Anti-mutant calreticulin (CALR) antibodies and uses thereof

JP2025500796A5Pending Publication Date: 2026-04-08INCYTE CORP
View PDF 0 Cites 0 Cited by

Patent Information

Authority / Receiving Office
JP · JP
Patent Type
Applications
Current Assignee / Owner
Filing Date
2022-12-07
Publication Date
2026-04-08

AI Technical Summary

Technical Problem

Current treatments for myeloproliferative neoplasms, such as chronic myeloid leukemia and polycythemia vera, are limited in effectively targeting mutant calreticulin (CALR) proteins that drive oncogenic signaling and proliferation.

Method used

Development of antibodies with high binding affinity and specificity for mutant CALR (mutCALR) proteins, inhibiting pathways downstream of the thrombopoietin receptor (MPL) and disrupting MPL dimerization to suppress oncogenic cell proliferation.

Benefits of technology

The antibodies effectively inhibit mutCALR-induced signaling and proliferation, offering therapeutic potential for myeloproliferative neoplasms by restoring normal blood cell counts and bone marrow environments.

✦ Generated by Eureka AI based on patent content.
Patent Text Reader

Abstract

Anti-mutant calreticulin (mutCALR) antibodies are disclosed. Also disclosed are related nucleic acids, vectors, cells, kits, and pharmaceutical compositions. Also disclosed are methods of treating or diagnosing myeloproliferative neoplasms using anti-mutCALR antibodies.
Need to check novelty before this filing date? Find Prior Art

Description

[Technical field]

[0001] Claiming priority This application claims the benefit of U.S. Provisional Patent Application No. 63 / 287,394, filed December 8, 2021, U.S. Provisional Patent Application No. 63 / 288,479, filed December 10, 2021, and U.S. Provisional Patent Application No. 63 / 421,052, filed October 31, 2022, each of which is incorporated by reference in its entirety herein.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically as an XML file named 20443-0736WO1_SL.xml. The size of the XML file, created on December 5, 2022, is 388,039 bytes. The material within the XML file is incorporated herein by reference in its entirety. [Background technology]

[0003] Calreticulin (CALR) is a highly conserved chaperone protein that resides primarily in the endoplasmic reticulum and is involved in a variety of cellular processes, including protein folding, calcium homeostasis, cell adhesion, and integrin signaling. CALR is also found in the nucleus, suggesting that it may be involved in transcriptional regulation. Mutations in the gene for CALR have been identified in patients with myeloproliferative neoplasms. Summary of the Invention

[0004] The present disclosure is based, at least in part, on the development of antibodies with high binding affinity and specificity for mutant CALR ("mutCALR").

[0005] Accordingly, aspects of the disclosure provide an antibody that binds to human mutant calreticulin (CALR), the antibody comprising a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein VH CDR1 comprises the amino acid sequence X1X2X3X4X5, where X1 is S, E, or D, X2 is Y, L, or S, X3 is A, S, or F, X4 is I or M, and X5 is S, Q, or H, and VH CDR2 comprises the amino acid sequence X6X7X8PX9X 10 X 11 X 12 X 13 X 14 YAX 15 X 16 X 17 X 18 X6 is L or G, X7 is V, F, or I, X8 is D or I, X9 is E, D, or I, and X 10 is D, G, F, A, S, or E, and X 11 is G or A, and X 12 is E or T, and X 13 is T or A, and X 14 is I, M, or N, and X 15 is E or Q, and X 16 is K or R, and X 17 is F or L, and X 18 is R or Q, and VH CDR3 is the amino acid sequence X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 X 35 X 36 X 37 X 38 (SEQ ID NO:98), wherein X 19is P, E, or absent, and X 20 is G, E, or non-existent, and X 21 is G, W, S, or non-existent, and X 22 is I, D, S, P, or non-existent, and X 23 is S, L, I, T, G, or absent, and X 24 is P, T, Q, I, D, R, or non-existent, and X 25 is G, D, or non-existent, and X 26 is E, Y, P, L, D, or S, and X 27 is E, D, A, or G, and X 28 is S, F, A, E, Y, or W, and X 29 is Y or F, and X 30 is G, D, or W, and X 31 is P, Y, I, or H, and X 32 is Y or absent, X 33 is Y or absent, X 34 is Y or absent, X 35 is G or non-existent, and X 36 is M or absent, and X 37 is D or non-existent, and X 38 is V or absent, and the antibody comprises a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3, and VL CDR1 has the amino acid sequence X 39 X 40 X 41 X 42 X 43 X 44 X 45 X 46 X 47 X 48 X 49 X 50 X 51 X 52 X 53 X 54 (SEQ ID NO: 99), wherein X 39 is T, A, or absent, and X 40 is G or non-existent, and X 41 is Q, G, V, T, or S, and X 42is A, G, S, or N, and X 43 is S, N, D, T, or Y, and X 44 is Q, Y, N, D, S, or K, and X 45 is D, I, F, V, S, or T, and X 46 is N or absent, and X 47 is I or absent, X 48 is I, G, or R, and X 49 is S, G, A, D, I, R, or T, and X 50 is Y or absent, X 51 is N, K, I, or E, and X 52 is Y, S, N, D, H, F, R, or G, and X 53 is L or V, and X 54 is N, H, S, D, or F, and VL CDR2 is the amino acid sequence X 55 X 56 X 57 X 58 X 59 X 60 X 61 where X 55 is T, D, E, Q, or R, and X 56 is A, D, V, or N, and X 57 is S, G, N, or R, and X 58 is I, N, D, or K, and X 59 is L, R, or W, and X 60 is E or P, and X 61 is S, T, or L, and the VL CDR3 is the amino acid sequence X 62 X 63 X 64 X 65 X 66 X 67 X 68 X 69 X 70 X 71 X 72 where X 62 is Q, S, C, or G, and X 63 is Q, V, S, T, or A, and X 64 is Q, L, W, or Y, and X 65is Q, N, D, I, T, A, or G, and X 66 is S, P, G, N, or A, and X 67 is N, Y, I, S, N, L, or D, and X 68 is E, P, S, I, N, H, L, or T, and X 69 is D, T, S, or absent, and X 70 is P, H, L, R, F, A, Q, or absent, and X 71 is W, L, V, Y, S, A, or E, and X 72 is T, V, or I.

[0006] In some embodiments, VH CDR1 comprises any one of the amino acid sequences of SEQ ID NOs: 1 to 6, VH CDR2 comprises any one of the amino acid sequences of SEQ ID NOs: 7 to 17 and 92 to 95, VH CDR3 comprises any one of the amino acid sequences of SEQ ID NOs: 18 to 25, VL CDR1 comprises any one of the amino acid sequences of SEQ ID NOs: 26 to 52 or 118, VL CDR2 comprises any one of the amino acid sequences of SEQ ID NOs: 53 to 68, and VL CDR3 comprises any one of the amino acid sequences of SEQ ID NOs: 69 to 91.

[0007] In some embodiments, VH CDR1, VH CDR2, and VH CDR3 each correspond to the VH CDRs shown in Tables 1-2 for a single VH clone, and VL CDR1, VL CDR2, and VL CDR3 each correspond to the VL CDRs shown in Tables 1-2 for a single VL clone.

[0008] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, the VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, the VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, the VL CDR1 comprises the amino acid sequence of SEQ ID NO:26, the VL CDR2 comprises the amino acid sequence of SEQ ID NO:53, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:69. VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:27, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:70; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:8, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:28, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:28, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:29, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:72; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:30, VL CDR2 comprises the amino acid sequence of SEQ ID NO:55, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:29, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:28, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:73; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:28, VL CDR2 comprises the amino acid sequence of SEQ ID NO:56, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:31, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:30, VL CDR2 comprises the amino acid sequence of SEQ ID NO:57, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:9, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:32, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:33, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:73; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:34, VL CDR2 comprises the amino acid sequence of SEQ ID NO:56, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:27, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:35, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:36, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:72; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:28, VL CDR2 comprises the amino acid sequence of SEQ ID NO:58, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:37, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:38, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:32, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:39, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:40, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:10, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:28, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:41, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:28, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:70; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:40, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:28, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:74; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:7, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:28, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:75; VH CDR1 comprises the amino acid sequence of SEQ ID NO: 1, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 7, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 18, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 118, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:2, VH CDR2 comprises the amino acid sequence of SEQ ID NO:11, VH CDR3 comprises the amino acid sequence of SEQ ID NO:19, VL CDR1 comprises the amino acid sequence of SEQ ID NO:30, VL CDR2 comprises the amino acid sequence of SEQ ID NO:59, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:76; VH CDR1 comprises the amino acid sequence of SEQ ID NO:2, VH CDR2 comprises the amino acid sequence of SEQ ID NO:11, VH CDR3 comprises the amino acid sequence of SEQ ID NO:19, VL CDR1 comprises the amino acid sequence of SEQ ID NO:30, VL CDR2 comprises the amino acid sequence of SEQ ID NO:55, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:77; VH CDR1 comprises the amino acid sequence of SEQ ID NO:2, VH CDR2 comprises the amino acid sequence of SEQ ID NO:11, VH CDR3 comprises the amino acid sequence of SEQ ID NO:19, VL CDR1 comprises the amino acid sequence of SEQ ID NO:42, VL CDR2 comprises the amino acid sequence of SEQ ID NO:59, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:76; VH CDR1 comprises the amino acid sequence of SEQ ID NO:2, VH CDR2 comprises the amino acid sequence of SEQ ID NO:11, VH CDR3 comprises the amino acid sequence of SEQ ID NO:19, VL CDR1 comprises the amino acid sequence of SEQ ID NO:30, VL CDR2 comprises the amino acid sequence of SEQ ID NO:55, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:78; VH CDR1 comprises the amino acid sequence of SEQ ID NO:2, VH CDR2 comprises the amino acid sequence of SEQ ID NO:12, VH CDR3 comprises the amino acid sequence of SEQ ID NO:19, VL CDR1 comprises the amino acid sequence of SEQ ID NO:30, VL CDR2 comprises the amino acid sequence of SEQ ID NO:55, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:77; VH CDR1 comprises the amino acid sequence of SEQ ID NO:2, VH CDR2 comprises the amino acid sequence of SEQ ID NO:11, VH CDR3 comprises the amino acid sequence of SEQ ID NO:19, VL CDR1 comprises the amino acid sequence of SEQ ID NO:43, VL CDR2 comprises the amino acid sequence of SEQ ID NO:59, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:79; VH CDR1 comprises the amino acid sequence of SEQ ID NO:2, VH CDR2 comprises the amino acid sequence of SEQ ID NO:11, VH CDR3 comprises the amino acid sequence of SEQ ID NO:19, VL CDR1 comprises the amino acid sequence of SEQ ID NO:30, VL CDR2 comprises the amino acid sequence of SEQ ID NO:60, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:79; VH CDR1 comprises the amino acid sequence of SEQ ID NO:3, VH CDR2 comprises the amino acid sequence of SEQ ID NO:13, VH CDR3 comprises the amino acid sequence of SEQ ID NO:20, VL CDR1 comprises the amino acid sequence of SEQ ID NO:44, VL CDR2 comprises the amino acid sequence of SEQ ID NO:61, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:80; VH CDR1 comprises the amino acid sequence of SEQ ID NO:3, VH CDR2 comprises the amino acid sequence of SEQ ID NO:11, VH CDR3 comprises the amino acid sequence of SEQ ID NO:20, VL CDR1 comprises the amino acid sequence of SEQ ID NO:45, VL CDR2 comprises the amino acid sequence of SEQ ID NO:61, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:80; VH CDR1 comprises the amino acid sequence of SEQ ID NO: 4, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 14, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 21, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 46, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 62, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 81; VH CDR1 comprises the amino acid sequence of SEQ ID NO:5, VH CDR2 comprises the amino acid sequence of SEQ ID NO:14, VH CDR3 comprises the amino acid sequence of SEQ ID NO:21, VL CDR1 comprises the amino acid sequence of SEQ ID NO:46, VL CDR2 comprises the amino acid sequence of SEQ ID NO:62, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:81; VH CDR1 comprises the amino acid sequence of SEQ ID NO:5, VH CDR2 comprises the amino acid sequence of SEQ ID NO:14, VH CDR3 comprises the amino acid sequence of SEQ ID NO:21, VL CDR1 comprises the amino acid sequence of SEQ ID NO:46, VL CDR2 comprises the amino acid sequence of SEQ ID NO:63, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:81; VH CDR1 comprises the amino acid sequence of SEQ ID NO:3, VH CDR2 comprises the amino acid sequence of SEQ ID NO:15, VH CDR3 comprises the amino acid sequence of SEQ ID NO:22, VL CDR1 comprises the amino acid sequence of SEQ ID NO:47, VL CDR2 comprises the amino acid sequence of SEQ ID NO:64, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:82; VH CDR1 comprises the amino acid sequence of SEQ ID NO:3, VH CDR2 comprises the amino acid sequence of SEQ ID NO:15, VH CDR3 comprises the amino acid sequence of SEQ ID NO:22, VL CDR1 comprises the amino acid sequence of SEQ ID NO:48, VL CDR2 comprises the amino acid sequence of SEQ ID NO:64, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:83; VH CDR1 comprises the amino acid sequence of SEQ ID NO:3, VH CDR2 comprises the amino acid sequence of SEQ ID NO:15, VH CDR3 comprises the amino acid sequence of SEQ ID NO:22, VL CDR1 comprises the amino acid sequence of SEQ ID NO:49, VL CDR2 comprises the amino acid sequence of SEQ ID NO:65, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:84; VH CDR1 comprises the amino acid sequence of SEQ ID NO:3, VH CDR2 comprises the amino acid sequence of SEQ ID NO:15, VH CDR3 comprises the amino acid sequence of SEQ ID NO:22, VL CDR1 comprises the amino acid sequence of SEQ ID NO:48, VL CDR2 comprises the amino acid sequence of SEQ ID NO:64, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:85; VH CDR1 comprises the amino acid sequence of SEQ ID NO:3, VH CDR2 comprises the amino acid sequence of SEQ ID NO:15, VH CDR3 comprises the amino acid sequence of SEQ ID NO:22, VL CDR1 comprises the amino acid sequence of SEQ ID NO:50, VL CDR2 comprises the amino acid sequence of SEQ ID NO:65, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:84; VH CDR1 comprises the amino acid sequence of SEQ ID NO:3, VH CDR2 comprises the amino acid sequence of SEQ ID NO:15, VH CDR3 comprises the amino acid sequence of SEQ ID NO:22, VL CDR1 comprises the amino acid sequence of SEQ ID NO:28, VL CDR2 comprises the amino acid sequence of SEQ ID NO:66, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:86; VH CDR1 comprises the amino acid sequence of SEQ ID NO:3, VH CDR2 comprises the amino acid sequence of SEQ ID NO:15, VH CDR3 comprises the amino acid sequence of SEQ ID NO:22, VL CDR1 comprises the amino acid sequence of SEQ ID NO:47, VL CDR2 comprises the amino acid sequence of SEQ ID NO:64, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:84; VH CDR1 comprises the amino acid sequence of SEQ ID NO:3, VH CDR2 comprises the amino acid sequence of SEQ ID NO:15, VH CDR3 comprises the amino acid sequence of SEQ ID NO:22, VL CDR1 comprises the amino acid sequence of SEQ ID NO:118, VL CDR2 comprises the amino acid sequence of SEQ ID NO:66, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:87; VH CDR1 comprises the amino acid sequence of SEQ ID NO:3, VH CDR2 comprises the amino acid sequence of SEQ ID NO:15, VH CDR3 comprises the amino acid sequence of SEQ ID NO:22, VL CDR1 comprises the amino acid sequence of SEQ ID NO:50, VL CDR2 comprises the amino acid sequence of SEQ ID NO:64, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:88; VH CDR1 comprises the amino acid sequence of SEQ ID NO:5, VH CDR2 comprises the amino acid sequence of SEQ ID NO:11, VH CDR3 comprises the amino acid sequence of SEQ ID NO:23, VL CDR1 comprises the amino acid sequence of SEQ ID NO:44, VL CDR2 comprises the amino acid sequence of SEQ ID NO:61, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:89; VH CDR1 comprises the amino acid sequence of SEQ ID NO:5, VH CDR2 comprises the amino acid sequence of SEQ ID NO:16, VH CDR3 comprises the amino acid sequence of SEQ ID NO:24, VL CDR1 comprises the amino acid sequence of SEQ ID NO:51, VL CDR2 comprises the amino acid sequence of SEQ ID NO:67, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:90; VH CDR1 comprises the amino acid sequence of SEQ ID NO:6, VH CDR2 comprises the amino acid sequence of SEQ ID NO:17, VH CDR3 comprises the amino acid sequence of SEQ ID NO:25, VL CDR1 comprises the amino acid sequence of SEQ ID NO:52, VL CDR2 comprises the amino acid sequence of SEQ ID NO:68, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:91; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:92, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:30, VL CDR2 comprises the amino acid sequence of SEQ ID NO:55, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:93, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:30, VL CDR2 comprises the amino acid sequence of SEQ ID NO:55, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:94, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:30, VL CDR2 comprises the amino acid sequence of SEQ ID NO:55, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO: 1, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 95, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 18, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 30, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 55, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:92, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:28, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:93, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:28, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:94, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:28, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:95, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:28, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO: 1, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 92, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 18, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 27, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:93, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:27, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:94, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:27, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:95, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:27, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; VH CDR1 comprises the amino acid sequence of SEQ ID NO: 1, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 92, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 18, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 36, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 72; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:93, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:36, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:72; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:94, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:36, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:72; VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, VH CDR2 comprises the amino acid sequence of SEQ ID NO:95, VH CDR3 comprises the amino acid sequence of SEQ ID NO:18, VL CDR1 comprises the amino acid sequence of SEQ ID NO:36, VL CDR2 comprises the amino acid sequence of SEQ ID NO:54, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:72, or VH CDR1 comprises the amino acid sequence of SEQ ID NO: 329, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 7, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 18, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 30, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 55, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 71.

[0009] In some embodiments, the VH is at least 80% identical to the amino acid sequence of any one of SEQ ID NOs: 165-208, and the VL is at least 80% identical to the amino acid sequence of any one of SEQ ID NOs: 264-318.

[0010] In some embodiments, the VH comprises any one of the amino acid sequences of SEQ ID NOs: 165-208, and the VL comprises any one of the amino acid sequences of SEQ ID NOs: 264-318.

[0011] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 165 and the VL comprises the amino acid sequence of SEQ ID NO: 264. VH comprises the amino acid sequence of SEQ ID NO: 165, and VL comprises the amino acid sequence of SEQ ID NO: 265; VH comprises the amino acid sequence of SEQ ID NO: 166, and VL comprises the amino acid sequence of SEQ ID NO: 266; VH comprises the amino acid sequence of SEQ ID NO: 165, and VL comprises the amino acid sequence of SEQ ID NO: 266; VH comprises the amino acid sequence of SEQ ID NO: 165, and VL comprises the amino acid sequence of SEQ ID NO: 267; VH comprises the amino acid sequence of SEQ ID NO: 165, and VL comprises the amino acid sequence of SEQ ID NO: 268; VH comprises the amino acid sequence of SEQ ID NO: 165, and VL comprises the amino acid sequence of SEQ ID NO: 269; VH comprises the amino acid sequence of SEQ ID NO: 167, and VL comprises the amino acid sequence of SEQ ID NO: 270; VH comprises the amino acid sequence of SEQ ID NO: 165, and VL comprises the amino acid sequence of SEQ ID NO: 271; VH comprises the amino acid sequence of SEQ ID NO: 165, and VL comprises the amino acid sequence of SEQ ID NO: 272; VH comprises the amino acid sequence of SEQ ID NO: 168, and VL comprises the amino acid sequence of SEQ ID NO: 273; VH comprises the amino acid sequence of SEQ ID NO: 169, and VL comprises the amino acid sequence of SEQ ID NO: 274; VH comprises the amino acid sequence of SEQ ID NO: 170, and VL comprises the amino acid sequence of SEQ ID NO: 275; VH comprises the amino acid sequence of SEQ ID NO: 171, and VL comprises the amino acid sequence of SEQ ID NO: 276; VH comprises the amino acid sequence of SEQ ID NO: 165, and VL comprises the amino acid sequence of SEQ ID NO: 277; VH comprises the amino acid sequence of SEQ ID NO: 165, and VL comprises the amino acid sequence of SEQ ID NO: 278; VH comprises the amino acid sequence of SEQ ID NO: 165, and VL comprises the amino acid sequence of SEQ ID NO: 279; VH comprises the amino acid sequence of SEQ ID NO: 165, and VL comprises the amino acid sequence of SEQ ID NO: 280; VH comprises the amino acid sequence of SEQ ID NO: 172, and VL comprises the amino acid sequence of SEQ ID NO: 281; VH comprises the amino acid sequence of SEQ ID NO: 165, and VL comprises the amino acid sequence of SEQ ID NO: 282; VH comprises the amino acid sequence of SEQ ID NO: 173, and VL comprises the amino acid sequence of SEQ ID NO: 283; VH comprises the amino acid sequence of SEQ ID NO: 165, and VL comprises the amino acid sequence of SEQ ID NO: 284; VH comprises the amino acid sequence of SEQ ID NO: 165, and VL comprises the amino acid sequence of SEQ ID NO: 285; VH comprises the amino acid sequence of SEQ ID NO: 174, and VL comprises the amino acid sequence of SEQ ID NO: 286; VH comprises the amino acid sequence of SEQ ID NO: 165, and VL comprises the amino acid sequence of SEQ ID NO: 287; VH comprises the amino acid sequence of SEQ ID NO: 165, and VL comprises the amino acid sequence of SEQ ID NO: 288; VH comprises the amino acid sequence of SEQ ID NO: 165, and VL comprises the amino acid sequence of SEQ ID NO: 289; VH comprises the amino acid sequence of SEQ ID NO: 165, and VL comprises the amino acid sequence of SEQ ID NO: 290; VH comprises the amino acid sequence of SEQ ID NO: 165, and VL comprises the amino acid sequence of SEQ ID NO: 291; VH comprises the amino acid sequence of SEQ ID NO: 165, and VL comprises the amino acid sequence of SEQ ID NO: 292; VH comprises the amino acid sequence of SEQ ID NO: 175, and VL comprises the amino acid sequence of SEQ ID NO: 293; VH comprises the amino acid sequence of SEQ ID NO: 175, and VL comprises the amino acid sequence of SEQ ID NO: 294; VH comprises the amino acid sequence of SEQ ID NO: 175, and VL comprises the amino acid sequence of SEQ ID NO: 295; VH comprises the amino acid sequence of SEQ ID NO: 175, and VL comprises the amino acid sequence of SEQ ID NO: 296; VH comprises the amino acid sequence of SEQ ID NO: 176, and VL comprises the amino acid sequence of SEQ ID NO: 294; VH comprises the amino acid sequence of SEQ ID NO: 175, and VL comprises the amino acid sequence of SEQ ID NO: 297; VH comprises the amino acid sequence of SEQ ID NO: 175, and VL comprises the amino acid sequence of SEQ ID NO: 298; VH comprises the amino acid sequence of SEQ ID NO: 177, and VL comprises the amino acid sequence of SEQ ID NO: 299; VH comprises the amino acid sequence of SEQ ID NO: 178, and VL comprises the amino acid sequence of SEQ ID NO: 300; VH comprises the amino acid sequence of SEQ ID NO: 179, and VL comprises the amino acid sequence of SEQ ID NO: 301; VH comprises the amino acid sequence of SEQ ID NO: 180, and VL comprises the amino acid sequence of SEQ ID NO: 301; VH comprises the amino acid sequence of SEQ ID NO: 180, and VL comprises the amino acid sequence of SEQ ID NO: 302; VH comprises the amino acid sequence of SEQ ID NO: 181, and VL comprises the amino acid sequence of SEQ ID NO: 303; VH comprises the amino acid sequence of SEQ ID NO: 182, and VL comprises the amino acid sequence of SEQ ID NO: 304; VH comprises the amino acid sequence of SEQ ID NO: 182, and VL comprises the amino acid sequence of SEQ ID NO: 305; VH comprises the amino acid sequence of SEQ ID NO: 183, and VL comprises the amino acid sequence of SEQ ID NO: 306; VH comprises the amino acid sequence of SEQ ID NO: 182, and VL comprises the amino acid sequence of SEQ ID NO: 307; VH comprises the amino acid sequence of SEQ ID NO: 182, and VL comprises the amino acid sequence of SEQ ID NO: 308; VH comprises the amino acid sequence of SEQ ID NO: 182, and VL comprises the amino acid sequence of SEQ ID NO: 309; VH comprises the amino acid sequence of SEQ ID NO: 184, and VL comprises the amino acid sequence of SEQ ID NO: 310; VH comprises the amino acid sequence of SEQ ID NO: 182, and VL comprises the amino acid sequence of SEQ ID NO: 311; VH comprises the amino acid sequence of SEQ ID NO: 185, and VL comprises the amino acid sequence of SEQ ID NO: 312; VH comprises the amino acid sequence of SEQ ID NO: 186, and VL comprises the amino acid sequence of SEQ ID NO: 313; VH comprises the amino acid sequence of SEQ ID NO: 187, and VL comprises the amino acid sequence of SEQ ID NO: 314; VH comprises the amino acid sequence of SEQ ID NO: 188, and VL comprises the amino acid sequence of SEQ ID NO: 268; VH comprises the amino acid sequence of SEQ ID NO: 189, and VL comprises the amino acid sequence of SEQ ID NO: 268; VH comprises the amino acid sequence of SEQ ID NO: 190, and VL comprises the amino acid sequence of SEQ ID NO: 268; VH comprises the amino acid sequence of SEQ ID NO: 191, and VL comprises the amino acid sequence of SEQ ID NO: 268; VH comprises the amino acid sequence of SEQ ID NO: 192, and VL comprises the amino acid sequence of SEQ ID NO: 268; VH comprises the amino acid sequence of SEQ ID NO: 193, and VL comprises the amino acid sequence of SEQ ID NO: 268; VH comprises the amino acid sequence of SEQ ID NO: 194, and VL comprises the amino acid sequence of SEQ ID NO: 268; VH comprises the amino acid sequence of SEQ ID NO: 195, and VL comprises the amino acid sequence of SEQ ID NO: 268; VH comprises the amino acid sequence of SEQ ID NO: 196, and VL comprises the amino acid sequence of SEQ ID NO: 268; VH comprises the amino acid sequence of SEQ ID NO: 165, and VL comprises the amino acid sequence of SEQ ID NO: 315; VH comprises the amino acid sequence of SEQ ID NO: 188, and VL comprises the amino acid sequence of SEQ ID NO: 315; VH comprises the amino acid sequence of SEQ ID NO: 189, and VL comprises the amino acid sequence of SEQ ID NO: 315; VH comprises the amino acid sequence of SEQ ID NO: 190, and VL comprises the amino acid sequence of SEQ ID NO: 315; VH comprises the amino acid sequence of SEQ ID NO: 191, and VL comprises the amino acid sequence of SEQ ID NO: 315; VH comprises the amino acid sequence of SEQ ID NO: 192, and VL comprises the amino acid sequence of SEQ ID NO: 315; VH comprises the amino acid sequence of SEQ ID NO: 193, and VL comprises the amino acid sequence of SEQ ID NO: 315; VH comprises the amino acid sequence of SEQ ID NO: 194, and VL comprises the amino acid sequence of SEQ ID NO: 315; VH comprises the amino acid sequence of SEQ ID NO: 195, and VL comprises the amino acid sequence of SEQ ID NO: 315; VH comprises the amino acid sequence of SEQ ID NO: 196, and VL comprises the amino acid sequence of SEQ ID NO: 315; VH comprises the amino acid sequence of SEQ ID NO: 165, and VL comprises the amino acid sequence of SEQ ID NO: 316; VH comprises the amino acid sequence of SEQ ID NO: 188, and VL comprises the amino acid sequence of SEQ ID NO: 316; VH comprises the amino acid sequence of SEQ ID NO: 189, and VL comprises the amino acid sequence of SEQ ID NO: 316; VH comprises the amino acid sequence of SEQ ID NO: 190, and VL comprises the amino acid sequence of SEQ ID NO: 316; VH comprises the amino acid sequence of SEQ ID NO: 191, and VL comprises the amino acid sequence of SEQ ID NO: 316; VH comprises the amino acid sequence of SEQ ID NO: 192, and VL comprises the amino acid sequence of SEQ ID NO: 316; VH comprises the amino acid sequence of SEQ ID NO: 193, and VL comprises the amino acid sequence of SEQ ID NO: 316; VH comprises the amino acid sequence of SEQ ID NO: 194, and VL comprises the amino acid sequence of SEQ ID NO: 316; VH comprises the amino acid sequence of SEQ ID NO: 195, and VL comprises the amino acid sequence of SEQ ID NO: 316; VH comprises the amino acid sequence of SEQ ID NO: 196, and VL comprises the amino acid sequence of SEQ ID NO: 316; VH comprises the amino acid sequence of SEQ ID NO: 197, and VL comprises the amino acid sequence of SEQ ID NO: 268; VH comprises the amino acid sequence of SEQ ID NO: 198, and VL comprises the amino acid sequence of SEQ ID NO: 268; VH comprises the amino acid sequence of SEQ ID NO: 199, and VL comprises the amino acid sequence of SEQ ID NO: 268; VH comprises the amino acid sequence of SEQ ID NO: 200, and VL comprises the amino acid sequence of SEQ ID NO: 268; VH comprises the amino acid sequence of SEQ ID NO: 201, and VL comprises the amino acid sequence of SEQ ID NO: 268; VH comprises the amino acid sequence of SEQ ID NO: 197, and VL comprises the amino acid sequence of SEQ ID NO: 315; VH comprises the amino acid sequence of SEQ ID NO: 198, and VL comprises the amino acid sequence of SEQ ID NO: 315; VH comprises the amino acid sequence of SEQ ID NO: 199, and VL comprises the amino acid sequence of SEQ ID NO: 315; VH comprises the amino acid sequence of SEQ ID NO: 200, and VL comprises the amino acid sequence of SEQ ID NO: 315; VH comprises the amino acid sequence of SEQ ID NO: 201, and VL comprises the amino acid sequence of SEQ ID NO: 315; VH comprises the amino acid sequence of SEQ ID NO: 197, and VL comprises the amino acid sequence of SEQ ID NO: 316; VH comprises the amino acid sequence of SEQ ID NO: 198, and VL comprises the amino acid sequence of SEQ ID NO: 316; VH comprises the amino acid sequence of SEQ ID NO: 199, and VL comprises the amino acid sequence of SEQ ID NO: 316; VH comprises the amino acid sequence of SEQ ID NO: 200, and VL comprises the amino acid sequence of SEQ ID NO: 316; VH comprises the amino acid sequence of SEQ ID NO: 201, and VL comprises the amino acid sequence of SEQ ID NO: 316; VH comprises the amino acid sequence of SEQ ID NO: 170, and VL comprises the amino acid sequence of SEQ ID NO: 268; VH comprises the amino acid sequence of SEQ ID NO: 202, and VL comprises the amino acid sequence of SEQ ID NO: 268; VH comprises the amino acid sequence of SEQ ID NO: 203, and VL comprises the amino acid sequence of SEQ ID NO: 268; VH comprises the amino acid sequence of SEQ ID NO: 204, and VL comprises the amino acid sequence of SEQ ID NO: 268; VH comprises the amino acid sequence of SEQ ID NO: 205, and VL comprises the amino acid sequence of SEQ ID NO: 268; VH comprises the amino acid sequence of SEQ ID NO: 206, and VL comprises the amino acid sequence of SEQ ID NO: 268; VH comprises the amino acid sequence of SEQ ID NO: 207, and VL comprises the amino acid sequence of SEQ ID NO: 268; VH comprises the amino acid sequence of SEQ ID NO: 208, and VL comprises the amino acid sequence of SEQ ID NO: 268; VH comprises the amino acid sequence of SEQ ID NO: 170, and VL comprises the amino acid sequence of SEQ ID NO: 315; VH comprises the amino acid sequence of SEQ ID NO: 202, and VL comprises the amino acid sequence of SEQ ID NO: 315; VH comprises the amino acid sequence of SEQ ID NO: 203, and VL comprises the amino acid sequence of SEQ ID NO: 315; VH comprises the amino acid sequence of SEQ ID NO: 204, and VL comprises the amino acid sequence of SEQ ID NO: 315; VH comprises the amino acid sequence of SEQ ID NO: 205, and VL comprises the amino acid sequence of SEQ ID NO: 315; VH comprises the amino acid sequence of SEQ ID NO: 206, and VL comprises the amino acid sequence of SEQ ID NO: 315; VH comprises the amino acid sequence of SEQ ID NO: 207, and VL comprises the amino acid sequence of SEQ ID NO: 315; VH comprises the amino acid sequence of SEQ ID NO: 208, and VL comprises the amino acid sequence of SEQ ID NO: 315; VH comprises the amino acid sequence of SEQ ID NO: 170, and VL comprises the amino acid sequence of SEQ ID NO: 316; VH comprises the amino acid sequence of SEQ ID NO: 202, and VL comprises the amino acid sequence of SEQ ID NO: 316; VH comprises the amino acid sequence of SEQ ID NO: 203, and VL comprises the amino acid sequence of SEQ ID NO: 316; VH comprises the amino acid sequence of SEQ ID NO: 204, and VL comprises the amino acid sequence of SEQ ID NO: 316; VH comprises the amino acid sequence of SEQ ID NO: 205, and VL comprises the amino acid sequence of SEQ ID NO: 316; VH comprises the amino acid sequence of SEQ ID NO: 206, and VL comprises the amino acid sequence of SEQ ID NO: 316; VH comprises the amino acid sequence of SEQ ID NO: 207, and VL comprises the amino acid sequence of SEQ ID NO: 316; VH comprises the amino acid sequence of SEQ ID NO: 208, and VL comprises the amino acid sequence of SEQ ID NO: 316; VH comprises the amino acid sequence of SEQ ID NO: 188, and VL comprises the amino acid sequence of SEQ ID NO: 277; VH comprises the amino acid sequence of SEQ ID NO: 189, and VL comprises the amino acid sequence of SEQ ID NO: 277; VH comprises the amino acid sequence of SEQ ID NO: 190, and VL comprises the amino acid sequence of SEQ ID NO: 277; VH comprises the amino acid sequence of SEQ ID NO: 191, and VL comprises the amino acid sequence of SEQ ID NO: 277; VH comprises the amino acid sequence of SEQ ID NO: 192, and VL comprises the amino acid sequence of SEQ ID NO: 277; VH comprises the amino acid sequence of SEQ ID NO: 193, and VL comprises the amino acid sequence of SEQ ID NO: 277; VH comprises the amino acid sequence of SEQ ID NO: 194, and VL comprises the amino acid sequence of SEQ ID NO: 277; VH comprises the amino acid sequence of SEQ ID NO: 195, and VL comprises the amino acid sequence of SEQ ID NO: 277; VH comprises the amino acid sequence of SEQ ID NO: 196, and VL comprises the amino acid sequence of SEQ ID NO: 277; VH comprises the amino acid sequence of SEQ ID NO: 165, and VL comprises the amino acid sequence of SEQ ID NO: 317; VH comprises the amino acid sequence of SEQ ID NO: 188, and VL comprises the amino acid sequence of SEQ ID NO: 317; VH comprises the amino acid sequence of SEQ ID NO: 189, and VL comprises the amino acid sequence of SEQ ID NO: 317; VH comprises the amino acid sequence of SEQ ID NO: 190, and VL comprises the amino acid sequence of SEQ ID NO: 317; VH comprises the amino acid sequence of SEQ ID NO: 191, and VL comprises the amino acid sequence of SEQ ID NO: 317; VH comprises the amino acid sequence of SEQ ID NO: 192, and VL comprises the amino acid sequence of SEQ ID NO: 317; VH comprises the amino acid sequence of SEQ ID NO: 193, and VL comprises the amino acid sequence of SEQ ID NO: 317; VH comprises the amino acid sequence of SEQ ID NO: 194, and VL comprises the amino acid sequence of SEQ ID NO: 317; VH comprises the amino acid sequence of SEQ ID NO: 195, and VL comprises the amino acid sequence of SEQ ID NO: 317; VH comprises the amino acid sequence of SEQ ID NO: 196, and VL comprises the amino acid sequence of SEQ ID NO: 317; VH comprises the amino acid sequence of SEQ ID NO: 197, and VL comprises the amino acid sequence of SEQ ID NO: 277; VH comprises the amino acid sequence of SEQ ID NO: 198, and VL comprises the amino acid sequence of SEQ ID NO: 277; VH comprises the amino acid sequence of SEQ ID NO: 199, and VL comprises the amino acid sequence of SEQ ID NO: 277; VH comprises the amino acid sequence of SEQ ID NO: 200, and VL comprises the amino acid sequence of SEQ ID NO: 277; VH comprises the amino acid sequence of SEQ ID NO: 201, and VL comprises the amino acid sequence of SEQ ID NO: 277; VH comprises the amino acid sequence of SEQ ID NO: 197, and VL comprises the amino acid sequence of SEQ ID NO: 317; VH comprises the amino acid sequence of SEQ ID NO: 198, and VL comprises the amino acid sequence of SEQ ID NO: 317; VH comprises the amino acid sequence of SEQ ID NO: 199, and VL comprises the amino acid sequence of SEQ ID NO: 317; VH comprises the amino acid sequence of SEQ ID NO: 200, and VL comprises the amino acid sequence of SEQ ID NO: 317; VH comprises the amino acid sequence of SEQ ID NO: 201, and VL comprises the amino acid sequence of SEQ ID NO: 317; VH comprises the amino acid sequence of SEQ ID NO: 170, and VL comprises the amino acid sequence of SEQ ID NO: 277; VH comprises the amino acid sequence of SEQ ID NO: 202, and VL comprises the amino acid sequence of SEQ ID NO: 277; VH comprises the amino acid sequence of SEQ ID NO: 203, and VL comprises the amino acid sequence of SEQ ID NO: 277; VH comprises the amino acid sequence of SEQ ID NO: 204, and VL comprises the amino acid sequence of SEQ ID NO: 277; VH comprises the amino acid sequence of SEQ ID NO: 205, and VL comprises the amino acid sequence of SEQ ID NO: 277; VH comprises the amino acid sequence of SEQ ID NO: 206, and VL comprises the amino acid sequence of SEQ ID NO: 277; VH comprises the amino acid sequence of SEQ ID NO: 207, and VL comprises the amino acid sequence of SEQ ID NO: 277; VH comprises the amino acid sequence of SEQ ID NO: 208, and VL comprises the amino acid sequence of SEQ ID NO: 277; VH comprises the amino acid sequence of SEQ ID NO: 170, and VL comprises the amino acid sequence of SEQ ID NO: 317; VH comprises the amino acid sequence of SEQ ID NO: 202, and VL comprises the amino acid sequence of SEQ ID NO: 317; VH comprises the amino acid sequence of SEQ ID NO: 203, and VL comprises the amino acid sequence of SEQ ID NO: 317; VH comprises the amino acid sequence of SEQ ID NO: 204, and VL comprises the amino acid sequence of SEQ ID NO: 317; VH comprises the amino acid sequence of SEQ ID NO: 205, and VL comprises the amino acid sequence of SEQ ID NO: 317; VH comprises the amino acid sequence of SEQ ID NO: 206, and VL comprises the amino acid sequence of SEQ ID NO: 317; VH comprises the amino acid sequence of SEQ ID NO: 207, and VL comprises the amino acid sequence of SEQ ID NO: 317; VH comprises the amino acid sequence of SEQ ID NO: 208, and VL comprises the amino acid sequence of SEQ ID NO: 317; VH comprises the amino acid sequence of SEQ ID NO: 188, and VL comprises the amino acid sequence of SEQ ID NO: 279; VH comprises the amino acid sequence of SEQ ID NO: 189, and VL comprises the amino acid sequence of SEQ ID NO: 279; VH comprises the amino acid sequence of SEQ ID NO: 190, and VL comprises the amino acid sequence of SEQ ID NO: 279; VH comprises the amino acid sequence of SEQ ID NO: 191, and VL comprises the amino acid sequence of SEQ ID NO: 279; VH comprises the amino acid sequence of SEQ ID NO: 192, and VL comprises the amino acid sequence of SEQ ID NO: 279; VH comprises the amino acid sequence of SEQ ID NO: 193, and VL comprises the amino acid sequence of SEQ ID NO: 279; VH comprises the amino acid sequence of SEQ ID NO: 194, and VL comprises the amino acid sequence of SEQ ID NO: 279; VH comprises the amino acid sequence of SEQ ID NO: 195, and VL comprises the amino acid sequence of SEQ ID NO: 279; VH comprises the amino acid sequence of SEQ ID NO: 196, and VL comprises the amino acid sequence of SEQ ID NO: 279; VH comprises the amino acid sequence of SEQ ID NO: 165, and VL comprises the amino acid sequence of SEQ ID NO: 318; VH comprises the amino acid sequence of SEQ ID NO: 188, and VL comprises the amino acid sequence of SEQ ID NO: 318; VH comprises the amino acid sequence of SEQ ID NO: 189, and VL comprises the amino acid sequence of SEQ ID NO: 318; VH comprises the amino acid sequence of SEQ ID NO: 190, and VL comprises the amino acid sequence of SEQ ID NO: 318; VH comprises the amino acid sequence of SEQ ID NO: 191, and VL comprises the amino acid sequence of SEQ ID NO: 318; VH comprises the amino acid sequence of SEQ ID NO: 192, and VL comprises the amino acid sequence of SEQ ID NO: 318; VH comprises the amino acid sequence of SEQ ID NO: 193, and VL comprises the amino acid sequence of SEQ ID NO: 318; VH comprises the amino acid sequence of SEQ ID NO: 194, and VL comprises the amino acid sequence of SEQ ID NO: 318; VH comprises the amino acid sequence of SEQ ID NO: 195, and VL comprises the amino acid sequence of SEQ ID NO: 318; VH comprises the amino acid sequence of SEQ ID NO: 196, and VL comprises the amino acid sequence of SEQ ID NO: 318; VH comprises the amino acid sequence of SEQ ID NO: 197, and VL comprises the amino acid sequence of SEQ ID NO: 279; VH comprises the amino acid sequence of SEQ ID NO: 198, and VL comprises the amino acid sequence of SEQ ID NO: 279; VH comprises the amino acid sequence of SEQ ID NO: 199, and VL comprises the amino acid sequence of SEQ ID NO: 279; VH comprises the amino acid sequence of SEQ ID NO: 200, and VL comprises the amino acid sequence of SEQ ID NO: 279; VH comprises the amino acid sequence of SEQ ID NO: 201, and VL comprises the amino acid sequence of SEQ ID NO: 279; VH comprises the amino acid sequence of SEQ ID NO: 197, and VL comprises the amino acid sequence of SEQ ID NO: 318; VH comprises the amino acid sequence of SEQ ID NO: 198, and VL comprises the amino acid sequence of SEQ ID NO: 318; VH comprises the amino acid sequence of SEQ ID NO: 199, and VL comprises the amino acid sequence of SEQ ID NO: 318; VH comprises the amino acid sequence of SEQ ID NO: 200, and VL comprises the amino acid sequence of SEQ ID NO: 318; VH comprises the amino acid sequence of SEQ ID NO: 201, and VL comprises the amino acid sequence of SEQ ID NO: 318; VH comprises the amino acid sequence of SEQ ID NO: 170, and VL comprises the amino acid sequence of SEQ ID NO: 279; VH comprises the amino acid sequence of SEQ ID NO: 202, and VL comprises the amino acid sequence of SEQ ID NO: 279; VH comprises the amino acid sequence of SEQ ID NO: 203, and VL comprises the amino acid sequence of SEQ ID NO: 279; VH comprises the amino acid sequence of SEQ ID NO: 204, and VL comprises the amino acid sequence of SEQ ID NO: 279; VH comprises the amino acid sequence of SEQ ID NO: 205, and VL comprises the amino acid sequence of SEQ ID NO: 279; VH comprises the amino acid sequence of SEQ ID NO: 206, and VL comprises the amino acid sequence of SEQ ID NO: 279; VH comprises the amino acid sequence of SEQ ID NO: 207, and VL comprises the amino acid sequence of SEQ ID NO: 279; VH comprises the amino acid sequence of SEQ ID NO: 208, and VL comprises the amino acid sequence of SEQ ID NO: 279; VH comprises the amino acid sequence of SEQ ID NO: 170, and VL comprises the amino acid sequence of SEQ ID NO: 318; VH comprises the amino acid sequence of SEQ ID NO: 202, and VL comprises the amino acid sequence of SEQ ID NO: 318; VH comprises the amino acid sequence of SEQ ID NO: 203, and VL comprises the amino acid sequence of SEQ ID NO: 318; VH comprises the amino acid sequence of SEQ ID NO: 204, and VL comprises the amino acid sequence of SEQ ID NO: 318; VH comprises the amino acid sequence of SEQ ID NO: 205, and VL comprises the amino acid sequence of SEQ ID NO: 318; VH comprises the amino acid sequence of SEQ ID NO: 206, and VL comprises the amino acid sequence of SEQ ID NO: 318; VH comprises the amino acid sequence of SEQ ID NO: 207 and VL comprises the amino acid sequence of SEQ ID NO: 318; or The VH comprises the amino acid sequence of SEQ ID NO:208, and the VL comprises the amino acid sequence of SEQ ID NO:318.

[0012] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 165 and the VL comprises the amino acid sequence of SEQ ID NO:268.

[0013] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 165 and the VL comprises the amino acid sequence of SEQ ID NO:277.

[0014] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 165 and the VL comprises the amino acid sequence of SEQ ID NO:279.

[0015] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 165 and the VL comprises the amino acid sequence of SEQ ID NO:268.

[0016] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 165 and the VL comprises the amino acid sequence of SEQ ID NO:315.

[0017] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:190 and the VL comprises the amino acid sequence of SEQ ID NO:315.

[0018] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:196 and the VL comprises the amino acid sequence of SEQ ID NO:315.

[0019] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 191 and the VL comprises the amino acid sequence of SEQ ID NO:277.

[0020] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 165 and the VL comprises the amino acid sequence of SEQ ID NO:317.

[0021] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:191 and the VL comprises the amino acid sequence of SEQ ID NO:318.

[0022] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:195 and the VL comprises the amino acid sequence of SEQ ID NO:318.

[0023] In some embodiments, the antibody comprises: A heavy chain comprising any one of the amino acid sequences of SEQ ID NOs: 119 to 135 and 137 to 164; and a light chain comprising any one of the amino acid sequences of SEQ ID NOs: 209 to 249 and 251 to 263.

[0024] In some embodiments, the antibody comprises: A single clone selected from the group consisting of clones 1 to 53 and 55 to 215 contains a heavy chain and a light chain corresponding to the heavy chain and the light chain shown in Tables 4 to 5, respectively.

[0025] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:119 and the light chain comprises the amino acid sequence of SEQ ID NO:213.

[0026] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:119 and the light chain comprises the amino acid sequence of SEQ ID NO:222.

[0027] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:119 and the light chain comprises the amino acid sequence of SEQ ID NO:224.

[0028] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 142 and the light chain comprises the amino acid sequence of SEQ ID NO:213.

[0029] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:142 and the light chain comprises the amino acid sequence of SEQ ID NO:260.

[0030] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:145 and the light chain comprises the amino acid sequence of SEQ ID NO:260.

[0031] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 151 and the light chain comprises the amino acid sequence of SEQ ID NO:260.

[0032] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 146 and the light chain comprises the amino acid sequence of SEQ ID NO:222.

[0033] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:142 and the light chain comprises the amino acid sequence of SEQ ID NO:262.

[0034] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 146 and the light chain comprises the amino acid sequence of SEQ ID NO:263.

[0035] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 150 and the light chain comprises the amino acid sequence of SEQ ID NO:263.

[0036] In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein VH CDR1 comprises the amino acid sequence ELSMQ (SEQ ID NO: 1) and VH CDR2 comprises the amino acid sequence GFDPDDX 101 ETMYAEX 102 X 103 QG (SEQ ID NO: 102; Group 1 clone), 101 is D or G, and X 102 is K or R, and X 103 is F or L, the VH CDR3 is SPGYDFFDY (SEQ ID NO: 18), and the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, and the VL CDR1 has the amino acid sequence GGX 104 X 105 X 106 GX 107 X 108 X 109VX 110 (SEQ ID NO: 103; Group 1 clone), 104 is N, D, or S, and X 105 is Y, N, or D, and X 106 is I or T, and X 107 is S, D, I, R or T, and X 108 is K, E, or I, and X 109 is S, I, R, G, N, or A, and X 110 is H, F, or N, and VL CDR2 is the amino acid sequence DDX 111 DRPX 112 (SEQ ID NO: 104; Group 1 clone), 111 is G, S, or R, and X 112 is S or L, and the VL CDR3 has the amino acid sequence QVWDX 113 X 114 X 115 DX 116 X 117 X 118 (SEQ ID NO: 105; Group 1 clone), 113 is S or A, and X 114 is I or S, and X 115 is S, I, or N, and X 116 is H, L, or Q, and X 117 is V or L, and X 118 is V or I.

[0037] In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein VH CDR1 comprises the amino acid sequence GYTLTELSMQ (SEQ ID NO: 329) and VH CDR2 comprises the amino acid sequence GFDPDDX 101 ETMYAEX 102 X 103 QG (SEQ ID NO: 102; Group 1 clone), 101 is D or G, and X 102 is K or R, and X 103is F or L, the VH CDR3 is SPGYDFFDY (SEQ ID NO: 18), and the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, and the VL CDR1 has the amino acid sequence GGX 104 X 105 X 106 GX 107 X 108 X 109 VX 110 (SEQ ID NO: 103; Group 1 clone), 104 is N, D, or S, and X 105 is Y, N, or D, and X 106 is I or T, and X 107 is S, D, I, R or T, and X 108 is K, E, or I, and X 109 is S, I, R, G, N, or A, and X 110 is H, F, or N, and VL CDR2 is the amino acid sequence DDX 111 DRPX 112 (SEQ ID NO: 104; Group 1 clone), 111 is G, S, or R, and X 112 is S or L, and the VL CDR3 has the amino acid sequence QVWDX 113 X 114 X 115 DX 116 X 117 X 118 (SEQ ID NO: 105; Group 1 clone), 113 is S or A, and X 114 is I or S, and X 115 is S, I, or N, and X 116 is H, L, or Q, and X 117 is V or L, and X 118 is V or I.

[0038] In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3, where VH CDR1 comprises the amino acid sequence ELSMQ (SEQ ID NO: 1) and VH CDR2 comprises the amino acid sequence GFDPDDX 101 ETMYAEX102 X 103 QG (SEQ ID NO: 102; Group 1 clone), 101 is D or G, and X 102 is K or R, and X 103 is F or L, VH CDR3 is SPGYDFFDY (SEQ ID NO: 18), and the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein VL CDR1 comprises the amino acid sequence TGTSSDVGGYNYVS (SEQ ID NO: 30) and VL CDR2 is the amino acid sequence X 119 VSX 120 RPS (SEQ ID NO: 106; Group 1 clone), 119 is E or D, and X 120 is N or K, and the VL CDR3 comprises the amino acid sequence QVWDSSNDLLI (SEQ ID NO:71).

[0039] In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3, where VH CDR1 comprises the amino acid sequence GYTLTELSMQ (SEQ ID NO: 329) and VH CDR2 comprises the amino acid sequence GFDPDDX 101 ETMYAEX 102 X 103 QG (SEQ ID NO: 102; Group 1 clone), 101 is D or G, and X 102 is K or R, and X 103 is F or L, VH CDR3 is SPGYDFFDY (SEQ ID NO: 18), and the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein VL CDR1 comprises the amino acid sequence TGTSSDVGGYNYVS (SEQ ID NO: 30) and VL CDR2 is the amino acid sequence X 119 VSX 120 RPS (SEQ ID NO: 106; Group 1 clone), 119 is E or D, and X 120 is N or K, and the VL CDR3 comprises the amino acid sequence QVWDSSNDLLI (SEQ ID NO:71).

[0040] In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein VH CDR1 comprises the amino acid sequence DYFIH (SEQ ID NO:2) and VH CDR2 comprises the amino acid sequence LVDPEDGETIYAEX 121 FQG (SEQ ID NO: 107; group 2 clone), where X 121 is K or R, the VH CDR3 comprises the amino acid sequence PGGILTDPDAFDI (SEQ ID NO: 19), and the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, and the VL CDR1 is 122 GTX 123 SDVGGYNX 124 VS (SEQ ID NO: 108; group 2 clone), where X 122 is T or A, and X 123 is S or G, and X 124 is Y or H, and VL CDR2 is the amino acid sequence X 125 VX 126 X 127 RPS (SEQ ID NO: 109; Group 2 clone), wherein X 125 is D or E, and X 126 is N or S, and X 127 is K or N, and the VL CDR3 has the amino acid sequence SSYX 128 X 129 SSTX 130 X 131 V (SEQ ID NO: 110; Group 2 clone), where X 128 is I or T, and X 129 is P or S, and X 130 is R, P, F, or absent, and X 131 is W or Y.

[0041] In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3, where VH CDR1 comprises the amino acid sequence SYAIS (SEQ ID NO:3) and VH CDR2 comprises the amino acid sequence LVDPEDGETIYAEKFX.132 G (SEQ ID NO: 111; group 3 clone), 132 is R or Q, the VH CDR3 comprises the amino acid sequence EESYGP (SEQ ID NO: 20), and the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, where the VL CDR1 is the amino acid sequence QASQDISNYLX 133 (SEQ ID NO: 112; group 3 clone), 133 is N or D, the VL CDR2 comprises the amino acid sequence DASNLET (SEQ ID NO:61), and the VL CDR3 comprises the amino acid sequence QQLNSYPLT (SEQ ID NO:80).

[0042] In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein VH CDR1 has the amino acid sequence EX 134 SMH (SEQ ID NO: 113, group 4 clone), 134 is S or L, VH CDR2 comprises the amino acid sequence LVDPEDGETIYAQKFQG (SEQ ID NO: 14) and VH CDR3 comprises the amino acid sequence EEWSGDGDDAFDI (SEQ ID NO: 21), and the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, where VL CDR1 comprises the amino acid sequence SGSSSNIGSYSVS (SEQ ID NO: 46) and VL CDR2 comprises the amino acid sequence DX 135 NKRPS (SEQ ID NO: 114, group 4 clone), 135 is N or D, and the VL CDR3 comprises the amino acid sequence GTWDSSLSAWV (SEQ ID NO:81).

[0043] In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein VH CDR1 comprises the amino acid sequence SYAIS (SEQ ID NO:3), VH CDR2 comprises the amino acid sequence GIIPIFGTANYAQKFQG (SEQ ID NO:15), and VH CDR3 comprises the amino acid sequence SPLRGSGWYWHYYYGMDV (SEQ ID NO:22), and the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein VL CDR1 is the amino acid sequence GGNX 136 IX 137 X 138 KX 139 VH (SEQ ID NO: 115; group 6 clone), 136 is N or K, and X 137 is R or G, and X 138 is A, S, R, or T, and X 139 is H or S, and VL CDR2 is the amino acid sequence X 140 DX 141 X 142 RPS (SEQ ID NO: 116; group 6 clone), 140 is Q or R, and X 141 is S or R, and X 142 is N or K, and the VL CDR3 is the amino acid sequence QX 143 WX 144 SX 145 TX 146 V (SEQ ID NO: 117, group 6 clone), 143 is A or V, and X 144 is D or G, and X 145 is S or N, and X 146 is V, A, or E.

[0044] In some embodiments, the human mutant CALR is human type 1 mutant CALR comprising the amino acid sequence of SEQ ID NO: 320. In some embodiments, the human mutant CALR is human type 2 mutant CALR comprising the amino acid sequence of SEQ ID NO: 321.

[0045] In some embodiments, the anti-mutCALR antibodies inhibit one or more signaling pathways downstream of the thrombopoietin receptor (MPL) in cells expressing human mutant CALR, inhibit oncogenic cell proliferation in cells expressing human mutant CALR; and / or inhibit dimerization of MPL in cells expressing human mutant CALR.

[0046] In some embodiments, the anti-mutCALR antibody inhibits one or more signaling pathways downstream of MPL in both a first cell expressing human type 1 mutant CALR and a second cell expressing human type 2 mutant CALR; inhibits oncogenic cell proliferation in both a first cell expressing human type 1 mutant CALR and a second cell expressing human type 2 mutant CALR; and / or inhibits dimerization of MPL in both a first cell expressing human type 1 mutant CALR and a second cell expressing human type 2 mutant CALR.

[0047] In some embodiments, the one or more signaling pathways downstream of MPL are selected from the group consisting of Janus tyrosine kinase (JAK) and signal transducer and activator of transcription (STAT) signaling, mitogen-activated protein kinase (MEK) and extracellular signal-regulated kinase (ERK) signaling, serine / threonine kinase (AKT) signaling, and mammalian target of rapamycin (mTOR) signaling.

[0048] In some embodiments, the anti-mutCALR antibody has a modulated Fc effector function. In some embodiments, the modulated Fc effector function is an increased Fc effector function or a reduced Fc effector function. In some embodiments, the anti-mutCALR antibody has a reduced Fc effector function. In some embodiments, the Fc effector function is antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), or antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the anti-mutCALR antibody with reduced Fc effector function has an increased binding affinity to human mutant CALR compared to an antibody without reduced Fc effector function. In some embodiments, the Fc effector function is ADCC.

[0049] In some embodiments, the anti-mutCALR antibody is a human or humanized antibody. In some embodiments, the anti-mutCALR antibody is a full-length antibody. In some embodiments, the anti-mutCALR antibody is an IgG1, IgG2, IgG3 or IgG4 antibody. In some embodiments, the anti-mutCALR antibody is an IgG1 antibody. In some embodiments, the anti-mutCALR antibody is a bispecific antibody, a biparatopic antibody, a single chain antibody (scFv), a Fab fragment, a F(ab')2 fragment, a Fab' fragment, an Fsc fragment, an Fv fragment, a scFv, a sc(Fv)2, or a diabody.

[0050] In some embodiments, the anti-mutCALR antibody is a biparatopic antibody comprising two heavy-light chain pairs or one heavy-light chain pair. In some embodiments, the biparatopic antibody is a full-length antibody. In some embodiments, the biparatopic antibody comprises one heavy-light chain pair.

[0051] In some embodiments, the anti-mutCALR antibody is conjugated to a toxic agent. In some embodiments, the toxic agent is a radioisotope or a cytotoxic drug.

[0052] An embodiment of the disclosure provides a nucleic acid or set of nucleic acids that collectively encode any one of the anti-mutCALR antibodies described herein.

[0053] An embodiment of the present disclosure provides an expression vector or a set of expression vectors comprising a nucleic acid or a set of nucleic acids encoding any one of the anti-mutCALR antibodies described herein, operably linked to a promoter.

[0054] An embodiment of the present disclosure provides an isolated cell comprising a nucleic acid or set of nucleic acids encoding any one of the anti-mutCALR antibodies, or an expression vector or set of expression vectors comprising a nucleic acid or set of nucleic acids encoding any one of the anti-mutCALR antibodies described herein operably linked to a promoter.

[0055] Aspects of the present disclosure provide a method of producing an anti-mutCALR antibody, comprising culturing a cell described herein and isolating the antibody.

[0056] An embodiment of the present disclosure provides a pharmaceutical composition comprising an anti-mutCALR antibody, a nucleic acid or set of nucleic acids, an expression vector or set of expression vectors, or an isolated cell, and a pharma- ceutically acceptable carrier.

[0057] An embodiment of the present disclosure provides a method of treating a myeloproliferative neoplasm in a human subject in need thereof, the method comprising administering to the human subject an effective amount of an anti-mutCALR antibody described herein or a pharmaceutical composition thereof. Another embodiment of the present disclosure provides an anti-mutCALR antibody described herein for use in treating a myeloproliferative neoplasm, or a use of an anti-mutCALR antibody described herein for the manufacture of a medicament for treating a myeloproliferative neoplasm.

[0058] An embodiment of the present disclosure provides a method for detecting a CALR exon 9 mutation in a biological sample, the method comprising obtaining a biological sample from a human subject having or suspected of having a myeloproliferative neoplasm, and contacting the sample with an anti-mutCALR antibody described herein, such that the anti-mutCALR antibody binds to the mutCALR protein, if present in the biological sample.

[0059] Another aspect of the present disclosure provides a method of diagnosing a human subject having a myeloproliferative neoplasm, the method comprising obtaining a biological sample from a human subject having or suspected of having a myeloproliferative neoplasm, and contacting the sample with an anti-mutCALR antibody described herein, such that if mutCALR protein is present in the biological sample, the anti-mutCALR antibody binds to the mutCALR protein.

[0060] In some embodiments, the myeloproliferative neoplasm is selected from the group consisting of chronic myelogenous leukemia, polycythemia vera, primary myelofibrosis, essential thrombocythemia, chronic neutrophilic leukemia, acute myelogenous leukemia, and chronic eosinophilic leukemia, chronic myelomonocytic leukemia, myeloproliferative neoplasms, and myelodysplastic syndromes (including myelodysplastic syndromes with refractory anemia with ringed sideroblasts, myelodysplastic syndromes with refractory anemia, and myelodysplastic syndromes with refractory anemia with excess blasts).

[0061] In some embodiments, the methods described herein further comprise administering to the human subject an additional therapy selected from the group consisting of a Janus tyrosine kinase (JAK) inhibitor, a phosphoinositide 3-kinase (PI3K) inhibitor, a standard of care, or a combination thereof. In some embodiments, the JAK inhibitor is ruxolitinib and itaticinib. In some embodiments, the PI3K inhibitor is palsaclisib. In some embodiments, the standard of care is selected from the group consisting of IFN-α, hydroxyurea, thalidomide, lenalidomide, androgens, erythropoietin stimulating agents, chemotherapeutic agents, or a combination thereof.

[0062] In some embodiments, administering the antibody or pharmaceutical composition thereof in combination with a JAK inhibitor produces a synergistic effect. In some embodiments, the JAK inhibitor is ruxolitinib.

[0063] An embodiment of the present disclosure provides a kit comprising an anti-mutCALR antibody, a nucleic acid or set of nucleic acids, an expression vector or set of expression vectors, or an isolated cell, and instructions for use in treating a myeloproliferative neoplasm in a human subject in need thereof, optionally together with instructions for use in combination with an additional therapy. [Brief description of the drawings]

[0064] [Figure 1] 1 includes graphs showing that anti-mutCALR antibodies inhibit STAT5 activation in Ba / F3 cells expressing MPL / mutCALR. [Diagram 2] 1 includes graphs showing that anti-mutCALR antibodies inhibit mutCALR-induced cell proliferation in Ba / F3 cells stably transfected with MPL / mutCALR. [Diagram 3] 1 includes graphs showing that anti-mutCALR antibodies are effective in inhibiting oncogenic cell proliferation in Ba / F3 cells stably transfected with either MPL / mutCALR type 1 (top) or MPL / mutCALR type 2 (bottom). [Figure 4A] 1 includes graphs showing that anti-mutCALR antibodies inhibit dimerization of the MPL protein. [Figure 4B] 1 includes graphs showing that anti-mutCALR antibodies inhibit dimerization of the MPL protein. [Figure 4C] 1 includes graphs showing that anti-mutCALR antibodies inhibit dimerization of the MPL protein. [Figure 4D] 1 includes graphs showing that anti-mutCALR antibodies inhibit dimerization of the MPL protein. [Figure 4E]1 includes graphs showing that anti-mutCALR antibodies inhibit dimerization of the MPL protein. [Figure 4F] 1 includes graphs showing that anti-mutCALR antibodies inhibit dimerization of the MPL protein. [Figure 4G] 1 includes graphs showing that anti-mutCALR antibodies inhibit dimerization of the MPL protein. [Diagram 5] 1 includes a schematic diagram of the dosing schedule of anti-mutCALR antibodies in mice injected with tumor cells expressing MPL / mutCALR. [Figure 6] Included is a graph of mouse survival following treatment with anti-mutCALR antibodies. [Figure 7] 2 includes a graph of spleen weight in mice following treatment with anti-mutCALR antibody. [Figure 8] 1 includes a graph of the number of platelets in the blood following treatment of mice with anti-mutCALR antibodies. [Figure 9] 1 includes a graph of the number of tumor cells in the blood following treatment of mice with anti-mutCALR antibodies. [Figure 10] Graphs showing the ability of anti-mutCALR antibodies to enhance the therapeutic response of ruxolitinib in inhibiting oncogenic cell proliferation caused by mutCALR type 1 (top) or type 2 (bottom). [Figure 11] 1 includes graphs showing that anti-mutCALR antibodies compete with MPL for binding to mutCALR. [Figure 12A] Contains structural data of the Fab-mutCALR-peptide complex. [Figure 12B] Contains structural data of the Fab-mutCALR-peptide complex. [Figure 12C] Contains structural data of the Fab-mutCALR-peptide complex. [Figure 13A] 1 includes structural data of the Fab fragment binding to the first mutCALR peptide in the asymmetric unit of the crystal structure. [Figure 13B]1 includes structural data of the Fab fragment binding to the first mutCALR peptide in the asymmetric unit of the crystal structure. [Figure 13C] 1 includes structural data of the Fab fragment binding to the first mutCALR peptide in the asymmetric unit of the crystal structure. [Figure 14A] Contains structural data of the Fab fragment binding to the second mutCALR peptide in the asymmetric unit of the crystal structure. [Figure 14B] Contains structural data of the Fab fragment binding to the second mutCALR peptide in the asymmetric unit of the crystal structure. [Figure 14C] Contains structural data of the Fab fragment binding to the second mutCALR peptide in the asymmetric unit of the crystal structure. [Figure 15] Included are images of the sequence of the mutant CALR peptide (SEQ ID NO: 328) showing CalR1 conformation-binding residues (top) and CalR2 conformation-binding residues (bottom) that are in close contact with Fab1 (<4.5 Å) and shaded in grey. Note that the CalR1 and CalR2 conformation-binding residues represent two opposing faces of the mutant CALR C-terminal helix, with more residues at the N-terminus covered in the CalR1-bound conformation than in the CalR2-bound conformation. Fab1 close contacts were assessed in MOE (Molecular Operating Environment). [Figure 16A] Contains structural data of an anti-mutCALR antibody Fab fragment bound to a 31-mer mutant CalR peptide. [Figure 16B] Contains structural data of an anti-mutCALR antibody Fab fragment bound to a 31-mer mutant CalR peptide. [Figure 16C] Contains structural data of an anti-mutCALR antibody Fab fragment bound to a 31-mer mutant CalR peptide. [Figure 17A] Contains structural data from antigen-antibody interaction analysis. [Figure 17B] Contains structural data from antigen-antibody interaction analysis. [Figure 17C] Contains structural data from antigen-antibody interaction analysis. [Figure 18A] Includes data showing that anti-mutCALR antibody treatment restored normal blood counts, spleen volume, and bone marrow environment in CALRDEL / DEL engineered mice. [Figure 18B] Includes data showing that anti-mutCALR antibody treatment restored normal blood counts, spleen volume, and bone marrow environment in CALRDEL / DEL engineered mice. [Figure 18C] Includes data showing that anti-mutCALR antibody treatment restored normal blood counts, spleen volume, and bone marrow environment in CALRDEL / DEL engineered mice. [Figure 19A] Includes data showing that anti-mutCALR antibodies inhibit mutCALR-derived oncogenic functions in CD34+ cells isolated from MPN patients harboring CALR mutations. [Figure 19B] Includes data showing that anti-mutCALR antibodies inhibit mutCALR-derived oncogenic functions in CD34+ cells isolated from MPN patients harboring CALR mutations. [Figure 19C] Includes data showing that anti-mutCALR antibodies inhibit mutCALR-derived oncogenic functions in CD34+ cells isolated from MPN patients harboring CALR mutations. [Figure 19D] Includes data showing that anti-mutCALR antibodies inhibit mutCALR-derived oncogenic functions in CD34+ cells isolated from MPN patients harboring CALR mutations. [Figure 20] 1 includes data showing cell cycle profiles of BaF3 cells with the indicated genotypes in the presence of different concentrations of anti-mutCALR antibodies. [Figure 21A] Graphs showing data on the binding of AB1-AB4 and clone 4 to mutCALR are included. [Figure 21B] 1 includes a graph showing data showing that clone 4 inhibits cell proliferation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] Anti-mutCALR antibodies and related nucleic acids, expression vectors, cells, kits, and pharmaceutical compositions are provided herein. The anti-mutCALR antibodies described herein are useful for treating, preventing, or diagnosing myeloproliferative neoplasms, such as chronic myeloid leukemia, polycythemia vera, primary myelofibrosis, essential thrombocythemia, chronic neutrophilic leukemia, acute myeloid leukemia, chronic eosinophilic leukemia, chronic myelomonocytic leukemia, myeloproliferative neoplasms, and myelodysplastic syndromes, including myelodysplastic syndrome with refractory anemia with ringed sideroblasts, myelodysplastic syndrome with refractory anemia, and myelodysplastic syndrome with refractory anemia with excess blasts.

[0066] Mutant CALR and anti-mutCALR antibodies CALR is a highly conserved chaperone protein that resides primarily in the endoplasmic reticulum and is involved in a variety of cellular processes, including protein folding, calcium homeostasis, cell adhesion, and integrin signaling. Mutations in the CALR gene have been identified in patients with myeloproliferative neoplasms. The two most frequent CALR mutations are a 52 base pair (bp) deletion and a 5 bp insertion, which are referred to as type 1 and type 2 mutations, respectively. Type 1 and type 2 mutations cause a +1 frameshift in exon 9 that generates a novel positively charged C-terminal amino acid sequence that lacks the KDEL domain (SEQ ID NO: 347) of the WT protein, thereby allowing mutCALR to escape the ER, activate the thrombopoietin receptor (MPL), and induce constitutive activation of Janus kinase 2 (JAK2) signaling. The amino acid sequences of human WT CALR, type 1 and type 2 mutCALR, as well as the novel C-terminal sequence, are shown below. The frameshifted amino acid residues in type 1 and type 2 mutCALR are shown in bold and the novel C-terminal sequences are underlined. Human WT CALR (GenBank accession number NP_004334.1) MLLSVPLLLGLLGLAVAEPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGLQTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCG GGYVKLFPNSLDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKDDEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDA SKPEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEPPVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGVLGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQDEEQRLKEEEEDKKRKEEEEAEDKEDDEDKDEDEEDEEDKEEDEEEDVPGQAKDEL (SEQ ID NO: 319) [ka] [ka] mutCALR C-terminal consensus mutant sequence RMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTEA (SEQ ID NO: 322)

[0067] The present disclosure provides anti-mutCALR antibodies that are useful in the treatment or diagnosis of myeloproliferative neoplasms. It should be noted that in this disclosure, unless otherwise stated, the amino acid positions assigned to the CDRs and frameworks in the variable regions of anti-mutCALR antibodies are designated by Kabat. See EA Kabat, Sequences of Proteins of Immunological Interest, USDept. of Health and Human Services, Public Health Service, National Institutes of Health, 1991, (OCoLC) 1138727707.

[0068] In some embodiments, the anti-mutCALR antibody is an anti-mutCALR antibody that comprises one, two, three, four, five, and / or six CDRs of any one of the antibodies described herein. In some embodiments, the anti-mutCALR antibody comprises (i) one, two, and / or three heavy chain CDRs of any one of the clones shown in Tables 1-2, and / or (ii) one, two, and / or three light chain CDRs from any one of the clones shown in Tables 1-2.

[0069] In some embodiments, the anti-mutCALR antibody comprises (i) three heavy chain CDRs derived from any one of the clones presented in Tables 4-5, and (ii) three light chain CDRs derived from any one of the clones presented in Tables 4-5.

[0070] In some embodiments, the anti-mutCALR antibody comprises heavy chain CDR1, CDR2, and CDR3, and / or light chain variable region CDR1, CDR2, and CDR3 from an antibody described herein. In some embodiments, the anti-mutCALR antibody comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 from an antibody described herein. In some embodiments, the anti-mutCALR antibody comprises a murine version, murine variant, human version, human variant, humanized version, humanized variant, or affinity matured variant of an antibody described herein.

[0071] In some embodiments, the anti-mutCALR antibody comprises heavy chain CDR1, CDR2, and CDR3, and / or light chain variable region CDR1, CDR2, and CDR3 from any clone disclosed herein, their humanized version, or variants thereof (including affinity matured variants). In some embodiments, the anti-mutCALR antibody comprises heavy chain CDR1, heavy chain variable region CDR2, and heavy chain variable region CDR3 from any clone disclosed herein. In other embodiments, the anti-mutCALR antibody comprises light chain variable region CDR1, light chain variable region CDR2, and light chain variable region CDR3 from any clone disclosed herein. In certain embodiments, the anti-mutCALR antibody comprises heavy chain CDR1, heavy chain variable region CDR2, heavy chain variable region CDR3, light chain variable region CDR1, light chain variable region CDR2, and light chain variable region CDR3 from any clone of the antibody disclosed herein. In some embodiments, the anti-mutCALR antibody is a murine version of any clone disclosed herein. In some embodiments, the anti-mutCALR antibody is a mouse variant of any of the clones disclosed herein. In some embodiments, the anti-mutCALR antibody is a human version of any of the clones disclosed herein. In some embodiments, the anti-mutCALR antibody is a human variant of any of the clones disclosed herein. In some embodiments, the anti-mutCALR antibody is a humanized version of any of the clones disclosed herein. In some embodiments, the anti-mutCALR antibody is a variant of any of the clones disclosed herein. In some embodiments, the anti-mutCALR antibody is an affinity matured variant of any of the clones disclosed herein.

[0072] In some embodiments, the anti-mutCALR antibody is a variant of an anti-mutCALR antibody described herein that comprises 1-30 conservative amino acid substitution(s), e.g., 1-25, 1-20, 1-15, 1-10, 1-5, or 1-3 conservative amino acid substitution(s). In some embodiments, the conservative amino acid substitution(s) are in the CDRs of the antibody. In some embodiments, the conservative amino acid substitution(s) are not in the CDRs of the antibody. In some embodiments, the conservative amino acid substitution(s) are in the framework regions of the antibody.

[0073] In some embodiments, the CDR comprises one amino acid substitution. In some embodiments, the CDR comprises two amino acid substitutions. In some embodiments, the CDR comprises three amino acid substitutions. In some embodiments, the CDR comprises four amino acid substitutions. In some embodiments, the one or more amino acid substitutions are conservative substitutions. In some embodiments, the CDR is a heavy chain CDR1. In some embodiments, the CDR is a heavy chain variable region CDR2. In some embodiments, the CDR is a heavy chain variable region CDR3. In some embodiments, the CDR is a light chain variable region CDR1. In some embodiments, the CDR is a light chain variable region CDR2. In some embodiments, the CDR is a light chain variable region CDR3. In some embodiments, the one or more substitutions are made as part of a humanization process. In some embodiments, the one or more substitutions are made as part of a germline humanization process. In some embodiments, the one or more substitutions are made as part of an affinity maturation process. In some embodiments, the one or more substitutions are made as part of an optimization process.

[0074] In some embodiments, the anti-mutCALR antibody comprises a heavy chain variable region CDR1, a heavy chain variable region CDR2, and a heavy chain variable region CDR3, each of which corresponds to a heavy chain variable region CDR shown in Tables 1-2 for a single clone, and a light chain variable region CDR1, a light chain variable region VL CDR2, and a light chain variable region VL CDR3, each of which corresponds to a VL CDR shown in Tables 1-2 for a single clone.

[0075] In some embodiments, the anti-mutCALR antibody comprises a heavy chain variable region CDR1, a heavy chain variable region CDR2, a heavy chain variable region CDR3, a light chain variable region CDR1, a light chain variable region CDR2, and a light chain variable region CDR3, each of which corresponds to the VH and VL CDRs shown in Tables 1-2 for a single clone.

[0076] In some embodiments, the anti-mutCALR antibody comprises a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprises the amino acid sequence X1X2X3X4X5, where X1 is S, E, or D, X2 is Y, L, or S, X3 is A, S, or F, X4 is I or M, and X5 is S, Q, or H, and VH CDR2 is X6X7X8PX9X 10 X 11 X 12 X 13 X 14 YAX 15 X 16 X 17 X 18 X6 is L or G, X7 is V, F, or I, X8 is D or I, X9 is E, D, or I, and X 10 is D, G, F, A, S, or E, and X 11 is G or A, and X 12 is E or T, and X 13 is T or A, and X 14 is I, M, or N, and X 15 is E or Q, and X 16 is K or R, and X 17is F or L, and X 18 is R or Q, and VH CDR3 is the amino acid sequence X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 X 35 X 36 X 37 X 38 (SEQ ID NO:98), wherein X 19 is P, E, or absent, and X 20 is G, E, or non-existent, and X 21 is G, W, S, or non-existent, and X 22 is I, D, S, P, or non-existent, and X 23 is S, L, I, T, G, or absent, and X 24 is P, T, Q, I, D, R, or non-existent, and X 25 is G, D, or non-existent, and X 26 is E, Y, P, L, D, or S, and X 27 is E, D, A, or G, and X 28 is S, F, A, E, Y, or W, and X 29 is Y or F, and X 30 is G, D, or W, and X 31 is P, Y, I, or H, and X 32 is Y or absent, X 33 is Y or absent, X 34 is Y or absent, X 35 is G or non-existent, and X 36 is M or absent, and X 37 is D or non-existent, and X 38is V or absent, and the antibody comprises a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3, and VL CDR1 has the amino acid sequence X 39 X 40 X 41 X 42 X 43 X 44 X 45 X 46 X 47 X 48 X 49 X 50 X 51 X 52 X 53 X 54 (SEQ ID NO: 99), wherein X 39 is T, A, or absent, and X 40 is G or non-existent, and X 41 , is Q, G, V, T, or S, and X 42 is A, G, S, or N, and X 43 is S, N, D, T, or Y, and X 44 is Q, Y, N, D, S, or K, and X 45 is D, I, F, V, S, or T, and X 46 is N or absent, and X 47 is I or absent, X 48 is I, G, or R, and X 49 is S, G, A, D, I, R, or T, and X 50 is Y or absent, X 51 is N, K, I, or E, and X 52 is Y, S, N, D, H, F, R, or G, and X 53 is L or V, and X 54 is N, H, S, D, or F, and VL CDR2 is the amino acid sequence X 55 X 56 X 57 X 58 X 59 X 60 X 61 where X 55 is T, D, E, Q, or R, and X 56 is A, D, V, or N, and X 57is S, G, N, or R, and X 58 is I, N, D, or K, and X 59 is L, R, or W, and X 60 is E or P, and X 61 is S, T, or L, and the VL CDR3 is the amino acid sequence X 62 X 63 X 64 X 65 X 66 X 67 X 68 X 69 X 70 X 71 X 72 where X 62 is Q, S, C, or G, and X 63 is Q, V, S, T, or A, and X 64 is Q, L, W, or Y, and X 65 is Q, N, D, I, T, A, or G, and X 66 is S, P, G, N, or A, and X 67 is N, Y, I, S, N, L, or D, and X 68 is E, P, S, I, N, H, L, or T, and X 69 is D, T, S, or absent, and X 70 is P, H, L, R, F, A, Q, or absent, and X 71 is W, L, V, Y, S, A, or E, and X 72 is T, V, or I.

[0077] In some embodiments, the anti-mutCALR antibody comprises a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein VH CDR1 comprises the amino acid sequence ELSMQ (SEQ ID NO: 1) and VH CDR2 comprises the amino acid sequence GFDPDDX 101 ETMYAEX 102 X 103 QG (SEQ ID NO: 102; Group 1 clone), 101 is D or G, and X 102 is K or R, and X 103is F or L, the VH CDR3 is SPGYDFFDY (SEQ ID NO: 18), and the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, and the VL CDR1 has the amino acid sequence GGX 104 X 105 X 106 GX 107 X 108 X 109 VX 110 (SEQ ID NO: 103; Group 1 clone), 104 is N, D, or S, and X 105 is Y, N, or D, and X 106 is I or T, and X 107 is S, D, I, R or T, and X 108 is K, E, or I, and X 109 is S, I, R, G, N, or A, and X 110 is H, F, or N, and VL CDR2 is the amino acid sequence DDX 111 DRPX 112 (SEQ ID NO: 104; Group 1 clone), 111 is G, S, or R, and X 112 is S or L, and the VL CDR3 has the amino acid sequence QVWDX 113 X 114 X 115 DX 116 X 117 X 118 (SEQ ID NO: 105; Group 1 clone), 113 is S or A, and X 114 is I or S, and X 115 is S, I, or N, and X 116 is H, L, or Q, and X 117 is V or L, and X 118 is V or I.

[0078] In some embodiments, the anti-mutCALR antibody comprises a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein VH CDR1 comprises the amino acid sequence ELSMQ (SEQ ID NO: 1) and VH CDR2 comprises the amino acid sequence GFDPDDX 101ETMYAEX 102 X 103 QG (SEQ ID NO: 102; Group 1 clone), 101 is D or G, and X 102 is K or R, and X 103 is F or L, VH CDR3 is SPGYDFFDY (SEQ ID NO: 18), and the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein VL CDR1 comprises the amino acid sequence TGTSSDVGGYNYVS (SEQ ID NO: 30) and VL CDR2 is the amino acid sequence X 119 VSX 120 RPS (SEQ ID NO: 106; Group 1 clone), 119 is E or D, and X 120 is N or K, and the VL CDR3 comprises the amino acid sequence QVWDSSNDLLI (SEQ ID NO:71).

[0079] In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3, where VH CDR1 comprises the amino acid sequence GYTLTELSMQ (SEQ ID NO: 329) and VH CDR2 comprises the amino acid sequence GFDPDDX 101 ETMYAEX 102 X 103 QG (SEQ ID NO: 102; Group 1 clone), 101 is D or G, and X 102 is K or R, and X 103 is F or L, VH CDR3 is SPGYDFFDY (SEQ ID NO: 18), and the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein VL CDR1 comprises the amino acid sequence TGTSSDVGGYNYVS (SEQ ID NO: 30) and VL CDR2 is the amino acid sequence X 119 VSX 120 RPS (SEQ ID NO: 106; Group 1 clone), 119 is E or D, and X 120 is N or K, and the VL CDR3 comprises the amino acid sequence QVWDSSNDLLI (SEQ ID NO:71).

[0080] In some embodiments, the anti-mutCALR antibody comprises a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein VH CDR1 comprises the amino acid sequence DYFIH (SEQ ID NO: 2) and VH CDR2 comprises the amino acid sequence LVDPEDGETIYAEX 121 FQG (SEQ ID NO: 107; group 2 clone), where X 121 is K or R, the VH CDR3 comprises the amino acid sequence PGGILTDPDAFDI (SEQ ID NO: 19), and the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, and the VL CDR1 is 122 GTX 123 SDVGGYNX 124 VS (SEQ ID NO: 108; group 2 clone), where X 122 is T or A, and X 123 is S or G, and X 124 is Y or H, and VL CDR2 is the amino acid sequence X 125 VX 126 X 127 RPS (SEQ ID NO: 109; Group 2 clone), wherein X 125 is D or E, and X 126 is N or S, and X 127 is K or N, and the VL CDR3 has the amino acid sequence SSYX 128 X 129 SSTX 130 X 131 V (SEQ ID NO: 110; Group 2 clone), where X 128 is I or T, and X 129 is P or S, and X 130 is R, P, F, or absent, and X 131 is W or Y.

[0081] In some embodiments, the anti-mutCALR antibody comprises a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3, where VH CDR1 comprises the amino acid sequence SYAIS (SEQ ID NO:3) and VH CDR2 comprises the amino acid sequence LVDPEDGETIYAEKFX. 132 G (SEQ ID NO: 111; group 3 clone), 132 is R or Q, the VH CDR3 comprises the amino acid sequence EESYGP (SEQ ID NO: 20), and the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, where the VL CDR1 is the amino acid sequence QASQDISNYLX 133 (SEQ ID NO: 112; group 3 clone), 133 is N or D, the VL CDR2 comprises the amino acid sequence DASNLET (SEQ ID NO:61), and the VL CDR3 comprises the amino acid sequence QQLNSYPLT (SEQ ID NO:80).

[0082] In some embodiments, the anti-mutCALR antibody comprises a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein VH CDR1 has the amino acid sequence EX 134 SMH (SEQ ID NO: 113, group 4 clone), 134 is S or L, VH CDR2 comprises the amino acid sequence LVDPEDGETIYAQKFQG (SEQ ID NO: 14) and VH CDR3 comprises the amino acid sequence EEWSGDGDDAFDI (SEQ ID NO: 21), and the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, where VL CDR1 comprises the amino acid sequence SGSSSNIGSYSVS (SEQ ID NO: 46) and VL CDR2 comprises the amino acid sequence DX 135 NKRPS (SEQ ID NO: 114, group 4 clone), 135 is N or D, and the VL CDR3 comprises the amino acid sequence GTWDSSLSAWV (SEQ ID NO:81).

[0083] In some embodiments, the anti-mutCALR antibody comprises a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein VH CDR1 comprises the amino acid sequence SYAIS (SEQ ID NO: 3), VH CDR2 comprises the amino acid sequence GIIPIFGTANYAQKFQG (SEQ ID NO: 15), and VH CDR3 comprises the amino acid sequence SPLRGSGWYWHYYYGMDV (SEQ ID NO: 22), and the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein VL CDR1 is the amino acid sequence GGNX 136 IX 137 X 138 KX 139 VH (SEQ ID NO: 115; group 6 clone), 136 is N or K, and X 137 is R or G, and X 138 is A, S, R, or T, and X 139 is H or S, and VL CDR2 is the amino acid sequence X 140 DX 141 X 142 RPS (SEQ ID NO: 116; group 6 clone), 140 is Q or R, and X 141 is S or R, and X 142 is N or K, and the VL CDR3 is the amino acid sequence QX 143 WX 144 SX 145 TX 146 V (SEQ ID NO: 117, group 6 clone), 143 is A or V, and X 144 is D or G, and X 145 is S or N, and X 146 is V, A, or E.

[0084] In some embodiments, the anti-mutCALR antibody comprises a heavy chain variable region with the C-terminal lysine removed. In some embodiments, the anti-mutCALR antibody comprises a heavy chain variable region having three VH CDRs of any anti-mutCALR clone disclosed herein and comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the VH sequences shown in Tables 4-5, and a light chain variable region having three VL CDRs of any anti-mutCALR clone disclosed herein and comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the VL sequences shown in Tables 4-5.

[0085] In some embodiments, the anti-mutCALR antibody comprises a heavy chain variable region comprising any one of the VH sequences shown in Tables 4-5. In some embodiments, the anti-mutCALR antibody comprises a light chain variable region comprising any one of the VL sequences shown in Tables 4-5. In some embodiments, the anti-mutCALR antibody comprises a heavy chain variable region comprising any one of the VH sequences shown in Tables 4-5 and a light chain variable region comprising any one of the VL sequences shown in Tables 4-5.

[0086] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 165 and a VL comprising the amino acid sequence of SEQ ID NO: 264.

[0087] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 165, and a VL comprising the amino acid sequence of SEQ ID NO: 265.

[0088] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 166 and a VL comprising the amino acid sequence of SEQ ID NO: 266.

[0089] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 165 and a VL comprising the amino acid sequence of SEQ ID NO: 266.

[0090] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 165, and a VL comprising the amino acid sequence of SEQ ID NO: 267.

[0091] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 165, and a VL comprising the amino acid sequence of SEQ ID NO: 268.

[0092] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 165 and a VL comprising the amino acid sequence of SEQ ID NO: 269.

[0093] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 167 and a VL comprising the amino acid sequence of SEQ ID NO: 270.

[0094] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 165 and a VL comprising the amino acid sequence of SEQ ID NO: 271.

[0095] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 165 and a VL comprising the amino acid sequence of SEQ ID NO: 272.

[0096] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 168 and a VL comprising the amino acid sequence of SEQ ID NO: 273.

[0097] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 169 and a VL comprising the amino acid sequence of SEQ ID NO: 274.

[0098] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 170, and a VL comprising the amino acid sequence of SEQ ID NO: 275.

[0099] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 171 and a VL comprising the amino acid sequence of SEQ ID NO: 276.

[0100] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 165, and a VL comprising the amino acid sequence of SEQ ID NO: 277.

[0101] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 165, and a VL comprising the amino acid sequence of SEQ ID NO: 278.

[0102] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 165 and a VL comprising the amino acid sequence of SEQ ID NO: 279.

[0103] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 165 and a VL comprising the amino acid sequence of SEQ ID NO: 280.

[0104] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 172 and a VL comprising the amino acid sequence of SEQ ID NO: 281.

[0105] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 165 and a VL comprising the amino acid sequence of SEQ ID NO: 282.

[0106] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 173, and a VL comprising the amino acid sequence of SEQ ID NO: 283.

[0107] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 165 and a VL comprising the amino acid sequence of SEQ ID NO: 284.

[0108] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 165, and a VL comprising the amino acid sequence of SEQ ID NO: 285.

[0109] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 174 and a VL comprising the amino acid sequence of SEQ ID NO: 286.

[0110] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 165, and a VL comprising the amino acid sequence of SEQ ID NO: 287.

[0111] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 165, and a VL comprising the amino acid sequence of SEQ ID NO: 288.

[0112] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 165, and a VL comprising the amino acid sequence of SEQ ID NO: 289.

[0113] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 165 and a VL comprising the amino acid sequence of SEQ ID NO: 290.

[0114] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 165 and a VL comprising the amino acid sequence of SEQ ID NO: 291.

[0115] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 165 and a VL comprising the amino acid sequence of SEQ ID NO: 292.

[0116] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 175 and a VL comprising the amino acid sequence of SEQ ID NO: 293.

[0117] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 175 and a VL comprising the amino acid sequence of SEQ ID NO: 294.

[0118] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 175, and a VL comprising the amino acid sequence of SEQ ID NO: 295.

[0119] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 175 and a VL comprising the amino acid sequence of SEQ ID NO: 296.

[0120] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 176 and a VL comprising the amino acid sequence of SEQ ID NO: 294.

[0121] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 175 and a VL comprising the amino acid sequence of SEQ ID NO: 297.

[0122] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 175 and a VL comprising the amino acid sequence of SEQ ID NO:298.

[0123] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 177 and a VL comprising the amino acid sequence of SEQ ID NO:299.

[0124] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 178 and a VL comprising the amino acid sequence of SEQ ID NO: 300.

[0125] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 179 and a VL comprising the amino acid sequence of SEQ ID NO: 301.

[0126] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 180, and a VL comprising the amino acid sequence of SEQ ID NO: 301.

[0127] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 180, and a VL comprising the amino acid sequence of SEQ ID NO: 302.

[0128] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 181 and a VL comprising the amino acid sequence of SEQ ID NO: 303.

[0129] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 182 and a VL comprising the amino acid sequence of SEQ ID NO: 304.

[0130] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 182 and a VL comprising the amino acid sequence of SEQ ID NO: 305.

[0131] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 183 and a VL comprising the amino acid sequence of SEQ ID NO: 306.

[0132] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 182 and a VL comprising the amino acid sequence of SEQ ID NO: 307.

[0133] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 182 and a VL comprising the amino acid sequence of SEQ ID NO: 308.

[0134] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 182 and a VL comprising the amino acid sequence of SEQ ID NO: 309.

[0135] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 184 and a VL comprising the amino acid sequence of SEQ ID NO: 310.

[0136] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 182 and a VL comprising the amino acid sequence of SEQ ID NO: 311.

[0137] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 185 and a VL comprising the amino acid sequence of SEQ ID NO: 312.

[0138] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 186 and a VL comprising the amino acid sequence of SEQ ID NO: 313.

[0139] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 187 and a VL comprising the amino acid sequence of SEQ ID NO: 314.

[0140] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 188 and a VL comprising the amino acid sequence of SEQ ID NO: 268.

[0141] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 189 and a VL comprising the amino acid sequence of SEQ ID NO: 268.

[0142] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 190, and a VL comprising the amino acid sequence of SEQ ID NO: 268.

[0143] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 191, and a VL comprising the amino acid sequence of SEQ ID NO: 268.

[0144] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 192 and a VL comprising the amino acid sequence of SEQ ID NO: 268.

[0145] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 193, and a VL comprising the amino acid sequence of SEQ ID NO: 268.

[0146] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 194, and a VL comprising the amino acid sequence of SEQ ID NO: 268.

[0147] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 195, and a VL comprising the amino acid sequence of SEQ ID NO: 268.

[0148] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 196 and a VL comprising the amino acid sequence of SEQ ID NO: 268.

[0149] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 165, and a VL comprising the amino acid sequence of SEQ ID NO: 315.

[0150] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 188 and a VL comprising the amino acid sequence of SEQ ID NO: 315.

[0151] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 189, and a VL comprising the amino acid sequence of SEQ ID NO: 315.

[0152] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 190, and a VL comprising the amino acid sequence of SEQ ID NO: 315.

[0153] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 191, and a VL comprising the amino acid sequence of SEQ ID NO: 315.

[0154] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 192 and a VL comprising the amino acid sequence of SEQ ID NO: 315.

[0155] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 193 and a VL comprising the amino acid sequence of SEQ ID NO: 315.

[0156] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 194 and a VL comprising the amino acid sequence of SEQ ID NO: 315.

[0157] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 195, and a VL comprising the amino acid sequence of SEQ ID NO: 315.

[0158] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 196 and a VL comprising the amino acid sequence of SEQ ID NO: 315.

[0159] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 165 and a VL comprising the amino acid sequence of SEQ ID NO: 316.

[0160] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 188 and a VL comprising the amino acid sequence of SEQ ID NO: 316.

[0161] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 189 and a VL comprising the amino acid sequence of SEQ ID NO: 316.

[0162] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 190 and a VL comprising the amino acid sequence of SEQ ID NO: 316.

[0163] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 191 and a VL comprising the amino acid sequence of SEQ ID NO: 316.

[0164] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 192 and a VL comprising the amino acid sequence of SEQ ID NO: 316.

[0165] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 193 and a VL comprising the amino acid sequence of SEQ ID NO: 316.

[0166] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 194 and a VL comprising the amino acid sequence of SEQ ID NO: 316.

[0167] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 195 and a VL comprising the amino acid sequence of SEQ ID NO: 316.

[0168] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 196 and a VL comprising the amino acid sequence of SEQ ID NO: 316.

[0169] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 197, and a VL comprising the amino acid sequence of SEQ ID NO: 268.

[0170] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 198 and a VL comprising the amino acid sequence of SEQ ID NO: 268.

[0171] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 199 and a VL comprising the amino acid sequence of SEQ ID NO: 268.

[0172] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 200 and a VL comprising the amino acid sequence of SEQ ID NO: 268.

[0173] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 201, and a VL comprising the amino acid sequence of SEQ ID NO: 268.

[0174] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 197, and a VL comprising the amino acid sequence of SEQ ID NO: 315.

[0175] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 198 and a VL comprising the amino acid sequence of SEQ ID NO: 315.

[0176] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 199 and a VL comprising the amino acid sequence of SEQ ID NO: 315.

[0177] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 200 and a VL comprising the amino acid sequence of SEQ ID NO: 315.

[0178] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 201 and a VL comprising the amino acid sequence of SEQ ID NO: 315.

[0179] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 197, and a VL comprising the amino acid sequence of SEQ ID NO: 316.

[0180] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 198 and a VL comprising the amino acid sequence of SEQ ID NO: 316.

[0181] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 199 and a VL comprising the amino acid sequence of SEQ ID NO: 316.

[0182] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 200 and a VL comprising the amino acid sequence of SEQ ID NO: 316.

[0183] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 201 and a VL comprising the amino acid sequence of SEQ ID NO: 316.

[0184] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 170, and a VL comprising the amino acid sequence of SEQ ID NO: 268.

[0185] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 202 and a VL comprising the amino acid sequence of SEQ ID NO: 268.

[0186] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 203 and a VL comprising the amino acid sequence of SEQ ID NO: 268.

[0187] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 204 and a VL comprising the amino acid sequence of SEQ ID NO: 268.

[0188] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 205 and a VL comprising the amino acid sequence of SEQ ID NO: 268.

[0189] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 206 and a VL comprising the amino acid sequence of SEQ ID NO: 268.

[0190] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 207 and a VL comprising the amino acid sequence of SEQ ID NO: 268.

[0191] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 208 and a VL comprising the amino acid sequence of SEQ ID NO: 268.

[0192] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 170, and a VL comprising the amino acid sequence of SEQ ID NO: 315.

[0193] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 202 and a VL comprising the amino acid sequence of SEQ ID NO: 315.

[0194] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 203 and a VL comprising the amino acid sequence of SEQ ID NO: 315.

[0195] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 204 and a VL comprising the amino acid sequence of SEQ ID NO: 315.

[0196] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 205 and a VL comprising the amino acid sequence of SEQ ID NO: 315.

[0197] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 206 and a VL comprising the amino acid sequence of SEQ ID NO: 315.

[0198] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 207 and a VL comprising the amino acid sequence of SEQ ID NO: 315.

[0199] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 208 and a VL comprising the amino acid sequence of SEQ ID NO: 315.

[0200] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 170, and a VL comprising the amino acid sequence of SEQ ID NO: 316.

[0201] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 202 and a VL comprising the amino acid sequence of SEQ ID NO: 316.

[0202] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 203 and a VL comprising the amino acid sequence of SEQ ID NO: 316.

[0203] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 204 and a VL comprising the amino acid sequence of SEQ ID NO: 316.

[0204] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 205 and a VL comprising the amino acid sequence of SEQ ID NO: 316.

[0205] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 206 and a VL comprising the amino acid sequence of SEQ ID NO: 316.

[0206] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 207 and a VL comprising the amino acid sequence of SEQ ID NO: 316.

[0207] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 208 and a VL comprising the amino acid sequence of SEQ ID NO: 316.

[0208] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 188 and a VL comprising the amino acid sequence of SEQ ID NO: 277.

[0209] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 189, and a VL comprising the amino acid sequence of SEQ ID NO: 277.

[0210] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 190, and a VL comprising the amino acid sequence of SEQ ID NO: 277.

[0211] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 191, and a VL comprising the amino acid sequence of SEQ ID NO: 277.

[0212] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 192 and a VL comprising the amino acid sequence of SEQ ID NO: 277.

[0213] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 193, and a VL comprising the amino acid sequence of SEQ ID NO: 277.

[0214] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 194, and a VL comprising the amino acid sequence of SEQ ID NO: 277.

[0215] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 195, and a VL comprising the amino acid sequence of SEQ ID NO: 277.

[0216] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 196 and a VL comprising the amino acid sequence of SEQ ID NO: 277.

[0217] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 165 and a VL comprising the amino acid sequence of SEQ ID NO: 317.

[0218] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 188 and a VL comprising the amino acid sequence of SEQ ID NO: 317.

[0219] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 189 and a VL comprising the amino acid sequence of SEQ ID NO: 317.

[0220] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 190, and a VL comprising the amino acid sequence of SEQ ID NO: 317.

[0221] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 191, and a VL comprising the amino acid sequence of SEQ ID NO: 317.

[0222] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 192 and a VL comprising the amino acid sequence of SEQ ID NO: 317.

[0223] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 193, and a VL comprising the amino acid sequence of SEQ ID NO: 317.

[0224] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 194 and a VL comprising the amino acid sequence of SEQ ID NO: 317.

[0225] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 195 and a VL comprising the amino acid sequence of SEQ ID NO: 317.

[0226] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 196 and a VL comprising the amino acid sequence of SEQ ID NO: 317.

[0227] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 197, and a VL comprising the amino acid sequence of SEQ ID NO: 277.

[0228] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 198 and a VL comprising the amino acid sequence of SEQ ID NO: 277.

[0229] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 199 and a VL comprising the amino acid sequence of SEQ ID NO: 277.

[0230] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 200 and a VL comprising the amino acid sequence of SEQ ID NO: 277.

[0231] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 201 and a VL comprising the amino acid sequence of SEQ ID NO: 277.

[0232] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 197, and a VL comprising the amino acid sequence of SEQ ID NO: 317.

[0233] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 198 and a VL comprising the amino acid sequence of SEQ ID NO: 317.

[0234] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 199 and a VL comprising the amino acid sequence of SEQ ID NO: 317.

[0235] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 200 and a VL comprising the amino acid sequence of SEQ ID NO: 317.

[0236] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 201 and a VL comprising the amino acid sequence of SEQ ID NO: 317.

[0237] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 170, and a VL comprising the amino acid sequence of SEQ ID NO: 277.

[0238] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 202 and a VL comprising the amino acid sequence of SEQ ID NO: 277.

[0239] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 203 and a VL comprising the amino acid sequence of SEQ ID NO: 277.

[0240] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 204 and a VL comprising the amino acid sequence of SEQ ID NO: 277.

[0241] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 205 and a VL comprising the amino acid sequence of SEQ ID NO: 277.

[0242] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 206 and a VL comprising the amino acid sequence of SEQ ID NO: 277.

[0243] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 207, and a VL comprising the amino acid sequence of SEQ ID NO: 277.

[0244] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 208 and a VL comprising the amino acid sequence of SEQ ID NO: 277.

[0245] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 170, and a VL comprising the amino acid sequence of SEQ ID NO: 317.

[0246] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 202 and a VL comprising the amino acid sequence of SEQ ID NO: 317.

[0247] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 203 and a VL comprising the amino acid sequence of SEQ ID NO: 317.

[0248] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 204 and a VL comprising the amino acid sequence of SEQ ID NO: 317.

[0249] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 205 and a VL comprising the amino acid sequence of SEQ ID NO: 317.

[0250] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 206 and a VL comprising the amino acid sequence of SEQ ID NO: 317.

[0251] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 207 and a VL comprising the amino acid sequence of SEQ ID NO: 317.

[0252] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 208 and a VL comprising the amino acid sequence of SEQ ID NO: 317.

[0253] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 188 and a VL comprising the amino acid sequence of SEQ ID NO: 279.

[0254] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 189 and a VL comprising the amino acid sequence of SEQ ID NO: 279.

[0255] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 190, and a VL comprising the amino acid sequence of SEQ ID NO: 279.

[0256] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 191, and a VL comprising the amino acid sequence of SEQ ID NO: 279.

[0257] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 192 and a VL comprising the amino acid sequence of SEQ ID NO: 279.

[0258] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 193, and a VL comprising the amino acid sequence of SEQ ID NO: 279.

[0259] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 194 and a VL comprising the amino acid sequence of SEQ ID NO: 279.

[0260] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 195, and a VL comprising the amino acid sequence of SEQ ID NO: 279.

[0261] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 196 and a VL comprising the amino acid sequence of SEQ ID NO: 279.

[0262] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 165 and a VL comprising the amino acid sequence of SEQ ID NO: 318.

[0263] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 188 and a VL comprising the amino acid sequence of SEQ ID NO: 318.

[0264] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 189 and a VL comprising the amino acid sequence of SEQ ID NO: 318.

[0265] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 190, and a VL comprising the amino acid sequence of SEQ ID NO: 318.

[0266] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 191, and a VL comprising the amino acid sequence of SEQ ID NO: 318.

[0267] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 192 and a VL comprising the amino acid sequence of SEQ ID NO: 318.

[0268] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 193 and a VL comprising the amino acid sequence of SEQ ID NO: 318.

[0269] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 194 and a VL comprising the amino acid sequence of SEQ ID NO: 318.

[0270] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 195 and a VL comprising the amino acid sequence of SEQ ID NO: 318.

[0271] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 196 and a VL comprising the amino acid sequence of SEQ ID NO: 318.

[0272] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 197, and a VL comprising the amino acid sequence of SEQ ID NO: 279.

[0273] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 198 and a VL comprising the amino acid sequence of SEQ ID NO: 279.

[0274] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 199, and a VL comprising the amino acid sequence of SEQ ID NO: 279.

[0275] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 200 and a VL comprising the amino acid sequence of SEQ ID NO: 279.

[0276] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 201 and a VL comprising the amino acid sequence of SEQ ID NO: 279.

[0277] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 197, and a VL comprising the amino acid sequence of SEQ ID NO: 318.

[0278] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 198 and a VL comprising the amino acid sequence of SEQ ID NO: 318.

[0279] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 199 and a VL comprising the amino acid sequence of SEQ ID NO: 318.

[0280] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 200 and a VL comprising the amino acid sequence of SEQ ID NO: 318.

[0281] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 201 and a VL comprising the amino acid sequence of SEQ ID NO: 318.

[0282] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 170, and a VL comprising the amino acid sequence of SEQ ID NO: 279.

[0283] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 202 and a VL comprising the amino acid sequence of SEQ ID NO: 279.

[0284] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 203 and a VL comprising the amino acid sequence of SEQ ID NO: 279.

[0285] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 204 and a VL comprising the amino acid sequence of SEQ ID NO: 279.

[0286] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 205 and a VL comprising the amino acid sequence of SEQ ID NO: 279.

[0287] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 206 and a VL comprising the amino acid sequence of SEQ ID NO: 279.

[0288] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 207 and a VL comprising the amino acid sequence of SEQ ID NO: 279.

[0289] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 208 and a VL comprising the amino acid sequence of SEQ ID NO: 279.

[0290] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 170, and a VL comprising the amino acid sequence of SEQ ID NO: 318.

[0291] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 202 and a VL comprising the amino acid sequence of SEQ ID NO: 318.

[0292] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 203 and a VL comprising the amino acid sequence of SEQ ID NO: 318.

[0293] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 204 and a VL comprising the amino acid sequence of SEQ ID NO: 318.

[0294] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 205 and a VL comprising the amino acid sequence of SEQ ID NO: 318.

[0295] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 206 and a VL comprising the amino acid sequence of SEQ ID NO: 318.

[0296] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 207 and a VL comprising the amino acid sequence of SEQ ID NO: 318.

[0297] In some embodiments, the anti-mutCALR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 208 and a VL comprising the amino acid sequence of SEQ ID NO: 318.

[0298] In some embodiments, anti-mutCALR antibodies include modifications that modulate (e.g., reduce or increase) Fc region-mediated effector functions such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). Depending on the application of the therapeutic antibody or Fc fusion protein, it may be desirable to reduce or increase effector function.

[0299] In certain embodiments, the anti-mutCALR antibody has Fc effector function. In certain embodiments, the anti-mutCALR antibody has enhanced Fc effector function. In certain embodiments, the anti-mutCALR antibody exhibits antibody-dependent cell-mediated cytotoxicity (ADCC). The anti-mutCALR antibody can be engineered to enhance ADCC activity (for review, see Kubota T et al. Cancer Sci. 2009; 100(9): 1566-72). For example, if the antibody itself has low ADCC activity, the ADCC activity of the antibody can be improved by slightly modifying the constant region of the antibody (Junttila TT. et al. Cancer Res. 2010; 70(11) 4481-9). Changes may also be made to improve storage or production, or to remove C-terminal lysines (Kubota T et al. Cancer Sci. 2009; 100(9): 1566-72). Another suitable method of improving the ADCC activity of antibodies is by enzymatically interfering with the glycosylation pathway resulting in reduced fucose (von Horsten HH.et al.Glycobiology.2010;20(12):1607-18). Alternatively or additionally, other suitable methods can be used to achieve enhanced ADCC, including, for example, glycoengineering (Kyowa Hakko / Biowa, GlycArt (Roche) and Eureka Therapeutics) and mutagenesis, all of which aim to improve Fc binding to the low affinity activating FcγRIIIa and / or reduce binding to the low affinity inhibitory FcγRIIb. In certain embodiments, the binding moieties of the present disclosure exhibit enhanced antibody-dependent cell-mediated cytotoxicity (ADCC). In certain embodiments, the binding moieties of the present disclosure are afucosylated.

[0300] In certain embodiments, the anti-mutCALR antibody has reduced Fc effector function. In certain embodiments, the anti-mutCALR antibody exhibits reduced or substantially no complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or antibody-dependent cellular phagocytosis (ADCP). In certain embodiments, the anti-mutCALR antibody exhibits reduced or substantially no antibody-dependent cell-mediated cytotoxicity (ADCC). The anti-mutCALR antibody can be engineered to reduce effector function, e.g., ADCC activity, by any suitable method, including removal of glycosylation sites in the Fc region. In certain embodiments, the anti-mutCALR antibody with reduced Fc effector function (e.g., reduced ADCC effector function) comprises a N297A mutation on the heavy chain.

[0301] In some embodiments, the anti-mutCALR antibody is an IgG1 isotype (e.g., IgG1, IgG2, IgG3 or IgG4). In some embodiments, the anti-mutCALR antibody is IgG1. In some embodiments, the IgG1, IgG2, IgG3 or IgG4 anti-mutCALR antibody has Fc effector function. In some embodiments, the IgG1, IgG2, IgG3 or IgG4 anti-mutCALR antibody is Fc effector function null. In some embodiments, the IgG1 anti-mutCALR antibody has Fc effector function. In some embodiments, the IgG1 anti-mutCALR antibody is Fc effector function null.

[0302] In some examples, the anti-mutCALR antibody is an antibody fragment. Fragments of the antibodies described herein (e.g., Fab, Fab', F(ab')2, Facb, and Fv) may be prepared by proteolytic digestion of intact antibodies. For example, antibody fragments can be obtained by treating whole antibodies with enzymes such as papain, pepsin, or plasmin, or FabRICATOR® (IdeS) recombinant enzyme (Genovis AB), which digests IgG antibodies to produce a homogenous pool of F(ab')2 and Fc / 2 fragments. Papain digestion of whole antibodies produces F(ab)2 or Fab fragments, pepsin digestion of whole antibodies produces F(ab')2 or Fab', and plasmin digestion of whole antibodies produces Facb fragments.

[0303] Alternatively, antibody fragments can be produced recombinantly: for example, a nucleic acid encoding the antibody fragment of interest can be constructed, introduced into an expression vector, and expressed in a suitable host cell. See, e.g., Co, MS et al., J. Immunol., 152:2968-2976 (1994); Better, M. and Horwitz, AH, Methods in Enzymology, 178:476-496 (1989); Plueckthun, A. and Skerra, A., Methods in Enzymology, 178:476-496 (1989); Lamoyi, E., Methods in Enzymology, 121:652-663 (1989); Rousseaux, J. et al., Methods in Enzymology, (1989) 121:663-669 (1989); and Bird, RE et al., TIBTECH, 9:132-137 (1991)). Antibody fragments can be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab)2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). According to another approach, F(ab')2 fragments can be directly isolated from recombinant host cell culture. Fab and F(ab')2 fragments with increased in vivo half-lives that contain salvage receptor binding epitope residues are described in U.S. Pat. No. 5,869,046.

[0304] In some examples, the anti-mutCALR antibody is a minibody. Minibodies of anti-mutCALR antibodies include diabodies, single chain (scFv), and single chain (Fv)2 (sc(Fv)2).

[0305] A "diabody" is a bivalent minibody constructed by gene fusion (e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993), EP404,097, WO93 / 11161). A diabody is a dimer consisting of two polypeptide chains. The VL and VH domains of each polypeptide chain of a diabody are linked by a linker. The number of amino acid residues constituting the linker can be 2 to 12 residues (e.g., 3 to 10 residues or 5 or about 5 residues). The linker of the polypeptides in a diabody is typically too short to allow the VL and VH to bind to each other. Thus, the VL and VH encoded by the same polypeptide chain cannot form a single chain variable region fragment, but instead form a dimer with different single chain variable region fragments. As a result, the diabody has two antigen binding sites.

[0306] scFv is a single-chain polypeptide antibody obtained by linking VH and VL with a linker (see, for example, Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988), and Plickthun, "The Pharmacology of Monoclonal Antibodies" Vol. 113, Ed Resenburg and Moore, Springer Verlag, New York, pp. 269-315, (1994)). The order of VH and VL to be linked is not particularly limited and they can be arranged in any order. Examples of arrangements include the following: [VH] linker [VL] or [VL] linker [VH]. The heavy chain variable domain and light chain variable domain in the scFv can be derived from any anti-mutCALR antibody described herein.

[0307] sc(Fv)2 is a minibody in which two VHs and two VLs are linked by a linker to form a single chain (Hudson, et al., J. Immunol. Methods, (1999) 231:177-189 (1999)). sc(Fv)2 can be prepared, for example, by connecting scFvs with a linker. In the sc(Fv)2 of the present invention, the two VHs and two VLs are preferably arranged in the order of VH, VL, VH, and VL ([VH] linker [VL] linker [VH] linker [VL]) starting from the N-terminus of the single-chain polypeptide, but the order of the two VHs and two VLs is not limited to the above arrangement, and they can be arranged in any order.

[0308] In some examples, the anti-mutCALR antibody is a bispecific antibody. A bispecific antibody is an antibody that has binding specificity for at least two different epitopes. An exemplary bispecific antibody may bind to two different epitopes of the mutCALR protein. Other such antibodies may combine a mutCALR binding site with a binding site of another antigen. Bispecific antibodies can be prepared as full-length antibodies or low molecular weight forms thereof (e.g., F(ab')2 bispecific antibodies, sc(Fv)2 bispecific antibodies, diabody bispecific antibodies).

[0309] Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where these two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). According to a different approach, antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host cell. This provides greater flexibility in adjusting the ratio of the three polypeptide fragments. However, it is possible to insert the coding sequences for two or all three polypeptide chains into one expression vector, provided that expression of at least two polypeptide chains in equal ratios results in high yields.

[0310] According to another approach described in US Patent No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. A preferred interface comprises at least a portion of the CH3 domain. In this method, one or more small amino acid side chains from the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of the same or similar size as the large side chain(s) are created on the interface of a second antibody molecule by replacing the large amino acid side chain with a small amino acid side chain (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers over other undesired end products such as homodimers.

[0311] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Heteroconjugate antibodies can be made using any convenient cross-linking method.

[0312] "Diabody" technology provides an alternative mechanism for the production of bispecific antibody fragments. These fragments contain a VH connected to a VL by a linker that is too short to allow pairing between the two domains on the same chain. Thus, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.

[0313] In certain embodiments, the bispecific anti-mutCALR antibody is a biparatopic antibody. A biparatopic antibody is an antibody that recognizes two non-identical epitopes (overlapping or non-overlapping epitopes) on the same target antigen (e.g., the C-terminus of the mutCALR domain). A biparatopic anti-mutCALR antibody can comprise two immunoglobulin heavy chain-light chain pairs or one immunoglobulin heavy chain-light chain pair. In some embodiments, a biparatopic anti-mutCALR antibody comprises one immunoglobulin heavy chain-light chain pair. In some embodiments, a biparatopic anti-mutCALR antibody is a full-length antibody comprising one immunoglobulin heavy chain-light chain pair.

[0314] In some examples, the anti-mutCALR antibody is a multivalent antibody. A multivalent antibody may be internalized (and / or catabolized) faster than a bivalent antibody by a cell expressing the antigen to which the antibody binds. The antibodies described herein may be multivalent antibodies (e.g., tetravalent antibodies) having three or more antigen binding sites, which may be readily produced by recombinant expression of a nucleic acid encoding the polypeptide chains of the antibody. A multivalent antibody may comprise a dimerization domain and three or more antigen binding sites. Exemplary dimerization domains include (or consist of) an Fc region or a hinge region. A multivalent antibody may comprise (or consist of) from three to about eight (e.g., four) antigen binding sites. A multivalent antibody optionally comprises at least one polypeptide chain (e.g., at least two polypeptide chains), where the polypeptide chain(s) comprise two or more variable domains. For example, the polypeptide chain(s) may comprise VD1-(X1) n -VD2-(X2) n-Fc, where VD1 is a first variable domain, VD2 is a second variable domain, Fc is the polypeptide chain of the Fc region, X1 and X2 represent amino acids or peptide spacers, and n is 0 or 1.

[0315] In some examples, the anti-mutCALR antibody is a conjugated antibody. The antibodies disclosed herein can be conjugated to macromolecular substances, such as polymers (e.g., polyethylene glycol (PEG), polyethyleneimine (PEI) modified with PEG (PEI-PEG), polyglutamic acid (PGA) (N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer), hyaluronic acid, radioactive substances (e.g., 90 Y, 131 I), fluorescent substances, luminescent substances, haptens, enzymes, metal chelates, drugs, and toxins (e.g., calicheamicin, Pseudomonas exotoxin A, ricin (e.g., deglycosylated ricin A chain), and auristatin (e.g., auristatin E or auristatin F)).

[0316] In one embodiment, to improve the cytotoxic effect of anti-mutCALR antibodies and therefore their therapeutic efficacy, the antibodies are conjugated with highly toxic substances, including radioisotopes and cytotoxic drugs. These conjugates can selectively deliver a toxic load to the target site (i.e., cells expressing the antigen recognized by the antibody), while sparing cells not recognized by the antibody. To minimize toxicity, conjugates are generally engineered based on molecules with short serum half-lives (hence the use of mouse sequences and IgG3 or IgG4 isotypes).

[0317] In certain embodiments, the anti-mutCALR antibody is modified with a moiety that improves its stabilization and / or retention in the circulation, e.g., blood, serum, or other tissues, e.g., at least 1.5, 2, 5, 10, or 50 fold. For example, the anti-mutCALR antibody can be associated (e.g., conjugated) with a polymer, e.g., a substantially non-antigenic polymer, such as a polyalkylene oxide or polyethylene oxide. Suitable polymers vary substantially by weight. Polymers having molecular number average weights ranging from about 200 to about 35,000 daltons (or about 1,000 to about 15,000, and 2,000 to about 12,500) can be used. For example, the anti-mutCALR antibody can be conjugated to a water-soluble polymer, e.g., a hydrophilic polyvinyl polymer, e.g., polyvinyl alcohol or polyvinylpyrrolidone. Examples of such polymers include polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycol, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained. Further useful polymers include polyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene, polymethacrylates, carbomers, and branched or unbranched polysaccharides.

[0318] Each of the above conjugated antibodies can be prepared by chemically modifying the antibodies described herein or their low molecular weight forms. Methods for modifying antibodies are well known in the art (see, for example, US 5,057,313 and US 5,156,840).

[0319] Methods for producing antibodies Antibodies can be produced in bacteria or eukaryotic cells. Some antibodies, e.g., Fab, can be produced in bacterial cells, e.g., E. coli cells. Antibodies can also be produced in eukaryotic cells, such as transformed cell lines (e.g., CHO, 293E, COS). In addition, antibodies (e.g., scFv) can be expressed in yeast cells, such as Pichia (e.g., Powers et al., J Immunol Methods. 251:123-35 (2001)), Hansenula, or Saccharomyces. To produce the antibody of interest, a polynucleotide encoding the antibody is constructed, introduced into an expression vector, and then expressed in a suitable host cell. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cell, select the transformant, culture the host cell, and recover the antibody.

[0320] If the antibody is expressed in bacterial cells (eg, E. coli), the expression vector should possess features that allow for the amplification of the vector in the bacterial cell. Additionally, when E. coli such as JM109, DH5α, HB101, or XL1-Blue is used as the host, the vector has a promoter, such as the lacZ promoter (Ward et al., 341:544-546 (1989)), the araB promoter (Better et al., Science, 240:1041-1043 (1988)), or the T7 promoter, which can allow efficient expression in E. coli. Examples of such vectors include, for example, M13 series vectors, pUC series vectors, pBR322, pBluescript, pCR-Script, pGEX-5X-1 (Pharmacia), "QIAexpress system" (QIAGEN), pEGFP, and pET (when this expression vector is used, the host is preferably BL21, which expresses T7 RNA polymerase). The expression vector may contain a signal sequence for antibody secretion. For production into the periplasm of E. coli, the pelB signal sequence (Lei et al., 2002) is used. al., J. Bacteriol., 169:4379 (1987) may be used as a signal sequence for antibody secretion. For bacterial expression, the calcium chloride method or electroporation may be used to introduce the expression vector into bacterial cells.

[0321] When the antibody is expressed in animal cells such as CHO, COS, and NIH3T3 cells, the expression vector contains a promoter necessary for expression in these cells, such as the SV40 promoter (Mulligan et al., Nature, 277:108 (1979)), the MMLV-LTR promoter, the EF1α promoter (Mizushima et al., Nucleic Acids Res., 18:5322 (1990)), or the CMV promoter. In addition to the nucleic acid sequence encoding an immunoglobulin or a domain thereof, the recombinant expression vector may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017). For example, the selectable marker gene usually confers resistance to drugs such as G418, hygromycin, methotrexate, etc. to a host cell into which the vector has been introduced. Examples of vectors having selectable markers include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0322] In one embodiment, the antibody is produced in a mammalian cell. Exemplary mammalian host cells for expressing the antibody include Chinese hamster ovary (CHO cells) (including dhfr-CHO cells as described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, e.g., used with a DHFR selectable marker as described in Kaufman and Sharp (1982) Mol. Biol. 159:601 621), human embryonic kidney 293 cells (e.g., 293, 293E, 293T), COS cells, NIH3T3 cells, lymphocytic cell lines, e.g., NS0 myeloma cells and SP2 cells, and cells from transgenic animals, e.g., transgenic mammals. For example, the cell is a mammary epithelial cell.

[0323] In an exemplary system for antibody expression, a recombinant expression vector encoding both the antibody heavy and light chains of an anti-mutCALR antibody is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operably linked to enhancer / promoter regulatory elements (e.g., derived from SV40, CMV, adenovirus, etc., such as the CMV enhancer / AdMLP promoter regulatory element or the SV40 enhancer / AdMLP promoter regulatory element) to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for the selection of CHO cells transfected with the vector using methotrexate selection / amplification. The selected transformed host cells are cultured to allow expression of the antibody heavy and light chains, and the antibody is recovered from the culture medium.

[0324] Antibodies can also be produced by transgenic animals. For example, US Patent No. 5,849,992 describes a method for expressing antibodies in the mammary gland of a transgenic mammal. A transgene is constructed that includes a milk-specific promoter, a nucleic acid encoding the antibody of interest, and a signal sequence for secretion. The milk produced by the female of such a transgenic mammal contains the antibody of interest secreted therein. The antibody can be purified from the milk or used directly for some applications. Animals that include one or more of the nucleic acids described herein are also provided.

[0325] The antibody of the present disclosure can be isolated from inside or outside the host cell (e.g., from the medium) and purified as a substantially pure and homogeneous antibody. Isolation and purification methods commonly used for antibody purification may be used for antibody isolation and purification, and are not limited to any particular method. The antibody can be isolated and purified by appropriately selecting and combining, for example, column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization. Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reversed phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). Chromatography can be performed using liquid phase chromatography such as HPLC and FPLC. Columns used for affinity chromatography include protein A columns and protein G columns. Examples of columns that use Protein A columns include Hyper D, POROS, and Sepharose FF (GE Healthcare Biosciences). The present disclosure also includes antibodies that are highly purified using these purification methods.

[0326] Polynucleotides, expression vectors, and cells The present disclosure also provides polynucleotides and vectors encoding the anti-mutCALR antibodies or portions thereof (e.g., VH, VL, HC, or LC) described herein. The polynucleotides of the present disclosure can be in the form of RNA or in the form of DNA. In some examples, the polynucleotide is DNA. In some examples, the polynucleotide is complementary DNA (cDNA). In some examples, the polynucleotide is RNA. In some embodiments, the polynucleotides described herein are isolated. In some embodiments, the polynucleotides described herein are purified.

[0327] In some examples, the polynucleotide encodes a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 of any antibody described herein (see, e.g., Tables 1-5). In some examples, the polynucleotide encodes a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 of any antibody described herein (see, e.g., Tables 1-5). In some examples, the polynucleotide encodes a heavy chain comprising a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 of any antibody described herein (see, e.g., Tables 1-5). In some examples, the polynucleotide encodes a light chain comprising a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 of any antibody described herein (see, e.g., Tables 1-5). In some examples, the polynucleotide is operably linked to a promoter.

[0328] In some examples, the polynucleotide comprises: (i) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 of any antibody described herein (see, e.g., Tables 1-5); and (ii) a second nucleic acid sequence encoding a second polypeptide, the first polypeptide comprising a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 of any antibody described herein (see, e.g., Tables 1-5). In some examples, the polynucleotide comprises (i) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a heavy chain comprising a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 of any antibody described herein (see, e.g., Tables 1-5), and (ii) a second nucleic acid sequence encoding a second polypeptide, the first polypeptide comprising a light chain comprising a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 of any antibody described herein (see, e.g., Tables 1-5). In some examples, the first nucleic acid is operably linked to a first promoter and the second nucleic acid is operably linked to a second promoter.

[0329] In some examples, the polynucleotide encodes a VH described herein (see, e.g., Tables 4-5) or a variant thereof. In some examples, the polynucleotide encodes a polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 165-208. In some examples, the polynucleotide encodes a polypeptide comprising an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions, additions, and / or deletions to the amino acid sequence set forth in any one of SEQ ID NOs: 165-208. In some examples, the polynucleotide encodes a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 165-208. In some examples, the polynucleotide is operably linked to a promoter.

[0330] In some examples, the polynucleotide encodes a VL described herein (see, e.g., Tables 4-5) or a variant thereof. In some examples, the polynucleotide encodes a polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 264-318. In some examples, the polynucleotide encodes a polypeptide comprising an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions, additions, and / or deletions to the amino acid sequence set forth in any one of SEQ ID NOs: 264-318. In some examples, the polynucleotide encodes a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 264-318. In some examples, the polynucleotide is operably linked to a promoter.

[0331] In some examples, the polynucleotide comprises: (i) a first nucleic acid encoding a first polypeptide, wherein the first polypeptide comprises a VH described herein (see, e.g., Tables 4-5) or a variant thereof; and (ii) a second nucleic acid encoding a second polypeptide, wherein the second polypeptide comprises a VL described herein (see, e.g., Tables 4-5) or a variant thereof. In some examples, the polynucleotide comprises: (i) a first nucleic acid sequence encoding a first polypeptide, the first nucleic acid sequence comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 165-208; and (ii) a second nucleic acid sequence encoding a second polypeptide, the second nucleic acid sequence comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 264-318. In some examples, the polynucleotide comprises: (i) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions, additions, and / or deletions relative to the amino acid sequence set forth in any one of SEQ ID NOs: 165-208; and (ii) a second nucleic acid sequence encoding a second polypeptide, the second polypeptide comprising an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions, additions, and / or deletions relative to the amino acid sequence set forth in any one of SEQ ID NOs: 264-318. In some examples, the first nucleic acid encodes the amino acid sequence set forth in any one of SEQ ID NOs: 165-208, and the second nucleic acid encodes the amino acid sequence set forth in any one of SEQ ID NOs: 264-318.In some examples, the first nucleic acid is operably linked to a first promoter and the second nucleic acid is operably linked to a second promoter.

[0332] Also provided herein are expression vectors encoding the anti-mutCALR antibodies or portions thereof (e.g., VH, VL, HC, and / or LC) described herein. Also provided herein are expression vectors comprising one or more polynucleotides described herein. Various types of expression vectors are known in the art and described herein.

[0333] Also provided herein is a cell comprising the anti-mutCALR antibody described herein.Also provided herein is a cell comprising one or more polynucleotides described herein.Also provided herein is a cell comprising one or more expression vectors described herein.Various types of cells are known in the art and described herein.

[0334] efficacy The anti-mutCALR antibodies of the present disclosure may inhibit the activity of mutCALR, inhibit the activity of one or more signaling pathways downstream of MPL, inhibit oncogenic cell proliferation, inhibit dimerization of MPL, compete with MPL for binding to mutCALR, or combinations thereof.

[0335] As used herein, an anti-mutCALR antibody that competes with MPL for binding to mutCALR means that the anti-mutCALR antibody binds to mutCALR with a higher affinity than MPL. In some embodiments, the anti-mutCALR antibody binds to mutCALR with an affinity that is about 10-fold, 50-fold, 100-fold, 500-fold, or 1000-fold higher than MPL. In some embodiments, the anti-mutCALR antibody has an IC that is about 10-fold, 50-fold, 100-fold, 500-fold, or 1000-fold lower than MPL. 50 In some embodiments, the anti-mutCALR antibody binds to mutCALR with an IC of about 0.1 to 1 nM. 50In some embodiments, the anti-mutCALR antibody binds to mutCALR with an IC of about 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM or 1 nM. 50 Binds to mutCALR.

[0336] Thus, the antibodies or compositions described herein can be used in a method of inhibiting the activity of mutCALR, inhibiting the activity of one or more signaling pathways downstream of MPL, inhibiting oncogenic cell proliferation, inhibiting dimerization of MPL, inhibiting binding of MPL to mutCALR, or combinations thereof, in an individual / patient in need of inhibition by administering an effective amount of an antibody described herein.

[0337] Non-limiting examples of signaling pathways downstream of MPL include Janus tyrosine kinase (JAK) and signal transducer and activator of transcription (STAT) signaling, mitogen-activated protein kinase (MEK) and extracellular signal-regulated kinase (ERK) signaling, serine / threonine kinase (AKT) signaling, and mammalian target of rapamycin (mTOR) signaling.

[0338] Another aspect of the present disclosure relates to a method of treating a mutCALR-associated disease or disorder in an individual (e.g., a patient) in need of such treatment by administering a therapeutically effective amount or dose of one or more antibodies of the present disclosure or a pharmaceutical composition thereof. The mutCALR-associated disease or disorder can include any disease, disorder, or condition that is directly or indirectly associated with the expression or activity of mutCALR.

[0339] Another aspect of the present disclosure relates to a method of treating a myeloproliferative neoplasm in an individual (e.g., a patient) in need of such treatment by administering a therapeutically effective amount or dose of one or more antibodies of the present disclosure or a pharmaceutical composition thereof.

[0340] Non-limiting examples of myeloproliferative neoplasms include chronic myelogenous leukemia, polycythemia vera, primary myelofibrosis, essential thrombocythemia, chronic neutrophilic leukemia, acute myelogenous leukemia, chronic eosinophilic leukemia, chronic myelomonocytic leukemia, myeloproliferative neoplasms, and myelodysplastic syndromes (including myelodysplastic syndrome with refractory anemia with ringed sideroblasts, myelodysplastic syndrome with refractory anemia, and myelodysplastic syndrome with refractory anemia with excess blasts).

[0341] The anti-mutCALR antibodies disclosed herein can be used to treat myeloproliferative neoplasms, or can be used for the manufacture of medicaments for the treatment of myeloproliferative neoplasms, alone or in combination with other therapies. Non-limiting examples of other therapies include JAK inhibitors (e.g., ruxolitinib, itaticinib), PI3K inhibitors (e.g., palsaclisib), standard therapies (e.g., IFN-alpha, hydroxyurea, thalidomide, lenalidomide, androgens, erythropoietin stimulating agents, chemotherapeutic agents), or combinations thereof.

[0342] Non-limiting examples of JAK inhibitors for use as described herein are provided in U.S. Pat. No. 7,335,667, U.S. Pat. No. 9,359,358, U.S. Pat. No. 8,691,807, U.S. Pat. No. 9,181,271, and U.S. Pat. No. 9,034,884, each of which is incorporated by reference herein in its entirety.

[0343] Non-limiting examples of PI3K inhibitors for use as described herein are provided in U.S. Pat. No. 9,108,984, U.S. Pat. No. 9,062,055, U.S. Pat. No. 8,759,359, and U.S. Pat. No. 9,434,746, each of which is incorporated by reference in its entirety.

[0344] The terms "individual" or "patient" or "subject" are used interchangeably and refer to any animal, including a mammal, preferably a mouse, rat, other rodent, rabbit, dog, cat, pig, cow, sheep, horse, or primate, and most preferably a human (i.e., a human subject).

[0345] The phrase "therapeutically effective amount" refers to that amount of active antibody or pharmaceutical agent that elicits the biological or pharmacological response in a tissue, system, animal, individual, or human that is being sought by a researcher, veterinarian, physician, or other clinician.

[0346] As used herein, the term "treating" or "treatment" refers to one or more of: (1) inhibiting the disease, e.g., inhibiting a disease, condition, or disorder in an individual experiencing or exhibiting a pathology or symptom of the disease, condition, or disorder (i.e., halting further development of the pathology and / or symptoms), and (2) alleviating the disease, condition, or disorder (i.e., reducing the severity of the disease), in an individual experiencing or exhibiting a pathology or symptom of the disease, condition, or disorder (i.e., reversing the pathology and / or symptoms).

[0347] In some embodiments, the antibodies of the invention are useful for preventing or reducing the risk of developing any of the diseases referred to herein, e.g., for preventing or reducing the risk of developing a disease, condition or disorder in an individual who is predisposed to the disease, condition or disorder but has not yet experienced or exhibited the pathology or symptoms of the disease.

[0348] Pharmaceutical Compositions The anti-mutCALR antibodies described herein can be formulated as pharmaceutical compositions for administration to a subject, e.g., for treating a disease or disorder described herein. Typically, a pharmaceutical composition includes a pharma- ceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are physiologically compatible. The compositions can include pharma- ceutically acceptable salts, e.g., acid addition salts or base addition salts (see, e.g., Berge, SM, et al. (1977) J. Pharm. Sci. 66:1-19).

[0349] Pharmaceutical formulation is a well-established art and is further described, for example, in Gennaro (ed.), Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott, Williams & Wilkins (2000) (ISBN: 0683306472), Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Ed., Lippincott Williams & Wilkins Publishers (1999) (ISBN: 0683305727), and Kibbe (ed.), Handbook of Pharmaceutical Excipients American Pharmaceutical Association, 3rd ed. (2000) (ISBN: 091733096X).

[0350] Pharmaceutical compositions can be in various forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form can depend on the intended mode of administration and therapeutic application. Typically, the compositions for the agents described herein are in the form of injectable or infusible solutions.

[0351] Pharmaceutical compositions can be in various forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, and liposomes. The preferred form can depend on the intended mode of administration and therapeutic application. Typically, the compositions for the agents described herein are in the form of injectable or infusible solutions.

[0352] The composition may be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for stable storage at high concentration or as a lyophilized preparation. Sterile injectable solutions can be prepared by incorporating the required amount of the anti-mutCALR antibody described herein in an appropriate solvent with one or a combination of ingredients listed above, as required, followed by filtered sterilization. In general, dispersions are prepared by incorporating the agent described herein into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization, which yield a powder of the agent described herein plus any additional desired ingredients from a previously sterile-filtered solution of the agent, plus any additional desired ingredients. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, a monostearate salt.

[0353] The anti-mutCALR antibodies can also be formulated as liposomes prepared by any suitable method known in the art.

[0354] Pharmaceutical compositions formulated for subcutaneous administration may be preferred in some situations because the subject may self-administer the pharmaceutical composition. Pharmaceutical formulations for subcutaneous administration may further include a protein formulation comprising arginine-HCl, histidine, and / or polysorbate, which may confer increased potency, improved serum half-life, or enhanced solubility to the anti-mutCALR antibody.

[0355] Route of administration The anti-mutCALR antibody can be administered to a subject, e.g., a subject in need thereof, e.g., a human subject, by a variety of methods. For many applications, the route of administration can be intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal (IP), or intramuscular injection.

[0356] The route and / or mode of administration of the antibody can also be tailored to individual cases, e.g., by monitoring the subject, e.g., by visualizing the tumor, e.g., using tomographic imaging.

[0357] The anti-mutCALR antibody can be administered as a fixed dose or in mg / kg patient weight dose. The dose can also be selected to reduce or avoid the production of antibodies against the anti-mutCALR antibody. The dosing regimen is adjusted to provide the desired response, e.g., a therapeutic response or a combined therapeutic effect. In general, the dose of the anti-mutCALR antibody (and optionally the second agent) can be used to provide a bioavailable amount of the agent to the subject.

[0358] As used herein, dosage unit form or "fixed dose" or "uniform dose" refers to physically discrete units suitable as a unitary dosage for treating a subject, each unit containing a predetermined amount of active compound calculated to produce a desired therapeutic effect in association with the necessary pharmaceutical carrier, and optionally in association with other agents. Single or multiple doses may be administered. Alternatively, or in addition, the antibody may be administered via continuous infusion.

[0359] The present disclosure also provides kits comprising one or more containers of an anti-mutCALR antibody or a pharmaceutical formulation thereof, optionally comprising one or more other prophylactic or therapeutic agents useful for treating a disease or disorder, and optionally instructions for using the anti-mutCALR antibody or a pharmaceutical formulation thereof.

[0360] The instructions for use of the anti-mutCALR antibody generally include information regarding dosage, administration schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or subunit doses. The instructions provided with the kits of the present disclosure are typically written instructions on a label or insert. The label insert indicates that the anti-mutCALR antibody is used to treat, delay the onset of, and / or ameliorate myeloproliferative neoplasms.

[0361] Detection and Diagnosis Mutant CALR can be detected in a biological sample of a subject having myeloproliferative neoplasm.Accordingly, an embodiment of the present disclosure provides a method for detecting CALR exon 9 mutation in a biological sample of a subject, the method includes obtaining a biological sample from a subject having or suspected of having myeloproliferative neoplasm, and contacting the sample with an anti-mutCALR antibody described herein, such that the anti-mutCALR antibody binds to mutCALR protein if mutCALR protein is present in the biological sample.

[0362] Another aspect of the present disclosure provides a method of diagnosing a subject having a myeloproliferative neoplasm, the method comprising obtaining a biological sample from a subject having or suspected of having a myeloproliferative neoplasm, and contacting the sample with an anti-mutCALR antibody described herein, such that if a mutCALR antibody is present in the biological sample, the anti-mutCALR antibody binds to the mutCALR protein.

[0363] The biological sample may be a blood sample, a bone marrow sample, or a serum sample. In some embodiments, the biological sample is fresh or frozen. In some embodiments, the biological sample is fixed, for example, in formaldehyde or paraformaldehyde.

[0364] Non-limiting examples of myeloproliferative neoplasms that may be diagnosed by the present methods include chronic myelogenous leukemia, polycythemia vera, primary myelofibrosis, essential thrombocythemia, chronic neutrophilic leukemia, acute myelogenous leukemia, chronic eosinophilic leukemia, chronic myelomonocytic leukemia, myeloproliferative neoplasms, and myelodysplastic syndromes (including myelodysplastic syndrome with refractory anemia with ringed sideroblasts, myelodysplastic syndrome with refractory anemia, and myelodysplastic syndrome with refractory anemia with excess blasts).

[0365] The anti-mutCALR antibodies described herein for use in the detection or diagnosis methods of the present invention can also include (e.g., be conjugated to) a detectable label. Suitable detectable labels include radioisotopes, nanoparticles, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelators, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., used in ELISA), biotin, digoxigenin, or haptens. Suitable techniques for conjugating diagnostic agents to antibodies are known in the art (e.g., Jazayeri etl al., Sensing and Bio-Sensing Research (2016); 9: 17-22 and Balasubramanya, Mater. Methods (2018); 8: 2670).

[0366] Whether labeled or unlabeled, the anti-mutCALR antibodies described herein bound to mutCALR proteins can be detected by any appropriate detection method, including immunological techniques such as immunohistochemistry (IHC), immunocytochemistry, Western blot, or ELISA immunoassays, gel- or blot-based methods, mass spectrometry, flow cytometry, or fluorescence-activated cell sorting (FACS).

[0367] The present disclosure also provides a kit for the diagnosis of a myeloproliferative neoplasm comprising one or more containers of an anti-mutCALR antibody or diagnostic preparation thereof described herein, and optionally instructions for using the anti-mutCALR antibody or diagnostic preparation thereof to detect a mutCALR exon 9 mutation or diagnose a myeloproliferative neoplasm.

[0368] The following are examples of the practice of the present invention, which should not be construed in any way as limiting the scope of the invention. EXAMPLES

[0369] The following examples are provided to better illustrate the claimed invention and are not intended to limit the scope of the invention. To the extent that specific materials are mentioned, they are merely for illustrative purposes and are not intended to limit the invention. Those skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

[0370] Example 1: Generation of anti-mutCALR antibodies To generate anti-human mutCALR monoclonal antibodies, mice were immunized with one plasmid encoding human MPL and a second plasmid encoding human type 1 mutCalR. The sequences of mutant CalR and MPL were cloned into the pVAC2 expression plasmid (Invivogen). The nucleotide sequences used in the vectors, as well as the sequences of the encoded proteins, are shown below. Human MPL nucleotide sequence: Human MPL amino acid sequence: (SEQ ID NO:324) Human type 1 mutCALR nucleotide sequence: Human type I mutCALR amino acid sequence: MLLSVPLLLGLLGLAVAEPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGLQTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDC GGGYVKLFPNSLDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKDDEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKD PDASKPEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEPPVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGVLGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQDEEQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTEA (SEQ ID NO: 320)

[0371] Plasma cells were isolated from mouse spleens and lymph nodes by flow cytometry. Antibody sequences of plasmablasts were determined using 10x Genomics VH / VL paired B cell sequencing. Mouse VH / VL pairs were expressed as chimeras with huIgG1 Fc and tested for binding and functionality. This process produced an antibody designated clone 54.

[0372] In addition, multiple rounds of selection of single-donor and multi-donor phage display libraries were performed according to the method of Erasmus et al., Communications Biology 4, article no. 350 (2021). Phage libraries were enriched on biotinylated MBP-mutCalR type 1 fusion protein (Cepter Biopartners) for either 2 or 3 rounds with MBP (Rockland Immunochemicals) and streptavidin beads (Thermo Fisher) for each round (see, e.g., Example 2). Then, scFv cassettes from library pools showing the strongest specific enrichment for mutCalR were recombined into a yeast display vector to generate yeast display libraries. These were selected over 4 rounds using decreasing concentrations of MBP-mutCalR with or without blocking with either scrambled or oligolysine peptides (Alamanda Polymers). In the final round of selection, yeast were also selected for binding to biotinylated mutCalR long peptide (Biot-LC-MKDKQDEEQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSSREASLQGWTEA, SEQ ID NO: 332) or short peptide (Biot-LC-MKDKQDEEQRTRRMMRTKMRMRRMRRTRRKM, SEQ ID NO: 333). Unique sequences were obtained from the final sorting output by Sanger sequencing of yeast colonies. For screening, these scFvs were reformatted by cloning into a human IgG1 expression vector and then purified from Expi293F cell (Thermo Fisher, Cat. No. A14635) supernatant. The MBP-mutCalR type 1 fusion proteins used in this experiment are: (SEQ ID NO:334)

[0373] The amino acid sequences of the six CDRs for each of the 54 unique clones are shown in Table 1. The heavy chain, VH, light chain, and VL sequences for each clone are shown in Table 4.

[0374] Mutations were introduced into three of the identified clones (clones 6, 15, and 17) to generate 161 unique mutant clones (clones 55-215). The amino acid sequences of the six CDRs for each of the mutant clones are shown in Table 2. The light and heavy chain sequences of the parent clones (clones 6, 15, and 17) and mutant clones (clones 55-215) are shown in Table 5. One such mutation to the heavy chain, N297A, resulted in an Fc effector function "null" mutant in which Fc effector function, particularly ADCC, was eliminated or substantially eliminated. As can be seen from the activity data in the Examples, these antibodies with the N297A Fc effector null mutation generally have increased binding affinity to mutCALR over those antibodies without the Fc effector null mutation. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 2-1] [Table 2-2]

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 2-18

Table 2-19

Table 2-20

Table 2-21

Table 2-22

Table 2-23

Table 2-24

Table 2-25

Table 2-26

Table 2-27

Table 2-28

Table 2-29

Table 2-30

Table 2-31

Table 2-32

Table 2-33

Table 2-34

Table 2-35

Table 2-36

Table 2-37

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

Table 4-6

Table 4-7

Table 4-8

Table 4-9

Table 4-10

Table 4-11

Table 4-12

Table 4-13

Table 4-14

Table 4-15

Table 4-16

Table 4-17

Table 4-18

Table 4-19

Table 4-20

Table 4-21

Table 4-22

Table 4-23

Table 4-24

Table 4-25

Table 4-26

Table 4-27

Table 4-28

Table 4-29

Table 4-30

Table 4-31

Table 4-32

Table 4-33

Table 4-34

Table 4-35

Table 4-36

Table 4-37

Table 4-38

Table 4-39

Table 4-40

Table 4-41

Table 4-42

Table 4-43

Table 4-44

Table 4-45

Table 4-46

Table 4-47

Table 4-48

Table 4-49

Table 4-50

Table 4-51

Table 4-52

Table 4-53

Table 4-54

Table 5-1

Table 5-2

Table 5-3

Table 5-4

Table 5-5

Table 5-6

Table 5-7

Table 5-8

Table 5-9

Table 5-10

Table 5-11

Table 5-12

Table 5-13

Table 5-14

Table 5-15

Table 5-16

Table 5-17

Table 5-18

Table 5-19

Table 5-20

Table 5-21

Table 5-22

Table 5-23

Table 5-24

Table 5-25

Table 5-26

Table 5-27

Table 5-28

Table 5-29

Table 5-30

Table 5-31

Table 5-32

Table 5-33

Table 5-34

Table 5-35

Table 5-36

Table 5-37

Table 5-38

Table 5-39

Table 5-40

Table 5-41

Table 5-42

Table 5-43

Table 5-44

Table 5-45

Table 5-46

Table 5-47

Table 5-48

Table 5-49

Table 5-50

Table 5-51

Table 5-52

Table 5-53

Table 5-54

Table 5-55

Table 5-56

Table 5-57

Table 5-58

Table 5-59

Table 5-60

Table 5-61

Table 5-62

Table 5-63

Table 5-64

Table 5-65

Table 5-66

Table 5-67

Table 5-68

Table 5-69

Table 5-70

Table 5-71

Table 5-72

Table 5-73

Table 5-74

Table 5-75

Table 5-76

Table 5-77

Table 5-78

Table 5-79

Table 5-80

Table 5-81

Table 5-82

Table 5-83

Table 5-84

Table 5-85

Table 5-86

Table 5-87

Table 5-88

Table 5-89

Table 5-90

Table 5-91

Table 5-92

Table 5-93

Table 5-94

Table 5-95

Table 5-96

Table 5-97

Table 5-98

Table 5-99

Table 5-100

Table 5-101

Table 5-102

Table 5-103

Table 5-104

Table 5-105

Table 5-106

Table 5-107

Table 5-108

Table 5-109

Table 5-110

Table 5-111

Table 5-112

Table 5-113

Table 5-114

Table 5-115

Table 5-116

Table 5-117

Table 5-118

Table 5-119

Table 5-120

Table 5-121

Table 5-122

Table 5-123

Table 5-124

Table 5-125

Table 5-126

Table 5-127

Table 5-128

Table 5-129

Table 5-130

Table 5-131

Table 5-132

Table 5-133

Table 5-134

Table 5-135

Table 5-136

Table 5-137

Table 5-138

Table 5-139

Table 5-140

Table 5-141

Table 5-142

Table 5-143

Table 5-144

Table 5-145

Table 5-146

Table 5-147

Table 5-148

Table 5-149

Table 5-150

Table 5-151

Table 5-152

Table 5-153

Table 5-154

Table 5-155

Table 5-156

Table 5-157

Table 5-158

[0375] Example 2: Binding Affinity The amino acid sequence of the recombinant MBP-mutCALR type 1 fusion protein used in Experiments 1 and 2 is as follows: (SEQ ID NO:335)

[0376] Experiment 1: Recombinant MBP-mutCALR type 1 fusion protein was custom made at Cepter (cepterbiopartners.com). Data on antibody interactions with recombinant MBP-mutCALR type 1 fusion protein were collected at 25°C on a Biacore 8K instrument (Cytiva, Marlborough, MA) and all reagents were obtained from Cytiva unless otherwise stated. Experiments were performed using Tris-buffered saline (pH 7.2), 0.005% surfactant P20, 1 mM CaCl2 as running buffer. To prepare the capture surface, anti-human IgG (Fc) antibody (product no. 29234600) was amine-coupled onto a CM4 chip (product no. BR100534) under standard conditions using the Amine Coupling Kit (product no. BR100050). Anti-mutCALR antibody was captured onto the chip surface by injecting only for 30 seconds over flow cell 2 at a flow rate of 30 μL / min. Typical capture levels ranged from 10 to 30 RU. Recombinant MBP-mutCALR type 1 fusion protein was prepared at nominal concentrations of 0, 3.1, 9.3, 27.8, 83.3, and 250 nM in running buffer and injected into both flow cells 1 and 2 at a flow rate of 50 μL / min for 210 s, followed by a 510 s dissociation phase at the same flow rate. Kinetic parameters were obtained by applying a 1:1 binding model to fit the data from the multi-cycle injection experiments using Biacore Insight Evaluation software. A 250 nM concentration of MBP protein was injected as a control, which showed no binding to the anti-mutCALR antibody. [Table 6-1] [Table 6-2]

[0377] Experiment 2: Recombinant MBP-mutCALR type 1 fusion protein was custom made at Cepter (cepterbiopartners.com). Data on antibody interactions with recombinant mutCALR protein were collected at 25°C on a Biacore 8K instrument (Cytiva, Marlborough, MA) and all reagents were obtained from Cytiva unless otherwise stated. Experiments were performed using Tris-buffered saline (pH 7.2), 0.005% surfactant P20, 1 mM CaCl2 as running buffer. To prepare the capture surface, anti-human IgG (Fc) antibody (product no. 29234600) was amine-coupled onto a CM4 chip (product no. BR100534) under standard conditions using an amine coupling kit (product no. BR100050). Anti-mutCALR antibody was captured onto the chip surface by injecting only for 30 seconds at a flow rate of 10 μL / min over flow cell 2. Typical capture levels ranged from 15-25 RU. Recombinant MBP-mutCALR type 1 fusion protein was prepared at nominal concentrations of 0, 0.75, 2.22, 6.67, 20, and 60 nM and injected into both flow cells 1 and 2 at a flow rate of 69 μL / min for 150 s, followed by a 230 s dissociation phase at the same flow rate. Kinetic parameters were obtained by applying a 1:1 binding model to fit the data from the multi-cycle injection experiments using Biacore Insight Evaluation software. [Table 7-1] [Table 7-2] [Table 7-3]

[0378] Experiment 3: The amino acid sequence of the recombinant MBP-mutCALR type 2 fusion protein used in this experiment 3 is as follows: (SEQ ID NO:331)

[0379] Recombinant MBP-mutCALR type 2 fusion protein was custom made at Cepter (cepterbiopartners.com). Data on antibody interaction with recombinant MBP-mutCALR type 2 fusion protein was collected as described in experiment 2. The chip used was a Biacore Series S sensor chip CM4 with anti-huFc immobilized on the chip surface. Recombinant MBP-mutCALR type 2 fusion protein was prepared at nominal concentrations of 1.1, 3.3, 10, 30, and 90 nM. Typical capture levels ranged from 16 to 37 RU. The results are shown in Table 8. [Table 8]

[0380] Example 3: Binding of anti-mutCALR antibodies to cell surface MPL and mutCALR To evaluate the binding of CALR antibodies to cell surface CALR, parental BaF3 (DSMZ) cells or BaF3 cells stably expressing human MPL alone or human MPL (Uniprot number: P40238-1) and both human mutCALR types 1 and 2 were used. On the day of the assay, cells were washed and resuspended in assay buffer. Approximately 200,000 cells / well were added to a 96-well plate and stained with the indicated concentrations of antibodies for 30 minutes on ice. Cells were then washed and stained with goat anti-human secondary conjugated to R-phycoerythrin (R-PE) (Jackson Immuno Research Laboratories) for 30 minutes on ice. Cells were then washed and analyzed by flow cytometry. Geometric mean fluorescence intensity (GMFI) of cell binding was graphed and EC50, hillslope, and area under the curve (AUC) were determined after four parameter curve fitting using GraphPad Prism software (version 7.04). [Table 9-1] [Table 9-2] [Table 10-1] [Table 10-2]

[0381] Example 4: Anti-mutCALR antibody-mediated inhibition of pSTAT5 To test the ability of anti-mutCALR antibodies to inhibit STAT5 phosphorylation, Ba / F3 cells (DSMZ) expressing MPL and type 1 mutCALR variants were generated by nucleofection (Amaxa Cell Line Nucleofection Kit V, Lonza, Basel, Switzerland) and cultured in RPMI 1640 + 10% FBS + selection antibiotics. Prior to pSTAT5 evaluation (24 h), cells were cultured in selection-free medium and then plated at 200,000 cells per well (96-well plates) in RPMI 1640, 10% FBS. Antibodies were added to cells and incubated for 2 h before cell lysis and quantification of pSTAT-5 levels by MSD (Phospho-STAT5a,b Whole Cell Lysate Kit, MSD, Kenilworth, NJ). Anti-mutCALR antibodies inhibit STAT5 phosphorylation in a dose-dependent manner (Figure 1). [Table 11-1] [Table 11-2]

[0382] Example 5: Anti-mutCALR antibody-mediated inhibition of cell proliferation To test the ability of anti-mutCALR antibodies to inhibit cell proliferation, engineered Ba / F3 cells transfected with MPL and mutCALR type 1 were plated at 5,000 cells per well in RPMI 1640 + 2% FBS, antibodies were added, incubated for 72 h, followed by evaluation of cell viability using a CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI) for luminescence quantification and Top Count (Perkin Elmer, Boston, MA) or Pherastar (BMG Labtech, Ortenberg, Germany). Anti-mutCALR antibodies inhibit mutCALR-induced oncogenic cell proliferation in a dose-dependent manner in both Ba / F3 engineered cells (Figure 2). [Table 12-1] [Table 12-2]

[0383] Example 6: Anti-mutCALR antibodies inhibit oncogenic cell proliferation induced by both type 1 and type 2 CALR mutations. To test the ability of anti-mutCALR antibodies to inhibit oncogenic cell proliferation induced by type 1 and type 2 CALR mutations, Ba / F3 cells (DSMZ) were engineered to express MPL+mutCALR type 1 (52 bp deletion, SEQ ID NO: 320) or MPL+mutCALR type 2 (5 bp insertion, SEQ ID NO: 321). Cells were plated at 5,000 cells per well in RPMI 1640+2% FBS, antibodies were added, incubated for 72 hours, followed by assessment of cell viability using a CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI) and Top Count (Perkin Elmer, Boston, MA) or Pherastar (BMG Labtech, Ortenberg, Germany) for luminescence quantification. Anti-mutant CALR antibodies show the ability to inhibit mutCALR-induced oncogenic cell proliferation associated with both type 1 and type 2 mutations (Figure 3).

[0384] Example 7: Anti-mutCALR antibody-mediated inhibition of MPL dimerization Inhibition of MPL dimerization was tested as a potential mechanism of action of anti-mutCALR antibodies. HAP1 cells knocked out for human JAK2 (Horizon Discovery, Ltd.) were transiently transfected with vectors encoding MPL-LgBiT and MPL-smBiT fusion proteins (Promega Corp.). Transfections also included vectors encoding full-length human JAK2 and full-length WT or mutant CALR type 1 proteins (pD2529 vector, full-length cds cloned into ATUM Bio. Cells were transfected in 96-well plates with equal amounts of each plasmid using Trans-IT 2020 reagent (Mirus Bio LLC). Cells were then incubated at 37°C, 5% CO2. Six hours after transfection, antibodies were diluted in growth medium (IMDM, 10% FBS) and added to the cells at the indicated concentrations. Plates were incubated overnight and growth medium was replaced with 100 μl OPTI-MEM I without phenol red containing the same concentrations of antibodies. After 1 hour at 37°C, 5% CO2, 25 μl NanoGlo Live Cell Reagent (Promega) was added to each well. Plates were returned to the incubator for 30 minutes before luminescence was read on a PHERAstar FSX (BMG Labtech). GraphPad Data were analyzed using PRISM software (version 7.04) and expressed as percent inhibition compared to isotype control. As shown in Figures 4A-4G, anti-mutCALR antibodies (clones 55, 65, 68, 74, 134, 184, and 188) can inhibit MPL dimerization.

[0385] The amino acid sequences of the MPL-LgBiT and MPL-smBiT fusion proteins used in this example are as follows: MPL-smBiT (SEQ ID NO:336) MPL-LgBiT (SEQ ID NO:337)

[0386] Example 8: Effect of anti-mutCALR antibodies in mice injected with tumor cells expressing MPL-mutCALR To test the functional capacity of anti-mutCALR antibodies in vivo, the antibodies were evaluated in a mouse model of tumor growth. Engineered Ba / F3 tumor cells expressing MPL / mutCALR type 1 were transfected into NSG immunodeficient mice (NOD-scid IL2Rgamma null Mice were inoculated intravenously at 100 ng / mL incubators (The Jackson Laboratories, Bar Harbor, ME). Tumors were allowed to grow for 10 days when mice were randomized into antibody or isotype control treatment groups. Different doses of antibody were administered intraperitoneally and tumor growth was followed over time by assessing the presence and number of tumor cells in the blood (Sysmex, Kobe, Japan). In addition, complete hematology and tumor infiltration in the spleen and bone marrow were evaluated for evaluation of antibody efficacy. A representative in vivo study is shown in Figure 5.

[0387] Anti-mutCALR antibodies prolonged mouse survival (Figure 6) and prevented splenomegaly (Figure 7), thrombocytopenia (Figure 8), and proliferation of tumor cells in the blood (Figure 9).

[0388] Example 9: Anti-mutCALR antibodies enhance the therapeutic response of ruxolitinib in inhibiting oncogenic cell proliferation caused by mutCALR type 1 or 2. To test the ability of anti-mutCALR antibodies to enhance the therapeutic response of the JAK1 / 2 inhibitor ruxolitinib, Ba / F3 cells were engineered to express MPL+mutCALR type 1 (52 bp deletion) or MPL+mutCALR type 2 (5 bp insertion) as described above. Cells were plated at 5,000 cells per well in RPMI 1640+2% FBS and treated with 50 nM ruxolitinib and / or anti-mutCALR antibodies. Cells were incubated for 72 hours, followed by evaluation of cell viability using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI) and Top Count (Perkin Elmer, Boston, MA) or Pherastar (BMG Labtech, Ortenberg, Germany) for luminescence quantification. Ruxolitinib used at a concentration of 50 nM inhibited the level of oncogenic cell proliferation by approximately 20% (dotted line). The anti-mutant CALR antibody clone 6 enhanced the ability of ruxolitinib to inhibit cell proliferation in cells harboring CALR type 1 (Figure 10, top) or type 2 (Figure 10, bottom) mutations. IC of various clones 50 The values ​​are shown in Table 13. [Table 13]

[0389] Example 10: Anti-mutCALR antibodies compete with MPL for binding to mutCALR in vitro. The amino acid sequence of the recombinant His tag_hMPL used in this example is: SQDVSLASDSEPLKCFSRTFEDLTCFWDEEEAAPSGTYQLLYAYPREKPRACPLSSQSMPHFGTRYVCQFPDQEEVRLFFPLHLWVKNVFLNQTRTQRVLFVDSVGLPAPPSIIKAMGG SQPGELQISWEEPAPEISDFLRYELRYGPRDPKNSTGPTVIQLIATETCCPALQRPHSASALDQSPCAQPTMPWQDGPKQTSPSREASALTAEGGSCLISGLQPGNSYWLQLRSEPDGISL GGSWGSWSLPVTVDLPGDAVALGLQCFTLDLKNVTCQWQQQDHASSQGFFYHSRARCCPRDRYPIWENCEEEEKTNPGLQTPQFSRCHFKSRNDSIIHILVEVTTAPGTVHSYLGSPFWIHQAVRLPTPNLHWREISSGHLELEWQHPSSWAAQETCYQLRYTGEGHQDWKVLEPPLGARGGTLELRPRSRYRLQLRARLNGPTYQGPWSSWSDPTRVETATETAWHHHHH (SEQ ID NO: 338)

[0390] The amino acid sequence of the recombinant Flag tag_GFP_tev_hmutCALR type 1 used in this example is: (SEQ ID NO:326)

[0391] Recombinant His tag_hMPL and recombinant Flag tag_GFP_tev_hmutCALR type 1 proteins (80nM MPL + 5nM mutCALR) were incubated in assay buffer (HEPES, pH7.5 50nM; Prionex 0.05%; NaCl 100nM; Pluoronic F-127 0.01%; CaCl2 1mM; MgCl2 1mM; DTT) for 1h at room temperature to allow the formation of mutCALR / MPL complexes. In a 384-well plate, 5μl of anti-mutCALR antibodies 6 and 32, non-functional CAL2 antibody (Dianova), or untagged mutCALR (competitor) were incubated with 5μl of mutCALR / MPL protein mixture for 1h at room temperature. HTRF detection solution (2 nM anti-FLAG-Europium and 100 nM anti-6xHis-SureLight® APC) was then added and the plate was read on a BMG PHERAstar FSX using a kinetic HTRF protocol, with readings taken every 15 min for a total of 120 min. Data showed that binding equilibrium was reached by 90 min, and the HTRF ratio value at 90 min was used for analysis. HTRF ratios from each test well containing FLAG-GFP-tev-mutCALR+MPL-6xHis solution were background subtracted using control wells (no MPL_His tag). Percentage of control values ​​were obtained by dividing the background subtracted HTRF ratio of each test well by the buffer control wells in the same plate row. Data shows that isotype does not interfere with mutCALR / MPL interaction (negative control), while untagged mutCALR competes with FLAG tag_mutCALR for binding to MPL. Although the non-functional CAL2 antibody showed some signal inhibition at high concentrations (>100 nM), the anti-mutCALR antibodies 6 and 32 completely inhibited the interaction of MPL and mutCALR, indicating that the anti-mutCALR antibodies compete with MPL for binding to mutCALR (Figure 11).

[0392] Example 11: Structure determination of Fab fragments from anti-mutCALR antibodies bound to the mutCALR 21-mer peptide For Examples 11 and 12, and Figures 12, 13, 14, 15, 16, and 17, the CCG numbering scheme from MOE (Molecular Operating Environment, 2019.01; Chemical Computing Group ULC, 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2021) was used for the CDR definitions and antibody sequences.

[0393] The Fab portion of the anti-mutCALR antibody (clone 55; also referred to as antibody 55) was expressed and purified as follows. The Fab portion of antibody 55 was expressed in Expi293F cells (Thermo Fisher, Cat. No. A14635) by transient transfection for 5 days. The Fab was purified from the clarified supernatant by binding to CaptureSelect CH1-XL Affinity Matrix (ThermoFisher), washing with PBS buffer, and eluting with 50 mM sodium acetate, pH 4.0. After elution, the Fab was buffer exchanged into PBS and concentrated using Ultra-15 centrifugal filter units, 10 kDa MWCO (Amicon), then stored at -80°C.

[0394] array: Fab heavy chain: EVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMQWVRQAPGKGLEWMGGFDPDDGETMYAEKFQGRLTVTEDTSTDTVYMELRSLTSEDTALYFCATSPGYDFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT (SEQ ID NO: 327) Fab light chain: QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSNSGNTATLTINRVEAGDEADYYCQVWDSSNDLLIFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 213)

[0395] The crystallization was carried out as follows: Fab: The Fab portion of antibody 55 was concentrated to 10 mg / mL in TBS buffer. A sparse matrix crystallization screen was set up using an NT8 crystallization robot (Formulatrix, Bedford, MA). Drops containing 200 nl (Fab fragment) + 200 nl (reservoir) were used for the set up and plates were incubated at 4°C, 13°C, and 20°C. Crystals of the Fab portion of antibody 55 appeared in condition C10 of a JCSG Top96 screen (0.1 M HEPES pH 7.5, 20% (w / v) PEG 8,000 (Rigaku, Bainbridge Island, WA) after 5 days of incubation at 4°C.

[0396] Fab-mutCALR peptide: The Fab portion of antibody 55 was mixed with the mutCALR peptide (acetyl-DEEQRTRRMMRTKMRMRRMRR-NH2, SEQ ID NO:339) in a 1:1.5 molar excess ratio and then concentrated to a final concentration of 35 mg / mL. A sparse matrix crystallization screen was set up using an NT8 crystallization robot (Formulatrix, Bedford, MA). Drops containing 200 nl (Fab+mutCALR peptide)+200 nl (reservoir) were used for the set up and plates were incubated at 4°C, 13°C, and 20°C. The first crystal hit was grown from condition C7 of the JCSG Top96 screen (Rigaku, Bainbridge Island, WA) after 3 days of incubation at 4°C. This initial hit condition was further purified resulting in mutCALR peptide-bound crystals. The final conditions for Fab-mutCALR peptide were 0.1 M Bicine pH 7.94, 19% w / v PEG 6000. These crystals grew in approximately 5 days.

[0397] All crystals were flash-cooled in liquid nitrogen for X-ray data collection.

[0398] Data collection, processing and purification were performed as follows: Diffraction data were collected at 100K using synchrotron radiation at the Advanced Photon Source (IMCA-CAT beamline 17-ID). Indexing, integration, and scaling of the diffraction data were performed using the AutoPROC package. Data collection statistics, phasing, and refinement are shown in Table 14.

[0399] For the Fab-Apo data, two copies of the Fab fragment were placed as a template using MoRda (Keegan and Winn. (2007) Acta Cryst. D63, 447-57, Keegan et al. (2018) Acta Cryst. D74, 167-82, and Winn et al. (2011) Acta. Cryst. D67, 235-42) (PDB ID: 5AZE). For the Fab-mutCALR peptide data, the fragments of the Fab-Apo model were placed using Phaser, and a subsequent round of manual model building was performed, resulting in a model containing two Fabs and three peptides. For both data sets, subsequent cycles of density correction, model building and refinement were performed using Refmac (Murshudov et al. (1997) Acta Cryst. D53, 240-255), Coot (Emsley et al. (2010) Acta Crystallographica Section D-Biological Crystallography, 66, 486-501) and Phenix (Liebschner et al. (2019) Acta Cryst. (2019). D75, 861-77) until the structures were converged with reasonable R-work and R-free (Table 14). The final models were analyzed for favorable stereochemistry, geometry and clash scores using MolProbity (Table 14).

[0400] result: The Fab-Apo structure was determined to 2.9 Å and consists of residues 2-213 of the light chain and 2-219 of the heavy chain (2Fab) with good electron density throughout, except for residues strand L: Asp28-Tyr34, strand H: Ser99-Gly101, Lys134-Gly139, strand M: Gly24-Tyr34, strand I: Ser133-Gly138, which were not modeled due to poor electron density. The structure was refined to 23% and 30% R-work / R-free, respectively, and has good stereochemistry throughout, with four Ramachandran outliers allowed for this resolution (48th percentile for this residue range, Table 14).

[0401] The structure of the Fab-mutCALR peptide was resolved to 3.2 Å. The two light chains consist of residues L: 2-215, M: 2-216, and the two heavy chains H: 2-219 and I: 2-185. There are two Fabs and two peptides in the asymmetric unit. The model has good electron density throughout, except for the following residues that were not modeled due to poor electron density: H, Ser132-Gly139, M, Thr25-Gly31, and I, Ala130-Ala142. The structure was refined to 23% and 32% R-work / R-free, respectively, and has good stereochemistry throughout, with seven Ramachandran outliers allowed for this resolution (56th percentile for this residue range, Table 14). [Table 14]

[0402] As shown in Figure 12A, the asymmetric unit of the crystal structure contains one Fab molecule bound to two mutCALR peptides (designated CalR1 and CalR2). The structure contains two light chains (shown in white), two heavy chains (shown in black), and two identical peptides (CalR1, grey and CalR2, white) representing parts of the mutant CALR C-terminal domain. The Fab binds to the two mutCALR peptides in two different binding profiles (Figure 12A). For clarity, only the Fv region of the Fab is displayed.

[0403] FIG. 12B shows the composition of the CDR loops (L1, L2, L3, H1, H2, and H3), amino acid composition, and length of the Fab. CDR predictions performed using the CCG scheme in MOE (Molecular Operation Environment). Amino acids highlighted in bold / italics are noted for their significant contribution to CalR1 peptide binding, as detailed in FIG. 13B-13C. Amino acids highlighted in bold / underlined are noted for their important contribution to CalR2 peptide binding, as detailed in FIG. 14B-14C. Amino acids indicated by asterix contribute to both CalR1 and CalR2 binding. FIG. 12C shows the arrangement of CalR1 (gray) and CalR2 (white) in the CDR region of Fab1 (top), as well as the arrangement of the CDR loops (L1, L2, L3, H1, H2, and H3) and the orientation of CalR1 (gray) and CalR2 (white) in the CDR region of Fab1 (bottom).

[0404] Figures 13A-13B show residues involved in binding of Fab to CalR1. A magnified image of selected CalR1-Fab1 interacting residues is shown in Figure 13B. Figure 13C provides details of selected CalR1-Fab1 interacting residues shown in Figure 13B. Figures 14A-14B show residues involved in binding of Fab to CalR2. A magnified image of selected CalR2-Fab1 interacting residues is shown in Figure 14B. Figure 14C provides details of selected CalR2-Fab1 interacting residues shown in Figure 14B. In Figures 13A-13C and Figures 14A-14C, residues were selected based on distances and interaction types determined by MOE (Molecular Operation Environment). Heavy chain residues are shown in black and light chain residues in white. Angstrom distances are indicated by dotted lines. Interacting residues are shown as sticks and were calculated using the InterfaceResidues.py script and Pymol (protein.osaka-u.ac.jp / rcsfp / supracryst / suzuki / jpxtal / Katsutani / en / interface.php).

[0405] FIG. 15 shows the sequences of mutant CALR peptides with CalR1 conformation-binding residues (top) and CalR2 conformation-binding residues (bottom) shaded in grey.

[0406] Example 12: Structure determination of Fab fragments from anti-mutCALR antibodies bound to type 1 mutant CalR 31-mer peptide The Fab fragment having the sequence shown below was purified as described above for Example 11.

[0407] array: Fab heavy chain EVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMQWVRQAPGKGLEWMGGFDPDDAETMYAEKFQGRLTVTEDTSTDTVYMELSSLRSEDTAVYFCATSPGYDFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT (SEQ ID NO: 330) Fab light chain SYVLTQPPSVSVAPGKTARITCTGTSSDVGGYNYVSWYQQKPGQAPVLVVYEVSNRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSNDLLIFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 260) 31-mer mutCALR peptide: H-DEEQRTRRMMRTKMRMRRMRRTRRKMRRKMS-OH (SEQ ID NO: 340)

[0408] Crystallization was performed as follows: the recombinant Fab fragment was mixed with the 31-mer peptide in a 1:3 molar excess ratio and then concentrated to a final concentration of 21 mg / mL. A sparse matrix crystallization screen was set up using an NT8 crystallization robot (Formulatrix, Bedford, MA). Drops containing 200 nl (Fab fragment + CalR peptide) + 200 nl (reservoir) were used for the set up and plates were incubated at 4°C, 13°C, and 20°C. Crystals were grown from condition E7 of the JCSG Top96 Screen (Rigaku, Bainbridge Island, WA) after 1 day of incubation at 20°C. Final crystallization conditions for the antibody X:31-mer peptide complex were TRIS 0.1 M, pH 8.5, polyethylene glycol 400 40% v / v, and 0.2 M lithium sulfate.

[0409] All crystals were flash-cooled in liquid nitrogen for X-ray data collection.

[0410] Data collection, processing and refinement were performed as follows: Diffraction data were collected at 100K using synchrotron radiation at beamline 17-ID-1 at the Center for BioMolecular Structure, National Synchrotron Light Source II, Brookhaven, NY, USA, using an Eiger 9M detector (λ=0.9201 Å). 1800 images were collected using a rotation of 0.1° per image. Indexing, integration and scaling of the diffraction data were performed using Fast_dp. Data collection statistics, phasing and refinement are shown in Table 15.

[0411] For the Fab fragment-31-mer peptide data, Phaser was used to place the fragments of the Fab fragment model, and a subsequent round of manual model building was performed to obtain a model containing one Fab and one peptide. Subsequent cycles of density modification, model building and refinement were performed using Refmac, Coot and Phenix until the structure was converged with reasonable R-work and R-free (Table 15). The final model was analyzed for favorable stereochemistry, geometry and clash scores using MolProbity (Table 15). [Table 15]

[0412] result: The structure of the Fab fragment-31-mer mutCalR peptide was determined at 2.0 Å using space group C121. Each asymmetric unit contains one Fab molecule and one 31-mer peptide molecule. The Fab molecule consists of a light chain, strand L: 2-215, and a heavy chain, strand H: 1-221. The model has well-defined electron density throughout, except for the following residues: strand H, Gly42-Lys43, which were not modeled due to weak electron density. The structure was refined to 20.1% and 22.6% R-work / R-free, respectively, and has good stereochemistry throughout, with two Ramachandran outliers.

[0413] FIG. 16A shows a diagram of the protein structure in the asymmetric unit. The asymmetric unit consists of one light chain, one heavy chain from the Fab fragment, and a 31 amino acid peptide from type 1 mutant CalR. FIG. 16B shows a zoomed-in view of the CDRs and the region of the mutant CalR peptide. The constant region of the Fab is omitted for clarity. FIG. 16C shows the CDR composition of the Fab fragment. Bold font indicates the amino acids that contribute to epitope recognition in the mutant CalR. The CCG numbering scheme was used to define the CDRs in MOE (Molecular Operating Environment, 2019.01; Chemical Computing Group ULC, 1010 Sherbooke St. West, Suite #910, Montreal, QC Canada, H3A 2R7, 2021).

[0414] Figure 17A shows a diagram of interacting residues located across the CDR regions from the heavy and light chains. Figure 17B shows selected residues for interaction analysis between the antibody and antigen CalR31 (31-mer mutant CalR peptide). Residues were selected based on distances and interaction types determined by MOE (Molecular Operating Environment, 2019.01; Chemical Computing Group ULC, 1010 Sherbooke St. West, Suite #910, Montreal, QC Canada, H3A 2R7, 2021). Side chains of residues are shown in stick representation with numbering adopted from the CCG scheme in MOE. Figure 17C shows results from a detailed interaction analysis between CalR31 and the CDRs of the antibody. Distances between interacting pairs were calculated in MOE. Distances in angstroms are shown as dotted lines.

[0415] Example 13: Efficacy of anti-mutCALR antibodies in a mouse model of essential thrombocythemia To test the therapeutic potential of anti-mutCALR antibodies in vivo, the antibodies were evaluated in a mouse model of essential thrombocythemia (ET). In this mouse model, a conditional allele expressing a mutCALR protein with a C-terminal sequence (DEL52) identical to that found in MPN patients was knocked into the mouse CALR sequence. The engineered mice (CALRdel / del) develop an ET-like disease with marked thrombocytosis, splenomegaly, and abnormal megakaryocytosis (Li et.al.Blood 2018;131:649). Expression of mutCALR in the engineered mice was induced with intraperitoneal injections of polyinosinic:polycytidylic acid (Poly I:C) (250 μg / dose; every other day for a total of three injections). Treatment began 19 weeks after Poly I:C induction and consisted of intravenous injections of anti-mutCALR antibody (clone 74) at 50 mg / kg QW for a total of four weeks. The ET phenotype was confirmed by assessing platelet counts in blood (Sysmex, Kobe, Japan), spleen size and bone marrow histology. Representative studies are shown in Figures 18A-18C.

[0416] Anti-mutCALR antibodies restored normal platelet counts (Figure 18A), spleen volume (Figure 18B), and bone marrow cellular environment (Figure 18C).

[0417] Taken together, these results demonstrate the efficacy of anti-mutCALR antibodies in treating ET.

[0418] Example 14: Efficacy of anti-mutCALR antibodies in primary patient cells CD34 isolated from MPN patients with CALR mutations + Cells were used to characterize the ability of anti-mutCALR antibodies to inhibit mutCALR-derived oncogenic functions. Peripheral blood mononuclear cells (PBMCs) were isolated from de-identified blood samples from MPN patients by Ficoll gradient extraction (Fisher Scientific) and were cloned using CD34 + Cells were enriched using magnetic enrichment (Miltenyi Biotec). +Cells were cultured for 7 days in SFEM-II medium (STEMCELL Technologies) containing hSCF, hFLT3L, TPO, LDL2698, SR1, and UM171.

[0419] CD34+ cells (50,000 cells / well) were then seeded in 96-well plates and treated with mutCALR or isotype control antibodies for 2 h. After treatment, plates were centrifuged and the supernatant was aspirated, followed by washing with PBS. After centrifugation, cell pellets were lysed using lysis buffer (Cell Signaling Technologies) supplemented with 1×Halt™ protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Lysates were added to a phospho(Tyr694) / Total STAT5a,b whole cell lysate kit (Meso Scale Diagnostics), and phospho-STAT5 levels were then quantified using a Meso Sector S600 (Meso Scale Diagnostics). Clone 74 selectively inhibited pSTAT5 in CD34+ cells bearing mutCALR in a dose-dependent manner, whereas 10 μg / mL of isotype control (IgG) inhibited pSTAT5 in CD34+ cells bearing mutCALR. + Furthermore, pSTAT5 was not inhibited in CD34+ cells harboring the V617F JAK2 mutation (Figure 19A).

[0420] To evaluate the ability of the antibodies to inhibit the oncogenic function of mutCALR and the disproportionate proliferation of megakaryocytes, CD34 +Cells (50,000 cells / well) were added to 12-well plates containing SFEM-II supplemented with hSCF, hGCSF, hIL3, and hIL6 and treated with mutCALR antibody or isotype control for 6 days. Cells were stained and analyzed by flow cytometry (LSRFortessa™ X-20 analyzer, BD Biosciences). Antibodies used were: APC anti-human CD38 antibody (BioLegend), FITC anti-human lineage cocktail (BioLegend), PE / Cyanine 7 anti-human CD34 antibody (BioLegend), PE anti-human CD41 antibody (BioLegend), APC mouse anti-human CD42b antibody (BD Pharmingen). Megakaryocytes were stained with CD41 + CD42b + Anti-mutCALR antibodies (clones 74 and 65) inhibited mutCALR in a dose-dependent manner. + CD34 + The cells were selectively prevented from differentiating into mature megakaryocytes, whereas the isotype control (IgG) had no effect on this population. A representative experiment is shown in Figure 19B.

[0421] In a separate experiment, the CD34 +Cells were added at 50,000 cells / well to 12-well plates containing 2.0 mL of culture medium with the specified concentrations of clone 74 or isotype control. The treatment period was 12 h. After 12 h, cells were harvested, washed, and lysed using lysis buffer (Cell Signaling Technologies) supplemented with 1× Halt™ protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Protein samples (6 μg) were separated on pre-cast 4-12% TrisGlycine gels (Thermo Fisher Scientific) and transferred to nitrocellulose membranes using the iBlot 2 Dry Blotting System and iBlot™ 2 Transfer Stacks (Thermo Fisher Scientific). Nitrocellulose membranes were blocked with StartingBlock (Thermo Fisher Scientific) for 1 hour and probed with antibodies to detect pSTAT5 (Cell Signaling), STAT5 (Cell Signaling), pSTAT3 (Cell Signaling), STAT3 (Cell Signaling), and β-actin (Cell Signaling). Detection was performed using horseradish peroxidase (HRP)-conjugated secondary rabbit antibodies (Cell Signaling) and chemiluminescent HRP substrate (Thermo Scientific). Clone 74 selectively inhibited pSTAT3 and pSTAT5 in CD34+ cells bearing mutCALR in a dose-dependent manner, whereas 10 μg / mL of isotype control (IgG) inhibited CD34+ cells. + It had no effect on the cells (Figure 19C).

[0422] In a separate experiment, clone 74 and ruxolitinib were combined to treat CD34+ cells from MPN patients with human umbilical cord blood (WT cells) or mutCALR (mutCALR cells). + The effect of co-treatment on the megakaryogenesis produced by cells was determined. After 6 days in culture, 25 nM ruxolitinib alone did not significantly increase the number of WT or mutCALR-expressing CD34+ In contrast, 25 μg / mL of clone 74 alone did not affect the frequency of megakaryocytes produced by either of the mutCALR + CD34 + Selectively reduced megakaryocyte production from WT CD34 cells + Furthermore, the combination of 25 nM ruxolitinib and 25 μg / mL clone 74 did not reduce megakaryocyte production from mutCALR cells. + CD34 + This resulted in further depletion of pathogenic megakaryocytes produced by the cells (Figure 19D).

[0423] Taken together, these results demonstrate the ability of anti-mutCALR antibodies to inhibit mutCALR-derived oncogenic functions in MPN patient cells.

[0424] Example 15: Effect of anti-mutCALR antibodies on the cell cycle Engineered BaF3 cells (10,000 cells / well) were added to 12-well plates containing 2.0 mL of culture medium with serially diluted antibodies. The treatment period was 24 hours. After 22 hours of incubation, Ba / F3 cells were pulse-labeled with BrdU for 2 hours. After 24 hours, Ba / F3 cells were harvested, washed with PBS, and incubated with BD Cytofix / Cytoperm buffer for 20 minutes on ice. The cells were then washed with BD Perm / Wash buffer, and the cell pellet was resuspended in 100 μL of BD Cytoperm and Permeabilization stabilization buffer and incubated on ice for 10 minutes. The cells were then washed, resuspended in 100 μl of BD Cytofix / Cytoperm buffer, and incubated at room temperature for 5 minutes. The cells were washed, treated with 100 μL of a 300 μg / mL solution of DNase, incubated for 1 hour at room temperature, then washed and resuspended in 50 μL of BD Perm / Wash buffer containing anti-BrdU-APC antibody for 20 minutes at room temperature in the dark. After an additional wash, the cells were resuspended in PBS containing 2% FBS and 7-AAD and analyzed for cell cycle profile on an LSRFortessa™ X-20 analyzer (BD Biosciences). Clone 74 was screened to determine its effect on cell cycle using WT BaF3 cells or Ba / F3-TPOR / mutCALR type 1. Clone 74 selectively induced apoptosis in BaF3 cells carrying mutCALR in a dose-dependent manner, whereas the isotype control (IgG) had no effect on these cells. In contrast, Clone 74 did not affect the cell cycle profile of WT BaF3 cells (FIG. 20).

[0425] Example 16: Activity of anti-mutCALR antibodies The cell binding of various anti-mutCALR antibodies compared to clone 4 was evaluated using engineered BaF3 cells stably expressing human MPL and human mutCALR type 1. The antibodies used in this experiment were either commercially obtained (AB1: CAL2 antibody, Dianova) or synthesized from sequences found in the literature (AB2: anti-mutCALR antibody comprising the VH and VL regions of antibody B3 from WO2020175689 fused to a human constant region; AB3: anti-mutCALR antibody comprising the BJ095 VH and BJ097 VL regions from WO2019178362 fused to a human constant region; and AB4: anti-mutCALR antibody comprising the VH and VL regions of antibody 8B2-H6 from WO2016087514 fused to a human constant region).

[0426] array: AB2 heavy chain: QVQLQQSGAELVKPGSSVKISKASGYTFTRNFIHWIKQQPGNGLEWIGWIFPGDGDTEYNQKFNGKATLTADKSSSTAYMQLSSLTSEDSAVYFCARGNYNYEYFDYWGQGVM VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 341) AB2 light chain: DIQMTQSPASLSASLGETVSIECLASEDIYSYLAWYQQKPGKSPQLLIFAANRLQDGVPSRFSGSGSGTQFSLKISGMQPEDEGDYFCLQGSKFPYTFGPGTKLELNRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 342) AB3 heavy chain: QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYYIHWVRQAPGQGLEWIGYISAYNGASSYNQKFKGRATFTVDTSISTAYMELSRLRSDDTAVYYCASSMDYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 343) *AB3 light chain: DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYHCWQGTHFPYTFGQGTKLIEKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 344)

[0427] *There was an EI → IE transposition when synthesizing the VL sequence at the end of the variable region. Otherwise, the VL sequence is identical to the BJ097 VL region.

[0428] AB4 heavy chain: EVQLKQSGPELVKTGASVKISCKASGYSFTGYYYIHWVKQSHGKSLEWIGYISCYNGASSYNQKFKGKATFTVDTSSSTAYMQFNSLTSGDSAVYYCASSMDYWGQGTSVTVS SASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPNNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 345) AB4 light chain: DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYHCWQGTHFPYTFGGGTKLEIKRTVAAPSVFIFPPSDQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 346)

[0429] Approximately 200,000 cells / well were added to a 96-well plate and stained with the indicated concentrations of antibodies for 30 minutes on ice. Cells were then washed and stained with goat anti-human secondary conjugated to R-phycoerythrin (R-PE) (Jackson Immuno Research Laboratories) for 30 minutes on ice. Cells were then washed again and analyzed by flow cytometry. Geometric mean fluorescence intensity (GMFI) of cell binding was graphed using GraphPad Prism software (version 7.04). As shown in Figure 21A, anti-mutant CALR antibodies show differential levels of binding to cells expressing mutant CALR.

[0430] The ability of the five antibodies shown here to inhibit cell proliferation was tested. Inhibition of cell proliferation was assessed using engineered BaF3 cells expressing MPL / mutCALR type 1. Cells were plated at 5,000 cells per well in RPMI 1640 + 2% FBS, antibodies were added, incubated for 72 hours, followed by assessment of cell viability using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI) and Top Count (Perkin Elmer, Boston, MA) or Pherastar (BMG Labtech, Ortenberg, Germany) for luminescence quantification. As shown in Figure 21B, among the antibodies shown to bind to mutant CALR, only clone 4 is able to substantially inhibit cell proliferation. The data shown in Figures 21A and 21B were compiled from different experiments.

[0431] The ability of other antibodies of the present disclosure to inhibit cell proliferation compared to AB1, AB2, AB3 and AB4 was tested in the same manner as described above. The identified anti-mutCALR antibody clones listed below inhibited mutCALR-induced cancer cell proliferation in a dose-dependent manner, whereas AB1, AB2, AB3 and AB4 showed no functional activity. IC 50 The values ​​are shown in Table 16. [Table 16]

[0432] Other embodiments While the present invention has been described in conjunction with its detailed description, it is to be understood that the foregoing description is intended to be illustrative, and not limiting, of the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An antibody that binds to human variant calreticrin (CALR), (A) The antibody comprises a heavy chain variable region (VH) including VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) including VL CDR1, VL CDR2, and VL CDR3, where, The aforementioned VH CDR1 contains the amino acid sequence of SEQ ID NO: 1, The aforementioned VH CDR2 contains the amino acid sequence of SEQ ID NO: 7, The aforementioned VH CDR3 contains the amino acid sequence of SEQ ID NO: 18, The aforementioned VL CDR1 contains the amino acid sequence of SEQ ID NO: 30, The VL CDR2 contains the amino acid sequence of SEQ ID NO: 55, and The VL CDR3 contains the amino acid sequence of SEQ ID NO: 71, (B) The antibody comprises a heavy chain variable region (VH) including VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) including VL CDR1, VL CDR2, and VL CDR3, where, The aforementioned VH CDR1 contains the amino acid sequence of SEQ ID NO: 1, The VH CDR2 contains one of the amino acid sequences from sequence numbers 92 to 95, The aforementioned VH CDR3 contains the amino acid sequence of SEQ ID NO: 18, The aforementioned VL CDR1 contains the amino acid sequence of SEQ ID NO: 30, The VL CDR2 contains the amino acid sequence of SEQ ID NO: 55, and The VL CDR3 contains the amino acid sequence of SEQ ID NO: 71, or (C) The antibody comprises a heavy chain variable region (VH) including VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) including VL CDR1, VL CDR2, and VL CDR3, wherein, The aforementioned VH CDR1 contains the amino acid sequence of SEQ ID NO: 1, The VH CDR2 comprises one amino acid sequence from sequence number 7 or 92-95, The aforementioned VH CDR3 contains the amino acid sequence of SEQ ID NO: 18, The VL CDR1 comprises the amino acid sequence of SEQ ID NO: 28 or 30, The VL CDR2 contains the amino acid sequence of SEQ ID NO: 54 or 55, and The aforementioned VL CDR3 is an antibody containing the amino acid sequence of SEQ ID NO:

71.

2. The antibody according to claim 1, which is any of the following: i) The VH CDR1 contains the amino acid sequence of SEQ ID NO: 1, the VH CDR2 contains the amino acid sequence of SEQ ID NO: 7, the VH CDR3 contains the amino acid sequence of SEQ ID NO: 18, the VL CDR1 contains the amino acid sequence of SEQ ID NO: 30, the VL CDR2 contains the amino acid sequence of SEQ ID NO: 55, and the VL CDR3 contains the amino acid sequence of SEQ ID NO:

71. ii) The VH CDR1 contains the amino acid sequence of SEQ ID NO: 1, the VH CDR2 contains the amino acid sequence of SEQ ID NO: 92, the VH CDR3 contains the amino acid sequence of SEQ ID NO: 18, the VL CDR1 contains the amino acid sequence of SEQ ID NO: 30, the VL CDR2 contains the amino acid sequence of SEQ ID NO: 55, and the VL CDR3 contains the amino acid sequence of SEQ ID NO:

71. iii) The VH CDR1 contains the amino acid sequence of SEQ ID NO: 1, the VH CDR2 contains the amino acid sequence of SEQ ID NO: 93, the VH CDR3 contains the amino acid sequence of SEQ ID NO: 18, the VL CDR1 contains the amino acid sequence of SEQ ID NO: 30, the VL CDR2 contains the amino acid sequence of SEQ ID NO: 55, and the VL CDR3 contains the amino acid sequence of SEQ ID NO:

71. iv) The VH CDR1 contains the amino acid sequence of SEQ ID NO: 1, the VH CDR2 contains the amino acid sequence of SEQ ID NO: 94, the VH CDR3 contains the amino acid sequence of SEQ ID NO: 18, the VL CDR1 contains the amino acid sequence of SEQ ID NO: 30, the VL CDR2 contains the amino acid sequence of SEQ ID NO: 55, and the VL CDR3 contains the amino acid sequence of SEQ ID NO:

71. v) The VH CDR1 contains the amino acid sequence of SEQ ID NO: 1, the VH CDR2 contains the amino acid sequence of SEQ ID NO: 95, the VH CDR3 contains the amino acid sequence of SEQ ID NO: 18, the VL CDR1 contains the amino acid sequence of SEQ ID NO: 30, the VL CDR2 contains the amino acid sequence of SEQ ID NO: 55, and the VL CDR3 contains the amino acid sequence of SEQ ID NO:

71. vi) The VH CDR1 contains the amino acid sequence of SEQ ID NO: 1, the VH CDR2 contains the amino acid sequence of SEQ ID NO: 7, the VH CDR3 contains the amino acid sequence of SEQ ID NO: 18, the VL CDR1 contains the amino acid sequence of SEQ ID NO: 28, the VL CDR2 contains the amino acid sequence of SEQ ID NO: 54, and the VL CDR3 contains the amino acid sequence of SEQ ID NO:

71. vii) The VH CDR1 contains the amino acid sequence of SEQ ID NO: 1, the VH CDR2 contains the amino acid sequence of SEQ ID NO: 92, the VH CDR3 contains the amino acid sequence of SEQ ID NO: 18, the VL CDR1 contains the amino acid sequence of SEQ ID NO: 28, the VL CDR2 contains the amino acid sequence of SEQ ID NO: 54, and the VL CDR3 contains the amino acid sequence of SEQ ID NO:

71. viiii) The VH CDR1 contains the amino acid sequence of SEQ ID NO: 1, the VH CDR2 contains the amino acid sequence of SEQ ID NO: 93, the VH CDR3 contains the amino acid sequence of SEQ ID NO: 18, the VL CDR1 contains the amino acid sequence of SEQ ID NO: 28, the VL CDR2 contains the amino acid sequence of SEQ ID NO: 54, and the VL CDR3 contains the amino acid sequence of SEQ ID NO:

71. ix) The VH CDR1 contains the amino acid sequence of SEQ ID NO: 1, the VH CDR2 contains the amino acid sequence of SEQ ID NO: 94, the VH CDR3 contains the amino acid sequence of SEQ ID NO: 18, the VL CDR1 contains the amino acid sequence of SEQ ID NO: 28, the VL CDR2 contains the amino acid sequence of SEQ ID NO: 54, the VL CDR3 contains the amino acid sequence of SEQ ID NO: 71, or x) The VH CDR1 contains the amino acid sequence of SEQ ID NO: 1, the VH CDR2 contains the amino acid sequence of SEQ ID NO: 95, the VH CDR3 contains the amino acid sequence of SEQ ID NO: 18, the VL CDR1 contains the amino acid sequence of SEQ ID NO: 28, the VL CDR2 contains the amino acid sequence of SEQ ID NO: 54, and the VL CDR3 contains the amino acid sequence of SEQ ID NO:

71.

3. The VH contains any one of the amino acid sequences among sequence numbers 165, 170, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, and 208, or an amino acid sequence that is at least 90% identical to this, The antibody according to claim 1, wherein the VL comprises any one of the amino acid sequences of SEQ ID NOs. 268, 315, and 316, or an amino acid sequence that is at least 90% identical thereto.

4. The antibody according to claim 1, which is any of the following: 1) The VH contains the amino acid sequence of SEQ ID NO: 165, and the VL contains the amino acid sequence of SEQ ID NO:

268. 2) The VH contains the amino acid sequence of SEQ ID NO: 188, and the VL contains the amino acid sequence of SEQ ID NO:

268. 3) The VH contains the amino acid sequence of SEQ ID NO: 189, and the VL contains the amino acid sequence of SEQ ID NO:

268. 4) The VH contains the amino acid sequence of SEQ ID NO: 190, and the VL contains the amino acid sequence of SEQ ID NO:

268. 5) The VH contains the amino acid sequence of SEQ ID NO: 191, and the VL contains the amino acid sequence of SEQ ID NO:

268. 6) The VH contains the amino acid sequence of SEQ ID NO: 192, and the VL contains the amino acid sequence of SEQ ID NO:

268. 7) The VH comprises the amino acid sequence of SEQ ID NO: 193, and the VL comprises the amino acid sequence of SEQ ID NO:

268. 8) The VH comprises the amino acid sequence of SEQ ID NO: 194, and the VL comprises the amino acid sequence of SEQ ID NO:

268. 9) The VH comprises the amino acid sequence of SEQ ID NO: 195, and the VL comprises the amino acid sequence of SEQ ID NO:

268. 10) The VH comprises the amino acid sequence of SEQ ID NO: 196, and the VL comprises the amino acid sequence of SEQ ID NO:

268. 11) The VH comprises the amino acid sequence of SEQ ID NO: 165, and the VL comprises the amino acid sequence of SEQ ID NO:

315. 12) The VH comprises the amino acid sequence of SEQ ID NO: 188, and the VL comprises the amino acid sequence of SEQ ID NO:

315. 13) The VH comprises the amino acid sequence of SEQ ID NO: 189, and the VL comprises the amino acid sequence of SEQ ID NO:

315. 14) The VH comprises the amino acid sequence of SEQ ID NO: 190, and the VL comprises the amino acid sequence of SEQ ID NO:

315. 15) The VH comprises the amino acid sequence of SEQ ID NO: 191, and the VL comprises the amino acid sequence of SEQ ID NO:

315. 16) The VH comprises the amino acid sequence of SEQ ID NO: 192, and the VL comprises the amino acid sequence of SEQ ID NO:

315. 17) The VH comprises the amino acid sequence of SEQ ID NO: 193, and the VL comprises the amino acid sequence of SEQ ID NO:

315. 18) The VH comprises the amino acid sequence of SEQ ID NO: 194, and the VL comprises the amino acid sequence of SEQ ID NO:

315. 19) The VH comprises the amino acid sequence of SEQ ID NO: 195, and the VL comprises the amino acid sequence of SEQ ID NO:

315. 20) The VH comprises the amino acid sequence of SEQ ID NO: 196, and the VL comprises the amino acid sequence of SEQ ID NO:

315. 21) The VH comprises the amino acid sequence of SEQ ID NO: 165, and the VL comprises the amino acid sequence of SEQ ID NO:

316. 22) The VH comprises the amino acid sequence of SEQ ID NO: 188, and the VL comprises the amino acid sequence of SEQ ID NO:

316. 23) The VH comprises the amino acid sequence of SEQ ID NO: 189, and the VL comprises the amino acid sequence of SEQ ID NO:

316. 24) The VH comprises the amino acid sequence of SEQ ID NO: 190, and the VL comprises the amino acid sequence of SEQ ID NO:

316. 25) The VH comprises the amino acid sequence of SEQ ID NO: 191, and the VL comprises the amino acid sequence of SEQ ID NO:

316. 26) The VH comprises the amino acid sequence of SEQ ID NO: 192, and the VL comprises the amino acid sequence of SEQ ID NO:

316. 27) The VH comprises the amino acid sequence of SEQ ID NO: 193, and the VL comprises the amino acid sequence of SEQ ID NO:

316. 28) The VH comprises the amino acid sequence of SEQ ID NO: 194, and the VL comprises the amino acid sequence of SEQ ID NO:

316. 29) The VH comprises the amino acid sequence of SEQ ID NO: 195, and the VL comprises the amino acid sequence of SEQ ID NO:

316. 30) The VH comprises the amino acid sequence of SEQ ID NO: 196, and the VL comprises the amino acid sequence of SEQ ID NO:

316. 31) The VH comprises the amino acid sequence of SEQ ID NO: 197, and the VL comprises the amino acid sequence of SEQ ID NO:

268. 32) The VH comprises the amino acid sequence of SEQ ID NO: 198, and the VL comprises the amino acid sequence of SEQ ID NO:

268. 33) The VH comprises the amino acid sequence of SEQ ID NO: 199, and the VL comprises the amino acid sequence of SEQ ID NO:

268. 34) The VH comprises the amino acid sequence of SEQ ID NO: 200, and the VL comprises the amino acid sequence of SEQ ID NO:

268. 35) The VH comprises the amino acid sequence of SEQ ID NO: 201, and the VL comprises the amino acid sequence of SEQ ID NO:

268. 36) The VH comprises the amino acid sequence of SEQ ID NO: 197, and the VL comprises the amino acid sequence of SEQ ID NO:

315. 37) The VH comprises the amino acid sequence of SEQ ID NO: 198, and the VL comprises the amino acid sequence of SEQ ID NO:

315. 38) The VH comprises the amino acid sequence of SEQ ID NO: 199, and the VL comprises the amino acid sequence of SEQ ID NO:

315. 39) The VH comprises the amino acid sequence of SEQ ID NO: 200, and the VL comprises the amino acid sequence of SEQ ID NO:

315. 40) The VH comprises the amino acid sequence of SEQ ID NO: 201, and the VL comprises the amino acid sequence of SEQ ID NO:

315. 41) The VH comprises the amino acid sequence of SEQ ID NO: 197, and the VL comprises the amino acid sequence of SEQ ID NO:

316. 42) The VH comprises the amino acid sequence of SEQ ID NO: 198, and the VL comprises the amino acid sequence of SEQ ID NO:

316. 43) The VH comprises the amino acid sequence of SEQ ID NO: 199, and the VL comprises the amino acid sequence of SEQ ID NO:

316. 44) The VH comprises the amino acid sequence of SEQ ID NO: 200, and the VL comprises the amino acid sequence of SEQ ID NO:

316. 45) The VH comprises the amino acid sequence of SEQ ID NO: 201, and the VL comprises the amino acid sequence of SEQ ID NO:

316. 46) The VH comprises the amino acid sequence of SEQ ID NO: 170, and the VL comprises the amino acid sequence of SEQ ID NO:

268. 47) The VH comprises the amino acid sequence of SEQ ID NO: 202, and the VL comprises the amino acid sequence of SEQ ID NO:

268. 48) The VH comprises the amino acid sequence of SEQ ID NO: 203, and the VL comprises the amino acid sequence of SEQ ID NO:

268. 49) The VH comprises the amino acid sequence of SEQ ID NO: 204, and the VL comprises the amino acid sequence of SEQ ID NO:

268. 50) The VH comprises the amino acid sequence of SEQ ID NO: 205, and the VL comprises the amino acid sequence of SEQ ID NO:

268. 51) The VH comprises the amino acid sequence of SEQ ID NO: 206, and the VL comprises the amino acid sequence of SEQ ID NO:

268. 52) The VH comprises the amino acid sequence of SEQ ID NO: 207, and the VL comprises the amino acid sequence of SEQ ID NO:

268. 53) The VH comprises the amino acid sequence of SEQ ID NO: 208, and the VL comprises the amino acid sequence of SEQ ID NO:

268. 54) The VH comprises the amino acid sequence of SEQ ID NO: 170, and the VL comprises the amino acid sequence of SEQ ID NO:

315. 55) The VH comprises the amino acid sequence of SEQ ID NO: 202, and the VL comprises the amino acid sequence of SEQ ID NO:

315. 56) The VH comprises the amino acid sequence of SEQ ID NO: 203, and the VL comprises the amino acid sequence of SEQ ID NO:

315. 57) The VH comprises the amino acid sequence of SEQ ID NO: 204, and the VL comprises the amino acid sequence of SEQ ID NO:

315. 58) The VH comprises the amino acid sequence of SEQ ID NO: 205, and the VL comprises the amino acid sequence of SEQ ID NO:

315. 59) The VH comprises the amino acid sequence of SEQ ID NO: 206, and the VL comprises the amino acid sequence of SEQ ID NO:

315. 60) The VH comprises the amino acid sequence of SEQ ID NO: 207, and the VL comprises the amino acid sequence of SEQ ID NO:

315. 61) The VH comprises the amino acid sequence of SEQ ID NO: 208, and the VL comprises the amino acid sequence of SEQ ID NO:

315. 62) The VH comprises the amino acid sequence of SEQ ID NO: 170, and the VL comprises the amino acid sequence of SEQ ID NO:

316. 63) The VH comprises the amino acid sequence of SEQ ID NO: 202, and the VL comprises the amino acid sequence of SEQ ID NO:

316. 64) The VH comprises the amino acid sequence of SEQ ID NO: 203, and the VL comprises the amino acid sequence of SEQ ID NO:

316. 65) The VH comprises the amino acid sequence of SEQ ID NO: 204, and the VL comprises the amino acid sequence of SEQ ID NO:

316. 66) The VH comprises the amino acid sequence of SEQ ID NO: 205, and the VL comprises the amino acid sequence of SEQ ID NO:

316. 67) The VH comprises the amino acid sequence of SEQ ID NO: 206, and the VL comprises the amino acid sequence of SEQ ID NO:

316. 68) The VH comprises the amino acid sequence of SEQ ID NO: 207, and the VL comprises the amino acid sequence of SEQ ID NO: 316, or 69) The VH comprises the amino acid sequence of SEQ ID NO: 208, and the VL comprises the amino acid sequence of SEQ ID NO:

316.

5. The antibody according to claim 1, wherein VH comprises the amino acid sequence of SEQ ID NO: 165, and VL comprises the amino acid sequence of SEQ ID NO:

268.

6. The antibody according to claim 1, wherein VH comprises the amino acid sequence of SEQ ID NO: 165, and VL comprises the amino acid sequence of SEQ ID NO:

315.

7. The antibody according to claim 1, wherein VH comprises the amino acid sequence of SEQ ID NO: 190, and VL comprises the amino acid sequence of SEQ ID NO:

315.

8. The antibody according to claim 1, wherein VH comprises the amino acid sequence of SEQ ID NO: 196, and VL comprises the amino acid sequence of SEQ ID NO:

315.

9. The aforementioned antibody A heavy chain containing one of the amino acid sequences from sequence numbers 119, 142-164, The antibody according to claim 1, comprising a light chain having any one of the amino acid sequences of SEQ ID NOs: 213, 260, and 261.

10. The antibody according to claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 119, and the light chain comprises the amino acid sequence of SEQ ID NO:

213.

11. The antibody according to claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 142, and the light chain comprises the amino acid sequence of SEQ ID NO:

213.

12. The antibody according to claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 142, and the light chain comprises the amino acid sequence of SEQ ID NO:

260.

13. The antibody according to claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 145, and the light chain comprises the amino acid sequence of SEQ ID NO:

260.

14. The antibody according to claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 151, and the light chain comprises the amino acid sequence of SEQ ID NO:

260.

15. The antibody according to claim 1, wherein the human variant CALR is a human type 1 variant CALR containing the amino acid sequence of SEQ ID NO: 320, or a human type 2 variant CALR containing the amino acid sequence of SEQ ID NO:

321.

16. The aforementioned antibody i. Human antibodies or humanized antibodies, ii. It is a full-length antibody, and / or iii. An IgG1, IgG2, IgG3, or IgG4 antibody, wherein the antibody is optionally an IgG1 antibody. The antibody according to claim 1.

17. The aforementioned antibodies include bispecific antibodies, biparatopic antibodies, single-chain antibodies (scFv), Fab fragments, and F(ab'). ２ Fragment, Fab' fragment, Fsc fragment, Fv fragment, scFv, sc(Fv) ２ , or a diabody, and optionally the antibody is i. A biparatopic antibody containing two heavy-chain-light chain pairs or one heavy-chain-light chain pair, and / or ii. A double paratopic antibody that is a full-length antibody, The antibody according to claim 1.

18. An antibody according to claim 1, which is conjugated with a toxic substance, wherein the antibody is optionally conjugated with a radioisotope or a cytotoxic agent.

19. A nucleic acid or set of nucleic acids that collectively encodes the antibody described in claim 1.

20. An expression vector or set of expression vectors comprising the nucleic acid or set of nucleic acids according to claim 19, operably linked to a promoter.

21. Isolated cells comprising the nucleic acid or set of nucleic acids described in claim 19, or the expression vector or set of expression vectors described in claim 20.

22. A method for producing an antibody according to any one of claims 1 to 17, comprising culturing isolated cells containing a nucleic acid or set of nucleic acids according to claim 19, or an expression vector or set of expression vectors according to claim 20, and isolating the antibody.

23. (a) A method for detecting CALR exon 9 mutations in a biological sample, or (b) A method for diagnosing a human subject having a myeloproliferative neoplasm, The method comprising contacting a biological sample from a human subject having or suspected of having a myeloproliferative neoplasm with an antibody according to any one of claims 1 to 18, thereby causing the antibody to bind to the mutCALR protein if the mutCALR protein is present in the biological sample.