Materials and methods for binding Siglec-3 / CD33

Abstract

The invention provides antigen binding domains that bind myeloid cell surface antigen CD33 protein comprising the antigen binding domains that bind CD33, polynucleotides encoding them, vectors, host cells, methods of making and using them.

Description

[0001] MATERIALS AND METHODS FOR BINDING SIGLEC-3 / CD33

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] The present application claims the priority of U.S. provisional patent application serial number 62 / 989093 filed on March 13, 2020, U.S. provisional patent application serial number 62 / 989071 filed on March 13, 2020, U.S. provisional patent application serial number 62 / 989120 filed on March 13, 2020, U.S. provisional patent application serial number 62 / 989187 filed on March 13, 2020 and U.S. provisional patent application serial number 62 / 989230 filed on March 13, 2020.

[0004] TECHNICAL FIELD

[0005] The invention provides antigen binding domains that bind myeloid cell surface antigen CD33 protein comprising the antigen binding domains that bind CD33, polynucleotides encoding them, vectors, host cells, methods of making and using them.

[0006] BACKGROUND

[0007] Myeloid cell surface antigen CD33, also known as Sialic acid-binding immunoglobulin-like lectin 3 (Siglec-3), is a member of the sialic acid binding family of proteins. CD33 is a type-I single pass transmembrane protein with an N-terminal sialic acid binding domain in its extracellular domain (ECD) and intracellular immunoreceptor tyrosine-based inhibitor motif (ITIM) motifs (Uniprot.org) [4]

[0008] In normal cells, CD33 in involved in immune cell maintenance and cell-cell interactions [5] CD33 protein is expressed in at least the bone marrow, lung, skin, appendix, spleen, lymph nodes, and tonsils. Expression of CD33 mRNA is found in a wide variety of tissues and organs, including but not limited to, bone marrow, the brain, eye, lung, endocrine tissues, salivary glands, esophagus, stomach, colon, rectum, liver, gallbladder, pancreas, kidney, bladder, male and female reproductive tissues, prostate, breast, heart, smooth muscle, skeletal muscle, adipose tissue, skin, thymus, appendix, spleen, lymph nodes, tonsil, granulocytes, and blood (e.g., PBMC, dendritic cells, and lymphocytes). See, e.g., www.proteinatlas. org / ENSGOOOOO 105383 -CD33 / tissue.

[0009] CD33 is expressed in 90% of AML patients [6] and is one of the most widely explored surface antigens for AML immunotherapy. Several approaches have been explored to target CD33, including antibody -drug / antibody -toxin conjugates, chimeric antigen receptors (CARs) and T-cell redirector bispecific antibodies [7] There is a need for additional CD33 binding domains for therapeutic and diagnostic purposes.

[0010] Acute Myeloid Leukemia (AML) is a type of leukemia of the myeloid cell lineage characterized by rapid development and progression [1] AML is a heterogeneous disease with widely varying molecular changes along with different clinical outcomes. Moreover, AML is characterized by the ability of AML blasts, or leukemia cells, to self-renew, continuously proliferate and escape the immune system resulting in disease relapse from minor clones [2] Accordingly, AML has historically been considered to have one of the lowest long-term survival rates, with an overall 5 -year survival rate of adult AML at about 27%. Survival rates have a strong inverse relationship with age [3] Historically, the primary AML treatment strategies involve chemotherapy, along with stem cell transplantation. In recent years however, there has been aggressive effort to develop immunotherapy for AML exploring key surface antigens. One of the surface antigens highly expressed in AML cells is CD33.

[0011] SUMMARY

[0012] In one aspect, the disclosure provides an isolated protein that binds CD33, wherein the isolated protein comprises: a) a heavy chain complementarity determining region (HCDR) 1, an HCDR2 and an

[0013] HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 18; b) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 19; c) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 20; d) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 21; e) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 22; f) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 23; g) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 24; h) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 25; i) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 26; or j) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 27, or a CD33 binding fragment thereof.

[0014] In some embodiments, the isolated protein comprises the HCDR1, the HCDR2, the HCDR3 of a) SEQ ID NOs: 1, 6, and 12, respectively; b) SEQ ID NOs: 2, 7, and 13, respectively; c) SEQ ID NOs: 1, 8, and 14, respectively; d) SEQ ID NOs: 3, 9, and 13, respectively; e) SEQ ID NOs: 4, 10, and 15, respectively; f) SEQ ID NOs: 5, 11, and 16, respectively; or g) SEQ ID NOs: 5, 11, and 17, respectively.

[0015] In some embodiments, the isolated protein is a scFv, a (scFv)2, a Fv, a Fab, a F(ab’)2, a Fd, a dAb or a VHH. In some embodiments, the isolated protein is the VHH. In some embodiments, the isolated protein is the Fab. In some embodiments, the isolated protein is the scFv. In some embodiments, the isolated protein comprises the VH of any one of SEQ ID NOs: 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27.

[0016] In another aspect, the disclosure provides an isolated protein that binds CD33, where the isolated protein comprises a VHH of any one of SEQ ID NOs: 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27.

[0017] In another aspect, the disclosure provides an isolated protein that binds CD33, wherein the isolated protein comprises a) an HCDR1, an HCDR2 and an HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 52; b) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 53; c) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 54; d) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 55; e) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 56; f) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 57; g) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 58; h) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 59; i) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 60; j) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 61; k) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 262; l) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 263; or m) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 264.

[0018] In some embodiments, the isolated protein comprises the HCDR1, the HCDR2, the HCDR3 of a) SEQ ID NOs: 28, 36, and 45, respectively; b) SEQ ID NOs: 29, 37, and 46, respectively; c) SEQ ID NOs: 30, 38, and 47, respectively; d) SEQ ID NOs: 31, 39, and 48, respectively; e) SEQ ID NOs: 32, 40, and 49, respectively; f) SEQ ID NOs: 33, 41, and 50, respectively; g) SEQ ID NOs: 34, 42, and 51, respectively; h) SEQ ID NOs: 34, 43, and 51, respectively; i) SEQ ID NOs: 34, 44, and 51, respectively; j) SEQ ID NOs: 35, 42, and 51, respectively k) SEQ ID NOs: 253, 256, and 259, respectively: l) SEQ ID NOs: 254, 257 and 260, respectively: or m) SEQ ID NOs: 255, 258 and 261, respectively. In another aspect, the disclosure provides an isolated protein that binds CD33, wherein the isolated protein comprises the HCDR1, the HCDR2, and the HCDR3 of a) SEQ ID NOs: 29, 37, and 46, respectively; b) SEQ ID NOs: 32, 40, and 49, respectively; c) SEQ ID NOs: 34, 43, and 51, respectively; d) SEQ ID NOs: 253, 256, and 259, respectively; e) SEQ ID NOs: 254, 257, and 260, respectively; or f) SEQ ID NOs: 255, 258, and 261, respectively.

[0019] In some embodiments, the isolated protein comprises a) an HCDR1, an HCDR2 and an HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 52 and a light chain complementarity determining region (LCDR) 1, an LCDR2 and an LCDR3 of a light chain variable region (VL) of SEQ ID NO: 81; b) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 53 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 82; c) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 54 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 83; d) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 55 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 84; e) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 56 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 85; f) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 57 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 86; g) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 58 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 87; h) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 59 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 87; i) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 60 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 87; j) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 61 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 88; k) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 262 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 271; l) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 263 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 272; or m) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 264 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 273.

[0020] In some embodiments, the isolated protein comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of a) SEQ ID NOs: 28, 36, 45, 62, 69, and 75, respectively; b) SEQ ID NOs: 29, 37, 46, 63, 70, and 76, respectively; c) SEQ ID NOs: 30, 38, 47, 64, 71, and 77, respectively; d) SEQ ID NOs: 31, 39, 48, 65, 72, and 78, respectively; e) SEQ ID NOs: 32, 40, 49, 66, 70, and 76, respectively; f) SEQ ID NOs: 33, 41, 50, 67, 73, and 79, respectively; g) SEQ ID NOs: 34, 42, 51, 68, 74, and 80, respectively; h) SEQ ID NOs: 34, 43, 51, 68, 74, and 80, respectively; i) SEQ ID NOs: 34, 44, 51, 68, 74, and 80, respectively; j) SEQ ID NOs: 35, 42, 51, 68, 74, and 80, respectively: k) SEQ ID NOs: 253, 256, 259, 265, 74 and 269, respectively: l) SEQ ID NOs: 254, 257, 260, 66, 267 and 76, respectively: or m) SEQ ID NOs: 255, 258, 261, 266, 268 and 270, respectively.

[0021] In some embodiments, the isolated protein is a scFv, a (scFv)2, a Fv, a Fab, a F(ab’)2, a Fd, a dAb or a VHH. In some embodiments, the isolated protein is the Fab. In some embodiments, the isolated protein is the VHH. In some embodiments, the isolated protein is the scFv. In some embodiments, the scFv comprises, from the N- to C-terminus, a VH, a first linker (LI) and a VL (VH-L1-VL) or the VL, the LI and the VH (VL-L1-VH).

[0022] In some embodiments, the LI comprises a) about 5-50 amino acids; b) about 5-40 amino acids; c) about 10-30 amino acids; or d) about 10-20 amino acids.

[0023] In some embodiments, the LI comprises an amino acid sequence of any one of SEQ ID NOs:

[0024] 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, or 140. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 108.

[0025] In some embodiments, the isolated protein comprises the VH of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 262, 263, 264, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351,

[0026] 352, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, or 382, and the VL of SEQ ID NOs: 81, 82, 83, 84, 85, 86, 87, 88, 271, 272,273, 311, 312, 313, 314, 315, 316, 317, 318, 319 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 353, 354, 355, 356,

[0027] 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393,

[0028] 394, 395, 396, 397 or 398.

[0029] In some embodiments, the isolated protein comprises: a) the VH of SEQ ID NO: 52 and the VL of SEQ ID NO: 81; b) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 82; c) the VH of SEQ ID NO: 54 and the VL of SEQ ID NO: 83; d) the VH of SEQ ID NO: 55 and the VL of SEQ ID NO: 84; e) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 85; f) the VH of SEQ ID NO: 57 and the VL of SEQ ID NO: 86; g) the VH of SEQ ID NO: 58 and the VL of SEQ ID NO: 87; h) the VH of SEQ ID NO: 59 and the VL of SEQ ID NO: 87; i) the VH of SEQ ID NO: 60 and the VL of SEQ ID NO: 87; j) the VH of SEQ ID NO: 61 and the VL of SEQ ID NO: 88; k) the VH of SEQ ID NO: 262 and the VL of SEQ ID NO: 271; l) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 272; m) the VH of SEQ ID NO: 264 and the VL of SEQ ID NO: 273; n) the VH of SEQ ID NO: 291 and the VL of SEQ ID NO: 271; o) the VH of SEQ ID NO: 292 and the VL of SEQ ID NO: 271; p) the VH of SEQ ID NO: 293 and the VL of SEQ ID NO: 271; q) the VH of SEQ ID NO: 294 and the VL of SEQ ID NO: 271; r) the VH of SEQ ID NO: 295 and the VL of SEQ ID NO: 271; s) the VH of SEQ ID NO: 296 and the VL of SEQ ID NO: 271; t) the VH of SEQ ID NO: 297 and the VL of SEQ ID NO: 271; u) the VH of SEQ ID NO: 298 and the VL of SEQ ID NO: 271; v) the VH of SEQ ID NO: 299 and the VL of SEQ ID NO: 271; w) the VH of SEQ ID NO: 300 and the VL of SEQ ID NO: 271; x) the VH of SEQ ID NO: 301 and the VL of SEQ ID NO: 271; y) the VH of SEQ ID NO: 302 and the VL of SEQ ID NO: 271; z) the VH of SEQ ID NO: 303 and the VL of SEQ ID NO: 271; aa) the VH of SEQ ID NO: 304 and the VL of SEQ ID NO: 271; ab) the VH of SEQ ID NO: 305 and the VL of SEQ ID NO: 271; ac) the VH of SEQ ID NO: 306 and the VL of SEQ ID NO: 271; ad) the VH of SEQ ID NO: 307 and the VL of SEQ ID NO: 271; ae) the VH of SEQ ID NO: 308 and the VL of SEQ ID NO: 271; af) the VH of SEQ ID NO: 309 and the VL of SEQ ID NO: 271; ag) the VH of SEQ ID NO: 310 and the VL of SEQ ID NO: 271; ah) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 311 ; ai) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 312. aj) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 313; ak) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 314; al) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 315; am) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 316; an) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 317; ao) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 318; ap) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 319; aq) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 320; ar) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 321; as) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 322; at) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 323; au) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 324; av) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 325; aw) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 326; ax) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 327; ay) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 328; az) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 329; ba) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 330; bb) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 331 ; be) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 332; bd) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 333; be) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 334; bf) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 335; bg) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 336; bh) the VH of SEQ ID NO: 337 and the VL of SEQ ID NO: 272; bi) the VH of SEQ ID NO: 338 and the VL of SEQ ID NO: 272; bj) the VH of SEQ ID NO: 339 and the VL of SEQ ID NO: 272; bk) the VH of SEQ ID NO: 340 and the VL of SEQ ID NO: 272; bl) the VH of SEQ ID NO: 341 and the VL of SEQ ID NO: 272; bm) the VH of SEQ ID NO: 342 and the VL of SEQ ID NO: 272; bn) the VH of SEQ ID NO: 343 and the VL of SEQ ID NO: 272; bo) the VH of SEQ ID NO: 344 and the VL of SEQ ID NO: 272; bp) the VH of SEQ ID NO: 345 and the VL of SEQ ID NO: 272; bq) the VH of SEQ ID NO: 346 and the VL of SEQ ID NO: 272; br) the VH of SEQ ID NO: 347 and the VL of SEQ ID NO: 272; bs) the VH of SEQ ID NO: 348 and the VL of SEQ ID NO: 272; bt) the VH of SEQ ID NO: 349 and the VL of SEQ ID NO: 272; bu) the VH of SEQ ID NO: 350 and the VL of SEQ ID NO: 272; bv) the VH of SEQ ID NO: 351 and the VL of SEQ ID NO: 272; bw) the VH of SEQ ID NO: 352 and the VL of SEQ ID NO: 272. bx) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 353; by) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 354; bz) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 355; ca) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 356; cb) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 357; cc) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 358; cd) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 359; ce) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 360; cf) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 361; eg) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 362; ch) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 363; ci) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 364; cj) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 365; ck) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 366; cl) the VH of SEQ ID NO: 367 and the VL of SEQ ID NO: 85; cm) the VH of SEQ ID NO: 368 and the VL of SEQ ID NO: 85; cn) the VH of SEQ ID NO: 369 and the VL of SEQ ID NO: 85; co) the VH of SEQ ID NO: 370 and the VL of SEQ ID NO: 85; cp) the VH of SEQ ID NO: 371 and the VL of SEQ ID NO: 85; cq) the VH of SEQ ID NO: 372 and the VL of SEQ ID NO: 85; cr) the VH of SEQ ID NO: 373 and the VL of SEQ ID NO: 85; cs) the VH of SEQ ID NO: 374 and the VL of SEQ ID NO: 85; ct) the VH of SEQ ID NO: 375 and the VL of SEQ ID NO: 85; cu) the VH of SEQ ID NO: 376 and the VL of SEQ ID NO: 85; cv) the VH of SEQ ID NO: 377 and the VL of SEQ ID NO: 85; cw) the VH of SEQ ID NO: 378 and the VL of SEQ ID NO: 85; ex) the VH of SEQ ID NO: 379 and the VL of SEQ ID NO: 85; cy) the VH of SEQ ID NO: 380 and the VL of SEQ ID NO: 85; cz) the VH of SEQ ID NO: 381 and the VL of SEQ ID NO: 85; da) the VH of SEQ ID NO: 382 and the VL of SEQ ID NO: 85; db) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 383; dc) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 384; dd) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 385; de) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 386; df) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 387; dg) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 388; dh) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 389; de) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 390; df) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 391; dg) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 392; dh) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 393; di) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 394; dj) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 395; dk) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 396; dl) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 397; or dm) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 398.

[0030] In another aspect, the disclosure provides an isolated protein that binds CD33, where the isolated protein comprises a) the VH of SEQ ID NO: 52 and the VL of SEQ ID NO: 81; b) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 82; c) the VH of SEQ ID NO: 54 and the VL of SEQ ID NO: 83; d) the VH of SEQ ID NO: 55 and the VL of SEQ ID NO: 84; e) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 85; f) the VH of SEQ ID NO: 57 and the VL of SEQ ID NO: 86; g) the VH of SEQ ID NO: 58 and the VL of SEQ ID NO: 87; h) the VH of SEQ ID NO: 59 and the VL of SEQ ID NO: 87; i) the VH of SEQ ID NO: 60 and the VL of SEQ ID NO: 87; j) the VH of SEQ ID NO: 61 and the VL of SEQ ID NO: 88; k) the VH of SEQ ID NO: 262 and the VL of SEQ ID NO: 271; l) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 272; m) the VH of SEQ ID NO: 264 and the VL of SEQ ID NO: 273; n) the VH of SEQ ID NO: 291 and the VL of SEQ ID NO: 271; o) the VH of SEQ ID NO: 292 and the VL of SEQ ID NO: 271; p) the VH of SEQ ID NO: 293 and the VL of SEQ ID NO: 271; q) the VH of SEQ ID NO: 294 and the VL of SEQ ID NO: 271; r) the VH of SEQ ID NO: 295 and the VL of SEQ ID NO: 271; s) the VH of SEQ ID NO: 296 and the VL of SEQ ID NO: 271; t) the VH of SEQ ID NO: 297 and the VL of SEQ ID NO: 271; u) the VH of SEQ ID NO: 298 and the VL of SEQ ID NO: 271; v) the VH of SEQ ID NO: 299 and the VL of SEQ ID NO: 271; w) the VH of SEQ ID NO: 300 and the VL of SEQ ID NO: 271; x) the VH of SEQ ID NO: 301 and the VL of SEQ ID NO: 271; y) the VH of SEQ ID NO: 302 and the VL of SEQ ID NO: 271; z) the VH of SEQ ID NO: 303 and the VL of SEQ ID NO: 271; aa) the VH of SEQ ID NO: 304 and the VL of SEQ ID NO: 271; ab) the VH of SEQ ID NO: 305 and the VL of SEQ ID NO: 271; ac) the VH of SEQ ID NO: 306 and the VL of SEQ ID NO: 271; ad) the VH of SEQ ID NO: 307 and the VL of SEQ ID NO: 271; ae) the VH of SEQ ID NO: 308 and the VL of SEQ ID NO: 271; af) the VH of SEQ ID NO: 309 and the VL of SEQ ID NO: 271; ag) the VH of SEQ ID NO: 310 and the VL of SEQ ID NO: 271; ah) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 311 ; ai) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 312. aj) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 313; ak) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 314; al) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 315; am) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 316; an) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 317; ao) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 318; ap) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 319; aq) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 320; ar) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 321; as) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 322; at) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 323; au) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 324; av) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 325; aw) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 326; ax) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 327; ay) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 328; az) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 329; ba) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 330; bb) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 331 ; be) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 332; bd) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 333; be) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 334; bf) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 335; bg) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 336; bh) the VH of SEQ ID NO: 337 and the VL of SEQ ID NO: 272; bi) the VH of SEQ ID NO: 338 and the VL of SEQ ID NO: 272; bj) the VH of SEQ ID NO: 339 and the VL of SEQ ID NO: 272; bk) the VH of SEQ ID NO: 340 and the VL of SEQ ID NO: 272; bl) the VH of SEQ ID NO: 341 and the VL of SEQ ID NO: 272; bm) the VH of SEQ ID NO: 342 and the VL of SEQ ID NO: 272; bn) the VH of SEQ ID NO: 343 and the VL of SEQ ID NO: 272; bo) the VH of SEQ ID NO: 344 and the VL of SEQ ID NO: 272; bp) the VH of SEQ ID NO: 345 and the VL of SEQ ID NO: 272; bq) the VH of SEQ ID NO: 346 and the VL of SEQ ID NO: 272; br) the VH of SEQ ID NO: 347 and the VL of SEQ ID NO: 272; bs) the VH of SEQ ID NO: 348 and the VL of SEQ ID NO: 272; bt) the VH of SEQ ID NO: 349 and the VL of SEQ ID NO: 272; bu) the VH of SEQ ID NO: 350 and the VL of SEQ ID NO: 272; bv) the VH of SEQ ID NO: 351 and the VL of SEQ ID NO: 272; bw) the VH of SEQ ID NO: 352 and the VL of SEQ ID NO: 272. bx) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 353; by) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 354; bz) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 355; ca) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 356; cb) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 357; cc) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 358; cd) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 359; ce) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 360; cf) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 361; eg) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 362; ch) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 363; ci) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 364; cj) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 365; ck) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 366; cl) the VH of SEQ ID NO: 367 and the VL of SEQ ID NO: 85; cm) the VH of SEQ ID NO: 368 and the VL of SEQ ID NO: 85; cn) the VH of SEQ ID NO: 369 and the VL of SEQ ID NO: 85; co) the VH of SEQ ID NO: 370 and the VL of SEQ ID NO: 85; cp) the VH of SEQ ID NO: 371 and the VL of SEQ ID NO: 85; cq) the VH of SEQ ID NO: 372 and the VL of SEQ ID NO: 85; cr) the VH of SEQ ID NO: 373 and the VL of SEQ ID NO: 85; cs) the VH of SEQ ID NO: 374 and the VL of SEQ ID NO: 85; ct) the VH of SEQ ID NO: 375 and the VL of SEQ ID NO: 85; cu) the VH of SEQ ID NO: 376 and the VL of SEQ ID NO: 85; cv) the VH of SEQ ID NO: 377 and the VL of SEQ ID NO: 85; cw) the VH of SEQ ID NO: 378 and the VL of SEQ ID NO: 85; ex) the VH of SEQ ID NO: 379 and the VL of SEQ ID NO: 85; cy) the VH of SEQ ID NO: 380 and the VL of SEQ ID NO: 85; cz) the VH of SEQ ID NO: 381 and the VL of SEQ ID NO: 85; da) the VH of SEQ ID NO: 382 and the VL of SEQ ID NO: 85; db) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 383; dc) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 384; dd) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 385; de) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 386; df) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 387; dg) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 388; dh) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 389; de) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 390; df) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 391; dg) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 392; dh) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 393; di) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 394; dj) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 395; dk) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 396; dl) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 397; or dm) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 398.

[0031] In some embodiments, the isolated protein is a scFv, a (scFv)2, a Fv, a Fab, a F(ab’)2, a Fd, a dAb or a VHH. In some embodiments, the isolated protein is the Fab. In some embodiments, the isolated protein is the VHH. In some embodiments, the isolated protein is the scFv. In some embodiments, the scFv comprises, from the N- to C-terminus, a VH, a first linker (LI) and a VL (VH-L1-VL) or the VL, the LI and the VH (VL-L1-VH).

[0032] In some embodiments, the LI comprises a) about 5-50 amino acids; b) about 5-40 amino acids; c) about 10-30 amino acids; or d) about 10-20 amino acids.

[0033] In some embodiments, the LI comprises an amino acid sequence of any one of SEQ ID NOs:

[0034] 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,

[0035] 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, or 140. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 108.

[0036] In some embodiments, the isolated protein comprises the amino acid sequence of any one of SEQ

[0037] ID NO: 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221

[0038] 274, 275 or 276. In particular, the amino acid sequence of SEQ ID NO: 213, 216, 219, 274, 275 or 276.

[0039] In another aspect the disclosure provides an isolated protein that binds myeloid CD33, where the isolated protein comprises: a) a heavy chain complementarity determining region (HCDR) 1, an HCDR2 and an HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 95; b) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 96; c) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 97; d) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 170; e) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 171; f) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 175; g) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 179; h) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 181; or i) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 196.

[0040] In some embodiments, the isolated protein comprises the HCDR1, the HCDR2, and the HCDR3 of a) SEQ ID NOs: 89, 90, and 92, respectively; b) SEQ ID NOs: 89, 90, and 93, respectively; c) SEQ ID NOs: 33, 91, and 94, respectively d) SEQ ID NOs: 167, 168, and 169, respectively; e) SEQ ID NOs: 172, 173, and 174, respectively; f) SEQ ID NOs: 176, 177, and 178, respectively; g) SEQ ID NOs: 89, 90, and 180, respectively; or h) SEQ ID NOs: 89, 90, and 93, respectively.

[0041] In another aspect the disclosure provides an isolated protein of claim 32 or 33, wherein the isolated protein comprises a) an HCDR1, an HCDR2 and an HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 95 and a light chain complementarity determining region (LCDR) 1, an LCDR2 and an LCDR3 of a light chain variable region (VL) of SEQ ID NO: 105; b) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 96 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 106; c) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 97 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 107; d) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 170 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 185; e) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 171 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 188; f) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 175 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 192; g) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 179 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 196; h) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 181 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 200; or i) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 96 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 202.

[0042] In some embodiments, the isolated protein comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of a) SEQ ID NOs: 89, 90, 92, 98, 101, and 104, respectively; b) SEQ ID NOs: 89, 90, 93, 99, 102, and 104, respectively; c) SEQ ID NOs: 33, 91, 94, 100, 103, and 104, respectively; d) SEQ ID NOs: 167, 168, 169, 182, 183, and 184, respectively; e) SEQ ID NOs: 33, 91, 94, 186, 187, and 104, respectively; f) SEQ ID NOs: 172, 173, 174, 189, 190, and 191, respectively; g) SEQ ID NOs: 176, 177, 178, 193, 194, and 195, respectively; h) SEQ ID NOs: 89, 90, 180, 197, 198, and 199, respectively; i) SEQ ID NOs: 89, 90, 93, 201, 102, and 104, respectively.

[0043] In some embodiments, the isolated protein is a scFv, a (scFv)2, a Fv, a Fab, a F(ab’)2, a Fd, a dAb or a VHH.

[0044] In some embodiments, the isolated protein is the Fab.

[0045] In some embodiments, the isolated protein is the VHH.

[0046] In some embodiments, the isolated protein is the scFv.

[0047] In some embodiments, the scFv comprises, from the N- to C-terminus, a VH, a first linker (LI) and a VL (VH-L1-VL) or the VL, the LI and the VH (VL-L1-VH).

[0048] In some embodiments, the LI comprises a) about 5-50 amino acids; b) about 5-40 amino acids; c) about 10-30 amino acids; or d) about 10-20 amino acids.

[0049] In some embodiments, the LI comprises an amino acid sequence of any one of SEQ ID NOs:

[0050] 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, or 140.

[0051] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 108. In some embodiments, the isolated protein comprises the VH of SEQ ID NOs: 95, 96, 97, 170, 171, 175, 179, 181, or 96, and the VL of SEQ ID NOs: 105, 106, 107, 185, 188, 192, 196, 200, or 202. In some embodiments, the isolated protein comprises: a) the VH of SEQ ID NO: 95 and the VL of SEQ ID NO: 105; b) the VH of SEQ ID NO: 96 and the VL of SEQ ID NO: 106; c) the VH of SEQ ID NO: 97 and the VL of SEQ ID NO: 107; d) the VH of SEQ ID NO: 170 and the VL of SEQ ID NO: 185; e) the VH of SEQ ID NO: 171 and the VL of SEQ ID NO: 188; f) the VH of SEQ ID NO: 175 and the VL of SEQ ID NO: 192; g) the VH of SEQ ID NO: 179 and the VL of SEQ ID NO: 196; h) the VH of SEQ ID NO: 181 and the VL of SEQ ID NO: 200; or i) the VH of SEQ ID NO: 96 and the VL of SEQ ID NO: 202.

[0052] In another aspect, the disclosure provides an isolated protein that that binds CD33, where the isolated protein comprises: a) the VH of SEQ ID NO: 95 and the VL of SEQ ID NO: 105; b) the VH of SEQ ID NO: 96 and the VL of SEQ ID NO: 106; c) the VH of SEQ ID NO: 97 and the VL of SEQ ID NO: 107; d) the VH of SEQ ID NO: 170 and the VL of SEQ ID NO: 185; e) the VH of SEQ ID NO: 171 and the VL of SEQ ID NO: 188; f) the VH of SEQ ID NO: 175 and the VL of SEQ ID NO: 192; g) the VH of SEQ ID NO: 179 and the VL of SEQ ID NO: 196; h) the VH of SEQ ID NO: 181 and the VL of SEQ ID NO: 200; or i) the VH of SEQ ID NO: 96 and the VL of SEQ ID NO: 202.

[0053] In some embodiments, the isolated protein is a scFv, a (scFv)2, a Fv, a Fab, a F(ab’)2, a Fd, a dAb or a VHH. In some embodiments, the isolated protein is the Fab. In some embodiments, the isolated protein is the VHH. In some embodiments, the isolated protein is the scFv. In some embodiments, the scFv comprises, from the N- to C-terminus, a VH, a first linker (LI) and a VL (VH-L1-VL) or the VL, the LI and the VH (VL-L1-VH). In some embodiments, the LI comprises a) about 5-50 amino acids; b) about 5-40 amino acids; c) about 10-30 amino acids; or d) about 10-20 amino acids.

[0054] In some embodiments, the LI comprises an amino acid sequence of any one of SEQ ID NOs:

[0055] 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, or 140. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 108.

[0056] The disclosure provides any isolated protein described above, where the protein is conjugated to a half-life extending moiety. In some embodiments, the half-life extending moiety is an immunoglobulin (Ig), a fragment of the Ig, an Ig constant region, a fragment of the Ig constant region, a Fc region, transferrin, albumin, an albumin binding domain or polyethylene glycol. In some embodiments, the isolated protein is a monospecific protein. In some embodiments, the isolated protein is a multispecific protein. In some embodiments, the multispecific protein is a bispecific protein. In some embodiments, the multispecific protein is a trispecific protein. In some embodiments, the multispecific protein further comprises an immunoglobulin (Ig) constant region or a fragment of the Ig constant region thereof. In some embodiments, the fragment of the Ig constant region comprises a Fc region. In some embodiments, the fragment of the Ig constant region comprises a CH2 domain. In some embodiments, the fragment of the Ig constant region comprises a CH3 domain. In some embodiments, the fragment of the Ig constant region comprises the CH2 domain and the CH3 domain. In some embodiments, the fragment of the Ig constant region comprises at least portion of a hinge, the CH2 domain and the CH3 domain. In some embodiments, the fragment of the Ig constant region comprises a hinge, the CH2 domain and the CH3 domain. In some embodiments, the isolated protein is conjugated to the N-terminus of the Ig constant region or the fragment of the Ig constant region. In some embodiments, the isolated protein is conjugated to the C-terminus of the Ig constant region or the fragment of the Ig constant region. In some embodiments, the isolated protein is conjugated to the Ig constant region or the fragment of the Ig constant region via a second linker (L2). In some embodiments, the L2 comprises the amino acid sequence of any one of SEQ ID NOs:108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,

[0057] 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, or 140.

[0058] In some embodiments, the multispecific protein comprises an antigen binding domain that binds an antigen on a lymphocyte. In some embodiments, the lymphocyte is a T cell. In some embodiments, the T cell is a CD8+T cell. In some embodiments, the lymphocyte is a natural killer (NK) cell. In some embodiments, the multispecific protein comprises an antigen binding domain that binds CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, TRGV9, or NKG2C. In some embodiments, the multispecific protein comprises an antigen binding domain that binds CD3ε. In some embodiments, the antigen binding domain that binds CD3ε comprises: a) a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 141, a HCDR2 of SEQ ID NO: 142, a HCDR3 of SEQ ID NO: 143, a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO: 144, a LCDR2 of SEQ ID NO: 145 and a LCDR3 of SEQ ID NO: 146; b) the VH of SEQ ID NO: 147 and the VL of SEQ ID NO: 148; c) the HCDR1 of SEQ ID NO: 149, the HCDR2 of SEQ ID NO: 150, the HCDR3 of SEQ ID NO:

[0059] 151, the LCDR1 of SEQ ID NO: 152, the LCDR2 of SEQ ID NO: 153 and the LCDR3 of SEQ ID NO: 154; or d) the VH of SEQ ID NO: 155 and the VL of SEQ ID NO: 156.

[0060] In some embodiments, the multispecific protein comprises an antigen binding domain that binds TRGV9. In some embodiments, the antigen binding domain that binds TRGV9 comprises: a) a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 236, a HCDR2 of SEQ ID NO: 237, a HCDR3 of SEQ ID NO: 238, a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO: 239, a LCDR2 of SEQ ID NO: 240 and a LCDR3 of SEQ ID NO: 241; b) a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 236, a HCDR2 of SEQ ID NO: 237, a HCDR3 of SEQ ID NO: 242, a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO: 239, a LCDR2 of SEQ ID NO: 240 and a LCDR3 of SEQ ID NO: 241; c) a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 236, a HCDR2 of SEQ ID NO: 237, a HCDR3 of SEQ ID NO: 243, a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO: 239, a LCDR2 of SEQ ID NO: 240 and a LCDR3 of SEQ ID NO: 241; d) a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 236, a HCDR2 of SEQ ID NO: 237, a HCDR3 of SEQ ID NO: 244, a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO: 239, a LCDR2 of SEQ ID NO: 240 and a LCDR3 of SEQ ID NO: 241; e) the VH of SEQ ID NO: 245 and the VL of SEQ ID NO: 249; f) the VH of SEQ ID NO: 246 and the VL of SEQ ID NO: 249; g) the VH of SEQ ID NO: 247 and the VL of SEQ ID NO: 249; or h) the VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 249.

[0061] In some embodiments, the Ig constant region or the fragment of the Ig constant region is an IgGl, an IgG2, an IgG3 or an IgG4 isotype. In some embodiments, the Ig constant region or the fragment of the Ig constant region comprises at least one mutation that results in reduced binding of the protein to a Fey receptor (FcyR). In some embodiments, the at least one mutation that results in reduced binding of the protein to the FcyR is selected from the group consisting of F234A / L235A, L234A / L235A, L234A / L235A / D265S, V234A / G237A / P238S / H268A / V309L / A330S / P331S, F234A / L235A, S228P / F234A / L235A, N297A, V234A / G237A, K214T / E233P / L234V / L235A / G236- deleted / A327G / P331A / D365E / L358M, H268Q / V309L / A330S / P331S, S267E / L328F, L234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S and S228P / F234A / L235A / G236-deleted / G237A / P238S, wherein residue numbering is according to the EU index. In some embodiments, the Ig constant region or the fragment of the Ig constant region comprises at least one mutation that results in enhanced binding of the protein to the FcyR. In some embodiments, the at least one mutation that results in enhanced binding of the protein to the FcyR is selected from the group consisting of S239D / I332E, S298A / E333A / K334A, F243L / R292P / Y300L, F243L / R292P / Y300L / P396L, F243L / R292P / Y300L / V305I / P396L and G236A / S239D / I332E, wherein residue numbering is according to the EU index. In some embodiments, the FcyR is FcyRI, FcyRIIA, FcyRIIB or FcyRIII, or any combination thereof. In some embodiments, the Ig constant region of the fragment of the Ig constant region comprises at least one mutation that modulates a half-life of the protein. In some embodiments, the at least one mutation that modulates the half-life of the protein is selected from the group consisting of H435A, P257I / N434H, D376V / N434H, M252Y / S254T / T256E / H433K / N434F, T308P / N434A and H435R, wherein residue numbering is according to the EU index. In some embodiments, the protein comprises at least one mutation in a CH3 domain of the Ig constant region. In some embodiments, the at least one mutation in the CH3 domain of the Ig constant region is selected from the group consisting of T350V, L351 Y, F405A, Y407V, T366Y, T366W, F405W, T394W, T394S, Y407T, Y407A, T366S / L368A / Y407V, L351 Y / F405A / Y407V, T366I / K392M / T394W, F405A / Y407V, T366L / K392M / T394W, L351Y / Y407A, T366A / K409F, L351Y / Y407A, T366V / K409F, T366A / K409F, T350V / L351Y / F405A / Y407V and T350V / T366L / K392L / T394W, wherein residue numbering is according to the EU index.

[0062] The disclosure provides any isolated protein described above, where the protein is a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises a) an extracellular domain comprising any of the isolated proteins described above; b) a transmembrane domain; and c) an intracellular signaling domain optionally comprising at least one co stimulatory domain.

[0063] In some embodiments, the CAR further comprises a CD8a-hinge region. In some embodiments, a) the transmembrane domain comprises a CD8a transmembrane region (CD8a- TM) polypeptide; and b) the intracellular signaling domain comprises a co-stimulatory domain comprising a TNF receptor superfamily member 9 (CD 137) component and a primary signaling domain comprising a T-cell surface glycoprotein CD3 zeta chain (CD3z) component.

[0064] In some embodiments, a) the CD8a-hinge region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 157; b) the transmembrane domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 162; and / or c) the intracellular signaling domain comprises a co-stimulatory domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 163 and a primary signaling domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 164.

[0065] In some embodiments, the intracellular signaling domain comprises a polypeptide component selected from the group consisting of a TNF receptor superfamily member 9 (CD137) component, a T- cell surface glycoprotein CD3 zeta chain (CD3z) component, a cluster of differentiation (CD27) component, a cluster of differentiation superfamily member component, or any combinations thereof.

[0066] In another aspect, the disclosure provides an isolated multispecific protein comprising a first antigen binding domain that binds CD33 and a second antigen binding domain that binds a lymphocyte antigen. In some embodiments, the lymphocyte antigen is a T cell antigen. In some embodiments, the T cell antigen is a CD8+T cell antigen. In some embodiments, the lymphocyte antigen is a NK cell antigen. In some embodiments, the lymphocyte antigen is CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, TRGV9, or NKG2C. In certain embodiments, the lymphocyte antigen is CD3ε. In some embodiments, the first antigen binding domain that binds CD33 and / or the second antigen binding domain that binds the lymphocyte antigen comprise a scFv, a (SCFV)2, a Fv, a Fab, a F(ab’)2, a Fd, a dAb or a VHH. In some embodiments, the first antigen binding domain that binds CD33 and / or the second antigen binding domain that binds the lymphocyte antigen comprise the Fab. In some embodiments, the first antigen binding domain that binds CD33 and / or the second antigen binding domain that binds the lymphocyte antigen comprise the VHH. In some embodiments, the first antigen binding domain that binds CD33 and / or the second antigen binding domain that binds the lymphocyte antigen comprise the scFv. In some embodiments, the scFv comprises, from the N- to C-terminus, a VH, a first linker (LI) and a VL (VH-L1-VL) or the VL, the LI and the VH (VL-L1-VH). In some embodiments, the LI comprises a) about 5-50 amino acids; b) about 5-40 amino acids; c) about 10-30 amino acids; or d) about 10-20 amino acids.

[0067] In some embodiments, the LI comprises the amino acid sequence of any one of SEQ ID NOs: 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, or 140. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 108. In some embodiments, the first antigen binding domain that binds CD33 comprises the HCDR1 of any one of SEQ ID NOs: 1, 2, 3, 4, or 5, the HCDR2 of any one of SEQ ID NOs: 6, 7, 8, 9, 10, or 11, and the HCDR3 of any one of SEQ ID NOs: 12, 13, 14, 15, 16, or 17. In some embodiments, the first antigen binding domain that binds CD33 comprises the HCDR1, the HCDR2, the HCDR3 of a) SEQ ID NOs: 1, 6, and 12, respectively; b) SEQ ID NOs: 2, 7, and 13, respectively; c) SEQ ID NOs: 1, 8, and 14, respectively; d) SEQ ID NOs: 3, 9, and 13, respectively; e) SEQ ID NOs: 4, 10, and 15, respectively; f) SEQ ID NOs: 5, 11, and 16, respectively; or g) SEQ ID NOs: 5, 11, and 17, respectively.

[0068] In some embodiments, the first antigen binding domain that binds CD33 comprises the VH of any one of SEQ ID NOs: 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27. In some embodiments, the first antigen binding domain that binds CD33 comprises the HCDR1 of SEQ ID NOs: 28, 29, 30, 31, 32, 33, 34, or 35, the HCDR2 of SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, or 44, the HCDR3 of SEQ ID NOs: 45, 46,

[0069] 47, 48, 49, 50, or 51, the LCDR1 of SEQ ID NOs: 62, 63, 64, 65, 66, 67, or 68, the LCDR2 of SEQ ID NOs: 69, 70, 71, 72, 73, or 74, and the LCDR3 of SEQ ID NOs: 75, 76, 77, 78, 79, or 80. In some embodiments, the first antigen binding domain that binds CD33 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of a) SEQ ID NOs: 28, 36, 45, 62, 69, and 75, respectively; b) SEQ ID NOs: 29, 37, 46, 63, 70, and 76, respectively; c) SEQ ID NOs: 30, 38, 47, 64, 71, and 77, respectively; d) SEQ ID NOs: 31, 39, 48, 65, 72, and 78, respectively; e) SEQ ID NOs: 32, 40, 49, 66, 70, and 76, respectively; f) SEQ ID NOs: 33, 41, 50, 67, 73, and 79, respectively; g) SEQ ID NOs: 34, 42, 51, 68, 74, and 80, respectively; h) SEQ ID NOs: 34, 43, 51, 68, 74, and 80, respectively; i) SEQ ID NOs: 34, 44, 51, 68, 74, and 80, respectively; j) SEQ ID NOs: 35, 42, 51, 68, 74, and 80, respectively: k) SEQ ID NOs: 253, 256, 259, 265, 74 and 269, respectively: l) SEQ ID NOs: 254, 257, 260, 66, 267 and 76, respectively: or m) SEQ ID NOs: 255, 258, 261, 266, 268 and 270, respectively. In some embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 262, 263 or 264 and the VL of SEQ ID NOs: 81, 82, 83, 84,

[0070] 85, 86, 87, 88, 271, 272 or 273. In some embodiments, the first antigen binding domain that binds CD33 comprises a) the VH of SEQ ID NO: 52 and the VL of SEQ ID NO: 81; b) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 82; c) the VH of SEQ ID NO: 54 and the VL of SEQ ID NO: 83; d) the VH of SEQ ID NO: 55 and the VL of SEQ ID NO: 84; e) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 85; f) the VH of SEQ ID NO: 57 and the VL of SEQ ID NO: 86; g) the VH of SEQ ID NO: 58 and the VL of SEQ ID NO: 87; h) the VH of SEQ ID NO: 59 and the VL of SEQ ID NO: 87; i) the VH of SEQ ID NO: 60 and the VL of SEQ ID NO: 87; j) the VH of SEQ ID NO: 61 and the VL of SEQ ID NO: 88; k) the VH of SEQ ID NO: 262 and the VL of SEQ ID NO: 271; l) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 272; or m) the VH of SEQ ID NO: 264 and the VL of SEQ ID NO: 273.

[0071] In some embodiments, the first antigen binding domain that binds CD33 comprises the HCDR1 of SEQ ID NOs: 33, 89, 167, 172 or 176, the HCDR2 of SEQ ID NOs: 90, 91, 168, 173, or 177, the

[0072] HCDR3 of SEQ ID NOs: 92, 93, 94, 169, 174, 178, or 180, the LCDR1 of SEQ ID NOs: 98, 99, 100, 182,

[0073] 186, 189, 193, 197, or 201, the LCDR2 of SEQ ID NOs: 101, 102, 103, 183, 187, 190, 194, or 198, and the LCDR3 of SEQ ID NO: 104, 184, 191, 195, or 199. In some embodiments, the first antigen binding domain that binds CD33 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of a) SEQ ID NOs: 89, 90, 92, 98, 101, and 104, respectively; b) SEQ ID NOs: 89, 90, 93, 99, 102, and 104, respectively; c) SEQ ID NOs: 33, 91, 94, 100, 103, and 104, respectively; d) SEQ ID NOs: 167, 168, 169, 182, 183, and 184, respectively; e) SEQ ID NOs: 33, 91, 94, 186, 187, and 104, respectively; f) SEQ ID NOs: 172, 173, 174, 189, 190, and 191, respectively; g) SEQ ID NOs: 176, 177, 178, 193, 194, and 195, respectively; h) SEQ ID NOs: 89, 90, 180, 197, 198, and 199, respectively; or i) SEQ ID NOs: 89, 90, 93, 201, 102, and 104, respectively. In some embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NOs: 95, 96, 97, 170, 171, 175, 179, 181, or 96, and the VL of SEQ ID NOs 105, 106, 107, 185, 188,

[0074] 192, 196, 200, or 202. In some embodiments, the first antigen binding domain that binds CD33 comprises a) the VH of SEQ ID NO: 95 and the VL of SEQ ID NO: 105; b) the VH of SEQ ID NO: 96 and the VL of SEQ ID NO: 106; c) the VH of SEQ ID NO: 97 and the VL of SEQ ID NO: 107; d) the VH of SEQ ID NO: 170 and the VL of SEQ ID NO: 185; e) the VH of SEQ ID NO: 171 and the VL of SEQ ID NO: 188; f) the VH of SEQ ID NO: 175 and the VL of SEQ ID NO: 192; g) the VH of SEQ ID NO: 179 and the VL of SEQ ID NO: 196; h) the VH of SEQ ID NO: 181 and the VL of SEQ ID NO: 200; or i) the VH of SEQ ID NO: 96 and the VL of SEQ ID NO: 202.

[0075] In some embodiments, the second antigen binding domain that binds the lymphocyte antigen comprises a) the HCDR1 of SEQ ID NO: 141, the HCDR2 of SEQ ID NO: 142, the HCDR3 of SEQ ID NO: 143, the LCDR1 of SEQ ID NO: 144, the LCDR2 of SEQ ID NO: 145 and the LCDR3 of SEQ ID NO: 146; b) the VH of SEQ ID NO: 147 and the VL of SEQ ID NO: 148; c) the HCDR1 of SEQ ID NO: 149, the HCDR2 of SEQ ID NO: 150, the HCDR3 of SEQ ID NO: 151, the LCDR1 of SEQ ID NO: 152, the LCDR2 of SEQ ID NO: 153 and the LCDR3 of SEQ ID NO: 154; or d) the VH of SEQ ID NO: 155 and the VL of SEQ ID NO: 156.

[0076] In some embodiments, the second antigen binding domain that binds the lymphocyte antigen comprises: a) a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 236, a HCDR2 of SEQ ID NO: 237, a HCDR3 of SEQ ID NO: 238, a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO: 239, a LCDR2 of SEQ ID NO: 240 and a LCDR3 of SEQ ID NO: 241; b) a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 236, a HCDR2 of SEQ ID NO: 237, a HCDR3 of SEQ ID NO: 242, a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO: 239, a LCDR2 of SEQ ID NO: 240 and a LCDR3 of SEQ ID NO: 241; c) a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 236, a HCDR2 of SEQ ID NO: 237, a HCDR3 of SEQ ID NO: 243, a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO: 239, a LCDR2 of SEQ ID NO: 240 and a LCDR3 of SEQ ID NO: 241; d) a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 236, a HCDR2 of SEQ ID NO: 237, a HCDR3 of SEQ ID NO: 244, a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO: 239, a LCDR2 of SEQ ID NO: 240 and a LCDR3 of SEQ ID NO: 241; e) the VH of SEQ ID NO: 245 and the VL of SEQ ID NO: 249; f) the VH of SEQ ID NO: 246 and the VL of SEQ ID NO: 249; g) the VH of SEQ ID NO: 247 and the VL of SEQ ID NO: 249; or h) the VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 249.

[0077] In some embodiments, the first antigen binding domain that binds CD33 is conjugated to a first immunoglobulin (Ig) constant region or a fragment of the first Ig constant region and / or the second antigen binding domain that binds the lymphocyte antigen is conjugated to a second immunoglobulin (Ig) constant region or a fragment of the second Ig constant region. In certain embodiments, the multispecific protein further comprising a second linker (L2) between the first antigen binding domain that binds CD33 and the first Ig constant region or the fragment of the first Ig constant region and the second antigen binding domain that binds the lymphocyte antigen and the second Ig constant region or the fragment of the second Ig constant region. In some embodiments, the L2 comprises the amino acid sequence of any one of SEQ ID NOs: 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, or 140. In some embodiments, the first Ig constant region or the fragment of the first Ig constant region and the second Ig constant region or the fragment of the second Ig constant region is an IgGl, an IgG2, and IgG3 or an IgG4 isotype. In some embodiments, the first Ig constant region or the fragment of the first Ig constant region and the second Ig constant region or the fragment of the second Ig constant region comprises at least one mutation that results in reduced binding of the multispecific protein to a FcyR. In some embodiments, the at least one mutation that results in reduced binding of the multispecific protein to the FcyR is selected from the group consisting of F234A / L235A, L234A / L235A, L234A / L235A / D265S, V234A / G237A / P238S / H268A / V309L / A330S / P331S, F234A / L235A, S228P / F234A / L235A, N297A, V234A / G237A, K214T / E233P / L234V / L235A / G236-deleted / A327G / P331A / D365E / L358M,

[0078] H268Q / V309L / A330S / P33 IS, S267E / L328F, L234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P33 IS, S228P / F234A / L235A / G237A / P238S and S228P / F234A / L235A / G236-deleted / G237A / P238S, wherein residue numbering is according to the EU index.

[0079] In some embodiments, the first Ig constant region or the fragment of the first Ig constant region and the second Ig constant region or the fragment of the second Ig constant region comprises at least one mutation that results in enhanced binding of the multispecific protein to a Fey receptor (FcyR). In some embodiments, the at least one mutation that results in enhanced binding of the multispecific protein to the FcyR is selected from the group consisting of S239D / I332E, S298A / E333A / K334A, F243L / R292P / Y300L, F243L / R292P / Y300L / P396L, F243L / R292P / Y300L / V305I / P396L and G236A / S239D / I332E, wherein residue numbering is according to the EU index. In some embodiments, the FcyR is FcyRI, FcyRIIA, FcyRIIB or FcyRIII, or any combination thereof. In some embodiments, the first Ig constant region or the fragment of the first Ig constant region and the second Ig constant region or the fragment of the second Ig constant region comprises at least one mutation that modulates a half-life of the multispecific protein. In some embodiments, the at least one mutation that modulates the half-life of the multispecific protein is selected from the group consisting of H435A, P257I / N434H, D376V / N434H, M252Y / S254T / T256E / H433K / N434F, T308P / N434A and H435R, wherein residue numbering is according to the EU index. In some embodiments, the isolated multispecific protein comprises at least one mutation in a CH3 domain of the first Ig constant region or in a CH3 domain of the fragment of the first Ig constant region and / or at least one mutation in a CH3 domain of the second Ig constant region or in a CH3 domain of the fragment of the second Ig constant region. In some embodiments, the at least one mutation in a CH3 domain of the first Ig constant region or in a CH3 domain of the fragment of the first Ig constant region and / or at least one mutation in a CH3 domain of the second Ig constant region or in a CH3 domain of the fragment of the second Ig constant region is selected from the group consisting of T350V, L351Y, F405A,Y407V, T366Y, T366W, F405W, T394W, T394S, Y407T, Y407A, T366S / L368A / Y407V, L351Y / F405A / Y407V, T366I / K392M / T394W, F405A / Y407V,

[0080] T366L / K392M / T394 W, L351Y / Y407A, T366A / K409F, L351Y / Y407A, T366V / K409F, T366A / K409F, T350V / L351 Y / F405A / Y407V and T350V / T366L / K392L / T394W, wherein residue numbering is according to the EU index. In some embodiments, the first Ig constant region or the fragment of the first Ig constant region and the second Ig constant region or the fragment of the second Ig constant region comprise the following mutations: a) L235A_L235A_D265S_T350V_L351Y_F405A_Y407V in the first Ig constant region and L235A_L235A_D265S_T350V_T366L_K392L_T394W in the second Ig constant region; or b) L235A_L235A_D265S_T350V_T366L_K392L_T394W in the first Ig constant region and L235A_L235A_D265S_T350V_L351Y_F405A_Y407V in the second Ig constant region.

[0081] The disclosure provides an immunoconjugate comprising any of the above isolated proteins conjugated to a therapeutic agent or an imaging agent. The disclosure provides a pharmaceutical composition comprising any of the above isolated proteins and a pharmaceutically acceptable carrier.

[0082] The disclosure provides a polynucleotide encoding any of the above isolated proteins. The disclosure also provides a vector comprising a polynucleotide encoding any of the above isolated proteins. The disclosure also provides a host cell comprising a vector comprising a polynucleotide encoding any of the above isolated proteins.

[0083] In another aspect the disclosure provides a method of producing any of the above isolated proteins, the method comprising culturing the host cell in conditions that the protein is expressed, and recovering the protein produced by the host cell. In another aspect the disclosure provides an immunoconjugate comprising any of the above isolated multispecific proteins conjugated to a therapeutic agent or an imaging agent.

[0084] In another aspect the disclosure provides a method of producing any of the above isolated multispecific proteins, comprising culturing any host cell described above in conditions that the multispecific protein is expressed, and recovering the multispecific protein produced by the host cell.

[0085] In another aspect the disclosure provides a method of treating a CD33 expressing cancer in a subject, comprising administering a therapeutically effective amount of any of the above isolated proteins, any of the above isolated multispecific proteins, any of the above immunoconjugates, or any of the above pharmaceutical compositions to the subject for a time sufficient to treat the CD33 expressing cancer.

[0086] In another aspect the disclosure provides a method of reducing the amount of CD33 expressing cells in a subject, comprising administering any of the above isolated proteins, any of the above isolated multispecific proteins, any of the above immunoconjugates, or any of the above pharmaceutical compositions to the subject for a time sufficient to reduce the amount of CD33 expressing cells. In some embodiments, the CD33 expressing cells are cancerous cells. In some embodiments, the CD33 expressing cells are not cancerous cells.

[0087] In another aspect the disclosure provides a method of preventing establishment of a CD33 expressing cancer in a subject, comprising administering any of the above isolated proteins, any of the above isolated multispecific proteins, any of the above immunoconjugates, or any of the above pharmaceutical compositions to the subject to prevent establishment of the CD33 expressing cancer in the subject. In another aspect the disclosure provides a method of treating a noncancerous condition in a subject at risk of developing a CD33 expressing cancer, comprising administering any of the above isolated proteins, any of the above isolated multispecific proteins, any of the above immunoconjugates, or any of the above pharmaceutical compositions to the subject to the subject to treat the noncancerous condition.

[0088] In some embodiments of the above three aspects, the CD33 expressing cancer is a hematologic cancer. In some embodiments, the hematologic cancer is a leukemia, a lymphoma, or a multiple myeloma. In some embodiments, the hematologic cancer is acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML) or blastic plasmacytoid dendritic cell neoplasm (DPDCN). In some embodiments, the isolated protein or the isolated multispecific protein is administered in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent is surgery, chemotherapy, or radiation, or any combination thereof. The disclosure also provides a method of detecting the presence of a hematologic cancer in a subject, comprising administering any of the above immunoconjugates to a subject suspected to have the hematologic cancer and visualizing the biological structures to which the immunoconjugate is bound, thereby detecting the presence of the hematologic cancer.

[0089] The disclosure also provides a kit comprising any of the above isolated proteins, any of the above isolated multispecific proteins, any of the above immunoconjugates, or any of the above pharmaceutical compositions.

[0090] The disclosure also provides an anti-idiotypic antibody binding to any of the above isolated proteins.

[0091] The disclosure also provides a chimeric antigen receptor (CAR) comprising: a) an extracellular domain comprising an antigen binding domain that binds CD33; b) a transmembrane domain; and c) an intracellular signaling domain optionally comprising at least one co-stimulatory domain.

[0092] In some embodiments, the CAR further comprises a CD8a-hinge region. a) In some embodiments, the transmembrane domain comprises a CD8a transmembrane region (CD8a-TM) polypeptide; and b) the intracellular signaling domain comprises a co-stimulatory domain comprising a

[0093] TNF receptor superfamily member 9 (CD 137) component and a primary signaling domain comprising a T-cell surface glycoprotein CD3 zeta chain (CD3z) component.

[0094] In some embodiments, a) the CD8a-hinge region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 157; b) the transmembrane domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 162; and / or c) the intracellular signaling domain comprises a co-stimulatory domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 163, and a primary signaling domain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 164.

[0095] In some embodiments, the antigen binding domain that binds CD33 comprises the HCDR1 of any one of SEQ ID NOs: 1, 2, 3, 4, or 5, the HCDR2 of any one of SEQ ID NOs: 6, 7, 8, 9, 10, or 11, and the HCDR3 of any one of SEQ ID NOs: 12, 13, 14, 15, 16, or 17.

[0096] In some embodiments, the antigen binding domain that binds CD33 comprises the HCDR1, the HCDR2, the HCDR3 of a) SEQ ID NOs: 1, 6, and 12, respectively; b) SEQ ID NOs: 2, 7, and 13, respectively; c) SEQ ID NOs: 1, 8, and 14, respectively; d) SEQ ID NOs: 3, 9, and 13, respectively; e) SEQ ID NOs: 4, 10, and 15, respectively; f) SEQ ID NOs: 5, 11, and 16, respectively; or g) SEQ ID NOs: 5, 11, and 17, respectively.

[0097] In some embodiments, the antigen binding domain that binds CD33 comprises the VH of any one of SEQ ID NOs: 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27. In some embodiments, the antigen binding domain that binds CD33 comprises the HCDR1 of SEQ ID NOs: 28, 29, 30, 31, 32, 33, 34, 35, 253, 254 or 255, the HCDR2 of SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, 44, 256, 257 or 258, the HCDR3 of SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51, 259, 260 or 261, the LCDR1 of SEQ ID NOs: 62, 63, 64, 65, 66, 67, 68, 265, or 266, the LCDR2 of SEQ ID NOs: 69, 70, 71, 72, 73, 74, 267 or 268, and the LCDR3 of SEQ ID NOs: 75, 76, 77, 78, 79, 80, 269 or 270. In some embodiments, the antigen binding domain that binds CD33 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of a) SEQ ID NOs: 28, 36, 45, 62, 69, and 75, respectively; b) SEQ ID NOs: 29, 37, 46, 63, 70, and 76, respectively; c) SEQ ID NOs: 30, 38, 47, 64, 71, and 77, respectively; d) SEQ ID NOs: 31, 39, 48, 65, 72, and 78, respectively; e) SEQ ID NOs: 32, 40, 49, 66, 70, and 76, respectively; f) SEQ ID NOs: 33, 41, 50, 67, 73, and 79, respectively; g) SEQ ID NOs: 34, 42, 51, 68, 74, and 80, respectively; h) SEQ ID NOs: 34, 43, 51, 68, 74, and 80, respectively; i) SEQ ID NOs: 34, 44, 51, 68, 74, and 80, respectively; j) SEQ ID NOs: 35, 42, 51, 68, 74, and 80, respectively; k) SEQ ID NOs: 253, 256, 259, 265, 74 and 269, respectively: l) SEQ ID NOs: 254, 257, 260, 66, 267 and 76, respectively: or m) SEQ ID NOs: 255, 258, 261, 266, 268 and 270, respectively.

[0098] In some embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 262, 263 or 264 and the VL of SEQ ID NOs: 81, 82, 83, 84, 85, 86, 87, 88, 271, 272, 273. In some embodiments, the antigen binding domain that binds CD33 comprises a) the VH of SEQ ID NO: 52 and the VL of SEQ ID NO: 81; b) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 82; c) the VH of SEQ ID NO: 54 and the VL of SEQ ID NO: 83; d) the VH of SEQ ID NO: 55 and the VL of SEQ ID NO: 84; e) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 85; f) the VH of SEQ ID NO: 57 and the VL of SEQ ID NO: 86; g) the VH of SEQ ID NO: 58 and the VL of SEQ ID NO: 87; h) the VH of SEQ ID NO: 59 and the VL of SEQ ID NO: 87; i) the VH of SEQ ID NO: 60 and the VL of SEQ ID NO: 87; j) the VH of SEQ ID NO: 61 and the VL of SEQ ID NO: 88 k) the VH of SEQ ID NO: 262 and the VL of SEQ ID NO: 271; l) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 272; or m) the VH of SEQ ID NO: 264 and the VL of SEQ ID NO: 273.

[0099] In some embodiments, the antigen binding domain that binds CD33 comprises the HCDR1 of SEQ ID NOs: 33, 89, 167, 172 or 176, the HCDR2 of SEQ ID NOs: 90, 91, 168, 173, or 177, the HCDR3 of SEQ ID NOs: 92, 93, 94, 169, 174, 178, or 180, the LCDR1 of SEQ ID NOs: 98, 99, 100, 182, 186, 189, 193, 197, or 201, the LCDR2 of SEQ ID NOs: 101, 102, 103, 183, 187, 190, 194, or 198, and the LCDR3 of SEQ ID NO: 104, 184, 191, 195, or 199. In some embodiments, the antigen binding domain that binds CD33 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of a) SEQ ID NOs: 89, 90, 92, 98, 101, and 104, respectively; b) SEQ ID NOs: 89, 90, 93, 99, 102, and 104, respectively; c) SEQ ID NOs: 33, 91, 94, 100, 103, and 104, respectively; d) SEQ ID NOs: 167, 168, 169, 182, 183, and 184, respectively; e) SEQ ID NOs: 33, 91, 94, 186, 187, and 104, respectively; f) SEQ ID NOs: 172, 173, 174, 189, 190, and 191, respectively; g) SEQ ID NOs: 176, 177, 178, 193, 194, and 195, respectively; h) SEQ ID NOs: 89, 90, 180, 197, 198, and 199, respectively; or i) SEQ ID NOs: 89, 90, 93, 201, 102, and 104, respectively.

[0100] In some embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NOs: 95, 96, 97, 170, 171, 175, 179, 181, or 96, and the VL of SEQ ID NOs 105, 106, 107, 185, 188,

[0101] 192, 196, 200, or 202. In some embodiments, the antigen binding domain that binds CD33 comprises a) the VH of SEQ ID NO: 95 and the VL of SEQ ID NO: 105; b) the VH of SEQ ID NO: 96 and the VL of SEQ ID NO: 106; c) the VH of SEQ ID NO: 97 and the VL of SEQ ID NO: 107; d) the VH of SEQ ID NO: 170 and the VL of SEQ ID NO: 185; e) the VH of SEQ ID NO: 171 and the VL of SEQ ID NO: 188; f) the VH of SEQ ID NO: 175 and the VL of SEQ ID NO: 192; g) the VH of SEQ ID NO: 179 and the VL of SEQ ID NO: 196; h) the VH of SEQ ID NO: 181 and the VL of SEQ ID NO: 200; or i) the VH of SEQ ID NO: 96 and the VL of SEQ ID NO: 202.

[0102] In some embodiments, the antigen binding domain that binds CD33 is a VHH.

[0103] In some embodiments, the antigen binding domain that binds CD33 is a scFv.

[0104] In some embodiments, the scFv comprises a first linker (LI) between the VL and the VH.

[0105] In certain embodiments, the LI comprises an amino acid sequence of any one of SEQ ID NOs:

[0106] 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, or 140.

[0107] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 108.

[0108] In some embodiments, the extracellular domain of the CAR comprising the antigen binding domain that binds CD33 further comprises a signal polypeptide. In some embodiments, the intracellular signaling domain comprises a polypeptide component selected from the group consisting of a TNF receptor superfamily member 9 (CD 137) component, a T-cell surface glycoprotein CD3 zeta chain (CD3z) component, a cluster of differentiation (CD27) component, a cluster of differentiation superfamily member component, and a combination thereof. In some embodiments, the CD 137 component comprises the amino acid sequence of SEQ ID NO: 163. In some embodiments, the CD3z component comprises an amino acid sequence of SEQ ID NO: 164. In some embodiments, the intracellular signaling domain comprises an amino acid sequence of SEQ ID NO: 165. In some embodiments, the CD8a-TM polypeptide comprises an amino acid sequence of SEQ ID NO: 162. In some embodiments, the CD8a- hinge region comprises an amino acid sequence of SEQ ID NO: 157.

[0109] In some embodiments, the CAR is a multispecific protein. In some embodiments, the multispecific protein is a bispecific protein. In some embodiments, the multispecific protein is a trispecific protein. In some embodiments, the multispecific protein comprises an antigen binding domain that binds an antigen on a lymphocyte. In some embodiments, the lymphocyte is a T cell. In some embodiments, the T cell is a CD8+T cell. In some embodiments, the lymphocyte is a natural killer (NK) cell. In some embodiments, the multispecific protein comprises an antigen binding domain that binds CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD 186, BTNL8, PD-1, CD195, TRGV9, or NKG2C. In some embodiments, the multispecific protein comprises an antigen binding domain that binds CD3ε. In some embodiments, the antigen binding domain that binds CD3ε comprises: a) a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 141, a HCDR2 of SEQ ID NO: 142, a HCDR3 of SEQ ID NO: 143, a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO: 144, a LCDR2 of SEQ ID NO: 145 and a LCDR3 of SEQ ID NO: 146; b) the VH of SEQ ID NO: 147 and the VL of SEQ ID NO: 148; c) the HCDR1 of SEQ ID NO: 149, the HCDR2 of SEQ ID NO: 150, the HCDR3 of SEQ ID NO:

[0110] 151, the LCDR1 of SEQ ID NO: 152, the LCDR2 of SEQ ID NO: 153 and the LCDR3 of SEQ ID NO: 154; or d) the VH of SEQ ID NO: 155 and the VL of SEQ ID NO: 156.

[0111] In some embodiments, the multispecific protein comprises an antigen binding domain that binds TRGV9. In some embodiments, the antigen binding domain that binds TRGV9 comprises: a) a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 236, a HCDR2 of SEQ ID NO: 237, a HCDR3 of SEQ ID NO: 238, a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO: 239, a LCDR2 of SEQ ID NO: 240 and a LCDR3 of SEQ ID NO: 241; b) a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 236, a HCDR2 of SEQ ID NO: 237, a HCDR3 of SEQ ID NO: 242, a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO: 239, a LCDR2 of SEQ ID NO: 240 and a LCDR3 of SEQ ID NO: 241; c) a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 236, a HCDR2 of SEQ ID NO: 237, a HCDR3 of SEQ ID NO: 243, a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO: 239, a LCDR2 of SEQ ID NO: 240 and a LCDR3 of SEQ ID NO: 241; d) a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 236, a HCDR2 of SEQ ID NO: 237, a HCDR3 of SEQ ID NO: 244, a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO: 239, a LCDR2 of SEQ ID NO: 240 and a LCDR3 of SEQ ID NO: 241; e) the VH of SEQ ID NO: 245 and the VL of SEQ ID NO: 249; f) the VH of SEQ ID NO: 246 and the VL of SEQ ID NO: 249; g) the VH of SEQ ID NO: 247 and the VL of SEQ ID NO: 249; or h) the VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 249.

[0112] In another aspect, the disclosure provides an isolated lymphocyte expressing any of the CARs above. In some embodiments, the lymphocyte is a T lymphocyte. In some embodiments, the lymphocyte is a natural killer (NK) cell. In another aspect, the disclosure provides an isolated polynucleotide encoding any of the above CARs. The disclosure also provides a vector comprising an isolated polynucleotide encoding any of the above CARs. The disclosure provides a host cell comprising the isolated polynucleotide or the vector. The disclosure also provides a pharmaceutical composition comprising any of the above lymphocytes and a pharmaceutically acceptable excipient.

[0113] In another aspect, the disclosure provides a method of treating a subject having a CD33 expressing cancer, the method comprising: administering a therapeutically effective amount of any of the above lymphocytes to a subject in need thereof, whereby the lymphocyte mediates killing of the CD33 expressing cancer in the subject. In some embodiments, the CD33 expressing cancer is a hematologic cancer. In some embodiments, the CD33 expressing cancer is a leukemia, a lymphoma, or a multiple myeloma. In some embodiments, the CD33 expressing cancer is acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML) or blastic plasmacytoid dendritic cell neoplasm (DPDCN).

[0114] In another aspect is provided a method of targeted killing of a cancer cell, the method comprising: contacting the cancer cell with any of the above lymphocytes, whereby the lymphocyte induces targeted killing of the cancer cell. In some embodiments, the cancer cell is a hematologic cancer. In some embodiments, the CD33 expressing cancer is a leukemia, a lymphoma, or a multiple myeloma. In some embodiments, the CD33 expressing cancer is acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML) or blastic plasmacytoid dendritic cell neoplasm (DPDCN). In another aspect is provided a method of detecting the presence of a cancer in a subject, comprising: a) contacting a cell sample obtained from the subject with any of the above CARs, thereby forming a CAR-cell complex, and b) detecting the CAR-cell complex, wherein detection of the CAR-cell complex is indicative of the presence of the cancer in the subject.

[0115] In some embodiments, the cancer cell is a hematologic cancer. In some embodiments, the CD33 expressing cancer is a leukemia, a lymphoma, or a multiple myeloma. In some embodiments, the CD33 expressing cancer is acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML) or blastic plasmacytoid dendritic cell neoplasm (DPDCN).

[0116] In another aspect is provided an immunoconjugate comprising any of the above isolated proteins conjugated to a radioactive agent. In some embodiments, the radioactive agent comprises a radiometal ion. In some embodiments, the radiometal ion is32P,47Sc,67Cu,77As,89Sr,90Y,99Tc,105Rh,109Pd,111Ag,131I,153Sm,159Gd,165Dy,166Ho,169Er,177Lu,186Re,188Re,194Ir,198Au,199Au,211At,212Pb,212Bi,213Bi,223Ra,225Ac,255Fm,227Th,62Cu,64Cu,67Ga,68Ga,86Y,89Zr, or111In. In certain embodiments, the radiometal ion is225Ac,1 1 1In or89Zr. In certain embodiments, the radiometal ion is225Ac. In some embodiments, the radiometal ion of the immunoconjugate is conjugated to a chelating moiety. In some embodiments, the chelating moiety comprises a macrocycle having the structure of formula (I): wherein each of Ri, R2, R3and Rus independently CHQCO2X, wherein Q is independently hydrogen, C1-C4alkyl or (C1-C2alkyl) phenyl, and X is independently hydrogen, benzyl, C1-C4alkyl; and Z is (CH2)nY, wherein n is 1-10, and

[0117] Y is an electrophilic or nucleophilic moiety covalently linked to the second click reaction partner; alternatively, Z is hydrogen; and each of Ri, R2, R3and R4is independently CHQCO2X, wherein Q is independently hydrogen, C1-C4alkyl or (C1-C2alkyl) phenyl, and X is independently hydrogen, benzyl, C1-C4alkyl, or an electrophilic or nucleophilic moiety covalently linked to the second click reaction partner; alternatively, the chelant comprises an open chain ligand.

[0118] In some embodiments, the chelating moiety comprises a macrocycle having the structure of formula (II): or a structure of formula (III):

[0119] The disclosure also provides an immunoconjugate of formula (IV), formula (IV) where the protein is any of the above isolated proteins.

[0120] The disclosure also provides an immunoconjugate of formula (V),

[0121] formula (V) where the protein is any of the above isolated proteins.

[0122] The disclosure provides a pharmaceutical composition comprising a means for treating a CD33 expressing cancer in a subject.

[0123] The disclosure provides a pharmaceutical composition comprising a means for reducing the amount of CD33 expressing tumor cells in a subject.

[0124] The disclosure provides a pharmaceutical composition comprising a means for preventing establishment of a CD33 expressing cancer in a subject.

[0125] The disclosure provides a pharmaceutical composition comprising a means for treating a noncancerous condition in a subject at risk of developing a CD33 expressing cancer.

[0126] The disclosure provides a method of beating a CD33 expressing cancer in a subject, the method comprising administering to the subject a means for targeting a therapeutic agent to a CD33 expressing cancer cell in the subject.

[0127] The disclosure provides a method of reducing the amount of CD33 expressing tumor cells in a subject, the method comprising administering to the subject a means for targeting a therapeutic agent to one or more of the CD33 expressing tumor cells in the subject.

[0128] The disclosure provides a pharmaceutical composition comprising a means for preventing establishment of a CD33 expressing cancer in a subject, the method comprising administering to the subject a means for targeting a therapeutic agent to a CD33 expressing cell in the subject.

[0129] The disclosure provides a pharmaceutical composition comprising a means for treating a noncancerous condition in a subject at risk of developing a CD33 expressing cancer, the method comprising administering to the subject a means for targeting a therapeutic agent to a CD33 expressing cell in the subject.

[0130] BRIEF DESCRIPTION OF THE DRAWINGS

[0131] This patent application fde contains at least one drawing executed in color. Copies of this patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. FIG. 1 shows representative BIAcore 8K sensorgrams of anti-CD33 vHHs interactions against hu CD33 antigens (FL ECD and IgC2 domain).

[0132] FIGS. 2A and 2B show binding of CD33 antibodies to target cell lines. Graphs show the non linear regression profiles of anti-human CD33 antibodies at 4°C. Median fluorescence intensity (MFI) versus log concentration of antibody are plotted. Binding was performed by incubating the cells with the serial dilutions of the antibody at 4°C followed by detection with PE conjugated Goat F(ab')2 Anti- Human IgG - Fc antibody. The top hit for each cell line is indicated in red, and Lintuzumab binding is shown in blue curves.

[0133] FIGS. 3 show time and temperature dependent binding of different CD33 antibodies to target cell lines. Graphs show the non-linear regression profiles of binding of anti-human CD33 antibodies at 37°C and 4°C. Median fluorescence intensity (MFI) versus log concentrations of antibody are plotted. Binding was performed by incubating the cells with the serial dilutions of the directly conjugated antibodies at 37°C and 4°C.

[0134] FIGS. 4A-4D show internalization of CD33 antibodies on high, medium and low expression target cell lines. Cells were cultured with serial dilutions of the CD33 antibodies along with Protein A- MMAF for 96hrs. After 96 hours, CellTitre-Glo reagent was added to cells to measure cell viability. Wells with Protein A-MMAF alone were considered as 100% viable. HDLM-2 cells were used as a no expression cell line. A decrease in cell viability indicates antibody internalization. Graphs indicate non linear regression analysis for cell viability against log concentration of antibody.

[0135] FIG. 5 shows kinetics of CD33 antibody internalization. CD33 antibodies were labeled with pHAb dyes and incubated with the cells at a concentration of lOOnM at 37°C for the indicated time points following which the cells were washed and acquired on the flow cytometer. Graphs show MFI values at indicated time points.

[0136] FIG. 6 shows CD33 expression on target cell lines. High, medium and low CD33 expression cell lines were identified using anti-human CD33 antibody from Biolegend. MOLM-13, Kasumi-1, OCI- AML-3 and HDLM2 cells were stained with the antibody (orange histograms) or the mouse IgG isotype control (blue histograms) and fold change over isotype for each cell line was calculated.

[0137] FIG. 7 shows antibody integrity and DAR values post conjugation with pHAb dyes. Antibodies were labeled with pHAb dyes and analyzed on SDS-PAGE. Drug to antibody ratios (DAR) were calculated by measuring the OD at 280 and 530 nm and correcting for the dye absorbance at 280nm.

[0138] FIGS. 8A and 8B show specificity of pHAb mediated antibody internalization kinetics determination. In FIG. 8A, CD33 antibodies were labeled with pHAb dyes and incubated with the Kasumi-1 or OCI-AML-3 cells at a concentration of lOOnM at 4°C for the indicated time points following which the cells were washed and acquired on the flow cytometer. Graphs show MFI values at indicated time points. No internalization was observed at 4C. In FIG. 8B, antibodies were also incubated with HDLM-2 cells at 37°C. No increase in MFI was observed.

[0139] FIG. 9 shows a summary of the binding EC50 values of CD33 antibodies to high (MOLM-13), medium (Kasumi-1) and low (OCI -AML-3) cell lines. Binding was carried out at 4°C with serial dilutions of the antibody.

[0140] FIG. 10 shows a summary of CD33 antibody internalization on various target cell lines. For MOLM-13 and OCI-AML-3, EC50 values are indicated. For Kasumi-1, % viability at lOOnM concentration.

[0141] FIG. 11 shows a schematic demonstrating the binding of an anti-TRGV9 / anti-CD33 bispecific antibody to recruit gd T-cells to a cancer cell that is CD33+ and to induce cancer cell death.

[0142] FIG. 12 shows the results of an assay of the integrity of the VG4 bispecific antibody by an SDS- PAGE (non-reducing) gel.

[0143] FIGS. 13-15 show the results of assays of the binding of anti-CD33 antibody (clone C33B904) to a panel of tumor cell lines were measured by FACS. The EC50 for: MOLM-13 (high receptor density) was 134.3 nM, Kasumi-1 (moderate density) was 82.2 nM and OCI-AML-3 (low surface density) was 16.4 nM.

[0144] FIGS. 16-17 show the results of an experiment demonstrating that the anti-TRGV9 / anti-CD33 bispecific antibody mediates gd T cell cytotoxicity against CD33 expressing Kasumi-3 cells in vitro. In the experiment, enriched gd T cells (Effectors), isolated from PBMCs cultured with Zoledronic acid + IL- 2 + IL-15 for 12 days, were co-cultured with CFSE labelled Kasumi-3 cells (Targets) at 1:1 and 5:1 E:T ratios in the presence of various concentrations of the bispecific antibody for 24 hours. Dose response curves show anti-TRGV9 / anti-CD33 and anti-TRGV9 / anti-NULL bispecific antibodies mediated gd T cell cytotoxicity against CD33 expressing kasumi-3 cells in a dose dependent manner at 1 : 1 and 5: 1 E:T ratios. Cytotoxicity values represented here were subtracted of basal cytotoxicity value observed in the absence of bispecific antibody. EC50 values were calculated as described in methods. Representative data are shown here are from a single experiment.

[0145] FIG. 18 shows the results of experiments demonstrating that the anti-TRGV9 / anti-CD33 bispecific antibody mediates gd T cell cytotoxicity against CD33 expressing Kasumi-3 cells in vitro. Healthy donor derived PBMCs (Effectors), cultured with Zoledronic acid + IL-2 + IL-15 for 12 days, were co-cultured with CFSE labelled Kasumi-3 cells (Targets) at 1: 1 E:T ratios in the presence of various concentrations of the bispecific antibody for 24 hours.

[0146] FIG. 19 shows in silico analysis of potential Post-Translational Modifications (PTMs) risk.

[0147] Some potential PTM risk motifs (DG, NG, DP) are highlighted. FIG. 20 shows the results of assays to evaluate the thermostability of scFvs generated from NNK library based on C33B1475 (SEQ ID NO: 274). ELISAs were performed on each variant in order to assess thermostability. E. coli supernatants were heat treated at 55C, 60C, 65C and luminescence signals were normalized to untreated room temperature of the same clone.

[0148] FIG. 21 shows the results of assay to evaluate the thermostability of scFv generated from NNK library based on C33B1516 (SEQ ID NO: 275). ELISAs were performed on each variant in order to assess thermostability. E. coli supernatants were heat treated at 55C, 60C, 65C and luminescence signals were normalized to untreated room temperature of the same clone.

[0149] FIG. 22 shows the results of assay to evaluate the thermostability of scFv generated from NNK library based on C33B1517 (SEQ ID NO: 216). ELISAs were performed on each variant in order to assess thermostability. E. coli supernatants were heat treated at 55C, 60C, 65C and luminescence signals were normalized to untreated room temperature of the same clone.

[0150] FIG. 23 shows the results of assay to evaluate the thermostability of scFv generated from NNK library based on C33B1522 (SEQ ID NO: 213). ELISAs were performed on each variant in order to assess thermostability. E. coli supernatants were heat treated at 55C, 60C, 65C and luminescence signals were normalized to untreated room temperature of the same clone.

[0151] FIG. 24 is a table listing variant VH and VL sequences identified during library screen of NNK library.

[0152] DETAILED DESCRIPTION

[0153] The disclosed methods may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures, which form a part of this disclosure. It is to be understood that the disclosed methods are not limited to the specific methods described and / or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed methods.

[0154] All patents, published patent applications and publications cited herein are incorporated by reference as if set forth fully herein.

[0155] When a list is presented, unless stated otherwise, it is to be understood that each individual element of that list, and every combination of that list, is a separate embodiment. For example, a list of embodiments presented as “A, B, or C” is to be interpreted as including the embodiments, “A,” “B,” “C,” “A or B,” “A or C,” “B or C,” or “A, B, or C.”

[0156] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like. The transitional terms “comprising,” “consisting essentially of,” and “consisting of’ are intended to connote their generally accepted meanings in the patent vernacular; that is, (i) “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; (ii) “consisting of’ excludes any element, step, or ingredient not specified in the claim; and (iii) “consisting essentially of’ limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. Embodiments described in terms of the phrase “comprising” (or its equivalents) also provide as embodiments those independently described in terms of “consisting of’ and “consisting essentially of.”

[0157] “About” means 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, i.e., the limitations of the measurement system. Unless explicitly stated otherwise within the Examples or elsewhere in the Specification in the context of a particular assay, result or embodiment, “about” means within one standard deviation per the practice in the art, or a range of up to 5%, whichever is larger.

[0158] “Activation” or “stimulation” or “activated” or “stimulated” refers to induction of a change in the biologic state of a cell resulting in expression of activation markers, cytokine production, proliferation or mediating cytotoxicity of target cells. Cells may be activated by primary stimulatory signals. Co stimulatory signals can amplify the magnitude of the primary signals and suppress cell death following initial stimulation resulting in a more durable activation state and thus a higher cytotoxic capacity. A “co stimulatory signal” refers to a signal, which in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell and / or NK cell proliferation and / or upregulation or downregulation of key molecules.

[0159] “Alternative scaffold” refers to a single chain protein framework that contains a structured core associated with variable domains of high conformational tolerance. The variable domains tolerate variation to be introduced without compromising scaffold integrity, and hence the variable domains can be engineered and selected for binding to a specific antigen.

[0160] “Antibody-dependent cellular cytotoxicity”, “antibody-dependent cell-mediated cytotoxicity” or “ADCC" refers to the mechanism of inducing cell death that depends upon the interaction of antibody -coated target cells with effector cells possessing lytic activity, such as natural killer cells (NK), monocytes, macrophages and neutrophils via Fc gamma receptors (FcyR) expressed on effector cells.

[0161] “Antibody-dependent cellular phagocytosis” or “ADCP” refers to the mechanism of elimination of antibody -coated target cells by internalization by phagocytic cells, such as macrophages or dendritic cells. “Antigen” refers to any molecule (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, portions thereof, or combinations thereof) capable of being bound by an antigen binding domain or a T-cell receptor that is capable of mediating an immune response. Exemplary immune responses include antibody production and activation of immune cells, such as T cells, B cells or NK cells. Antigens may be expressed by genes, synthetized, or purified from biological samples such as a tissue sample, a tumor sample, a cell or a fluid with other biological components, organisms, subunits of proteins / antigens, killed or inactivated whole cells or lysates.

[0162] “Antigen binding fragment” or “antigen binding domain” refers to a portion of the protein that binds an antigen. Antigen binding fragments may be synthetic, enzymatically obtainable or genetically engineered polypeptides and include portions of an immunoglobulin that bind an antigen, such as VH, the VL, the VH and the VL, Fab, Fab’, F(ab')2, Fd and Fv fragments, domain antibodies (dAb) consisting of one VH domain or one VL domain, shark variable IgNAR domains, camelized VH domains, VHH domains, minimal recognition units consisting of the amino acid residues that mimic the CDRs of an antibody, such as FR3-CDR3-FR4 portions, the HCDR1, the HCDR2 and / or the HCDR3 and the LCDR1, the LCDR2 and / or the LCDR3, alternative scaffolds that bind an antigen, and multispecific proteins comprising the antigen binding fragments. Antigen binding fragments (such as VH and VL) may be linked together via a synthetic linker to form various types of single antibody designs where the VH / VL domains may pair intramolecularly, or intermolecularly in those cases when the VH and VL domains are expressed by separate single chains, to form a monovalent antigen binding domain, such as single chain Fv (scFv) or diabody. Antigen binding fragments may also be conjugated to other antibodies, proteins, antigen binding fragments or alternative scaffolds which may be monospecific or multispecific to engineer bispecific and multispecific proteins.

[0163] “Antibodies” is meant in a broad sense and includes immunoglobulin molecules including monoclonal antibodies including murine, human, humanized and chimeric monoclonal antibodies, antigen binding fragments, multispecific antibodies, such as bispecific, trispecific, tetraspecific etc., dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies and any other modified configuration of the immunoglobulin molecule that comprises an antigen binding site of the required specificity. “Full length antibodies” are comprised of two heavy chains (HC) and two light chains (LC) inter-connected by disulfide bonds as well as multimers thereof (e.g. IgM). Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (comprised of domains CHI, hinge, CH2 and CH3). Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The VH and the VL regions may be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with framework regions (FR). Each VH and VL is composed of three CDRs and four FR segments, arranged from amino-to-carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Immunoglobulins may be assigned to five major classes, IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgAl, IgA2, IgGl, IgG2, IgG3 and IgG4. Antibody light chains of any vertebrate species may be assigned to one of two clearly distinct types, namely kappa (K) and lambda (l), based on the amino acid sequences of their constant domains.

[0164] “Bispecific” refers to a molecule (such as an antibody) that specifically binds two distinct antigens or two distinct epitopes within the same antigen. The bispecific molecule may have cross reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca cynomolgus (cynomolgus, cyno) or Pan troglodytes, or may bind an epitope that is shared between two or more distinct antigens.

[0165] “Cancer” refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and may also metastasize to distant parts of the body through the lymphatic system or bloodstream. A “cancer” or “cancer tissue” can include a tumor.

[0166] “Chimeric antigen receptor” (CAR) as used herein is defined as a cell-surface receptor comprising an extracellular target-binding domain, a transmembrane domain and an intracellular signaling domain, all in a combination that is not naturally found together on a single protein. This includes receptors wherein the extracellular domain and the intracellular signaling domain are not naturally found together on a single receptor protein. CARs are intended primarily for use with lymphocyte such as T cells and natural killer (NK) cells.

[0167] “Complement-dependent cytotoxicity” or ”CDC”, refers to the mechanism of inducing cell death in which the Fc effector domain of a target-bound protein binds and activates complement component Clq which in turn activates the complement cascade leading to target cell death. Activation of complement may also result in deposition of complement components on the target cell surface that facilitate CDC by binding complement receptors (e.g., CR3) on leukocytes

[0168] “Complementarity determining regions” (CDR) are antibody regions that bind an antigen. There are three CDRs in the VH (HCDR1, HCDR2, HCDR3) and three CDRs in the VL (LCDR1, LCDR2, LCDR3). CDRs may be defined using various delineations such as Rabat (Wu et al. (1970) J Exp Med 132: 211-50; Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al. (1987) J Mol Biol 196: 901-17), IMGT (Leffanc et al. (2003) Dev Comp Immunol 27: 55-77), AbM (Martin and Thornton J Bmol Biol 263: 800-15, 1996), and Contact, which is based on an analysis of the available complex crystal structures (MacCallum, R. M., Martin, A. C. R. and Thornton, J. T. “Antibody-antigen interactions: Contact analysis and binding site topography” J. Mol. Biol. 262:732-745). The correspondence between the various delineations and variable region numbering is described (see e.g. Lefranc et al. (2003) Dev Comp Immunol 27: 55-77; Honegger and Pluckthun, J Mol Biol (2001) 309:657-70; International ImMunoGeneTics (IMGT) database; Web resources, www.imgt.org).

[0169] Available programs such as abYsis by UCL Business PLC may be used to delineate CDRs. The term “CDR”, “HCDRT”, “HCDR2”, “HCDR3”, “LCDRT”, “LCDR2” and “LCDR3” as used herein includes CDRs defined by any of the methods described supra, Rabat, Chothia, IMGT, AbM, or Contact, unless otherwise explicitly stated in the specification. The CDRs of the sequences with SEQ ID NO. 1 to 17, 28 to 51, 62 to 80, 89 to 94, 98 to 104, 141 to 146, 149 to 154, 167 to 169, 172 to 174, 176 to 178, 180, 182 to 184, 186, 187, 189 to 191, 193 to 195, 197 to 199, 201, 236 to 244, 253 to 261 and 265 to 270 are defined according to Rabat. The CDRs of the sequences with SEQ ID NO. 399 to 434 are defined according to AbM. The CDRs of the sequences with SEQ ID NO. 435 to 470, are defined according to Chothia. The CDRs of the sequences with SEQ ID NO. 471 to 506are defined according to IMGT. The CDRs of the sequences with SEQ ID NO. 507 to 542are defined according to Contact.

[0170] “Decrease,” “lower,” “lessen,” “reduce,” or “abate” refers generally to the ability of a test molecule to mediate a reduced response (i.e., downstream effect) when compared to the response mediated by a control or a vehicle. Exemplary responses are T cell expansion, T cell activation or T-cell mediated tumor cell killing or binding of a protein to its antigen or receptor, enhanced binding to a Fey or enhanced Fc effector functions such as enhanced ADCC, CDC and / or ADCP. Decrease may be a statistically significant difference in the measured response between the test molecule and the control (or the vehicle), or a decrease in the measured response, such as a decrease of about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 30 fold or more, such as 500, 600, 700, 800, 900 or 1000 fold or more (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.).

[0171] “Differentiation” refers to a method of decreasing the potency or proliferation of a cell or moving the cell to a more developmental^ restricted state.

[0172] “Encode” or “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence, and the non coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. “Enhance,” “promote,” “increase,” “expand” or “improve” refers generally to the ability of a test molecule to mediate a greater response (i.e., downstream effect) when compared to the response mediated by a control or a vehicle. Exemplary responses are T cell expansion, T cell activation or T-cell mediated tumor cell killing or binding of a protein to its antigen or receptor, enhanced binding to a Fey or enhanced Fc effector functions such as enhanced ADCC, CDC and / or ADCP. Enhance may be a statistically significant difference in the measured response between the test molecule and control (or vehicle), or an increase in the measured response, such as an increase of about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 30 fold or more, such as 500, 600, 700, 800, 900 or 1000 fold or more (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.).

[0173] “Expansion” refers to the outcome of cell division and cell death.

[0174] “Express” and “expression” refers the to the well-known transcription and translation occurring in cells or in vitro. The expression product, e.g., the protein, is thus expressed by the cell or in vitro and may be an intracellular, extracellular or a transmembrane protein.

[0175] “Expression vector” refers to a vector that can be utilized in a biological system or in a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0176] “dAb” or “dAb fragment" refers to an antibody fragment composed of a VH domain (Ward et al., Nature 341:544546 (1989)).

[0177] “Fab” or "Fab fragment" refers to an antibody fragment composed of VH, CHI, VL and CL domains.

[0178] “F(ab')2” or “F(ab')2fragment" refers to an antibody fragment containing two Fab fragments connected by a disulfide bridge in the hinge region.

[0179] “Fd” or “Fd fragment" refers to an antibody fragment composed of VH and CHI domains.

[0180] “Fv” or "Fv fragment" refers to an antibody fragment composed of the VH and the VL domains from a single arm of the antibody.

[0181] “Full length antibody” is comprised of two heavy chains (HC) and two light chains (LC) inter connected by disulfide bonds as well as multimers thereof (e.g. IgM). Each heavy chain is comprised of a heavy chain variable domain (VH) and a heavy chain constant domain, the heavy chain constant domain comprised of subdomains CHI, hinge, CH2 and CH3. Each light chain is comprised of a light chain variable domain (VL) and a light chain constant domain (CL). The VH and the VLmay be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with framework regions (FR). Each VH and VL is composed of three CDRs and four FR segments, arranged from amino-to-carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. “Genetic modification” refers to the introduction of a “foreign” (i.e.. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. The introduced gene or sequence may also be called a “cloned” or “foreign” gene or sequence, may include regulatory or control sequences operably linked to polynucleotide encoding the chimeric antigen receptor, such as start, stop, promoter, signal, secretion, or other sequences used by a cell's genetic machinery. The gene or sequence may include nonfunctional sequences or sequences with no known function. A host cell that receives and expresses introduced DNA or RNA has been “genetically engineered.” The DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell, or from a different genus or species.

[0182] “Heterologous” refers to two or more polynucleotides or two or more polypeptides that are not found in the same relationship to each other in nature.

[0183] “Heterologous polynucleotide” refers to a non-naturally occurring polynucleotide that encodes two or more neoantigens as described herein.

[0184] “Heterologous polypeptide” refers to a non-naturally occurring polypeptide comprising two or more neoantigen polypeptides as described herein.

[0185] “Host cell” refers to any cell that contains a heterologous nucleic acid. An exemplary heterologous nucleic acid is a vector (e.g., an expression vector).

[0186] “Human antibody” refers to an antibody that is optimized to have minimal immune response when administered to a human subject. Variable regions of human antibody are derived from human immunoglobulin sequences. If human antibody contains a constant region or a portion of the constant region, the constant region is also derived from human immunoglobulin sequences. Human antibody comprises heavy and light chain variable regions that are “derived from” sequences of human origin if the variable regions of the human antibody are obtained from a system that uses human germline immunoglobulin or rearranged immunoglobulin genes. Such exemplary systems are human immunoglobulin gene libraries displayed on phage, and transgenic non-human animals such as mice or rats carrying human immunoglobulin loci. “Human antibody” typically contains amino acid differences when compared to the immunoglobulins expressed in humans due to differences between the systems used to obtain the human antibody and human immunoglobulin loci, introduction of somatic mutations or intentional introduction of substitutions into the frameworks or CDRs, or both. Typically, “human antibody” is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical in amino acid sequence to an amino acid sequence encoded by human germline immunoglobulin or rearranged immunoglobulin genes. In some cases, “human antibody” may contain consensus framework sequences derived from human framework sequence analyses, for example as described in Knappik et al., (2000) J Mol Biol 296:57-86, or a synthetic HCDR3 incorporated into human immunoglobulin gene libraries displayed on phage, for example as described in Shi et al., (2010) J Mol Biol 397:385-96, and in Int. Patent Publ. No. W02009 / 085462. Antibodies in which at least one CDR is derived from a non-human species are not included in the definition of “human antibody”.

[0187] “Humanized antibody” refers to an antibody in which at least one CDR is derived from non human species and at least one framework is derived from human immunoglobulin sequences.

[0188] Humanized antibody may include substitutions in the frameworks so that the frameworks may not be exact copies of expressed human immunoglobulin or human immunoglobulin germline gene sequences.

[0189] “In combination with” means that two or more therapeutic agents are be administered to a subject together in a mixture, concurrently as single agents or sequentially as single agents in any order.

[0190] “Intracellular signaling domain” or “cytoplasmic signaling domain” refers to an intracellular portion of a molecule. It is the functional portion of the protein which acts by transmitting information within the cell to regulate cellular activity via defined signaling pathways by generating second messengers or functioning as effectors by responding to such messengers. The intracellular signaling domain generates a signal that promotes an immune effector function of the CAR containing cell, e.g., a CAR-T cell.

[0191] “Isolated” refers to a homogenous population of molecules (such as synthetic polynucleotides or polypeptides) which have been substantially separated and / or purified away from other components of the system the molecules are produced in, such as a recombinant cell, as well as a protein that has been subjected to at least one purification or isolation step. “Isolated” refers to a molecule that is substantially free of other cellular material and / or chemicals and encompasses molecules that are isolated to a higher purity, such as to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% purity.

[0192] “Modulate” refers to either enhanced or decreased ability of a test molecule to mediate an enhanced or a reduced response (i.e., downstream effect) when compared to the response mediated by a control or a vehicle.

[0193] “Monoclonal antibody” refers to an antibody obtained from a substantially homogenous population of antibody molecules, i.e., the individual antibodies comprising the population are identical except for possible well-known alterations such as removal of C-terminal lysine from the antibody heavy chain or post-translational modifications such as amino acid isomerization or deamidation, methionine oxidation or asparagine or glutamine deamidation. Monoclonal antibodies typically bind one antigenic epitope. A bispecific monoclonal antibody binds two distinct antigenic epitopes. Monoclonal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibody may be monospecific or multispecific such as bispecific, monovalent, bivalent or multivalent.

[0194] “Multispecific” refers to a molecule, such as an antibody that specifically binds two or more distinct antigens or two or more distinct epitopes within the same antigen. Multispecific molecule may have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca fascicularis (cynomolgus, cyno) or Pan troglodytes, or may bind an epitope that is shared between two or more distinct antigens.

[0195] “Natural killer cell” and “NK cell” are used interchangeably and synonymously herein. NK cell refers to a differentiated lymphocyte with a CD16+CD56+and / or CD57+TCR- phenotype. NK cells are characterized by their ability to bind to and kill cells that fail to express “self’ MHC / HLA antigens by the activation of specific cytolytic enzymes, the ability to kill tumor cells or other diseased cells that express a ligand for NK activating receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit the immune response.

[0196] “Operatively linked” and similar phrases, when used in reference to nucleic acids or amino acids, refers to the operational linkage of nucleic acid sequences or amino acid sequence, respectively, placed in functional relationships with each other. For example, an operatively linked promoter, enhancer elements, open reading frame, 5' and 3' UTR, and terminator sequences result in the accurate production of a nucleic acid molecule (e.g., RNA) and in some instances to the production of a polypeptide (i.e., expression of the open reading frame). Operatively linked peptide refers to a peptide in which the functional domains of the peptide are placed with appropriate distance from each other to impart the intended function of each domain.

[0197] “Pharmaceutical combination” refers to a combination of two or more active ingredients administered either together or separately.

[0198] “Pharmaceutical composition” refers to a composition that results from combining an active ingredient and a pharmaceutically acceptable carrier.

[0199] “Pharmaceutically acceptable carrier” or “excipient” refers to an ingredient in a pharmaceutical composition, other than the active ingredient, which is nontoxic to a subject. Exemplary pharmaceutically acceptable carriers are a buffer, stabilizer or preservative.

[0200] “Polynucleotide” or “nucleic acid” refers to a synthetic molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. cDNA is a typical example of a polynucleotide. Polynucleotide may be a DNA or a RNA molecule.

[0201] “Prevent,” “preventing,” “prevention,” or “prophylaxis” of a disease or disorder means preventing that a disorder occurs in a subject. “Proliferation” refers to an increase in cell division, either symmetric or asymmetric division of cells.

[0202] “Promoter” refers to the minimal sequences required to initiate transcription. Promoter may also include enhancers or repressor elements which enhance or suppress transcription, respectively.

[0203] “Protein” or “polypeptide” are used interchangeably herein are refers to a molecule that comprises one or more polypeptides each comprised of at least two amino acid residues linked by a peptide bond. Protein may be a monomer, or may be protein complex of two or more subunits, the subunits being identical or distinct. Small polypeptides of less than 50 amino acids may be referred to as “peptides”. Protein may be a heterologous fusion protein, a glycoprotein, or a protein modified by post- translational modifications such as phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, citrullination, polyglutamylation, ADP-ribosylation, pegylation or biotinylation. Protein may be recombinantly expressed.

[0204] “Recombinant” refers to polynucleotides, polypeptides, vectors, viruses and other macromolecules that are prepared, expressed, created or isolated by recombinant means.

[0205] “Regulatory element” refers to any cis-or trans acting genetic element that controls some aspect of the expression of nucleic acid sequences.

[0206] “Relapsed” refers to the return of a disease or the signs and symptoms of a disease after a period of improvement after prior treatment with a therapeutic.

[0207] “Refractory” refers to a disease that does not respond to a treatment. A refractory disease can be resistant to a treatment before or at the beginning of the treatment, or a refractory disease can become resistant during a treatment.

[0208] “Single chain Fv” or “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region (VL) and at least one antibody fragment comprising a heavy chain variable region (VH), wherein the VL and the VH are contiguously linked via a polypeptide linker, and capable of being expressed as a single chain polypeptide. Unless specified, as used herein, a scFv may have the VL and VH variable regions in either order, e.g., with respect to the N- terminal and C- terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0209] “Specifically binds,” “specific binding,” “specifically binding” or “binds” refer to a proteinaceous molecule binding to an antigen or an epitope within the antigen with greater affinity than for other antigens. Typically, the proteinaceous molecule binds to the antigen or the epitope within the antigen with an equilibrium dissociation constant (KD) of about lxlO-7M or less, for example about 5x10-8M or less, about lxlO-8M or less, about lxlO-9M or less, about lxlO-10M or less, about lxlO-11M or less, or about lxlO12M or less, typically with the KDthat is at least one hundred fold less than its KDfor binding to a non-specific antigen (e.g., BSA, casein). “Subject” includes any human or nonhuman animal. “Nonhuman animal” includes all vertebrates, e.g., mammals and non -mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. The terms “subject” and “patient” can be used interchangeably herein.

[0210] “T cell” and “T lymphocyte” are interchangeable and used synonymously herein. T cell includes thymocytes, naive T lymphocytes, memory T cells, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. A T cell can be a T helper (Th) cell, for example a T helper 1 (Thl) or a T helper 2 (Th2) cell. The T cell can be a helper T cell (HTL; CD4+T cell) CD4+T cell, a cytotoxic T cell (CTL; CD8+T cell), a tumor infiltrating cytotoxic T cell (TIL; CD8+T cell), CD4+CD8+T cell, or any other subset of T cells. Also included are “NKT cells”, which refer to a specialized population of T cells that express a semi -invariant ab T-cell receptor, but also express a variety of molecular markers that are typically associated with NK cells, such as NK1.1. NKT cells include NK1.1+and NK1.1-, as well as CD4+, CD4- CD8+and CDS- cells. The TCR on NKT cells is unique in that it recognizes glycolipid antigens presented by the MHC I-like molecule CD Id. NKT cells can have either protective or deleterious effects due to their abilities to produce cytokines that promote either inflammation or immune tolerance. Also included are “gamma-delta T cells (gd T cells),” which refer to a specialized population that to a small subset of T cells possessing a distinct TCR on their surface, and unlike the majority of T cells in which the TCR is composed of two glycoprotein chains designated α- and β-TCR chains, the TCR in gd T cells is made up of a g-chain and a d-chain . gd T cells can play a role in immunosurveillance and immunoregulation, and were found to be an important source of IL-17 and to induce robust CD8+cytotoxic T cell response. Also included are “regulatory T cells” or “Tregs” which refer to T cells that suppress an abnormal or excessive immune response and play a role in immune tolerance. Tregs are typically transcription factor Foxp3 -positive CD4+T cells and can also include transcription factor Foxp3-negative regulatory T cells that are IL-10-producing CD4+T cells.

[0211] “Therapeutically effective amount” or “effective amount” used interchangeably herein, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of a therapeutic or a combination of therapeutics to elicit a desired response in the individual. Example indicators of an effective therapeutic or combination of therapeutics that include, for example, improved wellbeing of the patient, reduction of a tumor burden, arrested or slowed growth of a tumor, and / or absence of metastasis of cancer cells to other locations in the body.

[0212] “T ransduction” refers to the introduction of a foreign nucleic acid into a cell using a viral vector. “T reat,” “treating” or “treatment” of a disease or disorder such as cancer refers to accomplishing one or more of the following: reducing the severity and / or duration of the disorder, inhibiting worsening of symptoms characteristic of the disorder being treated, limiting or preventing recurrence of the disorder in subjects that have previously had the disorder, or limiting or preventing recurrence of symptoms in subjects that were previously symptomatic for the disorder.

[0213] “Tumor cell” or a “cancer cell” refers to a cancerous, pre-cancerous or transformed cell, either in vivo, ex vivo, or in tissue culture, that has spontaneous or induced phenotypic changes. These changes do not necessarily involve the uptake of new genetic material. Although transformation may arise from infection with a transforming virus and incorporation of new genomic nucleic acid, uptake of exogenous nucleic acid or it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene. Transformation / cancer is exemplified by morphological changes, immortalization of cells, aberrant growth control, foci formation, proliferation, malignancy, modulation of tumor specific marker levels, invasiveness, tumor growth in suitable animal hosts such as nude mice, and the like, in vitro, in vivo, and ex vivo.

[0214] “Variant,” “mutant” or “altered” refers to a polypeptide or a polynucleotide that differs from a reference polypeptide or a reference polynucleotide by one or more modifications, for example one or more substitutions, insertions or deletions.

[0215] The numbering of amino acid residues in the antibody constant region throughout the specification is according to the EU index as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), unless otherwise explicitly stated.

[0216] Mutations in the Ig constant regions are referred to as follows: L351Y_F405A_Y407V refers to L351Y, F405A and Y407V mutations in one immunoglobulin constant region.

[0217] L351 Y_F405A_Y407V / T394W refers to L351 Y, F405A and Y407V mutations in the first Ig constant region and T394W mutation in the second Ig constant region, which are present in one multimeric protein.

[0218] COMPOSITIONS OF MATTER Antigen binding domains that bind CD33

[0219] The disclosure provides antigen binding domains that bind CD33, monospecific and multispecific proteins comprising the antigen binding domains that bind CD33, chimeric antigen receptors (CAR) comprising the antigen binding domains that bind CD33, polynucleotides encoding the foregoing, vectors, host cells and methods of making and using the foregoing. The antigen binding domains that bind CD33 identified herein demonstrated improved properties in terms of improved thermostability. The disclosure provides an isolated protein comprising an antigen binding domain that binds CD33, wherein the antigen binding domain that binds CD33comprises a heavy chain complementarity determining region (HCDR) 1, a HCDR2 and a HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 18; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 19; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 20; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 21; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 22; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 23; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 24; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 25; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 26; or an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 27.

[0220] Methods and techniques for identifying CDRs (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) are known in the art and can be used to identify CDRs within any of the heavy chain variable regions and / or any of the light chain variable regions disclosed herein. Conventions that can be used to identify the boundaries of CDRs include, but are not limited to, the Rabat definition, the Chothia definition, the AbM definition, the IMGT definition and the Contact definition. Without wishing to be bound by theory, the Rabat definition is based on sequence variability, the Chothia definition is based on location of structural loop regions, and the AbM definition is a compromise between the Chothia and Rabat approaches.

[0221] In some embodiments, the antigen binding domain that binds CD33 comprises the VH of any one of SEQ ID NOs: 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27. In some embodiments, the antigen binding domain that binds CD33 is an scFv. In some embodiments, the antigen binding domain that binds CD33 is an (scFvfy In some embodiments, the antigen binding domain that binds CD33 is an Fv. In some embodiments, the antigen binding domain that binds CD33 is an Fab. In some embodiments, the antigen binding domain that binds CD33 is an F(ab’)2. In some embodiments, the antigen binding domain that binds CD33 is an Fd. In some embodiments, the CD33 antigen binding domain is a dAb. In some embodiments, the CD33 antigen binding domain is a VHH.

[0222] The disclosure also provides an isolated protein isolated protein comprises the HCDR1, the HCDR2, the HCDR3 of SEQ ID NOs: 1, 6, and 12, respectively; SEQ ID NOs: 2, 7, and 13, respectively; SEQ ID NOs: 1, 8, and 14, respectively; SEQ ID NOs: 3, 9, and 13, respectively; SEQ ID NOs: 4, 10, and 15, respectively; SEQ ID NOs: 5, 11, and 16, respectively; or SEQ ID NOs: 5, 11, and 17, respectively. In some embodiments, the antigen binding domain that binds CD33 is an scFv. In some embodiments, the antigen binding domain that binds CD33 is an (scFvfy In some embodiments, the antigen binding domain that binds CD33 is an Fv. In some embodiments, the antigen binding domain that binds CD33 is a Fab. In some embodiments, the antigen binding domain that binds CD33 is an F(ab’)2. In some embodiments, the antigen binding domain that binds CD33 is an Fd. In some embodiments, the CD33 antigen binding domain is a dAb. In some embodiments, the CD33 antigen binding domain is a VHH.

[0223] The disclosure also provides an isolated protein comprising an antigen binding domain that binds myeloid cell surface antigen CD33, wherein the antigen binding domain that binds CD33 comprises a) an HCDR1, an HCDR2 and an HCDR3 of a heavy chain variable region (VH) of SEQ ID

[0224] NO: 52; b) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 53; c) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 54; d) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 55; e) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 56; f) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 57; g) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 58; h) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 59; i) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 60; j) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 61; k) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 262; l) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 263; or m) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 264.

[0225] In certain embodiments, the antigen binding domain that binds CD33 comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 53.

[0226] In other embodiments, the antigen binding domain that binds CD33 comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 56.

[0227] In other embodiments, the the antigen binding domain that binds CD33 comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 59.

[0228] In other embodiments, the antigen binding domain that binds CD33 comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 262.

[0229] In other embodiments, the the antigen binding domain that binds CD33 comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 263.

[0230] In other embodiments, the antigen binding domain that binds CD33 comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 264.

[0231] Exemplary HCDR sequences are described in Tables 4a, 4b, 4c, 4d, 4e and 4f.

[0232] In some embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 262, 263 or 264. In certain embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 53.

[0233] In other embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID

[0234] NO: 56.

[0235] In other embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID

[0236] NO: 59.

[0237] In other embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 262.

[0238] In other embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 263.

[0239] In other embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 264.

[0240] In some embodiments, the antigen binding domain that binds CD33 comprises the VL of SEQ ID NOs: 81, 82, 83, 84, 85, 86, 87, 88, 271, 272 or 273.

[0241] In certain embodiments, the antigen binding domain that binds CD33 comprises the VL of SEQ ID NO: 82.

[0242] In other embodiments, the antigen binding domain that binds CD33 comprises the VL of SEQ ID

[0243] NO: 85.

[0244] In other embodiments, the antigen binding domain that binds CD33 comprises the VL of SEQ ID

[0245] NO: 87.

[0246] In other embodiments, the antigen binding domain that binds CD33 comprises the VL of SEQ ID NO: 271.

[0247] In other embodiments, the antigen binding domain that binds CD33 comprises the VL of SEQ ID NO: 272

[0248] In other embodiments, the antigen binding domain that binds CD33 comprises the VL of SEQ ID NO: 273.

[0249] In some embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 262, 263 or 264 and the VL of SEQ ID NOs: 81, 82, 83, 84, 85, 86, 87, 88, 271, 272 or 273.

[0250] In certain embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 82.

[0251] In other embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 85. In other embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 59 and the VL of SEQ ID NO: 87.

[0252] In other embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 262 and the VL of SEQ ID NO: 271.

[0253] In other embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 272.

[0254] In other embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 264 and the VL of SEQ ID NO: 273.

[0255] In some embodiments, the antigen binding domain that binds CD33 is an scFv. In some embodiments, the antigen binding domain that binds CD33 is an (scFv)2. In some embodiments, the antigen binding domain that binds CD33 is an Fv. In some embodiments, the antigen binding domain that binds CD33 is an Fab. In some embodiments, the antigen binding domain that binds CD33 is an F(ab’)2. In some embodiments, the antigen binding domain that binds CD33 is an Fd. In some embodiments, the CD33 antigen binding domain is a dAb. In some embodiments, the CD33 antigen binding domain is a VHH.

[0256] The disclosure also provides an isolated protein comprising an HCDR1, an HCDR2, and an HCDR3 of a) SEQ ID NOs: 28, 36, and 45, respectively; b) SEQ ID NOs: 29, 37, and 46, respectively; c) SEQ ID NOs: 30, 38, and 47, respectively; d) SEQ ID NOs: 31, 39, and 48, respectively; e) SEQ ID NOs: 32, 40, and 49, respectively; f) SEQ ID NOs: 33, 41, and 50, respectively; g) SEQ ID NOs: 34, 42, and 51, respectively; h) SEQ ID NOs: 34, 43, and 51, respectively; i) SEQ ID NOs: 34, 44, and 51, respectively; or j) SEQ ID NOs: 35, 42, and 51, respectively; k) SEQ ID NOs: 253, 256 and 259, respectively; l) SEQ ID NOs: 254, 257 and 260, respectively; or m) SEQ ID NOs: 255, 258 and 261 respectively.

[0257] In certain embodiments, the isolated protein comprises an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 29, 37, and 46, respectively.

[0258] In other embodiments, the isolated protein comprises an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 32, 40, and 49, respectively. In other embodiments, the isolated protein comprises an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 34, 43, and 51, respectively.

[0259] In other embodiments, the isolated protein comprises an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 253, 256, and 259, respectively.

[0260] In other embodiments, the isolated protein comprises an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 254, 257, and 260, respectively.

[0261] In other embodiments, the isolated protein comprises an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 255, 258, and 361, respectively.

[0262] In some embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 262, 263 or 264.

[0263] In certain embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 53.

[0264] In other embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID

[0265] NO: 56.

[0266] In other embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID

[0267] NO: 59.

[0268] In other embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 262.

[0269] In other embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 263.

[0270] In other embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 264.

[0271] In some embodiments, the antigen binding domain that binds CD33 comprises the VL of SEQ ID NOs: 81, 82, 83, 84, 85, 86, 87, 88, 271, 272 or 273.

[0272] In certain embodiments, the antigen binding domain that binds CD33 comprises the VL of SEQ ID NO: 82.

[0273] In other embodiments, the antigen binding domain that binds CD33 comprises the VL of SEQ ID

[0274] NO: 85.

[0275] In other embodiments, the antigen binding domain that binds CD33 comprises the VL of SEQ ID

[0276] NO: 87.

[0277] In other embodiments, the antigen binding domain that binds CD33 comprises the VL of SEQ ID NO: 271.

[0278] In other embodiments, the antigen binding domain that binds CD33 comprises the VL of SEQ ID

[0279] NO: 272 In other embodiments, the antigen binding domain that binds CD33 comprises the VL of SEQ ID NO: 273.

[0280] In some embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 262, 263 or 264 and the VL of SEQ ID NOs: 81, 82, 83, 84,

[0281] 85, 86, 87, 88, 271, 272 or 273.

[0282] In certain embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 82.

[0283] In other embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 85.

[0284] In other embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 59 and the VL of SEQ ID NO: 87.

[0285] In other embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 262 and the VL of SEQ ID NO: 271.

[0286] In other embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 272.

[0287] In other embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 264 and the VL of SEQ ID NO: 273.

[0288] In some embodiments, the antigen binding domain that binds CD33 is an scFv. In some embodiments, the antigen binding domain that binds CD33 is an (scFv)2. In some embodiments, the antigen binding domain that binds CD33 is an Fv. In some embodiments, the antigen binding domain that binds CD33 is an Fab. In some embodiments, the antigen binding domain that binds CD33 is an F(ab’)2. In some embodiments, the antigen binding domain that binds CD33 is an Fd. In some embodiments, the CD33 antigen binding domain is a dAb. In some embodiments, the CD33 antigen binding domain is a VHH.

[0289] The disclosure also provides an antigen binding domain that binds myeloid cell surface antigen CD33, wherein the antigen binding domain that binds CD33 comprises a heavy chain complementarity determining region (HCDR) 1, an HCDR2 and an HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 52 and a light chain complementarity determining region (LCDR) 1, an LCDR2 and an LCDR3 of a light chain variable region (VL) of SEQ ID NO: 81; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 53 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 82; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 54 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 83; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 55 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 84; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 56 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 85; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 57 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 86; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 58 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 87; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 59 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 87; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 60 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 87; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 61 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 88; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 262 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 271; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 263 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 272; or an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 264 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 273.

[0290] Exemplary HCDR and LCDR sequences are described in Tables 4a, 4b, 4c, 4d, 4e and 4f.

[0291] In certain embodiments, the antigen binding domain that binds CD33 comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 53 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 82.

[0292] In other embodiments, the antigen binding domain that binds CD33 comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 56 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 85.

[0293] In other embodiments, the antigen binding domain that binds CD33 comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 59 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 87.

[0294] In other embodiments, the antigen binding domain that binds CD33 comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 262 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 271.

[0295] In other embodiments, the antigen binding domain that binds CD33 comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 263 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 272.

[0296] In other embodiments, the antigen binding domain that binds CD33 comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 264 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 273.

[0297] In some embodiments, the antigen binding domain that binds CD33 is an scFv. In some embodiments, the antigen binding domain that binds CD33 is an (scFv)2. In some embodiments, the antigen binding domain that binds CD33 is an Fv. In some embodiments, the antigen binding domain that binds CD33 is an Fab. In some embodiments, the antigen binding domain that binds CD33 is an F(ab’)2. In some embodiments, the antigen binding domain that binds CD33 is an Fd. In some embodiments, the CD33 antigen binding domain is a dAb. In some embodiments, the CD33 antigen binding domain is a VHH.

[0298] The disclosure also provides an antigen binding domain that binds myeloid cell surface antigen CD33, wherein the antigen binding domain that binds CD33 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of a) SEQ ID NOs: 28, 36, 45, 62, 69, and 75, respectively; b) SEQ ID NOs: 29, 37, 46, 63, 70, and 76, respectively; c) SEQ ID NOs: 30, 38, 47, 64, 71, and 77, respectively; d) SEQ ID NOs: 31, 39, 48, 65, 72, and 78, respectively; e) SEQ ID NOs: 32, 40, 49, 66, 70, and 76, respectively; f) SEQ ID NOs: 33, 41, 50, 67, 73, and 79, respectively; g) SEQ ID NOs: 34, 42, 51, 68, 74, and 80, respectively; h) SEQ ID NOs: 34, 43, 51, 68, 74, and 80, respectively; i) SEQ ID NOs: 34, 44, 51, 68, 74, and 80, respectively; j) SEQ ID NOs: 35, 42, 51, 68, 74, and 80, respectively; k) SEQ ID NOs: 253, 256, 259, 265, 74 and 269, respectively; l) SEQ ID NOs: 254, 257, 260, 66, 267 and 76, respectively; m) SEQ ID NOs: 255, 258, 261, 266, 268 and 270, respectively.

[0299] In certain embodiments, the antigen binding domain that binds CD33 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 29, 37, 46, 63, 70, and 76, respectively.

[0300] In other embodiments, the antigen binding domain that binds CD33 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 32, 40, 49, 66, 70, and 76, respectively.

[0301] In other embodiments, the antigen binding domain that binds CD33 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 34, 43, 51, 68, 74, and 80, respectively.

[0302] In other embodiments, the antigen binding domain that binds CD33 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 253, 256, 259, 265, 74 and 269, respectively.

[0303] In other embodiments, the antigen binding domain that binds CD33 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 254, 257, 260, 66, 267 and 76, respectively. In other embodiments, the antigen binding domain that binds CD33 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 255, 258, 261, 266, 268 and 270, respectively.

[0304] In some embodiments, the antigen binding domain that binds CD33 comprises: a) the VH of SEQ ID NO: 52 and the VL of SEQ ID NO: 81; b) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 82; c) the VH of SEQ ID NO: 54 and the VL of SEQ ID NO: 83; d) the VH of SEQ ID NO: 55 and the VL of SEQ ID NO: 84; e) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 85; f) the VH of SEQ ID NO: 57 and the VL of SEQ ID NO: 86; g) the VH of SEQ ID NO: 58 and the VL of SEQ ID NO: 87; h) the VH of SEQ ID NO: 59 and the VL of SEQ ID NO: 87; i) the VH of SEQ ID NO: 60 and the VL of SEQ ID NO: 87; j) the VH of SEQ ID NO: 61 and the VL of SEQ ID NO: 88; k) the VH of SEQ ID NO: 262 and the VL of SEQ ID NO: 271; l) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 272; or m) the VH of SEQ ID NO: 264 and the VL of SEQ ID NO: 273.

[0305] In certain embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 82.

[0306] In other embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 85.

[0307] In other embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 59 and the VL of SEQ ID NO: 87.

[0308] In other embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 262 and the VL of SEQ ID NO: 271.

[0309] In other embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 272.

[0310] In other embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 264 and the VL of SEQ ID NO: 273.

[0311] In some embodiments, the antigen binding domain that binds CD33 is an scFv. In some embodiments, the antigen binding domain that binds CD33 is an (scFv)2. In some embodiments, the antigen binding domain that binds CD33 is an Fv. In some embodiments, the antigen binding domain that binds CD33 is an Fab. In some embodiments, the antigen binding domain that binds CD33 is an F(ab’)2. In some embodiments, the antigen binding domain that binds CD33 is an Fd. In some embodiments, the CD33 antigen binding domain is a dAb. In some embodiments, the CD33 antigen binding domain is a VHH.

[0312] The disclosure also provides an isolated protein comprising an antigen binding domain that binds myeloid cell surface antigen CD33, where the antigen binding domain that binds CD33 comprises a) the VH of SEQ ID NO: 52 and the VL of SEQ ID NO: 81; b) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 82; c) the VH of SEQ ID NO: 54 and the VL of SEQ ID NO: 83; d) the VH of SEQ ID NO: 55 and the VL of SEQ ID NO: 84; e) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 85; f) the VH of SEQ ID NO: 57 and the VL of SEQ ID NO: 86; g) the VH of SEQ ID NO: 58 and the VL of SEQ ID NO: 87; h) the VH of SEQ ID NO: 59 and the VL of SEQ ID NO: 87; i) the VH of SEQ ID NO: 60 and the VL of SEQ ID NO: 87; j) the VH of SEQ ID NO: 61 and the VL of SEQ ID NO: 88; k) the VH of SEQ ID NO: 262 and the VL of SEQ ID NO: 271; l) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 272; or m) the VH of SEQ ID NO: 264 and the VL of SEQ ID NO: 273.

[0313] In certain embodiments, the isolated protein comprises the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 82.

[0314] In other embodiments, the isolated protein comprises the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 85.

[0315] In other embodiments, the isolated protein comprises the VH of SEQ ID NO: 59 and the VL of SEQ ID NO: 87.

[0316] In other embodiments, the isolated protein comprises the VH of SEQ ID NO: 262 and the VL of SEQ ID NO: 271.

[0317] In other embodiments, the isolated protein comprises the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 272.

[0318] In other embodiments, the isolated protein comprises the VH of SEQ ID NO: 264 and the VL of SEQ ID NO: 273.

[0319] In some embodiments, the antigen binding domain that binds CD33 is an scFv. In some embodiments, the antigen binding domain that binds CD33 is an (scFv)2. In some embodiments, the antigen binding domain that binds CD33 is an Fv. In some embodiments, the antigen binding domain that binds CD33 is an Fab. In some embodiments, the antigen binding domain that binds CD33 is an F(ab’)2. In some embodiments, the antigen binding domain that binds CD33 is an Fd. In some embodiments, the CD33 antigen binding domain is a dAb. In some embodiments, the CD33 antigen binding domain is a VHH.

[0320] The disclosure also provides an isolated protein comprising an antigen binding domain that binds myeloid cell surface antigen CD33, where the antigen binding domain that binds CD33 comprises an HCDR1, an HCDR2 and an HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 95; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 96; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 97; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 170; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 171; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 175; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 179; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 181; or an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 96. In some embodiments, the antigen binding domain that binds CD33 is an scFv. In some embodiments, the antigen binding domain that binds CD33 is an (scFv)2. In some embodiments, the antigen binding domain that binds CD33 is an Fv. In some embodiments, the antigen binding domain that binds CD33 is an Fab. In some embodiments, the antigen binding domain that binds CD33 is an F(ab’)2. In some embodiments, the antigen binding domain that binds CD33 is an Fd. In some embodiments, the CD33 antigen binding domain is a dAb. In some embodiments, the CD33 antigen binding domain is a VHH.

[0321] The disclosure also provides an isolated protein comprising an antigen binding domain that binds myeloid cell surface antigen CD33, where the antigen binding domain that binds CD33 comprises an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 89, 90, and 92, respectively; an HCDR1, an HCDR2, and the HCDR3 of SEQ ID NOs: 89, 90, and 93, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 33, 91, and 94, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 167, 168, and 169, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 172, 173, and 174, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 176, 177, and 178, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 89, 90, and 180, respectively; or an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 89, 90, and 93, respectively. In some embodiments, the antigen binding domain that binds CD33 is an scFv. In some embodiments, the antigen binding domain that binds CD33 is an (scFv)2. In some embodiments, the antigen binding domain that binds CD33 is an Fv. In some embodiments, the antigen binding domain that binds CD33 is an Fab. In some embodiments, the antigen binding domain that binds CD33 is an F(ab’)2. In some embodiments, the antigen binding domain that binds CD33 is an Fd. In some embodiments, the CD33 antigen binding domain is a dAb. In some embodiments, the CD33 antigen binding domain is a VHH.

[0322] The disclosure also provides an isolated protein comprising an antigen binding domain that binds myeloid cell surface antigen CD33, where the antigen binding domain that binds CD33 comprises a) an HCDR1, an HCDR2 and an HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 95 and a light chain complementarity determining region (LCDR) 1, an LCDR2 and an LCDR3 of a light chain variable region (VL) of SEQ ID NO: 105; b) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 96 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 106; c) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 97 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 107; d) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 170 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 185; e) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 171 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 188; f) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 175 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 192; g) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 179 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 196; h) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 181 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 200; or i) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 96 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 202. In some embodiments, the antigen binding domain that binds CD33 is an scFv. In some embodiments, the antigen binding domain that binds CD33 is an (SCFV)2. In some embodiments, the antigen binding domain that binds CD33 is an Fv. In some embodiments, the antigen binding domain that binds CD33 is an Fab. In some embodiments, the antigen binding domain that binds CD33 is an F(ab’)2. In some embodiments, the antigen binding domain that binds CD33 is an Fd. In some embodiments, the CD33 antigen binding domain is a dAb. In some embodiments, the CD33 antigen binding domain is a VHH.

[0323] The disclosure also provides an isolated protein comprising an antigen binding domain that binds myeloid cell surface antigen CD33, where the antigen binding domain that binds CD33 comprises an HCDR1, an HCDR2, an HCDR3, an LCDR1, an LCDR2 and an LCDR3 of a) SEQ ID NOs: 89, 90, 92, 98, 101, and 104, respectively; b) SEQ ID NOs: 89, 90, 93, 99, 102, and 104, respectively; c) SEQ ID NOs: 33, 91, 94, 100, 103, and 104, respectively; d) SEQ ID NOs: 167, 168, 169, 182, 183, and 184, respectively; e) SEQ ID NOs: 33, 91, 94, 186, 187, and 104, respectively; f) SEQ ID NOs: 172, 173, 174, 189, 190, and 191, respectively; g) SEQ ID NOs: 176, 177, 178, 193, 194, and 195, respectively; h) SEQ ID NOs: 89, 90, 180, 197, 198, and 199, respectively; i) SEQ ID NOs: 89, 90, 93, 201, 102, and 104, respectively.

[0324] In some embodiments, the antigen binding domain that binds CD33 is an scFv. In some embodiments, the antigen binding domain that binds CD33 is an (scFv)2. In some embodiments, the antigen binding domain that binds CD33 is an Fv. In some embodiments, the antigen binding domain that binds CD33 is an Fab. In some embodiments, the antigen binding domain that binds CD33 is an F(ab’)2. In some embodiments, the antigen binding domain that binds CD33 is an Fd. In some embodiments, the CD33 antigen binding domain is a dAb. In some embodiments, the CD33 antigen binding domain is a VHH.

[0325] The disclosure also provides an isolated protein comprising an antigen binding domain that binds myeloid cell surface antigen CD33, where the antigen binding domain that binds CD33 comprises a VH of SEQ ID NOs: 95, 96, 97, 170, 171, 175, 179, 181, or 96, and a VL of SEQ ID NOs: 105, 106, 107,

[0326] 185, 188, 192, 196, 200, or 202. In some embodiments, the antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 95 and the VL of SEQ ID NO: 105; the VH of SEQ ID NO: 96 and the VL of SEQ ID NO: 106; the VH of SEQ ID NO: 97 and the VL of SEQ ID NO: 107; the VH of SEQ ID NO: 170 and the VL of SEQ ID NO: 185; the VH of SEQ ID NO: 171 and the VL of SEQ ID NO: 188; the VH of SEQ ID NO: 175 and the VL of SEQ ID NO: 192; the VH of SEQ ID NO: 179 and the VL of SEQ ID NO: 196; the VH of SEQ ID NO: 181 and the VL of SEQ ID NO: 200; or the VH of SEQ ID NO: 96 and the VL of SEQ ID NO: 202. In some embodiments, the antigen binding domain that binds CD33 is an scFv. In some embodiments, the antigen binding domain that binds CD33 is an (scFv)2. In some embodiments, the antigen binding domain that binds CD33 is an Fv. In some embodiments, the antigen binding domain that binds CD33 is an Fab. In some embodiments, the antigen binding domain that binds CD33 is an F(ab’)2. In some embodiments, the antigen binding domain that binds CD33 is an Fd. In some embodiments, the CD33 antigen binding domain is a dAb. In some embodiments, the CD33 antigen binding domain is a VHH.

[0327] The disclosure also provides an isolated protein comprising an antigen binding domain that binds myeloid cell surface antigen CD33, where the antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 95 and the VL of SEQ ID NO: 105; the VH of SEQ ID NO: 96 and the VL of SEQ ID NO: 106; the VH of SEQ ID NO: 97 and the VL of SEQ ID NO: 107; the VH of SEQ ID NO: 170 and the VL of SEQ ID NO: 185; the VH of SEQ ID NO: 171 and the VL of SEQ ID NO: 188; the VH of SEQ ID NO: 175 and the VL of SEQ ID NO: 192; the VH of SEQ ID NO: 179 and the VL of SEQ ID NO: 196; the VH of SEQ ID NO: 181 and the VL of SEQ ID NO: 200; or the VH of SEQ ID NO: 96 and the VL of SEQ ID NO: 202. In some embodiments, the antigen binding domain that binds CD33 is an scFv. In some embodiments, the antigen binding domain that binds CD33 is an (scFv)2. In some embodiments, the antigen binding domain that binds CD33 is an Fv. In some embodiments, the antigen binding domain that binds CD33 is an Fab. In some embodiments, the antigen binding domain that binds CD33 is an F(ab’)2. In some embodiments, the antigen binding domain that binds CD33 is an Fd. In some embodiments, the CD33 antigen binding domain is a dAb. In some embodiments, the CD33 antigen binding domain is a VHH.

[0328] CD33 binding scFvs

[0329] Any of the VH and the VL domains identified herein that bind CD33 may be engineered into scFv format in either VH-linker-VL or VL-linker-VH orientation. Any of the VH and the VL domains identified herein may also be used to generate sc(Fv)2 structures, such as VH-linker-VL-linker- VL- linker-VH, VH-linker-VL-linker- VH-linker-VL. VH-linker-VH-linker-VL-linker-VL. VL-1 inker- VH- linker- VH-linker-VL. VL-linker-VH-linker- VL-linker-VH or VL-1 inkcr-VL-1 inker- VH-1 inker- VH.

[0330] The VH and the VL domains identified herein may be incorporated into a scFv format and the binding and thermostability of the resulting scFv to CD33 may be assessed using known methods. Binding may be assessed using ProteOn XPR36, Biacore 3000 or KinExA instrumentation, ELISA or competitive binding assays known to those skilled in the art. Binding may be evaluated using purified scFvs or E. coli supernatants or lysed cells containing the expressed scFv. The measured affinity of a test scFv to CD33 may vary if measured under different conditions (e.g., osmolarity, pH). Thus, measurements of affinity and other binding parameters (e.g., KD, Kon, K0ff) are typically made with standardized conditions and standardized buffers. Thermostability may be evaluated by heating the test scFv at elevated temperatures, such as at 50°C, 55°C or 60°C for a period of time, such as 5 minutes (min), 10 min, 15 min, 20 min, 25 min or 30 min and measuring binding of the test scFv to CD33. The scFvs retaining comparable binding to CD33 when compared to a non-heated scFv sample are referred to as being thermostable.

[0331] In recombinant expression systems, the linker is a peptide linker and may include any naturally occurring amino acid. Exemplary amino acids that may be included into the linker are Gly, Ser Pro, Thr, Glu, Lys, Arg, lie, Leu, His and The. The linker should have a length that is adequate to link the VH and the VL in such a way that they form the correct conformation relative to one another so that they retain the desired activity, such as binding to CD33.

[0332] The linker may be about 5-50 amino acids long. In some embodiments, the linker is about 10-40 amino acids long. In some embodiments, the linker is about 10-35 amino acids long. In some embodiments, the linker is about 10-30 amino acids long. In some embodiments, the linker is about 10- 25 amino acids long. In some embodiments, the linker is about 10-20 amino acids long. In some embodiments, the linker is about 15-20 amino acids long. In some embodiments, the linker is 6 amino acids long. In some embodiments, the linker is 7 amino acids long. In some embodiments, the linker is 8 amino acids long. In some embodiments, the linker is 9 amino acids long. In some embodiments, the linker is 10 amino acids long. In some embodiments, the linker is 11 amino acids long. In some embodiments, the linker is 12 amino acids long. In some embodiments, the linker is 13 amino acids long. In some embodiments, the linker is 14 amino acids long. In some embodiments, the linker is 15 amino acids long. In some embodiments, the linker is 16 amino acids long. In some embodiments, the linker is 17 amino acids long. In some embodiments, the linker is 18 amino acids long. In some embodiments, the linker is 19 amino acids long. In some embodiments, the linker is 20 amino acids long. In some embodiments, the linker is 21 amino acids long. In some embodiments, the linker is 22 amino acids long. In some embodiments, the linker is 23 amino acids long. In some embodiments, the linker is 24 amino acids long. In some embodiments, the linker is 25 amino acids long. In some embodiments, the linker is 26 amino acids long. In some embodiments, the linker is 27 amino acids long. In some embodiments, the linker is 28 amino acids long. In some embodiments, the linker is 29 amino acids long. In some embodiments, the linker is 30 amino acids long. In some embodiments, the linker is 31 amino acids long. In some embodiments, the linker is 32 amino acids long. In some embodiments, the linker is 33 amino acids long. In some embodiments, the linker is 34 amino acids long. In some embodiments, the linker is 35 amino acids long. In some embodiments, the linker is 36 amino acids long. In some embodiments, the linker is 37 amino acids long. In some embodiments, the linker is 38 amino acids long. In some embodiments, the linker is 39 amino acids long. In some embodiments, the linker is 40 amino acids long. Exemplary linkers that may be used are Gly rich linkers, Gly and Ser containing linkers, Gly and Ala containing linkers, Ala and Ser containing linkers, and other flexible linkers.

[0333] Other linker sequences may include portions of immunoglobulin hinge area, CL or CHI derived from any immunoglobulin heavy or light chain isotype. Alternatively, a variety of non-proteinaceous polymers, including polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, may find use as linkers. Exemplary linkers that may be used are shown in Table 1. Additional linkers are described for example in International Patent Publication No. WO2019 / 060695, which is incorporated by reference herein in its entirety.

[0334] In some embodiments, the scFv comprises, from the N- to C-terminus, a VH, a first linker (LI) and a VL (VH-L1-VL).

[0335] In some embodiments, the scFv comprises, from the N-to C-terminus, the VL, the LI and the VH (VL-L1-VH).

[0336] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 108.

[0337] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 109.

[0338] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 110.

[0339] In some embodiments, the L 1 comprises the amino acid sequence of SEQ ID NO: 111.

[0340] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 112.

[0341] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 113.

[0342] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 114. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 115.

[0343] In some embodiments, the L 1 comprises the amino acid sequence of SEQ ID NO: 116.

[0344] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 117.

[0345] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 118.

[0346] In some embodiments, the L 1 comprises the amino acid sequence of SEQ ID NO: 119.

[0347] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 120.

[0348] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 121.

[0349] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 122.

[0350] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 123.

[0351] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 124.

[0352] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 125.

[0353] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 126.

[0354] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 127.

[0355] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 128.

[0356] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 129.

[0357] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 130.

[0358] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 131.

[0359] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 132.

[0360] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 133.

[0361] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 134.

[0362] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 135.

[0363] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 136.

[0364] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 137.

[0365] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 138.

[0366] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 139.

[0367] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 140.

[0368] Table 1.

[0369]

[0370] In certain embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 108 (e.g. consists of the amino acid sequence of SEQ ID NO: 108). In some embodiments, the scFv comprises a heavy chain complementarity determining region (HCDR) 1, a HCDR2 and a HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 18; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 19; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 20; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 21 ; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 22; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 23; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 24; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 25; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 26; or an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 27. In some embodiments, the antigen binding domain that binds CD33 comprises the VH of any one of SEQ ID NOs: 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27.

[0371] In some embodiments, the scFv comprises an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 1, 6, and 12, respectively; SEQ ID NOs: 2, 7, and 13, respectively; SEQ ID NOs: 1, 8, and 14, respectively; SEQ ID NOs: 3, 9, and 13, respectively; SEQ ID NOs: 4, 10, and 15, respectively; SEQ ID NOs: 5, 11, and 16, respectively; or SEQ ID NOs: 5, 11, and 17, respectively.

[0372] In some embodiments, the scFv comprises a) an HCDR1, an HCDR2 and an HCDR3 of a heavy chain variable region (VH) of SEQ

[0373] ID NO: 52; b) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 53; c) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 54; d) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 55; e) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 56; f) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 57; g) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 58; h) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 59; i) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 60; j) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 61; k) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 262; l) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 263; or m) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 264.

[0374] In certain embodiments, the scFv comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 53.

[0375] In other embodiments, the scFv comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 56. In other embodiments, the scFv comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 59.

[0376] In other embodiments, the scFv comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 262.

[0377] In other embodiments, the scFv comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 263.

[0378] In other embodiments, the scFv comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 264.

[0379] Exemplary HCDR sequences are described in Tables 4a, 4b, 4c, 4d, 4e and 4f.

[0380] In some embodiments, the scFv comprises the VH of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58,

[0381] 59, 60, 61, 262, 263 or 264.

[0382] In certain embodiments, the scFv comprises the VH of SEQ ID NO: 53.

[0383] In other embodiments, the scFv comprises the VH of SEQ ID NO: 56.

[0384] In other embodiments, the scFv comprises the VH of SEQ ID NO: 59.

[0385] In other embodiments, the scFv comprises the VH of SEQ ID NO: 262.

[0386] In other embodiments, the scFv comprises the VH of SEQ ID NO: 263.

[0387] In other embodiments, the scFv comprises the VH of SEQ ID NO: 264.

[0388] In some embodiments, the scFv comprises the VL of SEQ ID NOs: 81, 82, 83, 84, 85, 86, 87, 88, 271, 272 or 273.

[0389] In certain embodiments, the scFv comprises the VL of SEQ ID NO: 82.

[0390] In other embodiments, the scFv comprises the VL of SEQ ID NO: 85.

[0391] In other embodiments, the scFv comprises the VL of SEQ ID NO: 87.

[0392] In other embodiments, the scFv comprises the VL of SEQ ID NO: 271.

[0393] In other embodiments, the scFv comprises the VL of SEQ ID NO: 272.

[0394] In other embodiments, the scFv comprises the VL of SEQ ID NO: 273.

[0395] In some embodiments, the scFv comprises the VH of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, 59,

[0396] 60, 61, 262, 263 or 264, and the VL of SEQ ID NOs: 81, 82, 83, 84, 85, 86, 87, 88, 271, 272 or 273. In some embodiments, the scFv comprises an HCDR1, an HCDR2, and an HCDR3 of a) SEQ ID NOs: 28, 36, and 45, respectively; b) SEQ ID NOs: 29, 37, and 46, respectively; c) SEQ ID NOs: 30, 38, and 47, respectively; d) SEQ ID NOs: 31, 39, and 48, respectively; e) SEQ ID NOs: 32, 40, and 49, respectively; f) SEQ ID NOs: 33, 41, and 50, respectively; g) SEQ ID NOs: 34, 42, and 51, respectively; h) SEQ ID NOs: 34, 43, and 51, respectively; i) SEQ ID NOs: 34, 44, and 51, respectively; j) SEQ ID NOs: 35, 42, and 51, respectively; k) SEQ ID NOs: 253, 256 and 259, respectively; l) SEQ ID NOs: 254, 257 and 260, respectively; or m) SEQ ID NOs: 255, 258 and 261, respectively.

[0397] In certain embodiments, the scFv comprises an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 29, 37, and 46, respectively.

[0398] In other embodiments, the scFv comprises an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 32, 40, and 49, respectively.

[0399] In other embodiments, the scFv comprises an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 34, 43, and 51, respectively.

[0400] In other embodiments, the scFv comprises an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 253, 256, and 259, respectively.

[0401] In other embodiments, the scFv comprises an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 254, 257, and 260, respectively.

[0402] In other embodiments, the scFv comprises an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 255, 258, and 261, respectively. In some embodiments, the scFv comprises an HCDR1, an HCDR2 and an HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 52 and a light chain complementarity determining region (LCDR) 1, an LCDR2 and an LCDR3 of a light chain variable region (VL) of SEQ ID NO: 81; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 53 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 82; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 54 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 83; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 55 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 84; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 56 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 85; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 57 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 86; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 58 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 87; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 59 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 87; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 60 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 87; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 61 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 88; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 262 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 271; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 263 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 272; or an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 264 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 273.

[0403] In certain embodiments, the scFv comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 53 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 82.

[0404] In other embodiments, the scFv comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 56 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 85.

[0405] In other embodiments, the scFv comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 59 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 87.

[0406] In other embodiments, the scFv comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 262 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 271.

[0407] In other embodiments, the scFv comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 263 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 272.

[0408] In other embodiments, the scFv comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 264 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 273.

[0409] Exemplary HCDR and LCDR sequences are described in Tables 4a, 4b, 4c, 4d, 4e and 4f.

[0410] In some embodiments, the scFv comprises an HCDR1, an HCDR2, an HCDR3, an LCDR1, an LCDR2 and an LCDR3 of a) SEQ ID NOs: 28, 36, 45, 62, 69, and 75, respectively; b) SEQ ID NOs: 29, 37, 46, 63, 70, and 76, respectively; c) SEQ ID NOs: 30, 38, 47, 64, 71, and 77, respectively; d) SEQ ID NOs: 31, 39, 48, 65, 72, and 78, respectively; e) SEQ ID NOs: 32, 40, 49, 66, 70, and 76, respectively; f) SEQ ID NOs: 33, 41, 50, 67, 73, and 79, respectively; g) SEQ ID NOs: 34, 42, 51, 68, 74, and 80, respectively; h) SEQ ID NOs: 34, 43, 51, 68, 74, and 80, respectively; i) SEQ ID NOs: 34, 44, 51, 68, 74, and 80, respectively; or j) SEQ ID NOs: 35, 42, 51, 68, 74, and 80, respectively; k) SEQ ID NOs: 253, 256, 259, 265, 74 and 269, respectively; l) SEQ ID NOs: 254, 257, 260, 66, 267 and 76, respectively; m) SEQ ID NOs: 255, 258, 261, 266, 268 and 270, respectively.

[0411] In certain embodiments, the scFv comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 29, 37, 46, 63, 70, and 76, respectively. In other embodiments, the scFv comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 32, 40, 49, 66, 70, and 76, respectively.

[0412] In other embodiments, the scFv comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 34, 43, 51, 68, 74, and 80, respectively.

[0413] In other embodiments, the scFv comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 253, 256, 259, 265, 74 and 269, respectively.

[0414] In other embodiments, the scFv comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 254, 257, 260, 66, 267 and 76, respectively.

[0415] In other embodiments, the scFv comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 255, 258, 261, 266, 268 and 270, respectively.

[0416] In some embodiments, the scFv comprises: a) the VH of SEQ ID NO: 52 and the VL of SEQ ID NO: 81; b) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 82; c) the VH of SEQ ID NO: 54 and the VL of SEQ ID NO: 83; d) the VH of SEQ ID NO: 55 and the VL of SEQ ID NO: 84; e) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 85; f) the VH of SEQ ID NO: 57 and the VL of SEQ ID NO: 86; g) the VH of SEQ ID NO: 58 and the VL of SEQ ID NO: 87; h) the VH of SEQ ID NO: 59 and the VL of SEQ ID NO: 87; i) the VH of SEQ ID NO: 60 and the VL of SEQ ID NO: 87; j) the VH of SEQ ID NO: 61 and the VL of SEQ ID NO: 88; k) the VH of SEQ ID NO: 262 and the VL of SEQ ID NO: 271; l) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 272; or m) the VH of SEQ ID NO: 264 and the VL of SEQ ID NO: 273.

[0417] In certain embodiments, the scFv comprises the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 82. In certain such embodiments, the scFv comprises a linker sequence of the amino acid sequence of SEQ ID NO: 108. Thus, in some embodiments, the scFv comprises, from the N- to C-terminus, a VH of SEQ ID NO: 53, a first linker (LI) of SEQ ID NO: 108 and a VL of SEQ ID: 82 (VH-L1-VL). In other embodiments, the scFv comprises from the N- to C- terminus, a VL of SEQ ID NO: 82, a first linker of SEQ ID NO: 108 and a VL of SEQ ID NO: 53 (VL-L1-VH).

[0418] In other embodiments, the scFv comprises the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 85. In certain such embodiments, the scFv comprises a linker sequence of the amino acid sequence of SEQ ID NO: 108. Thus, in some embodiments, the scFv comprises, from the N- to C-terminus, a VH of SEQ ID NO: 56, a first linker (LI) of SEQ ID NO: 108 and a VL of SEQ ID: 85 (VH-L1-VL). In other embodiments, the scFv comprises from the N- to C- terminus, a VL of SEQ ID NO: 56, a first linker of SEQ ID NO: 108 and a VL of SEQ ID NO: 85 (VL-L1-VH).

[0419] In other embodiments, the scFv comprises the VH of SEQ ID NO: 59 and the VL of SEQ ID NO: 87. In certain such embodiments, the scFv comprises a linker sequence of the amino acid sequence of SEQ ID NO: 108. Thus, in some embodiments, the scFv comprises, from the N- to C-terminus, a VH of SEQ ID NO: 59, a first linker (LI) of SEQ ID NO: 108 and a VL of SEQ ID: 87 (VH-L1-VL). In other embodiments, the scFv comprises from the N- to C- terminus, a VL of SEQ ID NO: 59, a first linker of SEQ ID NO: 108 and a VL of SEQ ID NO: 87 (VL-L1-VH).

[0420] In other embodiments, the scFv comprises the VH of SEQ ID NO: 262 and the VL of SEQ ID NO: 271. In certain such embodiments, the scFv comprises a linker sequence of the amino acid sequence of SEQ ID NO: 108. Thus, in some embodiments, the scFv comprises, from the N- to C-terminus, a VH of SEQ ID NO: 262, a first linker (LI) of SEQ ID NO: 108 and a VL of SEQ ID: 271 (VH-L1-VL). In other embodiments, the scFv comprises from the N- to C- terminus, a VL of SEQ ID NO: 271, a first linker of SEQ ID NO: 108 and a VL of SEQ ID NO: 262 (VL-L1-VH).

[0421] In other embodiments, the scFv comprises the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 272. In certain such embodiments, the scFv comprises a linker sequence of the amino acid sequence of SEQ ID NO: 108. Thus, in some embodiments, the scFv comprises, from the N- to C-terminus, a VH of SEQ ID NO: 263, a first linker (LI) of SEQ ID NO: 108 and a VL of SEQ ID: 272 (VH-L1-VL). In other embodiments, the scFv comprises from the N- to C- terminus, a VL of SEQ ID NO: 272, a first linker of SEQ ID NO: 108 and a VL of SEQ ID NO: 263 (VL-L1-VH).

[0422] In other embodiments, the scFv comprises the VH of SEQ ID NO: 264 and the VL of SEQ ID NO: 273. In certain such embodiments, the scFv comprises a linker sequence of the amino acid sequence of SEQ ID NO: 108. Thus, in some embodiments, the scFv comprises, from the N- to C-terminus, a VH of SEQ ID NO: 264, a first linker (LI) of SEQ ID NO: 108 and a VL of SEQ ID: 273 (VH-L1-VL). In other embodiments, the scFv comprises from the N- to C- terminus, a VL of SEQ ID NO: 273, a first linker of SEQ ID NO: 108 and a VL of SEQ ID NO: 264 (VL-L1-VH).

[0423] In some embodiments, the scFv comprises an HCDR1, an HCDR2 and an HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 95; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 96; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 97; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 170; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 171; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 175; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 179; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 181; or an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 96. In some embodiments, the scFv comprises an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 89, 90, and 92, respectively; an HCDR1, an HCDR2, and the HCDR3 of SEQ ID NOs: 89, 90, and 93, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 33, 91, and 94, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 167, 168, and 169, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 172, 173, and 174, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 176, 177, and 178, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 89, 90, and 180, respectively; or an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 89, 90, and 93, respectively.

[0424] In some embodiments, the scFv comprises an HCDR1, an HCDR2 and an HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 95 and an LCDR1, an LCDR2 and an LCDR3 of a light chain variable region (VL) of SEQ ID NO: 105; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 96 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 106; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 97 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 107; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 170 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 185; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 171 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 188; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 175 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 192; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 179 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 196; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 181 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 200; or an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 96 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 202.

[0425] In some embodiments, the scFv comprises an HCDR1, an HCDR2, an HCDR3, an LCDR1, an LCDR2 and an LCDR3 of a) SEQ ID NOs: 89, 90, 92, 98, 101, and 104, respectively; b) SEQ ID NOs: 89, 90, 93, 99, 102, and 104, respectively; c) SEQ ID NOs: 33, 91, 94, 100, 103, and 104, respectively; d) SEQ ID NOs: 167, 168, 169, 182, 183, and 184, respectively; e) SEQ ID NOs: 33, 91, 94, 186, 187, and 104, respectively; f) SEQ ID NOs: 172, 173, 174, 189, 190, and 191, respectively; g) SEQ ID NOs: 176, 177, 178, 193, 194, and 195, respectively; h) SEQ ID NOs: 89, 90, 180, 197, 198, and 199, respectively; i) SEQ ID NOs: 89, 90, 93, 201, 102, and 104, respectively. In some embodiments, the scFv comprises a VH of SEQ ID NOs: 95, 96, 97, 170, 171, 175, 179, 181, or 96, and a VL of SEQ ID NOs: 105, 106, 107, 185, 188, 192, 196, 200, or 202. In some embodiments, the scFv comprises the VH of SEQ ID NO: 95 and the VL of SEQ ID NO: 105; the VH of SEQ ID NO: 96 and the VL of SEQ ID NO: 106; the VH of SEQ ID NO: 97 and the VL of SEQ ID NO: 107; the VH of SEQ ID NO: 170 and the VL of SEQ ID NO: 185; the VH of SEQ ID NO: 171 and the VL of SEQ ID NO: 188; the VH of SEQ ID NO: 175 and the VL of SEQ ID NO: 192; the VH of SEQ ID NO: 179 and the VL of SEQ ID NO: 196; the VH of SEQ ID NO: 181 and the VL of SEQ ID NO: 200; or the VH of SEQ ID NO: 96 and the VL of SEQ ID NO: 202.

[0426] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 203.

[0427] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 204.

[0428] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 205.

[0429] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 206.

[0430] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 207.

[0431] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 208.

[0432] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 209.

[0433] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 210.

[0434] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 211.

[0435] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 212.

[0436] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 213.

[0437] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 214.

[0438] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 215.

[0439] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 216.

[0440] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 217.

[0441] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 218.

[0442] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 219.

[0443] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 220.

[0444] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 221.

[0445] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 274.

[0446] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 275.

[0447] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 276.

[0448] In certain embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 213.

[0449] In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 216.

[0450] In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 219.

[0451] In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 274. In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 275.

[0452] In other embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 276.

[0453] Other antigen binding domains that bind CD33

[0454] Any of the VH and the VL domains identified herein that bind CD33 may also be engineered into Fab, F(ab’)2, Fd or Fv format and their binding to CD33 and thermostability may be assessed using the assays described herein. In certain embodiments, the VH and VL domains identified herein are engineered into a Fab.

[0455] In some embodiments, the Fab comprises an HCDR1, a HCDR2 and a HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 18; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 19; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 20; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 21; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 22; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 23; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 24; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 25; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 26; or an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 27. In some embodiments, the antigen binding domain that binds CD33 comprises the VH of any one of SEQ ID NOs: 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27.

[0456] In some embodiments, the Fab comprises an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 1, 6, and 12, respectively; SEQ ID NOs: 2, 7, and 13, respectively; SEQ ID NOs: 1, 8, and 14, respectively; SEQ ID NOs: 3, 9, and 13, respectively; SEQ ID NOs: 4, 10, and 15, respectively; SEQ ID NOs: 5, 11, and 16, respectively; or SEQ ID NOs: 5, 11, and 17, respectively.

[0457] In some embodiments, the Fab comprises a) an HCDR1, an HCDR2 and an HCDR3 of a heavy chain variable region (VH) of SEQ

[0458] ID NO: 52; b) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 53; c) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 54; d) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 55; e) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 56; f) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 57; g) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 58; h) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 59; i) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 60; j) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 61; k) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 262; l) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 263; or m) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 264.

[0459] In certain embodiments, the Fab comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 53.

[0460] In other embodiments, the Fab comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 56.

[0461] In other embodiments, the Fab comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 59.

[0462] In other embodiments, the Fab comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 262.

[0463] In other embodiments, the Fab comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 263.

[0464] In other embodiments, the Fab comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 264.

[0465] Exemplary HCDR sequences are described in Tables 4a, 4b, 4c, 4d, 4e and 4f.

[0466] In some embodiments, the Fab comprises the VH of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 262, 263 or 264. In some embodiments, the Fab comprises the VL of SEQ ID NOs: 81, 82, 83, 84, 85, 86, 87, 88, 271, 272 or 273. In some embodiments, the Fab comprises the VH of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 262, 263 or 264, and the VL of SEQ ID NOs: 81, 82, 83, 84, 85, 86, 87, or 88, 271, 272 or 273.

[0467] In some embodiments, the Fab comprises an HCDR1, an HCDR2, and an HCDR3 of a) SEQ ID NOs: 28, 36, and 45, respectively; b) SEQ ID NOs: 29, 37, and 46, respectively; c) SEQ ID NOs: 30, 38, and 47, respectively; d) SEQ ID NOs: 31, 39, and 48, respectively; e) SEQ ID NOs: 32, 40, and 49, respectively; f) SEQ ID NOs: 33, 41, and 50, respectively; g) SEQ ID NOs: 34, 42, and 51, respectively; h) SEQ ID NOs: 34, 43, and 51, respectively; i) SEQ ID NOs: 34, 44, and 51, respectively; or j) SEQ ID NOs: 35, 42, and 51, respectively; or l) SEQ ID NOs: 253, 256, and 259, respectively; m) SEQ ID NOs: 254, 257, and 260, respectively; n) SEQ ID NOs: 255, 258, and 261, respectively. In some embodiments, the Fab comprises an HCDR1, an HCDR2 and an HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 52 and a light chain complementarity determining region (LCDR) 1, an LCDR2 and an LCDR3 of a light chain variable region (VL) of SEQ ID NO: 81; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 53 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 82; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 54 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 83; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 55 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 84; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 56 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 85; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 57 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 86; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 58 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 87; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 59 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 87; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 60 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 87; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 61 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 88; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 262 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 271; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 263 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 272; or an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 264 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 273.

[0468] Exemplary HCDR and LCDR sequences are described in Tables 4a, 4b, 4c, 4d, 4e and 4f.

[0469] In certain embodiments, the Fab comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 53 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 82.

[0470] In other embodiments, the Fab comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 56 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 85.

[0471] In other embodiments, the Fab comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 59 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 87.

[0472] In other embodiments, the Fab comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 262 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 271.

[0473] In other embodiments, the Fab comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 263 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 272.

[0474] In other embodiments, the Fab comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 264 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 273. In some embodiments, the Fab comprises an HCDR1, an HCDR2, an HCDR3, an LCDR1, an LCDR2 and an LCDR3 of a) SEQ ID NOs: 28, 36, 45, 62, 69, and 75, respectively; b) SEQ ID NOs: 29, 37, 46, 63, 70, and 76, respectively; c) SEQ ID NOs: 30, 38, 47, 64, 71, and 77, respectively; d) SEQ ID NOs: 31, 39, 48, 65, 72, and 78, respectively; e) SEQ ID NOs: 32, 40, 49, 66, 70, and 76, respectively; f) SEQ ID NOs: 33, 41, 50, 67, 73, and 79, respectively; g) SEQ ID NOs: 34, 42, 51, 68, 74, and 80, respectively; h) SEQ ID NOs: 34, 43, 51, 68, 74, and 80, respectively; i) SEQ ID NOs: 34, 44, 51, 68, 74, and 80, respectively; j) SEQ ID NOs: 35, 42, 51, 68, 74, and 80, respectively; k) SEQ ID NOs: 253, 256, 259, 265, 74, and 269, respectively; l) SEQ ID NOs: 254, 257, 260, 66, 267, and 76, respectively; or m) SEQ ID NOs: 255, 258, 261, 266, 268, and 270, respectively.

[0475] In some embodiments, the Fab comprises: a) the VH of SEQ ID NO: 52 and the VL of SEQ ID NO: 81; b) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 82; c) the VH of SEQ ID NO: 54 and the VL of SEQ ID NO: 83; d) the VH of SEQ ID NO: 55 and the VL of SEQ ID NO: 84; e) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 85; f) the VH of SEQ ID NO: 57 and the VL of SEQ ID NO: 86; g) the VH of SEQ ID NO: 58 and the VL of SEQ ID NO: 87; h) the VH of SEQ ID NO: 59 and the VL of SEQ ID NO: 87; i) the VH of SEQ ID NO: 60 and the VL of SEQ ID NO: 87; j) the VH of SEQ ID NO: 61 and the VL of SEQ ID NO: 88; k) the VH of SEQ ID NO: 262 and the VL of SEQ ID NO: 271; l) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 272; or m) the VH of SEQ ID NO: 264 and the VL of SEQ ID NO: 273.

[0476] In some embodiments, the Fab comprises an HCDR1, an HCDR2 and an HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 95; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 96; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 97; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 170; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 171; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 175; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 179; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 181; or an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 96.

[0477] In some embodiments, the Fab comprises an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 89, 90, and 92, respectively; an HCDR1, an HCDR2, and the HCDR3 of SEQ ID NOs: 89, 90, and 93, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 33, 91, and 94, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 167, 168, and 169, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 172, 173, and 174, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 176, 177, and 178, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 89, 90, and 180, respectively; or an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 89, 90, and 93, respectively.

[0478] In some embodiments, the Fab comprises an HCDR1, an HCDR2 and an HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 95 and an LCDR1, an LCDR2 and an LCDR3 of a light chain variable region (VL) of SEQ ID NO: 105; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 96 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 106; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 97 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 107; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 170 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 185; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 171 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 188; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 175 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 192; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 179 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 196; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 181 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 200; or an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 96 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 202.

[0479] In some embodiments, the Fab comprises an HCDR1, an HCDR2, an HCDR3, an LCDR1, an LCDR2 and an LCDR3 of a) SEQ ID NOs: 89, 90, 92, 98, 101, and 104, respectively; b) SEQ ID NOs: 89, 90, 93, 99, 102, and 104, respectively; c) SEQ ID NOs: 33, 91, 94, 100, 103, and 104, respectively; d) SEQ ID NOs: 167, 168, 169, 182, 183, and 184, respectively; e) SEQ ID NOs: 33, 91, 94, 186, 187, and 104, respectively; f) SEQ ID NOs: 172, 173, 174, 189, 190, and 191, respectively; g) SEQ ID NOs: 176, 177, 178, 193, 194, and 195, respectively; h) SEQ ID NOs: 89, 90, 180, 197, 198, and 199, respectively; i) SEQ ID NOs: 89, 90, 93, 201, 102, and 104, respectively.

[0480] In some embodiments, the Fab comprises a VH of SEQ ID NOs: 95, 96, 97, 170, 171, 175, 179,

[0481] 181, or 96, and a VL of SEQ ID NOs: 105, 106, 107, 185, 188, 192, 196, 200, or 202. In some embodiments, the Fab comprises the VH of SEQ ID NO: 95 and the VL of SEQ ID NO: 105; the VH of SEQ ID NO: 96 and the VL of SEQ ID NO: 106; the VH of SEQ ID NO: 97 and the VL of SEQ ID NO: 107; the VH of SEQ ID NO: 170 and the VL of SEQ ID NO: 185; the VH of SEQ ID NO: 171 and the VL of SEQ ID NO: 188; the VH of SEQ ID NO: 175 and the VL of SEQ ID NO: 192; the VH of SEQ ID NO: 179 and the VL of SEQ ID NO: 196; the VH of SEQ ID NO: 181 and the VL of SEQ ID NO: 200; or the VH of SEQ ID NO: 96 and the VL of SEQ ID NO: 202.

[0482] In other embodiments, the VH and VL domains identified herein are engineered into a F(ab’) 2.

[0483] In some embodiments, the F(ab’)2 comprises an HCDR1, a HCDR2 and a HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 18; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 19; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 20; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 21; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 22; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 23; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 24; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 25; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 26; or an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 27. In some embodiments, the antigen binding domain that binds CD33 comprises the VH of any one of SEQ ID NOs: 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27.

[0484] In some embodiments, the F(ab’)2 comprises an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 1, 6, and 12, respectively; SEQ ID NOs: 2, 7, and 13, respectively; SEQ ID NOs: 1, 8, and 14, respectively; SEQ ID NOs: 3, 9, and 13, respectively; SEQ ID NOs: 4, 10, and 15, respectively; SEQ ID NOs: 5, 11, and 16, respectively; or SEQ ID NOs: 5, 11, and 17, respectively.

[0485] In some embodiments, the F(ab’)2 comprises a) an HCDR1, an HCDR2 and an HCDR3 of a heavy chain variable region (VH) of SEQ

[0486] ID NO: 52; b) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 53; c) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 54; d) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 55; e) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 56; f) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 57; g) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 58; h) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 59; i) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 60; j) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 61; k) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 262; l) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 263; or m) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 264.

[0487] In some embodiments, the F(ab’)2 comprises the VH of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 262, 263 or 264. In some embodiments, the F(ab’)2 comprises the VL of SEQ ID NOs: 81, 82, 83, 84, 85, 86, 87, 88, 271, 272 or 273. In some embodiments, the F(ab’)2 comprises the VH of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, 59, 60 61, 262, 263 or 264 and the VL of SEQ ID NOs: 81, 82, 83, 84,

[0488] 85, 86, 87, 88, 271, 272 or 273.

[0489] In some embodiments, the F(ab’)2 comprises an HCDR1, an HCDR2, and an HCDR3 of a) SEQ ID NOs: 28, 36, and 45, respectively; b) SEQ ID NOs: 29, 37, and 46, respectively; c) SEQ ID NOs: 30, 38, and 47, respectively; d) SEQ ID NOs: 31, 39, and 48, respectively; e) SEQ ID NOs: 32, 40, and 49, respectively; f) SEQ ID NOs: 33, 41, and 50, respectively; g) SEQ ID NOs: 34, 42, and 51, respectively; h) SEQ ID NOs: 34, 43, and 51, respectively; i) SEQ ID NOs: 34, 44, and 51, respectively; j) SEQ ID NOs: 35, 42, and 51, respectively; k) SEQ ID NOs: 253, 256 and 259, respectively l) SEQ ID NOs: 254, 257 and 260, respectively m) SEQ ID NOs: 255, 258 and 261 respectively.

[0490] In some embodiments, the F(ab’)2 comprises an HCDR1, an HCDR2 and an HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 52 and a light chain complementarity determining region (LCDR) 1, an LCDR2 and an LCDR3 of a light chain variable region (VL) of SEQ ID NO: 81; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 53 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 82; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 54 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 83; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 55 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 84; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 56 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 85; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 57 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 86; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 58 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 87; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 59 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 87; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 60 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 87; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 61 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 88 an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 262 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 271; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 263 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 272; or an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 264 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 273.

[0491] In some embodiments, the F(ab’)2 comprises an HCDR1, an HCDR2, an HCDR3, an LCDR1, an LCDR2 and an LCDR3 of a) SEQ ID NOs: 28, 36, 45, 62, 69, and 75, respectively; b) SEQ ID NOs: 29, 37, 46, 63, 70, and 76, respectively; c) SEQ ID NOs: 30, 38, 47, 64, 71, and 77, respectively; d) SEQ ID NOs: 31, 39, 48, 65, 72, and 78, respectively; e) SEQ ID NOs: 32, 40, 49, 66, 70, and 76, respectively; f) SEQ ID NOs: 33, 41, 50, 67, 73, and 79, respectively; g) SEQ ID NOs: 34, 42, 51, 68, 74, and 80, respectively; h) SEQ ID NOs: 34, 43, 51, 68, 74, and 80, respectively; i) SEQ ID NOs: 34, 44, 51, 68, 74, and 80, respectively; j) SEQ ID NOs: 35, 42, 51, 68, 74, and 80, respectively; k) SEQ ID NOs: 253, 256, 259, 265, 74, and 269, respectively; l) SEQ ID NOs: 254, 257, 260, 66, 267, and 76, respectively; or m) SEQ ID NOs: 255, 258, 261, 266, 268, and 270, respectively.

[0492] In some embodiments, the F(ab’)2 comprises: a) the VH of SEQ ID NO: 52 and the VL of SEQ ID NO: 81; b) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 82; c) the VH of SEQ ID NO: 54 and the VL of SEQ ID NO: 83; d) the VH of SEQ ID NO: 55 and the VL of SEQ ID NO: 84; e) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 85; f) the VH of SEQ ID NO: 57 and the VL of SEQ ID NO: 86; g) the VH of SEQ ID NO: 58 and the VL of SEQ ID NO: 87; h) the VH of SEQ ID NO: 59 and the VL of SEQ ID NO: 87; i) the VH of SEQ ID NO: 60 and the VL of SEQ ID NO: 87; j) the VH of SEQ ID NO: 61 and the VL of SEQ ID NO: 88; k) the VH of SEQ ID NO: 262 and the VL of SEQ ID NO: 271; l) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 272; or m) the VH of SEQ ID NO: 264 and the VL of SEQ ID NO: 273.

[0493] In some embodiments, the F(ab’)2 comprises an HCDR1, an HCDR2 and an HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 95; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 96; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 97; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 170; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 171; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 175; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 179; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 181; or an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 96.

[0494] In some embodiments, the F(ab’)2 comprises an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 89, 90, and 92, respectively; an HCDR1, an HCDR2, and the HCDR3 of SEQ ID NOs: 89, 90, and 93, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 33, 91, and 94, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 167, 168, and 169, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 172, 173, and 174, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 176, 177, and 178, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 89, 90, and 180, respectively; or an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 89, 90, and 93, respectively.

[0495] In some embodiments, the F(ab’)2 comprises an HCDR1, an HCDR2 and an HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 95 and an LCDR1, an LCDR2 and an LCDR3 of a light chain variable region (VL) of SEQ ID NO: 105; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 96 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 106; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 97 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 107; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 170 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 185; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 171 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 188; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 175 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 192; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 179 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 196; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 181 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 200; or an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 96 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 202. In some embodiments, the F(ab’)2 comprises an HCDR1, an HCDR2, an HCDR3, an LCDR1, an LCDR2 and an LCDR3 of a) SEQ ID NOs: 89, 90, 92, 98, 101, and 104, respectively; b) SEQ ID NOs: 89, 90, 93, 99, 102, and 104, respectively; c) SEQ ID NOs: 33, 91, 94, 100, 103, and 104, respectively; d) SEQ ID NOs: 167, 168, 169, 182, 183, and 184, respectively; e) SEQ ID NOs: 33, 91, 94, 186, 187, and 104, respectively; f) SEQ ID NOs: 172, 173, 174, 189, 190, and 191, respectively; g) SEQ ID NOs: 176, 177, 178, 193, 194, and 195, respectively; h) SEQ ID NOs: 89, 90, 180, 197, 198, and 199, respectively; i) SEQ ID NOs: 89, 90, 93, 201, 102, and 104, respectively.

[0496] In some embodiments, the F(ab’)2 comprises a VH of SEQ ID NOs: 95, 96, 97, 170, 171, 175, 179, 181, or 196, and a VL of SEQ ID NOs: 105, 106, 107, 185, 188, 192, 196, 200, or 202. In some embodiments, the F(ab’)2 comprises the VH of SEQ ID NO: 95 and the VL of SEQ ID NO: 105; the VH of SEQ ID NO: 96 and the VL of SEQ ID NO: 106; the VH of SEQ ID NO: 97 and the VL of SEQ ID NO: 107; the VH of SEQ ID NO: 170 and the VL of SEQ ID NO: 185; the VH of SEQ ID NO: 171 and the VL of SEQ ID NO: 188; the VH of SEQ ID NO: 175 and the VL of SEQ ID NO: 192; the VH of SEQ ID NO: 179 and the VL of SEQ ID NO: 196; the VH of SEQ ID NO: 181 and the VL of SEQ ID NO:

[0497] 200; or the VH of SEQ ID NO: 96 and the VL of SEQ ID NO: 202.

[0498] In certain embodiments, the VH and VL domains identified herein are engineered into a Fv.

[0499] In some embodiments, the Fv comprises a heavy chain complementarity determining region (HCDR) 1, a HCDR2 and a HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 18; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 19; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 20; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 21 ; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 22; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 23; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 24; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 25; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 26; or an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 27. In some embodiments, the antigen binding domain that binds CD33 comprises the VH of any one of SEQ ID NOs: 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27.

[0500] In some embodiments, the Fv comprises an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 1, 6, and 12, respectively; SEQ ID NOs: 2, 7, and 13, respectively; SEQ ID NOs: 1, 8, and 14, respectively; SEQ ID NOs: 3, 9, and 13, respectively; SEQ ID NOs: 4, 10, and 15, respectively; SEQ ID NOs: 5, 11, and 16, respectively; or SEQ ID NOs: 5, 11, and 17, respectively. In some embodiments, the Fv comprises a) an HCDR1, an HCDR2 and an HCDR3 of a heavy chain variable region (VH) of SEQ

[0501] ID NO: 52; b) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 53; c) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 54; d) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 55; e) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 56; f) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 57; g) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 58; h) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 59; i) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 60; j) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 61; k) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 262; l) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 263; or m) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 264.

[0502] In certain embodiments, the Fv comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 53.

[0503] In other embodiments, the Fv comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 56.

[0504] In other embodiments, the Fv comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 59.

[0505] In other embodiments, the Fv comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 262.

[0506] In other embodiments, the Fv comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 263.

[0507] In other embodiments, the Fv comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 264.

[0508] In some embodiments, the Fv comprises the VH of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 262, 263 or 264. In some embodiments, the Fv comprises the VL of SEQ ID NOs: 81, 82, 83, 84, 85, 86, 87, 88, 271, 272, or 273. In some embodiments, the Fv comprises the VH of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, 59, 60, r 61, 262, 263 or 264, and the VL of SEQ ID NOs: 81, 82, 83, 84, 85, 86, 87, 88, 271, 272, 273.

[0509] In some embodiments, the Fv comprises an HCDR1, an HCDR2, and an HCDR3 of a) SEQ ID NOs: 28, 36, and 45, respectively; b) SEQ ID NOs: 29, 37, and 46, respectively; c) SEQ ID NOs: 30, 38, and 47, respectively; d) SEQ ID NOs: 31, 39, and 48, respectively; e) SEQ ID NOs: 32, 40, and 49, respectively; f) SEQ ID NOs: 33, 41, and 50, respectively; g) SEQ ID NOs: 34, 42, and 51, respectively; h) SEQ ID NOs: 34, 43, and 51, respectively; i) SEQ ID NOs: 34, 44, and 51, respectively; j) SEQ ID NOs: 35, 42, and 51, respectively; k) SEQ ID NOs: 253, 256 and 259, respectively l) SEQ ID NOs: 254, 257 and 260, respectively m) SEQ ID NOs: 255, 258 and 261 respectively.

[0510] In some embodiments, the Fv comprises an HCDR1, an HCDR2 and an HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 52 and a light chain complementarity determining region (LCDR) 1, an LCDR2 and an LCDR3 of a light chain variable region (VL) of SEQ ID NO: 81; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 53 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 82; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 54 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 83; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 55 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 84; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 56 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 85; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 57 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 86; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 58 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 87; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 59 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 87; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 60 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 87; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 61 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 88; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 262 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 271; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 263 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 272; or an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 264 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 273.

[0511] In certain embodiment, the Fv comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 53 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 82. In other embodiments, the Fv comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 56 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 85.

[0512] In other embodiments, the Fv comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 59 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 87.

[0513] In other embodiments, the Fv comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 262 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 271.

[0514] In other embodiments the Fv comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 263 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 272.

[0515] In other embodiments, the Fv comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 264 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 273.

[0516] In some embodiments, the Fv comprises an HCDR1, an HCDR2, an HCDR3, an LCDR1, an LCDR2 and an LCDR3 of a) SEQ ID NOs: 28, 36, 45, 62, 69, and 75, respectively; b) SEQ ID NOs: 29, 37, 46, 63, 70, and 76, respectively; c) SEQ ID NOs: 30, 38, 47, 64, 71, and 77, respectively; d) SEQ ID NOs: 31, 39, 48, 65, 72, and 78, respectively; e) SEQ ID NOs: 32, 40, 49, 66, 70, and 76, respectively; f) SEQ ID NOs: 33, 41, 50, 67, 73, and 79, respectively; g) SEQ ID NOs: 34, 42, 51, 68, 74, and 80, respectively; h) SEQ ID NOs: 34, 43, 51, 68, 74, and 80, respectively; i) SEQ ID NOs: 34, 44, 51, 68, 74, and 80, respectively; or j) SEQ ID NOs: 35, 42, 51, 68, 74, and 80, respectively.

[0517] In some embodiments, the Fv comprises: a) the VH of SEQ ID NO: 52 and the VL of SEQ ID NO: 81; b) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 82; c) the VH of SEQ ID NO: 54 and the VL of SEQ ID NO: 83; d) the VH of SEQ ID NO: 55 and the VL of SEQ ID NO: 84; e) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 85; f) the VH of SEQ ID NO: 57 and the VL of SEQ ID NO: 86; g) the VH of SEQ ID NO: 58 and the VL of SEQ ID NO: 87; h) the VH of SEQ ID NO: 59 and the VL of SEQ ID NO: 87; i) the VH of SEQ ID NO: 60 and the VL of SEQ ID NO: 87; j) the VH of SEQ ID NO: 61 and the VL of SEQ ID NO: 88; k) the VH of SEQ ID NO: 262 and the VL of SEQ ID NO: 271; l) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 272; or m) the VH of SEQ ID NO: 264 and the VL of SEQ ID NO: 273.

[0518] In some embodiments, the Fv comprises an HCDR1, an HCDR2 and an HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 95; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 96; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 97; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 170; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 171; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 175; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 179; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO:

[0519] 181; or an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 96.

[0520] In some embodiments, the Fv comprises an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 89, 90, and 92, respectively; an HCDR1, an HCDR2, and the HCDR3 of SEQ ID NOs: 89, 90, and 93, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 33, 91, and 94, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 167, 168, and 169, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 172, 173, and 174, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 176, 177, and 178, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 89, 90, and 180, respectively; or an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 89, 90, and 93, respectively.

[0521] In some embodiments, the Fv comprises an HCDR1, an HCDR2 and an HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 95 and an LCDR1, an LCDR2 and an LCDR3 of a light chain variable region (VL) of SEQ ID NO: 105; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 96 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 106; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 97 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 107; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 170 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 185; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 171 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 188; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 175 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 192; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 179 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 196; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 181 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 200; or an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 96 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 202.

[0522] In certain embodiments, the VH and VL domains identified herein are engineered into a Fd.

[0523] In some embodiments, the Fd comprises an HCDR1, a HCDR2 and a HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 18; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 19; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 20; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 21; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 22; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 23; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 24; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 25; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 26; or an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 27. In some embodiments, the antigen binding domain that binds CD33 comprises the VH of any one of SEQ ID NOs: 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27.

[0524] In some embodiments, the Fd comprises an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 1, 6, and 12, respectively; SEQ ID NOs: 2, 7, and 13, respectively; SEQ ID NOs: 1, 8, and 14, respectively; SEQ ID NOs: 3, 9, and 13, respectively; SEQ ID NOs: 4, 10, and 15, respectively; SEQ ID NOs: 5, 11, and 16, respectively; or SEQ ID NOs: 5, 11, and 17, respectively.

[0525] In some embodiments, the Fd comprises a) an HCDR1, an HCDR2 and an HCDR3 of a heavy chain variable region (VH) of SEQ ID NO:

[0526] 52; b) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 53; c) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 54; d) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 55; e) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 56; f) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 57; g) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 58; h) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 59; i) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 60; j) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 61; k) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 262; l) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 263; or m) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 264.

[0527] In certain embodiments, the Fd comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 53.

[0528] In other embodiments, the Fd comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 56.

[0529] In other embodiments, the Fd comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 59.

[0530] In other embodiments, the Fd comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 262. In other embodiments, the Fd comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 263.

[0531] In other embodiments, the Fd comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 264.

[0532] In some embodiments, the Fd comprises the VH of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 262, 263 or 264.

[0533] In some embodiments, the Fd comprises an HCDR1, an HCDR2, and an HCDR3 of a) SEQ ID NOs: 28, 36, and 45, respectively; b) SEQ ID NOs: 29, 37, and 46, respectively; c) SEQ ID NOs: 30, 38, and 47, respectively; d) SEQ ID NOs: 31, 39, and 48, respectively; e) SEQ ID NOs: 32, 40, and 49, respectively; f) SEQ ID NOs: 33, 41, and 50, respectively; g) SEQ ID NOs: 34, 42, and 51, respectively; h) SEQ ID NOs: 34, 43, and 51, respectively; i) SEQ ID NOs: 34, 44, and 51, respectively; j) SEQ ID NOs: 35, 42, and 51, respectively; k) SEQ ID NOs: 253, 256 and 259, respectively l) SEQ ID NOs: 254, 257 and 260, respectively m) SEQ ID NOs: 255, 258 and 261 respectively.

[0534] In some embodiments, the Fd comprises an HCDR1, an HCDR2 and an HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 52; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 53; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 54; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 55; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 56; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 57; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 58; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 59; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 60; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 61; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO:

[0535] 262 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 271; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 263 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 272; or an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 264 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 273.

[0536] In certain embodiment, the Fd comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 53 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 82. In other embodiments, the Fd comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 56 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 85.

[0537] In other embodiments, the Fd comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 59 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 87.

[0538] In other embodiments, the Fd comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 262 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 271.

[0539] In other embodiments the Fd comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 263 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 272.

[0540] In other embodiments, the Fd comprises an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 264 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 273.

[0541] In some embodiments, the Fd comprises: a) the VH of SEQ ID NO: 52; b) the VH of SEQ ID NO: 53; c) the VH of SEQ ID NO: 54; d) the VH of SEQ ID NO: 55; e) the VH of SEQ ID NO: 56; f) the VH of SEQ ID NO: 57; g) the VH of SEQ ID NO: 58; h) the VH of SEQ ID NO: 59; i) the VH of SEQ ID NO: 60; j) the VH of SEQ ID NO: 61; k) the VH of SEQ ID NO: 262; l) the VH of SEQ ID NO: 263; or m) the VH of SEQ ID NO: 264.

[0542] In some embodiments, the Fd comprises an HCDR1, an HCDR2 and an HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 95; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 96; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 97; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 170; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 171; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 175; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 179; an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO:

[0543] 181; or an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 96.

[0544] In some embodiments, the Fd comprises an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 89, 90, and 92, respectively; an HCDR1, an HCDR2, and the HCDR3 of SEQ ID NOs: 89, 90, and 93, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 33, 91, and 94, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 167, 168, and 169, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 172, 173, and 174, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 176, 177, and 178, respectively; an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 89, 90, and 180, respectively; or an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOs: 89, 90, and 93, respectively.

[0545] Homologous antigen binding domains and antigen binding domains with conservative substitutions

[0546] Variants of the antigen binding domains that bind CD33 are within the scope of the disclosure. For example, variants may comprise 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 or 29 amino acid substitutions in the antigen binding domain that bind CD33 as long as they retain or have improved functional properties when compared to the parent antigen binding domains. For instance, in some embodiments, the variant antigen binding domain that binds CD33 has improved thermostability when compared to the parent antigen binding domain in the same assay. In some embodiments, the variant antigen binding domain that binds CD33 has reduced likelihood of post- translational modification when compared to the parent binding domain. A reduced likelihood of post translation modification may arise due to the substitution of a motif such as NG, DG or DP. In some embodiments, the sequence identity may be about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to the antigen binding domains that bind CD33 of the disclosure. In some embodiments, the variation is in the framework regions. In some embodiments, variants are generated by conservative substitutions.

[0547] For example, the antigen binding domains that bind CD33 may comprise substitutions at residue positions P41, 149, M70, and A88 in the VH (residue numbering according to the hul 1B6_VH of SEQ ID NO: 5) and S80, L82, A88 and Y91 in the VL (residue numbering according to the hul 1B6_VL of SEQ ID NO: 2). Conservative substitutions may be made at any indicated positions and the resulting variant antigen binding domains that bind CD33 are tested for their desired characteristics in the assays described herein.

[0548] Also provided are antigen binding domains that bind CD33 comprising the VH which are at least 80% identical to: the VH of SEQ ID NO: 18; the VH of SEQ ID NO: 19; the VH of SEQ ID NO: 20; the VH of SEQ ID NO: 21; the VH of SEQ ID NO: 22; the VH of SEQ ID NO: 23; the VH of SEQ ID NO: 24; the VH of SEQ ID NO: 25; the VH of SEQ ID NO: 26; the VH of SEQ ID NO: 27; the VH of SEQ ID NO: 52; the VH of SEQ ID NO: 53; the VH of SEQ ID NO: 54; the VH of SEQ ID NO: 55; the VH of SEQ ID NO: 56; the VH of SEQ ID NO: 57; the VH of SEQ ID NO: 58; the VH of SEQ ID NO: 59; the VH of SEQ ID NO: 60; the VH of SEQ ID NO: 61; the VH of SEQ ID NO: 95; the VH of SEQ ID NO: 96; the VH of SEQ ID NO: 97; the VH of SEQ ID NO: 170; the VH of SEQ ID NO: 171; the VH of SEQ ID NO: 175; the VH of SEQ ID NO: 179; the VH of SEQ ID NO: 181; the VH of SEQ ID NO: 96 the VH of SEQ ID NO: 262; the VH of SEQ ID NO: 263; or the VH of SEQ ID NO: 264.

[0549] In certain embodiments, the antigen binding domain that binds to CD33 comprises a VH that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the VH of SEQ ID NO: 53.

[0550] In other embodiments, the antigen binding domain that binds to CD33 comprises a VH that is least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the VH of SEQ ID NO: 56.

[0551] In other embodiments, the antigen binding domain that binds to CD33 comprises a VH that is least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the VH of SEQ ID NO: 59. In other embodiments, the antigen binding domain that binds to CD33 comprises a VH that is least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the VH of SEQ ID NO: 262.

[0552] In other embodiments, the antigen binding domain that binds to CD33 comprises a VH that is least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the VH of SEQ ID NO: 263.

[0553] In other embodiments, the antigen binding domain that binds to CD33 comprises a VH that is least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the VH of SEQ ID NO: 264.

[0554] In some embodiments, the identity is 85%. In some embodiments, the identity is 90%. In some embodiments, the identity is 91%. In some embodiments, the identity is 91%. In some embodiments, the identity is 92%. In some embodiments, the identity is 93%. In some embodiments, the identity is 94%.

[0555] In some embodiments, the identity is 94%. In some embodiments, the identity is 95%. In some embodiments, the identity is 96%. In some embodiments, the identity is 97%. In some embodiments, the identity is 98%. In some embodiments, the identity is 99%.

[0556] Also provided are antigen binding domains that bind CD33 comprising the VH and the VL which are at least 80% identical to the VH of SEQ ID NO: 52 and the VL of SEQ ID NO: 81 ; the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 82; the VH of SEQ ID NO: 54 and the VL of SEQ ID NO: 83; the VH of SEQ ID NO: 55 and the VL of SEQ ID NO: 84; the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 85; the VH of SEQ ID NO: 57 and the VL of SEQ ID NO: 86; the VH of SEQ ID NO: 58 and the VL of SEQ ID NO: 87; the VH of SEQ ID NO: 59 and the VL of SEQ ID NO: 87; the VH of SEQ ID NO: 60 and the VL of SEQ ID NO: 87; the VH of SEQ ID NO: 61 and the VL of SEQ ID NO: 88; the VH of SEQ ID NO: 95 and the VL of SEQ ID NO: 105; the VH of SEQ ID NO: 96 and the VL of SEQ ID NO: 106; the VH of SEQ ID NO: 97 and the VL of SEQ ID NO: 107; the VH of SEQ ID NO: 170 and the VL of SEQ ID NO: 185; the VH of SEQ ID NO: 171 and the VL of SEQ ID NO: 188; the VH of SEQ ID NO: 175 and the VL of SEQ ID NO: 192; the VH of SEQ ID NO: 179 and the VL of SEQ ID NO: 196; the VH of SEQ ID NO: 181 and the VL of SEQ ID NO: 200; the VH of SEQ ID NO: 96 and the VL of SEQ ID NO: 202; the VH of SEQ ID NO: 262 and the VL of SEQ ID NO: 271; the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 272; or the VH of SEQ ID NO: 264 and the VL of SEQ ID NO: 273.

[0557] In certain embodiments, the antigen binding domain that binds to CD33 comprises a VH and VL that are at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 82. For instance, the antigen binding domain that binds to CD33 comprises a VH of SEQ ID NO: 53 and a VL that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the VL of SEQ ID NO: 82. For instance, the antigen binding domain that binds to CD33 comprises a VL of SEQ ID NO: 82 and a VH that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the VH of SEQ ID NO: 53.

[0558] In other embodiments, the antigen binding domain that binds to CD33 comprises a VH that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 85. For instance, the antigen binding domain that binds to CD33 comprises a VH of SEQ ID NO: 56 and a VL that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the VL of SEQ ID NO: 85. For instance, the antigen binding domain that binds to CD33 comprises a VL of SEQ ID NO: 85 and a VH that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the VH of SEQ ID NO: 56.

[0559] In other embodiments, the antigen binding domain that binds to CD33 comprises a VH that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the VH of SEQ ID NO: 59 and the VL of SEQ ID NO: 87. For instance, the antigen binding domain that binds to CD33 comprises a VH of SEQ ID NO: 59 and a VL that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the VL of SEQ ID NO: 87. For instance, the antigen binding domain that binds to CD33 comprises a VL of SEQ ID NO: 87 and a VH that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the VH of SEQ ID NO: 59.

[0560] In other embodiments, the antigen binding domain that binds to CD33 comprises a VH that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the VH of SEQ ID NO: 262 and the VL of SEQ ID NO: 271. For instance, the antigen binding domain that binds to CD33 comprises a VH of SEQ ID NO: 262 and a VL that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the VL of SEQ ID NO: 271. For instance, the antigen binding domain that binds to CD33 comprises a VL of SEQ ID NO: 271 and a VH that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the VH of SEQ ID NO: 262.

[0561] In other embodiments, the antigen binding domain that binds to CD33 comprises a VH that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 272. For instance, the antigen binding domain that binds to CD33 comprises a VH of SEQ ID NO: 263 and a VL that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the VL of SEQ ID NO: 272. For instance, the antigen binding domain that binds to CD33 comprises a VL of SEQ ID NO: 272 and a VH that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the VH of SEQ ID NO: 263.

[0562] In other embodiments, the antigen binding domain that binds to CD33 comprises a VH that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the VH of SEQ ID NO: 264 and the VL of SEQ ID NO: 273. For instance, the antigen binding domain that binds to CD33 comprises a VH of SEQ ID NO: 264 and a VL that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the VL of SEQ ID NO: 273. For instance, the antigen binding domain that binds to CD33 comprises a VL of SEQ ID NO: 273 and a VH that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the VH of SEQ ID NO: 264.

[0563] The inventors have found that positions in the VH and / or VL domains may be mutated to remove motifs that are liable to post-translational modification. Post-translational modification may increase the likelihood of, for example, deamidation, isomerization and / or fragmentation of the antigen binding domain that binds to CD33. These mutations may also enhance thermostability of an antigen binding domain that binds to CD33.

[0564] Examples of sequence motifs that are liable to post-translation modification, for example, NG, DG, and DP.

[0565] Accordingly, in some embodiments, the antigen binding domain that binds to CD33 comprises a VH and / or VL domain comprising one or two amino acid substitutions in one or more NG motifs. In some embodiments, the N residue of the NG motif is substituted. In other embodiments, the G residue of the NG motif is substituted. In some embodiments the N and G residues of the NG motif are substituted. In certain such embodiments, the one or two amino acid substitutions in the one or more NG motifs are the only substitutions within the VH and / or VL domain.

[0566] In some embodiments, the antigen binding domain that binds to CD33 comprises a VH and / or VL domain comprising one or two amino acid substitutions in one or more DG motifs. In some embodiments, the D residue of the DG motif is substituted. In other embodiments, the G residue of the DG motif is substituted. In some embodiments the D and G residues of the DG motif are substituted. In certain such embodiments, the one or two amino acid substitutions in the one or more DG motifs are the only substitutions within the VH and / or VL domain.

[0567] In some embodiments, the antigen binding domain that binds to CD33 comprises a VH and / or VL domain comprising one or two amino acid substitutions in one or more DP motifs. In some embodiments, the D residue of the DP motif is substituted. In other embodiments, the P residue of the DP motif is substituted. In some embodiments the D and P residues of the DP motif are substituted. In certain such embodiments, the one or two amino acid substitutions in the one or more DP motifs are the only substitutions within the VH and / or VL domain.

[0568] An exemplary antigen binding domain that binds to CD33 of the invention is a protein (for example, an scFv) comprising the VH of SEQ ID NO: 262 and VL of SEQ ID NO: 271. The inventors have found that mutating the DG motif within the VH of SEQ ID NO: 262 can reduce the likelihood of post-translation modification whilst maintaining CD33 binding properties. These mutations may also enhance thermostability.

[0569] An antigen binding domain that binds to CD33 having the VH of SEQ ID NO: 262 and VL of SEQ ID NO: 271 may be mutated to remove a DG motif. The VH of SEQ ID NO: 262 has a DG motif at positions 54-55 (using residue numbering according to EU index).

[0570] Thus, in some embodiments, an antigen binding domain that binds to CD33 (for example, an scFv) comprises a VH of SEQ ID NO: 262 in which the D residue at position 54 is substituted (for example, D54G, D54A, D54Y, D54P, D54N, D54S, D54R, D54L, D54V, D54T, D54F or D54W). In other embodiments, an antigen binding domain that binds to CD33 (for example, an scFv) comprises a VH of SEQ ID NO: 262 in which the G residue at position 55 is substituted (for example, G55V, G55P, G55R, G55A, G55L, G55Q, G55T or G55H). In other embodiments, the antigen binding domain that binds to CD33 (for example, an scFv) comprises a VH of SEQ ID NO: 262 in which the D residue at position 54 (for example, D54G, D54A, D54Y, D54P, D54N, D54S, D54R, D54L, D54V, D54T, D54F or D54W) is substituted and the G residue at position 55 is substituted (for example, G55V, G55P, G55R, G55A, G55L, G55Q, G55T or G55H). In some embodiments, the antigen binding domain that binds to CD33 (for example, an scFv) comprises a VH of SEQ ID NO: 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309 or 310.

[0571] In certain embodiments, the antigen binding domain that binds to CD33 (for example, an scFv) comprises:the VH of SEQ ID NO: 291 and the VL of SEQ ID NO: 271; the VH of SEQ ID NO: 292 and the VL of SEQ ID NO: 271; the VH of SEQ ID NO: 293 and the VL of SEQ ID NO: 271; the VH of SEQ ID NO: 294 and the VL of SEQ ID NO: 271 ; the VH of SEQ ID NO: 295 and the VL of SEQ ID NO: 271; the VH of SEQ ID NO: 296 and the VL of SEQ ID NO: 271 ; the VH of SEQ ID NO: 297 and the VL of SEQ ID NO: 271 ; the VH of SEQ ID NO: 298 and the VL of SEQ ID NO: 271; the VH of SEQ ID NO: 299 and the VL of SEQ ID NO: 271 ; the VH of SEQ ID NO: 300 and the VL of SEQ ID NO: 271; the VH of SEQ ID NO: 301 and the VL of SEQ ID NO: 271; the VH of SEQ ID NO: 302 and the VL of SEQ ID NO: 271; the VH of SEQ ID NO: 303 and the VL of SEQ ID NO: 271; the VH of SEQ ID NO: 304 and the VL of SEQ ID NO: 271; the VH of SEQ ID NO: 305 and the VL of SEQ ID NO: 271; the VH of SEQ ID NO: 306 and the VL of SEQ ID NO: 271; the VH of SEQ ID NO: 307 and the VL of SEQ ID NO: 271; the VH of SEQ ID NO: 308 and the VL of SEQ ID NO: 271; the VH of SEQ ID NO: 309 and the VL of SEQ ID NO: 271; or the VH of SEQ ID NO: 310 and the VL of SEQ ID NO: 271.

[0572] Another exemplary antigen binding domain that binds to CD33 of the invention is a protein (for example, an scFv) comprising the VH of SEQ ID NO: 263 and VL of SEQ ID NO: 272. The inventors have found that mutating one or more NG motifs within the VH of SEQ ID NO: 263 and VL of SEQ ID NO: 272 can reduce the likelihood of post-translation modification whilst maintaining CD33 binding properties. These mutations may also enhance thermostability.

[0573] An antigen binding domain that binds to CD33 having the VH of SEQ ID NO: 263 and VL of SEQ ID NO: 272 may be mutated to remove an NG motif. The VH of SEQ ID NO: 263 has a NG motif at positions 99-100 (using residue numbering according to EU index) and the VL of SEQ ID NO: 272 has a NG motif at positions 97-98 (using residue numbering according to EU index).

[0574] Thus, in some embodiments, the antigen binding domain that binds to CD33 (for example, an scFv) comprises a VH of SEQ ID NO: 263 in which the N residue at position 99 is substituted (for example, N99V, N99S, N99E, N99H, N99A, N99G, N99K, N99T, N99F, N99V, N99R, N99L, N99H or N99I). In other embodiments, an antigen binding domain that binds to CD33 (for example, an scFv) comprises a VH of SEQ ID NO: 263 in which the G residue at position 100 is substituted (for example, G100S, G100A). In other embodiments, the antigen binding domain that binds to CD33 (for example, an scFv) comprises a VH of SEQ ID NO: 263 in which the N residue at position 99 is substituted (for example, N99V, N99S, N99E, N99H, N99A, N99G, N99K, N99T, N99F, N99V, N99R, N99L, N99H or N99I) and the G residue at position 100 is substituted (for example, G100S, G100A). In some embodiments, the antigen binding domain that binds to CD33 comprises a VH of SEQ ID NO: 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, or 352.

[0575] In some embodiments, an antigen binding domain that binds to CD33 (for example, an scFv) comprises a VL of SEQ ID NO: 272 in which the N residue at position 97 is substituted (for example, N97V, N97R, N97P, N97T, N97G, N97Q, N97S, N97A, N97L, N97Y, N97E, N97F, N97D, N97I or N97H). In other embodiments, an antigen binding domain that binds to CD33 (for example, an scFv) comprises a VL of SEQ ID NO: 272 in which the G residue at position 98 is substituted (for example, G98V, G98P, G98R, G98D, G98E, G98W, G98Y, G98A, G98N, G98K, G98L). In other embodiments, the antigen binding domain that binds to CD33 (for example, an scFv) comprises a VL of SEQ ID NO: 272 in which the N residue at position 97 is substituted and the G residue at position 98 is substituted. In some embodiments, the antigen binding domain that binds to CD33 (for example, an scFv) comprises a VL of SEQ ID NO: 311, 312 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335 or 336.

[0576] In certain embodiments, the antigen binding domain that binds to CD33 (for example, an scFv) comprises: the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 311; the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 312. the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 313; the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 314; the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 315; the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 316; the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 317; the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 318; the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 319; the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 320; the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 321; the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 322; the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 323 ; the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 324; the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 325 ; the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 326; the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 327; the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 328; the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 329; the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 330; the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 331 ; the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 332; the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 333; the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 334; the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 335; the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 336; the VH of SEQ ID NO: 337 and the VL of SEQ ID NO: 272; the VH of SEQ ID NO: 338 and the VL of SEQ ID NO: 272; the VH of SEQ ID NO: 339 and the VL of SEQ ID NO: 272; the VH of SEQ ID NO: 340 and the VL of SEQ ID NO: 272; the VH of SEQ ID NO: 341 and the VL of SEQ ID NO: 272; the VH of SEQ ID NO: 342 and the VL of SEQ ID NO: 272; the VH of SEQ ID NO: 343 and the VL of SEQ ID NO: 272; the VH of SEQ ID NO: 344 and the VL of SEQ ID NO: 272; the VH of SEQ ID NO: 345 and the VL of SEQ ID NO: 272; the VH of SEQ ID NO: 346 and the VL of SEQ ID NO: 272; the VH of SEQ ID NO: 347 and the VL of SEQ ID NO: 272; the VH of SEQ ID NO: 348 and the VL of SEQ ID NO: 272; the VH of SEQ ID NO: 349 and the VL of SEQ ID NO: 272; the VH of SEQ ID NO: 350 and the VL of SEQ ID NO: 272; the VH of SEQ ID NO: 351 and the VL of SEQ ID NO: 272; or the VH of SEQ ID NO: 352 and the VL of SEQ ID NO: 272.

[0577] Another exemplary antigen binding domain that binds to CD33 of the invention is a protein (for example, an scFv) comprising the VH of SEQ ID NO: 56 and VL of SEQ ID NO: 85. The inventors have found that mutating one or more NG motifs within the VH of SEQ ID NO: 56 and VL of SEQ ID NO: 85 can reduce the likelihood of post-translation modification whilst maintaining CD33 binding properties. These mutations may also enhance thermostability.

[0578] An antigen binding domain that binds to CD33 having the VH of SEQ ID NO: 56 and VL of SEQ ID NO: 85 may be mutated to remove an NG motif. The VH of SEQ ID NO: 56 has a NG motif at positions 99-100 (using residue numbering according to EU index) and the VL of SEQ ID NO: 85 has a NG motif at positions 97-98 (using residue numbering according to EU index).

[0579] Thus, in some embodiments, the antigen binding domain that binds to CD33 (for example, an scFv) comprises a VH of SEQ ID NO: 56 in which the N residue at position 99 is substituted (for example, N99R, N99P, N99G, N99V, N99L, N99D, N99E, N99A, N99Q or N99T). In other embodiments, an antigen binding domain that binds to CD33 (for example, an scFv) comprises a VH of SEQ ID NO: 56 in which the G residue at position 100 is substituted (for example, G100S, G100A, G100L, G100P, G100T or G100Q). In other embodiments, the antigen binding domain that binds to CD33 (for example, an scFv) comprises a VH of SEQ ID NO: 56 in which the N residue at position 99 is substituted (for example, N99R, N99P, N99G, N99V, N99L, N99D, N99E, N99A, N99Q or N99T) and the G residue at position 100 is substituted (for example, G100S, G100A, G100L, G100P, G100T or G100Q). In some embodiments, the antigen binding domain that binds to CD33 (for example, an scFv) comprises a VH of SEQ ID NO: 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380,

[0580] 381 or 382.

[0581] In some embodiments, an antigen binding domain that binds to CD33 (for example, an scFv) comprises a VL of SEQ ID NO: 85 in which the N residue at position 97 is substituted (for example, N97G, N97D, N97P, N97K, N97E, N97A, N97R, N97Q, N97I, N97V, N97H, N97K, N97W or N97S).

[0582] In other embodiments, an antigen binding domain that binds to CD33 (for example, an scFv) comprises a VL of SEQ ID NO: 85 in which the G residue at position 98 is substituted. In other embodiments, the antigen binding domain that binds to CD33 (for example, an scFv) comprises a VL of SEQ ID NO: 85 in which the N residue at position 97 is substituted (for example, N97G, N97D, N97P, N97K, N97E, N97A, N97R, N97Q, N97I, N97V, N97H, N97K, N97W or N97S) and the G residue at position 98 is substituted. In some embodiments, the antigen binding domain that binds to CD33 (for example, an scFv) comprises a VL of SEQ ID NO: 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, or 366.

[0583] In certain embodiments, the antigen binding domain that binds to CD33 (for example, an scFv) comprises: the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 353; the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 354; the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 355; the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 356; the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 357; the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 358; the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 359; the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 360; the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 361; the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 362; the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 363; the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 364; the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 365; the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 366; the VH of SEQ ID NO: 367 and the VL of SEQ ID NO: 85; the VH of SEQ ID NO: 368 and the VL of SEQ ID NO: 85; the VH of SEQ ID NO: 369 and the VL of SEQ ID NO: 85; the VH of SEQ ID NO: 370 and the VL of SEQ ID NO: 85; the VH of SEQ ID NO: 371 and the VL of SEQ ID NO: 85; the VH of SEQ ID NO: 372 and the VL of SEQ ID NO: 85; the VH of SEQ ID NO: 373 and the VL of SEQ ID NO: 85; the VH of SEQ ID NO: 374 and the VL of SEQ ID NO: 85; the VH of SEQ ID NO: 375 and the VL of SEQ ID NO: 85; the VH of SEQ ID NO: 376 and the VL of SEQ ID NO: 85; the VH of SEQ ID NO: 377 and the VL of SEQ ID NO: 85; the VH of SEQ ID NO: 378 and the VL of SEQ ID NO: 85; the VH of SEQ ID NO: 379 and the VL of SEQ ID NO: 85; the VH of SEQ ID NO: 380 and the VL of SEQ ID NO: 85; the VH of SEQ ID NO: 381 and the VL of SEQ ID NO: 85; or the VH of SEQ ID NO: 382 and the VL of SEQ ID NO: 85. Another exemplary antigen binding domain that binds to CD33 of the invention is a protein (for example, an scFv) comprising the VH of SEQ ID NO: 53 and VL of SEQ ID NO: 82. The inventors have found that mutating the NG motif within the VH of SEQ ID NO: 53 and VL of SEQ ID NO: 82 can reduce the likelihood of post-translation modification whilst maintaining CD33 binding properties. These mutations may also enhance thermostability.

[0584] An antigen binding domain that binds to CD33 having the VH of SEQ ID NO: 53 and VL of SEQ ID NO: 82 may be mutated to an NG motif. The VL of SEQ ID NO: 82 has a NG motif at positions 97- 98 (using residue numbering according to EU index).

[0585] In some embodiments, an antigen binding domain that binds to CD33 (for example, an scFv) comprises a VL of SEQ ID NO: 82 in which the N residue at position 97 is substituted (for example, N97S, N97Q, N97V, N97G, N97W, N97T, N97I, N97E, N97A, N97R or N97L). In other embodiments, an antigen binding domain that binds to CD33 (for example, an scFv) comprises a VL of SEQ ID NO:

[0586] 272 in which the G residue at position 98 is substituted (for example, G98V, G98A, G98P, G98L or G98T). In other embodiments, the antigen binding domain that binds to CD33 (for example, an scFv) comprises a VL of SEQ ID NO: 82 in which the N residue at position 97 is substituted (for example, N97S, N97Q, N97V, N97G, N97W, N97T, N97I, N97E, N97A, N97R or N97L) and the G residue at position 98 is substituted (for example, G98V, G98A, G98P, G98L or G98T). In some embodiments, the antigen binding domain that binds to CD33 (for example, an scFv) comprises a VL of SEQ ID NO: 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397 or 398.

[0587] In certain embodiments, the antigen binding domain that binds to CD33 (for example, an scFv) comprises: the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 383; the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 384; the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 385; the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 386; the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 387; the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 388; the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 389; the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 390; the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 391; the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 392; the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 393; the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 394; the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 395; the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 396; the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 397; or the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 398.

[0588] In some embodiments, the antigen binding domain that binds to CD33 (for example, an scFv) comprises a VH domain of SEQ ID NO: 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, or 382.

[0589] In some embodiments, the antigen binding domain that binds to CD33 (for example, an scFv) comprises a VL domain of SEQ ID NO: 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323,

[0590] 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 353, 354, 355, 356, 357, 358, 359, 360 361, 362, 363, 364, 365, 366, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397 or 398.

[0591] In some embodiments, the antigen binding domain that binds to CD33 (for example, an scFv) comprises a VH domain of SEQ ID NO: 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303 304, 305, 306, 307, 308, 309, 310, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350,

[0592] 351, 352, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, or 382 and a

[0593] VL domain of SEQ ID NO: 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362,

[0594] 363, 364, 365, 366, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397 or 398.

[0595] In some embodiments, the identity is 85%. In some embodiments, the identity is 90%. In some embodiments, the identity is 91%. In some embodiments, the identity is 91%. In some embodiments, the identity is 92%. In some embodiments, the identity is 93%. In some embodiments, the identity is 94%.

[0596] In some embodiments, the identity is 94%. In some embodiments, the identity is 95%. In some embodiments, the identity is 96%. In some embodiments, the identity is 97%. In some embodiments, the identity is 98%. In some embodiments, the identity is 99%.

[0597] The percent identity between the two sequences is a limction of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positionsc100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0598] The percent identity between two amino acid sequences may be determined using the algorithm of E. Meyers and W. Miller ( Comput . Appl. Biosci. 4: 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences may be determined using the Needleman and Wunsch (. / . Mol. Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at www gcg com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0599] In some embodiments, variant antigen binding domains that bind CD33 comprise one or two conservative substitutions in any of the CDR regions, while retaining desired functional properties of the parent antigen binding fragments that bind CD33.

[0600] “Conservative modifications” refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid modifications. Conservative modifications include amino acid substitutions, additions and deletions. Conservative amino acid substitutions are those in which the amino acid is replaced with an amino acid residue having a similar side chain. The families of amino acid residues having similar side chains are well defined and include amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amide (e.g., asparagine, glutamine), beta-branched side chains (e.g., threonine, valine, isoleucine) and sulfur-containing side chains (cysteine, methionine). Furthermore, any native residue in the polypeptide may also be substituted with alanine, as has been previously described for alanine scanning mutagenesis (MacLennan et al., (1988) Acta Physiol Scand Suppl 643:55-67; Sasaki etal., (1988 ) Adv Biophys 35: 1-24). Amino acid substitutions to the antibodies of the invention may be made by known methods for example by PCR mutagenesis (U.S. Patent No. 4,683,195). Alternatively, libraries of variants may be generated for example using random (NNK) or non-random codons, for example DVK codons, which encode 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp). The resulting variants may be tested for their characteristics using assays described herein.

[0601] Methods of generating antigen binding fragment that bind CD33

[0602] Antigen binding domains that bind CD33 provided in the disclosure may be generated using various technologies. For example, the hybridoma method of Kohler and Milstein may be used to identify VH / VL pairs that bind CD33. In the hybridoma method, a mouse or other host animal, such as a hamster, rat or chicken is immunized with human and / or cyno CD33, followed by fusion of spleen cells from immunized animals with myeloma cells using standard methods to form hybridoma cells. Colonies arising from single immortalized hybridoma cells may be screened for production of the antibodies containing the antigen binding domains that bind CD33 with desired properties, such as specificity of binding, cross-reactivity or lack thereof, affinity for the antigen, and any desired functionality. Antigen binding domains that bind CD33 generated by immunizing non-human animals may be humanized. Exemplary humanization techniques including selection of human acceptor frameworks include CDR grafting (U.S. Patent No. 5,225,539), SDR grafting (U.S. Patent No. 6,818,749),

[0603] Resurfacing (Padlan, (1991) Mol Immunol 28:489-499), Specificity Determining Residues Resurfacing (U.S. Patent Publ. No. 2010 / 0261620), human framework adaptation (U.S. Patent No. 8,748,356) or superhumanization (U.S. Patent No. 7,709,226). In these methods, CDRs or a subset of CDR residues of parental antibodies are transferred onto human frameworks that may be selected based on their overall homology to the parental frameworks, based on similarity in CDR length, or canonical structure identity, or a combination thereof.

[0604] Humanized antigen binding domains may be further optimized to improve their selectivity or affinity to a desired antigen by incorporating altered framework support residues to preserve binding affinity (backmutations) by techniques such as those described in Int. Patent Publ. Nos. W01990 / 007861 and WO 1992 / 22653, or by introducing variation at any of the CDRs for example to improve affinity of the antigen binding domain.

[0605] Transgenic animals, such as mice, rat or chicken carrying human immunoglobulin (Ig) loci in their genome may be used to generate antigen binding fragments that bind CD33, and are described in for example U.S. Patent No. 6,150,584, Int. Patent Publ. No. WO 1999 / 45962, Int. Patent Publ. Nos. W02002 / 066630, WO2002 / 43478, W02002 / 043478 and W01990 / 04036. The endogenous immunoglobulin loci in such animal may be disrupted or deleted, and at least one complete or partial human immunoglobulin locus may be inserted into the genome of the animal using homologous or non- homologous recombination, using transchromosomes, or using minigenes. Companies such as Regeneron (www_regeneron_com), Harbour Antibodies (www_harbourantibodies_com), Open Monoclonal Technology, Inc. (OMT) (www_omtinc_net), KyMab (www_kymab_com), Trianni (www.trianni_com) and Ablexis (www_ablexis_com) may be engaged to provide human antibodies directed against a selected antigen using technologies as described above.

[0606] Antigen binding domains that bind CD33 may be selected from a phage display library, where the phage is engineered to express human immunoglobulins or portions thereof such as Fabs, single chain antibodies (scFv), or unpaired or paired antibody variable regions. The antigen binding domains that bind CD33 may be isolated for example from phage display library expressing antibody heavy and light chain variable regions as fusion proteins with bacteriophage pIX coat protein as described in Shi et al., (2010) J Mol Biol 397:385-96, and Int. Patent Publ. No. WO09 / 085462). The libraries may be screened for phage binding to human and / or cyno CD33 and the obtained positive clones may be further characterized, the Fabs isolated from the clone lysates, and converted to scFvs or other configurations of antigen binding fragments. Preparation of immunogenic antigens and expression and production of antigen binding domains of the disclosure may be performed using any suitable technique, such as recombinant protein production. The immunogenic antigens may be administered to an animal in the form of purified protein, or protein mixtures including whole cells or cell or tissue extracts, or the antigen may be formed c / e novo in the animal’s body from nucleic acids encoding said antigen or a portion thereof.

[0607] Conjugation to half-life extending moieties

[0608] The antigen binding domains that bind CD33 of the disclosure may be conjugated to a half-life extending moiety. Exemplary half-life extending moieties are albumin, albumin variants, albumin binding proteins and / or domains, transferrin and fragments and analogues thereof, immunoglobulins (Ig) or fragments thereof, such as Fc regions. Amino acid sequences of the aforementioned half-life extending moieties are known. Ig or fragments thereof include all isotypes, i.e., IgGl, IgG2, IgG3, IgG4, IgM, IgA and IgE.

[0609] Additional half-life extending moieties that may be conjugated to the antigen binding domains that bind CD33 of the disclosure include polyethylene glycol (PEG) molecules, such as PEG5000 or PEG20,000, fatty acids and fatty acid esters of different chain lengths, for example laurate, myristate, stearate, arachidate, behenate, oleate, arachidonate, octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like, polylysine, octane, carbohydrates (dextran, cellulose, oligo- or polysaccharides) for desired properties. These moieties may be direct fusions with the antigen binding domains that bind CD33 of the disclosure and may be generated by standard cloning and expression techniques. Alternatively, well known chemical coupling methods may be used to attach the moieties to recombinantly produced antigen binding domains that bind CD33 of the disclosure.

[0610] A pegyl moiety may for example be conjugated to the antigen binding domain that bind CD33 of the disclosure by incorporating a cysteine residue to the C-terminus of the antigen binding domain that bind CD33 of the disclosure, or engineering cysteines into residue positions that face away from the CD33 binding site and attaching a pegyl group to the cysteine using well known methods.

[0611] In some embodiments, the antigen binding fragment that binds CD33 is conjugated to a half-life extending moiety.

[0612] In some embodiments, the half-life extending moiety is an immunoglobulin (Ig), a fragment of the Ig, an Ig constant region, a fragment of the Ig constant region, a Fc region, transferrin, albumin, an albumin binding domain or polyethylene glycol. In some embodiments, the half-life extending moiety is an Ig constant region.

[0613] In some embodiments, the half-life extending moiety is the Ig.

[0614] In some embodiments, the half-life extending moiety is the fragment of the Ig.

[0615] In some embodiments, the half-life extending moiety is the Ig constant region. In some embodiments, the half-life extending moiety is the fragment of the Ig constant region.

[0616] In some embodiments, the half-life extending moiety is the Fc region.

[0617] In some embodiments, the half-life extending moiety is albumin.

[0618] In some embodiments, the half-life extending moiety is the albumin binding domain.

[0619] In some embodiments, the half-life extending moiety is transferrin.

[0620] In some embodiments, the half-life extending moiety is polyethylene glycol.

[0621] The antigen binding domains that bind CD33 conjugated to a half-life extending moiety may be evaluated for their pharmacokinetic properties utilizing known in vivo models.

[0622] Conjugation to immunoglobulin (Ig) constant regions or fragments of the Ig constant regions

[0623] The antigen binding domains that bind CD33 of the disclosure may be conjugated to an Ig constant region or a fragment of the Ig constant region to impart antibody-like properties, including Fc effector functions Clq binding, complement dependent cytotoxicity (CDC), Fc receptor binding, antibody -dependent cell-mediated cytotoxicity (ADCC), phagocytosis or down regulation of cell surface receptors (e.g., B cell receptor; BCR). The Ig constant region or the fragment of the Ig constant region functions also as a half-life extending moiety as discussed herein. The antigen binding domains that bind CD33 of the disclosure may be engineered into conventional full length antibodies using standard methods. The full length antibodies comprising the antigen binding domain that binds CD33 may further be engineered as described herein.

[0624] Immunoglobulin heavy chain constant region comprised of subdomains CHI, hinge, CH2 and CH3. The CHI domain spans residues A 118-V215, the CH2 domain residues A231-K340 and the CH3 domain residues G341-K447 on the heavy chain, residue numbering according to the EU Index. In some instances G341 is referred as a CH2 domain residue. “Hinge” is generally defined as including E216 and terminating at P230 of human IgGl. Ig Fc region comprises at least the CH2 and the CH3 domains of the Ig constant region, and therefore comprises at least a region from about A231 to K447 of Ig heavy chain constant region.

[0625] The invention also provides an antigen binding domain that binds CD33 conjugated to an immunoglobulin (Ig) constant region or a fragment of the Ig constant region. In certain such embodiments, the Ig constant region or a fragment of the Ig constant region is derived from IgGl.

[0626] In some embodiments, the Ig constant region is a heavy chain constant region. In certain such embodiments, the Ig constant region is an IgGl heavy chain constant region.

[0627] In some embodiments, the Ig constant region is a light chain constant region.

[0628] In some embodiments, the fragment of the Ig constant region comprises a Fc region. In certain such embodiments, the Ig constant region comprises an IgGl Fc region. In some embodiments, the fragment of the Ig constant region comprises a CH2 domain. In certain such embodiments, the Ig constant region comprises an IgGl CH2 domain.

[0629] In some embodiments, the fragment of the Ig constant region comprises a CH3 domain. In certain such embodiments, the Ig constant region comprises an IgGl CH3 domain.

[0630] In some embodiments, the fragment of the Ig constant region comprises the CH2 domain and the CH3 domain. In certain such embodiments, the Ig constant region comprises an IgGl CH2 and CH3 domain.

[0631] In some embodiments, the fragment of the Ig constant region comprises at least portion of a hinge, the CH2 domain and the CH3 domain. In certain such embodiments, at least portion of a hinge, the CH2 domain and the CH3 domain from IgGl. Portion of the hinge refers to one or more amino acid residues of the Ig hinge.

[0632] In some embodiments, the fragment of the Ig constant region comprises the hinge, the CH2 domain and the CH3 domain. In certain such embodiment, the hinge, the CH2 domain and the CH3 domain from IgGl.

[0633] In certain embodiments, the fragment of the Ig constant region comprises an amino acid sequence of SEQ ID NO: 278.

[0634] The cysteine residue at position 220 of the heavy chain may be mutated to prevent the formation of a cysteine bridge between the constant region and a light chain. Thus, in certain embodiments, the Ig constant region comprises a residue that is not cysteine at position 220. In one embodiment, the Ig constant region comprises a serine at position 220.

[0635] In certain embodiments, the fragment of the Ig constant region comprises an amino acid sequence of SEQ ID NO: 279.

[0636] In some embodiments, the antigen binding domain that binds CD33 is conjugated to the N- terminus of the Ig constant region or the fragment of the Ig constant region.

[0637] In some embodiments, the antigen binding domain that binds CD33 is conjugated to the C- terminus of the Ig constant region or the fragment of the Ig constant region.

[0638] In some embodiments, the antigen binding domain that binds CD33 is conjugated to the Ig constant region or the fragment of the Ig constant region via a second linker (L2).

[0639] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NOs: 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, or 140.

[0640] The antigen binding domains that binds CD33 of the disclosure conjugated to Ig constant region or the fragment of the Ig constant region may be assessed for their functionality using several known assays. Binding to CD33 may be assessed using methods described herein. Altered properties imparted by the Ig constant domain or the fragment of the Ig constant region such as Fc region may be assayed in Fc receptor binding assays using soluble forms of the receptors, such as the FcyRI, FcyRII, FcyRIII or FcRn receptors, or using cell-based assays measuring for example ADCC, CDC or ADCP.

[0641] ADCC may be assessed using an in vitro assay using CD33 expressing cells as target cells and NK cells as effector cells. Cytolysis may be detected by the release of label (e.g. radioactive substrates, fluorescent dyes or natural intracellular proteins) from the lysed cells. In an exemplary assay, target cells are used with a ratio of 1 target cell to 4 effector cells. Target cells are pre-labeled with BATDA and combined with effector cells and the test antibody. The samples are incubated for 2 hours and cell lysis measured by measuring released BATDA into the supernatant. Data is normalized to maximal cytotoxicity with 0.67% Triton X-100 (Sigma Aldrich) and minimal control determined by spontaneous release of BATDA from target cells in the absence of any antibody.

[0642] ADCP may be evaluated by using monocyte-derived macrophages as effector cells and any CD33 expressing cells as target cells which are engineered to express GFP or other labeled molecule. In an exemplary assay, effector: target cell ratio may be for example 4: 1. Effector cells may be incubated with target cells for 4 hours with or without the antibody of the invention. After incubation, cells may be detached using accutase. Macrophages may be identified with anti-CD 1 lb and anti-CD 14 antibodies coupled to a fluorescent label, and percent phagocytosis may be determined based on % GFP fluorescence in the CD11+CD14+macrophages using standard methods.

[0643] CDC of cells may be measured for example by plating Daudi cells at 1c105cells / well (50 μL / well) in RPMI-B (RPMI supplemented with 1% BSA), adding 50 μL of test protein to the wells at final concentration between 0-100 μg / mL, incubating the reaction for 15 min at room temperature, adding 11 μL of pooled human serum to the wells, and incubation the reaction for 45 min at 37° C. Percentage (%) lysed cells may be detected as % propidium iodide stained cells in FACS assay using standard methods.

[0644] Proteins comprising the antigen binding domains that bind CD33 of the disclosure

[0645] The antigen binding domains that bind CD33 of the disclosure may be engineered into monospecific or multispecific proteins of various designs using standard methods.

[0646] The disclosure also provides a monospecific protein comprising the antigen binding domain that binds CD33 of the disclosure.

[0647] In some embodiments, the monospecific protein is an antibody.

[0648] The disclosure also provides a multispecific protein comprising the antigen binding domain that binds CD33 of the disclosure.

[0649] In some embodiments, the multispecific protein is bispecific.

[0650] In some embodiments, the multispecific protein is trispecific. In some embodiments, the multispecific protein is tetraspecific.

[0651] In some embodiments, the multispecific protein is monovalent for binding to CD33.

[0652] In some embodiments, the multispecific protein is bivalent for binding to CD33.

[0653] The disclosure also provides an isolated multispecific protein comprising a first antigen binding domain that binds CD33 and a second antigen binding domain that binds a lymphocyte antigen.

[0654] In some embodiments, the lymphocyte antigen is a T cell antigen.

[0655] In some embodiments, the T cell antigen is a CD8+T cell antigen.

[0656] In some embodiments, the lymphocyte antigen is a NK cell antigen.

[0657] In some embodiments, the lymphocyte antigen is CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, TRGV9, or NKG2C.

[0658] In a certain embodiments, the multispecific protein is a bispecific protein comprising a first antigen binding domain that binds CD33 and second antigen binding domain that binds to TRGV9.TRGV9 is also known as Vy9, and is expressed on gdT cells. As used herein, the term “TRGV9” refers to a polypeptide capable of forming a T cell receptor when expressed on the surface of gd T cells. TRGV9-expressing gd T cells are among the first T cells to develop in the human fetus and are the predominant gd T cell subset in healthy adult peripheral blood cells. The term “TRGV9” includes any TRGV9 variant, isoform, and species homolog, which is naturally expressed by cells (including T cells) or can be expressed on cells transfected with genes or cDNA encoding the polypeptide. Unless noted, preferably the TRGV9 is a human TRGV9. A human TRGV9 amino acid sequence is provided by GenBank Accession Number NG_001336.2. Vy9Vd2 T lymphocytes, a major g / d T cell subset in humans, can recognize phosphoantigens, certain tumor cells, and cells treated with aminobisphosphonates. Vy9Vd2 T lymphocytes can display cytolytic activity against various tumor cells. Those having ordinary skill appreciate that g / d TCR is an heterodimeric TCR complex composed of covalently bound g and d chains involved in antigen recognition and the non-covalently associated monomorphic proteins CD3d, g, e, and z chains. The Vy9 TCR is a variant of the TCR g chain expressed on a subset of g / d T cells.

[0659] Without wishing to be bound by theory, a bispecific antibody expressing both TRGV9 antigen and a CD33 antigen could recruit gdT cells to the cancerous cells expressing CD33. A multispecific antibody (e.g., a bispecific antibody) can bridge the effector cell (e.g., gd T cell) and target cells together so as to resulting in cancer cell killing, gd T-cells may have innate immunity. Without wishing to be bound by theory, gd T-cells represent only a minor proportion of the peripheral CD3+T cells (about 1%- 5%), but constitute a major subset (about 20%— 50%) of T-cells in epithelial tissues. Circulating gd T- cells mainly express heterodimers of Vy9 (TRGV9) and Vd2 (TRVD2) chains whereas tissue gd T-cells preferentially express Vδ1 chains associated with different Vγ chains. In humans, gd T-cells are endowed with potent anti-cancer functions (high cytotoxicity and interferon g secretion). Moreover, gd T-cells are capable of phagocytosis, a function previously exclusive to innate myeloid lineage cells, and behave as efficient antigen-presenting cells for ab T-cells and induce adaptive immune response gd T-cells may infiltrate cancerous tissues, tumors and cancerous cells. A bispecific antibody expressing both TRGV9 antigen and a CD33 antigen may exhibit less or no significant T cell redirection, less pan activation of T cells, and have increased induction of potent cancer lysis via selective recruitment of gd T cells. Accordingly, a bispecific antibody expressing both TRGV9 antigen and a CD33 antigen may not lead to severe side effects that might arise by cytokine storm induction.

[0660] In some embodiments, the lymphocyte antigen is CD3ε.

[0661] In some embodiments, the first antigen binding domain that binds CD33 and / or the second antigen binding domain that binds the lymphocyte antigen (for example, TRGV9) comprise a scFv, a (SCFV)2, a Fv, a Fab, a F(ab’)2, a Fd, a dAb or a VHH.

[0662] In some embodiments, the first antigen binding domain that binds CD33 and / or the second antigen binding domain that binds the lymphocyte antigen (for example, TRGV9) comprise the Fab.

[0663] In some embodiments, the first antigen binding domain that binds CD33 and / or the second antigen binding domain that binds the lymphocyte antigen (for example, TRGV9) comprise the F(ab’)2.

[0664] In some embodiments, the first antigen binding domain that binds CD33 and / or the second antigen binding domain that binds the lymphocyte antigen (for example, TRGV9) comprise the VHH.

[0665] In some embodiments, the first antigen binding domain that binds CD33 and / or the second antigen binding domain that binds the lymphocyte antigen (for example, TRGV9) comprise the Fv.

[0666] In some embodiments, the first antigen binding domain that binds CD33 and / or the second antigen binding domain that binds the lymphocyte antigen (for example, TRGV9) comprise the Fd.

[0667] In some embodiments, the first antigen binding domain that binds CD33 and / or the second antigen binding domain that binds the lymphocyte antigen (for example, TRGV9) comprise the scFv.

[0668] In some embodiments, the first antigen binding domain that binds CD33 comprises an scFv and the second binding domain that binds the lymphocyte antigen (for example, TRGV9) comprises a VHH.

[0669] In some embodiments, the scFv comprises, from the N- to C-terminus, a VH, a first linker (LI) and a VL (VH-L1-VL) or the VL, the LI and the VH (VL-L1-VH).

[0670] In some embodiments, the LI comprises about 5-50 amino acids.

[0671] In some embodiments, the LI comprises about 5-40 amino acids.

[0672] In some embodiments, the LI comprises about 10-30 amino acids.

[0673] In some embodiments, the LI comprises about 10-20 amino acids. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NOs: 108, 109,, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 133, 134, 135, 136, 137, 138, 139, or 140.

[0674] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 108.

[0675] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 109. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 110. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 111. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 112. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 113. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 114. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 115. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 116. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 117. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 118. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 120. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 121. In some embodiments, the L 1 comprises the amino acid sequence of SEQ ID NO: 122. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 123. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 124. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 125. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 126. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 127. In some embodiments, the L 1 comprises the amino acid sequence of SEQ ID NO: 128. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 129. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 130. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 131. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 132. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 133. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 134. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 136. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 137. In some embodiments, the L 1 comprises the amino acid sequence of SEQ ID NO: 138. In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 139.

[0676] In some embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 140.

[0677] In certain embodiments, the LI comprises the amino acid sequence of SEQ ID NO: 108.

[0678] In some embodiments, the first antigen binding domain that binds CD33 comprises the HCDR1 of SEQ ID NOs: 1, 2, 3, 4, 5, 28, 29, 30, 31, 32, 33, 34, 35, 89, 167, 172, 176, 253, 254 or 255, the HCDR2 of SEQ ID NOs: 6, 7, 8, 9, 10, 11, 36, 37, 38, 39, 40, 41, 42, 43, 44, 90, 91, 168, 173, 177, 256, 257 or 258 the HCDR3 of SEQ ID NOs: 12, 13, 14, 15, 16, 17, 45, 46, 47, 48, 49, 50, 51, 92, 93, 94, 169, 174, 178, 180, 259, 260 or 261 the LCDR1 of SEQ ID NOs: 62, 63, 64, 65, 66, 67, 68, 98, 99, 100, 182, 186, 189, 193, 197, 201, 265 or 266 the LCDR2 of SEQ ID NOs: 69, 70, 71, 72, 73, 74, 101, 102, 103, 183, 187, 190, 194, 198, 267 or 268 and the LCDR3 of SEQ ID NOs: 75, 76, 77, 78, 79, 80, 104, 184, 191, 195, 199, 269 or 270.

[0679] In some embodiments, the first antigen binding domain that binds CD33 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 28, 36, 45, 62, 69, and 75, respectively;

[0680] SEQ ID NOs: 29, 37, 46, 63, 70, and 76, respectively;

[0681] SEQ ID NOs: 30, 38, 47, 64, 71, and 77, respectively;

[0682] SEQ ID NOs: 31, 39, 48, 65, 72, and 78, respectively;

[0683] SEQ ID NOs: 32, 40, 49, 66, 70, and 76, respectively;

[0684] SEQ ID NOs: 33, 41, 50, 67, 73, and 79, respectively;

[0685] SEQ ID NOs: 34, 42, 51, 68, 74, and 80, respectively;

[0686] SEQ ID NOs: 34, 43, 51, 68, 74, and 80, respectively;

[0687] SEQ ID NOs: 34, 44, 51, 68, 74, and 80, respectively;

[0688] SEQ ID NOs: 35, 42, 51, 68, 74, and 80, respectively;

[0689] SEQ ID NOs: 89, 90, 92, 98, 101, and 104, respectively;

[0690] SEQ ID NOs: 89, 90, 93, 99, 102, and 104, respectively;

[0691] SEQ ID NOs: 33, 91, 94, 100, 103, and 104, respectively;

[0692] SEQ ID NOs: 167, 168, 169, 182, 183, and 184, respectively;

[0693] SEQ ID NOs: 33, 91, 94, 186, 187, and 104, respectively;

[0694] SEQ ID NOs: 172, 173, 174, 189, 190, and 191, respectively;

[0695] SEQ ID NOs: 176, 177, 178, 193, 194, and 195, respectively;

[0696] SEQ ID NOs: 89, 90, 180, 197, 198, and 199, respectively;

[0697] SEQ ID NOs: 89, 90, 93, 201, 102, and 104, respectively;

[0698] SEQ ID NOs: 253, 256, 259, 265, 74 and 269 respectively;

[0699] SEQ ID NOs: 254, 257, 260, 66, 267 and 76 respectively SEQ ID NOs: 255, 258, 261, 266, 268 and 270 respectively.

[0700] In certain embodiments, the first antigen binding domain that binds CD33 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 29, 37, 46, 63, 70, and 76, respectively. In certain such embodiments, the first antigen binding domain is an scFv.

[0701] In other embodiments, the first antigen binding domain that binds CD33 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 32, 40, 49, 66, 70, and 76, respectively. In certain such embodiments, the first antigen binding domain is an scFv.

[0702] In other embodiments, the first antigen binding domain that binds CD33 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 34, 43, 51, 68, 74, and 80, respectively. In certain such embodiments, the first antigen binding domain is an scFv.

[0703] In other embodiments, the first antigen binding domain that binds CD33 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 253, 256, 259, 265, 74 and 269, respectively. In certain such embodiments, the first antigen binding domain is an scFv.

[0704] In other embodiments, the first antigen binding domain that binds CD33 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 254, 257, 260, 66,

[0705] 267 and 76, respectively. In certain such embodiments, the first antigen binding domain is an scFv.

[0706] In other embodiments, the first antigen binding domain that binds CD33 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 255, 258, 261, 266,

[0707] 268 and 270, respectively. In certain such embodiments, the first antigen binding domain is an scFv.

[0708] In certain embodiments, the first antigen binding domain that binds CD33 is an scFv comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 29, 37, 46, 63, 70, and 76, respectively and the second antigen binding domain binds to TRGV9 (for example, wherein the second binding domain is a VHH).

[0709] In other embodiments, the first antigen binding domain that binds CD33 is an scFv comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 32, 40, 49, 66, 70, and 76, respectively and the second antigen binding domain binds to TRGV9 (for example, wherein the second binding domain is a VHH).

[0710] In other embodiments, the first antigen binding domain that binds CD33 is an scFv comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 34, 43, 51, 68, 74, and 80, respectively and the second antigen binding domain binds to TRGV9 (for example, wherein the second binding domain is a VHH).

[0711] In other embodiments, the first antigen binding domain that binds CD33 is an scFv comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 253, 256, 259, 265, 74 and 269, respectively and the second antigen binding domain binds to TRGV9 (for example, wherein the second binding domain is a VHH).

[0712] In other embodiments, the first antigen binding domain that binds CD33 is an scFv comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 254,

[0713] 257, 260, 66, 267 and 76, respectively and the second antigen binding domain binds to TRGV9 (for example, wherein the second binding domain is a VHH).

[0714] In other embodiments, the first antigen binding domain that binds CD33 is an scFv comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 255, 258, 261, 266, 268 and 270, respectively and the second antigen binding domain binds to TRGV9 (for example, wherein the second binding domain is a VHH).

[0715] In some embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 52 and the VL of SEQ ID NO: 81.

[0716] In some embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 82.

[0717] In some embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 54 and the VL of SEQ ID NO: 83.

[0718] In some embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 55 and the VL of SEQ ID NO: 84.

[0719] In some embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 85.

[0720] In some embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 57 and the VL of SEQ ID NO: 86.

[0721] In some embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 58 and the VL of SEQ ID NO: 87.

[0722] In some embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 59 and the VL of SEQ ID NO: 87.

[0723] In some embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 60 and the VL of SEQ ID NO: 87.

[0724] In some embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 61 and the VL of SEQ ID NO: 88.

[0725] In some embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 95 and the VL of SEQ ID NO: 105.

[0726] In some embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 96 and the VL of SEQ ID NO: 106. In some embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 97 and the VL of SEQ ID NO: 107.

[0727] In some embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 170 and the VL of SEQ ID NO: 185.

[0728] In some embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 171 and the VL of SEQ ID NO: 188.

[0729] In some embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 175 and the VL of SEQ ID NO: 192.

[0730] In some embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 179 and the VL of SEQ ID NO: 196.

[0731] In some embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 181 and the VL of SEQ ID NO: 200.

[0732] In some embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 96 and the VL of SEQ ID NO: 202.

[0733] In some embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 262 and the VL of SEQ ID NO: 271.

[0734] In some embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 272. In some embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 264 and the VL of SEQ ID NO: 273.

[0735] In certain embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 82. In certain such embodiments, the first antigen binding domain is an scFv.

[0736] In other embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 85. In certain such embodiments, the first antigen binding domain is an scFv.

[0737] In other embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 59 and the VL of SEQ ID NO: 87. In certain such embodiments, the first antigen binding domain is an scFv.

[0738] In other embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 262 and the VL of SEQ ID NO: 271. In certain such embodiments, the first antigen binding domain is an scFv.

[0739] In other embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 272. In certain such embodiments, the first antigen binding domain is an scFv. In other embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NO: 264 and the VL of SEQ ID NO: 273. In certain such embodiments, the first antigen binding domain is an scFv. In certain embodiments, the first antigen binding domain that binds CD33 is an scFv comprising the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 82 and the second antigen binding domain binds to TRGV9 (for example, wherein the second binding domain is a VHH).

[0740] In other embodiments, the first antigen binding domain that binds CD33 is an scFv comprising the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 85 and the second antigen binding domain binds to TRGV9 (for example, wherein the second binding domain is a VHH).

[0741] In other embodiments, the first antigen binding domain that binds CD33 is an scFv comprising the VH of SEQ ID NO: 59 and the VL of SEQ ID NO: 87 and the second antigen binding binds to TRGV9 (for example, wherein the second binding domain is a VHH).

[0742] In other embodiments, the first antigen binding domain that binds CD33 is an scFv comprising the VH of SEQ ID NO: 262 and the VL of SEQ ID NO: 271 and the second antigen binding domain binds to TRGV9 (for example, wherein the second binding domain is a VHH).

[0743] In other embodiments, the first antigen binding domain that binds CD33 is an scFv comprising the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 272 and the second antigen binding domain binds to TRGV9 (for example, wherein the second binding domain is a VHH).

[0744] In other embodiments, the first antigen binding domain that binds CD33 is an scFv comprising the VH of SEQ ID NO: 264 and the VL of SEQ ID NO: 273 and the second antigen binding domain binds to TRGV9 (for example, wherein the second binding domain is a VHH).

[0745] In some embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 262, 263 or 264 and the VL of SEQ ID NOs: 81, 82, 83, 84, 85, 86, 87, 88, 271, 272 or 273.

[0746] In some embodiments, the first antigen binding domain that binds CD33 comprises the VH of SEQ ID NOs: 95, 96, 97, 170, 171, 175, 179, 181, or 96, and the VL of SEQ ID NOs: 105, 106, 107, 185, 188, 192, 196, 200, or 202.

[0747] In some embodiments, the first antigen binding domain that binds CD33 comprises: the VH of SEQ ID NO: 95 and the VL of SEQ ID NO: 105; the VH of SEQ ID NO: 96 and the VL of SEQ ID NO: 106; the VH of SEQ ID NO: 97 and the VL of SEQ ID NO: 107; the VH of SEQ ID NO: 170 and the VL of SEQ ID NO: 185; the VH of SEQ ID NO: 171 and the VL of SEQ ID NO: 188; the VH of SEQ ID NO: 175 and the VL of SEQ ID NO: 192; the VH of SEQ ID NO: 179 and the VL of SEQ ID NO: 196; the VH of SEQ ID NO: 181 and the VL of SEQ ID NO: 200; or the VH of SEQ ID NO: 96 and the VL of SEQ ID NO: 202.

[0748] In some embodiments, the first antigen binding domain that binds CD33 comprises the amino acid sequence of SEQ ID NOs: 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27.

[0749] In some embodiments, the first antigen binding domain that binds CD33 comprises the amino acid sequence of SEQ ID NO: 18.

[0750] In some embodiments, the first antigen binding domain that binds CD33 comprises the amino acid sequence of SEQ ID NO: 19.

[0751] In some embodiments, the first antigen binding domain that binds CD33 comprises the amino acid sequence of SEQ ID NO: 20.

[0752] In some embodiments, the first antigen binding domain that binds CD33 comprises the amino acid sequence of SEQ ID NO: 21.

[0753] In some embodiments, the first antigen binding domain that binds CD33 comprises the amino acid sequence of SEQ ID NO: 22.

[0754] In some embodiments, the first antigen binding domain that binds CD33 comprises the amino acid sequence of SEQ ID NO: 23.

[0755] In some embodiments, the first antigen binding domain that binds CD33 comprises the amino acid sequence of SEQ ID NO: 24.

[0756] In some embodiments, the first antigen binding domain that binds CD33 comprises the amino acid sequence of SEQ ID NO: 25.

[0757] In some embodiments, the first antigen binding domain that binds CD33 comprises the amino acid sequence of SEQ ID NO: 26.

[0758] In some embodiments, the first antigen binding domain that binds CD33 comprises the amino acid sequence of SEQ ID NO: 27.

[0759] In some embodiments, the first antigen binding domain that binds CD33 comprises the amino acid sequence of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, 59, 60 61, 262, 263 or 264.

[0760] In some embodiments, the first antigen binding domain that binds CD33 comprises the amino acid sequence of SEQ ID NO: 52.

[0761] In some embodiments, the first antigen binding domain that binds CD33 comprises the amino acid sequence of SEQ ID NO: 53.

[0762] In some embodiments, the first antigen binding domain that binds CD33 comprises the amino acid sequence of SEQ ID NO: 54.

[0763] In some embodiments, the first antigen binding domain that binds CD33 comprises the amino acid sequence of SEQ ID NO: 55. In some embodiments, the first antigen binding domain that binds CD33 comprises the amino acid sequence of SEQ ID NO: 56.

[0764] In some embodiments, the first antigen binding domain that binds CD33 comprises the amino acid sequence of SEQ ID NO: 57.

[0765] In some embodiments, the first antigen binding domain that binds CD33 comprises the amino acid sequence of SEQ ID NO: 58.

[0766] In some embodiments, the first antigen binding domain that binds CD33 comprises the amino acid sequence of SEQ...

Claims

CLAIMS1) An isolated protein that binds CD33, wherein the isolated protein comprises a) an HCDR1, an HCDR2 and an HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 53; b) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 56; c) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 59; d) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 262; e) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 263; or f) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 264.2) The isolated protein of claim 1, comprising the HCDR1, the HCDR2, and the HCDR3 of a) SEQ ID NOs: 29, 37, and 46, respectively; b) SEQ ID NOs: 32, 40, and 49, respectively; c) SEQ ID NOs: 34, 43, and 51, respectively; d) SEQ ID NOs: 253, 256, and 259, respectively; e) SEQ ID NOs: 254, 257, and 260, respectively; or f) SEQ ID NOs: 255, 258, and 261, respectively.3) An isolated protein that binds CD33, wherein the isolated protein comprises the HCDR1, the HCDR2, and the HCDR3 of a) SEQ ID NOs: 29, 37, and 46, respectively; b) SEQ ID NOs: 32, 40, and 49, respectively; c) SEQ ID NOs: 34, 43, and 51, respectively; d) SEQ ID NOs: 253, 256, and 259, respectively; e) SEQ ID NOs: 254, 257, and 260, respectively; or f) SEQ ID NOs: 255, 258, and 261, respectively.4) The isolated protein of claim 1 or 2, wherein the isolated protein comprises a) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 53 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 82; b) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 56 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 85; c) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 59 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 87; d) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 262 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 271;e) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 263 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 272; or f) an HCDR1, an HCDR2 and an HCDR3 of a VH of SEQ ID NO: 264 and an LCDR1, an LCDR2 and an LCDR3 of a VL of SEQ ID NO: 273.5) The isolated protein of claim 3 or 4, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of a) SEQ ID NOs: 29, 37, 46, 63, 70, and 76, respectively; b) SEQ ID NOs: 32, 40, 49, 66, 70, and 76, respectively; c) SEQ ID NOs: 34, 43, 51, 68, 74, and 80, respectively; d) SEQ ID NOs: 253, 256, 259, 265, 74 and 269, respectively e) SEQ ID NOs: 254, 257, 260, 66, 267 and 76, respectively; or f) SEQ ID NOs: 255, 258, 261, 266, 268 and 270, respectively.6) The isolated protein of any one of claims 1-5, wherein the isolated protein comprises the VH of SEQ ID NOs: 53, 56, 59, 262, 263, 264, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349,350, 351, 352, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, or 382, and the VL of SEQ ID NOs: 82, 85, 87, 271, 272, 273, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 353, 354, 355,356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 383, 384, 385, 386, 387, 388, 389, 390, 391,392, 393, 394, 395, 396, 397 or 398.7) The isolated protein of claim 6, wherein the isolated protein comprises: a) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 82; b) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 85; c) the VH of SEQ ID NO: 59 and the VL of SEQ ID NO: 87; d) the VH of SEQ ID NO: 262 and the VL of SEQ ID NO: 271; e) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 272; f) the VH of SEQ ID NO: 264 and the VL of SEQ ID NO: 273; g) the VH of SEQ ID NO: 291 and the VL of SEQ ID NO: 271; h) the VH of SEQ ID NO: 292 and the VL of SEQ ID NO: 271; i) the VH of SEQ ID NO: 293 and the VL of SEQ ID NO: 271 ; j) the VH of SEQ ID NO: 294 and the VL of SEQ ID NO: 271; k) the VH of SEQ ID NO: 295 and the VL of SEQ ID NO: 271; l) the VH of SEQ ID NO: 296 and the VL of SEQ ID NO: 271 ; m) the VH of SEQ ID NO: 297 and the VL of SEQ ID NO: 271 ;n) the VH of SEQ ID NO: 298 and the VL of SEQ ID NO: 271; o) the VH of SEQ ID NO: 299 and the VL of SEQ ID NO: 271; p) the VH of SEQ ID NO: 300 and the VL of SEQ ID NO: 271; q) the VH of SEQ ID NO: 301 and the VL of SEQ ID NO: 271; r) the VH of SEQ ID NO: 302 and the VL of SEQ ID NO: 271; s) the VH of SEQ ID NO: 303 and the VL of SEQ ID NO: 271; t) the VH of SEQ ID NO: 304 and the VL of SEQ ID NO: 271; u) the VH of SEQ ID NO: 305 and the VL of SEQ ID NO: 271; v) the VH of SEQ ID NO: 306 and the VL of SEQ ID NO: 271; w) the VH of SEQ ID NO: 307 and the VL of SEQ ID NO: 271; x) the VH of SEQ ID NO: 308 and the VL of SEQ ID NO: 271; y) the VH of SEQ ID NO: 309 and the VL of SEQ ID NO: 271; z) the VH of SEQ ID NO: 310 and the VL of SEQ ID NO: 271; aa) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 311 ; ab) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO:

312. ac) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 313; ad) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 314; ae) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 315; af) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 316; ag) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 317; ah) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 318; ai) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 319; aj) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 320; ak) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 321 ; al) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 322; am)the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 323; an) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 324; ao) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 325; ap) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 326; aq) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 327; ar) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 328; as) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 329; at) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 330; au) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 331 ;av) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 332; aw) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 333; ax) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 334; ay) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 335; az) the VH of SEQ ID NO: 263 and the VL of SEQ ID NO: 336; ba) the VH of SEQ ID NO: 337 and the VL of SEQ ID NO: 272; bb) the VH of SEQ ID NO: 338 and the VL of SEQ ID NO: 272; be) the VH of SEQ ID NO: 339 and the VL of SEQ ID NO: 272; bd) the VH of SEQ ID NO: 340 and the VL of SEQ ID NO: 272; be) the VH of SEQ ID NO: 341 and the VL of SEQ ID NO: 272; bf) the VH of SEQ ID NO: 342 and the VL of SEQ ID NO: 272; bg) the VH of SEQ ID NO: 343 and the VL of SEQ ID NO: 272; bh) the VH of SEQ ID NO: 344 and the VL of SEQ ID NO: 272; bi) the VH of SEQ ID NO: 345 and the VL of SEQ ID NO: 272; bj) the VH of SEQ ID NO: 346 and the VL of SEQ ID NO: 272; bk) the VH of SEQ ID NO: 347 and the VL of SEQ ID NO: 272; bl) the VH of SEQ ID NO: 348 and the VL of SEQ ID NO: 272; bm)the VH of SEQ ID NO: 349 and the VL of SEQ ID NO: 272; bn) the VH of SEQ ID NO: 350 and the VL of SEQ ID NO: 272; bo) the VH of SEQ ID NO: 351 and the VL of SEQ ID NO: 272; bp) the VH of SEQ ID NO: 352 and the VL of SEQ ID NO:

272. bq) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 353; br) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 354; bs) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 355; bt) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 356; bu) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 357; bv) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 358; bw)the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 359; bx) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 360; by) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 361; bz) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 362; ca) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 363; cb) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 364; cc) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 365;cd) the VH of SEQ ID NO: 56 and the VL of SEQ ID NO: 366; ce) the VH of SEQ ID NO: 367 and the VL of SEQ ID NO: 85; cf) the VH of SEQ ID NO: 368 and the VL of SEQ ID NO: 85; eg) the VH of SEQ ID NO: 369 and the VL of SEQ ID NO: 85; ch) the VH of SEQ ID NO: 370 and the VL of SEQ ID NO: 85; ci) the VH of SEQ ID NO: 371 and the VL of SEQ ID NO: 85; cj) the VH of SEQ ID NO: 372 and the VL of SEQ ID NO: 85; ck) the VH of SEQ ID NO: 373 and the VL of SEQ ID NO: 85; cl) the VH of SEQ ID NO: 374 and the VL of SEQ ID NO: 85; cm) the VH of SEQ ID NO: 375 and the VL of SEQ ID NO: 85; cn) the VH of SEQ ID NO: 376 and the VL of SEQ ID NO: 85; co) the VH of SEQ ID NO: 377 and the VL of SEQ ID NO: 85; cp) the VH of SEQ ID NO: 378 and the VL of SEQ ID NO: 85; cq) the VH of SEQ ID NO: 379 and the VL of SEQ ID NO: 85; cr) the VH of SEQ ID NO: 380 and the VL of SEQ ID NO: 85; cs) the VH of SEQ ID NO: 381 and the VL of SEQ ID NO: 85; ct) the VH of SEQ ID NO: 382 and the VL of SEQ ID NO: 85; cu) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 383; cv) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 384; cw)the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 385; ex) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 386; cy) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 387; cz) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 388; da) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 389; db) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 390; dc) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 391; dd) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 392; de) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 393; df) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 394; dg) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 395; dh) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 396; di) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 397; or dj) the VH of SEQ ID NO: 53 and the VL of SEQ ID NO: 398.8) The isolated protein of any one of claims 1-7, wherein the isolated protein is a Fab, a scFv, a (scFv)2, a Fv, a a F(ab’)2, a Fd, a dAb or a VHH.9) The isolated protein of claim 8, wherein the scFv comprises, from the N- to C-terminus, a VH, a first linker (LI) and a VL (VH-L1-VL) or the VL, the LI and the VH (VL-L1-VH).10) The isolated protein of claim 9, wherein the LI comprises: a) an amino acid sequence of any one of SEQ ID NOs: 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,136, 137, 138, 139, or 140; b) about 5-50 amino acids; about 5-40 amino acids; about 10-30 amino acids; or about 10-20 amino acids.11) The isolated protein of claim 10, wherein the isolated protein is a scFv comprising the amino acid sequence of SEQ ID NO: 213, 216, 219, 274, 275 or 276.12) The isolated protein of any one of claims 1-11, wherein the protein is conjugated to a half-life- extending or -modulating moiety selected from: an immunoglobulin (Ig); a fragment of the Ig; an Ig constant region; a fragment of the Ig constant region; a Fc region; a fragment of the Ig constant region comprising a Fc region; a fragment of the Ig constant region comprising a CH2 domain; a fragment of the Ig constant region comprising a CH3 domain; a fragment of the Ig constant region comprising a CH2 domain and a CH3 domain; a fragment of the Ig constant region comprising at least a portion of a hinge, a CH2 domain and a CH3 domain; a fragment of the Ig constant region comprising a hinge, a CH2 domain and a CH3 domain; transferrin; albumin; an albumin binding domain; or polyethylene glycol.13) The isolated protein of claims 12, comprising an antigen binding domain that binds CD33, wherein: a) said antigen binding domain is conjugated to the N-terminus of the Ig constant region or the fragment of the Ig constant region; b) said antigen binding domain is conjugated to the C-terminus of the Ig constant region or the fragment of the Ig constant region; or c) said antigen binding domain is conjugated to the Ig constant region or the fragment of the Ig constant region via a second linker (L2) comprising the amino acid sequence of any one of SEQ ID NOs: 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, or 140.14) The isolated protein of any one of claims 1-13, wherein the isolated protein is a monospecific protein, a multispecific protein, a bispecific protein, or a trispecific protein.15) The isolated protein of claim 14, wherein the multispecific protein, the bispecific protein, or the trispecific protein, each independently, comprises an antigen binding domain that binds an antigen on: a lymphocyte, a T cell, a CD8+T cell, or a natural killer (NK) cell.16) The isolated protein of claim 15, wherein the multispecific protein, the bispecific protein, or the trispecific protein, each independently, comprises an antigen binding domain that binds TRGV9, CD3 epsilon (CD3ε), CD3, CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1,CD 195, orNKG2C.17) The isolated protein of any one of claims 15-16, wherein a first antigen binding domain that binds CD33 and / or a second antigen binding domain that binds the lymphocyte antigen comprise a Fab, a scFv, a VHH, a (scFv)2, a Fv, a F(ab’)2, a Fd, or a dAb; or wherein the first antigen binding domain that binds CD33 comprises the scFv and the second antigen binding domain that binds the lymphocyte antigen comprises the VHH.18) The isolated protein of any one of claims 12-17, wherein the Ig constant region or the fragment of the Ig constant region is an IgGl, an IgG2, an IgG3 or an IgG4 isotype, wherein optionally the Ig constant region or the fragment of the Ig constant region is an IgGl.19) The isolated protein of claim 18, wherein the fragment of the Ig constant region has the sequence of SEQ ID NO: 277 or 278.20) The isolated protein of any one of claims 12-19, wherein the Ig constant region or the fragment of the Ig constant region comprises at least one mutation that results in reduced binding of the protein to a Fey receptor (FcyR); wherein the at least one mutation that results in reduced binding of the protein to the FcyR is selected from the group consisting of F234A / L235A, L234A / L235A, L234A / L235A / D265S, V234A / G237A / P238S / H268A / V309L / A330S / P331S, F234A / L235A, S228P / F234A / L235A, N297A, V234A / G237A, K214T / E233P / L234V / L235A / G236- deleted / A327G / P331A / D365E / L358M, H268Q / V309L / A330S / P331S, S267E / L328F, L234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S and S228P / F234A / L235A / G236-deleted / G237A / P238S, wherein residue numbering is according to the EU index.21) The isolated protein of any one of claims 12-19, wherein the Ig constant region or the fragment of the Ig constant region comprises at least one mutation that results in enhanced binding of the protein to the FcyR; wherein the at least one mutation that results in enhanced binding of the protein to the FcyR is selected from the group consisting of S239D / I332E, S298A / E333A / K334A, F243L / R292P / Y300L, F243L / R292P / Y300L / P396L, F243L / R292P / Y300L / V305I / P396L and G236A / S239D / I332E, wherein residue numbering is according to the EU index.22) The isolated protein of claims 20 or 21, wherein the FcyR is FcyRI, FcyRIIA, FcyRIIB or FcyRIII, or any combination thereof.23) The isolated protein of any one of claims 12-22, wherein the Ig constant region of the fragment of the Ig constant region comprises at least one mutation that modulates a half-life of the protein selected from the group consisting of H435A, P257I / N434H, D376V / N434H,M252Y / S254T / T256E / H433K / N434F, T308P / N434A and H435R, wherein residue numbering is according to the EU index.24) The isolated protein of any one of the claims 12-23, wherein the protein comprises at least one mutation in a CH3 domain of the Ig constant region selected from the group consisting of T350V, L351Y, F405A, Y407V, T366Y, T366W, F405W, T394W, T394S, Y407T, Y407A, T366S / L368A / Y407V, L351Y / F405A / Y407V, T366I / K392M / T394W, F405A / Y407V,T366L / K392M / T394 W, L351Y / Y407A, T366A / K409F, L351Y / Y407A, T366V / K409F, T366A / K409F, T350V / L351Y / F405A / Y407V and T350V / T366L / K392L / T394W, wherein residue numbering is according to the EU index.25) The isolated multispecific protein, the bispecific protein, or the trispecific protein, each independently, of any one of claims 14-24, wherein the first antigen binding domain that binds CD33 is conjugated to a first immunoglobulin (Ig) constant region or a fragment of the first Ig constant region and / or the second antigen binding domain that binds the lymphocyte antigen is conjugated to a second immunoglobulin (Ig) constant region or a fragment of the second Ig constant region.26) The isolated multispecific protein, the bispecific protein, or the trispecific protein, each independently, of claim 25, further comprising a second linker (L2) between the first antigen binding domain that binds CD33 and the first Ig constant region or the fragment of the first Ig constant region and the second antigen binding domain that binds the lymphocyte antigen and the second Ig constant region or the fragment of the second Ig constant region.27) The isolated multispecific protein, the bispecific protein, or the trispecific protein, each independently, of claim 26, wherein the L2 comprises the amino acid sequence of any one of SEQ ID NOs: 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, or 140.28) The isolated bispecific protein of any one of claims 25-27, wherein the first Ig constant region or the fragment of the first Ig constant region and the second Ig constant region or the fragment of the second Ig constant region is an IgGl, an IgG2, and IgG3 or an IgG4 isotype.29) The isolated bispecific protein of any one of claims 25-28, wherein the first Ig constant region or the fragment of the first Ig constant region and the second Ig constant region or the fragment of the second Ig constant region comprise the following mutations:L235A_L235A_D265S_T350V_L351Y_F405A_Y407V in the first Ig constant region and L235A_L235A_D265S_T350V_T366L_K392L_T394W in the second Ig constant region; or L235A_L235A_D265S_T350V_T366L_K392L_T394W in the first Ig constant region and L235A_L235A_D265S_T350V_L351Y_F405A_Y407V in the second Ig constant region.30) A chimeric antigen receptor (CAR) comprising: a) an extracellular domain comprising the antigen binding domain that binds CD33 of any one of claims 1-11; b) a transmembrane domain; and c) an intracellular signaling domain optionally comprising at least one co-stimulatory domain.31) The CAR of claim 30, wherein the CAR further comprises a CD8a-hinge region.32) The CAR of claims 30 or 31, wherein a) the transmembrane domain comprises a CD8a transmembrane region (CD8a-TM) polypeptide; and b) the intracellular signaling domain comprises a co-stimulatory domain comprising a TNF receptor superfamily member 9 (CD 137) component, a T-cell surface glycoprotein CD3 zeta chain (CD3z) component, a cluster of differentiation (CD27) component, a cluster of differentiation superfamily member component, and a combination thereof.33) The CAR of any one of claims 30-32, wherein a) the CD8a-hinge region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 157; b) the transmembrane domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 162; and / or c) the intracellular signaling domain comprises a co-stimulatory domain having a CD 137 component comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 163; a CD3z component comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 164; or an amino acid sequence of SEQ ID NO: 165.34) The CAR of any one of claims 30-33, wherein the extracellular domain comprising the antigen binding domain that binds CD33 further comprises a signal polypeptide.35) The CAR of claim 34, wherein the signal polypeptide comprises the amino acid sequence of SEQ IDNO: 166.36) The CAR of any one of claims 30-35, wherein the CD8a-TM polypeptide comprises an amino acid sequence of SEQ ID NO: 162.37) The CAR of any one of claims 30-36, wherein the CD8a-hinge region comprises an amino acid sequence of SEQ ID NO: 157.38) An isolated lymphocyte expressing the CAR of any one of claims 30-37.39) The isolated lymphocyte of claim 38, wherein the lymphocyte is a T lymphocyte or a natural killer (NK) cell.40) An immunoconjugate comprising the isolated protein of any one of claims 1-29 or the CAR of any one of claims 30-37, conjugated to a therapeutic agent, an imaging agent, or a radioactive agent.41) The immunoconjugate of claim 40, wherein the radioactive agent comprises a radiometal ion selected42) The immunoconjugate of claim 41, wherein the radiometal ion is conjugated to a chelating moiety, (a) wherein the chelating moiety comprises a macrocycle having the structure of formula (I):wherein each of R1, R2, R3and R4 is independently CHQCO2X, wherein Q is independently hydrogen, C1-C4alkyl or (C1-C2alkyl) phenyl, and X is independently hydrogen, benzyl, C1-C4alkyl; and Z is (CH2)nY, wherein n is 1-10, andY is an electrophilic or nucleophilic moiety covalently linked to the second click reaction partner; alternatively, Z is hydrogen; and each of R1, R2, R3and R4is independently CHQCO2X, wherein Q is independently hydrogen, C1-C4alkyl or (C1-C2alkyl) phenyl, and X is independently hydrogen, benzyl, C1-C4alkyl, or an electrophilic or nucleophilic moiety covalently linked to the second click reaction partner; alternatively, the chelant comprises an open chain ligand; or (b) wherein the chelating moiety comprises a macrocycle having the structure of formula (II):or a structure of formula (III):43) An immunoconjugate of formula (IV)formula (IV) wherein the protein is the isolated protein as defined in any one of claims 1-29, or the CAR as defined in any one of claims 30-37.44) An immunoconjugate of formula (V),formula (V) wherein the protein is the isolated protein as defined in any one of claims 1-29, or the CAR as defined in any one of claims 30-37.45) A pharmaceutical composition comprising the isolated protein of any one of claims 1-29, the CAR of any one of claims 30-37, the isolated lymphocyte of any one of claims 38-39, or the immunoconjugate of any one of claims 40-44, and a pharmaceutically acceptable carrier.46) A polynucleotide encoding the isolated protein of any one of claims 1-29, the CAR of any one of claims 30-37, or the immunoconjugate of any one of claims 40-44.47) A vector comprising the polynucleotide of claim 46.48) A host cell comprising the vector of claim 47 or polynucleotide of claim 46.49) A method of producing the isolated protein of any one of claims 1-29, comprising culturing the host cell of claim 48 in conditions that the protein is expressed, and recovering the protein produced by the host cell.50) A method of producing the immunoconjugate of any one of claims 40-44, comprising culturing the host cell of claim 48 in conditions that the protein is expressed, recovering the protein produced by the host cell, and conjugating the protein produced by the host cell with the agent.51) An anti-idiotypic antibody binding to the isolated protein of any one of claims 1-29.52) A kit comprising the isolated protein of any one of claims 1-29, or the CAR of any one of claims 30- 37.53) A method of treating a CD33 expressing cancer in a subject, comprising administering a therapeutically effective amount of the isolated protein of any one of claims 1-29, or immunoconjugate of any one of claims 40-44, to the subject for a time sufficient to beat the CD33 expressing cancer.54) A method of treating a subject having a CD33 expressing cancer, the method comprising: administering a therapeutically effective amount of the lymphocyte of any one of claims 38-39 to a subject in need thereof, whereby the lymphocyte mediates killing of the CD33 expressing cancer in the subject.55) The method of any one of claims 53-54, wherein the CD33 expressing cancer is a hematologic cancer.56) The method of claim 55, wherein the hematologic cancer is a leukemia, a lymphoma, a multiple myeloma, acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML) or blastic plasmacytoid dendritic cell neoplasm (DPDCN).57) The method of any one of claims 53-56, wherein the isolated protein, or the immunoconjugate, is administered in combination with a second therapeutic agent.58) The method of claim 57, wherein the second therapeutic agent is surgery, chemotherapy, androgen deprivation therapy or radiation, or any combination thereof.59) The isolated protein of any one of claims 1-29, or the immunoconjugate of any one of claims 40-44, for use in a method of treating a CD33 expressing cancer in a subject, comprising administering a therapeutically effective amount of the to the subject for a time sufficient to treat the CD33 expressing cancer.60) The lymphocyte of any one of claims 38-39, for use in a method of treating a subject having a CD33 expressing cancer, the method comprising: administering a therapeutically effective amount of the lymphocyte of any one of claims 38-39 to a subject in need thereof, whereby the lymphocyte mediates killing of the CD33 expressing cancer in the subject.61) The isolated protein, the immunoconjugate, or the lymphocyte for the use of claims 59 or 60, wherein the CD33 expressing cancer is a hematologic cancer.62) The isolated protein, the immunoconjugate, or the lymphocyte for the use of claim 61, wherein the hematologic cancer is a leukemia, a lymphoma, a multiple myeloma, acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML) or blastic plasmacytoid dendritic cell neoplasm (DPDCN).63) The isolated protein, the immunoconjugate, or the lymphocyte for the use of any one of claims 59-62, wherein the isolated protein, or the immunoconjugate, is administered in combination with a second therapeutic agent.64) The isolated protein, the immunoconjugate, or the lymphocyte for the use of claim 63, wherein the second therapeutic agent is surgery, chemotherapy, androgen deprivation therapy or radiation, or any combination thereof.65) A method of detecting the presence of a cancer in a subject, comprising: a) contacting a cell sample obtained from the subject with the CAR of any one of claims 30-37, thereby forming a CAR-cell complex, and b) detecting the CAR-cell complex, wherein detection of the CAR-cell complex is indicative of the presence of the cancer in the subject.