Immunotherapy targeting cell adhesion molecules

Abstract

Peptides, proteins, antibodies, antibody fragments, or antigen-binding fragments thereof, against cell adhesion molecules for use as immunotherapeutics.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 488,221, filed March 3, 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Array List This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy was created on February 23, 2024, is designated CWR-032502WO ORD.st.26, and is 126,180 bytes in size.

[0003] FIELD OF THE INVENTION The present disclosure relates to immunotherapy that targets cell adhesion molecules (CAMs) and, in particular, to peptides, proteins, antibodies, antibody fragments, and antigen-binding fragments thereof that bind to CAMs and their use in immunotherapy. [Background technology]

[0004] Brain tumors are one of the most deadly forms of cancer. This includes both primary brain cancer and tumors that metastasize to the brain. Grade 4 glioblastoma or glioblastoma neuriticum (GBM), the most aggressive brain tumor, has a median survival of one year. The sporadic and infiltrative cells characteristic of GBM contribute significantly to the poor prognosis associated with this disease. Unfortunately, no effective treatments exist, and novel therapies targeting these aggressive tumors are needed. Furthermore, tumors that metastasize to the brain also have an extremely poor prognosis. Summary of the Invention

[0005] The embodiments described herein relate to immunotherapy targeting cell adhesion molecules (CAMs), and in particular to peptides, proteins, antibodies, antibody fragments, and antigen-binding fragments thereof that bind to CAMs, and their uses in immunotherapy. Proteolysis of CAMs is a common phenomenon in cancer, and cleaved fragments of CAMs can serve as tumor biomarkers. Full-length CAMs, including the Ig superfamily receptor protein tyrosine phosphatase (PTPμ), function as tumor suppressors and contribute to cell-cell adhesion by normally binding homophilically to PTPμ on neighboring cells, linking them together. In the microenvironment of multiple tumor types, proteolysis of PTPμ generates extracellular domain (ECD) fragments, which result in the formation of unique epitopes (neoantigens) expressed only on the tumor cell surface.

[0006] We have developed immunotherapies based on peptides, proteins, antibodies, antibody fragments thereof, and antigen-binding fragments thereof that bind to CAMs such as PTPμ. These peptides, proteins, antibodies, antibody fragments, and antigen-binding fragments thereof may include peptides and proteins that bind to CAMs such as PTPμ, humanized and chimeric antibodies, antibody fragments thereof, and antigen-binding fragments that bind to CAMs such as PTPμ, scFv antibody fragments that bind to CAMs such as PTPμ, and Fc domain chimeric proteins in which the Fc domain of the human immunoglobulin heavy chain IgG1 is conjugated to a tumor-specific protein that binds to a CAM such as PTPμ.

[0007] The Fc domain binds to Fcγ receptors (FcγR) on innate immune cells, triggering antibody-dependent cellular cytotoxicity (ADCC) by peripheral natural killer (NK) cells and phagocytosis of tumor cells by macrophages or microglia in the brain. Furthermore, FcγR on dendritic cells (DCs) presents tumor antigens to T cells.

[0008] In some embodiments, peptides, proteins, antibodies, antibody fragments, or antigen-binding fragments thereof may comprise PTPμ-Fc chimeric agents that stimulate the immune system to target and kill tumor cells. By combining the tumor-targeting capabilities of PTPμ with the body's own immune recognition, PTPμ-Fc chimeric agents may induce ADCC.

[0009] In other embodiments, peptides, proteins, antibodies, antibody fragments, or antigen-binding fragments thereof may include humanized antibodies, chimeric antibodies, scFv, and peptide sequences that target PTPμ, which can be used as immunotherapeutics.

[0010] In some embodiments, the antibody, antibody fragment, or antigen-binding fragment thereof may comprise at least one of the following: a) CDR-H1 comprising the amino acid sequence of GFTFTDYY (SEQ ID NO: 3); CDR-H2 comprising the amino acid sequence of IRNKANGYRT (SEQ ID NO: 4), and CDR-H3 comprising the amino acid sequence of ASLSAY (SEQ ID NO: 5); b) CDR-L1 comprising the amino acid sequence of KSLLHSNGITY (SEQ ID NO: 8); CDR-L2 comprising the amino acid sequence of QMS (SEQ ID NO: 9), and CDR-L3 comprising the amino acid sequence of AQNLELWT (SEQ ID NO: 10); c) CDR-H1 comprising the amino acid sequence of GASITSGH (SEQ ID NO: 13); CDR-H2 comprising the amino acid sequence of IRDSGTT (SEQ ID NO: 14), and CDR-H3 comprising the amino acid sequence of VTTPFVY (SEQ ID NO: 15); d) CDR-L1 comprising the amino acid sequence of QDINSY (SEQ ID NO: 18); CDR-L2 comprising the amino acid sequence of RAN (SEQ ID NO: 19), and CDR-L3 comprising the amino acid sequence of LQYDEFPLT (SEQ ID NO: 20); e) CDR-H1 comprising the amino acid sequence of GFSLFNYG (SEQ ID NO: 23); CDR-H2 comprising the amino acid sequence of IWADGTI (SEQ ID NO: 24), and CDR-H3 comprising the amino acid sequence of ARHFDY (SEQ ID NO: 25); f) CDR-L1 comprising the amino acid sequence of QNIVHSNGNTY (SEQ ID NO: 28); CDR-L2 comprising the amino acid sequence of KVS (SEQ ID NO: 29); CDR-L3 comprising FQGSRLPLT (SEQ ID NO: 30); g) CDR-H1 comprising the amino acid sequence of GFIFSSYY (SEQ ID NO: 33); CDR-H2 comprising the amino acid sequence of ININGGST (SEQ ID NO: 34), and CDR-H3 comprising the amino acid sequence of GRHDHGYGWFAY (SEQ ID NO: 35); h) CDR-L1 comprising the amino acid sequence of QNVGRN (SEQ ID NO: 38); CDR-L2 comprising the amino acid sequence of SAS (SEQ ID NO: 39), and CDR-L3 comprising the amino acid sequence of QQYNSYPWT (SEQ ID NO: 40); i) CDR-H1 comprising the amino acid sequence of GYTFTTAG (SEQ ID NO: 43), CDR-H2 comprising the amino acid sequence of INTQSGVS (SEQ ID NO: 44), and CDR-H3 comprising the amino acid sequence of ARSNSGGFPY (SEQ ID NO: 45); j) CDR-L1 comprising the amino acid sequence of QSLFNSTTHKIY (SEQ ID NO: 48); CDR-L2 comprising the amino acid sequence of FAS (SEQ ID NO: 49), and CDR-L3 comprising the amino acid sequence of QQHYTAPWT (SEQ ID NO: 50); k) CDR-H1 comprising the amino acid sequence of GYTFTTVG (SEQ ID NO: 53); CDR-H2 comprising the amino acid sequence of INTHSGVS (SEQ ID NO: 54), and CDR-H3 comprising the amino acid sequence of ARGNSNYGFPY (SEQ ID NO: 55); l) CDR-L1 comprising the amino acid sequence of EDIYNR (SEQ ID NO: 58); CDR-L2 comprising the amino acid sequence of GAT (SEQ ID NO: 59), and CDR-L3 comprising the amino acid sequence of QQYWSTPWT (SEQ ID NO: 60); m) CDR-H1 comprising the amino acid sequence of GYSITSDYA (SEQ ID NO: 63); CDR-H2 comprising the amino acid sequence of ISYRSIT (SEQ ID NO: 64), and CDR-H3 comprising the amino acid sequence of ARFGYDGNY (SEQ ID NO: 65); n) CDR-L1 comprising the amino acid sequence of QNIVHSNGDTY (SEQ ID NO: 68); CDR-L2 comprising the amino acid sequence of RVS (SEQ ID NO: 69), and CDR-L3 comprising the amino acid sequence of FQGSHVPLT (SEQ ID NO: 70); o) CDR-H1 comprising the amino acid sequence of GYTFTDYN (SEQ ID NO: 73), CDR-H2 comprising the amino acid sequence of INPDNGAT (SEQ ID NO: 74), and CDR-H3 comprising the amino acid sequence of VRGFDY (SEQ ID NO: 75); p) CDR-L1 comprising the amino acid sequence of QAIVHSNGNTY (SEQ ID NO: 78); CDR-L2 comprising the amino acid sequence of KVS (SEQ ID NO: 79), and CDR-L3 comprising the amino acid sequence of FQSSHIPWT (SEQ ID NO: 80); or A heavy chain variable region and / or a light chain variable region that competitively inhibits the binding of a peptide, protein, isolated antibody, antibody fragment, or antigen-binding fragment thereof comprising at least one of a), b), c), d), e), f), g), h), i), j), k), l), m), n), o), or p) to a CAM such as PTPμ.

[0011] In some embodiments, the antibody, antibody fragment, or antigen-binding fragment thereof may comprise a heavy chain variable region comprising three CDRs of one of SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ ID NO:61, or SEQ ID NO:71; or a heavy chain variable region that competitively inhibits binding of an antibody, antibody fragment, or antigen-binding fragment comprising at least one heavy chain variable region comprising three CDRs of one of SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ ID NO:61, or SEQ ID NO:71.

[0012] In other embodiments, the antibody, antibody fragment, or antigen-binding fragment thereof may comprise a heavy chain variable region comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ ID NO:61, or SEQ ID NO:71.

[0013] In still other embodiments, the antibody, antibody fragment, or antigen-binding fragment thereof may comprise a light chain variable region that competitively inhibits binding of one of SEQ ID NO:6, SEQ ID NO:16, SEQ ID NO:26, SEQ ID NO:36, SEQ ID NO:46, SEQ ID NO:56, SEQ ID NO:66, or SEQ ID NO:76; or a light chain variable region that competitively inhibits binding of a peptide, protein, isolated antibody, antibody fragment, or antigen-binding fragment that comprises at least one light chain variable region that comprises three CDRs of one of SEQ ID NO:6, SEQ ID NO:16, SEQ ID NO:26, SEQ ID NO:36, SEQ ID NO:46, SEQ ID NO:56, SEQ ID NO:66, or SEQ ID NO:76.

[0014] In some embodiments, the antibody, antibody fragment, or antigen-binding fragment thereof may comprise a light chain variable region comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of one of SEQ ID NO:6, SEQ ID NO:16, SEQ ID NO:26, SEQ ID NO:36, SEQ ID NO:46, SEQ ID NO:56, SEQ ID NO:66, or SEQ ID NO:76.

[0015] In some embodiments, the antibody, antibody fragment, or antigen-binding fragment thereof may comprise at least one of the following: a) a heavy chain variable region comprising the three CDRs of SEQ ID NO: 1 and a light chain variable region comprising the three CDRs of SEQ ID NO: 6; b) a heavy chain variable region comprising the three CDRs of SEQ ID NO: 11 and a light chain variable region comprising the three CDRs of SEQ ID NO: 16; c) a heavy chain variable region comprising the three CDRs of SEQ ID NO: 21 and a light chain variable region comprising the three CDRs of SEQ ID NO: 26; d) a heavy chain variable region comprising the three CDRs of SEQ ID NO: 31 and a light chain variable region comprising the three CDRs of SEQ ID NO: 36; e) a heavy chain variable region comprising the three CDRs of SEQ ID NO: 41 and a light chain variable region comprising the three CDRs of SEQ ID NO: 46; f) a heavy chain variable region comprising the three CDRs of SEQ ID NO: 51 and a light chain variable region comprising the three CDRs of SEQ ID NO: 56; g) a heavy chain variable region comprising the three CDRs of SEQ ID NO: 61 and a light chain variable region comprising the three CDRs of SEQ ID NO: 66; or h) A heavy chain variable region comprising the three CDRs of SEQ ID NO: 71 and a light chain variable region comprising the three CDRs of SEQ ID NO: 76.

[0016] In some embodiments, the antibody, antibody fragment, or antigen-binding fragment thereof may comprise at least one of the following: a) a heavy chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:1; and a light chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:6; b) a heavy chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:11; and a light chain comprising an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO: 16; c) a heavy chain variable region comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:21. a light chain comprising a region, and an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:26; d) a heavy chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO: 31; and a light chain comprising an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:36; e) a heavy chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO: 41; and a light chain comprising an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:46; f) a heavy chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO: 51; and a light chain comprising an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:56; g) a heavy chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:61; and and a light chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:66; or h) a heavy chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO: 71; and a light chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:76.

[0017] In some embodiments, the peptide protein, antibody, antibody fragment, or antigen-binding fragment thereof may comprise a peptide linker.

[0018] In some embodiments, the peptide, protein, antibody, antibody fragment, or antigen-binding fragment thereof is humanized or chimeric.

[0019] In other embodiments, the peptide, protein, antibody, antibody fragment, or antigen-binding fragment thereof binds to PTP μ, PTP κ, PCP-2, PTP μ, or PTP ρ.

[0020] In other embodiments, the peptide, protein, antibody, antibody fragment, or antigen-binding fragment binds to amino acids 42-60 of PTP μ, amino acids 816-914 of PTP μ, amino acids 958-1161 of PTP μ, or amino acids 915-958 of PTP μ.

[0021] In some embodiments, the antibody, antibody fragment, or antigen-binding fragment has an identity identical to SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, or SEQ ID NO:111, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, or at least about 78% of SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, or SEQ ID NO:111. and have amino acid sequences that are about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical.

[0022] In other embodiments, the antibody, antibody fragment, or antigen-binding fragment has a sequence identity at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78% to SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, or SEQ ID NO:112. %, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the sequence of the nucleic acid sequence.

[0023] Yet other embodiments relate to chimeric antigen receptors (CARs) comprising an extracellular antigen binding domain comprising an scFv comprising at least one of the following: a) CDR-H1 comprising the amino acid sequence of GFTFTDYY (SEQ ID NO: 3); CDR-H2 comprising the amino acid sequence of IRNKANGYRT (SEQ ID NO: 4), and CDR-H3 comprising the amino acid sequence of ASLSAY (SEQ ID NO: 5); b) CDR-L1 comprising the amino acid sequence of KSLLHSNGITY (SEQ ID NO: 8); CDR-L2 comprising the amino acid sequence of QMS (SEQ ID NO: 9), and CDR-L3 comprising the amino acid sequence of AQNLELWT (SEQ ID NO: 10); c) CDR-H1 comprising the amino acid sequence of GASITSGH (SEQ ID NO: 13); CDR-H2 comprising the amino acid sequence of IRDSGTT (SEQ ID NO: 14), and CDR-H3 comprising the amino acid sequence of VTTPFVY (SEQ ID NO: 15); d) CDR-L1 comprising the amino acid sequence of QDINSY (SEQ ID NO: 18); CDR-L2 comprising the amino acid sequence of RAN (SEQ ID NO: 19), and CDR-L3 comprising the amino acid sequence of LQYDEFPLT (SEQ ID NO: 20); e) CDR-H1 comprising the amino acid sequence of GFSLFNYG (SEQ ID NO: 23); CDR-H2 comprising the amino acid sequence of IWADGTI (SEQ ID NO: 24), and CDR-H3 comprising the amino acid sequence of ARHFDY (SEQ ID NO: 25); f) CDR-L1 comprising the amino acid sequence of QNIVHSNGNTY (SEQ ID NO: 28); CDR-L2 comprising the amino acid sequence of KVS (SEQ ID NO: 29), and CDR-L3 comprising FQGSRLPLT (SEQ ID NO: 30); g) CDR-H1 comprising the amino acid sequence of GFIFSSYY (SEQ ID NO: 33); CDR-H2 comprising the amino acid sequence of ININGGST (SEQ ID NO: 34), and CDR-H3 comprising the amino acid sequence of GRHDHGYGWFAY (SEQ ID NO: 35); h) CDR-L1 comprising the amino acid sequence of QNVGRN (SEQ ID NO: 38); CDR-L2 comprising the amino acid sequence of SAS (SEQ ID NO: 39), and CDR-L3 comprising the amino acid sequence of QQYNSYPWT (SEQ ID NO: 40); i) CDR-H1 comprising the amino acid sequence of GYTFTTAG (SEQ ID NO: 43), CDR-H2 comprising the amino acid sequence of INTQSGVS (SEQ ID NO: 44), and CDR-H3 comprising the amino acid sequence of ARSNSGGFPY (SEQ ID NO: 45); j) CDR-L1 comprising the amino acid sequence of QSLFNSTTHKIY (SEQ ID NO: 48); CDR-L2 comprising the amino acid sequence of FAS (SEQ ID NO: 49), and CDR-L3 comprising the amino acid sequence of QQHYTAPWT (SEQ ID NO: 50); k) CDR-H1 comprising the amino acid sequence of GYTFTTVG (SEQ ID NO: 53); CDR-H2 comprising the amino acid sequence of INTHSGVS (SEQ ID NO: 54), and CDR-H3 comprising the amino acid sequence of ARGNSNYGFPY (SEQ ID NO: 55); l) CDR-L1 comprising the amino acid sequence of EDIYNR (SEQ ID NO: 58); CDR-L2 comprising the amino acid sequence of GAT (SEQ ID NO: 59), and CDR-L3 comprising the amino acid sequence of QQYWSTPWT (SEQ ID NO: 60); m) CDR-H1 comprising the amino acid sequence of GYSITSDYA (SEQ ID NO: 63); CDR-H2 comprising the amino acid sequence of ISYRSIT (SEQ ID NO: 64), and CDR-H3 comprising the amino acid sequence of ARFGYDGNY (SEQ ID NO: 65); n) CDR-L1 comprising the amino acid sequence of QNIVHSNGDTY (SEQ ID NO: 68); CDR-L2 comprising the amino acid sequence of RVS (SEQ ID NO: 69), and CDR-L3 comprising the amino acid sequence of FQGSHVPLT (SEQ ID NO: 70); o) CDR-H1 comprising the amino acid sequence of GYTFTDYN (SEQ ID NO: 73), CDR-H2 comprising the amino acid sequence of INPDNGAT (SEQ ID NO: 74), and CDR-H3 comprising the amino acid sequence of VRGFDY (SEQ ID NO: 75); p) CDR-L1 comprising the amino acid sequence of QAIVHSNGNTY (SEQ ID NO: 78); CDR-L2 comprising the amino acid sequence of KVS (SEQ ID NO: 79), and CDR-L3 comprising the amino acid sequence of FQSSHIPWT (SEQ ID NO: 80); or A heavy chain variable region and / or a light chain variable region that competitively inhibits the binding of a peptide, protein, isolated antibody, antibody fragment, or antigen-binding fragment thereof comprising at least one of a), b), c), d), e), f), g), h), i), j), k), l), m), n), o), or p) to a CAM such as PTPμ.

[0024] Still other embodiments relate to chimeric antigen receptors (CARs) comprising an extracellular antigen-binding domain comprising a PTP μ ligand. The PTP μ ligand can comprise an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to SEQ ID NO: 113. [Brief explanation of the drawings]

[0025] [Figure 1] 1 is a graph showing antibody-dependent cellular cytotoxicity (ADCC) by PTPμ-Fc chimeric agents. [Figure 2] 1 is a plot showing binding of mAB and its derivatives to PTPμ. [Figure 3] 10 is an image showing cells expressing PTPμ CAR, MAM-B scFv CAR, and MAM-D scFv CAR. [Figure 4]Figure 5 is an image showing a first adhesion spot assay of MAM-B scFv CAR-positive and CAR-negative cells. Figure 6 is an image showing a second adhesion spot assay of MAM-B scFv CAR-positive and CAR-negative cells. DETAILED DESCRIPTION OF THE INVENTION

[0026] As used herein, the term "acceptor human framework" refers to a framework comprising the amino acid sequence of a VL or VH framework derived from a human immunoglobulin framework or derived from a human consensus framework.

[0027] As used herein, the term "antibody" encompasses full-length monoclonal antibodies, polyclonal antibodies, nanobodies, and multispecific antibodies. Biological antibodies are typically heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains (L chains) and two identical heavy chains (H chains). The two heavy chains are linked together by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. Each full-length IgG molecule contains at least two binding sites for a specific target or antigen. The light chains are of either kappa or lambda type. Both light chains contain a domain of variable amino acid sequence called the variable region (also called the "VL," "Vκ," or "Vλ region") and a domain of relatively conserved amino acid sequence called the constant region ("CL region").

[0028] Similarly, each heavy chain comprises a variable region ("VH region"), three constant regions ("CH1 region," "CH2 region," and "CH3 region") and a hinge region.

[0029] As used herein, the term "antibody fragment" refers to a segment of a full-length antibody, typically referred to as the target-binding or variable region. Other antibodies include diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Examples include Fab, Fab', F(ab')2, Fv, or scFv fragments. An "Fv" fragment is the smallest antibody fragment that contains the complete target recognition and binding site.

[0030] As used herein, the term "antigen-binding fragment" refers to a fragment or fragments of an antibody molecule that contain the antibody variable region that is responsible for antigen binding.

[0031] Antigen-binding fragments can be prepared from full-length antibodies by protease digestion. Antigen-binding fragments can be produced by those skilled in the art using standard recombinant DNA techniques. Examples of antigen-binding fragments are: "Fab" fragment (a monovalent fragment consisting of one constant domain and one variable domain from each of the heavy and light chains) "Monovalent fragment" (antibody fragment consisting of VL, VH, CL and CH1 domains) "F(ab')2" fragment (a bivalent fragment containing two Fab' fragments linked by a disulfide bridge at the hinge region) "Fd" fragment (consisting of the VH and CH1 domains of an antibody) "Fv" fragments (consisting of the VL and VH domains of a single arm of an antibody); single domain antibodies ("dAbs") (consisting of a VH or VL domain); and Isolated complementarity determining regions ("CDRs").

[0032] As used herein, complementarity determining regions ("CDRs") are the primary binding regions of antibodies. CDRs refer to specific regions within the variable regions of heavy and light chains. Generally, a variable region consists of four framework regions (FR1, FR2, FR3, FR4) and three CDRs arranged as follows: NH2-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-COOH. The term "framework region" refers to variable domain residues other than the CDR residues as defined herein.

[0033] CDRs can also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, and bis-scFvs as antigen-binding fragments. Antigen-binding fragments of antibodies can be grafted into polypeptide-based scaffolds. Antigen-binding fragments can be incorporated into single-chain molecules containing a pair of tandem Fd segments (VH-CH1-VH-CH1), which, together with complementary light chain polypeptides, form a pair of antigen-binding regions.

[0034] As used herein, "single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.

[0035] As used herein, "effector function" refers to the biological activity attributable to the native Fc region of an antibody and varies depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cellular cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); lack of activation of platelets expressing Fc receptors; and B cell activation. It may be desirable to minimize or eliminate effector functions to minimize or eliminate side effects of therapeutic antibodies.

[0036] "Modified antibodies" are antibodies that are not produced in nature and that have been altered or engineered to achieve a particular purpose or have particular properties. For example, antibodies that have undergone deliberate modifications to their wild-type form to reduce effector function are modified antibodies.

[0037] As used herein, the term "Fc region" refers to the region of an antibody that provides protection against a given antigen.

[0038] As used herein, the term "first portion of an antibody" refers to a less than entire portion of a full-length antibody that includes the antigen-binding region of the antibody. A "second portion of an antibody" refers to a less than entire portion of a full-length antibody that consists of a portion of the antibody not included in the first portion.

[0039] The term "Fc receptor" or "FcγR" describes a receptor that binds to the Fc region of IgG. "FcγRI," "FcγRII," and "FcγRIII" are subclasses of FcγR.

[0040] A "modification" to an antibody, antibody fragment, antigen-binding fragment, and / or Fc region of an antibody refers to the substitution, insertion, or deletion of one or more amino acids in the wild-type polypeptide sequence of the protein. A modified antibody, antibody fragment, antigen-binding fragment, and / or Fc region is one in which the modification has been artificially introduced.

[0041] As used herein, "competitively inhibit" refers to the competitive inhibition of binding of an isolated antibody, or antigen-binding portion thereof, to a CAM, such as PTPμ, by any other molecule.

[0042] As used herein, the term "epitope" refers to the site on a CAM, such as PTPμ, to which antibodies and fragments thereof bind and exert their functional activity. The term epitope is synonymous with "antigenic site" and "antibody binding site." One skilled in the art can align the sequence of a CAM (such as PTPμ) from humans with the sequences of CAMs (such as PTPμ) from other animal species to determine the location of an epitope.

[0043] As used herein, "Fab fragment" refers to the variable (VL) and constant (CL) domains of the light chain, and the variable (VH) and first constant (CH1) domains of the heavy chain. Fab' fragments differ from Fab fragments by a few additional residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab' fragments are produced by cleavage of the disulfide bonds at the hinge cysteines of the pepsin digestion product of F(ab')2.

[0044] As used herein, the term "antigen-binding fragment thereof" of an antibody refers to an antibody fragment having qualitative biological activity in common with the full-length antibody. For example, an antigen-binding fragment thereof of an antibody is capable of binding to a CAM, such as PTPμ.

[0045] As used herein, a "human consensus framework" refers to a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences.

[0046] As used herein, a "humanized antibody" refers to an antibody that is composed primarily of human sequences, with the exception of CDR1, CDR2, and CDR3. All framework regions are also humanized. Chimeric antibodies contain murine CDRs, murine framework regions, and a human constant region. Generally, chimeric antibodies contain both murine variable regions and a human constant region.

[0047] As used herein, the term "monovalent antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment thereof that comprises a single binding domain, e.g., a VH or VHH, directed against an antigen, e.g., a single CAM such as a PTP μ molecule.

[0048] As used herein, the term "single domain antibody" is defined as a molecule in which the antigen-binding site resides on and is formed by a single immunoglobulin domain. Generally, the antigen-binding site of an immunoglobulin single variable domain is formed by three or fewer CDRs. A single variable domain may comprise, for example, a light chain variable domain sequence (VL sequence) or a suitable fragment thereof; or a heavy chain variable heavy chain variable domain sequence (e.g., a VH sequence or a VHH sequence) or a suitable fragment thereof, as long as it is capable of forming a single antigen-binding unit (i.e., a functional antigen-binding unit that is substantially a single variable domain, whereby the single antigen-binding domain does not need to interact with other variable domains to form a functional antigen-binding unit).

[0049] As used herein, the term "camelid antibody" refers to antibodies derived from camelid species, such as camel, dromedary, llama, alpaca, and guanaco. Camelid antibodies differ from most other mammalian antibodies in that they lack light chains and therefore contain only heavy chains with complete and diverse antigen-binding capacity (Hamers-Casterman, C. et al., Nature, 363:446-8, 1993).

[0050] As used herein, the term "VHH" refers to a single heavy chain variable domain antibody lacking a light chain. The VHH chain can be, for example, of the type found in camelids, which naturally lack light chains, or a synthetic, non-immunized VHH that can be engineered to suit. Each heavy chain contains variable regions encoded by V, D, and J exons. The VHH can be a natural VHH antibody, for example, a camelid-derived antibody, or a recombinant protein containing a heavy chain variable domain.

[0051] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that binds to a CAM, such as PTPμ, is substantially free of contaminants, e.g., antibodies that do not bind to a CAM, such as PTPμ). Furthermore, an "isolated" antibody refers to an antibody that has been identified, separated, and / or recovered from components of its natural environment, which are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.

[0052] As used herein, the term "specific binding" of an antibody or fragment thereof, polypeptide, or peptidomimetic refers to binding to a target molecule that is measurably different from binding to molecules that are not the target molecule. As used herein, specific binding refers to a preference of greater than 95% for binding a specific antigen over background ("non-specific") binding. "Substantially specific" binding refers to a preference of greater than about 80% for binding a specific antigen over background binding. Binding can be measured using a variety of methods, including, but not limited to, Western blot, immunoblot, enzyme-linked immunosorbent assay ("ELISA"), radioimmunoassay ("RIA"), immunoprecipitation, surface plasmon resonance, biolayer interferometry, chemiluminescence, fluorescence polarization, phosphorescence, immunohistochemistry, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF), microcytometry, microarray, microscopy, fluorescence-activated cell sorting (FACS), and flow cytometry.

[0053] The term "cancer" or "tumor" refers to any neoplastic growth in a subject, including the primary tumor and any metastasis. Cancer can be of liquid or solid tumor type. Liquid tumors include tumors of hematological origin, including, for example, myeloma (e.g., multiple myeloma), leukemia (e.g., Waldenstrom's syndrome, chronic lymphocytic leukemia, other leukemias), lymphoma (e.g., B-cell lymphoma, non-Hodgkin's lymphoma). Solid tumors originate from organs and include cancers of the lung, brain, breast, prostate, ovary, colon, kidney, and liver.

[0054] The term "cancer cell" or "tumor cell" can refer to a cell that divides at an abnormal (i.e., increased) rate. Cancer cells include carcinomas such as squamous cell carcinoma, non-small cell carcinoma (e.g., non-small cell lung cancer), small cell carcinoma (e.g., small cell lung cancer), basal cell carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, undifferentiated carcinoma, bronchogenic carcinoma, melanoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, papillary carcinoma, transitional cell carcinoma, choriocarcinoma, testicular tumor (seminoa), germ cell tumor, breast cancer, gastrointestinal cancer, colon cancer, bladder cancer, prostate cancer, and head and neck squamous cell carcinoma; fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chondrosarcoma, angiosarcoma, endothelial tumor, lymphangiosarcoma, synovial tumor, and mesothelioma. sarcoma); blood cancers (e.g., myeloma, leukemia (e.g., acute myeloid leukemia, chronic lymphocytic leukemia, granulocytic leukemia, monocytic leukemia, lymphocytic leukemia), lymphoma (e.g., follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, malignant lymphoma, plasmacytoma, reticulum sarcoma, or Hodgkin's disease), and tumors of the nervous system (including glioma, glioblastoma multiforme, meningioma, medulloblastoma, schwannoma, and ependymoma).

[0055] The term "chimeric protein" or "fusion protein" refers to a fusion of a first amino acid sequence encoding a polypeptide with a second amino acid sequence that defines a domain (e.g., a polypeptide portion) that is heterologous to and not substantially homologous to all domains of the first polypeptide. A chimeric protein may represent a foreign domain found in an organism that also expresses the first protein (albeit within a different protein), or it may be a "heterogeneous," "intergenic," etc. fusion of protein structures expressed by different types of organisms.

[0056] The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or CAR. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics, as well as specific charge characteristics.

[0057] The term "cancer" as used herein is defined as a disease characterized by the rapid and uncontrollable growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colon cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, melanoma, lung cancer, etc.

[0058] The term "chimeric antigen receptor" or "CAR" refers to a set of polypeptides (usually two in the simplest embodiments) that, when present in a T cell, provides the cell with specificity for a target cell (usually a cancer cell) and intracellular signal generation. In some embodiments, a CAR comprises at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") that comprises a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule. In some embodiments, the set of polypeptides is present on the same polypeptide chain (e.g., comprising a chimeric fusion protein). In some embodiments, the set of polypeptides is not contiguous with one another, e.g., present on different polypeptide chains. In some embodiments, the set of polypeptides comprises a dimerization switch that, in the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., couple the antigen-binding domain to the intracellular signaling domain. In one embodiment, the stimulatory molecule of the CAR is a zeta chain associated with the T cell receptor complex. In one aspect, the cytoplasmic signaling domain comprises a primary signaling domain (e.g., the primary signaling domain of CD3-zeta). In one embodiment, the cytoplasmic signaling domain further comprises one or more of the functional signaling domains of at least one costimulatory molecule as defined below.

[0059] In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain of a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain of a costimulatory molecule and a functional signaling domain of a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains of one or more costimulatory molecules and a functional signaling domain of a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains of one or more costimulatory molecules and a functional signaling domain of a stimulatory molecule.

[0060] The terms "T lymphocyte" and "T cell" are used interchangeably and refer to the major type of white blood cell that completes maturation in the thymus and plays various roles in the immune system, including identifying specific foreign antigens in the body and activating and inactivating other immune cells. T cells can be any T cell, such as cultured T cells (e.g., primary T cells), or T cells from cultured T cell lines (e.g., Jurkat, SupT1, etc.), or T cells obtained from a mammal. T cells can be CD3+ cells. T cells can be any type of T cell, including CD4+ / CD8+ double-positive T cells, CD4+ helper T cells (e.g., Th1 cells and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulatory T cells, gamma delta T cells, etc. Additional types of helper T cells can include those at any stage of development, including Th3 (Treg), Th17, Th9, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (Tcm cells) and effector memory T cells (Tem cells and TEMRA cells). T cells can also refer to genetically modified T cells, such as T cells modified to express a T cell receptor (TCR) or chimeric antigen receptor (CAR). T cells can also be differentiated from stem or progenitor cells.

[0061] "CD4+ T cells" refer to a subset of T cells that express CD4 on their surface and participate in cellular immune responses. They are characterized by their secretory profile after stimulation, which may include secretion of cytokines such as IFN-γ, TNF-α, IL2, IL4, and IL10. "CD4" is a 55-kD glycoprotein that was first defined as a differentiation antigen on T lymphocytes but is also found on other cells, including monocytes / macrophages. The CD4 antigen is a member of the immunoglobulin supergene family and participates as the relevant recognition sequence in MHC (major histocompatibility complex) class II-restricted immune responses. On T lymphocytes, they define helper / inducer subsets.

[0062] "CD8+ T cells" refer to a subset of T cells that express CD8 on their surface, are MHC class I-restricted, and function as cytotoxic T cells. The "CD8" molecule is a differentiation antigen found on thymocytes and cytotoxic / suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene family and is the relevant recognition element in major histocompatibility complex class I-restricted interactions.

[0063] The term "NK cells" or "natural killer cells" refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the lack of a T cell receptor (CD3). As used herein, the terms "adaptive NK cells" and "memory NK cells" are used interchangeably and refer to a subset of NK cells that are phenotypically CD3-negative and CD56-positive, express at least one of NKG2C and CD57, and optionally express CD16, but lack expression of one or more of PLZF, SYK, FcRγ, and EAT-2. In some embodiments, an isolated subpopulation of CD56+ NK cells contains expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIRs, NKG2A, and / or DNAM-1. CD56+ may be weakly or strongly expressed.

[0064] The term "NKT cell" or "natural killer T cell" refers to a CD1d-restricted T cell that expresses a T cell receptor (TCR). Unlike conventional T cells, which recognize peptide antigens presented by classical major histocompatibility (MHC) molecules, NKT cells recognize lipid antigens presented by the non-classical MHC molecule, CD1d. Two types of NKT cells have been recognized. Invariant or type I NKT cells express a very limited TCR repertoire—a canonical α chain (Vα24-Jα18 in humans) coupled with a limited spectrum of β chains (Vβ11 in humans). A second population of NKT cells, termed non-classical or non-invariant type II NKT cells, exhibits more diverse TCRαβ utilization. Type I NKT cells are considered suitable for immunotherapy. Adaptive or invariant (type I) NKT cells can be identified by the expression of at least one or more of the following markers: TCR Va24-Jα18, Vb11, CD1d, CD3, CD4, CD8, aGalCer, CD161, and CD56.

[0065] As used herein, the term "identical" or "substantially identical" with respect to an antibody chain polypeptide sequence may be interpreted as an antibody chain exhibiting at least 65%, 70%, 80%, 90%, or 95% sequence identity to a reference polypeptide sequence present in the variable region of an antigen-binding fragment. With respect to a nucleic acid sequence, the term may be interpreted as a nucleotide sequence exhibiting at least about 65%, 75%, 85%, 90%, 95%, or 97% sequence identity to a reference nucleic acid sequence.

[0066] As used herein, the term "individual" refers to a vertebrate, preferably a mammal, and more preferably a human. Individuals suitable for treatment include those who are currently asymptomatic but who are at risk of developing a symptomatic disease involving the alternative complement pathway or involving activation of the alternative complement pathway.

[0067] As used herein, the term "mammal" refers to any animal classified as a mammal, including humans, higher primates, domestic and farm animals, horses, pigs, cows, dogs, cats, polecats, etc. In one embodiment of the present invention, the mammal is a human.

[0068] As used herein, "monoclonal antibody" refers to a homogeneous population of antibodies. Such antibodies are highly specific and directed against a single target antigen. These monoclonal antibodies are produced homogeneously by hybridoma culture, uncontaminated by other immunoglobulins. Monoclonal antibodies can also be produced by other methods, such as phage display, by known methods.

[0069] As used herein, the term "treatment" refers to both therapeutic and prophylactic or preventative treatment.

[0070] The term "gene" or "recombinant gene" refers to a nucleic acid comprising an open reading frame that encodes a polypeptide, including both exon and (optionally) intron sequences.

[0071] The terms "homology" and "identity" are used synonymously throughout this specification and refer to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence, which are aligned for comparison. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous or identical at that position. The degree of homology or identity between sequences is a function of the number of matching or homologous positions shared by the sequences.

[0072] The term "mutant" refers to any change in the genetic material of an organism, and particularly to any change in the wild-type polynucleotide sequence (i.e., deletion, substitution, addition, or alteration) or any change in the wild-type protein. The term "variant" is used interchangeably with "mutant." While it is often assumed that changes in genetic material result in changes in the function of the protein, the terms "mutant" and "variant" refer to changes in the sequence of a wild-type protein, regardless of whether the change alters the function of the protein (e.g., increases, decreases, confers a new function), or whether the change does not affect the function of the protein (e.g., the mutation or change is silent).

[0073] The term "nucleic acid" refers to polynucleotides, such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). This term should also be understood to include single-stranded (sense or antisense) and double-stranded polynucleotides made from nucleotide analogs and equivalents, analogs of either RNA or DNA, as applicable to the described embodiments.

[0074] The phrases "parenteral administration" and "parenterally administered" are art-recognized terms and include modes of administration other than enteral and topical administration, such as injection, including, but not limited to, intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intraarticular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, and intraspinal injection and infusion.

[0075] As used herein, the terms "systemic administration," "systemically administered," "peripheral administration," and "peripherally administered" refer to the administration of a compound, drug, or other substance other than directly into a particular tissue, organ, or region of the subject being treated (e.g., the brain), whereby the compound, drug, or other substance enters the animal's system and is therefore subject to metabolism and other similar processes. For example, subcutaneous administration.

[0076] The terms "patient," "subject," "mammalian host," and the like are used interchangeably herein and refer to mammals, including human and veterinary subjects.

[0077] The terms "peptide," "protein," and "polypeptide" are used interchangeably herein. As used herein, "polypeptide" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds (i.e., peptide isomers). "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides, or oligomers, and longer chains, commonly referred to as proteins.

[0078] The terms "polynucleotide sequence" and "nucleotide sequence" are also used interchangeably herein.

[0079] As used herein, "recombinant" means that the protein is derived from a prokaryotic or eukaryotic expression system.

[0080] The terms "therapeutic agent," "pharmaceutical agent," "drug," and "bioactive agent" are art-recognized and include molecules and other agents that are biologically, physiologically, or pharmacologically active substances that act locally or systemically in a patient or subject to treat a disease or condition. These terms include, but are not limited to, pharmaceutically acceptable salts and prodrugs thereof. Such agents may be acidic, basic, or salts; they may be neutral molecules, polar molecules, or molecular complexes capable of forming hydrogen bonds; and they may be prodrugs in the form of ethers, esters, amides, etc., that become biologically activated upon administration into a patient or subject.

[0081] The phrases "therapeutically effective amount" or "pharmaceutically effective amount" are art-recognized terms. In certain embodiments, the term refers to an amount of a therapeutic agent that produces some desired effect at a reasonable benefit / risk ratio applicable to any medical treatment. In certain embodiments, the term refers to an amount necessary or sufficient to eliminate, reduce, or maintain the target of a particular therapeutic regimen. An effective amount may vary depending on factors such as the disease or condition being treated, the particular targeting construct being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular compound without necessitating undue experimentation. In certain embodiments, a therapeutically effective amount of a therapeutic agent for in vivo use will likely depend on several factors, including the release rate of the agent from the polymer matrix (which depends, in part, on the chemical and physical properties of the polymer); the identity of the agent; the mode and method of administration; and any other materials incorporated into the polymer matrix in addition to the agent.

[0082] The term "wild-type" refers to a naturally occurring polynucleotide sequence that encodes a protein or portion thereof, or a protein sequence or portion thereof, respectively, as it normally occurs in vivo.

[0083] Throughout the description, when a composition is described as having, including, or comprising particular components, it is assumed that the composition also "consist essentially of" or "consist of" the recited components. Similarly, when a method or process is described as having, including, or comprising particular process steps, it is assumed that the process also "consist essentially of" or "consist of" the recited processing steps. Further, it should be understood that the order of steps or order for performing certain actions is immaterial so long as the compositions and methods described herein remain operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0084] The embodiments described herein relate to immunotherapy targeting cell adhesion molecules (CAMs), particularly peptides, proteins, antibodies, antibody fragments, and antigen-binding fragments thereof that bind to CAMs, and their use in immunotherapy. Proteolysis of CAMs is a common phenomenon in cancer, and cleaved fragments of CAMs can serve as tumor biomarkers. Full-length CAMs, including the Ig superfamily receptor protein tyrosine phosphatase (PTPμ), function as tumor suppressors and contribute to cell-cell adhesion by normally binding homophilically to PTPμ on neighboring cells to link them together. In the microenvironment of multiple tumor types, proteolysis of PTPμ generates extracellular domain (ECD) fragments, which result in the formation of unique epitopes (neoantigens) expressed only on the surface of tumor cells.

[0085] We have developed immunotherapies based on peptides, proteins, antibodies, antibody fragments thereof, and antigen-binding fragments thereof that bind to CAMs such as PTPμ. These peptides, proteins, antibodies, antibody fragments, and antigen-binding fragments thereof may include peptides and proteins that bind to CAMs such as PTPμ, humanized and chimeric antibodies and antibody fragments thereof, antigen-binding fragments that bind to CAMs such as PTPμ, scFv antibody fragments that bind to CAMs such as PTPμ, and Fc domain chimeric proteins in which the Fc domain of a human immunoglobulin heavy chain IgG1 is conjugated to a tumor-specific protein that binds to a CAM such as PTPμ or a ligand of that protein.

[0086] The Fc domain binds to Fcγ receptors (FcγR) on innate immune cells, triggering antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells and phagocytosis of tumor cells by peripheral macrophages or microglia in the brain. Furthermore, FcγR on dendritic cells (DCs) presents tumor antigens to T cells.

[0087] In some embodiments, peptides, proteins, antibodies, antibody fragments, or antigen-binding fragments thereof may comprise PTPμ-Fc chimeric agents that stimulate the immune system to target and kill tumor cells. By combining the tumor-targeting capabilities of PTPμ with the body's own immune recognition, PTPμ-Fc chimeric agents may induce ADCC.

[0088] The peptides, proteins, antibodies, antibody fragments, and antigen-binding fragments thereof can include humanized and chimeric antibodies, antibody fragments, or antigen-binding fragments thereof that selectively bind to a CAM, such as the proteolytically cleaved extracellular fragment of PTP μ. The antibodies, antibody fragments, and antigen-binding fragments thereof can be used in immunotherapy to treat cancer in humans or other mammals.

[0089] Methods for creating humanized antibodies from non-human types are well known to those skilled in the art. Humanization is essentially performed by replacing rodent CDRs or CDR sequences with the corresponding sequences of a human antibody. The selection of human variable domains (both light and heavy chains) used in creating humanized antibodies can sometimes be important to reduce antigenicity and / or human anti-mouse antibody (HAMA) responses.

[0090] The variable domain of an antibody refers to a specific portion of the variable domain whose sequence differs between antibodies. The diversity in antibodies and their antigen-binding fragments can be concentrated in three CDR segments located in both the light and heavy chain variable domains. The highly conserved portions of the variable domain are called framework (FR) regions. In the antibodies described herein, there are four FR regions connected by the three CDRs comprising the variable chain. The CDRs in each of the light and heavy chains are held together in close proximity with the CDRs from the other chain by the FR regions and can contribute to the formation of the target binding site of the antibody.

[0091] Antibody humanization is a process that can generate modified human antibodies with variable region ("V region") sequences substantially similar to the actual human germline sequences while retaining the binding specificity and affinity of the reference antibody. This process can involve, for example, grafting the CDR1, CDR2, and CDR3 regions of heavy and light chain sequences into a humanized human framework that is optimized and pre-specified before the grafting process begins. The variable regions containing the humanized framework can be generated into Fab, Fab', F(ab')2, or single-chain antigen-binding fragments thereof. The resulting modified humanized antibody fragments can retain the binding specificity of the parent mouse antibody for the CAM and have binding affinity for a particular antigen that is comparable to or higher than that of the parent antibody. The modified antigen-binding fragments can have heavy and light chain V regions with high amino acid sequence identity compared to the closest human germline antibody gene. For example, additional maturation changes can be introduced into the CDR3 region of each chain during the construction process to identify antibodies with optimal binding kinetics.

[0092] In some embodiments, the antibody can be a single domain antibody, such as a VHH or V-NAR. Such antibodies naturally occur in camelids and sharks (Saerens, D. et al., Curr. Opin. Pharmacol., 8:600-8, 2008). Camelid antibodies are described, for example, in U.S. Patent Nos. 5,759,808; 5,800,988; 5,840,526; 5,874,541; 6,005,079; and 6,015,695, the entire contents of which are incorporated herein by reference. The cloned and isolated VHH domain is a stable polypeptide that possesses the full antigen-binding ability of the original heavy chain antibody. VHH domains, with their unique structural and functional properties, combine the advantages of conventional antibodies (high target specificity, high target affinity, and low intrinsic toxicity) with important features of small molecule drugs (the ability to inhibit enzymes and access receptor clefts). Furthermore, they are stable, potentially administrable by methods other than injection, and easy to manufacture.

[0093] In some embodiments, humanized antibodies, chimeric antibodies, antibody fragments, and antigen-binding fragments thereof can bind to the same epitope on a CAM as the antibodies described herein. Such humanized antibodies, chimeric antibodies, antibody fragments, and antigen-binding fragments thereof can be identified based on their ability to cross-compete with or competitively inhibit the binding of antibodies and their antigen-binding fragments in standard CAM binding assays. Thus, all humanized antibodies, chimeric antibodies, antibody fragments, and antigen-binding fragments thereof that competitively inhibit the binding of antibodies and their antigen-binding fragments are encompassed by the present disclosure.

[0094] Antigen-binding fragments of antibodies can be identified after protease digestion. These include, for example, "Fab fragments," "Fab' fragments" (Fab with heavy chain hinge regions), and "F(ab')2 fragments" (dimers of Fab' fragments linked at the heavy chain hinge regions). Recombinant methods have been used to produce such fragments and to generate even smaller antibody fragments, such as those called "single-chain Fv" (variable fragments) or "scFv," which consist of a VL and a VH linked by a synthetic peptide linker (VL-linker-VH or VH-linker-VL). Fab, Fab', and scFv fragments are monovalent or monospecific for antigen binding because they each contain only one antigen-binding domain, including one VH / VL dimer.

[0095] An even smaller monovalent antibody fragment is the dAb, which contains only a single immunoglobulin variable domain (e.g., VH or VL), which specifically binds antigen alone, i.e., without the need for a complementary VL or VH domain, respectively. dAbs bind antigen independently of other V domains, but dAbs can exist in homo- or heteromultimers with other VH or VL domains, in which case the other domains are not required for antigen binding by the dAb. i.e., dAbs bind antigen independently of additional VH or VL domains.

[0096] In some embodiments, the antibody, antibody fragment, or antigen-binding fragment thereof may comprise at least one of the following: a) CDR-H1 comprising the amino acid sequence of GFTFTDYY (SEQ ID NO: 3); CDR-H2 comprising the amino acid sequence of IRNKANGYRT (SEQ ID NO: 4), and CDR-H3 comprising the amino acid sequence of ASLSAY (SEQ ID NO: 5); b) CDR-L1 comprising the amino acid sequence of KSLLHSNGITY (SEQ ID NO: 8); CDR-L2 comprising the amino acid sequence of QMS (SEQ ID NO: 9), and CDR-L3 comprising the amino acid sequence of AQNLELWT (SEQ ID NO: 10); c) CDR-H1 comprising the amino acid sequence of GASITSGH (SEQ ID NO: 13); CDR-H2 comprising the amino acid sequence of IRDSGTT (SEQ ID NO: 14), and CDR-H3 comprising the amino acid sequence of VTTPFVY (SEQ ID NO: 15); d) CDR-L1 comprising the amino acid sequence of QDINSY (SEQ ID NO: 18); CDR-L2 comprising the amino acid sequence of RAN (SEQ ID NO: 19), and CDR-L3 comprising the amino acid sequence of LQYDEFPLT (SEQ ID NO: 20); e) CDR-H1 comprising the amino acid sequence of GFSLFNYG (SEQ ID NO: 23); CDR-H2 comprising the amino acid sequence of IWADGTI (SEQ ID NO: 24), and CDR-H3 comprising the amino acid sequence of ARHFDY (SEQ ID NO: 25); f) CDR-L1 comprising the amino acid sequence of QNIVHSNGNTY (SEQ ID NO: 28); CDR-L2 comprising the amino acid sequence of KVS (SEQ ID NO: 29), and CDR-L3 comprising FQGSRLPLT (SEQ ID NO: 30); g) CDR-H1 comprising the amino acid sequence of GFIFSSYY (SEQ ID NO: 33); CDR-H2 comprising the amino acid sequence of ININGGST (SEQ ID NO: 34), and CDR-H3 comprising the amino acid sequence of GRHDHGYGWFAY (SEQ ID NO: 35); h) CDR-L1 comprising the amino acid sequence of QNVGRN (SEQ ID NO: 38); CDR-L2 comprising the amino acid sequence of SAS (SEQ ID NO: 39), and CDR-L3 comprising the amino acid sequence of QQYNSYPWT (SEQ ID NO: 40); i) CDR-H1 comprising the amino acid sequence of GYTFTTAG (SEQ ID NO: 43), CDR-H2 comprising the amino acid sequence of INTQSGVS (SEQ ID NO: 44), and CDR-H3 comprising the amino acid sequence of ARSNSGGFPY (SEQ ID NO: 45); j) CDR-L1 comprising the amino acid sequence of QSLFNSTTHKIY (SEQ ID NO: 48); CDR-L2 comprising the amino acid sequence of FAS (SEQ ID NO: 49), and CDR-L3 comprising the amino acid sequence of QQHYTAPWT (SEQ ID NO: 50); k) CDR-H1 comprising the amino acid sequence of GYTFTTVG (SEQ ID NO: 53); CDR-H2 comprising the amino acid sequence of INTHSGVS (SEQ ID NO: 54), and CDR-H3 comprising the amino acid sequence of ARGNSNYGFPY (SEQ ID NO: 55); l) CDR-L1 comprising the amino acid sequence of EDIYNR (SEQ ID NO: 58); CDR-L2 comprising the amino acid sequence of GAT (SEQ ID NO: 59), and CDR-L3 comprising the amino acid sequence of QQYWSTPWT (SEQ ID NO: 60); m) CDR-H1 comprising the amino acid sequence of GYSITSDYA (SEQ ID NO: 63); CDR-H2 comprising the amino acid sequence of ISYRSIT (SEQ ID NO: 64), and CDR-H3 comprising the amino acid sequence of ARFGYDGNY (SEQ ID NO: 65); n) CDR-L1 comprising the amino acid sequence of QNIVHSNGDTY (SEQ ID NO: 68); CDR-L2 comprising the amino acid sequence of RVS (SEQ ID NO: 69), and CDR-L3 comprising the amino acid sequence of FQGSHVPLT (SEQ ID NO: 70); o) CDR-H1 comprising the amino acid sequence of GYTFTDYN (SEQ ID NO: 73), CDR-H2 comprising the amino acid sequence of INPDNGAT (SEQ ID NO: 74), and CDR-H3 comprising the amino acid sequence of VRGFDY (SEQ ID NO: 75); p) CDR-L1 comprising the amino acid sequence of QAIVHSNGNTY (SEQ ID NO: 78); CDR-L2 comprising the amino acid sequence of KVS (SEQ ID NO: 79), and CDR-L3 comprising the amino acid sequence of FQSSHIPWT (SEQ ID NO: 80); or A heavy chain variable region and / or a light chain variable region that competitively inhibits the binding of a peptide, protein, isolated antibody, antibody fragment, or antigen-binding fragment thereof comprising at least one of a), b), c), d), e), f), g), h), i), j), k), l), m), n), o), or p) to a CAM such as PTPμ.

[0097] In some embodiments, the antibody, antibody fragment, or antigen-binding fragment thereof may comprise a heavy chain variable region comprising three CDRs of one of SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ ID NO:61, or SEQ ID NO:71; or a heavy chain variable region that competitively inhibits binding of an antibody or antigen-binding fragment comprising at least one heavy chain variable region comprising three CDRs of one of SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ ID NO:61, or SEQ ID NO:71.

[0098] In other embodiments, the antibody, antibody fragment, or antigen-binding fragment thereof may comprise a heavy chain variable region comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ ID NO:61, or SEQ ID NO:71.

[0099] In yet other embodiments, the antibody, antibody fragment, or antigen-binding fragment thereof may comprise a light chain variable region comprising three CDRs of one of SEQ ID NO:6, SEQ ID NO:16, SEQ ID NO:26, SEQ ID NO:36, SEQ ID NO:46, SEQ ID NO:56, SEQ ID NO:66, or SEQ ID NO:76; or a light chain variable region that competitively inhibits binding of a peptide, protein, isolated antibody, antibody fragment, or antigen-binding fragment comprising at least one light chain variable region comprising three CDRs of one of SEQ ID NO:6, SEQ ID NO:16, SEQ ID NO:26, SEQ ID NO:36, SEQ ID NO:46, SEQ ID NO:56, SEQ ID NO:66, or SEQ ID NO:76.

[0100] In some embodiments, the antibody, antibody fragment, or antigen-binding fragment thereof may comprise a light chain variable region comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of one of SEQ ID NO:6, SEQ ID NO:16, SEQ ID NO:26, SEQ ID NO:36, SEQ ID NO:46, SEQ ID NO:56, SEQ ID NO:66, or SEQ ID NO:76.

[0101] In some embodiments, the antibody, antibody fragment, or antigen-binding fragment thereof may comprise at least one of the following: a) a heavy chain variable region comprising the three CDRs of SEQ ID NO: 1 and a light chain variable region comprising the three CDRs of SEQ ID NO: 6; b) a heavy chain variable region comprising the three CDRs of SEQ ID NO: 11 and a light chain variable region comprising the three CDRs of SEQ ID NO: 16; c) a heavy chain variable region comprising the three CDRs of SEQ ID NO: 21 and a light chain variable region comprising the three CDRs of SEQ ID NO: 26; d) a heavy chain variable region comprising the three CDRs of SEQ ID NO: 31 and a light chain variable region comprising the three CDRs of SEQ ID NO: 36; e) a heavy chain variable region comprising the three CDRs of SEQ ID NO: 41 and a light chain variable region comprising the three CDRs of SEQ ID NO: 46; f) a heavy chain variable region comprising the three CDRs of SEQ ID NO: 51 and a light chain variable region comprising the three CDRs of SEQ ID NO: 56; g) a heavy chain variable region comprising the three CDRs of SEQ ID NO: 61 and a light chain variable region comprising the three CDRs of SEQ ID NO: 66; or h) A heavy chain variable region comprising the three CDRs of SEQ ID NO: 71 and a light chain variable region comprising the three CDRs of SEQ ID NO: 76.

[0102] In some embodiments, the antibody, antibody fragment, or antigen-binding fragment thereof may comprise at least one of the following:

[0103] In some embodiments, the antibody, antibody fragment, or antigen-binding fragment thereof may comprise at least one of the following: a) a heavy chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:1; and a light chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:6; b) a heavy chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:11; and a light chain comprising an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO: 16; c) a heavy chain variable molecule comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:21. and a light chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:26. d) a heavy chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO: 31; and a light chain comprising an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:36; e) a heavy chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO: 41; and a light chain comprising an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:46; f) a heavy chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO: 51; and a light chain comprising an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:56; i) a heavy chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:61, and and a light chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:66; or j) a heavy chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO: 71; and a light chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:76.

[0104] In some embodiments, the peptide, protein, antibody, antibody fragment, or antigen-binding fragment thereof is humanized or chimeric.

[0105] In other embodiments, the peptide, protein, antibody, antibody fragment, or antigen-binding fragment thereof binds to PTP μ, PTP κ, PCP-2, PTP μ, or PTP ρ.

[0106] In other embodiments, the peptide, protein, antibody, antibody fragment, or antigen-binding fragment binds to amino acids 42-60 of PTP μ, amino acids 816-914 of PTP μ, amino acids 958-1161 of PTP μ, or amino acids 915-958 of PTP μ.

[0107] In some embodiments, the antibody, antibody fragment, or antigen-binding fragment has an identity identical to SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, or SEQ ID NO:111, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, or at least about 78% of SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, or SEQ ID NO:111. and have amino acid sequences that are about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical.

[0108] In other embodiments, the antibody, antibody fragment, or antigen-binding fragment has a sequence identity at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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%, at least about 99%, at least about 99%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 108%, at least about 109%, at least about 110%, or at least about 112. at least about 8%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the nucleotide sequence.

[0109] In some embodiments, a peptide, protein, antibody, antibody fragment, or antigen-binding fragment thereof may comprise a linker. A linker can be used as a link or connector between polypeptide or protein domains and / or related non-protein portions of a peptide, protein, antibody, antibody fragment, or antigen-binding fragment. In some embodiments, the linker is a link or connector between at least two polypeptide constructs, for example, whereby two polypeptide constructs are linked in tandem (e.g., an antibody or antigen-binding fragment thereof is linked to a second polypeptide or antibody). The linker can attach the N-terminus or C-terminus of one antibody construct to the N-terminus or C-terminus of a second polypeptide construct.

[0110] The linker can be any type of bond created by a simple covalent bond (e.g., a peptide bond), a synthetic polymer (e.g., a polyethylene glycol (PEG) polymer), or a chemical reaction (e.g., chemical bonding). When the linker is a peptide bond, a carboxylic acid group at the C-terminus of one protein domain can react with an amino group at the N-terminus of the other protein domain in a condensation reaction to form a peptide bond. Specifically, the peptide bond can be formed by synthetic means using conventional organic chemical reactions well known in the art or by natural production from host cells, and a polynucleotide sequence encoding the DNA sequences of both proteins (e.g., two antibody constructs) in tandem can be directly transcribed and translated into a continuous polypeptide encoding both proteins in the host cell by the necessary molecular machinery (e.g., DNA polymerase, RNA polymerase, and ribosomes).

[0111] When the linker is a synthetic polymer (eg, a PEG polymer), the polymer is functionalized at both ends with reactive chemical groups that can react with the terminal amino acids at the connecting ends of the two proteins.

[0112] When the linker (except for the aforementioned peptide bond) is produced by chemical reaction, a chemical functional group, such as an amine, a carboxylic acid, an ester, an azide, or other functional group commonly used in the art, can be synthetically attached to the C-terminus of one protein and the N-terminus of the other protein, respectively. The two functional groups can then be reacted by synthetic chemical methods to form a chemical bond, thus linking the two proteins together. Such chemical conjugation procedures are well known to those skilled in the art.

[0113] The linker between the two peptide constructs can be an amino acid linker containing 1-200 amino acids (e.g., 1-4, 1-10, 1-20, 1-30, 1-40, 2-10, 2-12, 2-16, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200). Suitable peptide linkers are known to those skilled in the art and include, for example, peptide linkers containing flexible amino acid residues such as glycine and serine.

[0114] In some embodiments, the antibody, antibody fragment, or antigen-binding fragment thereof uses a single-domain antibody, which is a heavy chain variable domain (VH, e.g., VHH) or a light chain domain (VL). Thus, one means of generating a single-domain antibody specific for a CAM is to amplify and express the VH and VL regions of heavy and light chain gene sequences isolated from, for example, a hybridoma (e.g., a murine hybridoma) expressing a CAM monoclonal antibody. The boundaries of the VH and VL domains are shown, for example, by Kabat et al. (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1991)). Information about the boundaries of the VH and VL domains of heavy and light chain genes is used to design PCR primers to amplify V domains from the coding sequence of a heavy or light chain encoding an antibody known to bind to a CAM, such as PTPμ.

[0115] The amplified V domains are inserted into an appropriate expression vector, such as pHEN-1 (Hoogenboom, H. et al., Nucleic Acids Res., 19:4133-7, 1991), and expressed, for example, as VH and VL fusions in scFv or other suitable monovalent formats. The resulting polypeptides can then be screened for high-affinity monovalent binding to a CAM, such as PTPμ. Screening for binding can be performed using methods known in the art. Single-domain antibodies can be generated using methods known in the art (WO2005118642; Ward, E. et al., Nature, 341:544-6, 1989; Holt, L. et al., Trends Biotechnol., 21:484-90, 2003). Each light chain domain is of either the kappa or lambda subgroup. Methods for separating VH and VL domains have been described in the art (EP0368684).

[0116] In one embodiment, the antibody, antibody fragment, or antigen-binding fragment thereof may comprise a single domain antibody derived from a human, humanized rodent, camelid, or shark. Any such single domain antibody can be humanized, if desired. Humanization of camelid single domain antibodies requires the introduction and mutagenesis of a limited number of amino acids in a single polypeptide chain. This contrasts with humanization of scFv, Fab, (Fab')2, and IgG, which requires the introduction of amino acid substitutions in both the light and heavy chains while preserving the assembly of both chains.

[0117] In some embodiments, the single domain antibody comprises a VHH domain. In some embodiments, the VHH domain corresponds to the VHH domain of a naturally occurring heavy chain antibody directed against a CAM such as PTPμ. Such a VHH sequence can be generated, for example, by suitably immunizing a camelid species with a CAM such as PTPμ (i.e., to generate an immune response and / or heavy chain antibodies directed against a CAM such as PTPμ), by obtaining a suitable biological sample from said camelid (such as a blood sample, serum sample, or B cell sample), and, starting from said sample, generating the VHH sequence directed against a CAM such as PTPμ using any suitable technique known in the art (e.g., a gene encoding the single domain antibody is cloned by PCR, or B cells encoding the single domain antibody are immortalized by EBV transformation or by fusion to an immortalized cell line).

[0118] Alternatively, such naturally occurring VHH domains against a CAM such as PTPμ can be obtained from a naive library of camelid VHH sequences, for example, by screening such a library with a CAM such as PTPμ, or at least a portion, fragment, antigenic determinant, or epitope thereof, using one or more screening techniques known in the art (WO99 / 37681, WO01 / 90190, WO03 / 025020, and WO03 / 035694). Alternatively, improved synthetic or semi-synthetic libraries derived from a naive VHH library, such as a VHH library obtained from a naive VHH library, can be used by techniques such as random mutagenesis and / or CDR shuffling (WO00 / 43507). In certain embodiments, a VHH library is constructed, transformed into host cells, and expressed on phage after infection of the host cells with helper phage. After several rounds of biopanning, single domain antibodies against human CAMs such as PTPμ can be isolated and efficiently expressed.

[0119] Libraries of fusion proteins comprising VHHs or VHH fragments can be displayed on phage or suitable microorganisms (such as yeast) for ease of screening.

[0120] Suitable methods, techniques and host organisms for displaying and screening (sets, collections or libraries of) fusion proteins comprising VHHs or VHH fragments are known in the art (WO03 / 054016; Hoogenboom, H., Nat. Biotechnol., 23:1105-16, 2005).

[0121] In a further embodiment, a method for producing a fusion protein comprising a VHH or VHH fragment sequence comprises at least the following steps: a) providing a collection or sample of cells from a camelid expressing immunoglobulin sequences; b) screening the collection or sample of cells for (i) cells expressing immunoglobulin sequences capable of binding to and / or having affinity for a CAM such as PTPμ; and (ii) cells expressing heavy chain antibodies, wherein substeps (i) and (ii) are essentially performed as a single screening step or as two separate screening steps in any suitable order to provide at least one cell expressing a heavy chain antibody capable of binding to and / or having affinity for a CAM such as PTPμ; and c) isolating from the cells VHH sequences present in heavy chain antibodies capable of binding to and / or having affinity for a CAM such as PTPμ, followed by expression of the VHH domains.

[0122] A method for generating amino acid sequences directed against a CAM may include at least the following steps: a) providing a set, collection, or library of nucleic acid sequences encoding heavy chain antibodies or VHH sequences; b) screening the set, collection, or library of nucleic acid sequences for nucleic acid sequences encoding heavy chain antibodies or fusion proteins comprising VHH sequences that are capable of binding to and / or have affinity for a CAM; and c) isolating the nucleic acid sequences, followed by expressing the VHH sequences present in the heavy chain antibodies or expressing fusion proteins comprising the VHH sequences, respectively.

[0123] Other suitable methods and techniques for obtaining single domain antibodies and / or nucleic acids encoding same, starting from naturally occurring VH or VHH sequences, may involve, for example, combining portions of one or more naturally occurring VH sequences (such as one or more framework region (FR) sequences and / or CDR sequences), portions of one or more naturally occurring VHH sequences (such as one or more framework region or CDR sequences), and / or one or more synthetic or semi-synthetic sequences in an appropriate manner to provide a monovalent single domain antibody or a nucleotide sequence or nucleic acid encoding same. Nucleotide sequences encoding the framework sequences of VHH or single domain antibodies are known in the art or can be obtained by polymerase chain reaction (PCR) starting from nucleotide sequences obtained using the methods described herein. Such compositions can be appropriately combined (e.g., by PCR assembly using overlapping primers) with nucleotide sequences encoding the desired CDRs to provide a single domain antibody or antibody fragment fused to a modulator of the alternative complement pathway or a fragment thereof.

[0124] Antibodies or antigen-binding fragments that recognize the same epitope as the parent antibody can be generated by known techniques. For example, antibodies or antigen-binding fragments can be prepared by proteolytic hydrolysis of the antibody or by expression of DNA encoding the fragment in E. coli. Antibodies or antigen-binding fragments are antigen-binding portions of antibodies, such as Fab, F(ab')2, and scFV, and can be obtained by conventional pepsin or papain digestion of whole antibodies or by genetic engineering techniques.

[0125] Antibodies or antigen-binding fragments can be produced by enzymatic cleavage of antibodies with pepsin to produce a 100 kDa fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide bonds, to produce 50 kDa Fab' monovalent fragments. Alternatively, enzymatic cleavage using papain directly produces two monovalent Fab fragments and an Fc fragment (U.S. Pat. Nos. 4,036,945 and 4,331,647; Nisonoff, A. et al., Arch. Biochem. Biophys., 89:230-44, 1960; Porter, R., Biochem. J., 73:119-26, 1959; Edelman et al., in Methods in Enzymology, Vol. I, page 422 (Academic Press, 1967), and Coligan et al., Current Protocols in Immunology, Vol. 1, pages 2.8.1-2.8.10 and 2.10-2.10.4 (John Wiley & Sons, 1991)).

[0126] Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of the fragments, or other enzymatic, chemical, or genetic techniques, can also be used so long as the fragments bind to the antigen recognized by the intact antibody.

[0127] Another form of an antibody or antigen-binding fragment is a peptide encoding a single complementarity-determining region (CDR). CDR peptides can be obtained by constructing genes encoding the CDRs of the antibody of interest. Such genes are prepared, for example, by synthesizing the variable region from RNA of antibody-producing cells using reverse transcriptase followed by the polymerase chain reaction (Larrick, J & Fry, K. METHODS—a companion to Methods in Enzymology Volume: New Techniques in Antibody Generation, 2:106-110, 1991); Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), pages 166-179 (Cambridge University Press 1995); and Ward et al., “Genetic Manipulation and Expression of Antibodies,” in Monoclonal Antibodies: Principles And Applications, Birch et al. (eds.), pages 137-185 (Wiley-Liss, Inc. 1995)).

[0128] Other antibodies or antigen-binding fragments, such as single-domain antibody fragments, are known in the art and can be used in the claimed constructs (Muyldermans, S. et al., Trends Biochem. Sci., 26:230-5, 2001; Yau, K. et al., J. Immunol. Methods, 281:161-75, 2003; Maass, D. et al., J. Immunol. Methods, 324:13-25, 2007). VHHs can have strong antigen-binding capabilities and can interact with novel epitopes that are inaccessible to conventional VH-VL pairs. Camelids can be immunized with a known antigen, such as a CAM, such as PTPμ, and VHHs can be isolated that bind to and neutralize the target antigen.

[0129] Yet other embodiments relate to chimeric antigen receptors (CARs), which comprise an extracellular antigen-binding domain comprising the antigen-binding domain of an antibody or antigen-binding fragment thereof (e.g., a single-chain variable fragment (scFv)) described herein linked to a T cell signaling domain via a transmembrane domain. Features of CARs include their ability to redirect T cell specificity and reactivity to selected targets in an MHC-unrestricted manner, and exploiting the antigen-binding properties of monoclonal antibodies. MHC-unrestricted antigen recognition confers on CAR-expressing T cells the ability to recognize antigens independent of antigen processing, thereby bypassing a major mechanism of tumor evasion. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with endogenous T cell receptor (TCR) α and β chains.

[0130] In some embodiments, the antigen binding domain may comprise an scFv having at least one of the following: a) CDR-H1 comprising the amino acid sequence of GFTFTDYY (SEQ ID NO: 3); CDR-H2 comprising the amino acid sequence of IRNKANGYRT (SEQ ID NO: 4), and CDR-H3 comprising the amino acid sequence of ASLSAY (SEQ ID NO: 5); b) CDR-L1 comprising the amino acid sequence of KSLLHSNGITY (SEQ ID NO: 8); CDR-L2 comprising the amino acid sequence of QMS (SEQ ID NO: 9), and CDR-L3 comprising the amino acid sequence of AQNLELWT (SEQ ID NO: 10); c) CDR-H1 comprising the amino acid sequence of GASITSGH (SEQ ID NO: 13); CDR-H2 comprising the amino acid sequence of IRDSGTT (SEQ ID NO: 14), and CDR-H3 comprising the amino acid sequence of VTTPFVY (SEQ ID NO: 15); d) CDR-L1 comprising the amino acid sequence of QDINSY (SEQ ID NO: 18); CDR-L2 comprising the amino acid sequence of RAN (SEQ ID NO: 19), and CDR-L3 comprising the amino acid sequence of LQYDEFPLT (SEQ ID NO: 20); e) CDR-H1 comprising the amino acid sequence of GFSLFNYG (SEQ ID NO: 23); CDR-H2 comprising the amino acid sequence of IWADGTI (SEQ ID NO: 24), and CDR-H3 comprising the amino acid sequence of ARHFDY (SEQ ID NO: 25); f) CDR-L1 comprising the amino acid sequence of QNIVHSNGNTY (SEQ ID NO: 28); CDR-L2 comprising the amino acid sequence of KVS (SEQ ID NO: 29), and CDR-L3 comprising FQGSRLPLT (SEQ ID NO: 30); g) CDR-H1 comprising the amino acid sequence of GFIFSSYY (SEQ ID NO: 33); CDR-H2 comprising the amino acid sequence of ININGGST (SEQ ID NO: 34), and CDR-H3 comprising the amino acid sequence of GRHDHGYGWFAY (SEQ ID NO: 35); h) CDR-L1 comprising the amino acid sequence of QNVGRN (SEQ ID NO: 38); CDR-L2 comprising the amino acid sequence of SAS (SEQ ID NO: 39), and CDR-L3 comprising the amino acid sequence of QQYNSYPWT (SEQ ID NO: 40); i) CDR-H1 comprising the amino acid sequence of GYTFTTAG (SEQ ID NO: 43), CDR-H2 comprising the amino acid sequence of INTQSGVS (SEQ ID NO: 44), and CDR-H3 comprising the amino acid sequence of ARSNSGGFPY (SEQ ID NO: 45); j) CDR-L1 comprising the amino acid sequence of QSLFNSTTHKIY (SEQ ID NO: 48); CDR-L2 comprising the amino acid sequence of FAS (SEQ ID NO: 49), and CDR-L3 comprising the amino acid sequence of QQHYTAPWT (SEQ ID NO: 50); k) CDR-H1 comprising the amino acid sequence of GYTFTTVG (SEQ ID NO: 53); CDR-H2 comprising the amino acid sequence of INTHSGVS (SEQ ID NO: 54), and CDR-H3 comprising the amino acid sequence of ARGNSNYGFPY (SEQ ID NO: 55); l) CDR-L1 comprising the amino acid sequence of EDIYNR (SEQ ID NO: 58); CDR-L2 comprising the amino acid sequence of GAT (SEQ ID NO: 59), and CDR-L3 comprising the amino acid sequence of QQYWSTPWT (SEQ ID NO: 60); m) CDR-H1 comprising the amino acid sequence of GYSITSDYA (SEQ ID NO: 63); CDR-H2 comprising the amino acid sequence of ISYRSIT (SEQ ID NO: 64), and CDR-H3 comprising the amino acid sequence of ARFGYDGNY (SEQ ID NO: 65); n) CDR-L1 comprising the amino acid sequence of QNIVHSNGDTY (SEQ ID NO: 68); CDR-L2 comprising the amino acid sequence of RVS (SEQ ID NO: 69), and CDR-L3 comprising the amino acid sequence of FQGSHVPLT (SEQ ID NO: 70); o) CDR-H1 comprising the amino acid sequence of GYTFTDYN (SEQ ID NO: 73), CDR-H2 comprising the amino acid sequence of INPDNGAT (SEQ ID NO: 74), and CDR-H3 comprising the amino acid sequence of VRGFDY (SEQ ID NO: 75); p) CDR-L1 comprising the amino acid sequence of QAIVHSNGNTY (SEQ ID NO: 78); CDR-L2 comprising the amino acid sequence of KVS (SEQ ID NO: 79), and CDR-L3 comprising the amino acid sequence of FQSSHIPWT (SEQ ID NO: 80); or A heavy chain variable region and / or a light chain variable region that competitively inhibits the binding of a peptide, protein, isolated antibody, antibody fragment, or antigen-binding fragment thereof comprising at least one of a), b), c), d), e), f), g), h), i), j), k), l), m), n), o), or p) to a CAM such as PTPμ.

[0131] In other embodiments, the antigen binding domain may comprise a PTP μ ligand. The PTP μ ligand may comprise an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to SEQ ID NO:113.

[0132] The PTP μ ligand can be encoded by a nucleotide sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to SEQ ID NO:114.

[0133] In other embodiments, a PTP μ ligand may comprise a PTP μ subsequence. A "subsequence" or "PTP μ subsequence" refers to a portion of PTP μ that maintains a PTP μ activity similar to that of the entire PTP μ sequence, and in particular, the extracellular portion of PTP μ that is involved in binding to PTP μ. In one example, a subsequence is a sequence that comprises at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% of a naturally occurring PTP μ sequence. Also contemplated are sequences that have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the entire subsequence. In one example, the polypeptide can include a BAFF ligand having an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to, for example, SEQ ID NO:113.

[0134] The CAR may further comprise a hinge region, a transmembrane domain, at least one costimulatory domain, and a signaling domain.

[0135] The hinge region is, for example, a sequence located between the antigen-binding domain and at least one of the transmembrane domain, the costimulatory domain, or the signal transduction domain. The hinge sequence can be obtained from any suitable sequence from any genus, including, for example, humans or a portion thereof. Such hinge regions are known in the art. In one embodiment, the hinge region comprises the hinge region of a human protein, including CD8α, CD28, 4-1BB, OX40, CD3-zeta, T cell receptor α chain or β chain, CD3-zeta chain, CD28, CD3c, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, and combinations thereof.

[0136] In some embodiments, the hinge region may comprise an immunoglobulin selected from IgG1, IgG2, IgG3, IgG4, or IgD.

[0137] A transmembrane domain comprises a hydrophobic polypeptide that spans the cell membrane. In particular, a transmembrane domain spans from one side of the cell membrane (extracellular) to the other side of the cell membrane (intracellular or cytoplasmic).

[0138] The transmembrane domain can be in the form of an alpha helix, a beta barrel, or a combination thereof. The transmembrane region may comprise a polytopic protein, which has many transmembrane segments, each of which is an alpha helix, a beta sheet, or a combination thereof.

[0139] In one embodiment, a transmembrane domain that naturally associates with one of the domains in the CAR is used. In another embodiment, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins in order to minimize interactions with other members of the receptor complex.

[0140] For example, transmembrane domains include the transmembrane domains of the T cell receptor alpha or beta chain, CD3 zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, and combinations thereof.

[0141] Artificially designed transmembrane domains are polypeptides that contain primarily hydrophobic residues, such as leucine and valine. In one embodiment, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain.

[0142] In one embodiment, the transmembrane domain is a CD8 transmembrane domain. In another embodiment, the transmembrane domain is a CD28 transmembrane domain. Such transmembrane domains are known in the art.

[0143] Signaling domains and costimulatory domains comprise polypeptides that stimulate or activate at least some aspect of an immune cell signaling pathway, leading to activation of the immune cell.

[0144] In some embodiments, the signaling domain comprises a polypeptide of a functional signaling domain of CD3 zeta, common FcR gamma (FCER1G), Fc gamma R11a, FcR beta (Fc epsilon rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DNAX activator protein 10 (DAP10), DNAX activator protein 12 (DAP12), active fragments thereof, functional derivatives thereof, and combinations thereof. Such signaling domains are known in the art.

[0145] In some embodiments, the CAR further comprises one or more costimulatory domains. In some embodiments, the costimulatory domain is a functional signaling domain selected from proteins including, but not limited to, at least the following: IL-15 receptor alpha; IL-15 receptor alpha cytoplasmic domain; B7-1 / CD80; CD28; 4-1BB, 4-1BBL, B7-2 / CD86; CTLA-4; B7-H1 / PD-L1; ICOS; B7-H2; PD-1; B7-H3; PD-L2; B7-H4; PDCD6; BTLA; 4-1BB / TNFRSF9 / CD137; CD40 ligand / TNFSFS; 4-1BB ligand / TNFSF9; GITR / TNFRSF18; BAFF / BLyS / TNFSF13B; GITR ligand / TNFSF18; BAFF R / TNFRSF13C; HVEM / TNFRSF14; CD27 / TNFRSF7; LIGHT / TNFSF14; CD27 ligand / TNFSF7; OX40 / TNFRSF4; CD30 / TNFRSF8; OX40 ligand / TNFSF4; CD30 ligand / TNFSF8; TACI / TNFRSF13B; CD40 / TNFRSF5; 2B4 / CD244 / SLAMF4; CD84 / SLAMFS; BLAME / SLAMF8; CD229 / SLAMF3; CD2,CD27,CRACC / SLAMF7; CD2F-10 / S LAMF9; NTB-A / SLAMF6; CD48 / SLAMF2; SLAM / CD150; CD58 / LFA-3; Ikaros; CD53; integrin α4 / CD49d; CD82 / Kai-1; integrin α4β1; CD90 / Thyl; integrin α4β7 / LPAM-1; CD96; LAG-3; CD160; LMIR1 / CD300A; CRTAM; TCL1A; DAP12; TIM-1 / KIM-1 / HAVCR; Dectin-1 / CLEC7A; TIM-4; DPPIV / CD26; TSLP; EphB6; TSLP R; and HLA-DR.

[0146] Other embodiments relate to nucleic acids comprising a nucleotide sequence encoding a CAR described herein. Nucleic acids encoding CARs can be easily prepared from the amino acid sequence of a given CAR by any conventional method. The base sequence encoding the amino acid sequence can be obtained from the aforementioned NCBI RefSeq ID or GenBank accession number for the amino acid sequence of each domain, and the nucleic acids of the present disclosure can be prepared using standard molecular biological and / or chemical techniques. For example, polynucleotides can be synthesized based on the base sequence, and the polynucleotides of the present disclosure can be prepared by combining DNA fragments obtained from a cDNA library using polymerase chain reaction (PCR).

[0147] The nucleotide sequence encoding CAR can be operably linked to a promoter and provided in an expression construct. The vector can be suitable for replication and integration into eukaryotes. Typical vectors include transcription and translation terminators, initiation sequences, and promoters that are useful for regulating the expression of desired nucleic acid sequences.

[0148] In some embodiments, the vector is a viral vector. Viral vector technology is known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY) and other virology and molecular biology manuals. In some embodiments, viruses useful as vectors include retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In some embodiments, the vector is a lentiviral vector. In general, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme cleavage sites, and one or more selectable markers (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0149] Vectors derived from viruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of the transgene and its propagation in daughter cells. Lentivirus vectors have the additional advantage over vectors derived from retroviruses (e.g., murine leukemia viruses) that they can transduce non-proliferating cells such as hepatocytes. They also have the additional advantage of low immunogenicity.

[0150] The retroviral vector may also be, for example, a gammaretroviral vector. A gammaretroviral vector may include, for example, a promoter, a packaging signal (ψ), a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTRs), and a transgene of interest (e.g., a gene encoding a CAR). A gammaretroviral vector may lack viral structural genes such as gag, pol, and env. Representative gammaretroviral vectors include murine leukemia virus (MLV), spleen focus-forming virus (SFFV), myeloproliferative sarcoma virus (MPSV), and vectors derived therefrom.

[0151] In some embodiments, a vector can express two or more genes, each expressed individually under the control of a different promoter region, for example, by using a bicistronic or tricistronic promoter. Expression of multiple genes from the same vector can be achieved by using a multi-promoter plasmid, for example, either a bicistronic or tricistronic promoter. Examples of lentiviral vectors containing multiple promoters are known in the literature. For example, the vector pLENTI-bi-cistronic uses the PKG promoter and the mini-CMV promoter in opposite orientations to drive the expression of two genes (Applied Biological Material Inc., Richmond, BC, Canada). Similarly, the tricistronic vector pLENTI-tri-cistronic drives the expression of three genes. In this configuration, one gene is driven by the mini-CMV promoter, while the second and third genes are driven by the PKG promoter, which separates the two genes at the T2A peptide cleavage site.

[0152] In another embodiment, bicistronic or tricistronic vectors can also be constructed using an internal ribosome entry site (IRES), such as a sequence derived from encephalomyocarditis virus (EMCV), for translation of two or more open reading frames (ORFs). Such vectors are designed to drive transcription of bicistronic or tricistronic messages under the control of a strong human promoter regulatory region (e.g., CMV or EF1 alpha). An IRES is a relatively short DNA sequence that can initiate RNA translation in a 5' cap-independent manner. The first cistron is translated in a cap-dependent manner by a strong mammalian promoter, while subsequent cistrons utilize a virus-derived intercistronic region, such as a poliovirus internal ribosome entry site or an encephalomyocarditis virus cap-independent translation enhancer for enhanced translation.

[0153] Additional promoter sequences, such as enhancers, can regulate the frequency of transcription initiation. Typically, these are located in the region 30–110 bp upstream of the start site, although many promoters have been shown to contain functional sequences downstream of the start site. The spacing between promoter sequences is often flexible, allowing promoter function to be maintained even when sequences are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter sequences can be increased to 50 bp before activity begins to decline. Depending on the promoter, individual sequences can function either cooperatively or independently to activate transcription.

[0154] Examples of other promoters include the SFFV promoter and the cytomegalovirus (CMV) promoter. Other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters such as the actin promoter, myosin promoter, elongation factor-1α promoter (EF1α), hemoglobin promoter, and creatine kinase promoter.

[0155] Furthermore, embodiments are not limited to the use of constitutive promoters, but may include, for example, inducible promoters. The use of an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, tetracycline promoters, and the like.

[0156] Vectors may also include, for example, a signal sequence facilitating secretion, a polyadenylation signal and a transcription terminator (e.g., from the bovine growth hormone (BGH) gene), elements allowing episomal replication and replication in prokaryotes (e.g., SV40 origin and ColE1, or others known to those of skill in the art), and / or elements allowing selection (e.g., a puromycin resistance gene, an ampicillin resistance gene, and / or a zeocin marker).

[0157] The sequences encoding various elements of CAR can be placed on the same nucleic acid molecule, for example, the same plasmid or vector, for example, a viral vector, for example, a lentiviral vector. For example, (i) the sequence encoding the extracellular antigen-binding domain and (ii) the sequence encoding the intracellular signaling member can both be present on the same nucleic acid, for example, a vector. The production of corresponding proteins can be achieved, for example, by using separate promoters or by using bicistronic transcription products (which can result in the production of two proteins by cleavage of a single translation product or translation of two separate protein products).

[0158] To assess the expression of a CAR polypeptide or a portion thereof, the expression vector introduced into the cells may also contain either a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells to be transfected or infected with a viral vector. In other embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection method. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as the neomycin resistance gene.

[0159] Reporter genes are likely used to identify transfected cells and evaluate the functionality of regulatory sequences. Generally, reporter genes are genes that are not present in or expressed by recipient organisms or tissues and encode polypeptides whose expression is manifested by some easily detectable property, such as enzymatic activity. Expression of the reporter gene is measured at an appropriate time after DNA is introduced into recipient cells. Suitable reporter genes include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and can be prepared using known techniques or commercially available. Generally, the construct with the smallest 5'-flanking region that exhibits the highest expression level of the reporter gene is identified as the promoter. Such promoter regions can be linked to reporter genes and used to evaluate drugs for their ability to modulate promoter-driven transcription.

[0160] Methods for introducing and expressing genes into cells are known in the art. In the case of expression vectors, the vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method known in the art. For example, the expression vector can be introduced into the host cell by physical, chemical, or biological means.

[0161] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, biolistics, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0162] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian (e.g., human) cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus type I, adenoviruses, adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0163] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems (polymer complexes, nanocapsules, microspheres, beads, etc.) and lipid systems (water-in-oil emulsions, micelles, mixed micelles, liposomes, etc.). An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., artificial membrane vesicle). When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, the nucleic acid can be combined with a lipid. Lipid-bound nucleic acids may be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes via linking molecules bound to both the liposomes and the oligonucleotides, encapsulated in liposomes, complexed with liposomes, dispersed in lipid-containing solutions, mixed with lipids, combined with lipids, contained as a suspension in lipids, contained or complexed with micelles, or otherwise associated with lipids. Lipid, lipid / DNA, or lipid / expression vector-associated compositions are not limited to a particular structure in solution. For example, they may exist in bilayer structures, micelles, or "collapsed" structures. They may also simply be dispersed in solution, forming aggregates that are not uniform in size and shape. Lipids are fatty substances that may be naturally occurring or synthetic. For example, lipids include lipid droplets naturally occurring in the cytoplasm, as well as classes of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0164] Lipids suitable for use are commercially available. For example, dimyristyl phosphatidylcholine (DMPC) is available from Sigma (St. Louis, Mo.), dicetyl phosphate (DCP) is available from K & K Laboratories (Plainview, NY), cholesterol ("Choi") is available from Calbiochem-Behring, and dimyristyl phosphatidylglycerol ("DMPG") and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Lipid stock solutions in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the solvent of choice because it is more volatile than methanol.

[0165] "Liposome" is a generic term encompassing a variety of unilamellar and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as vesicular structures with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5;505-10). However, compositions with structures in solution that differ from the typical vesicular structure are also encompassed. For example, lipids may adopt micellar structures or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0166] Regardless of the method used to introduce exogenous polynucleotides into host cells or otherwise expose cells to the polynucleotides of this disclosure, various assays can be performed to confirm the presence of the recombinant DNA sequence in the host cells. Such assays include, for example, "molecular biological" assays such as Southern blotting, Northern blotting, RT-PCR, PCR, etc., which are well known to those of skill in the art; and "biochemical" assays, such as detecting the presence or absence of specific peptides, for example, by immunological means (ELISA and Western blot) or by the assays described herein to identify agents within the scope of this disclosure.

[0167] In another embodiment, the present disclosure provides modified cells that express the chimeric antigen receptor polypeptide or the polynucleotide encoding it.

[0168] "Modified cells" refers to any cell of any organism that has been modified, transformed, or engineered by the addition or modification of a gene, DNA or RNA sequence, or protein or polypeptide. The isolated cells, host cells, and genetically modified cells of the present disclosure include isolated immune cells, such as NK cells and T cells, that contain a DNA or RNA sequence encoding a chimeric antigen receptor or chimeric antigen receptor complex and express the chimeric receptor on their cell surface. The isolated host cells and modified cells can be used, for example, to enhance NK cell activity or T lymphocyte activity, to treat cancer, and to treat infectious diseases.

[0169] Any cell capable of expressing and / or incorporating into its membrane a chimeric antigen receptor polypeptide as disclosed herein may be used.

[0170] In some embodiments, CAR can be expressed in at least one immune cell. In certain embodiments, the immune cell is T cell, for example, CD8+ T cell (for example, CD8+ naive T cell, central memory T cell, or effector memory T cell), CD4+ T cell, natural killer T cell (NKT cell), regulatory T cell (Treg), stem cell memory T cell, lymphoid progenitor cell, hematopoietic stem cell, natural killer cell (NK cell), or dendritic cell. In certain embodiments, the cell is monocyte or granulocyte, for example, myeloid cell, macrophage, neutrophil, dendritic cell, mast cell, eosinophil, and / or basophil.

[0171] In some embodiments, immune cells include one or more subsets of T cells or other cell types, such as the entire T cell population, CD4+ cells, CD8+ cells, and subpopulations thereof (e.g., defined by function, activation state, maturity, differentiation potential, proliferation potential, recirculation potential, localization potential, and / or persistence potential, antigen specificity, type of antigen receptor, presence in specific organs or compartments, marker or cytokine secretion profile, and / or degree of differentiation). The subtypes and subpopulations of T cells and / or CD4+ and / or CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes (such as stem cell memory T cells (TSCM), central memory T cells (TCM), effector memory T cells (TEM), and well-differentiated effector memory T cells), tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, spontaneous and adaptive regulatory T (Treg) cells, helper T cells (such as Th1 cells, Th2 cells, Th3 cells, Th17 cells, Th9 cells, Th22 cells, follicular helper T cells, α / β T cells, δ / γ T cells, etc.). In certain embodiments, any number of T cell lines available in the art may be used.

[0172] The modified cells may be obtained from peripheral blood, umbilical cord blood, bone marrow, tumor-infiltrating lymphocytes, lymph node tissue, or thymus tissue. Host cells may include placental cells, embryonic stem cells, induced pluripotent stem cells, or hematopoietic stem cells. Cells may be obtained from humans, monkeys, chimpanzees, dogs, cats, mice, rats, and transgenic species thereof. Cells may be obtained from established cell lines.

[0173] The cells may be obtained by any known method. The cells may be autologous, syngeneic, allogeneic, or xenogeneic to the recipient of the modified cells.

[0174] In certain embodiments, the T cells and NK cells are derived from human peripheral blood mononuclear cells (PBMCs), leukapheresis products (PBSCs), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, or umbilical cord blood.

[0175] The immunotherapies described herein, particularly peptides, proteins, antibodies, antibody fragments, antigen-binding fragments, and CAR immune cells, can be used in methods and compositions for treating cancer. In certain embodiments, immunotherapy can be used to treat virtually all types of cancer and precancerous conditions (e.g., myelodysplastic syndrome), including but not limited to carcinoma, sarcoma, melanoma, lymphoma, and leukemia, and having sites of origin including but not limited to colon, prostate, brain, breast, liver, lung, pancreas, bone, ovary, skin, pancreas, blood, etc. The methods disclosed herein contemplate the treatment of both metastatic cancer sites and primary tumor sites.

[0176] The immunotherapeutic agent may be included in a composition, such as a pharmaceutical composition, for immunotherapy, adoptive immunotherapy, and / or treating cancer or infectious diseases. The pharmaceutical compositions described herein may optionally include a pharmaceutically acceptable carrier. The active ingredient of the pharmaceutical composition includes at least an immunotherapy, such as a peptide, protein, antibody, antibody fragment, antigen-binding fragment, or CAR immune cell, as described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists essentially of a peptide, protein, antibody, antibody fragment, antigen-binding fragment, or CAR immune cell as described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists essentially of a peptide, protein, antibody, antibody fragment, antigen-binding fragment, or CAR immune cell as described herein. Pharmaceutically acceptable carriers for peptide-, protein-, or cell-based therapeutic formulations include saline and aqueous buffer solutions, Ringer's solution, and serum components (such as serum albumin, HDL, and LDL). Terms such as "excipient," "carrier," and "pharmaceutically acceptable carrier" are used interchangeably herein.

[0177] In some embodiments, pharmaceutical compositions comprising the immunotherapies described herein may be in parenteral dosage forms. Because parenteral administration typically bypasses a patient's natural defense mechanisms against contaminants, components other than the immunotherapeutic agent itself are preferably sterile or sterilizable prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions for injection, dry products that are ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions for injection, and emulsions. Any of these can be added to the immunotherapeutic formulation prior to administration.

[0178] Suitable vehicles that can be used to provide parenteral dosage forms of the immunotherapies disclosed herein are well known to those skilled in the art, and include, but are not limited to, aqueous vehicles such as saline, dextrose solution, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, and lactated Ringer's injection; aqueous vehicles such as ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0179] In some embodiments, the immunotherapies described herein can be administered as monotherapy, i.e., no other therapeutic agent for the disease is administered to the subject at the same time.

[0180] Methods of administration may include, for example, intravenous (iv) injection or infusion. The compositions described herein can be administered to a patient intraarterially, intratumorally, intranodally, or intramedullarily. In some embodiments, the immunotherapeutic composition may be injected directly into a tumor, lymph node, or site of infection. In one embodiment, the compositions described herein are administered into a body cavity or fluid (e.g., ascites, pleural effusion, peritoneal fluid, or cerebrospinal fluid).

[0181] The dosage of the above-described treatments administered to a patient will vary depending on the precise nature of the disease being treated and the recipient of the treatment. Adjustment of dosages for human administration can be performed according to art-recognized practices.

[0182] In some embodiments, only one treatment regimen is required.In other embodiments, one or more subsequent doses or treatment regimens can be administered.For example, after 3 months of biweekly treatment, treatment can be repeated once a month for 6 months or for more than 1 year.In some embodiments, no additional treatment is administered after the first treatment.

[0183] The dosage of the compositions described herein can be determined by a physician and adjusted as necessary to meet the observed effects of treatment.Regarding the duration and frequency of treatment, a skilled clinician will typically monitor the subject to determine when the treatment is producing a therapeutic effect and whether additional cells should be administered, treatment should be discontinued, treatment should be resumed, or other changes should be made to the treatment regimen.The dosage should not be so large as to cause harmful side effects, such as cytokine release syndrome.In general, the dosage varies depending on the age, condition, and sex of the patient, and can be determined by those skilled in the art.The dosage can also be adjusted by an individual physician if any complications occur.

[0184] In some embodiments, the immunotherapies described herein can be used in combination with other known drugs and therapies. As used herein, "administered in combination" means that two (or more) different therapies are administered to a subject during the period in which the subject is afflicted with a disease, i.e., after the subject is diagnosed with the disease and before the disease is cured or eliminated, or before treatment is terminated for other reasons. In some embodiments, administration of one therapy begins while administration of a second therapy is still ongoing, thereby resulting in an overlap in the administration period. This is sometimes referred to herein as "simultaneous" or "concurrent administration." In other embodiments, administration of one therapy ends before administration of another therapy begins. In some embodiments, in either case, the treatments are more effective due to the combined administration. For example, the second therapy is more effective; e.g., a comparable effect is seen with a lower dose of the second therapy, or the second therapy reduces symptoms to a greater extent than would be seen if the second therapy were administered in the absence of the first therapy, or if a similar situation were observed with the first therapy. In some embodiments, the administration is such that the reduction in symptoms or other parameters associated with the disease is greater than that which would be observed if one treatment were administered without the other. The effects of the two treatments may be partially additive, fully additive, or greater than additive. The administration may be such that the effect of the first treatment administered is still detectable at the time the second treatment is administered.

[0185] The immunotherapy described herein and at least one additional therapeutic agent can be administered simultaneously or sequentially in the same or separate compositions.When administered sequentially, the immunotherapy described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed.The immunotherapy and / or other therapeutic agents, treatments or modalities can be administered during the active phase of disease, or during remission or less active periods.The immunotherapy can be administered before, simultaneously with, after, or during the remission period of disease.

[0186] When administered in combination, the immunotherapy and additional agent (e.g., a second or third agent), or all of them, can be administered in amounts or dosages that are higher, lower, or the same as the amount or dosage of each agent used individually (e.g., as monotherapy). In certain embodiments, the amount or dosage of the immunotherapy, additional agent (e.g., a second or third agent), or all of them, is less (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent used individually. In other embodiments, the amount or dosage of the immunotherapy, additional agent (e.g., a second or third agent), or all of them that produces a desired effect (e.g., treatment of cancer) is less (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent individually required to achieve the same therapeutic effect. In further embodiments, the immunotherapies described herein can be used in treatment regimens in combination with surgery, chemotherapy, radiation therapy, immunosuppressants such as mTOR pathway inhibitors, cyclosporine, azathioprine, methotrexate, mycophenolic acid, FK506, antibodies, or immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytoxan, fludarabine, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, peptide vaccines.

[0187] In one embodiment, the immunotherapies described herein can be used in combination with checkpoint inhibitors. Exemplary checkpoint inhibitors include anti-PD-1 inhibitors (nivolumab, MK-3475, pembrolizumab, pidilizumab, AMP-224, AMP-514), anti-CTLA4 inhibitors (ipilimumab and tremelimumab), anti-PDL1 inhibitors (atezolizumab, aveloumab, MSB0010718C, MED14736, and MPDL3280A), and anti-TIM3 inhibitors.

[0188] In one embodiment, the immunotherapy described herein can be used in combination with a chemotherapeutic agent. Exemplary chemotherapeutic agents include anthracyclines (e.g., doxorubicin (e.g., liposomal doxorubicin)), vinca alkaloids (e.g., vinblastine, vincristine, vindesine, vinorelbine), alkylating agents (e.g., cyclophosphamide, dacarbazine, melphalan, ifosfamide, temozolomide), immune cell antibodies (e.g., alemtuzumab, gemtuzumab, rituximab, tositumomab), metabolic inhibitors (e.g., cyclophosphamide, dacarbazine, melphalan, ifosfamide, temozolomide), and steroids (e.g., steroids ... antagonists (including, for example, folate antagonists, pyrimidine analogs, purine analogs, adenosine deaminase inhibitors (e.g., fludarabine)), mTOR inhibitors, TNFR glucocorticoid-inducible TNFR-related protein (GITR) agonists, proteasome inhibitors (e.g., aclacinomycin A, gliotoxin, or bortezomib), immunomodulators such as thalidomide or its derivatives (e.g., lenalidomide). Common chemotherapy agents that may be considered for use in combination therapy include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxan®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leucine®), and cyclosporine. Tatin (registered trademark)), cyclophosphamide (Cytoxan (registered trademark) or Neosar (registered trademark)), cytarabine, cytosine arabinoside (Cytosar-U (registered trademark)), cytarabine liposome injection (Depocyte (registered trademark)), dacarbazine (DTIC-Dome (registered trademark)), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerbidine (registered trademark)), daunorubicin citrate liposome injection (Daunosome (registered trademark)), dexamethasone, docetaxel (Taxotere (registered trademark)), doxorubicin hydrochloride (Adriamycin (registered trademark), Rubex (registered trademark)),Etoposide (Bepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrsil®, Efudex®), flutamide (Eurexin®), tezacitibine, gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea®), idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), calcium leucovorin, melphalan (Alkeran®), 6-mercaptopurine (Prinesol®), methotrexate ( These include: Folex®), mitoxantrone (Novantron®), Mylotarg, paclitaxel (Taxol®), Phoenix (yttrium-90 / MX-DTPA), pentostatin, carmustine-containing polifeprosan 20 implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Bumone®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), injectable topotecan hydrochloride (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®). Exemplary alkylating agents include, but are not limited to, nitrogen mustards, ethylenimine derivatives, alkylsulfonic acids, nitrosoureas, triazenes: uracil mustard (Aminouracil Mustard®, Chloretaminacil®, Demethyldopan®, Desmethyldopan®, Hemanthamine®, Nordopan®, Uracil Nitrogen Mustard®, Uracillost®, Uracilmostaza®, Uramustine®, Uramustine®), chlormethine (Mastergen®), cyclophosphamide (Cytoxan®, Neosal®, Clafen®, Endoxan®, Prokitox®, Revimmun™), ifosfamide (Mitoxana®),These include melphalan (Alkeran®), chlorambucil (Leukeran®), pipobroman (Amedel®, Vercite®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®), triethylenethiophosphoramine, temozolomide (Temodar®), thiotepa (Thioplex®), busulfan (Busilbex®, Myleran®), carmustine (BiCNU®), lomustine (Cine®), streptozocin (Zanosar®), and dacarbazine (DTIC-Dome®). Additional exemplary alkylating agents include, but are not limited to, oxaliplatin (Eloxatin®); temozolomide (Temodar® and Temodar®); dactinomycin (Actinomycin-D, also known as Cosmegen®); melphalan (L-PAM, L-sarcolysin, phenylalanine mustard, also known as Alkeran®); altretamine (hexamethylmelamine (HMM), Hexalen®); carmustine (also known as BiCNU®); bendamustine (Treanda®); busulfan (also known as Busulfex® and Myleran®); carboplatin (Paraplatin®); loxacin (Lysulfan®); Mustine (CCNU, also known as CeeNU®); cisplatin (CDDP, also known as Platinol® and Platinol®-AQ); chlorambucil (Leukeran®); cyclophosphamide (Cytoxan® and Neosar®); dacarbazine (DTIC, DIC, imidazole carboxamide, also known as DTIC-Dome®); altretamine (hexamethylmelamine (HMM), also known as Hexalen®); ifosfamide (Ifex®); prednummustine; procarbazine (Matulane®); mechlorethamine (nitrogen mustard, mustine, mechlorethamine hydrochloride,Also known as Mastergen®); streptozocin (Zanosar®); thiotepa (thiophosphamide, also known as TESPA, TSPA, Thioplex®); cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Prokitox®, RevImmune®); and bendamustine hydrochloride (Treanda®). Exemplary mTOR inhibitors include, for example, temsirolimus; ridaforolimus (formerly known as deferolimus, (IR, 2R, 45)-4-[(2R)-2[(1R, 95, 125, 15R, 16E, 18R, 19R, 21R, 235, 24E, 26E, 28Z, 305, 325, 35R)-1,18-dihydroxy-19,30-dihydroxy-15,17,21,23,29,35-hexamethyl-2,3,1 0,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.04'9]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669, described in PCT Publication No. WO 03 / 064383); everolimus (Afinitor® or RADOOI; rapamycin (AY22989, Sirolimus®); simapimod (CAS 164301-51-3); emsirolimus, (5-{2,4-bis[(35)-3-methylmorpholin-4-yl]pyrido[2,3-(i]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-amino-8-[iraw5,-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502, CAS 1013101-36-4); and Exemplary immunomodulators include, for example, N2-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]L-arginylglycyl-La-aspartyl-L-serine-, inner salt (SF1126, CAS 936487-67-1), and XL765.These include afutuzumab (available from Roche®), pegfilgrastim (Neulasta®), lenalidomide (CC-5013, Revlimid®), thalidomide (Thalomid®), actimid (CC4047); and IRX-2 (a human cytokine mixture containing interleukin-1, interleukin-2, and interferon-gamma, CAS 951209-71-5, available from IRX Therapeutics). Exemplary anthracyclines include, for example, doxorubicin (Adriamycin® and Rubex®), bleomycin (Ieloxan®), daunorubicin (daunorubicin hydrochloride, daunomycin, rubidomycin hydrochloride, Cervizin®), liposomal daunorubicin (daunorubicin citrate liposome, Daunosome®), mitoxantrone (DHAD, Novantrone®); epirubicin (Elence™); idarubicin (Idamycin®, Idamycin PFS®); mitomycin C (Mutamycin®); geldanamycin; herbimycin; rabidomycin; and desacetylrabidomycin. Exemplary vinca alkaloids include, for example, vinorelbine tartrate (Navelbine®), vincristine (Oncovin®), vindesine (Eldisine®), vinblastine (vinblastine sulfate, vincaleukoblastine, VLB, Alkaban-AQ®, Velban®), vinorelbine (Navelbine®). Exemplary proteosome inhibitors include bortezomib (Velcade®), carfilzomib (PX-171-007,(2S)-N-((S)-l-((S)-4-methyl-l-((R)-2-methyloxiran-2-yl)-l-oxopentan-2-ylcarbamoyl)-2-phenylethyl-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)-4-methylpentanamide; marizomib (NPT0052); ixazomib citrate (MLN-9708); delanzomib (CEP-18770); and O-methyl-N-[(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O-methyl-N-[(1S)-2-[(2R)-2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide (ONX-0912).

[0189] Those skilled in the art can readily identify useful chemotherapeutic agents (see, e.g., Physicians' Cancer Chemotherapy Drug Manual 2014, Edward Chu, Vincent T. DeVita Jr., Jones & Bartlett Learning; Principles of Cancer Therapy, Chapter 85 in Harrison's Principles of Internal Medicine, 18th edition; Therapeutic Targeting of Cancer Cells: Era of Molecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 in Abeloff's Clinical Oncology, 2013 Elsevier; and Fischer DS (ed): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 2003).

[0190] The effectiveness of immunotherapy (e.g., in treating a condition described herein or for inducing a response described herein (e.g., reduction in cancer cells)) can be determined by a skilled clinician. However, a treatment, as that term is used herein, is considered "effective treatment" if one or more of the signs or symptoms of a condition described herein are beneficially altered, other clinically recognized symptoms are improved or even improved, or a desired response is induced, e.g., by at least 10%, following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring markers, indicators, symptoms, and / or incidence, or other suitable measurable parameters, of a condition treated according to the methods described herein. Treatment according to the methods described herein can reduce the level of a marker or symptom of a condition by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more.

[0191] Efficacy can also be measured by the absence of deterioration of an individual's condition (i.e., the prevention of disease progression) as assessed by the need for hospitalization or medical intervention. Methods for measuring these indicators are known to those of skill in the art or are described herein.

[0192] Example We have studied the role of intercellular adhesion molecules (CAMs), primarily members of the immunoglobulin (Ig) superfamily, and discovered that proteolysis of CAMs is a common event in cancer and that cleaved fragments of CAMs function as tumor biomarkers. Full-length CAMs containing the Ig superfamily receptor protein tyrosine phosphatase (PTPμ) function as tumor suppressors and normally contribute to cell-cell adhesion by homophilically binding to PTPμ on neighboring cells, linking them together. In the microenvironment of multiple tumor types, proteolysis of PTPμ generates extracellular CD fragments, which result in the formation of specific epitopes (neoantigens) expressed exclusively on the tumor cell surface. We have developed peptides, proteins, and antibodies that bind to this novel tumor biomarker. These antibodies include humanized antibodies and scFv antibody fragments. One of these agents is a novel type of reagent called PTPμ-Fc chimera (described below), which we initially utilized for adhesion and neurite outgrowth studies. Conjugating these agents to tags enables tumor recognition and tracking using imaging. These agents can label both primary tumors and their infiltrating and metastatic cells, especially in brain tumors.

[0193] Fc domain chimeric proteins, in which the Fc domain of the human immunoglobulin heavy chain IgG1 is conjugated to tumor-specific proteins, represent a promising new class of immunotherapeutic agents. The Fc domain binds to Fcγ receptors (FcγRs) on innate immune cells, triggering antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells and phagocytosis of tumor cells by peripheral macrophages or microglia in the brain, recruiting them to the tumor microenvironment and triggering an immune response. Furthermore, FcγRs on dendritic cells (DCs) present tumor antigens to T cells. We explored two approaches to utilize these tumor-specific targeting agents to target GBM. First, we used PTPμ-Fc chimeric agents to stimulate the immune system to target and kill tumor cells. By combining the tumor-targeting ability of PTPμ with the host's own immune recognition, we hypothesized that ADCC could be induced, as demonstrated by our data using NK cells (Figure 1). Therapeutic Fc fusions to CAM have not yet been developed, making our study of CAM cleavage as a mechanism for detecting and targeting cancer unique. A key innovation is that a "human" or "humanized" reagent is generated because both the PTPμ and Fc sequence are human, allowing for immediate application in patients. Second, we leveraged existing human scFV and peptide sequences targeting PTPμ to develop novel immunotherapies.

[0194] We have developed a novel immunotherapy involving Fc fusions to a CAM developed for cell adhesion studies. In GBM, proteolysis of PTPμ generates extracellular domain (ECD) fragments. Previously, we developed peptide and protein fragments that homophilically bind to this novel tumor biomarker and label both the primary tumor mass and distantly infiltrating glioma cells. We hypothesized that by combining the GBM targeting potential of PTPμ with immune recognition of the body's own Fc region, PTPμ-targeted agents would elicit ADCC. Leveraging PTPμ targeting would 1) enable tracking and identification of tumor cells, 2) penetrate the protected brain parenchyma, and 3) label both primary tumors and infiltrating and metastatic cells equally well. We are currently developing these agents as tumor-targeted immunotherapeutics. The Fc or scFv components of these agents add antibody functionality and, because they are significantly smaller than typical antibodies, we hypothesize they will further possess improved tumor tissue penetration. The PTPμ-targeted protein sequence is: They home to and attach to the tumor-associated PTPμ ECD on tumor cells, thereby triggering: the innate immune system, including the complement response by Fc binding to complement receptors, and antibody-induced cytotoxicity and antibody-induced cytophagocytosis by binding to FcγR on infiltrating immune cells; and the adaptive immune system, by presenting MHC class I antigens on tumor cells and inducing cytotoxic T lymphocyte attack of these tumor cells. These PTPμ agents have high potential to enhance NK cell activity via ADCC.

[0195] array [ka] TIFF2026507222000003.tif214159TIFF2026507222000004.tif209159TIFF202 6507222000005.tif223159TIFF2026507222000006.tif207159TIFF2026507222 000007.tif215159TIFF2026507222000008.tif212159TIFF2026507222000009. tif213159TIFF2026507222000010.tif209159TIFF2026507222000011.tif20715 9TIFF2026507222000012.tif211159TIFF2026507222000013.tif213159TIFF20 26507222000014.tif211159TIFF2026507222000015.tif213159TIFF2026507222 000016.tif217159TIFF2026507222000017.tif213159TIFF2026507222000018. tif216159TIFF2026507222000019.tif212159TIFF2026507222000020.tif79159 [Table 1]

[0196] From the above description of the invention, those skilled in the art will perceive improvements, changes, and modifications. Such improvements, changes, and modifications within the skill of those skilled in the art are intended to be covered by the appended claims. All references, publications, and patents cited in this application are incorporated herein by reference in their entirety.

Claims

1. An antibody, antibody fragment, or antigen-binding fragment thereof comprising at least one of the following: a) CDR-H1 comprising the amino acid sequence GFTFTDYY (SEQ ID NO: 3), CDR-H2 comprising the amino acid sequence of IRNKANGYRT (SEQ ID NO: 4), and CDR-H3 comprising the amino acid sequence of ASLSAY (SEQ ID NO: 5); b) CDR-L1 comprising the amino acid sequence KSLLHSNGITY (SEQ ID NO: 8); CDR-L2 comprising the amino acid sequence of QMS (SEQ ID NO: 9), and CDR-L3 comprising the amino acid sequence of AQNLELWT (SEQ ID NO: 10); c) CDR-H1 comprising the amino acid sequence of GASITSGH (SEQ ID NO: 13); CDR-H2 comprising the amino acid sequence IRDSGTT (SEQ ID NO: 14), and CDR-H3 comprising the amino acid sequence of VTTPFVY (SEQ ID NO: 15); d) CDR-L1 comprising the amino acid sequence of QDINSY (SEQ ID NO: 18); CDR-L2 comprising the amino acid sequence of RAN (SEQ ID NO: 19), and CDR-L3 comprising the amino acid sequence of LQYDEFPLT (SEQ ID NO: 20); e) CDR-H1 comprising the amino acid sequence of GFSLFNYG (SEQ ID NO: 23); CDR-H2 comprising the amino acid sequence of IWADGTI (SEQ ID NO: 24), and CDR-H3 comprising the amino acid sequence of ARHFDY (SEQ ID NO: 25); f) CDR-L1 comprising the amino acid sequence QNIVHSNGNTY (SEQ ID NO: 28); CDR-L2 comprising the amino acid sequence of KVS (SEQ ID NO: 29), and CDR-L3 comprising FQGSRLPLT (SEQ ID NO: 30); g) CDR-H1 comprising the amino acid sequence of GFIFSSYY (SEQ ID NO: 33); CDR-H2 comprising the amino acid sequence of ININGGST (SEQ ID NO: 34), and CDR-H3 comprising the amino acid sequence of GRHDHGYGWFAY (SEQ ID NO: 35); h) CDR-L1 comprising the amino acid sequence of QNVGRN (SEQ ID NO: 38); CDR-L2 comprising the amino acid sequence of SAS (SEQ ID NO: 39), and CDR-L3 comprising the amino acid sequence of QQYNSYPWT (SEQ ID NO: 40); i) CDR-H1 comprising the amino acid sequence GYTFTTAG (SEQ ID NO: 43); CDR-H2 comprising the amino acid sequence of INTQSGVS (SEQ ID NO: 44), and CDR-H3 comprising the amino acid sequence of ARSNSGGFPY (SEQ ID NO: 45); j) CDR-L1 comprising the amino acid sequence QSLFNSTTHKIY (SEQ ID NO: 48); CDR-L2 comprising the amino acid sequence of FAS (SEQ ID NO: 49), and CDR-L3 comprising the amino acid sequence of QQHYTAPWT (SEQ ID NO: 50); k) CDR-H1 comprising the amino acid sequence GYTFTTVG (SEQ ID NO: 53); CDR-H2 comprising the amino acid sequence of INTHSGVS (SEQ ID NO: 54), and CDR-H3 comprising the amino acid sequence of ARGNSNYGFPY (SEQ ID NO: 55); l) CDR-L1 comprising the amino acid sequence EDIYNR (SEQ ID NO: 58), CDR-L2 comprising the amino acid sequence of GAT (SEQ ID NO: 59), and CDR-L3 comprising the amino acid sequence of QQYWSTPWT (SEQ ID NO: 60); m) CDR-H1 comprising the amino acid sequence of GYSITSDYA (SEQ ID NO: 63); CDR-H2 comprising the amino acid sequence of ISYRSIT (SEQ ID NO: 64), and CDR-H3 comprising the amino acid sequence of ARFGYDGNY (SEQ ID NO: 65); n) CDR-L1 comprising the amino acid sequence of QNIVHSNGDTY (SEQ ID NO: 68); CDR-L2 comprising the amino acid sequence of RVS (SEQ ID NO: 69), and CDR-L3 comprising the amino acid sequence of FQGSHVPLT (SEQ ID NO: 70); o) CDR-H1 comprising the amino acid sequence of GYTFTDYN (SEQ ID NO: 73); CDR-H2 comprising the amino acid sequence of INPDNGAT (SEQ ID NO: 74), and CDR-H3 comprising the amino acid sequence of VRGFDY (SEQ ID NO: 75); p) CDR-L1 comprising the amino acid sequence of QAIVHSNGNTY (SEQ ID NO: 78); CDR-L2 comprising the amino acid sequence of KVS (SEQ ID NO: 79), and CDR-L3 comprising the amino acid sequence of FQSSHIPWT (SEQ ID NO: 80); or A heavy chain variable region and / or a light chain variable region that competitively inhibits the binding of a peptide, protein, isolated antibody, antibody fragment, or antigen-binding fragment thereof, comprising at least one of a), b), c), d), e), f), g), h), i), j), k), l), m), n), o), or p), to a CAM, such as PTPμ.

2. An antibody, antibody fragment, or antigen-binding fragment thereof, comprising a heavy chain variable region that competitively inhibits binding of a heavy chain variable region that comprises three CDRs of one of SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ ID NO:61, or SEQ ID NO:71; or a peptide, protein, isolated antibody, antibody fragment, or antigen-binding fragment that comprises at least one heavy chain variable region that comprises three CDRs of one of SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ ID NO:61, or SEQ ID NO:

71.

3. An antibody, antibody fragment, or antigen-binding fragment thereof, comprising a heavy chain variable region comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of one of SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ ID NO:61, or SEQ ID NO:

71.

4. an antibody, antibody fragment, or antigen-binding fragment thereof, comprising a light chain variable region that competitively inhibits binding of a peptide, protein, isolated antibody, antibody fragment, or antigen-binding fragment thereof; or a light chain variable region that competitively inhibits binding of a peptide, protein, isolated antibody, antibody fragment, or antigen-binding fragment comprising at least one light chain variable region that comprises three CDRs of one of SEQ ID NO:6, SEQ ID NO:16, SEQ ID NO:26, SEQ ID NO:36, SEQ ID NO:46, SEQ ID NO:56, SEQ ID NO:66, or SEQ ID NO:

76.

5. An antibody, antibody fragment, or antigen-binding fragment thereof, comprising a light chain variable region comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of one of SEQ ID NO:6, SEQ ID NO:16, SEQ ID NO:26, SEQ ID NO:36, SEQ ID NO:46, SEQ ID NO:56, SEQ ID NO:66, or SEQ ID NO:

76.

6. An antibody, antibody fragment, or antigen-binding fragment thereof, comprising at least one of the following: a) a heavy chain variable region comprising the three CDRs of SEQ ID NO: 1 and a light chain variable region comprising the three CDRs of SEQ ID NO: 6; b) a heavy chain variable region comprising the three CDRs of SEQ ID NO: 11 and a light chain variable region comprising the three CDRs of SEQ ID NO: 16; c) a heavy chain variable region comprising the three CDRs of SEQ ID NO: 21 and a light chain variable region comprising the three CDRs of SEQ ID NO: 26; d) a heavy chain variable region comprising the three CDRs of SEQ ID NO: 31 and a light chain variable region comprising the three CDRs of SEQ ID NO: 36; e) a heavy chain variable region comprising the three CDRs of SEQ ID NO: 41 and a light chain variable region comprising the three CDRs of SEQ ID NO: 46; f) a heavy chain variable region comprising the three CDRs of SEQ ID NO: 51 and a light chain variable region comprising the three CDRs of SEQ ID NO: 56; g) a heavy chain variable region comprising the three CDRs of SEQ ID NO: 61 and a light chain variable region comprising the three CDRs of SEQ ID NO: 66; or h) A heavy chain variable region comprising the three CDRs of SEQ ID NO: 71 and a light chain variable region comprising the three CDRs of SEQ ID NO:

76.

7. An antibody, antibody fragment, or antigen-binding fragment thereof, comprising at least one of the following: a) a heavy chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:1; and a light chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:6; b) a heavy chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:11; and a light chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO: 16; c) a heavy chain variable fragment comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:

21. and a light chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:

26. d) a heavy chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO: 31; and a light chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:36; e) a heavy chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:41; and a light chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:45; f) a heavy chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO: 51; and a light chain comprising an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:56; g) a heavy chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:61; and and a light chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:66; or h) a heavy chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO: 71; and a light chain comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the amino acid sequence of SEQ ID NO:

76.

8. The antibody, antibody fragment, or antigen-binding fragment thereof according to any one of claims 1 to 7, comprising a peptide linker.

9. The antibody, antibody fragment, or antigen-binding fragment thereof of any one of claims 1 to 8, which is humanized or chimeric.

10. The antibody, antibody fragment, or antigen-binding fragment thereof according to any one of claims 1 to 9, which binds to PTPμ, PTPκ, PCP-2, PTPμ, or PTPρ.

11. The antibody, antibody fragment, or antigen-binding fragment thereof according to any one of claims 1 to 10, which binds to amino acids 42 to 60 of PTPμ, amino acids 816 to 914 of PTPμ, amino acids 958 to 1161 of PTPμ, or amino acids 915 to 958 of PTPμ.

12. at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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%, at least about 99%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 107%, at least about 109%, at least about 110%, at least about 111%, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about 117%, at least about 118%, at least about 119%, at least about 120%, at least about 121%, at least about 122%, at least about 123%, at least about 124%, at least about 125%, at least about 126%, at least about 127%, at least about 128%, at least about 129%, at least about 130%, at least about 131%, at least about 132%, at least about 133%, at least about 134%, at least about 135%, at least about 136%, at least about 137%, at least about 13 an antibody, antibody fragment, or antigen-binding fragment comprising an amino acid sequence that is 0%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to the antibody, antibody fragment, or antigen-binding fragment.

13. at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80% of SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, or SEQ ID NO:112; An antibody, antibody fragment, or antigen-binding fragment encoded by a nucleotide sequence that is at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical.

14. A chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain comprising an scFv having at least one of the following: a) CDR-H1 comprising the amino acid sequence GFTFTDYY (SEQ ID NO: 3), CDR-H2 comprising the amino acid sequence of IRNKANGYRT (SEQ ID NO: 4), and CDR-H3 comprising the amino acid sequence of ASLSAY (SEQ ID NO: 5); b) CDR-L1 comprising the amino acid sequence KSLLHSNGITY (SEQ ID NO: 8); CDR-L2 comprising the amino acid sequence of QMS (SEQ ID NO: 9), and CDR-L3 comprising the amino acid sequence of AQNLELWT (SEQ ID NO: 10); c) CDR-H1 comprising the amino acid sequence of GASITSGH (SEQ ID NO: 13); CDR-H2 comprising the amino acid sequence IRDSGTT (SEQ ID NO: 14), and CDR-H3 comprising the amino acid sequence of VTTPFVY (SEQ ID NO: 15); d) CDR-L1 comprising the amino acid sequence of QDINSY (SEQ ID NO: 18); CDR-L2 comprising the amino acid sequence of RAN (SEQ ID NO: 19), and CDR-L3 comprising the amino acid sequence of LQYDEFPLT (SEQ ID NO: 20); e) CDR-H1 comprising the amino acid sequence of GFSLFNYG (SEQ ID NO: 23); CDR-H2 comprising the amino acid sequence of IWADGTI (SEQ ID NO: 24), and CDR-H3 comprising the amino acid sequence of ARHFDY (SEQ ID NO: 25); f) CDR-L1 comprising the amino acid sequence QNIVHSNGNTY (SEQ ID NO: 28); CDR-L2 comprising the amino acid sequence of KVS (SEQ ID NO: 29), and CDR-L3 comprising FQGSRLPLT (SEQ ID NO: 30); g) CDR-H1 comprising the amino acid sequence of GFIFSSYY (SEQ ID NO: 33); CDR-H2 comprising the amino acid sequence of ININGGST (SEQ ID NO: 34), and CDR-H3 comprising the amino acid sequence of GRHDHGYGWFAY (SEQ ID NO: 35); h) CDR-L1 comprising the amino acid sequence of QNVGRN (SEQ ID NO: 38); CDR-L2 comprising the amino acid sequence of SAS (SEQ ID NO: 39), and CDR-L3 comprising the amino acid sequence of QQYNSYPWT (SEQ ID NO: 40); i) CDR-H1 comprising the amino acid sequence GYTFTTAG (SEQ ID NO: 43); CDR-H2 comprising the amino acid sequence of INTQSGVS (SEQ ID NO: 44), and CDR-H3 comprising the amino acid sequence of ARSNSGGFPY (SEQ ID NO: 45); j) CDR-L1 comprising the amino acid sequence QSLFNSTTHKIY (SEQ ID NO: 48); CDR-L2 comprising the amino acid sequence of FAS (SEQ ID NO: 49), and CDR-L3 comprising the amino acid sequence of QQHYTAPWT (SEQ ID NO: 50); k) CDR-H1 comprising the amino acid sequence GYTFTTVG (SEQ ID NO: 53); CDR-H2 comprising the amino acid sequence of INTHSGVS (SEQ ID NO: 54), and CDR-H3 comprising the amino acid sequence of ARGNSNYGFPY (SEQ ID NO: 55); l) CDR-L1 comprising the amino acid sequence EDIYNR (SEQ ID NO: 58), CDR-L2 comprising the amino acid sequence of GAT (SEQ ID NO: 59), and CDR-L3 comprising the amino acid sequence of QQYWSTPWT (SEQ ID NO: 60); m) CDR-H1 comprising the amino acid sequence of GYSITSDYA (SEQ ID NO: 63); CDR-H2 comprising the amino acid sequence of ISYRSIT (SEQ ID NO: 64), and CDR-H3 comprising the amino acid sequence of ARFGYDGNY (SEQ ID NO: 65); n) CDR-L1 comprising the amino acid sequence of QNIVHSNGDTY (SEQ ID NO: 68); CDR-L2 comprising the amino acid sequence of RVS (SEQ ID NO: 69), and CDR-L3 comprising the amino acid sequence of FQGSHVPLT (SEQ ID NO: 70); o) CDR-H1 comprising the amino acid sequence of GYTFTDYN (SEQ ID NO: 73); CDR-H2 comprising the amino acid sequence of INPDNGAT (SEQ ID NO: 74), and CDR-H3 comprising the amino acid sequence of VRGFDY (SEQ ID NO: 75); q) CDR-L1 comprising the amino acid sequence of QAIVHSNGNTY (SEQ ID NO: 78); CDR-L2 comprising the amino acid sequence of KVS (SEQ ID NO: 79), and CDR-L3 comprising the amino acid sequence of FQSSHIPWT (SEQ ID NO: 80); or A heavy chain variable region and / or a light chain variable region that competitively inhibits the binding of a peptide, protein, isolated antibody, antibody fragment, or antigen-binding fragment thereof, comprising at least one of a), b), c), d), e), f), g), h), i), j), k), l), m), n), o), p), or q) to a CAM, such as PTPμ.

15. A chimeric antigen receptor (CAR) wherein the PTPμ ligand comprises an extracellular antigen-binding domain having an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to SEQ ID NO:

113.

16. A nucleic acid comprising a nucleotide sequence encoding the CAR of claim 14 or 15.

17. 17. The nucleic acid of claim 16, wherein the nucleotide is operably linked to a promoter.

18. 18. An expression construct comprising the nucleic acid of claim 16 or 17.

19. 20. The expression construct of claim 18, comprising a vector.

20. 20. The expression construct of claim 19, wherein the vector is a retroviral vector, a lentiviral vector, or an AAV vector.

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