. Results of aSEC.Docket No. 56146-753.601Example 2B - Hydrophobic Interaction ChromatographyProtocol for Hydrophobic Interaction Chromatography

[0262] Sample preparation Samples at Img / mL were spiked with some HIC mobile phase A in order to reach a concentration of IM of ammonium sulfate before injection (Example: 26.7uL of protein sample at Img / mL + 33.3uL of molbile phase A) before centrifugation at 20000xg, 4°C for lOmin.

[0263] The following Material was used: Ammonium sulfate (Emprove Expert, Granulated, MW= 132.14g / mol, Ref. 1.04161.1000, Sigma Aldrich); Sodium phosphate monobasic dihydrate (MW=156.01g / mol, Ref. 71505-1KG, Sigma Aldrich); Acetonitrile (for HPLC - Gradient grade, Ref. 20060.320, VWR chemicals). The Column was TSKgel Butyl-NPR column (4.6 x 35 m, 2.5 pm, Ref. 0014947, TOSOH BIOSCIENCE). References were Ustekinumab, Adalimumab. The following parameters were used: Flow rate 0.75 mL / min; Elution buffers Mobile phase A: 1.8M ammonium sulfate, 0.1M sodium phosphate pH6.5 (Filtered on 0.22um and sonicated); Mobile phase B: 0.1M sodium phosphate pH6.5 (Filtered on 0.22um and sonicated); Mobile phase C: 20% / 80% v / v Acetonitrile / H2O-MQ (Sonicated); Temperature 25°C; Sample loading 10 uL; Elution Hydrophobic chromatography elution gradient (See table); Data acquisition 45 min; Wavelengths 220nm and 280nm.

[0264] Table 7 Summarizes the Hydrophobic chromatography elution gradient for developability.Table 7. Hydrophobic chromatography elution gradient for developabilityDocket No. 56146-753.601

[0265] Samples at Img / mL were mixed with HIC mobile phase A containing 1.8M ammonium sulfate, 0.1M sodium phosphate pH6.5 in order to reach a concentration of IM of ammonium sulfate and promote hydrophobic interactions (Example: 26.7uL of protein sample at Img / mL + 33.3uL of mobile phase A). Samples were centrifuged at 20000xg at 4°C for lOmin before injection to remove any particles.

[0266] Hydrophobic interaction chromatography was performed using TSKgel Butyl-NPR column (4.6 x 35 m, 2.5 pm, Ref. 0014947, TOSOH BIOSCIENCE) equilibrated with 1.8M ammonium sulfate, 0.1M sodium phosphate pH6.5. 10 pL of sample was injected at a flow rate of 0.75 mL / min. Elution was achieved by applying a linear gradient of decreasing salt concentration using a mobile phase B containing 0.1M sodium phosphate pH6.5. Proteins were monitored by UV absorbance at 220 nm. The elution profile is recorded and compared to reference molecules (Ustekinumab, Adalimumab)Results of Hydrophobic Interaction Chromatography

[0267] Table 8 Summarizes the results of aSEC.Table 8. Results of aSEC.Docket No. 56146-753.601Example 2C- Analytical Heparin ChromatographyProtocol for Heparin Chromatography

[0268] Sample preparation: Samples are diluted to 0.2 mg / mL with some 20mM Histidine pH5.5 before centrifugation at 20 OOOxg at 4°C for 10 min. Column was TSKgel Heparin-5 PW (7.5mm x 7.5cm, 10pm, Ref. 13064, TOSOH BIOSCIENCE). References were Ustekinumab, Adalimumab, Briakinumab, Gantenerumab, Daratumumab, Obinutuzumab. The following parameters were used: Flow rate 0.5 mL / min; Elution buffers Mobile phase A: 50mM Tris pH7.4 (Filtered on 0.22um and sonicated); Mobile phase B: 20mM Tris pH7.4; IM NaCl (Filtered on 0.22um and sonicated); Temperature 25°C; Sample loading 50 uL (lOug); Elution Heparin chromatography elution gradient (See table); Data acquisition 45 min; Wavelengths 220nm and 280nm.

[0269] Due to different HPLC systems, samples were compared by using their relative retention

[0270] time (Rel. RT). It has been calculated by dividing their retention time (min) by the one of a control, Adalimumab.

[0271] Table 9 Summarizes the Hydrophobic chromatography elution gradient for developability for hepain chromatography.Table 9. Hydrophobic chromatography elution gradient for developability for hepain chromatographyDocket No. 56146-753.601

[0272] The propensity for charge-mediated heparin binding was assessed using Heparin chromatography. A higher affinity for heparin is indicative of increased charge-mediated interactions at the surface of endothelial cells, suggesting a greater risk of non-specific clearance via pinocytosis, which is an important consideration in evaluating molecule developability.

[0273] Samples were prepared by diluting to a concentration of 0.2 mg / mL in 20 mM Histidine buffer at pH 5.5. The diluted samples were then centrifuged at 20,000xg at 4°C for 10 minutes to remove any particulate matter prior to chromatography. 50 pL of sample is injected on a TSKgel Heparin-5 PW column (7.5 mm x 7.5 cm, 10 pm particles; Ref. 13064, TOSOH BIOSCIENCE) equilibrated with 50 mM Tris, pH 7.4 at a flow rate of 0.5 mL / min. Sample is eluted using a gradient of 20 mM Tris, pH 7.4, containing 1 M NaCl. Proteins were monitored by UV absorbance at 280 nm. The elution profile was recorded and compared to reference molecules (Ustekinumab, Briakinumab, Gantenerumab, Daratumumab, and Obinutuzumab). To facilitate comparison of molecules analyzed on different HPLC systems, the relative retention time (Heparin Rel. RT) compared to the reference antibody Adalimumab is provided.Results of Heparin Chromatography

[0274] Table 10 Summarizes the results of Heparin Chromatography. FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, and FIG. 5E, respectively, show the results of Heparin chromatography for composition 59, composition 74, composition 82, and composition 88, and composition 96, respectively. Table 10. Results of Heparin Chromatography.Example 2D - Accelerated Degradation Assay

[0275] Molecules were buffer exchanged into lOmM Histidine pH6.0, concentrated to 50 mg / mL and incubated for up to 48h at 52°C. Aggregation was stopped by diluting the molecule to lOmg / ml in ice-cold phosphate buffer. After 10 minutes on ice, the molecules wereDocket No. 56146-753.601centrifugated at 6.000rpm for 3min to remove large insoluble aggregates and molecules analyzed using aSEC. Percentage of monomer was calculated by measuring the area under the curve of the peak corresponding to the monomeric fraction.

[0276] Introduction of L154K mutation in bevacizumab light chain overcame aggregation liability with Bevacizumab.

[0277] FIG. 6 shows the accelerated degradation assay for Composition 10 (left) and composition 46 (right). Table 11 shows the results of the accelerated degradation assay for composition 10 and composition 46. Monomer and aggregates ratio were normalized to total peaks area.Table 11. Accelerated Degradation AssayExample 2E - VEGF Cooperativity Assessment by aSEC and Dynamic Light Scattering (DLS)

[0278] Samples were assessed for cooperativity effects by analyzing selected compositions by aSEC and DLS in the presence of VEGF. All samples were prepared without VEGF as well as with 2X of the VEGF dimer (Human VEGF165 Protein, His Tag, Cat. VE5-H5248, Aero Biosystems) before centrifugation at 20000xg, 4°C for 15min.Analytical Size Exclusion Chromatograohy for VEGF cooperativity assessment

[0279] Samples were assessed by aSEC using the following parameters. Sample preparation: All samples were prepared without VEGF as well as with 2X of the VEGF dimer (Human VEGF 165Docket No. 56146-753.601Protein, His Tag, Cat. VE5-H5248, Aero Biosystems) before centrifugation at 20000xg, 4°C for 15min. The Column was XBridge BEH200A SEC column (7.8 x 300 mm, 3.5 gm, 200A pore size, Waters). The following parameters were used. System Suitability Test (SST) BEH200 SEC Protein Standard mix (Ref. 186006518, Waters); Flow rate 0.86 mL / min; Elution buffer 50mM sodium phosphate dibasic dihydrate, 628mM Sodium chloride, 13.5mM potassium chloride, lOmM monobasic potassium phosphate pH7.2; Temperature 25°C; Sample loading lOuL; Elution 100% of elution buffer; Data acquisition 24 min; Wavelengths 220nm and 280nm. Dynamic Light Scattering (DLS) for VEGF cooperativity assessment

[0280] Samples were analyzed by DLS using the following parameters: System: Prometeus PANTA (NanoTemper) with Standard sensitivity capillaries (Ref. PR-C002); Temperature 25°C; DLS power 100%.Results of VEGF cooperativity Studies

[0281] Table 12 summarizes the results of the VEGF cooperativity Studies. FIG. 7A shows a DLS profile of purified composition 59 with and without VEGF addition (left) and a Zoomed Analytical SEC-HPLC chromatogram of purified composition 59 with and without VEGF addition (detection: 220 nm) (right). FIG. 7B shows a DLS profile of purified composition 74 with and without VEGF addition (left) and a Zoomed Analytical SEC-HPLC chromatogram of purified composition 74 with and without VEGF addition (detection: 220 nm) (right). FIG. 7C shows a DLS profile of purified composition 82 with and without VEGF addition (left) and a Zoomed Analytical SEC-HPLC chromatogram of purified composition 82 with and without VEGF addition (detection: 220 nm) (right). FIG. 7D shows a DLS profile of purified composition 88 with and without VEGF addition (left) and a Zoomed Analytical SEC-HPLC chromatogram of purified composition 88 with and without VEGF addition (detection: 220 nm) (right). FIG. 7E shows a DLS profile of purified composition 96 with and without VEGF addition (left) and a Zoomed Analytical SEC-HPLC chromatogram of purified composition 96 with and without VEGF addition (detection: 220 nm) (right).

[0282] Table 12. Results of the VEGF cooperativity Studies.Docket No. 56146-753.601Example 2F - Mass Spectrometry (performed by Functional Genomics Center Zurich) Protocol for LCMS

[0283] Sample preparation: All samples analysed under non reducing or reducing conditions are diluted with 1% TFA, passed through the AttractFiltra RC Micro Spin column and transferred to an autosampler vials for LC / MS. The Column was BioResolve Premier RP mAh (450 A, 2.7 pm, 2.1 mm x 20 mm, Waters). The System was Synapt G2-Si mass spectrometer coupled to an Acquity UPLC station. The following parameters were used: Flow rate 0.2mL / min; Elution buffers Buffer A: 0.1% DFA in water; Buffer B: 0.1% DFA in AN / 75% 2-PrOH; Temperature 80°C; Elution Gradient; Data acquisition 30 min; Mode Positive-ion mode ; m / z range From 400 to 5000 Da; Spray voltage 120V; Source temperature 100°C; Software MassLynx 4.2 (Waters).

[0284] The purity and identity of the recombinant protein from commercial source and the conjugated protein was confirmed by aSEC and LC-MS (performed at Functional Genomics Center Zurich).Docket No. 56146-753.601LCMS Results

[0285] Table 13. shows the results of the LCMS, with Non reduced LC-MS measured and expected molecular weight (Da).Table 13. Results of the LCMS.Example 3 - PD-1 VHH Screen for PD-1 Binding and ActivityExample 3A - Biolayer Interferometry (BLI)

[0286] BLI experiments were performed to assess the ability of anti -PD-1 binding domain VHHs using PD-1 as ligand bound to the biosensor (“PD1 as ligand”) and with PD-1 in solution with dual binding composition bound to the biosensor (“PD1 monovalent”).PD1 as ligand materials and methods

[0287] PD1 as ligand: Materials: Bio-Layer Interferometry experiments were performed on an Octet® R8 system (Sartorius) using High Precision Streptavidin Biosensors (SAX2) (Sartorius, Cat. No. 18-5136). The ligand was biotinylated human PD-1 / PDCD1 protein (Avi tag™, His Tag, 18.6 kDa; AcroBiosystems, #PD1-H82E4), prepared at a fixed concentration of 0.0375 pg / mL. Analytes consisted of various compositions prepared by serial dilution. The assay was conducted in lx Kinetic Buffer (PBS, 0.1% BSA, 0.02% Tween-20), with 10 mM glycine (pH 2.06) as the regeneration buffer. All measurements were performed in 96-well polypropylene flat-bottom plates (Greiner, #655209).

[0288] PD1 as ligand: Methods. Ligand loading solutions were prepared at a concentration of 0.0375 pg / mL in a total volume of 2mL, and 200 pL was dispensed into each well. Analyte solutions were prepared by 1:2 serial dilution, starting from 30 nM, to a final volume of 200 pL per well. Kinetic measurements were performed using the following BLI program: Loading: 600 s; Regeneration: 3 cycles of 6 s / 6 s; Association: 300 s; Dissociation: 500 s; Regeneration: 3 cyclesDocket No. 56146-753.601of 6 s / 6 s. Data were analyzed using Octet Analysis Software, applying a 1:1 global model with an unlinked curve fit to determine the equilibrium dissociation constant (K_D).PD1 monovalent materials and methods

[0289] PD1 monovalent: Materials. Experiments were performed on an Octet® R8 system (Sartorius) using Anti-Human Fc Capture (AHC) Biosensors (Sartorius, Cat. No. 18-5063). Ligands consisted of various compositions immobilized at a fixed concentration of 0.6 pg / mL. The analyte was recombinant human PD-1, His-tagged protein (R&D Systems, #8986-PD), prepared by serial dilution. The assay was conducted in lx Kinetic Buffer (PBS, 0.1% BSA, 0.02% Tween-20) in 96-well polypropylene flat-bottom plates (Greiner, #655209).

[0290] PD1 monovalent: Methods. Ligand loading solutions were prepared at 0.6 pg / mL in a total volume of 2 mL, and 200 pL was dispensed into each well. Analyte solutions were prepared by 1:2 serial dilution, starting from 100 nM, to a final volume of 200 pL per well. Kinetic measurements were performed using the following BLI program: Loading: 180 s; Association: 180 s; Dissociation: 300 s. Data were analyzed using Octet Analysis Software, applying a 1:1 global model with an unlinked curve fit to determine the equilibrium dissociation constant (K_D). Screen for PD-1 Nb binders - PD1 binding with BLI

[0291] Table 14 shows the KDs of all PD-1 Nb binders tested.Table 14 KDs of PD-1 Nb binders>>>Docket No. 56146-753.601Docket No. 56146-753.601Example 3B - anti-PD-1 ELISA

[0292] The interaction of the dual binder compositions with PD-1 (CD279) were measured by ELISA assay. For these studies, Corning high-binding half-area plates (Fisher Scientific, Reinach, Switzerland) were coated overnight at 4° C. with 25 pl of dual binder compositions at 2.5 pg / ml in PBS. Plates were then washed four times with 100 pl of PBS-0.2% Tween20 (wash buffer). Plate surfaces were blocked with 25 pl of PBS-1% BSA at 37° C. for Ih. Plates were then washed four times with 100 pl of wash buffer. Twenty-five microliters of recombinant biotinylated PD1 / CD279 protein (Biotinylated Recombinant Human PD-1 (CD279)-Fc Chimera (carrier-free), Biolegend #799506) were added in serial dilutions in PBS-T with 0.1% BSA and incubated at 37° C. for 2h. Plates were then washed four times with wash buffer. Twenty-five microliters of Streptavidin-Horseradish peroxidase (#RABHRP3, Merck, Buchs, Switzerland) diluted at 1:500 in PBS-0.02% Tween20-0.1% BSA were added to each well and incubated at Room Temperature for 30 min. Plates were then washed four times with 100 pl of wash buffer. Fifty microliters of TMB substrate reagent (#CL07, Merck, Buchs, Switzerland) were added to each well and incubated at 37° C. during 5 min. After 5 min at 37° C., Horseradish peroxidase reaction was stopped by adding 50 pl / well of 0.5M H2SO4 stop solution. ELISA signal was then measured at 450 nm on an EnSpire plate reader from Perkin Elmer (Schwerzenbach, Switzerland).Screen for PD-1 Nb binders - PD1 binding with ELISA:

[0293] Table 15 shows the KDs of all PD-1 Nb binders testedTable 15. KDs of PD-1 Nb bindersDocket No. 56146-753.601Docket No. 56146-753.601Example 3C - PD-1 / PD-L1 Blockade Assay

[0294] The ability of the control and dual binder compositions to interfere with PD1 / PDL1 pathway was measured using the PD-1 / PD-L1 Blockade Bioassay from Promega (Cat.# J1250, Madison, WI, USA). PD-1 / PD-L1 Blockade Bioassay is a bioluminescent cell-based assay based on the co-culture of effector cells with target cells mimicking an immunological synapse. Jurkat T cells expressing human PD-1 (Jurkat-Lucia-PDl) and a luciferase reporter driven by a NF AT response element (NFAT-RE) are activated by CHO-K1 cells expressing human PD-L1 (Raji-APC-L1, Cat#Rajkt-hpdl, Invivogen) and an engineered cell surface protein designed to activate Jurkat’ s cognate TCRs. Concurrent interaction PD-1 / PD-L1 inhibits TCR signaling and represses NFAT-RE-mediated luminescence. Addition of either an anti -PD-1 or anti-PD-Ll antibody that blocks the PD-1 / PD-L1 interaction releases the inhibitory signal, restoring TCR activation and resulting in a gain of signal of NFAT-RE luminescent reporter.Docket No. 56146-753.601

[0295] Briefly, PD-L1 aAPC / CHO-Kl Target cells were plated in white tissue culture 96-well plates and cultured 24h at 37°C / 5% CO2. Test molecules were measured in serial dilutions starting at and pre-incubated on target cells for lOmin before the addition of freshly thawed PD-1 Jurkat effector cells. Samples were incubated either with or without VEGFA (recombinant human VEGF165 (VE5-H5248, Aero Bio)) in a 2:1 molar ratio with dual binder composition. After 24h at 37°C / 5% CO2, activity NF AT -RE luminescent reporter was evaluated by removing a 20 microliter sample to which QUANTI-Lic reagent (#rep-qlc, Invivogen) was added. Bioluminescence was then immediately read with a plate readerScreen for PD-1 Nb binders - PD1 blocking assay:

[0296] Table 16 shows the IC50 of all PD-1 Nb binders tested + / -VEGFTable 16. IC50 of PD-1 Nb binders + / -VEGFDocket No. 56146-753.601Docket No. 56146-753.601Example 4 - Characterization of Additional Dual Binder Composition anti-PD-1 activity Example 4A - Biolayer Interferometry

[0297] BLI experiments were performed to assess the ability of dual binder compositions using PD-1 as ligand bound to the biosensor (“PD1 as ligand”) and with PD-1 in solution with dual binding composition bound to the biosensor (“PD1 monovalent”).PD1 as ligand materials and methods

[0298] PD1 as ligand: Materials. Bio-Layer Interferometry experiments were performed on an Octet® R8 system (Sartorius) using High Precision Streptavidin Biosensors (SAX2) (Sartorius, Cat. No. 18-5136). The ligand was biotinylated human PD-1 / PDCD1 protein (Avi tag™, His Tag, 18.6 kDa; AcroBiosystems, #PD1-H82E4), prepared at a fixed concentration of 0.0375 pg / mL. Analytes consisted of various compositions prepared by serial dilution. The assay was conducted in lx Kinetic Buffer (PBS, 0.1% BSA, 0.02% Tween-20), with 10 mM glycine (pH 2.06) as the regeneration buffer. All measurements were performed in 96-well polypropylene flat-bottom plates (Greiner, #655209).

[0299] PD1 as ligand: Methods. Ligand loading solutions were prepared at a concentration of 0.0375 pg / mL in a total volume of 2mL, and 200 pL was dispensed into each well. Analyte solutions were prepared by 1:2 serial dilution, starting from 30 nM, to a final volume of 200 pL per well. Kinetic measurements were performed using the following BLI program: Loading: 600 s; Regeneration: 3 cycles of 6 s / 6 s; Association: 300 s; Dissociation: 500 s; Regeneration: 3 cycles of 6 s / 6 s. Data were analyzed using Octet Analysis Software, applying a 1:1 global model with an unlinked curve fit to determine the equilibrium dissociation constant (K_D).PD1 monovalent materials and methods

[0300] PD1 monovalent: Materials. Experiments were performed on an Octet® R8 system (Sartorius) using Anti-Human Fc Capture (AHC) Biosensors (Sartorius, Cat. No. 18-5063). Ligands consisted of various compositions immobilized at a fixed concentration of 0.6 pg / mL. The analyte was recombinant human PD-1, His-tagged protein (R&D Systems, #8986-PD), prepared by serial dilution. The assay was conducted in lx Kinetic Buffer (PBS, 0.1% BSA, 0.02% Tween-20) in 96-well polypropylene flat-bottom plates (Greiner, #655209).Docket No. 56146-753.601

[0301] PD1 monovalent: Methods. Ligand loading solutions were prepared at 0.6 pg / mL in a total volume of 2 mL, and 200 pL was dispensed into each well. Analyte solutions were prepared by 1:2 serial dilution, starting from 100 nM, to a final volume of 200 pL per well. Kinetic measurements were performed using the following BLI program: Loading: 180 s; Association: 180 s; Dissociation: 300 s. Data were analyzed using Octet Analysis Software, applying a 1:1 global model with an unlinked curve fit to determine the equilibrium dissociation constant (K D). Data PD1 binding Assay by BLI (monovalent and PD1 ligand )

[0302] Table 17 shows the data for PD1 binding assay by BLI for PD1 ligand.Table 17. Data for PD1 binding assay for PD1 ligand.

[0303] Table 18 shows the data for PD1 binding assay by BLI for monovalent.Table 18. Data for PD1 binding assay for monovalent.Docket No. 56146-753.601Example 4B - anti-human and cynomolgus PD-1 ELISAProtocol

[0304] The interaction of the activatable dual binder compositions with both human and cynomolgus PD-1 (CD279) were measured by ELISA assay. For these studies, Corning high-binding half-area plates (Fisher Scientific, Reinach, Switzerland) were coated overnight at 4° C. with 25 pl of dual binder compositions at 2.5 pg / ml in PBS. Plates were then washed four times with 100 pl of PBS-0.2% Tween20 (wash buffer). Plate surfaces were blocked with 25 pl of PBS- 1% BSA at 37° C. for Ih. Plates were then washed four times with 100 pl of wash buffer. Twenty-five microliters of either recombinant biotinylated human PD1 / CD279 protein (Biotinylated Recombinant Human PD-1 (CD279)-Fc Chimera (carrier-free), Biolegend #799506) or recombinant biotinylated cynomolgus macaque (Biotyinylated Recombinant Cynomolgus Monkey PD-1 protein (Tagged), AB271617-1001 abeam) were added in serial dilutions in PBS-T with 0.1% BSA and incubated at 37° C. for 2h. Plates were then washed four times with wash buffer. Twenty-five microliters of Streptavidin-Horseradish peroxidase (#RABHRP3, Merck, Buchs, Switzerland) diluted at 1 :500 in PBS-0.02% Tween20-0.1% BSA were added to each well and incubated at Room Temperature for 30 min. Plates were then washed four times with 100 pl of wash buffer. Fifty microliters of TMB substrate reagent (#CL07, Merck, Buchs, Switzerland) were added to each well and incubated at 37° C. during 5 min. After 5 min at 37° C., Horseradish peroxidase reaction was stopped by adding 50 pl / well of 0.5M H2SO4 stop solution. ELISA signal was then measured at 450 nm on an EnSpire plate reader from Perkin Elmer (Schwerzenbach, Switzerland).Data for Selected Composition Characterization - PD-1 binding (ELISA)

[0305] FIG. 8 shows a summary of KDS of selected bispecific molecules (Pembro, ivonescimab, composition 59, composition 74, composition 82, and composition 88, from left to right). All tested molecules showed sufficient PD1 binding.Example 4C - PD-1 / PD-L1 Blockade AssayProtocol for PD1 blocking assay

[0306] The ability of the control and dual binder compositions to interfere with PD1 / PDL1 pathway was measured using the PD-1 / PD-L1 Blockade Bioassay from Promega (Cat.# J1250, Madison, WI, USA). PD-1 / PD-L1 Blockade Bioassay is a bioluminescent cell-based assay basedDocket No. 56146-753.601on the co-culture of effector cells with target cells mimicking an immunological synapse. Jurkat T cells expressing human PD-1 (Jurkat-Lucia-PDl) and a luciferase reporter driven by a NF AT response element (NFAT-RE) are activated by CH0-K1 cells expressing human PD-L1 (Raji-APC-L1, Cat#Rajkt-hpdl, Invivogen) and an engineered cell surface protein designed to activate Jurkat’ s cognate TCRs. Concurrent interaction PD-1 / PD-L1 inhibits TCR signaling and represses NFAT-RE-mediated luminescence. Addition of either an anti -PD-1 or anti-PD-Ll antibody that blocks the PD-1 / PD-L1 interaction releases the inhibitory signal, restoring TCR activation and resulting in a gain of signal of NFAT-RE luminescent reporter.

[0307] Briefly, PD-L1 aAPC / CHO-Kl Target cells were plated in white tissue culture 96-well plates and cultured 24h at 37°C / 5% CO2. Test molecules were measured in serial dilutions starting at and pre-incubated on target cells for lOmin before the addition of freshly thawed PD-1 Jurkat effector cells. Samples were incubated either with or without VEGFA (recombinant human VEGF165 (VE5-H5248, Aero Bio)) in a 2:1 molar ratio with dual binder composition. After 24h at 37°C / 5% CO2, activity NFAT-RE luminescent reporter was evaluated by removing a 20 microliter sample to which QUANTI-Lic reagent (#rep-qlc, Invivogen) was added. Bioluminescence was then immediately read with a plate reader.Lead panel characterization- PD-1 blocking assay

[0308] FIG. 9 shows average IC50s of PD1 blocking by selected molecules (Pembrolizumab, Pembrolizumab+VEGF 2x, Composition 5, Composition 5 + VEGF 2x, Composition 59, Composition 59 + VEGF 2x, Composition 74, Composition 74 + VEGF 2x, Composition 82, Composition 82 + VEGF 2x. Composition 88, Composition 88 + VEGF 2x, Composition 96, Composition 96 + VEGF 2x, from left to right. Blocking capacity increased for all molecules by the addition of VEGF (cooperativity).

[0309] Example 5 - Characterization of Additional Dual Binder Composition anti- VEGF activityExample 5A - VEGF ELISAProtocol for human VEGF 165 binding by ELISA

[0310] The following Materials were used: Biotinylated Human VEGF 165 Protein (Source: Acrobiosystems, Catalog Number: VE5-H82Q0-25ug), Streptavidin-HRP Conjugate (Source: Sigma-Aldrich, Catalog Number: RABHRP3-600UL); TMB Substrate Solution (Source: Sigma-Aldrich, Catalog Number: CL07), STOP Solution (Composition: 1 M sulfuric acid (H2SO4)). The following Buffers were used: Coating Buffer: PBS without calcium and magnesium (PBS - / -);Docket No. 56146-753.601Wash Buffer: PBS + 0.02% Tween-20; Blocking Buffer: PBS + 0.02% Tween-20 + 1% BSA; Protein Diluent: PBS + 0.02% Tween-20 + 0.1% BSA.

[0311] High-binding 96-well ELISA plates were coated with 25 pL per well of capture antibody solution in PBS -I- and incubated overnight at 4°C under sealed conditions. Plates were washed four times with PBS containing 0.02% Tween-20.

[0312] Blocking was performed by adding 50 pL per well of blocking buffer (PBS + 0.02% Tween-20 + 1% BSA), followed by incubation for 1 hour at 37°C with orbital shaking at 600 rpm.

[0313] Detection antibody solutions were prepared at a top concentration of 500 ng / mL in protein diluent (PBS + 0.02% Tween-20 + 0.1% BSA). After blocking, plates were washed four times, and 25 pL of serial dilutions of biotinylated human VEGF165 were added to each well. Plates were incubated for 2 hours at 37°C with shaking at 600 rpm.

[0314] Subsequently, plates were washed four times and incubated with 25 pL per well of Streptavidin-HRP conjugate diluted 1:500 in protein diluent. Incubation was carried out for 30 minutes at room temperature with shaking at 600 rpm.

[0315] After a final wash step, 50 pL of TMB substrate solution was added to each well and incubated for approximately 5 minutes at room temperature in the dark. The enzymatic reaction was terminated by the addition of 50 pL per well of 1 M H2SO4. Absorbance was measured at 450 nm using an Enspire plate reader.Data for human VEGF 165 binding by ELISA

[0316] FIG. 10 shows a summary of characterization of selected compositions - VEFG165 binding (ELISA) with Kds of selected bispecific molecules (Bevacizumab, Composition 5, composition 59, composition 74, composition 82, and composition 88, from left to right), all molecules tested showed good VEGF binding.

[0317] Example 5B - VEGFR Blocking AssayProtocol for VEGFR blocking Assay

[0318] The following Materials were used: VEGF Bioassay Kit (Promega, Cat.# GA2001), containing: KDR / NFAT-RE HEK293 cells, Assay Medium, VEGF Standard, Control Antibody, Bio-Gio™ Luciferase Assay Reagent. Additionally, 96-well white tissue culture plates (recommended for luminescence), CO2 incubator (37°C, 5% CO2), Luminometer (e.g., GloMax® Discover, Promega),

[0319] The VEGF Bioassay was performed according to the manufacturer’s instructions (Promega Technical Manual TM544). Briefly, cryopreserved KDR / NFAT-RE HEK293 cellsDocket No. 56146-753.601were thawed and resuspended in assay medium. Cells were plated into 96-well white plates at the recommended density and incubated overnight at 37°C in a humidified atmosphere with 5% CO2.

[0320] On the following day, serial dilutions of VEGF standard or test samples were prepared in assay medium. For neutralization studies, VEGF was pre-incubated with anti-VEGF antibodies for 30 minutes at room temperature before addition to the cells. Cells were treated with 100 pL of VEGF dilutions or sample mixtures and incubated for 6 hours at 37°C, 5% CO2.

[0321] After incubation, 100 pL of Bio-Gio™ Luciferase Assay Reagent was added directly to each well. Plates were incubated for 5-10 minutes at room temperature to stabilize the luminescent signal. Luminescence was measured using a GloMax® Discover luminometer. Data were analyzed by plotting relative luminescence units (RLU) versus VEGF concentration to generate a dose-response curve.Data for VEGFR blocking Assay

[0322] FIG. 11 shows a summary of the characterization of selected composition - VEFG165 blocking assay (ELISA) with average IC50s of VEGFR blocking by selected molecules (Bevacizumab, Composition 5, composition 59, composition 74, composition 82, composition 88, and composition 96, from left to right), All tested molecules showed strong blockade of VEGFR signaling.Example 5C - Biolayer Interferometry Analysis of VEGF Binding of Additional anti-VEGF VHHsProtocol for BLI

[0323] The ability of additional anti-VEGF VHH binders (those provided in Table 2B) to bind to human VEGF was assessed by BLI. In the experiments provided below, each indicated VHH binder was tested in the format depicted in FIG. 1G, but the anti-PD-1 Fab on the chain which does not comprise the anti-VEGF VHH domains was replaced with an IL-2 polypeptide. In the molecules tested, the first VHH domain is attached to the C-terminus of the Fc domain to which it is attached by a (GGGGS)4 peptide linker (SEQ ID NO: 30) and the second VHH domain is attached to the C-terminus of the first VHH domain by a (GGGGS)2 peptide linker (SEQ ID NO: 22). It is contemplated that anti-VEGF VHH binding domains observed in this experiment to provide suitable binding characteristics could be incorporated into a dual binder composition as described herein. The experiment was performed as follows:

[0324] The following materials were used: BLI experiments were performed on an Octet® R8 system (Sartorius) using Anti -Human Fc Capture (AHC) Biosensors (Sartorius, Cat. No. 18-5063). Ligands consisted of various compositions immobilized at a fixed concentration ofDocket No. 56146-753.6010.6 pg / mL. The analyte was human VEGF165 protein, His-tagged (AcroBiosystems, #VE5-H5248), prepared by serial dilution. The assay was conducted in lx Kinetic Buffer (PBS, 0.1% BSA, 0.02% Tween-20) in 96-well polypropylene flat-bottom plates (Greiner, #655209).

[0325] Ligand loading solutions were prepared at 0.6 pg / mL in a total volume of 2mL, and 200 pL was dispensed into each well. Analyte solutions were prepared by 1:2 serial dilution, starting from 100 nM, to a final volume of 200 pL per well.

[0326] Kinetic measurements were performed using the following BLI program: Loading: 180 s; Association: 180 s; Dissociation: 300 s.

[0327] Data were analyzed using Octet Analysis Software, applying a 1:1 global model with an unlinked curve fit to determine the equilibrium dissociation constant (K D).Data for BLI

[0328] Table 19 shows the data for VEGF Nb- VEGF165 binding by BLI.Table 19. VEGF Nb- VEGF165 binding by BLIDocket No. 56146-753.601Docket No. 56146-753.601Example 6 - Fc Binding Activity of Dual Binder CompositionsHuman FcyR Binding Assay

[0329] The interaction of the dual binding compositions (e.g., antibody formats described herein) with human Fc gamma receptors I (FcyRI / CD64), with human Fc gamma receptors Ila (FcyRIIa / CD32a), with inhibitory human Fc gamma receptors lib (FcyRIIb / CD32b), and with human Fc gamma receptors III FcyRIIIa / CD16 is measured by ELISA.

[0330] Briefly, Corning high-binding half-area plates (Fisher Scientific, Reinach, Switzerland) are coated overnight at 4°C with 25 pl of unmodified and of conjugated anti-PDl antibodies at 2.5 pg / ml in PBS. Plates are then washed four times with 100 pl of PBS-0.02% Tween20. Plates surfaces are blocked with 25 pl of PBS-0.02% Tween20-l% BSA at 37°C during Ih. Plates are then washed four times with 100 pl of PBS-0.02% Tween20. Then twenty-five microliters of either recombinant Human Fc gamma RI / CD64 Protein (R&D systems, 1257-FC-050, CF), recombinant Human Fc gamma RIIA / CD32a (H167) Protein (R&D systems, 9595-CD-050, CF), recombinant Human Fc gamma RIIB / CD32b Avi-tag Protein (R&D systems, AVI! 875-050, CF),Docket No. 56146-753.601or recombinant Human Fc gamma RIIIA / CD16a Protein (R&D systems, 4325-FC-050; CF) are added in five-fold serial dilutions ranging from 1000 nM to 0.001 nM into PBS-0.02% Tween20-0.1% BSA and incubated at 37°C during 2h. Plates are then washed four times with 100 pl of PBS-0.02% Tween20. Twenty-five microliters of a 1 / 500 HRP-anti-His antibody in PBS - 0.02% Tween20- 0.1%BSA (R&D systems, anti-HIS-HRP Ab, #MAB050H) ae added to each well and plates are incubated at Room Temperature during Ih. Plates are then washed four times with 100 pl of PBS-0.02% Tween20. Fifty microliters of TMB substrate reagent (#CL07, Merck, Buchs, Switzerland) are added to each well and incubated at 37°C during 5min. After 5min at 37°C, Horseradish peroxidase reaction is stopped by adding 50 pl / well of 0.5M H2SO4 stop solution. ELISA signal is then measured at 450 nm on an EnSpire plate reader from Perkin Elmer (Schwerzenbach, Switzerland).Human FcRn Binding Assay

[0331] The interaction of the unmodified and of conjugated anti-PDl antibodies with the human neonatal Fc receptor (FcRn) at pH 6 is measured using the AlphaLISA® Human FcRn Binding Kit (AL3095C) from Perkin Elmer (Schwerzenbach, Switzerland). The AlphaLISA® detection of FcRn and IgG binding uses IgG coated AlphaLISA® acceptor beads to interact with biotinylated human FcRn captured on Streptavidin-coated donor beads. When reference IgG binds to FcRn, donor and acceptor beads come into proximity enabling the transfer of singlet oxygen that trigger a cascade of energy transfer reactions in the acceptor beads, resulting in a sharp peak of light emission at 615 nm. Addition of a free IgG antibodies into the AlphaLISA® mixture creates a competition for the binding of FcRn to the reference antibody resulting in a loss of signal.

[0332] Briefly, test molecules arere measured in serial dilutions starting at 5uM down to 64 pM and incubated with AlphaLISA® reaction mixture consisting of 800 nM of recombinant biotinylated human FcRn, 40 pg / ml of human IgG conjugated Acceptor beads, and 40 pg / ml of Streptavidin coated Donor beads in pH 6 MES buffer. After 90min at 23 °C in the dark, AlphaLISA® signal is measured on an EnSpire plate reader (Excitation at 680 nm, Emission at 615 nm) from Perkin Elmer (Schwerzenbach, Switzerland).Example 7 - in vivo PK StudyProtocol for PK study in C57BL / 6 mice transgenic for huFcRn

[0333] Animals: Mice used were FcRn humanized C57BL / 6 mice, female, 6-8 weeks, weighing approximately 18-22 g purchased from Biocytogen Jiangsu Co., Ltd..Docket No. 56146-753.601

[0334] Table 20 shows groups and treatment for the PK study in C57BL / 6 mice transgenic for huFcRn.Table 20. Groups and treatment for the PK study in C57BL / 6 mice transgenic for huFcRn.

[0335] Table 21 shows Plasma sampling scheme for all groups:Table 21. Plasma sampling scheme for all groupsProtocol for BA Assay (ELISA) to detect bispecific molecules in mouse plasma

[0336] Plasma samples were assessed for Composition content by ELISA.

[0337] The following ELISA reagents were used: Coating Reagents: Human VEGF165 Protein, His tag, Aero Bio VE5-H5248; Detection Reagent: rH PD-1(CD279) biotinylated, fc tag R&D BT1086 #62; Buffers: Coating buffer: PBS - / -, Wash buffer: PBS-0.02% Tween20, Blocking buffer: PBS-0.02% Tween20 1% BSA, Protein diluent: PBS-0.02% Tween20 0.1% BSA, STOP solution: IM H2SO4.

[0338] The ELISA Procedure was as follows. High-binding ELISA plates were coated with 25 pL per well of human VEGF165 protein (Aero Biosystems, His-tag) diluted in PBS and followed by an overnight incubation at 4 °C. Plates were washed four times with PBS containing 0.02% Tween-20 and subsequently blocked with 50 pL per well of PBS supplemented with 1% BSA for 1 h at 37 °C. After blocking, plates were washed again four times with wash buffer. QualityDocket No. 56146-753.601controls, calibration standards, and samples were prepared in 10% matrix. 25 pL of each sample were added to the designated wells and incubated overnight at room temperature. Plates were then washed four times, and 25 pL of biotinylated recombinant human PD-1 (R&D Systems) was added to each well, followed by a 1 h incubation at room temperature. After washing, 25 pL of Streptavidin-HRP (1 :300 dilution in protein diluent) was added and incubated for 30 min at room temperature. Plates were washed four times before adding 25 pL of TMB substrate (Sigma) to each well and incubating for 5 min at room temperature. The reaction was stopped by adding 25 pL of 1 M H2SO4, and absorbance was measured at 450 nm using an EnSpire plate reader. Data for BA Assay (ELISA) to detect bispecific molecules in mouse plasma

[0339] FIG. 12 shows the mouse PK characterization of selected compositions. All tested bispecific molecules showed a favorable PK.Example 8 - Tumor Growth Inhibition (TGI) Study in MC38 tumor overexpressing VEGFA model in C57BL / 6 Mice transgenic for human PD-1 / PD-L1Protocol for TGI study in C57BL / 6 mice transgenic for human PD-1 / PDL1 bearing MC38 tumors overexpressing human VEGF- A

[0340] Experimental methods and procedures: The MC38 murine colon carcinoma cell line was purchased from Shunran Shanghai Biological Technology Co., Ltd. The MC38 cells were genetically modified to express human (h) VEGFA and named B-hVEGFA MC38 (clone#2-C05). Cells were maintained in vitro as monolayer culture in DMEM supplemented with 10% heat inactivated FBS at 37°C in a humidified atmosphere of 5% CO2.

[0341] Tumor inoculation and animal grouping: B-hPD-l / hPD-Ll mice were subcutaneously injected with B-hVEGFA MC38 (clone#2-C05) tumor cells (5* 105 / mouse) in 0.1 mL PBS in the right front flank for tumor development. When the average tumor volume reached approximately 80-120 mm3, mice were selected based on tumor volume and body weight and randomly enrolled into 12 study groups, with 8 mice in each group. On the grouping day (Day 0), treatment was initiated for all groups.

[0342] Tumor size was measured two times weekly in two dimensions using a caliper, and the volume is expressed in mm3using the formula: V = 0.5 a b2where a and b are the length and width of the tumor, respectively.

[0343] Table 22 shows the results of the TGI studyTable 22. Results of the TGI studyDocket No. 56146-753.601Data for TGI study in C57BL / 6 mice transgenic for human PD-1 / PDL1 bearing MC 38 tumors overexpressing human VEGF- A

[0344] FIG. 13 shows selected Bispecific molecules showed anti -tumor activity against MC38 tumors expressing human VEGFA.Example 9 - Tumor Growth Inhibition Study in SW620 CDX tumor model in BALB / c nude MiceProtocol for TGI study in Balb / C nude mice bearing SW620 CDX tumors

[0345] Cell Culture: The SW620 tumor cells (ATCC) were maintained in vitro as a monolayer culture in Leibovitz's L-15 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin and 100 pg / mL streptomycin at 37°C in a free gas exchange with atmospheric air. The tumor cells were routinely subcultured twice weekly. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation.

[0346] Animals: Mice used were BALB / c nude, female, 6-8 weeks, weighing approximately 18-22 g.

[0347] Tumor Inoculation: Each mouse was inoculated subcutaneously at the right upper flank with SW620 tumor cells (5x 106) in 0.1 mL of PBS for tumor development. The animals were randomized and treatment was started when the average tumor volume reaches approximately 100-150 mm3. Tumors were measured twice a week

[0348] Table 23 shows the protocol for the TGI study in Balb / C nude mice bearing SW620 CDX tumorsTable 23. Protocol for the TGI study in Balb / C nude mice bearing SW620 CDX tumorsDocket No. 56146-753.601Results of TGI study in Balb / C nude mice bearing SW620 CDX tumors

[0349] FIG. 14 shows the Results of TGI study in Balb / C nude mice bearing SW620 CDX tumors. The Bispecific molecule shows anti-tumor activity and increased survival in SW620 CDX bearing Balb / c nude mice for composition 59.

[0350] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the disclosure as defined in the appended claims.

Claims

1. Docket No. 56146-753.601CLAIMS WHAT IS CLAIMED IS:

1. A dual binding composition, comprising:a) a first binding domain targeting programmed cell death protein 1 (PD-1); b) a second binding domain targeting vascular endothelial growth factor A (VEGFA);wherein the first binding domain comprises a VHH having a VH CDR1, VH CDR2, and VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 each have sequences respectively selected from:510, 511, and 512; SEQ IDNOs: 502, 503, and 504; SEQ ID NOs: 506, 507, and 508; SEQ ID NOs: SEQ IDNOs: 514, 515, and 516; SEQ IDNOs: 518, 511, and 520; SEQ ID NOs: 522, 523, and 524; SEQ ID NOs: 526, 527, and 528; SEQ ID NOs: 530, 531, and 532; SEQ ID NOs: 534, 535, and 536; SEQ ID NOs: 538, 539, and 540; SEQ ID NOs: 542, 543, and 544; SEQ ID NOs: 546, 547, and 548; SEQ ID NOs: 550, 511, and 552; SEQ ID NOs: 554, 555, and 556; SEQ ID NOs: 558, 543, and 560; SEQ ID NOs: 562, 563, and 564; SEQ ID NOs: 566, 567, and 568; SEQ ID NOs: 570, 511, and 572; SEQ ID NOs: 574, 567, and 576; SEQ IDNOs: 578, 579, and 580; SEQ ID NOs: 582, 555, and 584; SEQ ID NOs: 586, 587, and 588; SEQ ID NOs: 590, 591, and 592; SEQ ID NOs: 594, 555, and 596; SEQ ID NOs: 578, 599, and 600; SEQ ID NOs: 602, 511, and 604; SEQ ID NOs: 606, 567, and 608; SEQ ID NOs: 610, 591, and 612; SEQ ID NOs: 614, 615, and 616; SEQ ID NOs: 618, 567, and 620; SEQ ID NOs: 622, 567, and 624; SEQ ID NOs: 626, 511, and 628; SEQ ID NOs: 630, 563, and 632; SEQ ID NOs: 634, 599, and 636; SEQ ID NOs: 638, 555, and 640; SEQ ID NOs: 642, 567, and 644; SEQ ID NOs: 646, 511, and 648; SEQ ID NOs: 650, 515, and 652; SEQ ID NOs: 654, 547, and 656; SEQ ID NOs: 658, 659, and 660; SEQ ID NOs: 662, 663, and 664; SEQ ID NOs: 421, 591, and 668; SEQ ID NOs: 670, 591, and 672; SEQ ID NOs: 674, 675, and 676; SEQ ID NOs: 678, 679, and 680; SEQ ID NOs: 682, 599, and 684; SEQ ID NOs: 686, 547, and 688; SEQ ID NOs: 690, 691, and 692; and SEQ IDNOs: 510, 699, and 512.

2. The composition of claim 1, wherein the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 509, 501, 505, 513, 517, 521, 525, 529, 533, 537, 541, 545, 549, 553, 557, 561, 565, 569, 573, 577, 581, 585, 589, 593, 597, 601, 605, 609, 613, 617, 621, 625, 629, 633, 637, 641, 645, 649, 653, 657, 661, 665, 669, 673, 677, 681, 685, 689, 693, 694, 695, 696, 697, and 698.Docket No. 56146-753.6013. The composition of claim 1 or 2, wherein the VHH comprises the amino acid sequence set forth in any one of SEQ ID NOs: 509, 501, 505, 513, 517, 521, 525, 529, 533, 537, 541, 545, 549, 553, 557, 561, 565, 569, 573, 577, 581, 585, 589, 593, 597, 601, 605, 609, 613, 617, 621, 625, 629, 633, 637, 641, 645, 649, 653, 657, 661, 665, 669, 673, 677, 681, 685, 689, 693, 694, 695, 696, 697, and 698.

4. The composition of claim 1, wherein the VH CDR1, VH CDR2, and VH CDR3 each have sequences respectively selected from: 510, 511, and 512; 570, 511, and 572; 602, 511, and 604; 626, 511, and 628; 658, 659, and 660; and 510, 699, and 512.

5. The composition of claim 4, wherein the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 509, 569, 601, 625, 657, 693, 694, 695, 696, 697, and 698.

6. The composition of claim 4, wherein the VHH comprises the amino acid sequence set forth in any one of SEQ ID NOs: 509, 569, 601, 625, 657, 693, 694, 695, 696, 697, and 698.

7. The composition of claim 1, wherein the VH CDR1 has a sequence of SEQ ID NO: 510, the VH CDR2 has a sequence of SEQ ID NO: 511, and the VH CDR3 has a sequence of SEQ ID NO: 512.

8. The composition of claim 7, wherein the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 509, 693, 694, 695, 696, and 698.

9. The composition of claim 7, wherein the VHH comprises the amino acid sequence set forth in any one of SEQ ID NOs: 509, 693, 694, 695, 696, and 698.

10. The composition of claim 7, wherein the VHH comprises the amino acid sequence of SEQ ID NO: 509.

11. The composition of claim 7, wherein the VHH comprises the amino acid sequence of SEQ ID NO: 693.

12. The composition of claim 7, wherein the VHH comprises the amino acid sequence of SEQ ID NO: 694.

13. The composition of claim 7, wherein the VHH comprises the amino acid sequence of SEQ ID NO: 695.

14. The composition of claim 7, wherein the VHH comprises the amino acid sequence of SEQ ID NO: 696.Docket No. 56146-753.60115. The composition of claim 7, wherein the VHH comprises the amino acid sequence of SEQ ID NO: 698.

16. The composition of claim 1, wherein the VH CDR1 has a sequence of SEQ ID NO: 570, the VH CDR2 has a sequence of SEQ ID NO: 511, and the VH CDR3 has a sequence of SEQ ID NO: 572.

17. The composition of claim 16, wherein the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

569.

18. The composition of claim 16, wherein the VHH comprises the amino acid sequence set forth in SEQ ID NO: 569.

19. The composition of claim 1, wherein the VH CDR1 has a sequence of SEQ ID NO: 602, the VH CDR2 has a sequence of SEQ ID NO: 511, and the VH CDR3 has a sequence of SEQ ID NO: 604.

20. The composition of claim 19, wherein the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

601.

21. The composition of claim 19, wherein the VHH comprises the amino acid sequence set forth in SEQ ID NO: 601.

22. The composition of claim 1, wherein the VH CDR1 has a sequence of SEQ ID NO:626, the VH CDR2 has a sequence of SEQ ID NO: 511, and the VH CDR3 has a sequence of SEQ ID NO: 628.

23. The composition of claim 22, wherein the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

625.

24. The composition of claim 22, wherein the VHH comprises the amino acid sequence set forth in SEQ ID NO: 625.

25. The composition of claim 1, wherein the VH CDR1 has a sequence of SEQ ID NO: 658, the VH CDR2 has a sequence of SEQ ID NO: 659, and the VH CDR3 has a sequence of SEQ ID NO: 660.

26. The composition of claim 25, wherein the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

657.

27. The composition of claim 25, wherein the VHH comprises the amino acid sequence set forth in SEQ ID NO: 657.

28. The composition of claim 1, wherein the VH CDR1 has a sequence of SEQ ID NO: 510, the VH CDR2 has a sequence of SEQ ID NO: 699, and the VH CDR3 has a sequence of SEQ ID NO: 512.Docket No. 56146-753.60129. The composition of claim 28, wherein the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

569.

30. The composition of claim 28, wherein the VHH comprises the amino acid sequence set forth in SEQ ID NO: 569.

31. The composition of claims 1-30, wherein the first binding domain targeting programmed cell death protein 1 (PD-1) is capable of disrupting the interaction of PD-1 with programmed cell death ligand 1 (PD-L1).

32. The composition of any one of claims 1-31, wherein the second binding domain targeting VEGFA is capable of disrupting the interaction of VEGFA with one or more of its receptors.

33. The composition of any one of claims 1-32, wherein the second binding domain is comprised in an antigen binding fragment derived from an antibody.

34. The composition of any one of claims 1-33, wherein the second binding domain comprises a heavy chain variable domain (VH) comprising a heavy chain first complementary determining region (VH CDR1), a heavy chain second complementary determining region (VH CDR2), and a heavy chain third complementary determining region (VH CDR3).

35. The composition of claim 34, wherein the VH is comprised in a Fab, a Fab’, F(ab')2, a bispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), a bispecific scFv, disulfide stabilized Fv (dsFv), a minibody, a diabody, a bispecific diabody, triabody, a tetrabody, a maxibody, a camelid, a VHH, a single domain light chain antibody, a Fab-Fc, a scFv-Fc, or a bispecific antibody.

36. The composition of claim 34 or 35, wherein the VH comprises a set of VH CDR1, VH CDR2, and VH CDR3 derived from an antibody in Table 2A or Table 2B.

37. The composition of any one of claims 34-36, wherein the VH comprise an amino acid sequence of a VH set forth in Table 2A or Table 2B.

38. The composition of any one of claims 34-37, wherein the VH comprises a VH CDR1 having a sequence GYTFTNYGMN (SEQ ID NO: 123), a VH CDR2 having a sequence WINTYTGEPTYAADFK (SEQ ID NO: 124), and a VH CDR3 having a sequence YPHYYGSSHWYFDV (SEQ ID NO: 125).

39. The composition of any one of claims 37-38, wherein the VH comprises a VH having a sequenceEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINDocket No. 56146-753.601TYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSS HWYFDVWGQGTLVTV (SEQ ID NO: 122).

40. The composition of any one of claims 1-39, wherein the second binding domain comprises a light chain variable domain (VL) comprising a light chain first complementary determining region (VL CDR1), a light chain second complementary determining region (VL CDR2), and a light chain third complementary determining region (VL CDR3).

41. The composition of claim 40, wherein the VL is comprised in the Fab, Fab’, F(ab')2, bispecific F(ab')2, variable fragment (Fv), single chain variable fragment (scFv), bispecific scFv, disulfide stabilized Fv (dsFv), minibody, diabody, bispecific diabody, triabody, tetrabody, maxibody, Fab-Fc, scFv-Fc, or bispecific antibody in which the VH of the second binding domain is comprised.

42. The composition of claim 40 or 41, wherein the VL comprises a set of VL CDR1, VL CDR2, and VL CDR3 derived from an antibody in Table 2 A.

43. The composition of any one of claims 39-42, wherein the VL comprises a VL CDR1 having a sequence SASQDISNYLN (SEQ ID NO: 128), a VL CDR2 having a sequence FTSSLHS (SEQ ID NO: 129), and a VL CDR3 having a sequence QQYSTVPWT (SEQ ID NO: 130).

44. The composition of any one of claims 39-43, wherein the VL comprises a sequence DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK (SEQ ID NO: 127).

45. The composition of any one of claims 1-44, wherein the second binding domain comprises a VH having a VH CDR1 having a sequence GYTFTNYGMN (SEQ ID NO: 123), a VH CDR2 having a sequence WINTYTGEPTYAADFK (SEQ ID NO: 124), and a VH CDR3 having a sequence YPHYYGSSHWYFDV (SEQ ID NO: 125), and a VL having a VL CDR1 having a sequence SASQDISNYLN (SEQ ID NO: 128), a VL CDR2 having a sequence FTSSLHS (SEQ ID NO: 129), and a VL CDR3 having a sequence QQYSTVPWT (SEQ ID NO: 130).

46. The composition of any one of claims 1-45, wherein the second binding domain comprises a VH having a sequence EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWIN TYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSDocket No. 56146-753.601HWYFDVWGQGTLVTV (SEQ ID NO: 122) and a VL having a sequence DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK (SEQ ID NO: 127).

47. The composition of any one of claims 1-46, wherein the second binding domain is an scFv.

48. The composition of any one of claims 1-47, wherein the second binding domain is a Fab.

49. The composition of any one of claims 1-33, wherein the second binding domain is a single domain antibody.

50. The composition of claim 49, wherein the second binding domain is a single domain heavy chain antibody (VHH).

51. The composition of claim 49 or 50, wherein the second binding domain comprises:a) a VH CDR1 sequence of AYPMM (SEQ ID NO: 202), a VH CDR2 sequence of EISPSGSYTYYADSVRG (SEQ ID NO: 203), and a VH CDR3 sequence of DPRKLDY (SEQ ID NO: 204);b) a VH CDR1 sequence of LYDMM (SEQ ID NO: 206), a VH CDR2 sequence of FIGGDGLNTYYADSVKG (SEQ ID NO: 207), and a VH CDR3 sequence of AGTQFDY (SEQ ID NO: 208);c) a VH CDR1 sequence of WYPMW (SEQ ID NO: 210), a VH CDR2 sequence of LIEGQGDRTYYADSVKG (SEQ ID NO: 211), and a VH CDR3 sequence of AGDRTAGSRGNSFDY (SEQ ID NO: 212);d) a VH CDR1 sequence of AYPMM (SEQ ID NO: 202), a VH CDR2 sequence of EISPSGSYTYYADSVKG (SEQ ID NO: 215), and a VH CDR3 sequence of DPRKFDY (SEQ ID NO: 216); ore) a VH CDR1 having the sequence SYSMG (SEQ ID NO: 218), a VH CDR2 having the sequence AISKGGYKYDAVSLEG (SEQ ID NO: 219) or the sequence of SEQ ID NO. 279, and a VH CDR3 having the sequence SRAYGSSRLRLADTYEY (SEQ ID NO: 220).

52. The composition of any one of claims 49-51, wherein the second binding domain comprises a sequence selected from:a)EVQLLVSGGGLVDPGGSLRLSCAASGFTFKAYPMMWVRQAPGKGLEWVDocket No. 56146-753.601SEISPSGSYTYYADSVRGRFFISRDNSKNTLYLQMNSLRAEDTAVYYCAK DPRKLDYWGQGTLWTVSS (SEQ ID NO: 201);b) EVQLLESGGGLVQPGGSLRLSCAASGFTFHLYDMMWVRQAPGKGLEWV SFIGGDGLNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA KAGTQFDYWGQGTLVTVSS (SEQ ID NO: 205);c) EVQLLESGGGLVQPGGSLRLSCAASGFTFQWYPMWWVRQAPGKGLEW VSLIEGQGDRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC AKAGDRTAGSRGNSFDYWGQGTLVTVSS (SEQ ID NO: 209); d) EVQLLESGGGLVQPGGSLRLSCAASGFTFGAYPMMWVRQAPGKGLEWV SEISPSGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DPRKFDYWGQGTLVTVSS (SEQ ID NO: 213);e)D VQLVESGGGLVQPGGSLRLSC AASGRTF S S YSMGWFRQ APGKEREF VV AISKGGYKYDAVSLEGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCASS RAYGSSRLRLADTYEYWGQGTLVTVSS (SEQ ID NO: 217);f)D VQLVESGGGLVQPGGSLRLSC AASGRTF S S YSMGWFRQ APGKEREF VV AISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSR AYGSSRLRLADTYEYWGQGTLVTVSS (SEQ ID NO: 221);g)D VQLVESGGGLVQPGGSLRLSC AASGRTF S S YSMGWFRQ APGKEREF VV AISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSR AYGSSRLRLADTYEYWGQGTLVTVSSPP (SEQ ID NO: 200); or h) any one of SEQ ID NOs: 280-284.

53. The composition of claim 49, wherein the second binding domain comprises a light chain single domain antibody.

54. The composition of claim 49 or 53, wherein the second binding domain comprises a VL CDR1 having the sequence RASQWIGPELS (SEQ ID NO: 223), a VL CDR2 having the sequence HTSILQS (SEQ ID NO: 224), and a VL CDR3 having the sequence QQYMFQPRT (SEQ ID NO: 225).Docket No. 56146-753.60155. The composition of any one of claims 49, 53, or 54, wherein the second binding domain comprises the sequence DIQMTQSPSSLSASVGDRVTITCRASQWIGPELSWYQQKPGKAPKLLIYHTSILQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYMFQPRTFGQGTKVEIRR (SEQ ID NO: 222).

56. The composition of any one of claims 1-32, wherein the second binding domain is an anti-VEGFA anticalin.

57. The composition of any one of claims 1-32 or 56, wherein the second binding domain comprises the sequence DGGGIRRSMSGTWYLKAMTVDREFPEMNLESVTPMTLTLLKGHNLEAKVTMLI SGRCQEVKAVLGRTKERKKYTADGGKHVAYIIPSAVRDHVIFYSEGQLHGKPVR GVKLVGRDPKNNLEALEDFEKAAGRLSTESILIPRQSETCSPG (SEQ ID NO: 226).

58. A dual binding composition, comprising:a) a first binding domain targeting programmed cell death protein 1 (PD-1); b) a second binding domain targeting vascular endothelial growth factor A (VEGFA);wherein the second binding domain comprises a VHH having a VH CDR1, VH CDR2, and VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 each have sequences respectively selected from:SEQ ID NOs: 301, 302, and 303; SEQ ID NOs: 305, 306, and 307; SEQ ID NOs: 309, 310, and 311; SEQ ID NOs: 313, 314, and 315; SEQ ID NOs: 317, 318, and 319; SEQ ID NOs: 321, 322, and 323; SEQ ID NOs: 325, 326, and 327; SEQ ID NOs: 329, 330, and 331; SEQ ID NOs: 333, 334, and 335; SEQ ID NOs: 337, 338, and 339; SEQ ID NOs: 341, 342, and 343; SEQ ID NOs: 345, 346, and 347; SEQ ID NOs: 349, 350, and 351; SEQ ID NOs: 353, 354, and 355; SEQ ID NOs: 357, 358, and 359; SEQ ID NOs: 361, 362, and 363; SEQ ID NOs: 365, 366, and 367; SEQ ID NOs: 369, 370, and 371; SEQ ID NOs: 373, 374, and 375; SEQ ID NOs: 377, 378, and 379; SEQ ID NOs: 381, 382, and 383; SEQ ID NOs: 385, 386, and 387; SEQ ID NOs: 389, 390, and 391; SEQ ID NOs: 393, 394, and 395; SEQ ID NOs: 397, 398, and 399; SEQ ID NOs: 401, 402, and 403; SEQ ID NOs: 405, 406, and 407; SEQ ID NOs: 409, 410, and 411; SEQ ID NOs: 413, 414, and 415; SEQ ID NOs: 417, 418, and 419; SEQ ID NOs: 421, 422, and 423; SEQ ID NOs: 425, 426, and 427; SEQ ID NOs: 429, 430, and 431; SEQ ID NOs: 433, 434, and 435; SEQ ID NOs: 437, 438, and 439; SEQ ID NOs: 441, 442, and 443;Docket No. 56146-753.601SEQ ID NOs: 445, 398, and 447; SEQ ID NOs: 449, 426, and 451; SEQ ID NOs: 453, 454, and 455; SEQ ID NOs: 457, 458, and 459; SEQ ID NOs: 461, 462, and 463; SEQ ID NOs: 465, 466, and 467; SEQ ID NOs: 469, 470, and 471; SEQ ID NOs: 473, 474, and 475; SEQ ID NOs: 477, 478, and 479; SEQ ID NOs: 481, 482, and 483; SEQ ID NOs: 485, 486, and 487; and SEQ ID NOs: 489, 490, and 491.

59. The composition of claim 58, wherein the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 300, 304, 308, 312, 316, 320, 324, 328, 332, 336, 340, 344, 348, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388, 392, 396, 400, 404, 408, 412, 416, 420, 424, 428, 432, 436, 440, 444, 448, 452, 456, 460, 464, 468, 472, 476, 480, 484, and 488.

60. The composition of claim 58 or 59, wherein the VHH comprises the amino acid sequence set forth in any one of SEQ ID NOs: 300, 304, 308, 312, 316, 320, 324, 328, 332, 336, 340, 344, 348, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388, 392, 396, 400, 404, 408, 412, 416, 420, 424, 428, 432, 436, 440, 444, 448, 452, 456, 460, 464, 468, 472, 476, 480, 484, and 488.

61. The composition of any one of claims 58-60, wherein the second binding domain targeting VEGFA is capable of disrupting the interaction of VEGFA with one or more of its receptors.

62. The composition of claim any one of claims 58-61, wherein the first binding domain is comprised in an antigen binding fragment derived from an antibody.

63. The composition of any one of claims 58-62, wherein the first binding domain comprises a heavy chain variable domain (VH) comprising a heavy chain first complementary determining region (VH CDR1), a heavy chain second complementary determining region (VH CDR2), and a heavy chain third complementary determining region (VH CDR3).

64. The composition of claim 63, wherein the VH is comprised in a Fab, a Fab’, F(ab')2, a bispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), a bispecific scFv, disulfide stabilized Fv (dsFv), a minibody, a diabody, a bispecific diabody, triabody, a tetrabody, a maxibody, a camelid, a VHH, a Fab-Fc, a scFv-Fc, or a bispecific antibody.

65. The composition of claim 63 or 64, wherein the VH comprises a set of VH CDR1, VH CDR2, and VH CDR3 derived from an antibody in Table 1 A or Table IB.Docket No. 56146-753.60166. The composition of any one of claims 63-65, wherein the VH comprise an amino acid sequence of a VH set forth in Table 1 A or Table IB.

67. The composition of any one of claims 63-66, wherein the first binding domain is a VHH.

68. The composition of claim 67, wherein the first binding domain comprises:a) a VH CDR1 sequence of SEQ ID NO: 2; a VH CDR2 sequence of SEQ ID NO: 3; and a VH CDR3 sequence of SEQ ID NO: 4;b) a VH CDR1 sequence of SEQ ID NO: 6; a VH CDR2 sequence of SEQ ID NO: 7; and a VH CDR3 sequence of SEQ ID NO: 8;c) a VH CDR1 sequence of SEQ ID NO: 14; a VH CDR2 sequence of SEQ ID NO: 15; and a VH CDR3 sequence of CDR3 SEQ ID NO: 16;d) a VH CDR1 sequence of SEQ ID NO: 18; a VH CDR2 sequence of SEQ ID NO: 19; and a VH CDR3 sequence of CDR 3 of SEQ ID NO: 20; ore) a VH CDR1 sequence of SEQ ID NO: 288, a VH CDR2 sequence of SEQ ID NO: 289, and a VH CDR3 sequence of SEQ ID NO: 290.

69. The composition of claim 68, wherein the VHH comprises an amino acid sequence selected from:a) SEQ ID NO: 1;b) SEQ ID NO: 5;c) SEQ ID NO: 13;d) SEQ ID NO: 17; ande) SEQ ID NO: 287.

70. The composition of any one of claims 63-66, wherein the VH comprises:a) a VH CDR1 sequence of NYYMY (SEQ ID NO: 80), a VH CDR2 sequence of GINPSNGGTNFNEKFKN (SEQ ID NO: 81), and a VH CDR3 sequence of RDYRFDMGFDY (SEQ ID NO: 82);b) a VH CDR1 sequence of NSGMH (SEQ ID NO:86), a VH CDR2 sequence of VIWYDGSKRYYADSVKG (SEQ ID NO: 87), and a VH CDR3 sequence of NDDY (SEQ ID NO: 88); orc) a VH CDR1 sequence of GYTFTSYYMY (SEQ ID NO: 113), a VH CDR2 sequence of GVNPSNGGTNFNEKFKS (SEQ ID NO: 114), and a VH CDR3 sequence of RDYRYDMGFDY (SEQ ID NO: 115).

71. The composition of any one of claims 63-66 or 70, wherein the VH comprises:Docket No. 56146-753.601a) a VH having a sequence QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEW MGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYC ARRDYRFDMGFDYWGQGTTVTV (SEQ ID NO: 48);b) a VH having a sequence QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWV AVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCA TNDDYWGQGTLVTVSS (SEQ ID NO: 50); orc) a VH having a sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMYWVRQAPGQGLEW MGGVNPSNGGTNFNEKFKSRVTITADKSTSTAYMELSSLRSEDTAVYYC ARRDYRYDMGFDYWGQGTTVTVSS (SEQ ID NO: 76).

72. The composition of any one of claims 58-66, 70 or 71, wherein the first binding domain comprises a light chain variable domain (VL) comprising a light chain first complementary determining region (VL CDR1), a light chain second complementary determining region (VL CDR2), and a light chain third complementary determining region (VL CDR3).

73. The composition of claim 72, wherein the first binding domain comprises a light chain variable domain (VL) comprising a light chain first complementary determining region (VL CDR1), a light chain second complementary determining region (VL CDR2), and a light chain third complementary determining region (VL CDR3).

74. The composition of claim 72 or 73, wherein the VL comprises a set of VL CDR1, VL CDR2, and VL CDR3 derived from an antibody in Table 1 A.

75. The composition of claim 15, wherein the VL comprisesa) a VL CDR1 sequence of RASKGVSTSGYSYLH (SEQ ID NO: 83), a VL CDR2 sequence of LASYLES (SEQ ID NO: 84), and a VL CDR3 sequence of QHSRDLPLT (SEQ ID NO: 85);b) a VL CDR1 sequence of RASQSVSSYLA (SEQ ID NO: 89), a VL CDR2 sequence of DASNRAT (SEQ ID NO: 90), and a VL CDR3 sequence of QQSSNWPRT (SEQ ID NO: 91); orc) a VL CDR1 sequence of RASKGVSTSGYSYLH (SEQ ID NO: 83), a VL CDR2 sequence of LASYLE (SEQ ID NO: 117), and a VL CDR3 sequence of QHSRELPLT (SEQ ID NO: 118).Docket No. 56146-753.60176. The composition of claim 75, wherein the VL comprises:a) a VL having a sequence EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRL LIYLAS YLESGVPARF SGSGSGTDFTLTIS SLEPEDF AVYYCQHSRDLPLTF GGGTKVEIK (SEQ ID NO: 49);b) a VL having a sequence EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDA SNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGT KVEIK (SEQ ID NO: 51); orc) a VL having a sequence EIVLTQSPATLSLSPGERATISCRASKGVSTSGYSYLHWYQQKPGQAPRLL IYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFATYYCQHSRELPLTFG TGTKVEIK (SEQ ID NO: 77).

77. The composition of any one of claims 58-66 or 70-76, wherein the first binding domain comprises:a) a VH having a VH CDR1 sequence of NYYMY (SEQ ID NO: 80), a VH CDR2 sequence of GINPSNGGTNFNEKFKN (SEQ ID NO: 81), and a VH CDR3 sequence of RDYRFDMGFDY (SEQ ID NO: 82), and a VL having a VL CDR1 sequence of RASKGVSTSGYSYLH (SEQ ID NO: 83), a VL CDR2 sequence of LASYLES (SEQ ID NO: 84), and a VL CDR3 sequence of QHSRDLPLT (SEQ ID NO: 85); orb) a VH having a VH CDR1 sequence of NSGMH (SEQ ID NO: 86), a VH CDR2 sequence of VIWYDGSKRYYADSVKG (SEQ ID NO: 87), and a VH CDR3 sequence of NDDY (SEQ ID NO: 88), and a VL having a VL CDR1 sequence of RASQSVSSYLA (SEQ ID NO: 89), a VL CDR2 sequence of DASNRAT (SEQ ID NO: 90), and a VL CDR3 sequence of QQSSNWPRT (SEQ ID NO: 91; orc) a VH having a VH CDR1 sequence of GYTFTSYYMY (SEQ ID NO: 113), a VH CDR2 sequence of GVNPSNGGTNFNEKFKS (SEQ ID NO: 114), and a VH CDR3 sequence of RDYRYDMGFDY (SEQ ID NO: 115), and a VL having a VL CDR1 sequence of RASKGVSTSGYSYLH (SEQ ID NO: 83), a VL CDR2 sequence of LASYLE (SEQ ID NO: 117), and a VL CDR3 sequence of QHSRELPLT (SEQ ID NO: 118).Docket No. 56146-753.60178. The composition of any one of claims 58-66 or 70-77, wherein the first binding domain comprises:a) a VH having a sequence QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEW MGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYC ARRDYRFDMGFDYWGQGTTVTV (SEQ ID NO: 48) and a VL having a sequence EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRL LIYLAS YLESGVPARF SGSGSGTDFTLTIS SLEPEDF AVYYCQHSRDLPLTF GGGTKVEIK (SEQ ID NO: 49);b) a VH having a sequence QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWV AVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCA TNDDYWGQGTLVTVSS (SEQ ID NO: 50) and a VL having a sequence EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDA SNRATGIPARF SGSGSGTDFTLTIS SLEPEDF AVYYCQQS SNWPRTFGQGT KVEIK (SEQ ID NO: 51); orc) a VH having a sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMYWVRQAPGQGLEW MGGVNPSNGGTNFNEKFKSRVTITADKSTSTAYMELSSLRSEDTAVYYC ARRDYRYDMGFDYWGQGTTVTVSS (SEQ ID NO: 76) and a VL having a sequence EIVLTQSPATLSLSPGERATISCRASKGVSTSGYSYLHWYQQKPGQAPRLL IYL AS YLESGVPARF SGSGSGTDFTLTIS SLEPEDF ATYYCQHSRELPLTFG TGTKVEIK (SEQ ID NO: 77).

79. The composition of any one of claims 58-66 or 70-78 wherein the first binding domain is an scFv.

80. The composition of any one of claims 58-66 or 70-79, wherein the first binding domain is a Fab.

81. The composition of any one of claims 1-80, wherein the composition comprises an Fc domain comprising first CH2 and CH3 domains on a first polypeptide chain and second CH2 and CH3 domains on a second polypeptide chain.

82. The composition of claim 81, wherein the Fc domain is derived from an IgG.Docket No. 56146-753.60183. The composition of claim 81, wherein the Fc domain is derived from an IgGl or IgG4.

84. The composition of any one of claims 81-83, wherein the composition comprises a structure of the formula:wherein:Y is the first CH2 and CH3 domains;Y’ is the second CH2 and CH3 domains;X and X’ are each independently the first binding domain, the second binding domain, a copy of the first binding domain, a copy of the second binding domain, or absent; Z and Z’ are each independently the first binding domain, the second binding domain, a copy of the first binding domain, a copy of the second binding domain, a third binding domain targeting PD-1, or a fourth binding domain targeting VEGFA, or absent;wherein X, Y, and Z and X’, Y’ and Z’ are depicted in an N-terminal to C-terminal direction; andwherein each of X, X’, Z, and Z’ are independently and optionally connected to Y or Y’ via a peptide linker.

85. The composition of claim 84, wherein X is the first binding domain; X’ is a copy of the first binding domain or absent; and one of Z or Z’ is the second binding domain and the other is absent or a copy of the second binding domain.

86. The composition of claim 85, wherein one of Z or Z’ is the second binding domain and the other is a copy of the second binding domain.

87. The composition of claim 84, wherein X is the second binding domain; X’ is a copy of the second binding domain or absent; and one of Z or Z’ is the first binding domain and the other is absent or a copy of the first binding domain.

88. The composition of claim 87, wherein one of Z or Z’ is the first binding domain and the other is a copy of the first binding domain.

89. The composition of claim 84, wherein:X is the first binding domain and is a Fab, X’ is a copy of the first binding domain, Z is the second binding domain and is an scFv, and Z’ is a copy of the second binding domain;Docket No. 56146-753.601X is the first binding domain and is a Fab, X’ is a copy of the first binding domain; Z is the second binding domain and is a single domain antibody, and Z’ is a copy of the second binding domain;X is the first binding domain and is a Fab, X’ is absent, Z is the second binding domain and is an scFv, and Z’ is a copy of the second binding domain;X is the first binding domain and is a Fab, X’ is absent, Z is the second binding domain and is a single domain antibody, and Z’ is a copy of the second binding domain;X is the first binding domain and is a Fab, X’ is the second biding domain and is a Fab, and Z and Z’ are absent;X is the first binding domain and is a Fab, X’ is the second binding domain and is an scFv, and Z and Z’ are absent;X is the first binding domain and is an scFv, X’ is the second binding domain and is a Fab, and Z and Z’ are absent;X is the second binding domain and is a Fab, X’ is a copy of the second binding domain, Z is the first binding domain and is an scFv, and Z’ is a copy of the first binding domain;X is the second binding domain and is a Fab, X’ is a copy of the second binding domain; Z is the first binding domain and is a single domain antibody, and Z’ is a copy of the first binding domain;X is the second binding domain and is a Fab, X’ is absent, Z is the first binding domain and is an scFv, and Z’ is a copy of the first binding domain;X is the second binding domain and is a Fab, X’ is absent, Z is the first binding domain and is a single domain antibody, and Z’ is a copy of the first binding domain;X is the second binding domain and is a Fab, X’ is the first binding domain and is a Fab, Z is a fourth binding domain targeting VEGFA and is an scFv, and Z’ is absent; orX is the second binding domain and is a Fab, X’ is the first binding domain and is a Fab, Z is a fourth binding domain targeting VEGFA and is a single domain antibody, and Z’ is absent.Docket No. 56146-753.60190. The composition of any one of claims 81-89, wherein the Fc domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in any one of SEQ ID NOs: 229-234 or 236-241.

91. A composition, comprising a binding domain targeting programmed cell death protein 1 (PD-1); wherein the first binding domain comprises a VHH having a VH CDR1, VH CDR2, and VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 each have sequences respectively selected from:510, 511, and 512; SEQ ID NOs: 502, 503, and 504; SEQ ID NOs: 506, 507, and 508; SEQ ID NOs: SEQ ID NOs: 514, 515, and 516; SEQ ID NOs: 518, 511, and 520; SEQ ID NOs: 522, 523, and 524; SEQ ID NOs: 526, 527, and 528; SEQ ID NOs: 530, 531, and 532; SEQ ID NOs: 534, 535, and 536; SEQ ID NOs: 538, 539, and 540; SEQ ID NOs: 542, 543, and 544; SEQ ID NOs: 546, 547, and 548; SEQ ID NOs: 550, 511, and 552; SEQ ID NOs: 554, 555, and 556; SEQ ID NOs: 558, 543, and 560; SEQ ID NOs: 562, 563, and 564; SEQ ID NOs: 566, 567, and 568; SEQ ID NOs: 570, 511, and 572; SEQ ID NOs: 574, 567, and 576; SEQ ID NOs: 578, 579, and 580; SEQ ID NOs: 582, 555, and 584; SEQ ID NOs: 586, 587, and 588; SEQ ID NOs: 590, 591, and 592; SEQ ID NOs: 594, 555, and 596; SEQ ID NOs: 578, 599, and 600; SEQ ID NOs: 602, 511, and 604; SEQ ID NOs: 606, 567, and 608; SEQ ID NOs: 610, 591, and 612; SEQ ID NOs: 614, 615, and 616; SEQ ID NOs: 618, 567, and 620; SEQ ID NOs: 622, 567, and 624; SEQ ID NOs: 626, 511, and 628; SEQ ID NOs: 630, 563, and 632; SEQ ID NOs: 634, 599, and 636; SEQ ID NOs: 638, 555, and 640; SEQ ID NOs: 642, 567, and 644; SEQ ID NOs: 646, 511, and 648; SEQ ID NOs: 650, 515, and 652; SEQ ID NOs: 654, 547, and 656; SEQ ID NOs: 658, 659, and 660; SEQ ID NOs: 662, 663, and 664; SEQ ID NOs: 421, 591, and 668; SEQ ID NOs: 670, 591, and 672; SEQ ID NOs: 674, 675, and 676; SEQ ID NOs: 678, 679, and 680; SEQ ID NOs: 682, 599, and 684; SEQ ID NOs: 686, 547, and 688; SEQ ID NOs: 690, 691, and 692; and SEQ ID NOs: 510, 699, and 512.

92. The composition of claim 91, wherein the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 509, 501, 505, 513, 517, 521, 525, 529, 533, 537, 541, 545, 549, 553, 557, 561, 565, 569, 573, 577, 581, 585, 589, 593, 597, 601, 605, 609, 613, 617, 621, 625, 629, 633, 637, 641, 645, 649, 653, 657, 661, 665, 669, 673, 677, 681, 685, 689, 693, 694, 695, 696, 697, and 698.

93. The composition of claim 91 or 92, wherein the VHH comprises the amino acid sequence set forth in any one of SEQ ID NOs: 509, 501, 505, 513, 517, 521, 525, 529,Docket No. 56146-753.601533, 537, 541, 545, 549, 553, 557, 561, 565, 569, 573, 577, 581, 585, 589, 593, 597, 601, 605, 609, 613, 617, 621, 625, 629, 633, 637, 641, 645, 649, 653, 657, 661, 665, 669, 673, 677, 681, 685, 689, 693, 694, 695, 696, 697, and 698.

94. The composition of claim 91, wherein the VH CDR1, VH CDR2, and VH CDR3 each have sequences respectively selected from: 510, 511, and 512; 570, 511, and 572; 602, 511, and 604; 626, 511, and 628; 658, 659, and 660; and 510, 699, and 512.

95. The composition of claim 94, wherein the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 509, 569, 601, 625, 657, 693, 694, 695, 696, 697, and 698.

96. The composition of claim 94, wherein the VHH comprises the amino acid sequence set forth in any one of SEQ ID NOs: 509, 569, 601, 625, 657, 693, 694, 695, 696, 697, and 698.

97. The composition of claim 91, wherein the VH CDR1 has a sequence of SEQ ID NO:510, the VH CDR2 has a sequence of SEQ ID NO: 511, and the VH CDR3 has a sequence of SEQ ID NO: 512.

98. The composition of claim 97, wherein the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 509, 693, 694, 695, 696, and 698.

99. The composition of claim 97, wherein the VHH comprises the amino acid sequence set forth in any one of SEQ ID NOs: 509, 693, 694, 695, 696, and 698.

100. The composition of claim 97, wherein the VHH comprises the amino acid sequence of SEQ ID NO: 509.

101. The composition of claim 97, wherein the VHH comprises the amino acid sequence of SEQ ID NO: 693.

102. The composition of claim 97, wherein the VHH comprises the amino acid sequence of SEQ ID NO: 694.

103. The composition of claim 97, wherein the VHH comprises the amino acid sequence of SEQ ID NO: 695.

104. The composition of claim 97, wherein the VHH comprises the amino acid sequence of SEQ ID NO: 696.

105. The composition of claim 97, wherein the VHH comprises the amino acid sequence of SEQ ID NO: 698.Docket No. 56146-753.601106. The composition of claim 91, wherein the VH CDR1 has a sequence of SEQ ID NO: 570, the VH CDR2 has a sequence of SEQ ID NO: 511, and the VH CDR3 has a sequence of SEQ ID NO: 572.

107. The composition of claim 106, wherein the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 569.

108. The composition of claim 106, wherein the VHH comprises the amino acid sequence set forth in SEQ ID NO: 569.

109. The composition of claim 91, wherein the VH CDR1 has a sequence of SEQ ID NO: 602, the VH CDR2 has a sequence of SEQ ID NO: 511, and the VH CDR3 has a sequence of SEQ ID NO: 604.

110. The composition of claim 109, wherein the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 601.

111. The composition of claim 109, wherein the VHH comprises the amino acid sequence set forth in SEQ ID NO: 601.

112. The composition of claim 91, wherein the VH CDR1 has a sequence of SEQ ID NO:626, the VH CDR2 has a sequence of SEQ ID NO: 511, and the VH CDR3 has a sequence of SEQ ID NO: 628.

113. The composition of claim 112, wherein the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 625.

114. The composition of claim 112, wherein the VHH comprises the amino acid sequence set forth in SEQ ID NO: 625.

115. The composition of claim 91, wherein the VH CDR1 has a sequence of SEQ ID NO: 658, the VH CDR2 has a sequence of SEQ ID NO: 659, and the VH CDR3 has a sequence of SEQ ID NO: 660.

116. The composition of claim 115, wherein the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 657.

117. The composition of claim 115, wherein the VHH comprises the amino acid sequence set forth in SEQ ID NO: 657.Docket No. 56146-753.601118. The composition of claim 91, wherein the VH CDR1 has a sequence of SEQ ID NO: 510, the VH CDR2 has a sequence of SEQ ID NO: 699, and the VH CDR3 has a sequence of SEQ ID NO: 512.

119. The composition of claim 118, wherein the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 569.

120. The composition of claim 118, wherein the VHH comprises the amino acid sequence set forth in SEQ ID NO: 569.

121. The composition of claims 91-120, wherein the first binding domain targeting programmed cell death protein 1 (PD-1) is capable of disrupting the interaction of PD-1 with programmed cell death ligand 1 (PD-L1).

122. A fusion protein comprising the composition of any one of claims 91-121.

123. A composition, comprising a binding domain targeting vascular endothelial growth factor A (VEGFA);wherein the binding domain comprises a VHH having a VH CDR1, VH CDR2, and VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 each have sequences respectively selected from:SEQ ID NOs: 301, 302, and 303; SEQ ID NOs: 305, 306, and 307; SEQ ID NOs: 309, 310, and 311; SEQ ID NOs: 313, 314, and 315; SEQ ID NOs: 317, 318, and 319; SEQ ID NOs: 321, 322, and 323; SEQ ID NOs: 325, 326, and 327; SEQ ID NOs: 329, 330, and 331; SEQ ID NOs: 333, 334, and 335; SEQ ID NOs: 337, 338, and 339; SEQ ID NOs: 341, 342, and 343; SEQ ID NOs: 345, 346, and 347; SEQ ID NOs: 349, 350, and 351; SEQ ID NOs: 353, 354, and 355; SEQ ID NOs: 357, 358, and 359; SEQ ID NOs: 361, 362, and 363; SEQ ID NOs: 365, 366, and 367; SEQ ID NOs: 369, 370, and 371; SEQ ID NOs: 373, 374, and 375; SEQ ID NOs: 377, 378, and 379; SEQ ID NOs: 381, 382, and 383; SEQ ID NOs: 385, 386, and 387; SEQ ID NOs: 389, 390, and 391; SEQ ID NOs: 393, 394, and 395; SEQ ID NOs: 397, 398, and 399; SEQ ID NOs: 401, 402, and 403; SEQ ID NOs: 405, 406, and 407; SEQ ID NOs: 409, 410, and 411; SEQ ID NOs: 413, 414, and 415; SEQ ID NOs: 417, 418, and 419; SEQ ID NOs: 421, 422, and 423; SEQ ID NOs: 425, 426, and 427; SEQ ID NOs: 429, 430, and 431; SEQ ID NOs: 433, 434, and 435; SEQ ID NOs: 437, 438, and 439; SEQ ID NOs: 441, 442, and 443; SEQ ID NOs: 445, 398, and 447; SEQ ID NOs: 449, 426, and 451; SEQ ID NOs: 453, 454, and 455; SEQ ID NOs: 457, 458, and 459; SEQ ID NOs: 461, 462, and 463; SEQDocket No. 56146-753.601ID NOs: 465, 466, and 467; SEQ ID NOs: 469, 470, and 471; SEQ ID NOs: 473, 474, and 475; SEQ ID NOs: 477, 478, and 479; SEQ ID NOs: 481, 482, and 483; SEQ ID NOs: 485, 486, and 487; and SEQ ID NOs: 489, 490, and 491.

124. The composition of claim 123, wherein the VHH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 300, 304, 308, 312, 316, 320, 324, 328, 332, 336, 340, 344, 348, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388, 392, 396, 400, 404, 408, 412, 416, 420, 424, 428, 432, 436, 440, 444, 448, 452, 456, 460, 464, 468, 472, 476, 480, 484, and 488.

125. The composition of claim 123 or 124, wherein the VHH comprises the amino acid sequence set forth in any one of SEQ ID NOs: 300, 304, 308, 312, 316, 320, 324, 328, 332, 336, 340, 344, 348, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388, 392, 396, 400, 404, 408, 412, 416, 420, 424, 428, 432, 436, 440, 444, 448, 452, 456, 460, 464, 468, 472, 476, 480, 484, and 488.

126. The composition of any one of claims 123-125, wherein the second binding domain targeting VEGFA is capable of disrupting the interaction of VEGFA with one or more of its receptors.

127. A fusion protein comprising the composition of any one of claims 123-126.

128. One or more polynucleotides encoding the composition of any one of claims 1- 121 or 123-126 or the fusion protein of claim 122 or 127.

129. A host cell comprising the composition of any one of claims 1-121 or 123-126 or the fusion protein of claim 122 or 127 or the one or more polynucleotides of claim 128.

130. A pharmaceutical composition comprising the composition of any one of claims 1-121 or 123-126 or the fusion protein of claim 122 or 127, and a pharmaceutically acceptable carrier or excipient.

131. A method of treating cancer in a subject in need thereof, comprising administering to the subject at therapeutically effective amount of a composition of any one of claims 1-121 or 123-126 or the fusion protein of claim 122 or 127 or a pharmaceutical composition of claim 130.

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