Anti-ADGRE2 antibodies and uses thereof

Abstract

Described herein are antibodies, fragments thereof, and fusion proteins that specifically bind to ADGRE2, as well as methods of making and using such antibodies. Such antibodies, fusion proteins, and fragments thereof are useful in the treatment and diagnosis of various autoimmune diseases and cancers, including, for example, acute myeloid leukemia.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 179,756, filed April 26, 2021, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on April 19, 2022, is named MIL-018WO_SL.txt and is 38,304 bytes in size. [Background technology]

[0003] Anti-ADGRE2 antibodies bind to the adhesion G protein-coupled receptor E2, or ADGRE2 antigen, also known as EMR2, CD312, VBU, or CD97. ADGRE2 is expressed on monocytes, macrophages, dendritic cells, and granulocytes. ADGRE2 is expressed on cancer cells in cancers, including acute myeloid leukemia. ADGRE2 binds to the chondroitin sulfate moiety of glycosaminoglycan chains and promotes cell adhesion. ADGRE2 is highly expressed in cancerous cells, including acute myeloid leukemia cells. Summary of the Invention

[0004] The present invention provides humanized anti-ADGRE2 antibodies and antigen-binding fragments thereof for the treatment of autoimmune diseases and cancers, including leukemia, lymphoma, or myeloma, including, for example, relapsed and refractory acute myeloid leukemia. The antibodies and fragments thereof of the present invention can be used alone, in fusion proteins, or conjugated to at least one diagnostic and / or therapeutic agent, or in combination with other therapies.

[0005] In some embodiments, provided herein are humanized anti-ADGRE2 antibodies or antigen-binding fragments thereof, including: (a) an immunoglobulin heavy chain variable (V) fragment comprising an amino acid sequence that is at least about 80% identical to QVQLQQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 7); H ) area, and An immunoglobulin light chain variable (V) molecule comprising an amino acid sequence that is at least about 80% identical to EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGLAPRLLIYDTSKLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSSNPLTFGQGTKVEIK (SEQ ID NO: 8). L )region; (b) an immunoglobulin heavy chain variable (V) fragment comprising an amino acid sequence at least about 80% identical to QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSISTAYMEVSRLRSDDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 9); H ) area, and An immunoglobulin light chain variable (V) molecule comprising an amino acid sequence that is at least about 80% identical to EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 10). L )region; (c) an immunoglobulin heavy chain variable (V) fragment comprising an amino acid sequence at least about 80% identical to QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTVYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 11); H ) area, and An immunoglobulin light chain variable (V) molecule comprising an amino acid sequence that is at least about 80% identical to EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 12). L ) area; or (d) an immunoglobulin heavy chain variable (V) fragment comprising an amino acid sequence at least about 80% identical to QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWMGAVYPGDGDTRHTQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSS (SEQ ID NO: 13); H ) area, and An immunoglobulin light chain variable (V) molecule comprising an amino acid sequence that is at least about 80% identical to EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGQSPKRWIYDTSKLASGVPARFSGSGSGTDYTFTISSMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 14). L )region; (e) an immunoglobulin heavy chain variable (V) fragment comprising an amino acid sequence at least about 80% identical to QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRHTQKFKGRVTMTADKSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSS (SEQ ID NO: 15); H ) area, and An immunoglobulin light chain variable (V) molecule comprising an amino acid sequence that is at least about 80% identical to QIVLTQSPATLSLSPGERATLTCSASSSVSYMHWYQQKPGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTIRRLEPEDFATYYCQQWSSNPLTFGQGTKVEIK (SEQ ID NO: 16). L )region; (f) an immunoglobulin heavy chain variable (V) polypeptide comprising an amino acid sequence at least about 80% identical to QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 17); H ) area, and An immunoglobulin light chain variable (V) molecule comprising an amino acid sequence that is at least about 80% identical to EIVLTQSPATLSASPGERVTMSCSASSSVSYMHWYQQKPGLAPRRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 18). L )region; (g) an immunoglobulin heavy chain variable (V) fragment comprising an amino acid sequence at least about 80% identical to QVQLVQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 30); H ) area, and An immunoglobulin light chain variable (V) molecule comprising an amino acid sequence at least about 80% identical to EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGQSPKRWIYDTSKLASGVPARFSGSGSGTDYTFTISSMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 31). L )region.

[0006] In some embodiments, provided herein is a humanized anti-ADGRE2 antibody or antigen-binding fragment thereof, comprising: (a) an immunoglobulin heavy chain variable (V) fragment comprising the amino acid sequence of QVQLQQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 7);H ) area, and An immunoglobulin light chain variable (V) antibody comprising the amino acid sequence of EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGLAPRLLIYDTSKLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSSNPLTFGQGTKVEIK (SEQ ID NO: 8) L )region; (b) an immunoglobulin heavy chain variable (V) fragment comprising the amino acid sequence of QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSISTAYMEVSRLRSDDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 9); H ) area, and An immunoglobulin light chain variable (V) antibody comprising the amino acid sequence of EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 10) L )region; (c) an immunoglobulin heavy chain variable (V) fragment comprising the amino acid sequence of QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTVYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 11); H ) area, and An immunoglobulin light chain variable (V) antibody comprising the amino acid sequence of EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 12) L ) area; or (d) an immunoglobulin heavy chain variable (V) fragment comprising the amino acid sequence of QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWMGAVYPGDGDTRHTQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSS (SEQ ID NO: 13); H ) area, and An immunoglobulin light chain variable (V) antibody comprising the amino acid sequence of EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGQSPKRWIYDTSKLASGVPARFSGSGSGTDYTFTISSMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 14) L )region; (e) an immunoglobulin heavy chain variable (V) fragment comprising the amino acid sequence of QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRHTQKFKGRVTMTADKSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSS (SEQ ID NO: 15); H ) area, and An immunoglobulin light chain variable (V) antibody comprising the amino acid sequence of QIVLTQSPATLSLSPGERATLTCSASSSVSYMHWYQQKPGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTIRRLEPEDFATYYCQQWSSNPLTFGQGTKVEIK (SEQ ID NO: 16) L )region; (f) an immunoglobulin heavy chain variable (V) polypeptide comprising the amino acid sequence of QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 17); H ) area, and An immunoglobulin light chain variable (V) antibody comprising the amino acid sequence of EIVLTQSPATLSASPGERVTMSCSASSSVSYMHWYQQKPGLAPRRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 18) L )region; (g) an immunoglobulin heavy chain variable (V) fragment comprising the amino acid sequence of QVQLVQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 30); H ) area, and An immunoglobulin light chain variable (V) antibody comprising the amino acid sequence of: EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGQSPKRWIYDTSKLASGVPARFSGSGSGTDYTFTISSMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 31). L )region.

[0007] In some embodiments, provided herein is a humanized anti-ADGRE2 antibody or antigen-binding fragment thereof, comprising: a heavy chain complementarity determining region (HCDR) 1 comprising the amino acid sequence of GYTFTNYW (SEQ ID NO: 1), an HCDR2 comprising the amino acid sequence of VYPGDGDT (SEQ ID NO: 2), and an HCDR3 comprising the amino acid sequence of ARGFTAYGMDY (SEQ ID NO: 3); and Light chain complementarity determining region (LCDR) 1 comprising the amino acid sequence of SSVSY (SEQ ID NO: 4), LCDR2 comprising the amino acid sequence of DTS (SEQ ID NO: 5), and LCDR3 comprising the amino acid sequence of QQWSSNPLT (SEQ ID NO: 6).

[0008] In some embodiments, provided herein is a humanized anti-ADGRE2 antibody or antigen-binding fragment thereof, wherein the anti-ADGRE2 antibody or fragment thereof is selected from the group consisting of an IgA antibody, an IgG antibody, an IgE antibody, an IgM antibody, a bispecific or multispecific antibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fd' fragment, an Fd fragment, an isolated CDR or set thereof; a single-chain variable fragment (scFv), a polypeptide-Fc fusion, a single domain antibody, a camelid antibody, a masked antibody, a small modular immunopharmaceutical ("SMIPs™"), a single chain, a tandem diabody, a VHH, anticalin, a nanobody, a minibody, a BiTE, an ankyrin repeat protein, a DARPIN, an Avimer, a DART, a TCR-like antibody, an adnectin, an affilin, a transbody, an affibody, a TrimerX, a MicroProtein, a Fynomer, a Centyrin, and a KALBITOR.

[0009] In some embodiments, the humanized anti-ADGRE2 antibody or antigen-binding fragment thereof is a monoclonal antibody or a single-chain variable fragment (scFv).

[0010] In some embodiments, the humanized anti-ADGRE2 antibody or fragment thereof is an antibody comprising an IgG constant region.

[0011] In some embodiments, the humanized anti-ADGRE2 antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv).

[0012] In some embodiments, the scFv comprises a signal sequence, a heavy chain variable sequence, a GS linker, and a light chain variable sequence.

[0013] In some embodiments, the scFv comprises a sequence having at least about 80% identity to: (a) QVQLQQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGQSPKRWIYDTSKLASGVPARFSGSGSGTDYTFTISSMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 19); (b) QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSISTAYMEVSRLRSDDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 20); (c) QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTVYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 21); or (d)QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWMGAVYPGDGDTRHTQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSSASTGGG GSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGLAPRLLIYDTSKLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSSNPLTFGQGTKVEIK (SEQ ID NO: 22).

[0014] In some embodiments, the single chain variable fragment (scFv) comprises the following sequence: QVQLQQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGQSPKRWIYDTSKLASGVPARFSGSGSGTDYTFTISSMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 19); QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSISTAYMEVSRLRSDDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 20); QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTVYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 21); or QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWMGAVYPGDGDTRHTQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSSASTGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGLAPRLLIYDTSKLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSSNPLTFGQGTKVEIK (SEQ ID NO: 22).

[0015] In some embodiments, the humanized anti-ADGRE2 antibody is about 10 -8 K less than M D , about 10 -9 K less than M D , about 10 -10 K less than M D , about 10 -11 K less than M D , about 10 -12 K less than M D , or about 10 -13 K less than M D The dissociation constant (K D )

[0016] In some embodiments, the humanized anti-ADGRE2 antibody has an EC50 of about 1-100 nM.

[0017] In some embodiments, the EC50 is about 10-95 nM.

[0018] In some embodiments, the EC50 is about 25-75 nM.

[0019] In some embodiments, provided herein are methods of treating cancer comprising administering a humanized anti-ADGRE2 antibody or antigen-binding fragment thereof.

[0020] In some embodiments, the cancer is selected from leukemia, lymphoma, and myeloma.

[0021] In some embodiments, a pharmaceutical composition comprising a humanized anti-ADGRE2 antibody or fragment thereof and a pharmaceutically acceptable carrier, wherein the humanized anti-ADGRE2 antibody or antibody fragment thereof comprises: (a) an immunoglobulin heavy chain variable (V) fragment comprising an amino acid sequence that is at least about 80% identical to QVQLQQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 7); H ) area, and An immunoglobulin light chain variable (V) molecule comprising an amino acid sequence that is at least about 80% identical to EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGLAPRLLIYDTSKLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSSNPLTFGQGTKVEIK (SEQ ID NO: 8). L )region; (b) an immunoglobulin heavy chain variable (V) fragment comprising an amino acid sequence at least about 80% identical to QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSISTAYMEVSRLRSDDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 9); H ) area, and An immunoglobulin light chain variable (V) molecule comprising an amino acid sequence that is at least about 80% identical to EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 10). L )region; (c) an immunoglobulin heavy chain variable (V) fragment comprising an amino acid sequence at least about 80% identical to QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTVYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 11); H ) area, and An immunoglobulin light chain variable (V) molecule comprising an amino acid sequence that is at least about 80% identical to EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 12). L )region; (d) an immunoglobulin heavy chain variable (V) fragment comprising an amino acid sequence at least about 80% identical to QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWMGAVYPGDGDTRHTQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSS (SEQ ID NO: 13); H ) area, and An immunoglobulin light chain variable (V) molecule comprising an amino acid sequence that is at least about 80% identical to EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGQSPKRWIYDTSKLASGVPARFSGSGSGTDYTFTISSMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 14). L )region; (e) an immunoglobulin heavy chain variable (V) fragment comprising an amino acid sequence at least about 80% identical to QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRHTQKFKGRVTMTADKSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSS (SEQ ID NO: 15); H ) area, and An immunoglobulin light chain variable (V) molecule comprising an amino acid sequence that is at least about 80% identical to QIVLTQSPATLSLSPGERATLTCSASSSVSYMHWYQQKPGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTIRRLEPEDFATYYCQQWSSNPLTFGQGTKVEIK (SEQ ID NO: 16). L ) area; or (f) an immunoglobulin heavy chain variable (V) polypeptide comprising an amino acid sequence at least about 80% identical to QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 17); H ) area, and An immunoglobulin light chain variable (V) molecule comprising an amino acid sequence that is at least about 80% identical to EIVLTQSPATLSASPGERVTMSCSASSSVSYMHWYQQKPGLAPRRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 18). L ) area; or (g) an immunoglobulin heavy chain variable (V) fragment comprising an amino acid sequence at least about 80% identical to QVQLVQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 30); H ) area, and An immunoglobulin light chain variable (V) molecule comprising an amino acid sequence at least about 80% identical to EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGQSPKRWIYDTSKLASGVPARFSGSGSGTDYTFTISSMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 31). L )region.

[0022] In some embodiments, the pharmaceutical composition comprises a humanized anti-ADGRE2 antibody or antigen-binding fragment, wherein the humanized anti-ADGRE2 antibody or antigen-binding fragment comprises: (a) an immunoglobulin heavy chain variable (V) fragment comprising the amino acid sequence of QVQLQQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 7); H ) area, and An immunoglobulin light chain variable (V) antibody comprising the amino acid sequence of EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGLAPRLLIYDTSKLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSSNPLTFGQGTKVEIK (SEQ ID NO: 8) L )region; (b) an immunoglobulin heavy chain variable (V) fragment comprising the amino acid sequence of QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSISTAYMEVSRLRSDDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 9); H ) area, and An immunoglobulin light chain variable (V) antibody comprising the amino acid sequence of EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 10) L )region; (c) an immunoglobulin heavy chain variable (V) fragment comprising the amino acid sequence of QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTVYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 11); H ) area, and An immunoglobulin light chain variable (V) antibody comprising the amino acid sequence of EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 12) L )region; (d) an immunoglobulin heavy chain variable (V) fragment comprising the amino acid sequence of QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWMGAVYPGDGDTRHTQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSS (SEQ ID NO: 13); H ) area, and An immunoglobulin light chain variable (V) antibody comprising the amino acid sequence of EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGQSPKRWIYDTSKLASGVPARFSGSGSGTDYTFTISSMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 14) L )region; (e) an immunoglobulin heavy chain variable (V) fragment comprising the amino acid sequence of QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRHTQKFKGRVTMTADKSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSS (SEQ ID NO: 15); H ) area, and An immunoglobulin light chain variable (V) antibody comprising the amino acid sequence of QIVLTQSPATLSLSPGERATLTCSASSSVSYMHWYQQKPGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTIRRLEPEDFATYYCQQWSSNPLTFGQGTKVEIK (SEQ ID NO: 16) L ) area; or (f) an immunoglobulin heavy chain variable (V) polypeptide comprising the amino acid sequence of QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 17); H ) area, and An immunoglobulin light chain variable (V) antibody comprising the amino acid sequence of EIVLTQSPATLSASPGERVTMSCSASSSVSYMHWYQQKPGLAPRRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 18) L ) area; or (g) an immunoglobulin heavy chain variable (V) fragment comprising an amino acid sequence at least about 80% identical to QVQLVQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 30); H ) area, and An immunoglobulin light chain variable (V) molecule comprising an amino acid sequence at least about 80% identical to EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGQSPKRWIYDTSKLASGVPARFSGSGSGTDYTFTISSMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 31). L )region.

[0023] In some embodiments, the pharmaceutical composition comprises a humanized anti-ADGRE2 antibody or antibody fragment thereof, comprising: a heavy chain complementarity determining region (HCDR) 1 comprising the amino acid sequence of GYTFTNYW (SEQ ID NO: 1), an HCDR2 comprising the amino acid sequence of VYPGDGDT (SEQ ID NO: 2), and an HCDR3 comprising the amino acid sequence of ARGFTAYGMDY (SEQ ID NO: 3); and Light chain complementarity determining region (LCDR) 1 comprising the amino acid sequence of SSVSY (SEQ ID NO: 4), LCDR2 comprising the amino acid sequence of DTS (SEQ ID NO: 5), and LCDR3 comprising the amino acid sequence of QQWSSNPLT (SEQ ID NO: 6).

[0024] In some embodiments, the pharmaceutical composition comprises a humanized anti-ADGRE2 antibody or antigen-binding fragment thereof, wherein the humanized anti-ADGRE2 antibody or antigen-binding fragment thereof is selected from the group consisting of an IgA antibody, an IgG antibody, an IgE antibody, an IgM antibody, a bispecific or multispecific antibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fd' fragment, an Fd fragment, an isolated CDR or a set thereof; a single-chain variable fragment (scFv), a polypeptide-Fc fusion, a single-domain antibody, a camelid antibody, a masked antibody, a small modular immunopharmaceutical ("SMIPs™"), a single chain, a tandem diabody, a VHH, anticalin, a nanobody, a minibody, a BiTE, an ankyrin repeat protein, a DARPIN, an Avimer, a DART, a TCR-like antibody, an adnectin, an affilin, a transbody, an affibody, TrimerX, a MicroProtein, a Fynomer, a Centyrin, and a KALBITOR.

[0025] In some embodiments, the pharmaceutical composition comprises a humanized anti-ADGRE2 antibody or antigen-binding fragment thereof, wherein the anti-ADGRE2 antibody or fragment thereof is a monoclonal antibody or a single chain variable fragment (scFv).

[0026] In some embodiments, the humanized anti-ADGRE2 antibody or antigen-binding fragment thereof is an antibody comprising an IgG constant region.

[0027] In some embodiments, the humanized anti-ADGRE2 antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv).

[0028] In some embodiments, the scFv comprises a signal sequence, a heavy chain variable sequence, a GS linker, and a light chain variable sequence.

[0029] In some embodiments, the scFv comprises a sequence having at least about 80% identity to: (a) QVQLQQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGQSPKRWIYDTSKLASGVPARFSGSGSGTDYTFTISSMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 19); (b) QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSISTAYMEVSRLRSDDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 20); (c) QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTVYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 21); or (d)QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWMGAVYPGDGDTRHTQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSSASTGGG GSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGLAPRLLIYDTSKLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSSNPLTFGQGTKVEIK (SEQ ID NO: 22).

[0030] In some embodiments, the single chain variable fragment (scFv) comprises the following sequence: (a) QVQLQQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGQSPKRWIYDTSKLASGVPARFSGSGSGTDYTFTISSMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 19); (b) QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSISTAYMEVSRLRSDDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 20); (c) QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTVYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 21); or (d)QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWMGAVYPGDGDTRHTQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSSASTGGG GSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGLAPRLLIYDTSKLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSSNPLTFGQGTKVEIK (SEQ ID NO: 22).

[0031] In some embodiments, provided herein are nucleic acid sequences that encode an amino acid sequence that is at least about 90% identical to any one of SEQ ID NOs: 7-18.

[0032] In some embodiments, provided herein are vectors comprising the nucleic acid sequences.

[0033] In some embodiments, provided herein are cells comprising the vectors.

[0034] In some embodiments, provided herein are methods of treating cancer, the methods comprising administering to a subject in need thereof a humanized anti-ADGRE2 antibody or antigen-binding fragment thereof, wherein the humanized anti-ADGRE2 antibody comprises: (a) an immunoglobulin heavy chain variable (V) fragment comprising an amino acid sequence that is at least about 80% identical to QVQLQQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 7); H ) area, and An immunoglobulin light chain variable (V) molecule comprising an amino acid sequence that is at least about 80% identical to EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGLAPRLLIYDTSKLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSSNPLTFGQGTKVEIK (SEQ ID NO: 8). L )region; (b) an immunoglobulin heavy chain variable (V) fragment comprising an amino acid sequence at least about 80% identical to QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSISTAYMEVSRLRSDDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 9); H ) area, and An immunoglobulin light chain variable (V) molecule comprising an amino acid sequence that is at least about 80% identical to EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 10). L )region; (c) an immunoglobulin heavy chain variable (V) fragment comprising an amino acid sequence at least about 80% identical to QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTVYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 11); H ) area, and An immunoglobulin light chain variable (V) molecule comprising an amino acid sequence that is at least about 80% identical to EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 12). L )region; (d) an immunoglobulin heavy chain variable (V) fragment comprising an amino acid sequence at least about 80% identical to QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWMGAVYPGDGDTRHTQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSS (SEQ ID NO: 13); H ) area, and An immunoglobulin light chain variable (V) molecule comprising an amino acid sequence that is at least about 80% identical to EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGQSPKRWIYDTSKLASGVPARFSGSGSGTDYTFTISSMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 14). L )region; (e) an immunoglobulin heavy chain variable (V) fragment comprising an amino acid sequence at least about 80% identical to QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRHTQKFKGRVTMTADKSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSS (SEQ ID NO: 15); H ) area, and An immunoglobulin light chain variable (V) molecule comprising an amino acid sequence that is at least about 80% identical to QIVLTQSPATLSLSPGERATLTCSASSSVSYMHWYQQKPGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTIRRLEPEDFATYYCQQWSSNPLTFGQGTKVEIK (SEQ ID NO: 16). L ) area; or (f) an immunoglobulin heavy chain variable (V) polypeptide comprising an amino acid sequence at least about 80% identical to QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 17); H ) area, and An immunoglobulin light chain variable (V) molecule comprising an amino acid sequence that is at least about 80% identical to EIVLTQSPATLSASPGERVTMSCSASSSVSYMHWYQQKPGLAPRRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 18). L ) area; or (g) an immunoglobulin heavy chain variable (V) fragment comprising an amino acid sequence at least about 80% identical to QVQLVQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 30); H ) area, and An immunoglobulin light chain variable (V) molecule comprising an amino acid sequence at least about 80% identical to EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGQSPKRWIYDTSKLASGVPARFSGSGSGTDYTFTISSMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 31). L )region.

[0035] In some embodiments, provided herein are methods of treating cancer, the methods comprising administering to a subject in need thereof a humanized anti-ADGRE2 antibody or antigen-binding fragment thereof, wherein the humanized anti-ADGRE2 antibody comprises: (a) an immunoglobulin heavy chain variable (V) fragment comprising the amino acid sequence of QVQLQQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 7); H ) area, and An immunoglobulin light chain variable (V) antibody comprising the amino acid sequence of EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGLAPRLLIYDTSKLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSSNPLTFGQGTKVEIK (SEQ ID NO: 8) L )region; (b) an immunoglobulin heavy chain variable (V) fragment comprising the amino acid sequence of QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSISTAYMEVSRLRSDDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 9); H ) area, and An immunoglobulin light chain variable (V) antibody comprising the amino acid sequence of EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 10) L )region; (c) an immunoglobulin heavy chain variable (V) fragment comprising the amino acid sequence of QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTVYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 11); H ) area, and An immunoglobulin light chain variable (V) antibody comprising the amino acid sequence of EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 12) L )region; (d) an immunoglobulin heavy chain variable (V) fragment comprising the amino acid sequence of QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWMGAVYPGDGDTRHTQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSS (SEQ ID NO: 13); H ) area, and An immunoglobulin light chain variable (V) antibody comprising the amino acid sequence of EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGQSPKRWIYDTSKLASGVPARFSGSGSGTDYTFTISSMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 14) L )region; (g) an immunoglobulin heavy chain variable (V) fragment comprising the amino acid sequence of QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRHTQKFKGRVTMTADKSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSS (SEQ ID NO: 15); H ) area, and An immunoglobulin light chain variable (V) antibody comprising the amino acid sequence of QIVLTQSPATLSLSPGERATLTCSASSSVSYMHWYQQKPGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTIRRLEPEDFATYYCQQWSSNPLTFGQGTKVEIK (SEQ ID NO: 16) L ) area; or (h) an immunoglobulin heavy chain variable (V) fragment comprising the amino acid sequence of QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 17); H ) area, and An immunoglobulin light chain variable (V) antibody comprising the amino acid sequence of EIVLTQSPATLSASPGERVTMSCSASSSVSYMHWYQQKPGLAPRRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 18) L ) area; or (g) an immunoglobulin heavy chain variable (V) fragment comprising the amino acid sequence of QVQLVQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 30); H ) area, and An immunoglobulin light chain variable (V) antibody comprising the amino acid sequence of: EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGQSPKRWIYDTSKLASGVPARFSGSGSGTDYTFTISSMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 31). L )region.

[0036] In some embodiments, provided herein are methods of treating cancer, the methods comprising administering to a subject in need thereof a humanized anti-ADGRE2 antibody or antigen-binding fragment thereof, wherein the humanized anti-ADGRE2 antibody comprises: a heavy chain complementarity determining region (HCDR) 1 comprising the amino acid sequence of GYTFTNYW (SEQ ID NO: 1), an HCDR2 comprising the amino acid sequence of VYPGDGDT (SEQ ID NO: 2), and an HCDR3 comprising the amino acid sequence of ARGFTAYGMDY (SEQ ID NO: 3); and Light chain complementarity determining region (LCDR) 1 comprising the amino acid sequence of SSVSY (SEQ ID NO: 4), LCDR2 comprising the amino acid sequence of DTS (SEQ ID NO: 5), and LCDR3 comprising the amino acid sequence of QQWSSNPLT (SEQ ID NO: 6).

[0037] In some embodiments, provided herein are methods for treating cancer, the methods comprising administering to a subject in need thereof a humanized anti-ADGRE2 antibody or antigen-binding fragment thereof, wherein the humanized anti-ADGRE2 antibody or antigen-binding fragment thereof is a monoclonal antibody or a single-chain variable fragment (scFv).

[0038] In some embodiments, provided herein are methods for treating cancer, the methods comprising administering to a subject in need thereof a humanized anti-ADGRE2 antibody or antigen-binding fragment thereof, wherein the humanized anti-ADGRE2 antibody or fragment thereof is an antibody comprising an IgG constant region.

[0039] In some embodiments, the anti-humanized ADGRE2 antibody or fragment thereof is a single chain variable fragment (scFv).

[0040] In some embodiments, the scFv comprises a leader sequence, a heavy chain variable sequence, a GS linker, and a light chain variable sequence.

[0041] In some embodiments, the scFv comprises a sequence having at least about 80% identity to: (a) QVQLQQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGQSPKRWIYDTSKLASGVPARFSGSGSGTDYTFTISSMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 19); (b) QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSISTAYMEVSRLRSDDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 20); (c) QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTVYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 21); or (d)QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWMGAVYPGDGDTRHTQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSSASTGGG GSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGLAPRLLIYDTSKLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSSNPLTFGQGTKVEIK (SEQ ID NO: 22).

[0042] In some embodiments, the single chain variable fragment (scFv) comprises the following sequence: QVQLQQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGQSPKRWIYDTSKLASGVPARFSGSGSGTDYTFTISSMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 19); QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSISTAYMEVSRLRSDDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 20); QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTVYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 21); or QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWMGAVYPGDGDTRHTQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSSASTGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGLAPRLLIYDTSKLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSSNPLTFGQGTKVEIK (SEQ ID NO: 22).

[0043] In some embodiments, the cancer is selected from leukemia, lymphoma, or myeloma.

[0044] In some embodiments, the treatment comprises the administration of one or more additional agents.

[0045] In some embodiments, the one or more additional agents are selected from an antibody, a chemotherapeutic agent, or radiation therapy.

[0046] definition "A" or "An": The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0047] Affinity: As used herein, the term "affinity" refers to a property of the binding interaction between a binding moiety (e.g., an antigen binding moiety (e.g., a variable domain described herein) and / or an Fc receptor binding moiety (e.g., an FcRn binding moiety described herein)) and a target (e.g., an antigen (e.g., ADGRE2) and / or an FcR (e.g., FcRn)), which indicates the strength of the binding interaction. In some embodiments, a measure of affinity is the dissociation constant (K D In some embodiments, the binding moiety has a high affinity for the target (e.g., about 10 -7 Less than M, about 10 -8Less than M or about 10 -9 K less than M D In some embodiments, the binding moiety has a low affinity for the target (e.g., about 10 -7 Higher than M, about 10 -6 Higher than M, about 10 -5 Higher than M, or about 10 -4 K is higher than M D In some embodiments, the binding moiety has a high affinity for the target at a first pH, a low affinity for the target at a second pH, and an intermediate affinity for the target at pH levels between the first and second pH.

[0048] Approximately or about: As used herein, when applied to one or more values ​​of interest, the term "approximately" or "about" refers to a value similar to the stated reference value. In certain embodiments, unless otherwise specified or otherwise clear from the context, the term "approximately" or "about" refers to a range of values ​​that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less) of the stated reference value (except where such number would exceed 100% of possible values).

[0049] Antibody: As used herein, the term "antibody" refers to a polypeptide comprising an amino acid sequence providing at least one immunoglobulin variable region, e.g., an immunoglobulin variable domain or immunoglobulin variable domain sequence. For example, an antibody can comprise a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody comprises two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody" encompasses antigen-binding fragments of antibodies (e.g., single-chain antibodies, Fab, F(ab'), Fd, Fv, and dAb fragments), as well as complete antibodies, e.g., intact immunoglobulins of types IgA, IgG, IgE, IgD, and IgM (and subtypes thereof). The light chains of the immunoglobulin may be of type kappa or lambda.

[0050] Binding moiety: As used herein, a "binding moiety" is any molecule or portion of a molecule that can specifically bind to a target, e.g., a target of interest (e.g., an antigen (e.g., ADGRE2) and / or an FcR (e.g., FcRn)). Binding moieties include, for example, antibodies, antigen-binding fragments thereof, Fc regions or Fc fragments thereof, antibody mimetics, peptides, and aptamers.

[0051] An antibody-binding fragment or antibody fragment thereof refers to a portion of an intact antibody. An antigen-binding fragment or antibody fragment thereof refers to a portion of an intact antibody that binds to an antigen (e.g., ADGRE2). An antigen-binding fragment may include the antigen-determining variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, antibody mimetics, scFv, and single-chain antibodies.

[0052] Complementarity-Determining Region (CDR): The "CDR" of a variable domain is the amino acid residues in the variable region identified according to Kabat, Chothia, or both Kabat and Chothia (i.e., AbM), contact, and / or conformational definitions, or any method of CDR determination known in the art. The CDRs of an antibody may be identified as hypervariable regions as originally defined by Kabat et al. See, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington, DC. The locations of CDRs may also be identified as structural loop structures originally described by Chothia et al. See, e.g., Chothia et al., Nature 342:877-883, 1989. Other approaches to CDR identification include the "AbM definition," a compromise between Kabat and Chothia, derived using Oxford Molecular's AbM antibody modeling software (now Accelrys®), or the "contact definition" of CDRs based on observed antigen contact sites, as described in MacCallum et al., J. Mol. Biol., 262:732-745, 1996. In another approach, referred to herein as the "conformational definition" of CDRs, the positions of CDRs may be identified as residues that contribute enthalpic-wise to antigen binding. See, for example, Makabe et al., Journal of Biological Chemistry, 283:1 156-1166, 2008. Still other CDR boundary definitions may not strictly adhere to one of the above approaches, but may be shortened or extended in light of predictions or experimental findings that certain residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding, yet still overlap with at least a portion of the Kabat CDRs. As used herein, CDR may refer to a CDR defined by any approach known in the art, including a combination of approaches.The methods used herein may utilize CDRs defined according to any of these approaches. For any given embodiment that includes more than one CDR, the CDRs may be defined according to the Kabat, Chothia, extended, AbM, contact, and / or conformational definitions.

[0053] Constant region: As used herein, the term "constant region" refers to a polypeptide corresponding to or derived from one or more constant region immunoglobulin domains of an antibody. The constant region can include any or all of the following immunoglobulin domains: CH1 domain, hinge region, CH2 domain, CH3 domain (derived from IgA, IgD, IgG, IgE, or IgM), and CH4 domain (derived from IgE or IgM).

[0054] Epitope: As used herein, "epitope" is a term of the art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, consecutive amino acids of a polypeptide (a linear or continuous epitope), or an epitope can be, for example, comprised of two or more non-contiguous regions of a polypeptide(s) (a conformational, non-linear, discontinuous, or discontinuous epitope). In certain embodiments, the epitope to which an antibody binds can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). In the case of X-ray crystallography, crystallization can be achieved using any method known in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen NE (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251:6300-6303). Antibody:antigen crystals can be studied using well-known X-ray diffraction techniques and refined using computer software known in the art, such as Refmac and Phenix. Mutation mapping studies can be achieved using any method known to those skilled in the art. See, for example, Champe M et al. (1995) J Biol Chem 270:1388-1394 and Cunningham BC & Wells JA (1989) Science 244:1081-1085 for a description of mutagenesis techniques, including alanine scanning mutagenesis techniques.

[0055] Fc region: As used herein, the term "Fc region" refers to a dimer of two "Fc polypeptides," each of which comprises the constant region of an antibody excluding the first constant region immunoglobulin domain. In some embodiments, an "Fc region" comprises two Fc polypeptides linked by one or more disulfide bonds, chemical linkers, or peptide linkers. "Fc polypeptide" refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and may also include part or all of the flexible hinge N-terminal to these domains. In the case of IgG, the "Fc polypeptide" includes immunoglobulin domains C gamma 2 (Cγ2) and C gamma 3 (Cγ3), as well as the lower part of the hinge between C gamma 1 (Cγ1) and Cγ2. Although the boundaries of the Fc polypeptide might vary, the human IgG heavy chain Fc polypeptide is usually defined to include residues starting from T223, or C226, or P230, to the carboxyl terminus, numbering according to the EU index of Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Services, Springfield, VA). For IgA, the Fc polypeptide includes immunoglobulin domains C alpha 2 (Cα2) and C alpha 3 (Cα3), as well as the lower portion of the hinge between C alpha 1 (Cα1) and Cα2. The Fc region may be synthetic, recombinant, or derived from a natural source, such as IVIG.

[0056] Humanized antibody: As used herein, a humanized antibody is an antibody derived from a non-human species whose protein sequence has been modified to increase its similarity to antibody variants naturally produced in humans. "Humanization" is typically applied to monoclonal antibodies developed for administration to humans (e.g., anti-ADGRE2 antibodies developed as anti-cancer drugs). In some embodiments, humanization is used when developing a specific antibody in a non-human immune system (such as an antibody in a mouse).

[0057] K a As used herein, "K a " refers to the rate of association of a particular binding moiety with a target to form a binding moiety / target complex.

[0058] K d As used herein, "K d " refers to the dissociation rate of a particular binding moiety / target complex.

[0059] K D As used herein, "K D " refers to the dissociation constant, which is K d K a to (i.e., K d / K a ) and expressed as molar concentration (M). D The value can be determined using methods well established in the art, for example using surface plasmon resonance or using a biosensor system such as a Biacore® system.

[0060] Reference: A "reference" entity, system, amount, set of conditions, etc., against which a test entity, system, amount, set of conditions, etc., is compared as described herein. For example, in some embodiments, a "reference" antibody is a control antibody that has not been engineered as described herein.

[0061] Selective binding: As used herein, "selective binding," "selectively binds," "specific binding," or "specifically binds," with respect to a binding moiety and a target, refers to the preferential association of the binding moiety with the target and not with non-target entities. Some non-specific binding may occur between the binding moiety and the non-target. In some embodiments, a binding moiety selectively binds to a target if the binding between the binding moiety and the target is more than 2-fold, more than 5-fold, more than 10-fold, or more than 100-fold greater than the binding between the binding moiety and the non-target. In some embodiments, a binding affinity is about 10 -5Less than M, about 10 -6 Less than M, about 10 -7 Less than M, about 10 -8 Less than M or about 10 -9 A binding moiety selectively binds to a target when M is less than M. In some embodiments, a molecule that specifically binds to an antigen may generally bind to other peptides or polypeptides with lower affinity, as determined, for example, by immunoassay, BIACORE®, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art.

[0062] Single-chain variable fragment (scFv): As used herein, the term "single-chain variable fragment" or "scFv" refers to a V H heavy chains (V) of immunoglobulins (e.g., murine or human) covalently linked to form a VL heterodimer. H ) and light chain (V L ) heavy chain (V H ) and light chain (V L ) are either directly bonded or V H N-terminus of V L or at the C-terminus of V H The C-terminus of V L The heavy and light chain variable regions of the extracellular antigen-binding domain are connected by a peptide-encoded linker (e.g., 10, 15, 20, or 25 amino acids) that connects the heavy and light chain variable regions to the N-terminus of the extracellular antigen-binding domain. The linker is typically rich in glycine for flexibility and serine or threonine for solubility. The linker can connect the heavy and light chain variable regions of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917 (2008) and WO2014 / 087010, the contents of which are incorporated herein by reference in their entirety.

[0063] Subject: As used herein, the term "subject" refers to any subject for whom diagnosis, prognosis, or treatment is desired. For example, the subject can be a mammal, such as a human or non-human primate (such as an ape, monkey, orangutan, or chimpanzee), dog, cat, guinea pig, rabbit, rat, mouse, horse, cattle, or cow.

[0064] Target: As used herein, a "target" is any molecule that is specifically bound by a binding portion of an antibody or antigen-binding fragment thereof. In some embodiments, the target is an antigen described herein (e.g., ADGRE2). In some embodiments, the target is an FcR (e.g., FcRn). The terms "primary target" and "secondary target" are used herein to refer to molecules of two different molecular species, rather than two molecules of the same molecular species. For example, in some embodiments, the primary target is a serum protein and the secondary target is FcRn.

[0065] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount of a therapeutic molecule (e.g., an anti-ADGRE2 antibody described herein) that confers a therapeutic effect on a treated subject at a reasonable benefit / risk ratio applicable to any medical treatment. The therapeutic effect can be objective (i.e., measurable by some test or marker) or subjective (i.e., the subject gives an indication of or feels an effect). In particular, a "therapeutically effective amount" refers to an amount of a therapeutic molecule or composition effective to treat, ameliorate, or prevent a particular disease or condition, or to exhibit a therapeutic or prophylactic effect that is detectable, for example, by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and / or reducing the severity or frequency of symptoms of the disease. A therapeutically effective amount can be administered in a dosing regimen that can be composed of multiple unit doses. For any particular therapeutic molecule, the therapeutically effective amount (and / or appropriate unit dose within an effective dosing regimen) can vary depending, for example, on the route of administration, combination with other pharmaceutical agents, etc. In addition, the specific therapeutically effective amount (and / or unit dose) for any particular subject will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the particular pharmaceutical agent used; the particular composition used; the age, weight, general health, sex, and diet of the subject; the time of administration, route of administration, and / or rate of excretion or metabolism of the particular therapeutic molecule used; the duration of treatment; and similar factors as are well known in the medical arts.

[0066] Treatment: As used herein, the term "treatment" (also "treat" or "treating") refers to any administration of a therapeutic molecule (e.g., an anti-ADGRE2 antibody described herein) that partially or completely relieves, improves, alleviates, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. Such treatment may be in subjects who do not exhibit symptoms of the associated disease, disorder, and / or condition and / or who exhibit only early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be in subjects who exhibit one or more established signs of the associated disease, disorder, and / or condition.

[0067] The drawings are for illustrative purposes only and not for limitation. [Brief explanation of the drawings]

[0068] [Figure 1] Illustrated is a table showing the number of residues that are changed in the humanized ADGRE2 antibody compared to the murine reference sequence. The table also shows the number of residues that differ from the germline sequence. [Figure 2] Illustrated is a table showing the number of residues that are changed in the humanized ADGRE2 antibody compared to the murine reference sequence. The table also shows the number of residues that differ from the germline sequence. [Figure 3-1]

[0023] Figure 1 shows a two-dimensional projection of an exemplary antibody VH sequence. The amino acid sequence is shown as forming nine antiparallel β-strands of the immunoglobulin fold. The N- to C-terminus of the primary sequence can be traced from the top left to the bottom right. The figure discloses SEQ ID NOS: 39-40, 43, 41-42, and 44, respectively, in the order shown. [Figure 3-2]

[0023] Figure 1 shows a two-dimensional projection of an exemplary antibody VH sequence. The amino acid sequence is shown as forming nine antiparallel β-strands of the immunoglobulin fold. The N- to C-terminus of the primary sequence can be traced from the top left to the bottom right. The figure discloses SEQ ID NOS: 39-40, 43, 41-42, and 44, respectively, in the order shown. [Figure 3-3]

[0023] Figure 1 shows a two-dimensional projection of an exemplary antibody VH sequence. The amino acid sequence is shown as forming nine antiparallel β-strands of the immunoglobulin fold. The N- to C-terminus of the primary sequence can be traced from the top left to the bottom right. The figure discloses SEQ ID NOS: 39-40, 43, 41-42, and 44, respectively, in the order shown. DETAILED DESCRIPTION OF THE INVENTION

[0069] The present disclosure is based, in part, on the discovery of engineered antibodies and antigen-binding fragments thereof that exhibit binding to the adhesion G protein-coupled receptor E2, i.e., ADGRE2 (e.g., human ADGRE2). In some embodiments, humanized anti-ADGRE2 antibodies and fragments thereof are provided herein. ADGRE2, also known as EMR2, CD312, VBU, or CD97, is a cell surface receptor that is a member of the adhesion G protein-coupled receptor (GPCR) family. ADGRE2 binds to chondroitin sulfate moieties of glycosaminoglycan chains and promotes cell attachment.

[0070] ADGRE2 is expressed in monocytes, macrophages, dendritic cells, and granulocytes and plays a role in chemotaxis, cell adhesion, and degranulation. In macrophages, ADGRE2 signals through G proteins to promote the release of proinflammatory cytokines, including IL8 and TNF.

[0071] ADGRE2 is highly expressed in cancerous cells, including, for example, relapsed and refractory acute myeloid leukemia. In some embodiments, inhibition of ADGRE2 is used to treat cancer. In some embodiments, inhibition of ADGRE2 is used to treat relapsed and refractory acute myeloid leukemia. antibody

[0072] The anti-ADGRE2 antibody binds to the ADGRE2 antigen. In some embodiments, the anti-ADGRE2 antibody binds to human ADGRE2. ADGRE2 has been described in the art, for example, in Uniprot sequence reference Q9UHX3-1.

[0073] In some embodiments, human ADGRE2 comprises an EGF-like 1 domain, an EGF-like 2 domain, an EGF-like 3 domain, an EGF-like 4 domain, an EGF-like 5 domain, and a GPS domain.

[0074] The anti-ADGRE2 antibodies described herein can be immunoglobulins, heavy chain antibodies, light chain antibodies, LRR-based antibodies, or other protein scaffolds with antibody-like properties, as well as other immunological binding moieties known in the art, including, for example, Fab, Fab', Fab'2, Fab2, Fab3, F(ab')2, Fd, Fv, Feb, scFv, SMIP, antibody, diabody, triabody, tetrabody, minibody, maxibody, tandab, DVD, BiTe, TandAb, etc., or any combination thereof. The subunit structures and three-dimensional configurations of different classes of antibodies are known in the art.

[0075] An antibody may be an immunoglobulin molecule of four polypeptide chains, e.g., two heavy (H) chains and two light (L) chains. The heavy chain may comprise a heavy chain variable domain and a heavy chain constant domain. The heavy chain constant domain may comprise a CH1, hinge, CH2, CH3, and, optionally, a CH4 region. A suitable heavy chain constant region may be derived from any immunoglobulin (e.g., IgA, IgG, or IgE). In some embodiments, a suitable heavy chain constant region may be derived from IgG1, IgG2, or IgG4. In certain embodiments, a suitable heavy chain constant region is derived from IgG1. The light chain may comprise a light chain variable domain and a light chain constant domain. The light chain constant domain may comprise either a kappa light chain or a lambda light chain. The heavy chain variable domain of the heavy chain and the light chain variable domain of the light chain may be further divided into regions of variability, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each of these heavy and light chain variable domains can comprise three CDRs and four framework regions, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. One or more of these can be engineered as described herein. The assignment of amino acids to each domain follows the definitions in the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987) and Chothia et al. Nature 342:878-883 (1989). As used herein, CDRs refer to the heavy (HCDR1, HCDR2, HCDR3) and light (LCDR1, LCDR2, LCDR3) chains, respectively.

[0076] Embodiments of the present invention include antibodies comprising the CDRs found in the VH and VL domains described herein, identified using conventional numbering systems, such as the IMGT, Kabat, and Clothia numbering systems. Such numbering systems are well known in the art. In certain embodiments, the CDRs are identified or numbered according to the IMGT numbering system. Exemplary Antibodies

[0077] In some embodiments, the anti-ADGRE2 antibodies or fragments thereof described herein comprise a common heavy chain variable region.

[0078] In certain embodiments, the CDRs are identified or numbered according to the IMGT numbering system.

[0079] In some embodiments, the anti-ADGRE2 antibody comprises the heavy chain variable region (VH) complementarity determining region (CDR) sequences of VH CDR1: GYTFTNYW (SEQ ID NO: 1), VH CDR2: VYPGDGDT (SEQ ID NO: 2), and VH CDR3: ARGFTAYGMDY (SEQ ID NO: 3).

[0080] In some embodiments, the heavy chain variable is It contains the amino acid sequence of QVQLQQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 7).

[0081] In some embodiments, the heavy chain variable is It contains the amino acid sequence of QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSISTAYMEVSRLRSDDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 9).

[0082] In some embodiments, the heavy chain variable is It contains the amino acid sequence of QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTVYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 11).

[0083] In some embodiments, the heavy chain variable is It contains the amino acid sequence of QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWMGAVYPGDGDTRHTQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSS (SEQ ID NO: 13).

[0084] In some embodiments, the heavy chain variable is It contains the amino acid sequence of QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRHTQKFKGRVTMTADKSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSS (SEQ ID NO: 15).

[0085] In some embodiments, the heavy chain variable is It contains the amino acid sequence of QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 17).

[0086] In some embodiments, the heavy chain variable is It contains the amino acid sequence of QVQLVQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 30).

[0087] In some embodiments, the anti-ADGRE2 antibody comprises a heavy chain amino acid sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 7, 9, 11, 13, 15, 17, or 30.

[0088] In some embodiments, the anti-ADGRE2 antibody comprises a heavy chain amino acid sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 7, 9, 11, 13, 15, 17, or 30, while also comprising one or more of the VH CDR1, vHCDR2, and / or vHCDR3 sequences described herein.

[0089] In some embodiments, the engineered antibody comprises a heavy chain amino acid sequence identical to SEQ ID NO: 7, 9, 11, 13, 15, 17, or 30. In certain embodiments, V H comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identical or homologous to the amino acid sequence set forth in SEQ ID NO: 7, 9, 11, 13, 15, 17, or 30. For example, V H comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 7, 9, 11, 13, 15, 17, or 30. In some embodiments, the anti-ADGRE2 antibody contains no more than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid substitutions relative to SEQ ID NO: 7, 9, 11, 13, 15, 17, or 30.

[0090] In some embodiments, the anti-ADGRE2 heavy chain variable is encoded by a polynucleotide comprising a nucleic acid sequence that encodes the amino acid sequence of humanized ADGRE2.

[0091] As will be appreciated by one of ordinary skill in the art, any such heavy chain CDR sequence can be readily combined, for example by molecular biology techniques, with any other antibody sequence or domain provided herein or otherwise known in the art, including any framework region, CDR, or constant domain, or portion thereof, disclosed herein or otherwise known in the art, as may be present in any format of antibody or antigen-binding fragment thereof disclosed herein or otherwise known in the art.

[0092] In the various engineered antibodies described herein, the heavy chain constant domain can be of any class (or subclass). In the various engineered antibodies described herein, the heavy chain constant domain can comprise the amino acid sequence of one or more of IgG, IgM, IgA, IgD, or IgE, including subclasses such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In various embodiments, the constant domain of the engineered antibodies described herein can comprise a mixture of two or more classes (or subclasses) of immunoglobulin heavy chain constant domains. For example, an anti-ADGRE2 antibody can comprise a first portion of a constant domain having the sequence of an immunoglobulin constant domain selected from the constant domains of the IgG, IgM, IgA, IgD, or IgE class, and a second portion of a constant domain having the sequence of an immunoglobulin constant domain selected from the constant domains of the IgG, IgM, IgA, IgD, or IgE class, different from the first portion. In some cases, the constant domains of the anti-ADGRE2 antibodies described herein can comprise a mixture of two or more subclasses of a particular class of constant domain, e.g., a first portion of a constant domain having an immunoglobulin constant domain sequence selected from IgG1, IgG2, IgG3, or IgG4 subclass constant domains, and a second portion of a constant domain having an immunoglobulin constant domain sequence selected from IgG1, IgG2, IgG3, or IgG4 subclass constant domains that is different from the first portion. In some particular embodiments, the constant domains comprise all or a portion of an IgG2 constant domain and all or a portion of an IgG4 constant domain.

[0093] In some cases, the anti-ADGRE2 antibody comprises an antibody constant region, Fc region, or Fc fragment that exhibits altered binding (compared to a reference constant region) to one or more Fc receptors (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB, FcγRIV, or FcRn receptors). In some embodiments, the constant region, Fc region, or Fc fragment is engineered to bind to a target (e.g., an FcRn receptor) in an altered manner (e.g., in a pH-sensitive manner (e.g., in a higher or lower pH-sensitive manner) and / or with decreased or increased binding) compared to the reference constant region, Fc region, or Fc fragment. In some embodiments, the anti-ADGRE2 antibody comprises an antibody constant region, Fc region, or Fc fragment that exhibits decreased binding (compared to a reference constant region) to one or more Fcγ receptors (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB, or FcγRIV). In some embodiments, the anti-ADGRE2 antibody comprises an antibody constant region, Fc region, or Fc fragment that exhibits increased binding (compared to a reference constant region) to the FcRn receptor at serum pH and / or intracellular pH.

[0094] For example, an anti-ADGRE2 antibody can comprise an IgG antibody constant region, Fc region, or Fc fragment engineered to contain amino acid additions, deletions, or substitutions of one or more of amino acid residues 251-256, 285-290, 308-314, 385-389, and 428-436 (Kabat numbering (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH)). Without wishing to be bound by theory, it is believed that one or more of the amino acids in these constant regions, Fc regions, or Fc fragments mediate interaction with Fc receptors, such as FcRn. In some embodiments, one or more of these disclosed amino acids is substituted with histidine, arginine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, or glutamine. In some embodiments, a non-histidine residue is substituted with a histidine residue. In some embodiments, the histidine residue is substituted with a non-histidine residue.

[0095] In some embodiments, the anti-ADGRE2 antibody comprises an IgG antibody constant region, Fc region, or Fc fragment having an amino acid modification at one or more of positions 308, 309, 311, 312, and 314, more specifically, a substitution of threonine, proline, serine, aspartic acid, and leucine at one or more of positions 308, 309, 311, 312, and 314, respectively. In some embodiments, one or more residues at positions 308, 309, and 311 are substituted with isoleucine, proline, and glutamic acid, respectively. In still other embodiments, one or more residues at positions 308, 309, 311, 312, and 314 are substituted with threonine, proline, serine, aspartic acid, and leucine, respectively.

[0096] In some embodiments, the anti-ADGRE2 antibody comprises an IgG antibody constant region, Fc region, or Fc fragment having an amino acid modification, more particularly a substitution, at one or more of positions 251, 252, 254, 255, and 256. In some embodiments, residue 251 is substituted with leucine or arginine, residue 252 is substituted with leucine, tyrosine, phenylalanine, serine, tryptophan, or threonine, residue 254 is substituted with threonine or serine, residue 255 is substituted with leucine, glycine, isoleucine, or arginine, and / or residue 256 is substituted with serine, phenylalanine, arginine, glutamine, glutamic acid, aspartic acid, alanine, asparagine, or threonine. In some embodiments, residue 251 is substituted with leucine, residue 252 is substituted with tyrosine or leucine, residue 254 is substituted with threonine or serine, and / or residue 255 is substituted with arginine. In still other embodiments, residue 252 is substituted with phenylalanine and / or residue 256 is substituted with aspartic acid. In some embodiments, residue 251 is substituted with leucine, residue 252 is substituted with tyrosine, residue 254 is substituted with threonine or serine, and / or residue 255 is substituted with arginine.

[0097] In some embodiments, the anti-ADGRE2 antibody comprises an IgG antibody constant region, Fc region, or Fc fragment having an amino acid modification, more particularly a substitution, at one or more of positions 428, 433, 434, 435, and 436. In some embodiments, residue 428 is substituted with methionine, threonine, leucine, phenylalanine, or serine, residue 433 is substituted with lysine, arginine, serine, isoleucine, proline, glutamine, or histidine, residue 434 is substituted with phenylalanine, tyrosine, or histidine, residue 435 is substituted with tyrosine, and / or residue 436 is substituted with histidine, asparagine, arginine, threonine, lysine, methionine, or threonine. In some embodiments, one or more of residues 433, 434, 435, and 436 are substituted with lysine, phenylalanine, tyrosine, and histidine, respectively. In some embodiments, residue 428 is substituted with methionine and / or residue 434 is substituted with tyrosine.

[0098] In some embodiments, the anti-ADGRE2 antibody comprises an IgG antibody constant region, Fc region, or Fc fragment having an amino acid modification, more particularly a substitution, at one or more of positions 385, 386, 387, and 389. In some embodiments, residue 385 is substituted with arginine, aspartic acid, serine, threonine, histidine, lysine, or alanine; residue 386 is substituted with threonine, proline, aspartic acid, serine, lysine, arginine, isoleucine, or methionine; residue 387 is substituted with arginine, histidine, serine, threonine, alanine, or proline; and / or residue 389 is substituted with proline or serine. In some embodiments, one or more of residues 385, 386, 387, and 389 are substituted with arginine, threonine, arginine, and proline, respectively. In some embodiments, one or more residues at positions 385, 386, and 389 are substituted with aspartic acid, proline, and serine, respectively.

[0099] In some embodiments, the anti-ADGRE2 antibody comprises an IgG antibody constant region, Fc region, or Fc fragment having one or more of the following substitutions: leucine at residue 251, tyrosine or leucine at residue 252, threonine or serine at residue 254, arginine at residue 255, threonine at residue 308, proline at residue 309, serine at residue 311, aspartic acid at residue 312, leucine at residue 314, arginine at residue 385, threonine at residue 386, arginine at residue 387, proline at residue 389, methionine at residue 428, lysine at residue 433, phenylalanine or tyrosine at residue 434, tyrosine at position 435, and / or tyrosine at position 436. Additional amino acid substitutions that can be included in the constant region, Fc region, or Fc fragment include those described in, for example, US Pat. Nos. 6,277,375, 8,012,476, and 8,163,881.

[0100] In some embodiments, the anti-ADGRE2 antibodies described herein comprise a heavy chain constant domain containing an Ala-Ala mutation, e.g., as described in PCT Publication Nos. WO94 / 28027 and WO98 / 47531, and Xu et al. (2000) Cell Immunol 200:16-26. Thus, in some embodiments, anti-ADGRE2 antibodies with one or more mutations in the heavy chain constant region, including an Ala-Ala mutation, have reduced or no effector function. In accordance with these embodiments, the constant region of the anti-ADGRE2 antibodies described herein may comprise an alanine substitution at position 234 and / or an alanine mutation at position 235 (EU numbering).

[0101] As will be appreciated by one of ordinary skill in the art, any such heavy chain constant domain sequence can be readily combined, for example by molecular biology techniques, with any other antibody sequence or domain provided herein or otherwise known in the art, including any framework region, CDR, or constant domain, or portion thereof, disclosed herein or otherwise known in the art, as may be present in any format of antibody or antigen-binding fragment thereof disclosed herein or otherwise known in the art.

[0102] The present invention further provides ADGRE2 antibodies or fragments thereof comprising various designated sequences in one or more light chain variable regions, including light chain complementarity determining regions LCDR1-3. In various embodiments, molecules having designated light chain variable regions are provided with heavy chain sequences as discussed above. In certain embodiments, the CDRs are identified or numbered according to the IMGH numbering system.

[0103] Thus, in one aspect, the present invention provides an anti-ADGRE2 antibody or fragment thereof comprising a light chain variable region having complementarity-determining region (CDR) sequences of SSVSY (sequence number 4), LCDR2 comprising the amino acid sequence of DTS (sequence number 5), and LCDR3 comprising the amino acid sequence of QQWSSNPLT (sequence number 6).

[0104] In some embodiments, the anti-ADGRE2 antibody or fragment thereof comprises an immunoglobulin light chain variable (VL) region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 8, 10, 12, 14, 16, 18, or 31, and an immunoglobulin heavy chain variable (VH) region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 7, 9, 11, 13, 15, 17, or 30.

[0105] In some embodiments, the VL region comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 8, 10, 12, 14, 16, 18, or 31.

[0106] In some embodiments, the anti-ADGRE2 antibody or fragment thereof It comprises a light chain variable region (VL) comprising the amino acid sequence of EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGLAPRLLIYDTSKLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSSNPLTFGQGTKVEIK (SEQ ID NO: 8).

[0107] In some embodiments, the light chain variable is It contains the amino acid sequence of EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 10).

[0108] In some embodiments, the light chain variable is It contains the amino acid sequence of EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 12).

[0109] In some embodiments, the light chain variable is It contains the amino acid sequence of EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGQSPKRWIYDTSKLASGVPARFSGSGSGTDYTFTISSMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 14).

[0110] In some embodiments, the light chain variable is It contains the amino acid sequence of QIVLTQSPATLSLSPGERATLTCSASSSVSYMHWYQQKPGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTIRRLEPEDFATYYCQQWSSNPLTFGQGTKVEIK (SEQ ID NO: 16).

[0111] In some embodiments, the light chain variable is It contains the amino acid sequence of EIVLTQSPATLSASPGERVTMSCSASSSVSYMHWYQQKPGLAPRRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 18).

[0112] In some embodiments, the light chain variable comprises the amino acid sequence of EIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGQSPKRWIYDTSKLASGVPARFSGSGSGTDYTFTISSMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 31).

[0113] In some embodiments, the anti-ADGRE2 antibody comprises a light chain amino acid sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 8, 10, 12, 14, 16, 18, or 31.

[0114] In some embodiments, the anti-ADGRE2 antibody comprises a light chain amino acid sequence having at least about 85%, about 90%, about 95%, about 98%, or about 99% sequence identity to SEQ ID NO: 8, 10, 12, 14, 16, 18, or 31, while also comprising one or more of the VL CDR1, vLCDR2, and / or vLCDR3 sequences described herein.

[0115] In some embodiments, the anti-ADGRE2 antibody or fragment thereof comprises a light chain amino acid sequence identical to SEQ ID NO: 8, 10, 12, 14, 16, 18, or 31. In some embodiments, the anti-ADGRE2 antibody comprises no more than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid substitutions relative to SEQ ID NO: 8, 10, 12, 14, 16, 18, or 31.

[0116] As will be appreciated by one of ordinary skill in the art, any such light chain CDR sequence can be readily combined, for example by molecular biology techniques, with any other antibody sequence or domain provided herein or otherwise known in the art, including any framework region, CDR, or constant domain, or portion thereof, disclosed herein or otherwise known in the art, as may be present in any format of antibody or antigen-binding fragment thereof disclosed herein or otherwise known in the art.

[0117] In some embodiments, the anti-ADGRE2 antibodies described herein comprise a light chain comprising any light chain constant domain sequence, for example, a light chain constant sequence known to those skilled in the art. As those skilled in the art will recognize, the light chain constant domain can be a kappa light chain constant domain or a lambda light chain constant domain. In certain embodiments, the light chain constant domain disclosed herein is a kappa light chain constant domain. In various embodiments, the anti-ADGRE2 antibodies described herein comprise a light chain constant domain.

[0118] Engineered antibodies can comprise various heavy and light chains as described herein. In some embodiments, an anti-ADGRE2 antibody can comprise two heavy and light chains. In various embodiments, the present disclosure encompasses antibodies comprising at least one heavy and / or light chain disclosed herein, at least one heavy chain framework domain and / or light chain framework domain disclosed herein, at least one heavy chain CDR domain and / or light chain CDR domain disclosed herein, and / or any heavy chain constant domain and / or light chain constant domain disclosed herein.

[0119] Thus, in one aspect, the present invention provides an anti-ADGRE2 antibody or fragment thereof comprising a heavy chain variable region having complementarity determining region (CDR) sequences of VH CDR1: GYTFTNYW (SEQ ID NO: 1), VH CDR2: VYPGDGDT (SEQ ID NO: 2), and VH CDR3: ARGFTAYGMDY (SEQ ID NO: 3), and a light chain variable region having complementarity determining region (CDR) sequences of SSVSY (SEQ ID NO: 4), LCDR2 comprising the amino acid sequence of DTS (SEQ ID NO: 5), and LCDR3 comprising the amino acid sequence of QQWSSNPLT (SEQ ID NO: 6).

[0120] The table below shows exemplary heavy chain variable region (VH) and light chain variable region sequences of the humanized ADGRE2 antibodies disclosed herein.

[0121] [Table 1-1]

[0122] [Table 1-2]

[0123] In various embodiments, the anti-ADGRE2 antibodies disclosed herein are homodimeric monoclonal antibodies. In various embodiments, the anti-ADGRE2 antibodies disclosed herein are heterodimeric antibodies. In various embodiments, the anti-ADGRE2 antibodies are, for example, a typical antibody, or a diabody, triabody, tetrabody, minibody, maxibody, tandab, DVD, BiTe, scFv, TandAb scFv, Fab, Fab2, Fab3, F(ab')2, etc., or any combination thereof.

[0124] In some embodiments, the present disclosure provides fusion proteins comprising one or more variable domains or engineered antibodies described herein, or portions thereof, and one or more additional polypeptides.

[0125] Exemplary Single-Chain Variable Fragments In some embodiments, the present disclosure provides single-chain variable fragments. In some embodiments, the scFv is a human scFv. The term "single-chain variable fragment" or "scFv" refers to a V H heavy chains (V) of immunoglobulins (e.g., murine or human) covalently linked to form a VL heterodimer. H ) and light chain (V L ) heavy chain (V H ) and light chain (V L ) are either directly bonded or V H N-terminus of V L or at the C-terminus of V H The C-terminus of V LThe heavy and light chain variable regions of the extracellular antigen-binding domain are connected by a peptide-encoded linker (e.g., 10, 15, 20, or 25 amino acids) connected to the N-terminus of the heavy chain variable region (e.g., 10, 15, 20, or 25 amino acids). The linker is typically rich in glycine for flexibility and rich in serine or threonine for solubility. The linker can connect the heavy and light chain variable regions of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917 (2008) and WO2014 / 087010, the contents of which are incorporated herein by reference in their entireties. In certain embodiments, the linker is a G4S linker (SEQ ID NO: 45).

[0126] Alternatively, or additionally, the scFv may be derived from a Fab' (e.g., rather than from an antibody obtained from a Fab library). In certain embodiments, the anti-ADGRE2 antibody or fragment thereof is a Fab. In certain embodiments, the Fab is cross-linked. In certain embodiments, the anti-ADGRE2 antibody or fragment thereof is a F(ab)2. Any of the foregoing molecules can be included in a fusion protein with a heterologous sequence to form an anti-ADGRE2 antigen antibody or antigen-binding fragment thereof.

[0127] In certain embodiments, the anti-ADGRE2 antibody or fragment thereof is at least about 1 x 10 -6 M, at least about 1 × 10 -7 M, at least about 1 × 10 -8 M, at least about 1 × 10 -9 M, or at least about 1 × 10 -10 Dissociation stationary state of M (K D In certain embodiments, the anti-ADGRE2 antibody or fragment thereof binds to ADGRE2 (e.g., human ADGRE2) at a concentration of at least about 2 x 10 -8 Dissociation stationary state of M (K D In certain embodiments, the anti-ADGRE2 antibody or fragment thereof binds to ADGRE2 (e.g., human ADGRE2) at about 2x 10-8 M ~ approx. 8×10 -9 Dissociation stationary state of M (K D) binds to ADGRE2 (e.g., human ADGRE2).

[0128] In some embodiments, the anti-ADGRE2 antibody or fragment thereof has a dissociation constant (K) of about 1 nM to 50 nM, about 5 nM to 30 nM, about 5 nM to 25 nM, or about 8 nM to 20 nM. D In some embodiments, the anti-ADGRE2 antibody or fragment thereof binds to ADGRE2 (e.g., human ADGRE2) with a dissociation constant (K) of at least about 50 nM, at least about 40 nM, at least about 35 nM, at least about 30 nM, at least about 25 nM, at least about 20 nM, at least about 19 nM, at least about 18 nM, at least about 17 nM, at least about 16 nM, at least about 15 nM, at least about 14 nM, at least about 13 nM, at least about 12 nM, at least about 11 nM, at least about 10 nM, at least about 9 nM, at least about 8 nM, at least about 7 nM, at least about 6 nM, or at least about 5 nM. D ) binds to ADGRE2 (e.g., human ADGRE2).

[0129] In some embodiments, the anti-ADGRE2 scFv comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22.

[0130] In some embodiments, the anti-ADGRE2 scFv comprises a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 24, provided below: GGGGSGGGGSGGGGS (SEQ ID NO: 24)

[0131] In some embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25, provided below: GGGGSGGGGSGGGSGGGGS (SEQ ID NO: 25).

[0132] In some embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:26, provided below: GGGGSGGGGSGGGGSGGGSGGGGS (SEQ ID NO: 26).

[0133] In some embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:27, provided below: GGGGSGGGGSGGGGSGGGGSGGGSGGGGS (SEQ ID NO: 27).

[0134] In some embodiments, an anti-ADGRE2 antibody or fragment thereof comprises conservative sequence modifications (e.g., an anti-ADGRE2 antibody or fragment thereof described herein). In some embodiments, conservative sequence modifications are amino acid modifications that do not significantly affect or alter the binding characteristics of an anti-ADGRE2 antibody or fragment thereof disclosed herein (e.g., an antibody or fragment thereof) comprising the amino acid sequence. Conservative modifications can include amino acid substitutions, additions, and deletions. Modifications can be introduced into an anti-ADGRE2 antibody or fragment thereof by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified into groups according to their physicochemical properties, such as charge and polarity. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid from the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, and histidine, negatively charged amino acids include aspartic acid and glutamic acid, and neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Amino acids can also be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; nonpolar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acid residues in a CDR region can be substituted with other amino acid residues from the same group, and the altered antibody can be tested for retained function. In certain embodiments, no more than one, two, three, four, or five residues in a given sequence or CDR region are altered.

[0135] In some embodiments, the light and / or heavy chains of the anti-ADGRE2 scFv comprise a signal peptide. In some embodiments, the signal peptide is one of the amino acid sequences MALPVTALLLPLALLLHA (SEQ ID NO: 32), METDTLLLWVLLLWVPGSTG (SEQ ID NO: 33), MYRMQLLSCIALSLALVTNS (SEQ ID NO: 34), METPAQLLFLLLLWLPDTTG (SEQ ID NO: 35), MALPVTALLLPLALLLHAARP (SEQ ID NO: 36), MKWVTFISLLFSSAYS (SEQ ID NO: 37), MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS (SEQ ID NO: 38). In some embodiments, the signal peptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to MEFGLSWVFLVALLRGVQC (SEQ ID NO: 29), MDMRVPAQLLGLLLLWLPDTRC (SEQ ID NO: 28), or MEFGLSWVFLVALLRGVQC (SEQ ID NO: 29). In some embodiments, the signal peptide comprises MALPVTALLLPLALLLHA (SEQ ID NO: 32). In some embodiments, the signal peptide comprises METDTLLLWVLLLWVPGSTG (SEQ ID NO: 33). In some embodiments, the signal peptide comprises MYRMQLLSCIALSLALVTNS (SEQ ID NO: 34). In some embodiments, the signal peptide comprises METPAQLLFLLLLWLPDTTG (SEQ ID NO: 35). In some embodiments, the signal peptide comprises MALPVTALLLPLALLLHAARP (SEQ ID NO: 36). In some embodiments, the signal peptide comprises MKWVTFISLLFSSAYS (SEQ ID NO: 37). In some embodiments, the signal peptide comprises MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS (SEQ ID NO: 38). In some embodiments, the signal peptide comprises MDMRVPAQLLGLLLLWLPDTRC (SEQ ID NO: 28).In some embodiments, the signal peptide comprises MEFGLSWVFLVALLRGVQC (SEQ ID NO: 29).

[0136] In some embodiments, the anti-ADGRE2 scFv comprises the following amino acid sequence: QVQLQQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGS GGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGQSPKRWIYDTSKLASGVPARFSGSGSGTDYTFTISSMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 19) (scFv “K”);

[0137] QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSISTAYMEVSRLRSDDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 20) (scFv "B");

[0138] QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTVYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 21) (scFv "N"); or

[0139] QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWMGAVYPGDGDTRHTQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSSASTGGGGSGGGG SGGGGSEIVLTQSPATLSLSPGERATLSCSCSASSSVSYMHWYQQKPGLAPRLLIYDTSKLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSSNPLTFGQGTKVEIK (SEQ ID NO: 22) (scFv "A").

[0140] In some embodiments, the anti-ADGRE2 scFv comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to SEQ ID NO: 19, 20, 21, or 22.

[0141] In some embodiments, the anti-ADGRE2 scFv comprises the amino acid sequence set forth in SEQ ID NO: 19, 20, 21, or 22. In some embodiments, the anti-ADGRE2 scFv comprises the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the anti-ADGRE2 scFv comprises the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the anti-ADGRE2 scFv comprises the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, the anti-ADGRE2 scFv comprises the amino acid sequence set forth in SEQ ID NO: 22.

[0142] Nucleotide sequence The present disclosure includes nucleotide sequences encoding one or more heavy chains, heavy chain variable domains, heavy chain framework regions, heavy chain CDRs, heavy chain constant domains, light chains, light chain variable domains, light chain framework regions, light chain CDRs, light chain constant domains, or other immunoglobulin-like sequences or antibodies disclosed herein. In various embodiments, such nucleotide sequences may be present in a vector. In various embodiments, such nucleotides may be present in the genome of a cell, e.g., a subject's cell in need of treatment, or a cell for antibody production, e.g., a mammalian cell for antibody production.

[0143] Engineered antibodies and fusion proteins In some embodiments, the present disclosure provides fusion proteins comprising (i) one or more antigen-binding regions described herein (e.g., antigen-binding regions of immunoglobulins, heavy chain antibodies, light chain antibodies, LRR-based antibodies, or other protein scaffolds with antibody-like properties, as well as other antigen-binding moieties known in the art, including, for example, Fab, Fab', Fab'2, Fab2, Fab3, F(ab')2, Fd, Fv, Feb, scFv, SMIP, antibody, diabody, triabody, tetrabody, minibody, maxibody, tandab, DVD, BiTe, TandAb, etc.), e.g., one or more variable domains described herein, or portions thereof (e.g., one or more CDRs described herein), and (ii) one or more additional polypeptides. For example, albumin is an abundant serum protein that is protected from degradation by pH-dependent recycling mediated by its interaction with FcRn. In some embodiments, one or more variable domains or engineered antibodies described herein, or portions thereof (e.g., one or more CDRs described herein) are fused to albumin, a portion thereof (e.g., a portion of albumin that binds to FcRn), and / or an engineered variant of albumin that binds to FcRn with improved affinity. In other examples, one or more variable domains or engineered antibodies described herein, or portions thereof (e.g., one or more CDRs described herein) are fused to a polypeptide that binds to albumin to form a fusion protein-albumin complex, thereby capable of binding to FcRn. In some embodiments, the polypeptide that binds to albumin is a single-chain variable fragment (scFv). Albumin or a portion thereof can contain one or more amino acid mutations that can alter binding to FcRn. Such mutations are known in the art (see, e.g., Andersen et al., Nature Communications 3:610 doi:10.1038 / nocmms1607(2012)). In other examples, one or more variable domains or engineered antibodies described herein, or portions thereof (e.g., one or more CDRs described herein) are fused to transferrin.Transferrin is recycled by binding to the transferrin receptor (see, eg, Widera et al., Adv. Drug Deliv. Rev. 55:1439-66 (2003)).

[0144] Engineered antibodies and fragments thereof Anti-ADGRE2 antibodies and antigen-binding fragments thereof according to the present disclosure are engineered to contain one or more binding moieties that specifically bind to one or more targets of interest. ADGRE2 antibodies and fragments thereof include nucleic acids (e.g., RNA and DNA), proteins (e.g., antibodies), and combinations thereof. In some embodiments, the pH-dependent binding moiety can be or include, for example, nucleic acids (e.g., RNA and DNA), as well as aptamers, polypeptides (e.g., antibodies or fragments thereof, albumin, receptors, ligands, signal peptides, avidin, and protein A), polysaccharides, biotin, hydrophobic groups, hydrophilic groups, drugs, and any organic molecule that binds to a receptor.

[0145] The ADGRE2 antibodies disclosed herein are humanized antibodies and fragments thereof. Various humanization methods are known in the art, including, for example, the Xoma humanization method, and other bioinformatics-based methods.

[0146] Antibodies or fragments thereof as binding moieties In some embodiments, the antibody or fragment thereof described herein is an anti-ADGRE2 antibody. In some cases, one or more binding moieties described herein are or include an antibody, an antigen-binding fragment thereof, and / or an Fc region (or Fc fragment) thereof. The basic structure of an IgG antibody consists of two identical light polypeptide chains and two identical heavy polypeptide chains linked together by disulfide bonds. The first domain, located at the amino terminus of each chain, is variable in amino acid sequence and provides the antibody binding specificity found in individual antibodies. These are known as the heavy chain variable (VH) region and the light chain variable (VL) region. The other domains of each chain are relatively invariant in amino acid sequence and are known as the constant heavy (CH) region and the constant light (CL) region. In the case of an IgG antibody, the light chain contains one variable region (VL) and one constant region (CL). The IgG heavy chain contains a variable region (VH), a first constant region (CH1), a hinge region, a second constant region (CH2), and a third constant region (CH3). In IgE and IgM antibodies, the heavy chain contains an additional constant region (CH4).

[0147] Antibodies may include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fvs (scFvs), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to as "antibody mimetics"), and antigen-binding fragments of any of the above. In certain embodiments, the antibodies described herein refer to polyclonal antibody populations.

[0148] As used herein, the term "Fc fragment" refers to one or more fragments of the Fc region that retain an Fc function and / or activity described herein, such as binding to an Fc receptor. As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, scFv fragments, dAb fragments (Ward et al., (1989) Nature 341:544-546), and isolated complementarity-determining regions (CDRs). These antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments can be screened for utility in the same manner as intact antibodies.

[0149] In some aspects, the present invention provides an antibody or fragment thereof that binds to human ADGRE2, comprising a human heavy constant region and / or a human light constant region, wherein the human heavy constant region comprises an isotype variant comprising the Fc region of human IgG1, human IgG2, human IgG3, or human IgG4.

[0150] In a further aspect, the present invention provides a humanized antibody or fragment thereof that binds to human ADGRE2, wherein the antibody comprises a variant human IgG Fc region that includes the amino acid substitution S324N, which replaces serine at amino acid position 324 of the parent antibody with asparagine, whereas the antibody comprising the variant human IgG Fc region exhibits improved complement-dependent cytotoxicity (CDC) compared to the parent antibody.

[0151] The antibody or fragment can be produced by any method known in the art for synthesizing antibodies (see, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Brinkman et al., 1995, J. Immunol. Methods 182:41-50; WO92 / 22324; WO98 / 46645). Chimeric antibodies can be produced, for example, using the method described in Morrison, 1985, Science 229:1202, and humanized antibodies can be produced, for example, by the method described in U.S. Pat. No. 6,180,370.

[0152] Further compositions and methods described herein are bispecific and multivalent antibodies, e.g., as described in Segal et al., J. Immunol. Methods 248:1-6 (2001), and Tutt et al., J. Immunol. 147:60 (1991).

[0153] Engineered antigen-binding regions In some embodiments, the binding moiety is or comprises an antibody (e.g., an IgG antibody, e.g., an IgG1, IgG2, or IgG3 antibody), or antigen-binding fragment, that has been engineered to bind to a target (i.e., an antigen) in an altered manner (e.g., in a pH-sensitive manner, e.g., in a higher or lower pH-sensitive manner) compared to a reference antibody or antigen-binding fragment. For example, an antibody can be engineered by modifying (e.g., by adding, deleting, or substituting) amino acids within one or more antibody CDRs and / or at positions involved in the antibody's CDR structure. Exemplary, non-limiting sites in an antibody that can be modified include the following (amino acid positions are indicated according to Kabat numbering (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH)):

[0154] Heavy chain: H27, H31, H32, H33, H35, H50, H58, H59, H61, H62, H63, H64, H65, H99, H100b, and H102.

[0155] Light chain: L24, L27, L28, L32, L53, L54, L56, L90, L92, and L94.

[0156] In some embodiments, one or more of these disclosed amino acids can be substituted with histidine, arginine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, or glutamine. Without wishing to be bound by theory, it is believed that substituting histidine for one or more of the amino acids at these positions can result in antibodies with pH-dependent antigen binding properties. In some embodiments, non-histidine residues are substituted with histidine residues. In some embodiments, histidine residues are substituted with non-histidine residues. Additional engineered antigen-binding regions include, for example, those described in US Patent Application Publication No. 20110229489.

[0157] Engineered constant regions In some cases, the binding moiety is or comprises an antibody constant region, Fc region, or Fc fragment that binds to one or more Fc receptors (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB, FcγRIV, or FcRn receptors). In some embodiments, the constant region, Fc region, or Fc fragment is engineered to bind to a target (e.g., an Fc receptor) in an altered manner (e.g., in a pH-sensitive manner, e.g., in a higher or lower pH-sensitive manner) relative to a reference constant region, Fc region, or Fc fragment.

[0158] In some examples, the binding moiety may be or comprise the constant region, Fc region, or Fc fragment of an IgG antibody that has been engineered to contain amino acid additions, deletions, or substitutions of one or more of the amino acid residues described herein (e.g., 251-256, 285-290, 308-314, 385-389, and 428-436 (Kabat numbering (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH))).

[0159] Production of ADGRE2 antibodies and their fragments In some embodiments, the antibodies or fragments thereof described herein are engineered by mutagenesis using known techniques to include one or more binding moieties that exhibit binding to one or more targets. For example, the sequence of a reference polypeptide (e.g., a therapeutic antibody or a therapeutic fusion protein) can be obtained, and one or more amino acid residues can be added, deleted, or substituted. In some embodiments, one or more amino acid residues are substituted with histidine, arginine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, or glutamine. In some embodiments, one or more amino acids are substituted with histidine.

[0160] In some embodiments, without wishing to be bound by theory, it is believed that substituting an amino acid residue with histidine can insert a protonation site and increase the pH sensitivity of the binding moiety. Polypeptides can be produced using standard methods and assayed for binding to the target of interest described herein. Additional methods for increasing the pH sensitivity of binding moieties are described, for example, in Sarkar et al., Nature Biotechnology 20:908-913 (2002), Murtaugh et al., Protein Science 20:1619-1631 (2011), and U.S. Patent Application Publication No. 20110229489.

[0161] In some embodiments, a first target of interest is selected, an antibody that selectively binds to the target is provided, obtained, and / or produced (e.g., using known methods described herein), one or more amino acids in the antigen binding region and / or Fc region are substituted (e.g., with histidine, arginine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, or glutamine), and the pH sensitivity of binding to the target (and additionally or alternatively, to FcRn) is determined.

[0162] In some embodiments, a polypeptide that naturally binds to a target of interest is provided, obtained, and / or produced. The polypeptide is conjugated to an Fc region or Fc fragment described herein (e.g., that binds to FcRn with the desired binding affinity) using known methods. For example, the polypeptide and Fc region or Fc fragment can be conjugated by chemical means or by recombinant expression as a fusion protein. Additionally or alternatively, one or more amino acids of the polypeptide can be substituted (e.g., with histidine, arginine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, or glutamine), and the pH sensitivity of the binding of the polypeptide and the target is determined.

[0163] In some embodiments, the antibodies or fragments thereof described herein are engineered to contain one or more binding moieties identified and / or selected by screening. For example, antigen-binding moieties that bind to an antigen can be identified using libraries that express antigen-binding moieties, such as phage libraries. Methods for screening recombinant antibody libraries are known (see, e.g., Hoogenboom, Nature Biotech. 23:1105-1116 (2005); U.S. Patent No. 5,837,500; U.S. Patent No. 5,571,698; WO2012 / 044831).

[0164] PEGylation In certain embodiments, the anti-ADGRE2 antibodies described herein can be PEGylated to contain mono- or poly (e.g., 2-4) PEG moieties. Such PEGylated antibodies can exhibit increased half-life compared to a non-PEGylated reference antibody, e.g., an antibody having the same amino acid sequence but a different, different amount of PEGylation, or no PEGylation.

[0165] PEGylation can be carried out by any suitable reaction known in the art. A method for preparing a PEGylated protein generally includes: (a) reacting a polypeptide with polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions that allow the polypeptide to be bound to one or more PEG groups; and (b) obtaining a reaction product(s). Generally, the reaction conditions can be determined on a case-by-case basis based on known parameters and desired results.

[0166] There are numerous methods of PEG attachment available to one of skill in the art. For example, PEGylation of the antibodies or fragments thereof described herein can be carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule.

[0167] Measuring binding moiety-target interactions The binding properties of an antibody or fragment thereof described herein (e.g., an anti-ADGRE2 antibody described herein) to a target (e.g., ADGRE2) can be measured by methods known in the art, such as one of the following: BIACORE analysis, enzyme-linked immunosorbent assay (ELISA), X-ray crystallography, sequence analysis, and scanning mutagenesis. The binding interaction of an antibody with ADGRE2 and / or FcRn can be analyzed using surface plasmon resonance (SPR). SPR, or biomolecular interaction analysis (BIA), detects biospecific interactions in real time, without labeling any of the interactants. A change in mass at the binding surface of the BIA chip (indicative of a binding event) results in a change in the refractive index of light near the surface. The change in refractive index generates a detectable signal, which is measured as an indicator of a real-time reaction between biomolecules. Methods for using SPR are described, for example, in U.S. Patent No. 5,641,640, Raether (1988) Surface Plasmons Springer Verlag, Sjolander and Urbaniczky (1991) Anal. Chem. 63:2338-2345, Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705, and online materials provided by BIAcore International AB (Uppsala, Sweden). In addition, the KinExA® (Kinexchange Exclusion Assay) assay available from Sapidyne Instruments (Boise, Id.) can also be used.

[0168] Information from SPR can be used to determine the equilibrium dissociation constant (K) for the binding of a binding moiety (e.g., ADGRE2 and / or an anti-ADGRE2 antibody to FcRn) to a target. D ), and K on and K. offIt can be used to provide accurate and quantitative measurements of kinetic parameters, including K. Such data can be used to compare different molecules. Information from SPR can also be used to develop structure-activity relationships (SAR). For example, the kinetic and equilibrium binding parameters of a particular binding moiety to a target at various pH levels can be evaluated. At particular pH levels, specific binding parameters, e.g., high affinity, low affinity, and slow K, can be determined. off A variant amino acid at a given position that correlates with the amino acid sequence can be identified.

[0169] Treatment method In some embodiments, the antibodies or fragments thereof described herein (e.g., the anti-ADGRE2 antibodies described herein) are used in methods of treating one or more ADGRE2-associated conditions. In some embodiments, the antibodies or fragments thereof described herein (e.g., the anti-ADGRE2 antibodies described herein) are for use as pharmaceuticals. ADGRE2-associated conditions can include, but are not limited to, conditions caused by ADGRE2 expression, conditions involving symptoms attributable in whole or in part to ADGRE2 expression, or conditions known to occur in association with ADGRE2 expression.

[0170] In some aspects, the present invention provides methods for treating cancer, comprising administering an agent that specifically binds to ADGRE2 (e.g., an anti-ADGRE2 antibody or fragment thereof described herein). Cancer is a broad group of diverse diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth can lead to the formation of malignant tumors that can invade neighboring tissues and metastasize to distant parts of the body via the lymphatic system or bloodstream. In some embodiments, "cancer" or "cancerous tissue" includes solid tumors. Examples of cancers that can be treated by the methods of the present invention include, but are not limited to, cancers of the immune system, including lymphoma, leukemia, myeloma, and other white blood cell malignancies. In some embodiments, the cancer is acute myeloid leukemia.

[0171] In some embodiments, the lymphoma is selected from the group consisting of acute lymphoblastic leukemia (ALL), AIDS-related lymphoma, ALK-positive large B-cell lymphoma, Burkitt's lymphoma, chronic lymphocytic leukemia (CLL), classical Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, intravascular large B-cell lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman's disease, lymphomatoid granulomatosis, lymphomatoid arthritis ... The lymphoplasmacytic lymphoma is selected from the group consisting of lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), nodal marginal zone B-cell lymphoma (NMZL), nodal lymphocyte-predominant Hodgkin lymphoma, non-Hodgkin lymphoma, plasmablastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, splenic marginal zone lymphoma (SMZL), and Waldenstrom's macroglobulinemia. In some embodiments, the lymphoma is selected from the group consisting of acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, mantle cell lymphoma (MCL), marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), and non-Hodgkin's lymphoma. In some embodiments, the lymphoma is non-Hodgkin's lymphoma. In some embodiments, the cancer is relapsed and refractory acute myeloid leukemia.

[0172] In certain embodiments, the tumor is cancer. In certain embodiments, the tumor is a hematological cancer. In certain embodiments, the tumor is selected from the group consisting of multiple myeloma, leukemia, lymphoma, and myeloid malignancies. Non-limiting examples of hematological cancers include multiple myeloma, leukemia, and lymphoma. Non-limiting examples of leukemia include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute promyelocytic leukemia (APL), mixed phenotype acute leukemia (MLL), hairy cell leukemia, and B-cell prolymphocytic leukemia. The lymphoma can be Hodgkin's lymphoma or non-Hodgkin's lymphoma. Non-limiting examples of myeloid malignancies include myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), myeloid / lymphoid neoplasms (e.g., myeloid / lymphoid neoplasms with eosinophilia and rearrangements of platelet-derived growth factor receptor alpha (PDGFRA), platelet-derived growth factor receptor beta (PDGFRB), or fibroblast growth receptor 1 (FGFR1), or myeloid / lymphoid neoplasms with PCM1-JAK2), acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasm, B-lymphoblastic leukemia / lymphoma, and T-lymphoblastic leukemia / lymphoma. In certain embodiments, the myeloid malignancy comprises a myelodysplastic syndrome.

[0173] In certain embodiments, the tumor is a B-cell malignancy. Non-limiting examples of B-cell malignancies include B-cell lymphoma (BCL), B-cell acute lymphocytic leukemia (ALL), B-cell chronic lymphocytic leukemia (CLL), multiple myeloma (MM), CLL with Richter's transformation, and CNS lymphoma. B-cell lymphomas include B-cell non-Hodgkin's lymphoma (NHL) and B-cell Hodgkin's lymphoma.

[0174] In various embodiments, administration of an antibody or fragment thereof described herein (e.g., an anti-ADGRE2 antibody or fragment thereof described herein) results in a reduction in the prevalence, frequency, level, and / or amount of one or more symptoms or biomarkers of an ADGRE2-associated condition described herein or known in the art, e.g., a reduction of at least about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, 95%, about 99%, or about 100% in one or more symptoms or biomarkers compared to a previous measurement or reference value in the subject.

[0175] In some embodiments, administration of an antibody or fragment thereof described herein (e.g., an anti-ADGRE2 antibody described herein) to a subject with cancer results in a greater reduction or amelioration of one or more symptoms or biomarkers of cancer than a reference antibody, e.g., an antibody that cross-competes for ADGRE2 binding, under comparable conditions.

[0176] In some embodiments, the antibodies or fragments thereof described herein (e.g., anti-ADGRE2 antibodies described herein) exhibit a reduced effective dose compared to a reference protein (e.g., an antibody that cross-competes for ADGRE2 binding). For example, the effective dose of an anti-ADGRE2 antibody described herein can be, for example, less than 1,000 mg / dose, e.g., 900 mg / dose, 800 mg / dose, 700 mg / dose, 600 mg / dose, 500 mg / dose, 550 mg / dose, 400 mg / dose, 350 mg / dose, 300 mg / dose, 200 mg / dose, 100 mg / dose, 50 mg / dose, 25 mg / dose, or less. In certain examples, the effective dose of an anti-ADGRE2 antibody disclosed herein is lower than the effective or recommended or approved dose of the reference antibody, which can be, for example, 900 mg / dose or 600 mg / dose. Alternatively, or in combination with the dosages disclosed herein, the anti-ADGRE2 antibodies described herein can be effectively or usefully administered less than once per week, e.g., less than once per week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or year. In certain instances, the effective or useful administration frequency of the anti-ADGRE2 antibodies disclosed herein is lower than the effective or recommended or approved administration frequency of a reference antibody, which can be administered weekly (e.g., at a dosage of 300-600 mg depending on the subject's weight) or every two weeks (e.g., at a dosage of 300-1200 mg depending on the subject's weight).

[0177] In some embodiments, an antibody or fragment thereof described herein (e.g., an anti-ADGRE2 antibody described herein) can be administered at a lower dose compared to a reference protein, e.g., an antibody that cross-competes for ADGRE2 binding, to achieve an equivalent, equivalently effective, comparably effective, or substantially effective outcome, when the anti-ADGRE2 antibody is administered in the same, equivalent, or substantially equivalent formulation and / or by the same, equivalent, or substantially equivalent route of administration as the reference (e.g., an antibody that cross-competes for ADGRE2 binding). In some embodiments, an anti-ADGRE2 antibody described herein can be administered at extended intervals compared to a reference antibody (e.g., an antibody that cross-competes for ADGRE2 binding) to achieve an equivalent, equivalently effective, comparably effective, or substantially effective outcome, when the anti-ADGRE2 antibody is administered in the same, equivalent, or substantially equivalent formulation and / or by the same, equivalent, or substantially equivalent route of administration as the reference. In some embodiments, the anti-ADGRE2 antibodies described herein can be administered at a reduced number of unit doses and / or for a shortened treatment period compared to a reference antibody to achieve an equivalent, equally effective, equally effective, or substantially effective outcome, when the anti-ADGRE2 antibody is administered in the same, equivalent, or substantially equivalent formulation as the reference (e.g., an antibody that cross-competes for ADGRE2 binding) and / or by the same, equivalent, or substantially equivalent route of administration as the reference.

[0178] According to some such embodiments, an administered dose of an anti-ADGRE2 antibody described herein may be less likely to induce an adverse reaction, e.g., an adverse immune reaction, when administered to a subject than an effective dose of a reference antibody, e.g., an antibody that cross-competes for ADGRE2 binding. Thus, in various embodiments, an anti-ADGRE2 antibody disclosed herein may be less likely to induce an adverse reaction or side effect per administered unit of activity than a reference antibody. In various embodiments, an anti-ADGRE2 antibody disclosed herein may be less likely to induce an adverse reaction or side effect of a particular severity per administered unit of activity than a reference antibody. In various embodiments, an anti-ADGRE2 antibody disclosed herein may induce one or more adverse reactions or side effects to a lesser extent or in fewer patients per administered unit of activity than a reference antibody. Examples of possible adverse reactions or side effects associated with the administration of antibodies capable of binding to ADGRE2 may include headache, nasopharyngitis, back pain, nausea, diarrhea, hypertension, upper respiratory tract infection, abdominal pain, vomiting, anemia, cough, peripheral edema, and / or urinary tract infection.

[0179] In some embodiments, upon administration to a subject (e.g., in a single dose), an antibody or fragment thereof described herein (e.g., an anti-ADGRE2 antibody described herein) is measured at an increased level in plasma at a defined time (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days) after administration compared to the level of a control (e.g., an antibody that cross-competes for ADGRE2 binding) at the same defined time. For example, at a given time point after administration of a single dose, the level of an anti-ADGRE2 antibody described herein is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, or about 500% higher than the corresponding level of the reference antibody.

[0180] In some embodiments, an antibody or fragment thereof described herein (e.g., an anti-ADGRE2 antibody described herein) is measured at an increased level in plasma at a defined time (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days) after administration (e.g., of a single dose) compared to a control level at the same defined time. For example, at a given time point after administration, the level of an anti-ADGRE2 antibody described herein is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, or about 500% higher than the corresponding level of the reference antibody.

[0181] In some embodiments, the anti-ADGRE2 antibodies described herein have an increased half-life (e.g., compared to a control, e.g., a reference antibody, e.g., an antibody that cross-competes for ADGRE2 binding), and therefore the anti-ADGRE2 antibody can be administered to a subject at extended intervals. For example, the anti-ADGRE2 antibody can be administered once every week, every two weeks, every three weeks, every four weeks, every six weeks, every eight weeks, or longer.

[0182] In some embodiments, a therapeutically effective amount of an antibody or fragment thereof described herein (e.g., an anti-ADGRE2 antibody described herein) is about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% of the effective amount of a reference therapeutic protein, e.g., an antibody that cross-competes for ADGRE2 binding. In some embodiments, a single dose of an anti-ADGRE2 antibody described herein achieves the same therapeutic effect as two or more doses of the reference antibody.

[0183] In some embodiments, an antibody or fragment thereof described herein (e.g., an anti-ADGRE2 antibody described herein) is administered at a dose that is approximately 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% of the concentration of the target antigen (e.g., ADGRE2) in the subject.

[0184] In some embodiments, an antibody or fragment thereof described herein (e.g., an anti-ADGRE2 antibody described herein) can be physically introduced into a subject using any of a variety of methods and delivery systems known to those of skill in the art. Exemplary routes of administration of the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" refers to a method of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. In some embodiments, a formulation is administered by a parenteral route, such as a topical, epidermal, or mucosal route of administration, e.g., intranasally, orally, intravaginally, rectally, sublingually, or topically. Also, administration can be, for example, once, multiple times, and / or over one or more extended periods of time.

[0185] In some embodiments, the antibodies described herein, or fragments thereof (e.g., anti-ADGRE2 antibodies described herein), can be used in a number of diagnostic and therapeutic applications. For example, detectably labeled versions of the engineered antibodies described herein can be used in assays to detect the presence or amount of ADGRE2 in a sample (e.g., a biological sample). The engineered antibodies described herein can be used in in vitro assays to study binding to ADGRE2. In some embodiments, the anti-ADGRE2 antibodies described herein can be used as positive controls in assays designed to identify additional novel compounds that would otherwise be useful in treating ADGRE2-associated disorders. For example, the anti-ADGRE2 antibodies described herein can be used as positive controls in assays to identify additional compounds (e.g., small molecules, aptamers, or antibodies) that bind to ADGRE2.

[0186] The antibodies or antigen-binding fragments thereof described herein can be used to monitor subjects, e.g., subjects who have, are suspected of having, are at risk of developing, or are undergoing treatment for one or more ADGRE2-associated conditions. Monitoring can include determining the amount or activity of ADGRE2 in the subject's serum, e.g., the subject's serum. In some embodiments, the evaluation is performed at least 1 hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 day, 2 days, 4 days, 10 days, 13 days, 20 days, or more, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks, or more, after administration of an anti-ADGRE2 antibody described herein. Subjects can be evaluated at one or more of the following time periods: before the start of treatment; during treatment; or after one or more components of treatment have been administered. Evaluation can include assessing the need for further treatment, e.g., assessing whether the dosage, frequency of administration, or duration of treatment should be changed. It can also include assessing the need to add or subtract selected therapies, for example, adding or subtracting any treatments for ADGRE2-associated disorders described herein.

[0187] Formulation and Administration In various embodiments, an antibody or antigen-binding fragment thereof described herein (e.g., an anti-ADGRE2 antibody described herein) can be incorporated into a pharmaceutical composition. Such a pharmaceutical composition can be useful, for example, for the prevention and / or treatment of a disease, e.g., an ADGRE2-associated disorder. Pharmaceutical compositions can be formulated by methods known to those skilled in the art (such as those described in Remington's Pharmaceutical Sciences, 17th edition, ed. Alfonso R. Gennaro, Mack Publishing Company, Easton, Pa. (1985)).

[0188] The appropriate means of administration can be selected based on the age and condition of the subject. A single dose of a pharmaceutical composition containing an antibody or fragment thereof described herein (e.g., an anti-ADGRE2 antibody described herein) can be selected from the range of 0.001 to 1,000 mg per kg of body weight. The dose can also be selected from the range of 0.001 to 100,000 mg per kg of body weight, although the present disclosure is not limited to such ranges. The dose and administration method vary depending on the patient's body weight, age, condition, etc., and can be appropriately selected by one skilled in the art as needed.

[0189] In various embodiments, pharmaceutical compositions can be formulated to include a pharmaceutically acceptable carrier or excipient. Examples of pharmaceutically acceptable carriers include, but are not limited to, any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. The compositions of the present invention can include pharmaceutically acceptable salts, such as acid addition salts or base addition salts.

[0190] In various embodiments, compositions comprising the antibodies described herein, e.g., sterile injectable preparations, can be formulated according to conventional pharmaceutical practice using distilled water for injection as a vehicle. For example, an isotonic solution containing saline or glucose and other supplements such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and the like, optionally in combination with a suitable solubilizing agent, e.g., alcohols such as ethanol and polyhydric alcohols such as propylene glycol or polyethylene glycol, non-ionic surfactants such as Polysorbate 80™, HCO-50, and the like, can be used as an injectable aqueous solution.

[0191] As disclosed herein, pharmaceutical compositions may be in any form known in the art, including, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories.

[0192] The selection or use of any particular form may depend, in part, on the intended mode of administration and therapeutic application. For example, compositions, including compositions intended for systemic or local delivery, may be in the form of an injection or infusion. For example, compositions may be formulated for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection). As used herein, parenteral administration refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intranasal, intraocular, intrapulmonary, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid, and intrasternal injection and infusion.

[0193] The route of administration can be parenteral, e.g., by injection, intranasal, pulmonary, or transdermal. Administration can be systemic or local, by intravenous, intramuscular, intraperitoneal, or subcutaneous injection.

[0194] In various embodiments, the pharmaceutical compositions of the present invention can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for stable storage at high concentrations. Sterile injectable solutions can be prepared by incorporating the required amount of the compositions described herein with one or a combination of the above-listed ingredients in an appropriate solvent, as needed, followed by filtration sterilization. Generally, dispersions are prepared by combining the compositions described herein with a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for preparing sterile injectable solutions, preparation methods include vacuum drying and freeze-drying, which yield a powder of the compositions described herein and any desired additional ingredients (see below) from a previously sterile-filtered solution of the compositions described herein and any desired additional ingredients. The proper fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prolonged absorption of injectable compositions can be achieved by including an agent that delays absorption, such as monostearate salts and gelatin, in the composition.

[0195] Pharmaceutical compositions can be administered parenterally in the form of injectable formulations, including sterile solutions or suspensions in water or another pharmaceutically acceptable liquid. For example, pharmaceutical compositions can be formulated by suitably combining a therapeutic molecule with a pharmaceutically acceptable vehicle or medium, such as sterile water and saline, vegetable oil, emulsifiers, suspending agents, surfactants, stabilizers, flavoring excipients, diluents, vehicles, preservatives, and binders, followed by mixing in a unit dosage form required by generally accepted pharmaceutical practice. The amount of active ingredient contained in the pharmaceutical formulation is such that a suitable dosage within the specified range is provided. Non-limiting examples of oily liquids include sesame oil and soybean oil, which may be combined with benzyl benzoate or benzyl alcohol as a solubilizer. Other items that may be included are buffers such as phosphate buffer or sodium acetate buffer, soothing agents such as procaine hydrochloride, stabilizers such as benzyl alcohol or phenol, and antioxidants. The formulated injections can be packaged in suitable ampoules.

[0196] In some embodiments, the compositions can be formulated for storage at temperatures below 0° C. (e.g., −20° C. or −80° C.). In some embodiments, the compositions can be formulated for storage at 2-8° C. (e.g., 4° C.) for up to 2 years (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 1½ years, or 2 years). Thus, in some embodiments, the compositions described herein are storage-stable at 2-8° C. (e.g., 4° C.) for at least 1 year.

[0197] In certain instances, the pharmaceutical composition can be formulated as a solution, e.g., in some embodiments, the composition can be formulated as a buffered solution at a suitable concentration, suitable for storage at 2-8°C (e.g., 4°C).

[0198] The composition comprising one or more engineered antibodies described herein can be formulated into an immunoliposome composition. Such formulations can be prepared by methods known in the art. Liposomes with enhanced circulation time are disclosed, for example, in U.S. Patent No. 5,013,556.

[0199] In certain embodiments, the composition can be formulated with a carrier that protects the compound from rapid release, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations are known in the art. See, for example, JR Robinson (1978) "Sustained and Controlled Release Drug Delivery Systems," Marcel Dekker, Inc., New York.

[0200] In some embodiments, the compositions can be formulated into compositions suitable for pulmonary administration (e.g., administration by inhaler or nebulizer) to mammals, such as humans. Methods for formulating such compositions are well known in the art. Dry powder inhalation formulations and systems suitable for administering the formulations are also known in the art. Pulmonary administration can be oral and / or nasal. Examples of pharmaceutical devices for pulmonary delivery include metered-dose inhalers, dry powder inhalers (DPIs), and nebulizers. For example, the compositions described herein can be administered to the lungs of a subject by a dry powder inhaler. These inhalers are propellant-free devices that deliver dispersible and stable dry powder formulations to the lungs. Dry powder inhalers are well known in the medical arts and include, but are not limited to, TURBOHALER® (AstraZeneca; London, England), AIR® inhaler (ALKERMES®; Cambridge, Mass.), ROTHALER® (GlaxoSmithKline; London, England), and ECLIPSE™ (Sanofi-Aventis; Paris, France). See also, e.g., PCT Publication Nos. WO 04 / 026380, WO 04 / 024156, and WO 01 / 78693. DPI devices have been used for pulmonary administration of polypeptides such as insulin and growth hormone. In some embodiments, the compositions described herein can be administered intrapulmonary by a metered-dose inhaler. These inhalers rely on propellants to deliver discrete doses of the compound to the lungs. Additional devices and methods of pulmonary administration are described, for example, in US Patent Application Publication Nos. 20050271660 and 20090110679, the disclosures of each of which are incorporated herein by reference in their entireties.

[0201] In some embodiments, the composition can be formulated for delivery to the eye, for example, in the form of a pharmaceutically acceptable solution, suspension or ointment.The preparation used for eye treatment can be in the form of a sterile aqueous solution, which contains additional components such as but not limited to preservatives, buffers, tonicity agents, antioxidants and stabilizers, non-ionic wetting or clarifying agents, and thickeners.The preparations described herein can be administered topically to the eye of the subject who needs treatment (for example, the subject who suffers from AMD) by conventional methods, for example, in the form of drops or by immersing the eye in a treatment solution containing one or more compositions.

[0202] In certain embodiments, various devices for introducing drugs into the vitreous cavity of the eye can be suitable for administering the compositions described herein.For example, US Patent Application Publication No. 2002 / 0026176 describes a drug-containing plug that can be inserted through the sclera, so that it protrudes into the vitreous cavity and delivers pharmaceuticals into the vitreous cavity.In another example, US Patent No. 5,443,505 describes an implantable device for introducing into the suprachoroidal space or avascular area for sustained drug release into the interior of the eye.US Patent No. 5,773,019 and US Patent No. 6,001,386 each disclose an implantable drug delivery device that can be attached to the scleral surface of the eye. Additional methods and devices for delivering therapeutic agents to the eye (e.g., delivery via transscleral patches and contact lenses) are described, for example, in Ambati and Adamis (2002) Prog Retin Eye Res 21(2):145-151, Ranta and Urtti (2006) Adv Drug Delivery Rev 58(11):1164-1181, Barocas and Balachandran (2008) Expert Opin Drug Delivery 5(1):1-10(10), Gulsen and Chauhan (2004) Invest Opthalmol Vis Sci 45:2342-2347, Kim et al. (2007) Ophthalmic Res 39:244-254, and PCT Publication No. WO 04 / 073551, the disclosures of which are incorporated herein by reference in their entireties.

[0203] In certain embodiments, administration of an antibody described herein is achieved by administering to a subject a nucleic acid encoding the antibody. Nucleic acids encoding the therapeutic antibodies described herein can be incorporated into genetic constructs used as part of gene therapy protocols to deliver nucleic acids that can be used to express and produce the antibody intracellularly. Such component expression constructs can be administered in a therapeutically effective carrier, e.g., any formulation or composition that can effectively deliver the component gene to cells in vivo. Approaches include viral vectors, including recombinant retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, and herpes simplex virus-1 (HSV-1), or insertion of the gene of interest into recombinant bacterial or eukaryotic plasmids. Viral vectors can be directly transfected into cells. Plasmid DNA can be delivered with the aid of, for example, cationic liposomes (lipofectin) or derivatized polylysine conjugates, gramicidin S, artificial viral envelopes, or other such intracellular carriers, and can also be delivered by direct injection or CaPO precipitation of the genetic construct (see, e.g., WO 04 / 060407).Examples of suitable retroviruses include pLJ, pZIP, pWE, and pEM, which are known to those skilled in the art (see, e.g., Eglitis et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc Natl Acad Sci USA 85:6460-6464; Wilson et al. (1988) Proc Natl Acad Sci USA 85:3014-3018; Armentano et al. (1990) Proc Natl Acad Sci USA 87:6141-6145; Huber et al. (1991) Proc Natl Acad Sci USA 88:8039-8043; Ferry et al. (1991) Proc Natl Acad Sci USA 88:8377-8381; Chowdhury et al. (1991) Proc Natl Acad Sci USA 88:8377-8381). (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc Natl Acad Sci USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc Natl Acad Sci USA 89:10892-10895; Hwu et al. (1993) J Immunol 150:4104-4115; U.S. Patent Nos. 4,868,116 and 4,980,286; and PCT Publication Nos. WO89 / 07136, WO89 / 02468, WO89 / 05345, and WO92 / 07573. Another viral gene delivery system utilizes adenovirus-derived vectors (see, e.g., Berkner et al. (1988) BioTechniques 6:616, Rosenfeld et al. (1991) Science 252:431-434, and Rosenfeld et al. (1992) Cell 68:143-155). Suitable adenovirus vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7, etc.) are known to those skilled in the art.Yet another viral vector system useful for delivering a gene of interest is the adeno-associated virus (AAV). See, e.g., Flotte et al. (1992) Am J Respir Cell Mol Biol 7:349-356, Samulski et al. (1989) J Virol 63:3822-3828, and McLaughlin et al. (1989) J Virol 62:1963-1973.

[0204] In various embodiments, subcutaneous administration may be achieved by a device such as a syringe, a prefilled syringe, an auto-injector (e.g., disposable or reusable), a pen-type injector, a patch injector, a wearable injector, a portable syringe infusion pump with a subcutaneous infusion set, or other device for combining with an antibody drug for subcutaneous injection.

[0205] The injection system of the present disclosure can use a delivery pen, as described in U.S. Patent No. 5,308,341. Pen devices, most commonly used for self-delivery of insulin to diabetic patients, are well known in the art. Such devices can include at least one injection needle (e.g., a 31-gauge needle approximately 5-8 mm in length) and are typically pre-filled with one or more therapeutic unit doses of a therapeutic solution, useful for rapid delivery of the solution to the subject with as little pain as possible. One drug delivery pen includes a vial holder that can receive a vial of a therapeutic or other medication. The pen can be a completely mechanical device or can be combined with electronic circuitry to accurately set and / or indicate to the user the dosage of medication to be injected. See, e.g., U.S. Patent No. 6,192,891. In some embodiments, the needle of the pen device is disposable, and the kit includes one or more disposable replacement needles. Pen devices suitable for delivering any one of the compositions described in the present invention are also described, for example, in U.S. Patent Nos. 6,277,099, 6,200,296, and 6,146,361, the disclosures of each of which are incorporated herein by reference in their entirety. Microneedle-based pen devices are described, for example, in U.S. Patent No. 7,556,615, the disclosure of which is incorporated herein by reference in its entirety. See also the Precision Pen Injector (PPI) device, MOLLY™, manufactured by Scandinavian Health Ltd.

[0206] In some embodiments, the compositions described herein can be therapeutically delivered to a subject by local administration. As used herein, "local administration" or "local delivery" can refer to delivery of a composition or agent that does not rely on transport to its intended target tissue or site via the vascular system. For example, the composition can be delivered by injection or implantation of the composition or agent, or by injection or implantation of a device containing the composition or agent. In certain embodiments, after local administration near the target tissue or site, the composition or agent, or one or more components thereof, can diffuse to the intended target tissue or site that is not the administration site.

[0207] In some embodiments, the compositions provided herein are in unit dosage form, and this unit dosage form can be suitable for self-administration.Such unit dosage form can typically be provided in a container such as a vial, a cartridge, a pre-filled syringe or a disposable pen.Administration device such as the administration device described in U.S. Patent No. 6,302,855 can also be used with the injection system described herein, for example.

[0208] The appropriate dose of a composition described herein capable of treating or preventing a subject's disorder may depend on various factors, including, for example, the age, sex, and weight of the subject being treated, as well as the particular inhibitor compound used. For example, a different dose of a composition comprising an antibody described herein may be required to treat a subject with an ADGRE2-associated disorder compared to a dose of a different formulation of that antibody. Other factors that affect the dose administered to a subject include, for example, the type or severity of the disorder. For example, a subject with one ADGRE2-associated disorder may require a different dosage than a subject with another ADGRE2-associated disorder. Other factors may include, for example, other medical disorders concurrently or previously affecting the subject, the subject's overall health, the subject's genetic predisposition, diet, duration of administration, excretion rate, drug combinations, and any other additional therapies administered to the subject. It should also be understood that the specific dosage and treatment regimen for any particular subject may also be adjusted based on the judgment of the treating physician.

[0209] The compositions described herein can be administered as a fixed dose or in milligrams per kilogram (mg / kg) doses. In some embodiments, the dose can be selected to reduce or avoid antibody production or other host immune responses to one or more of the antibodies or antigen-binding fragments thereof in the composition. While not intended to be limiting in any way, exemplary dosages of antibodies, such as the compositions described herein, include, for example, 1-1000 mg / kg, 1-100 mg / kg, 0.5-50 mg / kg, 0.1-100 mg / kg, 0.5-25 mg / kg, 1-20 mg / kg, and 1-10 mg / kg. Exemplary dosages of the compositions described herein include, but are not limited to, 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 4 mg / kg, 8 mg / kg, or 20 mg / kg.

[0210] The pharmaceutical solution can contain a therapeutically effective amount of a composition described herein. Such an effective amount can be readily determined by one of skill in the art based, in part, on the effect of the administered composition or, if multiple agents are used, on the combined effect of the composition and one or more additional active agents. A therapeutically effective amount of a composition described herein can also vary depending on factors such as the individual's medical condition, age, sex, and weight, as well as the ability of the composition (and one or more additional active agents) to elicit a desired response in the individual, e.g., improvement in at least one condition parameter, e.g., improvement in at least one symptom of an ADGRE2-associated disorder. For example, a therapeutically effective amount of a composition described herein can inhibit (reduce the severity or eliminate the onset of) and / or prevent a particular disorder and / or any one of the symptoms of a particular disorder known in the art or described herein. A therapeutically effective amount is also one in which any toxic or adverse effects of the composition are outweighed by the therapeutically beneficial effects.

[0211] Suitable human doses of any of the compositions described herein can be further evaluated, for example, in a Phase I dose-escalation study. See, e.g., van Gurp et al. (2008) Am J Transplantation 8(8):1711-1718, Hanouska et al. (2007) Clin Cancer Res 13(2, part 1):523-531, and Hetherington et al. (2006) Antimicrobial Agents and Chemotherapy 50(10):3499-3500.

[0212] Toxicity and therapeutic efficacy of the compositions can be determined by known pharmaceutical procedures in cell cultures or experimental animals (e.g., animal models of any ADGRE2-associated disorder). These procedures can be used, for example, in LD 50 (lethal dose for 50% of the population) and ED 50 It can be used to determine the therapeutically effective dose in 50% of the population. The dose ratio between toxic and therapeutic effects is the therapeutic index, which is the LD50 / ED 50 The therapeutic index can be expressed as a ratio of 0 to 1. Compositions described herein that exhibit a high therapeutic index are preferred. While compositions that exhibit toxic side effects may be used, care must be taken to design a delivery system that targets such compounds to the site of affected tissue, minimizing potential damage to normal cells and thereby mitigating side effects.

[0213] Those skilled in the art will appreciate that the data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. Appropriate dosages of the compositions described herein will generally be administered at an ED level with little or no toxicity. 50 The circulating concentration of the composition is within a range comprising 100 mg / mL of the compound. Dosages can vary within this range depending on the dosage form employed and / or the route of administration used. For the compositions described herein, a therapeutically effective amount can be initially estimated from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes 100 mg / mL (i.e., the concentration of antibody that achieves half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography. In some embodiments, for example, when local administration (e.g., to the eye or joint) is desired, cell culture or animal models can be used to determine the dose necessary to achieve a therapeutically effective concentration within the local site.

[0214] Combination therapy In various embodiments, the anti-ADGRE2 antibodies described herein can be included in a course of treatment that further includes administering to the subject at least one additional agent. In various embodiments, the additional agent administered in combination with the anti-ADGRE2 antibodies described herein can be a chemotherapeutic agent. In various embodiments, the additional agent administered in combination with the antibodies described herein can be an agent that inhibits inflammation.

[0215] In some embodiments, the anti-ADGRE2 antibody is a single-chain variable fragment (scFv) with specificity for human ADGRE2. In some embodiments, the anti-ADGRE2 scFv can be conjugated (e.g., linked) to a therapeutic agent (e.g., a chemotherapeutic agent and a radioactive atom) to bind to, deliver the therapeutic agent to, and kill cancer cells that express human ADGRE2. In some embodiments, the anti-ADGRE2 antibody is linked to a therapeutic agent. In some embodiments, the therapeutic agent is a chemotherapeutic agent, a cytokine, a radioactive atom, an siRNA, or a toxin. In some embodiments, the therapeutic agent is a chemotherapeutic agent. In some embodiments, the agent is a radioactive atom.

[0216] In some embodiments, the method can be carried out in conjunction with other treatments for ADGRE2-related disorders.For example, the composition can be administered to the subject at the same time, before, or after chemotherapy.In some embodiments, the composition can be administered to the subject at the same time, before, or after adoptive therapy.

[0217] In various embodiments, additional agents administered in combination with an anti-ADGRE2 antibody described herein can be administered simultaneously with the anti-ADGRE2 antibody, on the same day as the anti-ADGRE2 antibody, or in the same week as the anti-ADGRE2 antibody. In various embodiments, additional agents administered in combination with an anti-ADGRE2 antibody described herein can be administered in a single formulation with the anti-ADGRE2 antibody. In certain embodiments, the additional agents are administered in a time-distant manner from the administration of an anti-ADGRE2 antibody described herein, for example, one or more hours before or after, one or more days before or after, one or more weeks before or after, or one or more months before or after the administration of the anti-ADGRE2 antibody. In various embodiments, the administration frequency of one or more additional agents can be the same as, similar to, or different from the administration frequency of the anti-ADGRE2 antibody described herein.

[0218] Combination therapy includes therapeutic regimens that involve the administration of two different antibodies as described herein and / or therapeutic regimens that involve the administration of antibodies as described herein via multiple formulations and / or routes of administration.

[0219] In some embodiments, the compositions can be formulated with one or more additional therapeutic agents, such as additional therapeutic agents for treating or preventing an ADGRE2-associated disorder (e.g., cancer or an autoimmune disorder) in a subject. Additional agents for treating an ADGRE2-associated disorder in a subject will vary depending on the particular disorder being treated, but can include, but are not limited to, rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone, osmamide, carboplatin, etoposide, dexamethasone, cytarabine, cisplatin, cyclophosphamide, or fludarabine.

[0220] The compositions described herein can replace or enhance previous or currently administered therapies.For example, when treated with the compositions described herein, the administration of one or more additional active agents can be stopped or reduced following the administration of the anti-ADGRE2 antibody described herein, for example, at a lower level, for example, at a lower level of a reference antibody that cross-competes for ADGRE2 binding.In some embodiments, the administration of the previous therapy can be maintained.In some embodiments, the previous therapy is maintained until the level of the composition reaches a level sufficient to provide a therapeutic effect.The two therapies can be administered in combination.

[0221] recombinant gene technology In accordance with the present disclosure there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are described in the literature (e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, DNA Cloning: A Practical Approach, Volumes I and II (DNGlover ed.1985), Oligonucleotide Synthesis (MJGait ed.1984), Nucleic Acid Hybridization(BDHames&S.J.Higgins eds.(1985)), Transcription And Translation(BDHames&S.J.Higgins, eds.(1984)), Animal Cell Culture(RIFreshney, ed.(1986)), Immobilized Cells and Enzymes(IRL Press,(1986)), B.Perbal,A Practical Guide To Molecular Cloning (1984), FMAsubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

[0222] Recombinant expression of a gene, such as a nucleic acid, encoding a polypeptide, such as an anti-ADGRE2 antibody described herein, can involve the construction of an expression vector containing the nucleic acid encoding the polypeptide. Once a polynucleotide is obtained, a vector for the production of the polypeptide can be produced by recombinant DNA technology using techniques known in the art. Known methods can be used to construct expression vectors containing a polypeptide coding sequence and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

[0223] The expression vector can be transferred to a host cell by conventional techniques and the transfected cells can then be cultured by conventional techniques to produce the polypeptide.

[0224] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. [Example]

[0225] The following examples illustrate some of the preferred modes of making and practicing the present invention. However, it should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0226] Example 1. Identification and characterization of anti-ADGRE2 antibodies This example demonstrates the derivation and characterization of binding affinities of anti-ADGRE2 antibodies.

[0227] Antibodies were developed using hybridoma technology to include 24 humanized sequences of the mouse reference 1 clone. Antibodies were selected based on their expression as recombinant protein variants binding to ADGRE2-overexpressing mouse lymphoma EL4 cells as measured by FACS, and immunogenicity scores, resulting in the selection of 18 humanized recombinant antibodies representing a range of ADGRE2 binding affinities.

[0228] Reference 1 Sequencing of anti-ADGRE2 antibodies The amino acid sequence of the reference 1 anti-ADGRE2 antibody was determined by endoprotease digestion and subsequent analysis of peptide pools by LC-MS / MS. Briefly, the antibody heavy and light chains were separated by SDS-PAGE under reducing conditions. After staining with Coomassie blue, each band was excised from the gel and digested with AspN, chymotrypsin, trypsin, and elastase endopeptidase. The antibody was further digested in solution with pepsin. The peptide pools generated from the digestion were analyzed using an Orbitrap analyzer (LC-MS / MS Q-Exactive, ThermoFisher). LC-MS / MS data were processed using PEAKS AB antibody sequencing software.

[0229] The VH and VL coding sequences of Reference 1 anti-ADGRE2 were derived from the sequences of the respective antibody chains and cloned with the IgG2 constant region. Recombinant Reference 1 antibody was expressed in HEK293 cells, and purified antibody was analyzed by surface plasmon resonance (SPR) analysis using recombinantly produced protein containing the extracellular domain of ADGRE2. D Analytical comparisons were made with the commercially available Reference 1 antibody, as well as EC50 determinations for binding to cells expressing ADGRE2. For subsequent antibody screening work, purified recombinant Reference 1 antibody was used as the reference antibody.

[0230] Humanization of the variable domains of the anti-ADGRE2 (EMRE2) reference 1 antibody A predictive human engineering bioinformatics program called Xoma Humanization, which includes the "TSF Humanization" software, was used. This software program utilizes germline sequences from the Kabat database to engineer and humanize mouse clone reference 1. A panel of human sequence variants representing the best human germline match(s) from the Kabat database was generated by pairwise sequence alignment(s), and amino acid point mutations in the mouse sequence to the "surface" were introduced into the human sequence. This was performed for both the heavy and light chains of the mouse antibody, as shown in Figures 1 and 2. The original description of humanization or human engineering recommendations is discussed in the Xoma Software User's Guide and U.S. Pat. No. 5,766,886 A, which are incorporated herein by reference in their entireties.

[0231] Generation of humanized variants to clone reference 1 The best germline matches were selected, and Kabat CDR definitions were extracted from the alignment. Each was evaluated for its impact on binding and solvent exposure, and the "humanized" sequences were selected accordingly. A two-dimensional projection of the sequence-structure relationships was also performed (Figure 3).

[0232] An exemplary clone was humanized by CDR-grafting the largest CDRs into the human HC germline IGHV1-46*01 and Kappa germline IGKV3D-20*01. A longer GS linker was used to increase flexibility of the HV / LC pair. The linker utilized the first three amino acids of the HC constant domain followed by a standard 3xGS linker (SEQ ID NO:47): ASTGGGGSGGGGSGGGGS (SEQ ID NO:46).

[0233] Example 2. On-cell binding of anti-ADGRE2 antibodies This example demonstrates on-cell binding of anti-ADGRE2 antibodies as measured by flow cytometry.

[0234] On-cell binding of the anti-ADGRE2 antibodies was assessed by flow cytometry in E4 cells overexpressing ADGRE2. Each of the scFvs was tested for binding to ADGRE2 and compared to Reference 1 mAb.

[0235] Briefly, 100,000 cells per well were plated in a 96-well V-bottom plate and the scFv was diluted to 200 nM scFv, followed by a 1:4 serial dilution to 0.01 nM. Folding and binding of the recombinant scFv to ADGRE2 was verified by dose-dependent titration of the scFv.

[0236] To better interpret any differences seen in vivo that may be caused by affinity variations resulting from the humanization process, affinities at EC50 were compared. Data were analyzed using a four-parameter regression using Prism software.

[0237] Approximate EC50 values ​​were determined using the following equation: Y = Bottom + (Top - Bottom) / (1 + 10^((LogEC50-X) × HillSlope)), where the fitted parameters are defined as follows: Bottom, the lower plateau describing the minimum achievable binding; Top, the upper plateau describing the maximum achievable binding; LogEC50, the inflection point of the dose-response curve, also known as the concentration that produces half the maximal response; and Hill-Slope, the slope of the dose-response curve.

[0238] [Table 2]

[0239] As shown in Table 1, the EC50 values ​​calculated from the curves indicate that each of the antibodies binds to cells with an affinity ranging from 10.6 to 93.8.

[0240] Overall, these results showed that the humanized scFvs tested had EC50 values ​​comparable to the Reference 1 standard.

[0241] Example 3. In silico immunogenicity analysis This example illustrates in silico immunogenicity analysis.

[0242] Briefly, the mouse Ref. 1 scFv sequence along with the humanized scFv was analyzed by human MHC1 and MHCII presentation prediction software based on various prediction databases IEDB, SMN-Align, NN-Align.

[0243] [Table 3]

[0244] The immunogenicity of the antibodies was characterized based on binding to MHC I or MHC II or both MHC I and MHC II, as shown in Table 2. Overall, the data predict low immunogenicity for all tested humanized antibodies.

[0245] Example 4. Off-target screening panel assay This example demonstrates the specificity of anti-ADGRE2 scFv in an off-target binding assay.

[0246] Briefly, humanized scFv variants were tested for off-target binding. Three exemplary ADGRE2 scFv clones were tested in a "cut-down assay" to screen for binding to over 3,000 human receptors. In the cut-down assay, the higher the binding, the more likely the interaction is real. In general, hits labeled "V.weak" are less likely to be real interactions.

[0247] These results showed that all clones tested did not exhibit any off-target binding, and therefore were found to be highly specific for ADGRE2.

[0248] Example 5. Sequencing of anti-ADGRE2 antibody clones The anti-ADGRE2 antibody was sequenced and the CDR sequences are shown in Table 3 (heavy chain CDR sequences) and Table 4 (light chain CDR sequences). These analyses confirmed the CDR sequences of the humanized antibody.

[0249] [Table 4]

[0250] [Table 5]

[0251] Other embodiments While many embodiments of the present invention have been described herein, the disclosure and examples can be modified to provide other methods and compositions of the present invention. It will therefore be understood that the scope of the present invention is to be defined by the appended claims in addition to the specific embodiments that have been presented by way of example. All references cited herein are incorporated herein by reference.

Claims

1. A humanized anti-ADGRE2 antibody or antigen-binding fragment thereof, comprising: a heavy chain complementarity determining region (HCDR) 1 comprising the amino acid sequence of GYTFTNYW (SEQ ID NO: 1), a HCDR2 comprising the amino acid sequence of VYPGDGDT (SEQ ID NO: 2), and a HCDR3 comprising the amino acid sequence of ARGFTAYGMDY (SEQ ID NO: 3); and a light chain complementarity determining region (LCDR) 1 comprising the amino acid sequence of SSVSY (SEQ ID NO: 4), an LCDR2 comprising the amino acid sequence of DTS (SEQ ID NO: 5), and an LCDR3 comprising the amino acid sequence of QQWSSNPLT (SEQ ID NO: 6).

2. 2. The humanized anti-ADGRE2 antibody or antigen-binding fragment thereof of claim 1, comprising: (a) an immunoglobulin heavy chain variable (V) fragment comprising an amino acid sequence that is at least about 80% identical to QVQLQQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 7); Ｈ ) area, and An immunoglobulin light chain variable (V) comprising an amino acid sequence that is at least about 80% identical to Ｌ )region; (b) an immunoglobulin heavy chain variable (V) comprising an amino acid sequence that is at least about 80% identical to QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSISTAYMEVSRLRSDDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 9); Ｈ ) area, and An immunoglobulin light chain variable (V) comprising an amino acid sequence that is at least about 80% identical to Ｌ )region; (c) an immunoglobulin heavy chain variable (V) comprising an amino acid sequence that is at least about 80% identical to QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTVYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 11). Ｈ ) area, and An immunoglobulin light chain variable (V) comprising an amino acid sequence that is at least about 80% identical to Ｌ ) area; or (d) an immunoglobulin heavy chain variable (V) comprising an amino acid sequence that is at least about 80% identical to QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWMGAVYPGDGDTRHTQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSS (SEQ ID NO: 13). Ｈ ) area, and An immunoglobulin light chain variable (V) comprising an amino acid sequence that is at least about 80% identical to Ｌ )region; (e) an immunoglobulin heavy chain variable (V) fragment comprising an amino acid sequence that is at least about 80% identical to QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRHTQKFKGRVTMTADKSTSTTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSS (SEQ ID NO: 15). Ｈ ) area, and An immunoglobulin light chain variable (V) comprising an amino acid sequence that is at least about 80% identical to QIVLTQSPATLSLSPGERATLTCSASSSVSYMHWYQQKPGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTIRRLEPEDFATYYCQQWSSNPLTFGQGTKVEIK (SEQ ID NO: 16). Ｌ )region; (f) an immunoglobulin heavy chain variable (V) comprising an amino acid sequence that is at least about 80% identical to QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 17). Ｈ ) area, and An immunoglobulin light chain variable (V) comprising an amino acid sequence that is at least about 80% identical to Ｌ ) area; or (g) an immunoglobulin heavy chain variable (V) comprising an amino acid sequence that is at least about 80% identical to QVQLVQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 30). Ｈ ) area, and An immunoglobulin light chain variable (V) comprising an amino acid sequence that is at least about 80% identical to Ｌ )region.

3. 2. The humanized anti-ADGRE2 antibody or antigen-binding fragment thereof of claim 1, comprising: (a) an immunoglobulin heavy chain variable (V) comprising the amino acid sequence of QVQLQQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 7); Ｈ ) area, and Immunoglobulin light chain variable (V) comprising the amino acid sequence of Ｌ )region; (b) an immunoglobulin heavy chain variable (V) comprising the amino acid sequence of QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSISTAYMEVSRLRSDDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 9); Ｈ ) area, and An immunoglobulin light chain variable (V) comprising the amino acid sequence of: Ｌ )region; (c) an immunoglobulin heavy chain variable (V) comprising the amino acid sequence of QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSSTSTVYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 11); Ｈ ) area, and Immunoglobulin light chain variable (V) comprising the amino acid sequence of: Ｌ ) area; or (d) an immunoglobulin heavy chain variable (V) comprising the amino acid sequence of QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWMGAVYPGDGDTRHTQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSS (SEQ ID NO: 13); Ｈ ) area, and Immunoglobulin light chain variable (V) comprising the amino acid sequence of: Ｌ )region; (e) an immunoglobulin heavy chain variable (V) comprising the amino acid sequence of QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRHTQKFKGRVTMTADKSTSTTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSS (SEQ ID NO: 15); Ｈ ) area, and Immunoglobulin light chain variable (V) comprising the amino acid sequence of QIVLTQSPATLSLSPGERATLTCSASSSVSYMHWYQQKPGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTIRRLEPEDFATYYCQQWSSNPLTFGQGTKVEIK (SEQ ID NO: 16). Ｌ )region; (f) an immunoglobulin heavy chain variable (V) comprising the amino acid sequence of QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 17); Ｈ ) area, and Immunoglobulin light chain variable (V) comprising the amino acid sequence of EIVLTQSPATLSASPGERVTMSCSASSSVSYMHWYQQKPGLAPRRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 18). Ｌ ) area; or (g) an immunoglobulin heavy chain variable (V) comprising the amino acid sequence of QVQLVQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSS (SEQ ID NO: 30); Ｈ ) area, and Immunoglobulin light chain variable (V) comprising the amino acid sequence of: Ｌ )region.

4. The anti-ADGRE2 antibody or fragment thereof may be an IgA antibody, an IgG antibody, an IgE antibody, an IgM antibody, a bispecific or multispecific antibody, a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fd' fragment, an Fd fragment, an isolated CDR or a set thereof; a single chain variable fragment (scFv), a polypeptide-Fc fusion, a single domain antibody, a camelid antibody, a masked antibody, a small modular immunopharmaceutical ("SMIPs™"), a single chain, a tandem dye The humanized anti-ADGRE2 antibody or antigen-binding fragment thereof of claim 1, selected from the group consisting of an Abody, a VHH, an Anticalin, a Nanobody, a Minibody, a BiTE, an Ankyrin Repeat Protein, a DARPIN, an Avimer, a DART, a TCR-like antibody, an Adnectin, an Affilin, a Transbody, an Affibody, a TrimerX, a MicroProtein, a Fynomer, a Centyrin, and a KALBITOR.

5. The humanized anti-ADGRE2 antibody or antigen-binding fragment thereof of claim 1 , wherein the anti-ADGRE2 antibody or fragment thereof is a monoclonal antibody or a single-chain variable fragment (scFv).

6. The humanized anti-ADGRE2 antibody or antigen-binding fragment thereof of claim 1 , wherein the humanized anti-ADGRE2 antibody or antigen-binding fragment thereof is an antibody comprising an IgG constant region.

7. The humanized anti-ADGRE2 antibody or antigen-binding fragment thereof of claim 1 , wherein the ADGRE2 antibody or fragment thereof is a single-chain variable fragment (scFv).

8. The humanized anti-ADGRE2 antibody or antigen-binding fragment thereof of claim 7, wherein the scFv comprises a signal sequence, a heavy chain variable sequence, a GS linker, and a light chain variable sequence.

9. 9. The humanized anti-ADGRE2 antibody or antigen-binding fragment of claim 8, wherein the scFv comprises a sequence having at least about 80% identity to: (a) QVQLQQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGQSPKRWIYDTSKLASGVPARFSGSGSGTDYTFTISSMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 19); (b) QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSISTAYMEVSRLRSDDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 20); (c) QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTVYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 21); or (d)QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWMGAVYPGDGDTRH TQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSSASTGGG GSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSVSYMHWYQQKPGLAPRLLIYDTS KLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSSNPLTFGQGTKVEIK (SEQ ID NO: 22).

10. 9. The humanized anti-ADGRE2 antibody or antigen-binding fragment of claim 8, wherein the scFv comprises the following sequence: (a) QVQLQQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGQSPKRWIYDTSKLASGVPARFSGSGSGTDYTFTISSMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 19); (b) QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWIRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSISTAYMEVSRLRSDDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 20); (c) QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYWMQWVRQAPGQGLEWIGAVYPGDGDTRYTQKFQGRATLTADTSTSTVYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGLSPKRWIYDTSKLASGVPDRFSGSGSGTDYTFTISRMEPEDFATYYCQQWSSNPLTFGGGTKLEIK (SEQ ID NO: 21); or (d)QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQGLEWMGAVYPGDGDTRH TQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFTAYGMDYWGQGTLVTVSSASTGGG GSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSVSYMHWYQQKPGLAPRLLIYDTS KLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSSNPLTFGQGTKVEIK (SEQ ID NO: 22).

11. Dissociation constant (K Ｄ ) is about 10 －８ K less than M Ｄ , about 10 －９ K less than M Ｄ , about 10 －１０ K less than M Ｄ , about 10 －１１ K less than M Ｄ , about 10 －１２ K less than M Ｄ , or about 10 －１３ K less than M Ｄ The humanized anti-ADGRE2 antibody of claim 1,

12. The humanized anti-ADGRE2 antibody of claim 1, having an EC50 of about 1 to 100 nM.

13. The humanized anti-ADGRE2 antibody of claim 12, wherein the EC50 is about 10 to 95 nM.

14. The humanized anti-ADGRE2 antibody of claim 12, wherein the EC50 is about 25-75 nM.

15. A composition for treating cancer, comprising the humanized anti-ADGRE2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 14.

16. 16. The composition of claim 15, wherein the cancer is selected from leukemia, lymphoma, or myeloma.

17. The composition of claim 15, wherein the composition is administered together with one or more additional drugs.

18. The composition of claim 17, wherein the one or more additional agents are selected from an antibody, a chemotherapeutic agent, or radiation therapy.

19. A pharmaceutical composition comprising a humanized anti-ADGRE2 antibody or fragment thereof described in any one of claims 1 to 14 and a pharma- ceutically acceptable carrier.

20. A nucleic acid sequence encoding an amino acid sequence that is at least about 90% identical to any one of SEQ ID NOs:7-18.

21. A vector comprising the nucleic acid sequence of claim 20.

22. 22. An isolated cell comprising the vector of claim 21.

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