CD33-targeted antigen recognition receptor and uses thereof

Abstract

The subject matter disclosed herein provides an antigen-recognizing receptor that specifically targets CD33, and a cell comprising such a CD33-targeted antigen-recognizing receptor. The subject matter disclosed herein further provides a use of the CD33-targeted antigen-recognizing receptor for therapy. The subject matter disclosed herein provides an antigen-recognizing receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain specifically binds to CD33. In certain embodiments, the extracellular antigen-binding domain comprises a heavy chain variable region and a light chain variable region. In certain embodiments, the extracellular antigen-binding domain is a single chain variable fragment (scFv).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 306,395, filed February 2, 2022, and U.S. Provisional Patent Application No. 63 / 240,196, filed September 2, 2021, the contents of which are incorporated by reference in their entireties and priority thereto is claimed.

[0002] Sequence Listing This application contains a Sequence Listing submitted via EFS-Web, which is incorporated herein by reference in its entirety. The Sequence Listing, created on August 31, 2022, is named 0727341387.xml and is 164,036 bytes in size.

[0003] 1. Introduction The subject matter disclosed herein provides methods and compositions for immunotherapy, including antigen recognition receptors (e.g., chimeric antigen receptors (CARs)) that specifically target CD33, cells containing such receptors, and methods of using such cells for therapy. [Background technology]

[0004] 2. Background of the Invention Acute myeloid leukemia (AML) is the most common and most lethal form of acute leukemia in the United States. For the past 40 years, the standard of care has been chemotherapy, but in recent years, CD33 antibody-drug conjugates (ADCs) and targeted small molecules have been added to the suite. Despite these new additions, AML remains a devastating disease with a 5-year survival rate of less than 30%. Thus, there is a significant need for novel AML interventions.

[0005] Over the past decade, CAR T cells have emerged as one of the most powerful forms of immunotherapy. Many groups have deployed this technology in the treatment of AML via targeting CD33, but one major obstacle remains: heterogeneous density of this antigen within and between patient populations. This is of great importance since CAR T cell efficacy correlates with antigen density and reduction, rather than complete loss, of antigen surface expression has been shown to be a mechanism of immune evasion of CAR T cells. To overcome this, membrane proximal epitope targeting has emerged as a strategy to enhance CAR T cell efficacy, resulting in increased overall CAR T cell potency and the ability of effector cells to recognize low antigen density variants. As a result, developing CAR T cells targeting membrane proximal epitopes of CD33 would allow for enhanced elimination of diseases with variable CD33 expression, including splice variants that may lack the membrane distal domain that is the target of all currently known antibodies. Summary of the Invention

[0006] 3. Overview of the Invention The presently disclosed subject matter provides an antigen-recognizing receptor that specifically targets CD33, and a cell comprising such a CD33-targeted antigen-recognizing receptor. The presently disclosed subject matter further provides a use of the CD33-targeted antigen-recognizing receptor for therapy.

[0007] The subject matter disclosed herein provides an antigen-recognizing receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain specifically binds to CD33. In certain embodiments, the extracellular antigen-binding domain comprises a heavy chain variable region and a light chain variable region. In certain embodiments, the extracellular antigen-binding domain is a single chain variable fragment (scFv). In certain embodiments, the variable regions are arranged from the N-terminus to the C-terminus: H -V LIn certain embodiments, the extracellular antigen binding domain is a human scFv. In certain embodiments, the extracellular antigen binding domain is a Fab, which is optionally cross-linked. In certain embodiments, the extracellular antigen binding domain is a F(ab)2. In certain embodiments, one or more of an scFv, a Fab, and a F(ab)2 are included in a fusion protein with heterologous sequences to form the extracellular antigen binding domain.

[0008] In certain embodiments, the heavy chain variable region comprises: (a) CDR1 comprising the amino acid sequence set forth in SEQ ID NO:2 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:3 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:4 or a conservative modification thereof; (b) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14 or a conservative modification thereof; (c) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24 or a conservative modification thereof; (d) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 or a conservative modification thereof; (e) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39 or a conservative modification thereof; (f) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 47 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 48 or a conservative modification thereof; (g) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 56 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 57 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 58 or a conservative modification thereof; (h) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 66 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 67 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 68 or a conservative modification thereof; (i) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 76 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 57 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 77 or a conservative modification thereof; or (j) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 85 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 86 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 87 or a conservative modification thereof.

[0009] In certain embodiments, the heavy chain variable region comprises: (a) CDR1 comprising the amino acid sequence set forth in SEQ ID NO:2 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:3 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:4 or a conservative modification thereof; (b) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14 or a conservative modification thereof; or (c) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24 or a conservative modification thereof.

[0010] In certain embodiments, the light chain variable region comprises: (a) CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7 or a conservative modification thereof; (b) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16 or a conservative modification thereof, and CDR3 comprising SEQ ID NO: 17 or a conservative modification thereof; (c) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27 or a conservative modification thereof; (d) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 40 or a conservative modification thereof, CDR2 comprising SEQ ID NO: 41 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42 or a conservative modification thereof; (e) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 49 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 50 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 51 or a conservative modification thereof; (f) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 59 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 60 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 61 or a conservative modification thereof; (g) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 69 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 70 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 71 or a conservative modification thereof; (h) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 78 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 79 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 80 or a conservative modification thereof; or (i) It comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:41 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:42 or a conservative modification thereof.

[0011] In certain embodiments, the light chain variable region comprises: (a) CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7 or a conservative modification thereof; (b) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16 or a conservative modification thereof, and CDR3 comprising SEQ ID NO: 17 or a conservative modification thereof; (c) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27 or a conservative modification thereof.

[0012] In certain embodiments, (a) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:2, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:3, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:4, and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7; or (b) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17; or (c) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27; or (d) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27; or (e) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 40, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 41, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42; or (f) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 47, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 48, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 49, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 50, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 51; or (g) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 56, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 57, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 58, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 59, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 60, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 61; or (h) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 66, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 67, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 68, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 69, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 70, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 71; or (i) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 76, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 57, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 77, and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 78, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 79, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 80; or (j) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 85, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 86, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 87, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 41, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42.

[0013] In certain embodiments, (a) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:2, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:3, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:4, and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7; or (b) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14, and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17; or (c) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27.

[0014] In certain embodiments, the heavy chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:52, SEQ ID NO:62, SEQ ID NO:72, SEQ ID NO:81, or SEQ ID NO:88. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:52, SEQ ID NO:62, SEQ ID NO:72, SEQ ID NO:81, or SEQ ID NO:88. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:18, or SEQ ID NO:28.

[0015] In certain embodiments, the light chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:19, SEQ ID NO:29, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:63, SEQ ID NO:73, SEQ ID NO:82, SEQ ID NO:89, or SEQ ID NO:92. In certain embodiments, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:19, SEQ ID NO:29, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:63, SEQ ID NO:73, SEQ ID NO:82, SEQ ID NO:89, or SEQ ID NO:92. In certain embodiments, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:19, or SEQ ID NO:29.

[0016] In certain embodiments, (a) the heavy chain variable region is an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to a selected amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:52, SEQ ID NO:62, SEQ ID NO:72, SEQ ID NO:81, or SEQ ID NO:88. and (b) the light chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:19, SEQ ID NO:29, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:63, SEQ ID NO:73, SEQ ID NO:82, SEQ ID NO:89, or SEQ ID NO:92.

[0017] In certain embodiments, (a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:52, SEQ ID NO:62, SEQ ID NO:72, SEQ ID NO:81, or SEQ ID NO:88, and (b) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:19, SEQ ID NO:29, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:63, SEQ ID NO:73, SEQ ID NO:82, SEQ ID NO:89, or SEQ ID NO:92. In certain embodiments, (a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:18, or SEQ ID NO:28, and (b) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:19, or SEQ ID NO:29.

[0018] In certain embodiments, (a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:9; or (b) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 19; or (c) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:28 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:29; or (d) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 35 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 29; or (e) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 43 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 44; or (f) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:52 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:53; or (g) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:62 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:63; or (h) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 72 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 73; or (i) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 81 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 82; or (j) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 88 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 89; or (k) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:93.

[0019] In certain embodiments, (a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:9; or (b) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 19; or (c) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:28 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:29.

[0020] In certain embodiments, the extracellular antigen-binding domain comprises a linker between the heavy chain variable region and the light chain variable region. In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, or SEQ ID NO:100. In certain embodiments, a signal peptide is covalently linked to the 5' end of the extracellular antigen-binding domain.

[0021] In certain embodiments, the transmembrane domain comprises a CD8 polypeptide, a CD28 polypeptide, a CD3zeta polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, or a combination thereof.

[0022] In certain embodiments, the intracellular signaling domain comprises a CD3ζ polypeptide. In certain embodiments, the intracellular signaling domain further comprises at least one costimulatory signaling region. In certain embodiments, the at least one costimulatory signaling region comprises a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, or a combination thereof.

[0023] In certain embodiments, the antigen recognition receptor is a chimeric antigen receptor (CAR) or a T cell-like fusion protein. In certain embodiments, the antigen recognition receptor is a CAR.

[0024] In certain embodiments, the antigen recognition receptor is recombinantly expressed. In certain embodiments, the antigen recognition receptor is expressed from a vector. In certain embodiments, the vector is a gamma-retroviral vector.

[0025] The subject matter disclosed herein provides that a cell comprises an antigen recognition receptor disclosed herein. In certain embodiments, the cell is transduced with the antigen recognition receptor. In certain embodiments, the antigen recognition receptor is constitutively expressed on the surface of the cell.

[0026] In certain embodiments, the cell is an immunoresponsive cell. In certain embodiments, the cell is a lymphoid cell or a myeloid cell. In certain embodiments, the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, and a stem cell from which a lymphoid cell can differentiate. In certain embodiments, the cell is a T cell. In certain embodiments, the T cell is a cytotoxic T lymphocyte (CTL) or a regulatory T cell. In certain embodiments, the stem cell is a pluripotent stem cell. In certain embodiments, the pluripotent stem cell is an embryonic-like stem cell or an induced pluripotent stem cell.

[0027] The presently disclosed subject matter further provides a nucleic acid encoding an antigen-recognizing receptor disclosed herein. The presently disclosed subject matter further provides a vector comprising a nucleic acid molecule disclosed herein. In certain embodiments, the vector is a viral vector. In certain embodiments, the vector is a gamma-retroviral vector.

[0028] In addition, the presently disclosed subject matter provides a host cell expressing the nucleic acid molecules disclosed herein. In certain embodiments, the host cell is a T cell.

[0029] The presently disclosed subject matter further provides a composition comprising the cells disclosed herein. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier.

[0030] The subject matter disclosed herein also provides lipid nanoparticles comprising the nucleic acids disclosed herein.The subject matter disclosed herein also provides compositions comprising the lipid nanoparticles disclosed herein.In certain embodiments, the composition is a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier.

[0031] The presently disclosed subject matter further provides a method of treating or ameliorating a disease or disorder in a subject. In certain embodiments, the method comprises administering to the subject a cell or composition disclosed herein.

[0032] The subject matter disclosed herein also provides a method of reducing tumor burden in a subject. In certain embodiments, the method comprises administering to the subject a cell or composition disclosed herein. In certain embodiments, the method reduces the number of tumor cells, reduces tumor size, and / or eradicates tumors in the subject.

[0033] The subject matter disclosed herein provides methods of treating and / or preventing a tumor in a subject. In certain embodiments, the methods include administering to the subject a cell or composition disclosed herein. The subject matter disclosed herein provides methods of increasing or extending the survival time of a subject having a tumor. In certain embodiments, the methods include administering to the subject a cell or composition disclosed herein. In certain embodiments, the methods reduce or eradicate the tumor burden in the subject.

[0034] In certain embodiments, the disease or disorder is a tumor. In certain embodiments, the tumor is a hematological cancer or a solid tissue cancer. In certain embodiments, the tumor is selected from the group consisting of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), myeloproliferative neoplasms (MPN), and chronic myeloid neoplasms. In certain embodiments, the tumor is acute myeloid leukemia (AML). In certain embodiments, the subject is a human.

[0035] The subject matter disclosed herein further provides kits for treating or ameliorating a disease or disorder in a subject, reducing tumor burden in a subject, treating and / or preventing a tumor in a subject, and / or increasing or extending survival time of a subject having a tumor, the kit comprising a cell, nucleic acid, or composition disclosed herein. In certain embodiments, the kit further comprises written instructions for using the cell or composition disclosed herein to treat or ameliorate a disease or disorder in a subject, reducing tumor burden in a subject, treating and / or preventing a tumor in a subject, and / or increasing or extending survival time of a subject having a tumor.

[0036] In addition, the presently disclosed subject matter provides a method for producing a CD33-targeting antigen-recognizing receptor, comprising introducing into a cell a nucleic acid encoding the antigen-recognizing receptor.

[0037] Finally, the subject matter disclosed herein provides a cell or composition disclosed herein for use in treating or ameliorating a disease or disorder in a subject. In certain embodiments, the disease or disorder is a tumor. In certain embodiments, the tumor is a cancer. In certain embodiments, the disease or disorder is selected from the group consisting of neuroendocrine tumors of the lung, extrapulmonary neuroendocrine carcinoma, melanoma, neuroendocrine prostate cancer, breast cancer, neuroendocrine tumors of the gastrointestinal tract, pancreatic cancer, medullary thyroid carcinoma, small cell bladder cancer, small cell ovarian carcinoma, low-grade glioma, glioblastoma, and neuroblastoma. In certain embodiments, the neuroendocrine tumors of the lung are selected from the group consisting of lung neuroendocrine carcinoma, large cell neuroendocrine carcinoma, and small cell lung carcinoma. In certain embodiments, the tumor is small cell lung carcinoma. In certain embodiments, the subject is a human.

[0038] 4. Brief description of the drawings The following detailed description is given by way of example and is not intended to limit the invention to the particular embodiments described and may be understood in conjunction with the accompanying drawings. [Brief description of the drawings]

[0039] [Figure 1A] CD33 domain structure. Shown is full-length CD33 (CD33M) with two Ig-like domains (IgV and IgC2) in the extracellular region, a single transmembrane segment, immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and ITIM-like motifs in the cytoplasmic region. In the common CD33 variant (CD33m), the IgV domain is missing due to alternative splicing. [Figure 1B] CD33 domain structure is shown. CD33 gene structure with and without alternative splicing is shown. Removal of exon 2 results in loss of the IgV domain. Several antibodies that have progressed to the clinic in various formats (CAR, BiTE, radiotherapy) target the IgV domain. "GO" stands for gemtuzumab ozogamicin. [Diagram 2] 1 shows the structure of an exemplary CD33-targeted CAR according to the subject matter disclosed herein. The CD33-targeted CAR comprises a truncated EGFR (EGFRt), an anti-CD33 scFv, a Myc tag, a transmembrane domain comprising a CD28 polypeptide, and an intracellular signaling domain comprising a CD3ζ polypeptide, and a costimulatory signaling region comprising a CD28 polypeptide. T2A is the cleavage site. [Figure 3-1] Shown are surface expression levels and flow cytometry analysis of transduced PBMCs showing transduction efficiency and CAR surface expression of each construct detected by anti-Myc tag antibody. [Figure 3-2] Shown are surface expression levels and flow cytometry analysis of transduced PBMCs showing transduction efficiency and CAR surface expression of each construct detected by anti-Myc tag antibody. [Figure 4A] Cytotoxicity of membrane proximal CARs. 3P14 and 4B2 along with the H195 reference CAR were tested on the U937 cell line using the heavy and light chain variable regions of each antibody in VH-VL (HL) or VL-VH (LH) orientation. [Figure 4B]Cytotoxicity of membrane-proximal CARs. 3P14 and 4B2 along with the H195 reference CAR were tested on the OCi-AML3 cell line. [Figure 5A] Cytotoxicity of membrane-distal CARs. Cells from donor 2 are used to demonstrate in vitro killing of U937 cells by CAR T cells generated from five antibodies targeting membrane-distal epitopes within the CD33 IgV domain. [Figure 5B] Cytotoxicity of membrane-distal CARs. Cells from donor 3 are used to demonstrate in vitro killing of U937 cells by CAR T cells generated from five antibodies targeting membrane-distal epitopes within the CD33 IgV domain. [Figure 6A] Figure 1 shows in vitro cytotoxicity of TDI-Y-006 and TDI-Y-007 CARs. A-C show cytotoxicity of three AML target lines containing a GFP-firefly luciferase reporter (gL) from donor 1 with increasing effector-to-target ratios as indicated. Killing was assessed using a luciferase assay. Potent killing by TDI-Y-006 and TDI-Y-007 was observed in a variety of CD33+ target cells and PBMCs from different donors. [Figure 6B] Figure 1 shows in vitro cytotoxicity of TDI-Y-006 and TDI-Y-007 CARs. A-C show cytotoxicity of three AML target lines containing a GFP-firefly luciferase reporter (gL) from donor 1 with increasing effector-to-target ratios as indicated. Killing was assessed using a luciferase assay. Potent killing by TDI-Y-006 and TDI-Y-007 was observed in a variety of CD33+ target cells and PBMCs from different donors. [Figure 6C]Figure 1 shows in vitro cytotoxicity of TDI-Y-006 and TDI-Y-007 CARs. A-C show cytotoxicity of three AML target lines containing a GFP-firefly luciferase reporter (gL) from donor 1 with increasing effector-to-target ratios as indicated. Killing was assessed using a luciferase assay. Potent killing by TDI-Y-006 and TDI-Y-007 was observed in a variety of CD33+ target cells and PBMCs from different donors. [Figure 6D] Figure 1 shows in vitro cytotoxicity of TDI-Y-006 and TDI-Y-007 CARs. D-F show cytotoxicity of three AML target lines containing a GFP-firefly luciferase reporter (gL) from donor 2 with increasing effector-to-target ratios as indicated. Killing was assessed using a luciferase assay. Potent killing by TDI-Y-006 and TDI-Y-007 was observed in a variety of CD33+ target cells and PBMCs from different donors. [Figure 6E] Figure 1 shows in vitro cytotoxicity of TDI-Y-006 and TDI-Y-007 CARs. D-F show cytotoxicity of three AML target lines containing a GFP-firefly luciferase reporter (gL) from donor 2 with increasing effector-to-target ratios as indicated. Killing was assessed using a luciferase assay. Potent killing by TDI-Y-006 and TDI-Y-007 was observed in a variety of CD33+ target cells and PBMCs from different donors. [Figure 6F] Figure 1 shows in vitro cytotoxicity of TDI-Y-006 and TDI-Y-007 CARs. D-F show cytotoxicity of three AML target lines containing a GFP-firefly luciferase reporter (gL) from donor 2 with increasing effector-to-target ratios as indicated. Killing was assessed using a luciferase assay. Potent killing by TDI-Y-006 and TDI-Y-007 was observed in a variety of CD33+ target cells and PBMCs from different donors. [Figure 7A]Figure 1 shows the cytotoxicity of 1J19 CAR. Figure 2 shows the cytotoxicity as percent killing of HL60 cell line containing GFP-firefly luciferase reporter (gL) by donor 2 with increasing effector-to-target ratio as indicated. Killing was assessed using luciferase assay. Overall, strong killing by 1J19 was observed compared to H195 reference CAR. [Figure 7B] Figure 1 shows the cytotoxicity of 1J19 CAR. Figure 2 shows the cytotoxicity as percent killing of HL60 cell line containing GFP-firefly luciferase reporter (gL) by donor 3 with increasing effector-to-target ratio as indicated. Killing was assessed using luciferase assay. Overall, strong killing by 1J19 was observed compared to H195 reference CAR. [Figure 7C] Figure 1 shows the cytotoxicity of 1J19 CAR. Figure 2 shows the cytotoxicity as percent killing of U937 cell line containing GFP-firefly luciferase reporter (gL) by donor 2 with increasing effector-to-target ratio as indicated. Killing was assessed using luciferase assay. Overall, strong killing by 1J19 was observed compared to H195 reference CAR. [Figure 7D] Figure 1 shows the cytotoxicity of 1J19 CAR.D shows the cytotoxicity as percent killing of OCI-AML3 cell line containing GFP-firefly luciferase reporter (gL) by donor 1 with increasing effector-to-target ratio as indicated. Killing was assessed using luciferase assay. Overall, strong killing by 1J19 was observed compared to H195 reference CAR. [Figure 8A] Figure 1 shows CD33 CAR T cell proliferation upon binding to target cells. A and B show that human T cells from two donors transduced with the lead CAR were co-cultured with U937 cells (CD33high). Flow cytometry was used to detect the total number of remaining targets and T cells at 7, 14 and 21 days after co-culture. The total remaining human T cells per mL are shown in days over time. [Figure 8B]Figure 1 shows CD33 CAR T cell proliferation upon binding to target cells. A and B show that human T cells from two donors transduced with the lead CAR were co-cultured with U937 cells (CD33high). Flow cytometry was used to detect the total number of remaining targets and T cells at 7, 14 and 21 days after co-culture. The total remaining human T cells per mL are shown in days over time. [Figure 8C] CD33 CAR T cell proliferation upon binding to target cells. C and D show that human T cells from two donors transduced with the lead CAR were co-cultured with OCi-AML3 (CD33low) cells. Flow cytometry was used to detect the total number of remaining targets and T cells at 7, 14 and 21 days after co-culture. The total number of remaining human T cells per mL is shown in days over time. [Figure 8D] CD33 CAR T cell proliferation upon binding to target cells. C and D show that human T cells from two donors transduced with the lead CAR were co-cultured with OCi-AML3 (CD33low) cells. Flow cytometry was used to detect the total number of remaining targets and T cells at 7, 14 and 21 days after co-culture. The total number of remaining human T cells per mL is shown in days over time. [Figure 9A] Figure 1 shows the cytokine secretion profile of lead CD33 CAR T cells. A panel of four cytokines was analyzed using three different donors. Levels of GMCSF in supernatants collected after co-culture of target and effector cells at 24 hours were measured using a human 12-plex Luminex panel kit. Data from a representative donor 1 is shown. [Figure 9B] Figure 1 shows the cytokine secretion profile of lead CD33 CAR T cells. A panel of four cytokines was analyzed using three different donors. Levels of IFN-γ are shown. Data from a representative donor 1 are shown. [Figure 9C]Figure 1 shows the cytokine secretion profile of lead CD33 CAR T cells. A panel of four cytokines was analyzed using three different donors. Levels of TNF-α are shown. Data from a representative donor 1 are shown. [Figure 9D] Figure 1 shows the cytokine secretion profile of lead CD33 CAR T cells. A panel of four cytokines was analyzed using three different donors. Levels of IL-2 are shown. Data from a representative donor 1 is shown. [Figure 10A] Figure 1 shows in vivo efficacy of lead CAR T in a U937 AML xenograft mouse model. Time-lapse imaging and quantification of GFP-firefly luciferase tagged U937 cells (CD33high) in NCG mice treated IV with lead CAR T cells. [Figure 10B] Shows the in vivo efficacy of lead CAR T in U937 AML xenograft mouse model. Survival curves are shown. [Figure 11A] In vivo efficacy of lead CAR T with titrated dosing. Data shown are from animals injected with 5 105 CAR T cells and monitored for tumor growth and survival. [Figure 11B] In vivo efficacy of lead CAR T with titrated dosing. Data from animals injected with 2.5 105 CAR T cells are shown. [Figure 12A] Figure 1 shows in vivo efficacy of lead CAR T in an OCi-AML3 xenograft mouse model. Imaging and quantification of firefly luciferase tagged OCi-AML3 cells (CD33low) over time in NCG mice treated IV with lead CAR T cells. Mean survival time is shown for 5 animals per group. [Figure 12B] Figure 1 shows in vivo efficacy of lead CAR T in an OCi-AML3 xenograft mouse model. Imaging and quantification of firefly luciferase tagged OCi-AML3 cells (CD33low) over time in NCG mice treated IV with lead CAR T cells. Individual survival curves for each animal are shown. [Figure 13] Figure 1 shows a head-to-head comparison of in vivo efficacy of two lead and one backup CARs in a U937 AML xenograft mouse model. Imaging and quantification over time of firefly luciferase tagged U937 cells (CD33high) in NCG mice treated IV with lead and backup 1J19 CAR T cells. [Figure 14] Figure 1 shows the in vivo efficacy of TDI-Y-006 CAR T with patient-derived AML xenografts. Patient-derived tumor cells were injected intravenously and allowed to grow for 10 days. TDI-Y-006 CAR T was injected on day 14. After 7 days, the number of CD2-CD33+ cells was determined by flow cytometry. Mean data from 5 mice for H195_Del and 4 mice for TDI-Y-006 CAR T indicated with p-value *<0.05. [Figure 15A] Pro-inflammatory cytokine levels in PDX models. IFN-gamma levels measured using a human 12-plex Luminex panel kit from mice treated with TDI-Y-006 CAR T after patient-derived xenograft implantation. *p-value<0.05. [Figure 15B] Pro-inflammatory cytokine levels in PDX models. Levels of TNF-alpha are shown. *p-value<0.05. [Figure 16A] Figure 1 shows an in vitro evaluation of the toxicity of CD33 CAR T. Hematopoietic stem cells isolated from umbilical cords tested by colony forming units with and without CD33 lead CAR T. Gemtuzumab ozogamicin (GO, anti-CD33 ADC) and Del CAR were used as controls. [Figure 16B] In vitro evaluation of CD33 CAR T toxicity. Total colony counts are shown to assess toxicity of experimental agents. Gemtuzumab ozogamicin (GO, anti-CD33 ADC) and Del CAR were used as controls. [Figure 17] 1 shows a heatmap depicting the relative binding of selected antibodies to human full-length CD33, CD33-IgC, and the U937 CD33-expressing tumor line. [Figure 18A] Selection of membrane proximal CD33-IgC targeting scFv is shown. Quantification of cross-reactivity of hybridoma-derived monoclonal antibodies as measured by His-tag binding is shown. [Figure 18B] 1 shows the selection of membrane proximal CD33-IgC targeting scFv. Quantification of binding kinetics of hybridoma-derived monoclonal antibodies to CD33 in solution. [Figure 18C] Selection of membrane proximal CD33-IgC targeting scFv is shown. Quantification of EC50 of lead hybridoma-derived monoclonal antibodies on overexpressing (3T3) and AML cell lines (U937) is shown. [Figure 18D] Selection of membrane proximal CD33-IgC targeting scFv. Quantification of epitope binning of selected hybridoma-derived monoclonal antibodies. [Figure 19] Representative flow cytometry plots are shown demonstrating comparable retroviral transduction efficiencies as determined by flow cytometry. p values ​​determined by repeated measures one-way ANOVA. Data are mean ± SEM of three independent experiments. ns, not significant. [Figure 20A] Shown is membrane-proximal targeting of CAR T cells enhancing functionality and proliferation capacity in vitro. Shown is flow cytometry histograms and quantitative geometric mean fluorescence (MFI) showing expression of CD33 on wild-type or CD33 knockout (CD33KO) AML cells detected with a fluorescently labeled anti-CD33 antibody. [Figure 20B] Shows membrane-proximal targeting of CAR T cells enhancing functionality and proliferation capacity in vitro. Shows D-luciferin assay at 24 hours demonstrating lysis of U937-CD33highgfpLuc+ tumor cells (n=4, ****, P<0.0001, ***, P<0.001, *, P<0.05 by 2-way ANOVA). Data are mean ± SEM of 4 independent experiments. [Figure 20C]Shows membrane-proximal targeting of CAR T cells enhancing functionality and proliferation capacity in vitro. Shown is a 24-hour D-luciferin assay demonstrating lysis of OCiAML3-CD33lowgfpLuc+ tumor cells (n=4, *, P<0.05 by 2-way ANOVA). Data are the mean ± SEM of 4 independent experiments. [Figure 20D] Shows membrane-proximal targeting of CAR T cells enhancing functionality and proliferation capacity in vitro. Shown is a 138 hour (6 day) assay demonstrating lysis of U937-CD33highgfpLuc+ tumor cells at low effector-to-target ratios (n=2, **, P<0.01, *, P<0.05 by 2-way ANOVA). Data are representative of 4 independent experiments. [Figure 20E] Shows membrane-proximal targeting of CAR T cells enhancing functionality and proliferation capacity in vitro. Shown is a 138 hour assay demonstrating lysis of OCiAML3-CD33lowgfpLuc+ tumor cells at low effector-to-target ratios (n=2, **, P<0.01, *, P<0.05 by 2-way ANOVA). Data are representative of 4 independent experiments. [Figure 20F] Figure 1 shows membrane proximal targeting of CAR T cells enhances functionality and proliferation capacity in vitro. Quantification of flow cytometry analysis demonstrating enhanced proliferation by membrane proximal CD33 targeting of CAR T cells in the presence of U937-CD33high tumor cells (n=9, ****, P<0.0001 by 2-way ANOVA). Data are mean ± SEM of three independent experiments. [Figure 20G] Membrane proximal targeting of CAR T cells enhances functionality and proliferation capacity in vitro. Quantification of flow cytometry analysis demonstrating enhanced proliferation by membrane proximal CD33 targeting of CAR T cells in the presence of OCiAML3-CD33lowgfpLuc+ tumor cells (n=9, ****, P<0.0001 by 2-way ANOVA). Data are mean ± SEM of three independent experiments. ns, not significant. [Figure 21A]Shows membrane proximal specificity of targeting CAR T cells. Shows flow cytometry histograms showing FLAG expression on transduced U937-CD33IgCgfpLuc+ tumor cells. [Figure 21B] Shows membrane proximal specificity of targeting CAR T cells. Shows 24 hour D-luciferin assay demonstrating lysis of U937-CD33IgCgfpLuc+ tumor cells. Data are representative of two independent experiments. [Figure 21C] Shows membrane proximal specificity of targeting CAR T cells. Shows 24 hour D-luciferin assay demonstrating lysis of U937-CD33KOgfpLuc+ tumor cells. Data are representative of two independent experiments. [Figure 21D] Showing membrane-proximal specificity of targeting CAR T cells. Quantification of total colonies produced using colony forming unit (CFU) assay to measure hematopoietic killing is shown, where total colony numbers are a composite of BFU-E, GEMM-CFU, and GM-CFU (n=9, ****, P<0.0001, ***, P<0.001 by ordinary one-way ANOVA, ns, not significant). Data shown are a composite of the mean ± SEM of three independent experiments. [Figure 22A] Membrane proximal targeted CAR T cells characterized by unique activation profiles in vitro. Cytokine secretion profiles of Tc1 / Th1 cytokines upon co-culture with U937-CD33highgfpLuc+, OCiAML3-CD33lowgfpLuc+, U937-CD33IgCgfpLuc+ tumors, and U937-CD33KOgfpLuc+ detected by human 12-plex Luminex panel (n=3, ****, P<0.0001, *, P<0.05 by Dunnett's multiple comparison test vs. H195DEL control). Data are representative of three independent experiments. [Figure 22B]Membrane proximal targeted CAR T cells are characterized by a unique activation profile in vitro. Quantification of flow cytometry analysis showing CAR T cell intracellular production of Tc1 / Th1 activating cytokines is shown (n=4, ****, P<0.0001, **, P<0.01, *, P<0.05 by 2-way ANOVA, ns, not significant). Data are mean ± SEM of 4 independent experiments. [Figure 22C] Membrane proximal targeted CAR T cells characterized by unique activation profiles in vitro. Qualitative representation of CD4+CAR+ and CD8+CAR+Tc1 / Th1 activation cytokine secretion is shown. [Figure 22D] Membrane proximal targeted CAR T cells characterized by a unique activation profile in vitro. Quantification of flow cytometry analysis showing CD4+ CAR T cell intracellular production of Tc1 / Th1 activating cytokines is shown (n=4, ****, P<0.0001, *, P<0.05 by two-way ANOVA). Data are mean ± SEM of four independent experiments. [Figure 22E] Membrane proximal targeted CAR T cells characterized by a unique activation profile in vitro. Quantification of flow cytometry analysis showing CD8+ CAR T cell intracellular production of Tc1 / Th1 activating cytokines is shown (n=4, ***, P<0.001, **, P<0.01, *, P<0.05 by two-way ANOVA). Data are mean ± SEM of four independent experiments. [Figure 22F] Membrane proximal targeted CAR T cells characterized by a unique activation profile in vitro. Quantification of flow cytometry analysis demonstrating CAR T cell activation status 7 days after antigen stimulation is shown (n=4, ****, P<0.0001 by 2-way ANOVA). Data are the mean ± SEM of three independent experiments. [Figure 22G]1 shows membrane proximal targeted CAR T cells characterized by unique activation profiles in vitro. Qualitative representation of flow cytometry analysis comparing CAR T cell activation profiles. Data are pooled from three independent experiments. [Figure 23A] Figure 1 shows membrane-proximal CD33-targeted CAR T cells enhance survival time in a xenograft mouse model. Schematic diagram of in vivo experimental setup. NCG mice were inoculated with U937-CD33highgfpLuc+ tumors and subsequently treated with CAR T cells. [Figure 23B] Figure 1 shows that membrane-proximal CD33-targeted CAR T cells enhance survival time in a xenograft mouse model. Survival time of NCG mice bearing U937-CD33highgfpLuc+ tumors and treated with 5.0x105 CAR T cells is shown (n=5, **, P<0.01). p-values ​​for survival time determined by log-rank Mantel-Cox test with 95% confidence interval. In vivo data shown is pooled from 5 mice. [Figure 23C] Figure 1 shows that membrane-proximal CD33-targeted CAR T cells enhance survival time in a xenograft mouse model. Survival time of NCG mice bearing U937-CD33highgfpLuc+ tumors and treated with 2.5x105 CAR T cells is shown (n=5, **, P<0.01, *, P<0.05). p-values ​​for survival time determined by log-rank Mantel-Cox test with 95% confidence interval. In vivo data shown is pooled from 5 mice. [Figure 23D] Figure 1 shows that membrane-proximal CD33-targeted CAR T cells enhance survival time in xenograft mouse model. Survival time of NCG mice bearing U937-CD33highgfpLuc+ tumors and treated with 1.0x105 CAR T cells is shown (n=5, **, P<0.01, ns, not significant). p-values ​​for survival time determined by log-rank Mantel-Cox test with 95% confidence interval. [Figure 23E]Membrane-proximal CD33-targeted CAR T cells enhance survival time in xenograft mouse models. Survival time of NCG mice bearing U937-CD33highgfpLuc+ tumors and treated with 5.0x104 CAR T cells is shown (n=5, *, P<0.05). p-values ​​for survival time determined by log-rank Mantel-Cox test with 95% confidence interval. [Figure 23F] Figure 1 shows membrane proximal CD33 targeted CAR T cells enhance survival time in a xenograft mouse model. Figure 2 shows time course imaging of U937-CD33highgfpLuc+ in tumor-bearing NCG mice treated with 5.0x105 CAR T cells. [Figure 23G] Figure 1 shows membrane proximal CD33-targeted CAR T cells enhance survival time in a xenograft mouse model. Figure 2 shows tumor regression in NCG mice inoculated with U937-CD33highgfpLuc+ tumors and subsequently treated with 5.0x105 CAR T cells. [Figure 23H] Figure 1 shows membrane-proximal CD33-targeted CAR T cells enhance survival time in a xenograft mouse model. Schematic diagram of in vivo experimental setup. NCG mice were inoculated with OCiAML3-CD33lowgfpLuc+ tumors and subsequently treated with CAR T cells. [Figure 23I] Membrane-proximal CD33-targeted CAR T cells enhance survival in a xenograft mouse model. Survival time of NCG mice inoculated with OCiAML3-CD33lowgfpLuc+ tumors and treated with 5.0×105 CAR T cells is shown (n=5, *, P<0.05). p-values ​​for survival time determined by log-rank Mantel-Cox test with 95% confidence interval. [Figure 23J] Figure 1 shows membrane proximal CD33 targeted CAR T cells enhance survival time in a xenograft mouse model. Figure 2 shows time course imaging of OCiAML3-CD33lowgfpLuc+ in tumor-bearing NCG mice treated with 5.0x105 CAR T cells. [Figure 23K]Figure 1 shows membrane proximal CD33 targeted CAR T cells enhance survival time in a xenograft mouse model. Figure 2 shows tumor regression in NCG mice bearing OCiAML3-CD33lowgfpLuc+ tumors and treated with 5.0x105 CAR T cells. [Figure 24A] Figure 1 shows membrane-proximal CD33-targeted CAR T cells reducing tumor burden in a patient-derived AML xenograft model.Flow cytometry histograms and geometric median fluorescence intensity (MFI) of CD33 expression on AML60B patient samples detected with fluorescently labeled CD33-specific antibody and isotype control. [Figure 24B] Figure 1 shows membrane-proximal CD33-targeted CAR T cells reduce tumor burden in a patient-derived AML xenograft model. Schematic diagram of the experimental setup of the patient-derived xenograft model. NCG mice were inoculated with patient-derived AML blasts and treated with allogeneic CAR T cells. Bone marrow aspirates were analyzed 28 days after tumor inoculation. [Figure 24C] 1 shows membrane proximal CD33-targeted CAR T cells reduce tumor burden in a patient-derived AML xenograft model. Quantification of flow cytometry analysis demonstrating reduced tumor burden in mice treated with membrane proximal CD33-targeted CAR T cells is shown (n=6, *, P<0.05 by ordinary one-way ANOVA, ns, not significant). [Figure 24D] Figure 1 shows membrane proximal CD33 targeted CAR T cells reduce tumor burden in a patient derived AML xenograft model. Survival time of NCG mice bearing AML60B patient derived tumors and treated with membrane distal or membrane proximal CAR T cells is shown (n=6, **, P<0.01, *, P<0.05). p values ​​for survival time determined by log-rank Mantel-Cox test with 95% confidence interval. Data shown are pooled from two independent experiments using two different healthy donors. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] 5. Detailed Description of the Invention The presently disclosed subject matter provides antigen recognition receptors (e.g., chimeric antigen receptors (CARs)) that specifically target CD33. The presently disclosed subject matter further provides cells comprising such receptors. The cells can be immunoresponsive cells, e.g., genetically modified immunoresponsive cells (e.g., T cells or natural killer (NK) cells). The presently disclosed subject matter also provides methods of using such cells for therapy, e.g., treating and / or ameliorating a disease or disorder associated with CD33 (e.g., AML).

[0041] Non-limiting embodiments of the present disclosure are illustrated in the specification and examples.

[0042] For clarity of disclosure, and not by way of limitation, this detailed description is divided into the following sections. 5.1. Definition, 5.2.CD33, 5.3. Antigen Recognition Receptors, 5.4.Cells, 5.5. Compositions and Vectors 5.6. Polypeptides, 5.7. Formulation and Administration 5.8. Treatment method, 5.9. Kits, and 5.10. Exemplary Embodiments.

[0043] 5.1.Definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this invention belongs. The following references provide those skilled in the art with general definitions of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed.1994), The Cambridge Dictionary of Science and Technology (Walker ed.,1988), The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991), and Hale&Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless otherwise specified.

[0044] As used herein, the term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 or more than 3 standard deviations, as is customary in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, more preferably within 2-fold of a value.

[0045] By "immunoresponsive cell" is meant a cell that functions in the immune response or a precursor, or progeny thereof. In certain embodiments, the immunoresponsive cell is a cell of the lymphoid lineage. Non-limiting examples of cells of the lymphoid lineage include T cells, natural killer (NK) cells, B cells, and stem cells from which lymphoid cells can differentiate. In certain embodiments, the immunoresponsive cell is a cell of the myeloid lineage.

[0046] "Activating an immunoresponsive cell" refers to the induction of intracellular signaling or protein expression changes that result in the initiation of an immune response. For example, clustering of CD3 chains in response to ligand binding and immunoreceptor tyrosine-based inhibitory motifs (ITAMs) produces a signaling cascade. In certain embodiments, binding of an endogenous TCR or an exogenous CAR to an antigen results in the formation of an immunological synapse that involves the clustering of many molecules near the bound receptor (e.g., CD4 or CD8, CD3γ / δ / ε / ζ, etc.). This clustering of membrane-bound signaling molecules allows the ITAM motifs contained within the CD3 chains to be phosphorylated. This phosphorylation then initiates the T cell activation pathway, ultimately activating transcription factors such as NF-κB and AP-1. These transcription factors induce global gene expression in T cells to increase IL-2 production for proliferation and expression of master regulator T cell proteins to initiate a T cell-mediated immune response.

[0047] "Stimulating immunoresponsive cells" refers to signals that result in a robust and sustained immune response. In various embodiments, this is mediated after activation of immune cells (e.g., T cells) or simultaneously through receptors including, but not limited to, CD28, CD137 (4-1BB), OX40, CD40, and ICOS. Receiving multiple stimulatory signals may be important to mount a robust and long-lasting T cell-mediated immune response. T cells can quickly become inhibited and unresponsive to antigens. The effects of these costimulatory signals may vary, but they generally result in increased gene expression to generate long-lived, proliferative, and anti-apoptotic T cells that robustly respond to antigens for complete and sustained eradication.

[0048] As used herein, the term "antigen-recognizing receptor" refers to a receptor that can recognize a target antigen (e.g., CD33). In certain embodiments, the antigen-recognizing receptor can activate an immune cell or an immunoresponsive cell (e.g., a T cell) upon binding to the target antigen.

[0049] As used herein, the term "antibody" refers not only to intact antibody molecules, but also to fragments of antibody molecules that retain immunogen-binding ability. Such fragments are also well known in the art and are commonly used both in vitro and in vivo. Thus, as used herein, the term "antibody" refers not only to intact immunoglobulin molecules, but also to the well-known active fragments F(ab')2 and Fab. F(ab')2 and Fab fragments, which lack the Fe fragment of intact antibodies, are cleared from the circulation more rapidly and may exhibit less nonspecific tissue binding than intact antibodies (Wahl et al., Nucl Med (1983); 24: 316-325). As used herein, the term includes whole natural antibodies, bispecific antibodies, chimeric antibodies, Fab, Fab', single chain V region fragments (scFv), fusion polypeptides, and non-conventional antibodies. In certain embodiments, an antibody is a glycoprotein that includes at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (referred to herein as V H ) and heavy chain constant (C H The heavy chain constant region is composed of three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (herein referred to as V L (abbreviated as ) and light chain constant C L The light chain constant region consists of one domain, C L It consists of V H Area and V L The regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs). H and V Lis composed of three CDRs and four FRs arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0050] As used herein, "CDR" is defined as the complementarity determining region amino acid sequence of an antibody, which is the hypervariable region of the immunoglobulin heavy and light chains. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 4th USDepartment of Health and Human Services, National Institutes of Health (1987), or the IMGT numbering system (Lefranc, The Immunologist (1999); 7:132-136; Lefranc et al., Dev. Comp. Immunol. (2003); 27:55-77). Generally, an antibody contains three heavy chain and three light chain CDRs or CDR regions in the variable region. CDRs provide the majority of contact residues for the binding of the antibody to an antigen or epitope. In certain embodiments, the CDR regions are delineated using the IMGT numbering system. In certain embodiments, the CDR regions are delineated using the IMGT numbering system, accessible at http: / / www.imgt.org / IMGT_vquest / input.

[0051] As used herein, the term "single chain variable fragment" or "scFv" refers to a V H ::V L Heavy chains (V) of immunoglobulins (e.g., mouse or human) covalently linked to form heterodimers H ) and light chain (V L ) is a fusion protein of the variable regions of the heavy chain (V H) and light chain (V L ) are either directly connected or connected by a peptide-encoding linker (e.g., 10, 15, 20, 25 amino acids), H N-terminus of V L or V H The C-terminus of V L The linker is typically rich in glycine for flexibility and rich in serine or threonine for solubility. The linker may link the heavy and light chain variable regions of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917 (2008) and WO2014 / 087010, the contents of which are incorporated herein by reference in their entirety. In certain embodiments, the linker is a G4S linker.

[0052] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:95, provided below: GGGGSGGGGSGGGSGGGGS [SEQ ID NO: 95]

[0053] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:96, provided below: GGGGSGGGGSGGGGS [SEQ ID NO: 96]

[0054] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:97, provided below: GGGGSGGGGSGGGGSGGGSGGGGS [SEQ ID NO: 97]

[0055] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:98, provided below: GGGGSGGGGSGGGGSGGGGSGGGSGGGGS [SEQ ID NO: 98]

[0056] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:99, provided below: GGGGS [SEQ ID NO: 99]

[0057] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 100, provided below: GGGGSGGGGS [SEQ ID NO: 100]

[0058] Despite the removal of the constant region and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies can be synthesized using the VFv polypeptide as described in Huston, et al. Proc. Nat. Acad. Sci. USA, (1988); 85:5879-5883, U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778, and U.S. Patent Publication Nos. 2005 / 0196754 and 2005 / 0196754. H Coding sequence and V LThe scFv can be expressed from a nucleic acid containing the coding sequence. Antagonistic scFvs with inhibitory activity have been described (e.g., Zhao et al., Hyrbidoma (Larchmt) (2008); 27(6): 455-51; Peter et al., J Cachexia Sarcopenia Muscle (2012); August 12; Shieh et al., J Imunol (2009); 183(4): 2277-85; Giomarelli et al., Thromb Haemost (2007); 97(6): 955-63; Fife et al., J Clin Invst (2006); 116(8): 2252-61; Brocks et al., Immunotechnology 1997 3(3): 173-84; Moosmayer et al., Ther Immunol 1995). 2(10:31-40). Agonistic scFvs with stimulatory activity have been described (Peter et al., J Biol Chern (2003); 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol (1997); 17(5-6):427-55; Ho et al., BioChim Biophys Acta (2003); 1638(3):257-66).

[0059] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a molecule that comprises an extracellular antigen-binding domain fused to an intracellular signaling domain capable of activating or stimulating an immunoresponsive cell, and a transmembrane domain. In certain embodiments, the extracellular antigen-binding domain of a CAR comprises an scFv. The scFv may be derived from fusing the variable heavy and light chain regions of an antibody. Alternatively or additionally, the scFv may be derived from a Fab (e.g., instead of from an antibody obtained from a Fab library). In certain embodiments, the scFv is fused to a transmembrane domain and then to an intracellular signaling domain. "Substantially identical" or "substantially homologous" refers to a polypeptide or nucleic acid molecule that exhibits at least about 50% homology or identity to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or a reference nucleotide sequence (e.g., any of the nucleotide sequences described herein). In certain embodiments, such sequences are at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% homologous or identical to the amino acid or nucleic acid sequence used for comparison.

[0060] Sequence identity is measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, the BLAST program may be used, with a probability score of e-3 to e-100 indicating closely related sequences.

[0061] As used herein, the percentage of homology between two amino acid sequences is equal to the percentage of identity between the two sequences. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions x 100), taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. The comparison of sequences and the determination of the percentage of identity between two sequences can be accomplished using a mathematical algorithm.

[0062] The percent homology between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) as implemented in the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent homology between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) as implemented in the GAP program in the GCG software package (available at www.gcg.com) using either a Blossum 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0063] Additionally or alternatively, the amino acid sequences of the subject matter disclosed herein can further be used as a "query sequence" to perform searches against public databases, for example to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. To obtain amino acid sequences homologous to specific sequences disclosed herein (e.g., the heavy and light chain variable region sequences of scFv m903, m904, m905, m906, and m900), BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3. To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST, Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used.

[0064] An "effective amount" is an amount sufficient to produce a beneficial or desired clinical result upon treatment. An effective amount may be administered to a subject in one or more doses. In certain embodiments, an effective amount may be an amount sufficient to palliate, improve, stabilize, reverse, or slow the progression of a disease, or otherwise reduce the pathological consequences of a disease. An effective amount may be determined by a physician on a case-by-case basis and is within the skill of one of ordinary skill in the art. Several factors are typically considered in determining the appropriate dosage to achieve an effective amount. These factors include the age, sex, and weight of the subject, the condition being treated, the severity of the condition, and the form and effective concentration of the cells being administered.

[0065] As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or change the binding characteristics of the CD33-targeting CAR (e.g., extracellular antigen-binding domain) disclosed herein, including the amino acid sequence. Conservative modifications can include amino acid substitutions, additions, and deletions. Modifications can be introduced into the extracellular antigen-binding domain of the CAR disclosed herein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified into groups according to physicochemical properties, such as charge and polarity. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid within the same group. For example, amino acids can be classified by charge, with positively charged amino acids including lysine, arginine, and histidine, negatively charged amino acids including aspartic acid and glutamic acid, and neutrally charged amino acids including alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Additionally, amino acids can be classified by polarity, with polar amino acids including arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine, and non-polar amino acids including alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acid residues in a CDR region can be replaced with other amino acid residues from the same group, and the altered antibodies can be tested for retained function (i.e., the functions shown in (c)-(l) above) using the functional assays described herein. In certain embodiments, no more than one, no more than two, no more than three, no more than four, no more than five residues in a specified sequence or CDR region are altered.

[0066] As used herein, the term "endogenous" refers to a nucleic acid molecule or a polypeptide that is normally expressed in a cell or tissue.

[0067] As used herein, the term "exogenous" refers to a nucleic acid molecule or polypeptide that is not endogenously present in a cell. Thus, the term "exogenous" will encompass foreign, heterologous, and any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as overexpressed nucleic acid molecules and polypeptides. An "exogenous" nucleic acid refers to a nucleic acid that is not present in a natural wild-type cell, for example, an exogenous nucleic acid may vary from its endogenous counterpart by sequence, position / location, or both. For clarity, an exogenous nucleic acid may have the same or different sequence compared to its natural endogenous counterpart, it may be introduced into the cell itself or its precursor by genetic engineering, and may optionally be linked to alternative control sequences, such as non-native promoters or secretion sequences.

[0068] By "heterologous nucleic acid molecule or polypeptide" is meant a nucleic acid molecule (e.g., a cDNA, DNA, or RNA molecule) or a polypeptide that is not normally present in a cell or a sample obtained from a cell. The nucleic acid may be from another organism, or may be, for example, an mRNA molecule that is not normally expressed in the cell or sample.

[0069] By "increase" is meant a positive change of at least about 5%. The change can be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100%, or more.

[0070] By "decreasing" is meant a negative change of at least about 5%. The change can be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or even about 100%.

[0071] The terms "isolated," "purified," or "biologically pure" refer to material that is free to various degrees from components that normally accompany it as found in its native state. "Isolate" refers to a degree of separation from the original source or surroundings. "Purify" refers to a degree of separation greater than isolation. A "purified" or "biologically pure" protein is sufficiently free from other materials that any impurities do not substantially affect the biological properties of the protein or cause other deleterious consequences. That is, a nucleic acid or peptide is purified when it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can indicate that the nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For proteins that can be subject to modifications, such as phosphorylation or glycosylation, different modifications can give rise to different isolated proteins that can be purified separately.

[0072] The term "isolated cell" means a cell that is separated from molecules and / or cellular components that naturally accompany the cell.

[0073] As used herein, the term "antigen-binding domain" refers to a domain capable of specifically binding to a particular antigenic determinant or set of antigenic determinants present on a cell.

[0074] "Recognize" means selectively binding to a target. T cells that recognize a tumor can express a receptor (e.g., a CAR) that binds to a tumor antigen.

[0075] By "signal sequence" or "leader sequence" is meant a peptide sequence (e.g., 5, 10, 15, 20, 25 or 30 amino acids) present at the N-terminus of newly synthesized proteins that directs their entry into the secretory pathway.

[0076] By "specifically binds" or "specifically binds to" or "specifically targets" is meant a polypeptide or fragment thereof that recognizes and / or binds to a biological molecule of interest (e.g., a polypeptide, e.g., a CD33 polypeptide) but does not substantially recognize and / or bind to other molecules in a sample, e.g., a biological sample, which naturally includes a polypeptide disclosed herein (e.g., a CD33 polypeptide). In certain embodiments, the antigen-recognizing receptor disclosed herein is at least about 1×10 -8 M or less, about 5 x 10 -9 M or less, approximately 1×10 -9 M or less, about 5 x 10 -10 M or less, approximately 1×10 -10 M or less, about 5 x 10 -11 M or less, or about 1 x 10 -11 The dissociation constant (K D ) and binds to CD33 (e.g., human CD33).

[0077] As used herein, the term "derivative" refers to a compound that is derived from some other compound and maintains its general structure. For example, but not by way of limitation, trichloromethane (chloroform) is a derivative of methane.

[0078] The terms "comprises" and "comprising" are intended to have the broad meaning ascribed to them in U.S. patent law and can mean "includes," "including," etc.

[0079] As used herein, the term "treatment" refers to clinical intervention in an attempt to change the disease course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. The therapeutic effect of treatment includes, but is not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and remission or improved prognosis. By preventing the progression of a disease or disorder, treatment can not only prevent deterioration due to the disorder in a subject who has been affected or diagnosed, or a subject who is suspected of having the disorder, but also prevent the onset of the disorder or symptoms of the disorder in a subject who is at risk of the disorder or a subject who is suspected of having the disorder.

[0080] An "individual" or "subject" herein is a vertebrate, e.g., a human or a non-human animal, e.g., a mammal. Mammals include, but are not limited to, humans, primates, farm animals, sport animals, rodents, and pets. Non-limiting examples of non-human animal subjects include rodents, such as mice, rats, and hamsters, and guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cows, horses, and non-human primates, such as apes and monkeys.

[0081] Other aspects of the presently disclosed subject matter are described in the disclosure that follows and are within the scope of the presently disclosed subject matter.

[0082] 5.2.CD33 CD33 is a single-pass transmembrane molecule and a member of the sialic acid-binding immunoglobulin (Ig)-like lectin (Siglec) family. CD33 consists of two extracellular domains with an immunoglobulin-like fold, IgV and IgC2 (see FIG. 1A). CD33 has three isoforms produced by alternative splicing, with isoform 3 lacking the IgV domain (Ehninger et al., Blood Cancer J 4, e218 (2014); Sanford et al., Leuk Lymphoma 57, 1965-1968 (2016); and Haubner et al., Leukemia 33, 64-74 (2019)). Recent studies have shown that approximately 50% of AML patients have a CD33 single nucleotide polymorphism (SNP) (rs12459419C>T) that results in the expression of an alternatively spliced ​​CD33 isoform lacking exon 2, resulting in the elimination of the IgV domain (Bakker et al., Cancer Res 64, 8443-8450 (2004)). In these patients, the CD33-targeting antibody-drug conjugate (ADC), gemtuzumab ozogamicin (GO), had no effect and increased the risk of relapse, likely due to the inability of this ADC to kill AML cells expressing this IgV-deficient CD33 isoform, a problem encountered by all currently clinically available CD33-targeting products, given their epitopes in the IgV domain (see Figure 1B) (see Perna et al., Cancer Cell 32, 506-519 e505 (2017)).

[0083] In certain embodiments, the antigen recognition receptor binds to human CD33. In certain embodiments, human CD33 comprises or consists of an amino acid sequence having UniProt Reference Number: P20138-1 (SEQ ID NO: 1) or a fragment thereof. SEQ ID NO: 1 is provided below. In certain embodiments, CD33 comprises an extracellular domain, a transmembrane domain, and a cytoplasmic domain. In certain embodiments, the extracellular domain comprises or consists of amino acids 18 to 259 of SEQ ID NO: 1. In certain embodiments, the transmembrane domain comprises or consists of amino acids 260 to 282 of SEQ ID NO: 1. In certain embodiments, the cytoplasmic domain comprises or consists of amino acids 283 to 364 of SEQ ID NO: 1. MPLLLLLPLLWAGALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYW FREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRM ERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWL SAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTT GIFPGDGSGKQETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTH PTTGSASPKHQKKSKLHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSE VRTQ [SEQ ID NO: 1]

[0084] In certain embodiments, CD33 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% identical to the amino acid sequence set forth in SEQ ID NO:1, or a fragment thereof.

[0085] In certain embodiments, the antigen recognition receptor binds to a portion of human CD33. In certain embodiments, the antigen recognition receptor binds to the extracellular domain of CD33. In certain embodiments, the extracellular domain of CD33 comprises an Ig-like V-type domain and an Ig-like C2-type domain. In certain embodiments, the extracellular domain of CD33 comprises an Ig-like C2-type domain. In certain embodiments, the Ig-like V-type domain comprises or consists of amino acids 19-135 of SEQ ID NO:1. In certain embodiments, the Ig-like C2-type domain comprises or consists of amino acids 145-228 of SEQ ID NO:1.

[0086] 5.3. Antigen Recognition Receptors The antigen-recognizing receptor disclosed herein specifically targets or binds to CD33. In certain embodiments, the antigen-recognizing receptor is a chimeric antigen receptor (CAR). In certain embodiments, the antigen-recognizing receptor is a TCR-like fusion molecule.

[0087] The subject matter disclosed herein also provides a nucleic acid molecule encoding the antigen-recognizing receptor disclosed herein.In certain embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding the polypeptide of the CD33-targeting antigen-recognizing receptor disclosed herein.

[0088] 5.3.1. Extracellular Antigen-Binding Domains In certain embodiments, the extracellular antigen-binding domain of the antigen recognition receptor binds to CD33.

[0089] In certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, the scFv is a human scFv. In certain embodiments, the scFv is a humanized scFv. In certain embodiments, the scFv is a mouse scFv. In certain embodiments, the scFv is identified by screening an scFv phage library with an antigen-Fc fusion protein.

[0090] In certain embodiments, the extracellular antigen-binding domain is a Fab. In certain embodiments, the Fab is cross-linked. In certain embodiments, the extracellular antigen-binding domain is a F(ab)2.

[0091] Any of the aforementioned molecules can be included in a fusion protein with a heterologous sequence to form an extracellular antigen-binding domain. In certain non-limiting embodiments, the extracellular antigen-binding domain (e.g., the embodied scFv) has a binding affinity of, for example, 1×10 -8 M or less, for example, about 1×10 -8 M or less, about 5 x 10 -9 M or less, approximately 1×10 -9 M or less, about 5 x 10 -10 M or less, approximately 1×10 -10 M or less, or about 1 x 10 -11 The dissociation constant (K D In certain embodiments, the extracellular antigen-binding domains disclosed herein bind to CD33 (e.g., human CD33) at about 5×10 -9 The dissociation constant (K D In certain embodiments, the extracellular antigen-binding domain disclosed herein binds to CD33 (e.g., human CD33, e.g., soluble human CD33) at about 5×10 -9 Dissociation constant of M (K D In certain embodiments, the extracellular antigen-binding domain disclosed herein binds to CD33 (e.g., human CD33, e.g., soluble human CD33) at about 1×10 -9 Dissociation constant of M (K D In certain embodiments, the extracellular antigen-binding domain disclosed herein binds to CD33 (e.g., human CD33, e.g., soluble human CD33) at about 1×10 -9 M ~ approx. 5×10 -9 Dissociation constant of M (K D In certain embodiments, the extracellular antigen-binding domain disclosed herein binds to CD33 (e.g., human CD33, e.g., soluble human CD33) at about 1×10 -9 M ~ approx. 2×10 -9 Dissociation constant of M (K DIn certain embodiments, the extracellular antigen-binding domain disclosed herein binds to CD33 (e.g., human CD33, e.g., soluble human CD33) at about 5×10 -9 Dissociation constant of M (K D In certain embodiments, the extracellular antigen-binding domain disclosed herein binds to CD33 (e.g., human CD33, e.g., soluble human CD33) at about 1×10 -9 Dissociation constant of M (K D ) binds to CD33 (e.g., human CD33, e.g., soluble human CD33).

[0092] In certain embodiments, the extracellular antigen-binding domains disclosed herein bind to cells expressing CD33 (e.g., AML cells expressing CD33) with a half maximal effective concentration (EC50) value of about 1 nM to about 50 nM, about 5 nM to about 50 nM, about 10 nM to about 50 nM, about 20 nM to about 50 nM, about 30 nM to about 50 nM, about 40 nM to about 50 nM, or greater than about 50 nM. In certain embodiments, the extracellular antigen-binding domains disclosed herein bind to cells expressing CD33 (e.g., AML cells expressing CD33) with an EC50 value of about 1 nM to about 5 nM. In certain embodiments, the extracellular antigen-binding domains disclosed herein bind to cells expressing CD33 (e.g., AML cells expressing CD33) with an EC50 value of about 2 nM. In certain embodiments, the extracellular antigen binding domain disclosed herein binds to cells expressing CD33 (e.g., AML cells expressing CD33) with an EC50 value of about 2.16 nM. In certain embodiments, the extracellular antigen binding domain disclosed herein binds to cells expressing CD33 (e.g., AML cells expressing CD33) with an EC50 value of about 5 nM to about 10 nM. In certain embodiments, the extracellular antigen binding domain disclosed herein binds to cells expressing CD33 (e.g., AML cells expressing CD33) with an EC50 value of about 10 nM. In certain embodiments, the extracellular antigen binding domain disclosed herein binds to cells expressing CD33 (e.g., AML cells expressing CD33) with an EC50 value of about 8.5 nM. In certain embodiments, the extracellular antigen binding domains disclosed herein bind to cells expressing CD33 (e.g., AML cells expressing CD33) with an EC50 value of about 40 nM to about 50 nM. In certain embodiments, the extracellular antigen binding domains disclosed herein bind to cells expressing CD33 (e.g., AML cells expressing CD33) with an EC50 value of about 45 nM. In certain embodiments, the extracellular antigen binding domains disclosed herein bind to cells expressing CD33 (e.g., AML cells expressing CD33) with an EC50 value of about 45 nM.

[0093] Binding of the extracellular antigen-binding domain can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western blot assay. Each of these assays generally detects the presence of a particular protein-antibody complex of interest by using a labeled reagent (e.g., antibody, or scFv) specific for the complex of interest. For example, scFv can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, incorporated herein by reference). Radioisotopes can be detected by such means as the use of a gamma counter or scintillation counter, or autoradioactivity testing. In certain embodiments, the CD33-targeting extracellular antigen-binding domain is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, azurite, and mKalama1), cyan fluorescent protein (e.g., ECFP, cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, citrine, Venus, and YPet). In certain embodiments, the CD33-targeting human scFv is labeled with GFP.

[0094] In certain embodiments, the CDRs are identified according to the IMGT numbering system.

[0095] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:2 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:3 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:4 or a conservative modification thereof. H SEQ ID NOs: 2-4 are provided in Table 1.

[0096] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7 or a conservative modification thereof. L SEQ ID NOs: 5-7 are provided in Table 1.

[0097] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:2 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:3 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:4 or a conservative modification thereof. H and a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7 or a conservative modification thereof. L Includes.

[0098] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:2, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:3, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:4. H and CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7. L Includes.

[0099] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:8. HFor example, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to SEQ ID NO:8. H In certain embodiments, the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO:8. H An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 8 is set forth in SEQ ID NO: 10. SEQ ID NOs: 8 and 10 are provided in Table 1 below.

[0100] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:9. L For example, the extracellular antigen binding domain (e.g., scFv) comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to SEQ ID NO:9. L In certain embodiments, the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO:9. L An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 9 is set forth in SEQ ID NO: 11. SEQ ID NOs: 9 and 11 are provided in Table 1 below.

[0101] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO:8. H and V comprising the amino acid sequence set forth in SEQ ID NO:9. LIn certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, the scFv is designated "3-P14". In certain embodiments, the V H and V L are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:96.

[0102] In certain embodiments, the variable region within the extracellular antigen-binding domain comprises a heavy chain variable region (V H In certain embodiments, when the extracellular antigen-binding domain is an scFv, the variable regions are arranged from the N-terminus to the C-terminus: H -V L .

[0103] In certain embodiments, the variable region within the extracellular antigen-binding domain comprises a light chain variable region (V L In certain embodiments, the extracellular antigen-binding domain is an scFv, and the variable regions are arranged from the N-terminus to the C-terminus: L -V H . [Table 1]

[0104] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14 or a conservative modification thereof. H SEQ ID NOs: 12-14 are provided in Table 2.

[0105] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17 or a conservative modification thereof. L SEQ ID NOs: 15-17 are provided in Table 2.

[0106] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13 or a conservative modification thereof, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14 or a conservative modification thereof, H and CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17 or a conservative modification thereof. L Includes.

[0107] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14, and a CDR5 comprising the amino acid sequence set forth in SEQ ID NO: 15. H and CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17. L Includes.

[0108] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:18. HFor example, the extracellular antigen-binding domain (e.g., scFv) can comprise an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:18. H In certain embodiments, the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 18. H An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 18 is set forth in SEQ ID NO: 20. SEQ ID NOs: 18 and 20 are provided in Table 2 below.

[0109] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:19. L For example, the extracellular antigen-binding domain (e.g., scFv) can comprise an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:19. L In certain embodiments, the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 19. L An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 19 is set forth in SEQ ID NO: 21. SEQ ID NOs: 19 and 21 are provided in Table 2 below.

[0110] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO: 18. H and V comprising the amino acid sequence set forth in SEQ ID NO:19. LIn certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, the scFv is designated "4-B2". In certain embodiments, V H and V L are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:96.

[0111] In certain embodiments, the variable region within the extracellular antigen-binding domain comprises a heavy chain variable region (V H In certain embodiments, when the extracellular antigen-binding domain is an scFv, the variable regions are arranged from the N-terminus to the C-terminus: H -V L .

[0112] In certain embodiments, the variable region within the extracellular antigen-binding domain comprises a light chain variable region (V L In certain embodiments, when the extracellular antigen-binding domain is an scFv, the variable regions are arranged from the N-terminus to the C-terminus: L -V H . [Table 2]

[0113] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24 or a conservative modification thereof. H SEQ ID NOs:22-24 are provided in Table 3.

[0114] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27 or a conservative modification thereof. L SEQ ID NOs:25-27 are provided in Table 3.

[0115] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23 or a conservative modification thereof, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24 or a conservative modification thereof, H and a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:25 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:26 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:27 or a conservative modification thereof. L Includes.

[0116] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24, H and CDR1 comprising the amino acid sequence set forth in SEQ ID NO:25, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:26, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:27. L Includes.

[0117] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:28. HFor example, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:28. H In certain embodiments, the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO:28. H An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 28 is set forth in SEQ ID NO: 30. SEQ ID NOs: 28 and 30 are provided in Table 3 below.

[0118] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:29. L For example, the extracellular antigen-binding domain (e.g., scFv) can comprise an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:29. L In certain embodiments, the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO:29. L An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 29 is set forth in SEQ ID NO: 31. SEQ ID NOs: 29 and 31 are provided in Table 3 below.

[0119] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO:28. H and V comprising the amino acid sequence set forth in SEQ ID NO:29. LIn certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, the scFv is designated "1-J19". In certain embodiments, the V H and V L are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:96.

[0120] In certain embodiments, the variable region within the extracellular antigen-binding domain comprises a heavy chain variable region (V H In certain embodiments, when the extracellular antigen-binding domain is an scFv, the variable regions are arranged from the N-terminus to the C-terminus: H -V L .

[0121] In certain embodiments, the variable region within the extracellular antigen-binding domain comprises a light chain variable region (V L In certain embodiments, when the extracellular antigen-binding domain is an scFv, the variable regions are arranged from the N-terminus to the C-terminus: L -V H . [Table 3]

[0122] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 or a conservative modification thereof. H SEQ ID NOs:32-34 are provided in Table 4.

[0123] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27 or a conservative modification thereof. L SEQ ID NOs:25-27 are provided in Table 4.

[0124] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33 or a conservative modification thereof, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 or a conservative modification thereof, and a CDR4 comprising the amino acid sequence set forth in SEQ ID NO: 35 or a conservative modification thereof. H and a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:25 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:26 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:27 or a conservative modification thereof. L Includes.

[0125] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34, and a CDR5 comprising the amino acid sequence set forth in SEQ ID NO: 35. H and CDR1 comprising the amino acid sequence set forth in SEQ ID NO:25, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:26, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:27. L Includes.

[0126] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:35. HFor example, the extracellular antigen-binding domain (e.g., scFv) can comprise an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:35. H In certain embodiments, the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO:35. H An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 35 is set forth in SEQ ID NO: 36. SEQ ID NOs: 35 and 36 are provided in Table 4 below.

[0127] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:29. L For example, the extracellular antigen-binding domain (e.g., scFv) can comprise an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:29. L In certain embodiments, the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO:29. L An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 29 is set forth in SEQ ID NO: 31. SEQ ID NOs: 29 and 31 are provided in Table 4 below.

[0128] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO:35. H and V comprising the amino acid sequence set forth in SEQ ID NO:29. LIn certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, the scFv is designated "1-J19-2". In certain embodiments, the V H and V L are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:96.

[0129] In certain embodiments, the variable region within the extracellular antigen-binding domain comprises a heavy chain variable region (V H In certain embodiments, when the extracellular antigen-binding domain is an scFv, the variable regions are arranged from the N-terminus to the C-terminus: H -V L .

[0130] In certain embodiments, the variable region within the extracellular antigen-binding domain comprises a light chain variable region (V L In certain embodiments, when the extracellular antigen-binding domain is an scFv, the variable regions are arranged from the N-terminus to the C-terminus: L -V H . [Table 4]

[0131] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39 or a conservative modification thereof. H SEQ ID NOs:37-39 are provided in Table 5.

[0132] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 40 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 41 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42 or a conservative modification thereof. L SEQ ID NOs: 40-42 are provided in Table 5.

[0133] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38 or a conservative modification thereof, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39 or a conservative modification thereof, H and a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 40 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 41 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42 or a conservative modification thereof. L Includes.

[0134] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39, H and CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 40, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 41, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42. L Includes.

[0135] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:43. HFor example, the extracellular antigen-binding domain (e.g., scFv) can comprise an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:43. H In certain embodiments, the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO:43. H An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 43 is set forth in SEQ ID NO: 45. SEQ ID NOs: 43 and 45 are provided in Table 5 below.

[0136] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:44. L For example, the extracellular antigen-binding domain (e.g., scFv) can comprise an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:44. L In certain embodiments, the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO:44. L An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 44 is set forth in SEQ ID NO: 46. SEQ ID NOs: 44 and 46 are provided in Table 5 below.

[0137] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO:43. H and V comprising the amino acid sequence set forth in SEQ ID NO:44. LIn certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, the scFv is designated "1-P13". In certain embodiments, V H and V L are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:96.

[0138] In certain embodiments, the variable region within the extracellular antigen-binding domain comprises a heavy chain variable region (V H In certain embodiments, when the extracellular antigen-binding domain is an scFv, the variable regions are arranged from the N-terminus to the C-terminus: H -V L .

[0139] In certain embodiments, the variable region within the extracellular antigen-binding domain comprises a light chain variable region (V L In certain embodiments, when the extracellular antigen-binding domain is an scFv, the variable regions are arranged from the N-terminus to the C-terminus: L -V H . [Table 5]

[0140] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 47 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 48 or a conservative modification thereof. H SEQ ID NOs: 38, 47 and 48 are provided in Table 6.

[0141] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 49 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 50 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 51 or a conservative modification thereof. L SEQ ID NOs: 49-51 are provided in Table 6.

[0142] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 47 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38 or a conservative modification thereof, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 48 or a conservative modification thereof, H and a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 49 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 50 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 51 or a conservative modification thereof. L Includes.

[0143] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:47, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:38, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:48, H and CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 49, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 50, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 51. L Includes.

[0144] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:52. HFor example, the extracellular antigen-binding domain (e.g., scFv) can comprise an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:52. H In certain embodiments, the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO:52. H An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 52 is set forth in SEQ ID NO: 54. SEQ ID NOs: 52 and 54 are provided in Table 6 below.

[0145] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:53. L For example, the extracellular antigen-binding domain (e.g., scFv) can comprise an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:53. L In certain embodiments, the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO:53. L An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 53 is set forth in SEQ ID NO: 55. SEQ ID NOs: 53 and 55 are provided in Table 6 below.

[0146] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO:52. H and V comprising the amino acid sequence set forth in SEQ ID NO:53. LIn certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, the scFv is designated "1-P23". In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:96.

[0147] In certain embodiments, the variable region within the extracellular antigen-binding domain comprises a heavy chain variable region (V H In certain embodiments, when the extracellular antigen-binding domain is an scFv, the variable regions are arranged from the N-terminus to the C-terminus: H -V L .

[0148] In certain embodiments, the variable region within the extracellular antigen-binding domain comprises a light chain variable region (V L In certain embodiments, when the extracellular antigen-binding domain is an scFv, the variable regions are arranged from the N-terminus to the C-terminus: L -V H . [Table 6]

[0149] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 56 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 57 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 58 or a conservative modification thereof. H SEQ ID NOs:56-58 are provided in Table 7.

[0150] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:59 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:60 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:61 or a conservative modification thereof. L SEQ ID NOs: 59-61 are provided in Table 7.

[0151] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 56 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 57 or a conservative modification thereof, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 58 or a conservative modification thereof, H and a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:59 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:60 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:61 or a conservative modification thereof. L Includes.

[0152] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:56, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:57, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:58, H and CDR1 comprising the amino acid sequence set forth in SEQ ID NO:59, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:60, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:61. L Includes.

[0153] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:62. HFor example, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:62. H In certain embodiments, the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO:62. H An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 62 is set forth in SEQ ID NO: 64. SEQ ID NOs: 62 and 64 are provided in Table 7 below.

[0154] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:63. L For example, the extracellular antigen-binding domain (e.g., scFv) can comprise an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:63. L In certain embodiments, the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO:63. L An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 63 is set forth in SEQ ID NO: 65. SEQ ID NOs: 63 and 65 are provided in Table 7 below.

[0155] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO:62. H and V comprising the amino acid sequence set forth in SEQ ID NO:63. LIn certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, the scFv is designated "1-A20". In certain embodiments, V H and V L are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:96.

[0156] In certain embodiments, the variable region within the extracellular antigen-binding domain comprises a heavy chain variable region (V H In certain embodiments, when the extracellular antigen-binding domain is an scFv, the variable regions are arranged from the N-terminus to the C-terminus: H -V L .

[0157] In certain embodiments, the variable region within the extracellular antigen-binding domain comprises a light chain variable region (V L In certain embodiments, when the extracellular antigen-binding domain is an scFv, the variable regions are arranged from the N-terminus to the C-terminus: L -V H . [Table 7]

[0158] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 66 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 67 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 68 or a conservative modification thereof. H SEQ ID NOs:66-68 are provided in Table 8.

[0159] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 69 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 70 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 71 or a conservative modification thereof. L SEQ ID NOs: 69-71 are provided in Table 8.

[0160] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 66 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 67 or a conservative modification thereof, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 68 or a conservative modification thereof, H and a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:69 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:70 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:71 or a conservative modification thereof. L Includes.

[0161] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 66, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 67, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 68, H and CDR1 comprising the amino acid sequence set forth in SEQ ID NO:69, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:70, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:71. L Includes.

[0162] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:72. HFor example, the extracellular antigen-binding domain (e.g., scFv) can comprise an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:72. H In certain embodiments, the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO:72. H An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 72 is set forth in SEQ ID NO: 74. SEQ ID NOs: 72 and 74 are provided in Table 8 below.

[0163] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:73. L For example, the extracellular antigen-binding domain (e.g., scFv) can comprise an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:73. L In certain embodiments, the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO:73. L An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 73 is set forth in SEQ ID NO: 75. SEQ ID NOs: 73 and 75 are provided in Table 8 below.

[0164] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO:72. H and V comprising the amino acid sequence set forth in SEQ ID NO:73. LIn certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, the scFv is designated "1-N3". In certain embodiments, the V H and V L are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:96.

[0165] In certain embodiments, the variable region within the extracellular antigen-binding domain comprises a heavy chain variable region (V H In certain embodiments, when the extracellular antigen-binding domain is an scFv, the variable regions are arranged from the N-terminus to the C-terminus: H -V L .

[0166] In certain embodiments, the variable region within the extracellular antigen-binding domain comprises a light chain variable region (V L In certain embodiments, when the extracellular antigen-binding domain is an scFv, the variable regions are arranged from the N-terminus to the C-terminus: L -V H . [Table 8]

[0167] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 76 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 57 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 77 or a conservative modification thereof. H SEQ ID NOs: 57, 76 and 77 are provided in Table 9.

[0168] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 78 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 79 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 80 or a conservative modification thereof. L SEQ ID NOs:78-80 are provided in Table 9.

[0169] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 76 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 57 or a conservative modification thereof, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 77 or a conservative modification thereof, H and CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 78 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 79 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 80 or a conservative modification thereof. L Includes.

[0170] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 76, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 57, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 77, H and CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 78, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 79, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 80. L Includes.

[0171] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:81. HFor example, the extracellular antigen-binding domain (e.g., scFv) can comprise an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:81. H In certain embodiments, the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 81. H An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 81 is set forth in SEQ ID NO: 83. SEQ ID NOs: 81 and 83 are provided in Table 9 below.

[0172] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:82. L For example, the extracellular antigen-binding domain (e.g., scFv) can comprise an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:82. L In certain embodiments, the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 82. L An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 82 is set forth in SEQ ID NO: 84. SEQ ID NOs: 82 and 84 are provided in Table 9 below.

[0173] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO:81. H and V comprising the amino acid sequence set forth in SEQ ID NO:82. LIn certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, the scFv is designated "1-H19". In certain embodiments, the V H and V L are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:96.

[0174] In certain embodiments, the variable region within the extracellular antigen-binding domain comprises a heavy chain variable region (V H In certain embodiments, when the extracellular antigen-binding domain is an scFv, the variable regions are arranged from the N-terminus to the C-terminus: H -V L .

[0175] In certain embodiments, the variable region within the extracellular antigen-binding domain comprises a light chain variable region (V L In certain embodiments, when the extracellular antigen-binding domain is an scFv, the variable regions are arranged from the N-terminus to the C-terminus: L -V H . [Table 9]

[0176] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 85 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 86 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 87 or a conservative modification thereof. H SEQ ID NOs:85-87 are provided in Table 10.

[0177] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:41 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:42 or a conservative modification thereof. L SEQ ID NOs: 5, 41 and 42 are provided in Table 10.

[0178] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises a VFV comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 85, or a conservative modification thereof. H , a V comprising a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 86 or a conservative modification thereof H , a V comprising a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 87 or a conservative modification thereof H and a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:41 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:42 or a conservative modification thereof. L Includes.

[0179] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 85, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 86, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 87, H and CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:41, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:42. L Includes.

[0180] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:88. HFor example, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:88. H In certain embodiments, the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 88. H An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 88 is set forth in SEQ ID NO: 90. SEQ ID NOs: 88 and 90 are provided in Table 10 below.

[0181] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:89. L For example, the extracellular antigen-binding domain (e.g., scFv) can comprise an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:89. L In certain embodiments, the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO:89. L An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 89 is set forth in SEQ ID NO: 91. SEQ ID NOs: 89 and 91 are provided in Table 10 below.

[0182] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO:88. H and V comprising the amino acid sequence set forth in SEQ ID NO:89. LIn certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, the scFv is designated "2-F18". In certain embodiments, the V H and V L are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:96.

[0183] In certain embodiments, the variable region within the extracellular antigen-binding domain comprises a heavy chain variable region (V H In certain embodiments, when the extracellular antigen-binding domain is an scFv, the variable regions are arranged from the N-terminus to the C-terminus: H -V L .

[0184] In certain embodiments, the variable region within the extracellular antigen-binding domain comprises a light chain variable region (V L In certain embodiments, when the extracellular antigen-binding domain is an scFv, the variable regions are arranged from the N-terminus to the C-terminus: L -V H . [Table 10]

[0185] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:2 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:3 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:4 or a conservative modification thereof. H SEQ ID NOs: 2-4 are provided in Table 11.

[0186] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7 or a conservative modification thereof. L SEQ ID NOs: 5-7 are provided in Table 11.

[0187] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:2 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:3 or a conservative modification thereof, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:4 or a conservative modification thereof, H and a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7 or a conservative modification thereof. L Includes.

[0188] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises a V domain comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:2, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:3, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:4. H and CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7. L Includes.

[0189] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:8. HFor example, the extracellular antigen-binding domain (e.g., scFv) can comprise an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:8. H In certain embodiments, the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO:8. H An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 8 is set forth in SEQ ID NO: 93. SEQ ID NOs: 8 and 93 are provided in Table 11 below.

[0190] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:92. L For example, the extracellular antigen-binding domain (e.g., scFv) can comprise an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:92. L In certain embodiments, the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO:92. L An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 92 is set forth in SEQ ID NO: 94. SEQ ID NOs: 92 and 94 are provided in Table 11 below.

[0191] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO:8. H and V comprising the amino acid sequence set forth in SEQ ID NO:92. LIn certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, the scFv is designated "4-P3". In certain embodiments, the V H and V L are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:96.

[0192] In certain embodiments, the variable region within the extracellular antigen-binding domain comprises a heavy chain variable region (V H In certain embodiments, when the extracellular antigen-binding domain is an scFv, the variable regions are arranged from the N-terminus to the C-terminus: H -V L .

[0193] In certain embodiments, the variable region within the extracellular antigen-binding domain comprises a light chain variable region (V L In certain embodiments, when the extracellular antigen-binding domain is an scFv, the variable regions are arranged from the N-terminus to the C-terminus: L -V H . [Table 11]

[0194] A V that has at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homology or identity to a particular sequence (e.g., SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:92). H Amino acid sequence and / or V L The amino acid sequence may contain substitutions (eg, conservative substitutions), insertions, or deletions relative to the particular sequence(s), but retain the ability to bind to CD33.

[0195] In certain embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in a particular sequence (e.g., SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:92). In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the CDRs (e.g., within the FRs) of the extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain comprises a VD1 or VD2 selected from SEQ ID NOs:8, 9, 18, 19, 28, 29, 35, 43, 44, 52, 53, 62, 63, 72, 73, 81, 82, 88, 89, or 92. H Sequence and / or V L The sequence includes post-translational modifications of the sequence (SEQ ID NO: 8, 9, 18, 19, 28, 29, 35, 43, 44, 52, 53, 62, 63, 72, 73, 81, 82, 88, 89, or 92).

[0196] In certain embodiments, the extracellular antigen-binding domain of an antigen-recognizing receptor (e.g., CAR) disclosed herein is selected from the V of any one of the scFvs disclosed herein (e.g., 3-P14, 4-B2, 1-J19, 1-J19-2, 1-P13, 1-P23, 1-A20, 2-N3, 1-H19, 2-F18, and 4-P3) for binding to CD33 (e.g., human CD33). H CDR1 sequence, V H CDR2 sequence, and V H CDR3 sequence, and V L CDR1 sequence, V L CDR2 sequence, and V L In certain embodiments, the extracellular antigen-binding domain of a CAR disclosed herein cross-competes with a reference antibody or antigen-binding fragment thereof comprising the CDR3 sequence of any one of the scFvs disclosed herein (e.g., 3-P14, 4-B2, 1-J19, 1-J19-2, 1-P13, 1-P23, 1-A20, 2-N3, 1-H19, 2-F18, and 4-P3) for binding to CD33 (e.g., human CD33). H Sequence and V L The antibody or antigen-binding portion thereof may cross-compete with a reference antibody or antigen-binding portion thereof, comprising the sequence

[0197] In certain embodiments, the extracellular antigen-binding domain of an antigen-recognizing receptor (e.g., CAR) disclosed herein comprises, for binding to CD33 (e.g., human CD33), the V H CDR1 sequence, V H CDR2 sequence, and V H CDR3 sequence, and V L CDR1 sequence, V L CDR2 sequence, and V L For example, the extracellular antigen-binding domain of an antigen-recognizing receptor (e.g., CAR) disclosed herein comprises a V-amino acid sequence set forth in SEQ ID NO:2 for binding to CD33 (e.g., human CD33). H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:3 HCDR2, comprising the amino acid sequence set forth in SEQ ID NO:4 H CDR3, V comprising the amino acid sequence set forth in SEQ ID NO:5 L CDR1, V comprising an amino acid sequence having the sequence set forth in SEQ ID NO:6 L CDR2 and V comprising an amino acid sequence having the sequence set forth in SEQ ID NO:7 L In certain embodiments, the extracellular antigen-binding domain of an antigen-recognizing receptor (e.g., CAR) disclosed herein cross-competes with the V CDR3 of scFv 3-P14 for binding to CD33 (e.g., human CD33). H Sequence and V L For example, the extracellular antigen-binding domain of an antigen-recognizing receptor (e.g., CAR) disclosed herein may comprise a V-amino acid sequence having the sequence set forth in SEQ ID NO:8 for binding to CD33 (e.g., human CD33). H and V comprising an amino acid sequence having the sequence set forth in SEQ ID NO:9. L or an antigen-binding portion thereof.

[0198] In certain embodiments, the extracellular antigen-binding domain of an antigen-recognizing receptor (e.g., a CAR) disclosed herein binds to the same epitope region on CD33 (e.g., human CD33) as a reference antibody or antigen-binding portion thereof. For example, the extracellular antigen-binding domain of a CAR disclosed herein can be, for example, the V of any one of the scFvs disclosed herein (e.g., 3-P14, 4-B2, 1-J19, 1-J19-2, 1-P13, 1-P23, 1-A20, 2-N3, 1-H19, 2-F18, and 4-P3). H CDR1 sequence, V H CDR2 sequence, and V H CDR3 sequence, and V L CDR1 sequence, V L CDR2 sequence, and V LThe CDR3 sequence of the reference antibody or antigen-binding portion thereof binds to the same epitope region on CD33 (e.g., human CD33). In certain embodiments, the extracellular antigen-binding domain of an antigen-recognizing receptor (e.g., CAR) disclosed herein is selected from the V and VFv of any one of the scFvs disclosed herein (e.g., 3-P14, 4-B2, 1-J19, 1-J19-2, 1-P13, 1-P23, 1-A20, 2-N3, 1-H19, 2-F18, and 4-P3). H Sequence and V L It binds to the same epitope region on CD33 (e.g., human CD33) as a reference antibody or antigen-binding portion thereof that contains the sequence.

[0199] In certain embodiments, the extracellular antigen-binding domain of an antigen-recognizing receptor (e.g., CAR) disclosed herein cross-competes with a reference antibody or antigen-binding portion thereof for binding to CD33 (e.g., human CD33). For example, the extracellular antigen-binding domain of an antigen-recognizing receptor (e.g., CAR) disclosed herein cross-competes with a reference antibody or antigen-binding portion thereof for binding to CD33 (e.g., human CD33), for example, with the V of any one of the scFvs disclosed herein (e.g., 3-P14, 4-B2, 1-J19, 1-J19-2, 1-P13, 1-P23, 1-A20, 2-N3, 1-H19, 2-F18, and 4-P3). H CDR1 sequence, V H CDR2 sequence, and V H CDR3 sequence, and V L CDR1 sequence, V L CDR2 sequence, and V L In certain embodiments, the extracellular antigen-binding domain of an antigen-recognizing receptor (e.g., CAR) disclosed herein is selected from the group consisting of, for example, the V of any one of the scFvs disclosed herein (e.g., 3-P14, 4-B2, 1-J19, 1-J19-2, 1-P13, 1-P23, 1-A20, 2-N3, 1-H19, 2-F18, and 4-P3). H Sequence and V L It binds to the same epitope region on CD33 (e.g., human CD33) as a reference antibody or antigen-binding portion thereof that contains the sequence.

[0200] Extracellular antigen-binding domains that cross-compete or compete with a reference antibody or antigen-binding portion thereof for binding to CD33 (e.g., human CD33) can be identified by using routine methods known in the art, including, but not limited to, ELISA, radioimmunoassay (RIA), Biacore, flow cytometry, Western blotting, and any other suitable quantitative or qualitative antibody binding assay. Competitive ELISA is described in Morris, "Epitope Mapping of Protein Antigens by Competition ELISA", The Protein Protocols Handbook (1996), edited by J. Walker, pp595-600 (incorporated by reference in its entirety). In certain embodiments, the antibody binding assay comprises measuring initial binding of a reference antibody to a CD33 polypeptide, mixing the reference antibody with a test extracellular antigen binding domain, measuring a second binding of the reference antibody to a CD33 polypeptide in the presence of the test extracellular antigen binding domain, and comparing the initial binding to the second binding of the reference antibody, wherein a decrease in the second binding of the reference antibody to a CD33 polypeptide compared to the initial binding indicates that the test extracellular antigen binding domain cross-competes with the reference antibody, e.g., an antibody that recognizes the same or substantially the same epitope, an overlapping epitope, or an adjacent epitope, for binding to CD33. In certain embodiments, the reference antibody is labeled, e.g., with a fluorescent dye, biotin, or peroxidase. In certain embodiments, the CD33 polypeptide is expressed intracellularly, e.g., in a flow cytometry assay. In certain embodiments, the CD33 polypeptide is immobilized on a surface comprising a Biacore ship (e.g., in a Biacore assay) or other medium suitable for surface plasmon resonance analysis. Binding of the reference antibody in the presence of a completely irrelevant antibody (that does not bind CD33) can serve as a high control value. A low control value can be obtained by incubating a labeled reference antibody with an unlabeled reference antibody, which will result in competition and reduced binding of the labeled reference antibody.In certain embodiments, a test extracellular antigen-binding domain that reduces binding of the reference antibody to a CD33 polypeptide by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% is considered to be an extracellular antigen-binding domain that cross-competes with the reference antibody for binding to CD33. In certain embodiments, the assay is performed at room temperature.

[0201] In certain embodiments, the antibody binding assay comprises measuring initial binding of a test extracellular antigen binding domain to a CD33 polypeptide, mixing the test extracellular antigen binding domain with a reference antibody, measuring second binding of the test extracellular antigen binding domain to a CD33 polypeptide in the presence of the reference antibody, and comparing the initial binding of the test extracellular antigen binding domain to the second binding of the test extracellular antigen binding domain, where a decrease in the second binding of the test extracellular antigen binding domain to the CD33 polypeptide compared to the initial binding indicates that the test extracellular antigen binding domain cross-competes with the reference antibody, e.g., an antibody that recognizes the same or substantially the same epitope, an overlapping epitope, or an adjacent epitope, for binding to CD33. In certain embodiments, the test extracellular antigen binding domain is labeled, e.g., with a fluorescent dye, biotin, or peroxidase. In certain embodiments, the CD33 polypeptide is expressed intracellularly, e.g., in a flow cytometry assay. In certain embodiments, the CD33 polypeptide is immobilized on a surface comprising a Biacore ship (e.g., in a Biacore test) or other medium suitable for surface plasmon resonance analysis. Binding of the test extracellular antigen binding domain in the presence of a completely irrelevant antibody (that does not bind to CD33) can serve as a high control value. A low control value can be obtained by incubating a labeled test extracellular antigen binding domain with an unlabeled test extracellular antigen binding domain, which will result in competitive and reduced binding of the labeled test extracellular antigen binding domain. In certain embodiments, a test extracellular antigen binding domain whose binding to the CD33 polypeptide is reduced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% in the presence of the reference antibody is considered to be an extracellular antigen binding domain that cross-competes with the reference antibody for binding to CD33. In certain embodiments, the assay is performed at room temperature.

[0202] In certain embodiments, the extracellular antigen-binding domain of an antigen recognition receptor (e.g., CAR) disclosed herein comprises a linker connecting the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 95. In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 96. In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 97. In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 98. In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 99. In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 100.

[0203] In certain embodiments, the variable region within the extracellular antigen-binding domain comprises a heavy chain variable region (V H In certain embodiments, when the extracellular antigen-binding domain is an scFv, the variable regions are arranged from the N-terminus to the C-terminus: H -V L .

[0204] In certain embodiments, the variable region within the extracellular antigen-binding domain comprises a light chain variable region (V L In certain embodiments, when the extracellular antigen-binding domain is an scFv, the variable regions are arranged from the N-terminus to the C-terminus: L -V H .

[0205] Chimeric Antigen Receptors (CARs) In certain embodiments, the antigen recognition receptor is a CAR. A CAR is an engineered receptor that implants or confers a specificity of interest to immune effector cells. CARs can be used to implant the specificity of monoclonal antibodies into T cells, and the transfer of their coding sequences is facilitated by retroviral vectors.

[0206] There are three generations of CARs. "First generation" CARs are typically composed of an extracellular antigen binding domain (e.g., scFv) fused to a transmembrane domain that is fused to a cytoplasmic / intracellular signaling domain. "First generation" CARs provide novel antigen recognition and are independent of HLA-mediated antigen presentation and bind to CD4 through the CD3 ζ chain signaling domain in a single fusion molecule. + and CD8 + The "second generation" CARs add intracellular signaling domains from various costimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40) to the cytoplasmic tail of the CAR to provide additional signals to T cells. "Second generation" CARs include those that provide both costimulation (e.g., CD28 or 4-1BB) and activation (CD3ζ). "Third generation" CARs include those that provide multiple costimulations (e.g., CD28 and 4-1BB) and activation (CD3ζ). In certain embodiments, the antigen recognition receptor is a first generation CAR. In certain embodiments, the antigen recognition receptor is a CAR that does not include the intracellular signaling domain of a costimulatory molecule or a fragment thereof. In certain embodiments, the antigen recognition receptor is a second generation CAR.

[0207] In certain embodiments, the CAR comprises an extracellular antigen-binding domain that specifically binds CD33, a transmembrane domain, and an intracellular signaling domain.

[0208] 5.3.2.1. Extracellular Antigen-Binding Domain of the CAR The extracellular antigen binding domain of the CAR can be any extracellular antigen binding domain disclosed herein, e.g., in Section 5.3.1.

[0209] In certain embodiments, the CAR comprises an extracellular antigen-binding domain disclosed in Section 5.3.1.

[0210] In addition, the extracellular antigen binding domain can include a leader or signal peptide that directs the nascent protein into the endothelial endoplasmic reticulum. If the CAR is glycosylated and anchored in the cell membrane, the signal peptide or leader may be essential. The signal sequence or leader may be a peptide sequence (about 5, about 10, about 15, about 20, about 25, or about 30 amino acids long) present at the N-terminus of newly synthesized proteins, which directs their entry into the secretory pathway. In certain embodiments, the signal peptide is covalently attached to the 5' end of the extracellular antigen binding domain. In certain embodiments, the signal peptide comprises a CD8 polypeptide, e.g., the CAR comprises a truncated CD8 signal peptide. In certain embodiments, the CD8 signal peptide comprises or consists of amino acids 1-18 of SEQ ID NO: 101. An exemplary nucleotide sequence encoding amino acids 1-18 of SEQ ID NO: 101 is set forth in SEQ ID NO: 122, provided below. ATGGCTCTCCCAGTGACTGCCCTACTGCTTCCCCTAGCGCTTCTCCTGCATGCA [SEQ ID NO: 122]

[0211] 5.3.2.2. CAR Transmembrane Domain In certain embodiments, the transmembrane domain of the CAR comprises a hydrophobic alpha helix that spans at least a portion of the membrane. Different transmembrane domains result in different receptor stabilities. After antigen recognition, the receptors cluster and a signal is transmitted to the cell. According to the subject matter disclosed herein, the transmembrane domain of the CAR may comprise a native or modified transmembrane domain of CD8 or a fragment thereof, a native or modified transmembrane domain of CD28 or a fragment thereof, a native or modified transmembrane domain of CD3zeta or a fragment thereof, a native or modified transmembrane domain of CD4 or a fragment thereof, a native or modified transmembrane domain of 4-1BB or a fragment thereof, a native or modified transmembrane domain of OX40 or a fragment thereof, a native or modified transmembrane domain of ICOS or a fragment thereof, a native or modified transmembrane domain of CD84 or a fragment thereof, a native or modified transmembrane domain of CD166 or a fragment thereof, a native or modified transmembrane domain of CD8a or a fragment thereof, a native or modified transmembrane domain of CD8b or a fragment thereof, a native or modified transmembrane domain of ICAM-1 or a fragment thereof, a native or modified transmembrane domain of CTLA-4 or a fragment thereof, a native or modified transmembrane domain of CD27 or a fragment thereof, a native or modified transmembrane domain of CD40 or a fragment thereof, NKGD2 or a fragment thereof, or a combination thereof.

[0212] In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide (e.g., the transmembrane domain of CD8 or a fragment thereof). In certain embodiments, the transmembrane domain of the CAR comprises the transmembrane domain of human CD8 or a fragment thereof. In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to an amino acid sequence having NCBI reference number NP_001139345.1 (SEQ ID NO: 101), or a fragment thereof, and / or may optionally comprise up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 101 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 235 amino acids in length. In certain embodiments, the CD8 polypeptide comprises or consists of the amino acid sequence of amino acids 1-235, 1-50, 50-100, 100-150, 150-200, 137-209, or 200-235 of SEQ ID NO: 101. In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide comprising or consisting of amino acids 137-209 of SEQ ID NO: 101. SEQ ID NO: 101 is provided below. MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV [SEQ ID NO: 101]

[0213] In certain embodiments, the transmembrane domain of the CAR comprises the transmembrane domain of mouse CD8 or a fragment thereof. In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to an amino acid sequence having NCBI reference number AAA92533.1 (SEQ ID NO: 102) or a fragment thereof, and / or may optionally comprise up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 102, which is at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 100, or at least about 200, and up to 247 amino acids in length. In certain embodiments, the CD8 polypeptide comprises or consists of the amino acid sequence of amino acids 1-247, 1-50, 50-100, 100-150, 150-200, 151-219, or 200-247 of SEQ ID NO: 102. In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide comprising or consisting of amino acids 151-219 of SEQ ID NO: 102. SEQ ID NO: 102 is provided below. [ka]

[0214] In certain embodiments, the transmembrane domain of a CAR disclosed herein comprises a CD28 polypeptide (e.g., the transmembrane domain of CD28 or a fragment thereof).

[0215] In certain embodiments, the transmembrane domain of the CAR comprises the transmembrane domain of human CD28 or a fragment thereof. In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the amino acid sequence having NCBI reference number NP_006130 (SEQ ID NO: 103) or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 103 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acids in length. In certain embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 150-200, 153-179, or 200-220 of SEQ ID NO: 103. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 153-179 of SEQ ID NO: 103. SEQ ID NO: 103 is provided below: [ka]

[0216] An exemplary nucleotide sequence encoding amino acids 153-179 of SEQ ID NO:103 is set forth in SEQ ID NO:104, provided below. TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG [SEQ ID NO: 104]

[0217] In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide (e.g., the transmembrane domain of mouse CD28 or a fragment thereof). In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% homologous or identical to an amino acid sequence having NCBI reference number NP_031668.3 (SEQ ID NO: 105) or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 105 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 218 amino acids in length. In certain embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 150-200, 151-177, or 200-218 of SEQ ID NO: 105. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 151-177 of SEQ ID NO: 105. SEQ ID NO: 105 is provided below: [ka]

[0218] In certain non-limiting embodiments, the CAR further comprises a spacer region linking the extracellular antigen-binding domain to the transmembrane domain. The spacer region can be flexible enough to allow the antigen-binding domain to orient in different directions to facilitate antigen recognition while maintaining the active activity of the CAR.

[0219] In certain embodiments, the hinge / spacer region of the CAR is selected from the group consisting of a native or modified hinge region of CD8 or a fragment thereof, a native or modified hinge region of CD28 or a fragment thereof, a native or modified hinge region of CD3zeta or a fragment thereof, a native or modified hinge region of CD40 or a fragment thereof, a native or modified hinge region of 4-1BB or a fragment thereof, a native or modified hinge region of OX40 or a fragment thereof, a native or modified hinge region of CD84 or a fragment thereof, a native or modified hinge region of CD166 or a fragment thereof, a native or modified hinge region of CD8a ... The hinge region may be a modified hinge region or fragment thereof, a native or modified hinge region of CD8b or fragment thereof, a native or modified hinge region of ICOS or fragment thereof, a native or modified hinge region of ICAM-1 or fragment thereof, a native or modified hinge region of CTLA-4 or fragment thereof, a native or modified hinge region of CD27 or fragment thereof, a native or modified hinge region of CD40 or fragment thereof, a native or modified hinge region of NKGD2 or fragment thereof, a synthetic polypeptide (not based on a protein associated with an immune response), or a combination thereof. The hinge / spacer region can be a hinge region from IgG1, or an immunoglobulin CH2CH3 region and a portion of CD3, a portion of a CD8 polypeptide (e.g., a portion of SEQ ID NO: 101 or 102), a portion of a CD28 polypeptide (e.g., a portion of SEQ ID NO: 103 or 105), a variation of any of the foregoing that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% homologous or identical thereto, or a synthetic spacer sequence.

[0220] 5.3.2.3. CAR Intracellular Signaling Domain In certain embodiments, the CAR comprises an intracellular signaling domain. In certain non-limiting embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide. CD3ζ can activate or stimulate cells (e.g., cells of the lymphoid system, e.g., T cells). Wild-type ("native") CD3ζ comprises three functional immunoreceptor tyrosine-based activation motifs (ITAMs), three functional basic-rich extension (BRS) regions (BRS1, BRS2, ​​and BRS3). After antigen binding, CD3ζ transmits an activation signal to cells (e.g., cells of the lymphoid system, e.g., T cells). The intracellular signaling domain of the CD3ζ chain is the main transmitter of signals from endogenous TCR.

[0221] In certain embodiments, the intracellular signaling domain of the CAR comprises native CD3zeta. In certain embodiments, the CD3zeta polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence having NCBI reference number NP_932170 (SEQ ID NO: 106) or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the CD3zeta polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 106 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 164 amino acids in length. In certain embodiments, the CD3ζ polypeptide comprises or consists of the amino acid sequence of amino acids 1-164, 1-50, 50-100, 52-164, 100-150, or 150-164 of SEQ ID NO: 106. In certain embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide comprising or consisting of amino acids 52-164 of SEQ ID NO: 106. SEQ ID NO: 106 is provided below: [ka]

[0222] In certain embodiments, the intracellular signaling domain of the CAR comprises a CD3 polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 107. SEQ ID NO: 107 is provided below. RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR [SEQ ID NO: 107]

[0223] An exemplary nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:107 is set forth in SEQ ID NO:108, provided below. AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCC TGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC [SEQ ID NO: 108]

[0224] In certain embodiments, the intracellular signaling domain of the CAR further comprises at least a costimulatory signaling region. In certain embodiments, the costimulatory signaling region comprises at least one costimulatory molecule or a fragment thereof. In certain embodiments, the costimulatory signaling region comprises the intracellular domain of at least one costimulatory molecule or a fragment thereof.

[0225] As used herein, "costimulatory molecule" refers to a cell surface molecule other than an antigen receptor or its ligand that can provide an efficient response of lymphocytes to antigens. In certain embodiments, costimulatory molecules can provide optimal lymphocyte activation. Non-limiting examples of costimulatory molecules include CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, NKGD2, CD2, FN14, HVEM, LTBR, ​​CD28H, TNFR1, TNFR2, BAFF-R, BCMA, TACI, TROY, RANK, CD40, CD27, CD30, EDAR, XEDAR, GITR, DR6, and NGFR, and combinations thereof. Costimulatory molecules can bind to costimulatory ligands, which are proteins expressed on the cell surface that, when bound to their receptors, generate a costimulatory response, i.e., an intracellular response that affects the stimulation provided when an antigen-recognizing receptor (e.g., a chimeric antigen receptor (CAR)) binds to its target antigen. As an example, 4-1BB ligand (i.e., 4-1BBL) can be used in combination with a CAR signal to express CAR + It can bind 4-1BB to provide an intracellular signal that induces effector cell function of a T cell.

[0226] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a CD28 polypeptide, e.g., the intracellular domain of CD28 or a fragment thereof. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a CD28 polypeptide, e.g., the intracellular domain of human CD28 or a fragment thereof. In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 103 or a fragment thereof, and / or may optionally comprise up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 103 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acids in length. In certain embodiments, the CD28 polypeptide comprises, or consists of, the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 114-220, 150-200, 180-220, or 200-220 of SEQ ID NO: 103. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a CD28 polypeptide comprising, or consisting of, the amino acid sequence of amino acids 180-220 of SEQ ID NO: 103.

[0227] An exemplary nucleotide sequence encoding amino acids 180-220 of SEQ ID NO:103 is set forth in SEQ ID NO:109, provided below. AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC [SEQ ID NO: 109]

[0228] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a CD28 polypeptide, e.g., the intracellular domain of mouse CD28 or a fragment thereof. In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 104 or a fragment thereof, and / or may optionally comprise up to one, or up to two, or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 104 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to 218 amino acids in length. In certain embodiments, the CD28 polypeptide comprises, or consists of, the amino acid sequence of amino acids 1-218, 1-50, 50-100, 100-150, 150-218, 178-218, or 200-218 of SEQ ID NO: 105. In certain embodiments, a costimulatory signaling region of a CAR disclosed herein comprises a CD28 polypeptide comprising, or consisting of, amino acids 178-218 of SEQ ID NO: 105.

[0229] In certain embodiments, the costimulatory signaling region of the CAR disclosed herein comprises a CD28 polypeptide that includes a mutated YMNM motif. CD28 is a transmembrane protein that plays a key role in T cell activation through its function as a costimulatory molecule. CD28 has an intracellular domain that includes intracellular motifs that are important for effective signaling of CD28. In certain embodiments, the CD28 intracellular domain includes an intracellular subdomain (also known as an "intracellular motif") that regulates signaling pathways following TCR stimulation. CD28 includes three intracellular motifs: the YMNM motif, and two proline-rich motifs: the PRRP motif, and the PYAP motif. The CD28 intracellular motifs can function as docking sites for a number of adaptor molecules that interact with these motifs via their SH2 or SH3 domains. Such interactions transmit downstream signals that terminate on transcription factors that regulate gene expression. For example, the native YMNM motif binds to the p85 subunit of phosphoinositide 3-kinase (PI3K). The native YMNM motif also binds to growth factor receptor-bound protein 2 (Grb2) and / or Grb2-associated adaptor protein 2 (GADS). Grb2 binds to Gab1 and Gab2, which can then recruit the p85 subunit of PI3K.

[0230] In certain embodiments, the native YMNM motif consists of the amino acid sequence set forth in YMNM (SEQ ID NO: 123). In certain embodiments, the native YMNM motif binds to the p85 subunit of PI3K via the consensus sequence YMxM (SEQ ID NO: 124), where x is not aspartic acid (N). In certain embodiments, the native YMNM motif binds to Grb2 and / or GAD via the consensus sequence YxNx (SEQ ID NO: 125), where x is not methionine (M).

[0231] In certain embodiments, CD28 polypeptides comprising a mutated YMNM motif disclosed herein have reduced recruitment of the p85 subunit of PI3K compared to CD28 molecules comprising a native YMNM motif. In certain embodiments, the p85 subunit of PI3K does not bind to the mutated YMNM motif, thereby reducing recruitment of the p85 subunit of PI3K to the CD28 polypeptide. Mutated YMNM motifs that block binding of the p85 subunit of PI3K retain binding to Grb2 and / or GADS. Thus, downstream signaling of Grb2 / GADS remains intact, e.g., downstream signaling leading to IL-2 secretion remains intact. Such mutated YMNM motifs are referred to as "GADS / Grb2 permissive mutants."

[0232] In certain embodiments, the mutated YMNM binds to the p85 subunit of PI3K but not to Grb2 and / or GADS. Because PI3K p85 binding is retained, downstream signaling of PI3K remains intact. Because Grb2 / GADS binding is blocked, recruitment of the PI3K p85 subunit induced by binding of Grb2 to Gab1 and Gab2 is reduced or blocked. In addition, downstream signaling of Grb2 / GADS is blocked. Such mutated YMNM motifs are referred to as "PI3K permissive mutants."

[0233] In certain embodiments, the mutated YMNM does not bind to the p85 subunit of PI3K and does not bind to Grb2 and / or GADS. Such mutated YMNM motifs are referred to as "non-functional mutants." Non-functional mutants do not provide for binding of PI3K, Grb2, or GADS to CD28 at the YMNM motif, but do not prevent these signaling molecules from binding elsewhere in the CD28 molecule.

[0234] In certain embodiments, the mutated YMNM retains only one of the two methionine residues present in the YMNM motif, i.e., YMxx or YxxM. These motifs potentially regulate signaling through PI3K by limiting the number of methionine residues that can bind to the p85 subunit of PI3K. Such mutated YMNM motifs are referred to as "hybrid "HEMI" mutants."

[0235] In certain embodiments, the mutated YMNM motif is a GADS / Grb-2 permissive mutation. In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YxNx (SEQ ID NO: 125), where x is not methionine (M). In certain embodiments, x is selected from the group consisting of the amino acids A, R, N, D, C, E, Q, G, H, I, K, F, P, S, T, W, Y, V, and L. In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YENV (SEQ ID NO: 126), YSNV (SEQ ID NO: 127), YKNL (SEQ ID NO: 128), YENQ (SEQ ID NO: 129), YKNI (SEQ ID NO: 130), YINQ (SEQ ID NO: 131), YHNK (SEQ ID NO: 132), YVNQ (SEQ ID NO: 133), YLNP (SEQ ID NO: 134), YLNT (SEQ ID NO: 135), YDND (SEQ ID NO: 136), YENI (SEQ ID NO: 137), YENL (SEQ ID NO: 138), YKNQ (SEQ ID NO: 139), YKNV (SEQ ID NO: 140), or YANG (SEQ ID NO: 141). In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YSNV (SEQ ID NO: 127). In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YKNI (SEQ ID NO: 130). In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YENV (SEQ ID NO: 126). In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YKNL (SEQ ID NO: 128).

[0236] In certain embodiments, the mutated YMNM motif is a PI3K-permissive mutant. In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YMxM (SEQ ID NO: 124), where x is not aspartic acid (N). In certain embodiments, x is selected from the group consisting of the amino acids A, R, D, C, E, Q, G, H, I, K, M, F, P, S, T, W, Y, V, and L. In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YMDM (SEQ ID NO: 142), YMPM (SEQ ID NO: 143), YMRM (SEQ ID NO: 144), or YMSM (SEQ ID NO: 145). In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YMDM (SEQ ID NO: 142).

[0237] In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YbxM (SEQ ID NO: 146), where x is not aspartic acid (N) and b is not methionine (M). In certain embodiments, x is selected from the group consisting of the amino acids A, R, D, C, E, Q, G, H, I, K, M, F, P, S, T, W, Y, V, and L. In certain embodiments, b is selected from the group consisting of the amino acids A, R, N, C, E, Q, G, H, I, K, N, F, P, S, T, W, Y, V, and L. In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YTHM (SEQ ID NO: 147), YVLM (SEQ ID NO: 148), YIAM (SEQ ID NO: 149), YVEM (SEQ ID NO: 150), YVKM (SEQ ID NO: 151), or YVPM (SEQ ID NO: 152).

[0238] In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YMxb (SEQ ID NO: 153), where x is not aspartic acid (N) and b is not methionine (M). In certain embodiments, x is selected from the group consisting of the amino acids A, R, D, C, E, Q, G, H, I, K, M, F, P, S, T, W, Y, V, and L. In certain embodiments, b is selected from the group consisting of the amino acids A, R, N, C, E, Q, G, H, I, K, N, F, P, S, T, W, Y, V, and L. In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YMAP (SEQ ID NO: 154).

[0239] Certain mutated YMNM motifs are described in Mol Cell Proteomics. 2010 Nov;9(11):2391-404, Virology. 2015 May;0:568-577, both of which are incorporated by reference in their entireties.

[0240] In certain embodiments, the mutated YMNM motif is a hybrid "HEMI" mutant. In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YMNx (SEQ ID NO: 155) or YxNM (SEQ ID NO: 156), where x is not methionine (M). In certain embodiments, x is selected from the group consisting of the amino acids A, R, N, C, E, Q, G, H, I, K, N, F, P, S, T, W, Y, V, and L. In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YMNV (SEQ ID NO: 157), YENM (SEQ ID NO: 158), YMNQ (SEQ ID NO: 159), YMNL (SEQ ID NO: 160), or YSNM (SEQ ID NO: 161).

[0241] In certain embodiments, the mutated YMNM motif is a non-functional variant. In certain embodiments, the mutated YMNM motif consists of the amino acid sequence Ybxb (SEQ ID NO: 162), where x is not aspartic acid (N) and b is not methionine (M). In certain embodiments, x is selected from the group consisting of A, R, D, C, E, Q, G, H, I, K, M, F, P, S, T, W, Y, V, and L. In certain embodiments, b is selected from the group consisting of A, R, N, D, C, E, Q, G, H, I, K, F, P, S, T, W, Y, V, and L. In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YGGG (SEQ ID NO: 163), YAAA (SEQ ID NO: 164), YFFF (SEQ ID NO: 165), YETV (SEQ ID NO: 166), YQQQ (SEQ ID NO: 167), YHAE (SEQ ID NO: 168), YLDL (SEQ ID NO: 169), YLIP (SEQ ID NO: 170), YLRV (SEQ ID NO: 171), YTAV (SEQ ID NO: 172), or YVHV (SEQ ID NO: 173). In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YGGG (SEQ ID NO: 163).

[0242] In certain embodiments, the intracellular signaling domain of a chimeric receptor disclosed herein comprises a costimulatory signaling domain comprising a CD28 polypeptide comprising a mutated YMNM motif consisting of the amino acid sequence set forth in YENV (SEQ ID NO: 126), wherein the CD28 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 174. SEQ ID NO: 174 is provided below. RSKRSRLLHSDYENVTPRRPGPTRKHYQPYAPPRDFAAYRS [SEQ ID NO: 174]

[0243] In certain embodiments, the intracellular signaling domain of a chimeric receptor disclosed herein comprises a costimulatory signaling domain comprising a CD28 polypeptide comprising a mutated YMNM motif consisting of the amino acid sequence set forth in YKNI (SEQ ID NO: 130), wherein the CD28 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 175. SEQ ID NO: 175 is provided below. RSKRSRLLHSDYKNITPRRPGPTRKHYQPYAPPRDFAAYRS [SEQ ID NO: 175]

[0244] In certain embodiments, the intracellular signaling domain of a chimeric receptor disclosed herein comprises a costimulatory signaling domain comprising a CD28 polypeptide comprising a mutated YMNM motif consisting of the amino acid sequence set forth in YMDM (SEQ ID NO: 142), wherein the CD28 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 176. SEQ ID NO: 176 is provided below. RSKRSRLLHSDYMDMTPRRPGPTRKHYQPYAPPRDFAAYRS [SEQ ID NO: 176]

[0245] In certain embodiments, the intracellular signaling domain of a chimeric receptor disclosed herein comprises a costimulatory signaling domain comprising a CD28 polypeptide comprising a mutated YMNM motif consisting of the amino acid sequence set forth in YGGG (SEQ ID NO: 163), wherein the CD28 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 177. SEQ ID NO: 177 is provided below. RSKRSRLLHSDYGGGTPRRPGPTRKHYQPYAPPRDFAAYRS [SEQ ID NO: 177]

[0246] In certain embodiments, the intracellular signaling domain of a chimeric receptor disclosed herein comprises a costimulatory signaling domain comprising a CD28 polypeptide comprising a mutated YMNM motif consisting of the amino acid sequence set forth in YSNV (SEQ ID NO: 127), wherein the CD28 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 178. SEQ ID NO: 178 is provided below. RSKRSRLLHSDYSNVTPRRPGPTRKHYQPYAPPRDFAAYRS [SEQ ID NO: 178]

[0247] In certain embodiments, the intracellular signaling domain of a CAR disclosed herein comprises a first costimulatory signaling domain comprising a CD28 polypeptide comprising a mutated YMNM motif (disclosed herein), and a second costimulatory signaling domain comprising an intracellular domain of a costimulatory molecule. Additional information regarding CARs comprising a CD28 polypeptide comprising a mutated YMNM motif can be found in International Patent Publication No. WO 2021 / 158850, which is incorporated by reference in its entirety.

[0248] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a 4-1BB polypeptide, e.g., the intracellular domain of 4-1BB or a fragment thereof. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a 4-1BB polypeptide, e.g., the intracellular domain of human 4-1BB or a fragment thereof. In certain embodiments, the 4-1BB polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to an amino acid sequence having NCBI reference number NP_001552 (SEQ ID NO: 110) or a fragment thereof, and / or may optionally comprise up to one, or up to two, or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the 4-1BB polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 106 that is at least 20, or at least 30, or at least 40, or at least 50, or at least 100, or at least 150, or at least 150, and up to 255 amino acids in length. In certain embodiments, the 4-1BB polypeptide comprises or consists of an amino acid sequence of amino acids 1-255, 1-50, 50-100, 100-150, 150-200, or 200-255 of SEQ ID NO: 110. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region that comprises a 4-1BB polypeptide that comprises or consists of an amino acid sequence of amino acids 214-255 of SEQ ID NO: 110. SEQ ID NO: 110 is provided below. [ka]

[0249] An exemplary nucleotide sequence encoding amino acids 214-255 of SEQ ID NO:110 is set forth in SEQ ID NO:111, provided below. AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG [SEQ ID NO: 111]

[0250] In certain embodiments, the intracellular signaling domain of the CAR comprises an intracellular domain of two or more costimulatory molecules or portions thereof, for example, the intracellular domain of CD28 or a fragment thereof, the intracellular domain of 4-1BB or a fragment thereof, or the intracellular domain of CD28 or a fragment thereof, and the intracellular domain of OX40 or a fragment thereof.

[0251] In certain embodiments, the CARs disclosed herein further comprise an inducible promoter for expressing the nucleic acid sequence in human cells. The promoter used for expression of the CAR gene may be a constitutive promoter, such as the Ubiquitin C (UbiC) promoter.

[0252] 5.3.2.4. Example CAR In certain embodiments, the CAR is a CD33-targeting CAR. In certain embodiments, the CAR comprises: (a)(i) a VDR1 comprising the amino acid sequence set forth in SEQ ID NO:2, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:3, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:4. H and (ii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7. L(b) an extracellular antigen-binding domain comprising a CD3ζ polypeptide (e.g., a transmembrane domain comprising a CD28 polypeptide (e.g., a transmembrane domain of human CD28 or a fragment thereof); and (c) an intracellular signaling domain comprising (i) a CD3ζ polypeptide, and (ii) a costimulatory signaling region comprising a CD28 polypeptide (e.g., an intracellular domain of human CD28 or a fragment thereof). In certain embodiments, the transmembrane domain comprises a CD28 polypeptide comprising amino acids 153-179 of SEQ ID NO: 103. In certain embodiments, the intracellular signaling domain comprises (i) a CD3ζ polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 107, and (ii) a costimulatory signaling region comprising a CD28 polypeptide comprising amino acids 180-220 of the amino acid sequence set forth in SEQ ID NO: 103. In certain embodiments, the extracellular antigen-binding domain is an scFv designated as "3-P14". In certain embodiments, the V H and V L are linked via a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 96. H and V L are arranged from the N-terminus to the C-terminus: H -V L In certain embodiments, the CAR is designated as "TDI-Y-006_h28z". In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 112, provided below. EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVSAISGRGGSTYYTDSVKGRFTISRDNSKNTVSLQMNSLRAEDTAVYYCAGRGDYYYYYGMDVWGQGTTVTVSAG GGGSGGGGSGGGGSDIVMTQSPLSSPVTLGQPASFSCRSSQSLVYSDGNTYLSWLQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQSTQFPHTFGQGTK LEIKEQKLISEEDLAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR [SEQ ID NO: 112]

[0253] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:112 is set forth in SEQ ID NO:113, provided below.

[0254] In certain embodiments, the CAR is a CD33-targeting CAR. In certain embodiments, the CAR comprises: (a)(i) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a VDR comprising the amino acid sequence set forth in SEQ ID NO: 14. H V containing CDR3 H and (ii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a V comprising the amino acid sequence set forth in SEQ ID NO: 17. L V containing CDR3 L (b) an extracellular antigen-binding domain comprising a CD28 polypeptide (e.g., a transmembrane domain of human CD28 or a portion thereof); and (c) an intracellular signaling domain comprising (i) a CD3ζ polypeptide, and (ii) a costimulatory signaling region comprising a CD28 polypeptide (e.g., an intracellular domain of human CD28 or a fragment thereof). In certain embodiments, the transmembrane domain comprises a CD28 polypeptide comprising amino acids 153-179 of SEQ ID NO: 103. In certain embodiments, the intracellular signaling domain comprises (i) a CD3ζ polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 107, and (ii) a costimulatory signaling region comprising a CD28 polypeptide comprising amino acids 180-220 of the amino acid sequence set forth in SEQ ID NO: 103. In certain embodiments, the extracellular antigen-binding domain is an scFv designated as "4-B2". In certain embodiments, the V H and V L are linked via a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 96. H and V L are arranged from the N-terminus to the C-terminus: H -V L In certain embodiments, the CAR is designated as "TDI-Y-007_h28z". In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 114, provided below. EVHLLESGGGLVQPGGSLRLSCAASGFIFSSNAMSWVRQAPGKGLEWVSAISGYGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWGTYIVGATGDYWGQGTLVTVSS GGGGSGGGGSGGGGSQSALTQPPSASGSPGQSVTISCTGTSNDVGGYNYVSWYQQHPGKAPKLLIYEVSKRPSGVPDRFSGSQSGNTASLTVSGLQAEDEADYYCSSYAGSNNWVFGGGTKL TVLEQKLISEEDLAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR [SEQ ID NO: 114]

[0255] An exemplary nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:114 is set forth in SEQ ID NO:115, provided below.

[0256] In certain embodiments, the CAR is a CD33-targeting CAR. In certain embodiments, the CAR comprises: (a)(i) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, and a VDR comprising the amino acid sequence set forth in SEQ ID NO: 24. H V containing CDR3 H and (ii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and a V comprising the amino acid sequence set forth in SEQ ID NO: 27. L V containing CDR3 L (b) an extracellular antigen-binding domain comprising a CD28 polypeptide (e.g., a transmembrane domain of human CD28 or a portion thereof); and (c) an intracellular signaling domain comprising (i) a CD3ζ polypeptide, and (ii) a costimulatory signaling region comprising a CD28 polypeptide (e.g., an intracellular domain of human CD28 or a fragment thereof). In certain embodiments, the transmembrane domain comprises a CD28 polypeptide comprising amino acids 153-179 of SEQ ID NO: 103. In certain embodiments, the intracellular signaling domain comprises (i) a CD3ζ polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 107, and (ii) a costimulatory signaling region comprising a CD28 polypeptide comprising amino acids 180-220 of the amino acid sequence set forth in SEQ ID NO: 103. In certain embodiments, the extracellular antigen-binding domain is an scFv designated as "1-J19". In certain embodiments, the V H and V L are linked via a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 96. H and V L are arranged from the N-terminus to the C-terminus: H -V L In certain embodiments, the CAR is designed as "1J19HL_h28z". In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 116, provided below. QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYFRSKWYNVYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCASEGGSYYDHWGQGTLVTVSS GGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISNWLTWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQADSFPFTFGGPGTKVDI KEQKLISEEDLAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR [Sequence number 116]

[0257] An exemplary nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:116 is set forth in SEQ ID NO:117, provided below.

[0258] In certain embodiments, the CAR disclosed herein further comprises an inducible promoter for expressing the nucleotide sequence in human cells. The promoter used for expression of the CAR gene may be a constitutive promoter, such as the Ubiquitin C (UbiC) promoter.

[0259] 5.3.3. TCR-like fusion molecules In certain embodiments, the antigen recognizing receptor is a TCR-like fusion molecule. Non-limiting examples of TCR fusion molecules include HLA-independent TCR-based chimeric antigen receptors (also known as "HIT-CARs" and disclosed, for example, in International Patent Application No. PCT / US19 / 017525, which is incorporated by reference in its entirety), T cell receptor fusion constructs (TRuCs) (for example, as disclosed in Baeuerle et al., "Synthetic TRuC receptors engaging the complete T cell receptor for potent anti-tumor response," Nature Communications volume 10, Article number:2087 (2019), which is incorporated by reference in its entirety), and T cell antigen couplers (TACs), which are chimeric receptors that co-select endogenous TCRs (for example, as disclosed in Helsen et al., "The chimeric TAC receptor co-opts the T cell receptor yielding robust anti-tumor activity without toxicity," Nature Communications (2018);9:3049 (2018), which is incorporated by reference in its entirety).

[0260] In certain embodiments, the TCR-like fusion molecule comprises an antigen binding chain comprising an extracellular antigen binding domain and a constant domain, and the TCR-like fusion molecule binds to antigen in an HLA-independent manner. In certain embodiments, the constant domain comprises a T cell receptor constant region selected from the group consisting of a native or modified TRAC peptide, a native or modified TRBC peptide, a native or modified TRDC peptide, a native or modified TRGC peptide, and any variant or functional fragment thereof. In certain embodiments, the constant domain comprises a native or modified TRAC peptide. In certain embodiments, the constant domain comprises a native or modified TRBC peptide. In certain embodiments, the constant domain can form a homodimer or heterodimer with another constant domain. In certain embodiments, the antigen binding chain can associate with a CD3ζ polypeptide. In certain embodiments, the antigen binding chain can activate a CD3ζ polypeptide associated with the antigen binding chain upon binding to an antigen. In certain embodiments, activation of the CD3ζ polypeptide can activate an immunoresponsive cell. In certain embodiments, the TCR-like fusion molecule can incorporate with the CD3 complex to provide HLA-independent antigen recognition. In certain embodiments, the TCR-like fusion molecule replaces an endogenous TCR in the CD3 / TCR complex. In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule is capable of dimerizing with another extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule comprises a ligand for a cell surface receptor, a receptor for a cell surface ligand, an antigen-binding portion of an antibody or fragment thereof, or an antigen-binding portion of a TCR. In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule comprises one or two immunoglobulin variable regions. In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule comprises a heavy chain variable region (V) of an antibody. H In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule comprises the light chain variable region of an antibody (V L In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule is capable of dimerizing with another extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule is capable of dimerizing with the VH Including V H is the V of the antibody L To form a fragment variable (Fv), the extracellular antigen-binding domain of the TCR-like fusion molecule may be dimerized with another extracellular antigen-binding domain comprising the V of an antibody. L Including V L is the V of the antibody H to form a fragment variable (Fv).

[0261] 5.4.Cells The presently disclosed subject matter provides cells comprising a CD33-targeting antigen-recognizing receptor disclosed herein (e.g., those disclosed in Section 5.3). In certain embodiments, the cells are selected from the group consisting of cells of the lymphoid lineage, cells of the myeloid lineage, stem cells from which cells of the lymphoid lineage may be derived, and stem cells from which cells of the myeloid lineage may be derived. In certain embodiments, the cells are immunoresponsive cells. In certain embodiments, the immunoresponsive cells are cells of the lymphoid lineage.

[0262] In certain embodiments, the cell is a lymphoid cell. The lymphoid cell can provide antibody production, regulation of the cellular immune system, detection of foreign substances in the blood, detection of cells foreign to the host, etc. Non-limiting examples of lymphoid cells include T cells, natural killer (NK) cells, B cells, dendritic cells, and stem cells from which lymphoid cells can be differentiated. In certain embodiments, the stem cell is a pluripotent stem cell (e.g., an embryonic stem cell).

[0263] In certain embodiments, the cell is a T cell. T cells may be lymphocytes that mature in the thymus and are primarily involved in cell-mediated immunity. T cells are involved in the adaptive immune system. The T cells of the subject matter disclosed herein can be any type of T cell, including, but not limited to, helper T cells, cytotoxic T cells, memory T cells (central memory T cells, stem cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells: e.g., TEM cells and TEMRA cells, regulatory T cells (also known as suppressor T cells), tumor infiltrating lymphocytes (TILs), natural killer T cells, mucosal-associated invariant T cells, and γδ T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes that can induce the death of infected somatic or tumor cells. The patient's own T cells may be genetically modified to target specific antigens through the introduction of an antigen recognition receptor, e.g., a CAR. In certain embodiments, the immunoresponsive cells are T cells. The T cells are CD4 + T cells or CD8 + In certain embodiments, the T cells are CD4 + In certain embodiments, the T cells are CD8 + T cells.

[0264] In certain embodiments, the cell is a NK cell. Natural killer (NK) cells are part of cell-mediated immunity and can be lymphocytes that act during innate immune response. NK cells do not require prior activation to carry out cytotoxic effects on target cells.

[0265] Types of human lymphocytes of the presently disclosed subject matter include, but are not limited to, peripheral donor lymphocytes. See, e.g., Sadelain et al., Nat Rev Cancer (2003); 3:35-45 (disclosing peripheral blood donor lymphocytes genetically modified to express CARs), Morgan, RA, et al. 2006 Science 314:126-129 (disclosing peripheral blood donor lymphocytes genetically modified to express a full-length tumor antigen-recognizing T cell receptor complex containing α and β heterodimers), Panelli et al., J Immunol (2000); 164:495-504, Panelli et al., J Immunol (2000); 164:4382-4392 (disclosing lymphocyte cultures derived from tumor-infiltrating lymphocytes (TILs) in tumor biopsies), and Dupont et al., Cancer Res (2005); 65:5417-5427, Papanicolaou et al. al., Blood (2003); 102:2498-2505 (disclosing selective in vitro expanded antigen-specific peripheral blood leukocytes using artificial antigen presenting cells (AAPC) or pulsed dendritic cells).

[0266] Cells (eg, T cells) can be derived in vitro from autologous, non-autologous (eg, allogeneic), or engineered progenitor or stem cells.

[0267] The subject matter disclosed herein may be a myeloid cell. Non-limiting examples of myeloid cells include monocytes, macrophages, neutrophils, dendritic cells, basophils, neutrophils, eosinophils, megakaryocytes, mast cells, erythrocytes, platelets, and stem cells from which myeloid cells can be differentiated. In certain embodiments, the stem cells are pluripotent stem cells (e.g., embryonic stem cells or induced pluripotent stem cells).

[0268] In certain embodiments, the cells disclosed herein can modulate the tumor microenvironment. Tumors have a microenvironment hostile to the host immune response, involving a series of mechanisms by malignant cells to protect themselves from immune recognition and elimination. This "hostile tumor microenvironment" includes the infiltrating regulatory CD4 + The tumor microenvironment contains a variety of immunosuppressive factors, including T cells (Tregs), myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), immunosuppressive cytokines including TGF-β, and expression of ligands that target immunosuppressive receptors expressed by activated T cells (CTLA-4 and PD-1). Although these immunosuppressive mechanisms play a role in maintaining tolerance and suppressing inappropriate immune responses, within the tumor microenvironment, these mechanisms prevent effective antitumor immune responses. Collectively, these immunosuppressive factors can induce either significant anergy or apoptosis of adoptively transferred CAR-modified T cells upon encounter with target tumor cells.

[0269] In certain embodiments, cells can be transfected with a CD33-targeting antigen recognition receptor disclosed herein to express the antigen recognition receptor.

[0270] In certain embodiments, the cells further comprise a soluble single chain variable fragment (scFv) that binds to a polypeptide having immunosuppressive or immunostimulatory activity. In certain embodiments, immunosuppressive activity refers to induction of signaling or changes in protein expression in cells (e.g., activated immunoresponsive cells) that result in a decrease in immune response. Polypeptides known to suppress or decrease immune responses through binding include CD47, PD-1, CTLA-4, and their corresponding ligands, including SIRPa, PD-L1, PD-L2, B7-1, and B7-2. Such polypeptides are present in the tumor microenvironment and inhibit the immune response to tumor cells. In various embodiments, inhibiting, blocking, or antagonizing the interaction of immunosuppressive polypeptides and / or their ligands enhances the immune response of immunoresponsive cells.

[0271] In certain embodiments, immunostimulatory activity refers to the induction of signaling or changes in protein expression in cells (e.g., activated immunoresponsive cells) that result in an increased immune response. Immunostimulatory activity can include proinflammatory activity. Polypeptides known to stimulate or increase immune responses through binding include CD28, OX-40, 4-IBB, and their corresponding ligands, including B7-1, B7-2, OX-40L, and 4-1BBL. Such polypeptides are present in the tumor microenvironment and activate immune responses against tumor cells. In various embodiments, promoting, stimulating, or agonizing proinflammatory polypeptides and / or their ligands enhances the immune response of immunoresponsive cells.

[0272] Cells comprising a soluble scFv that binds to an antigen recognition receptor (e.g., a CAR) and a polypeptide having immunosuppressive or immunostimulatory activity are disclosed in International Patent Publication No. WO 2014 / 134165, which is incorporated by reference in its entirety.

[0273] In certain embodiments, the cells further comprise exogenous CD40L. Cells comprising an antigen recognition receptor (e.g., a CAR) and exogenous CD40L are disclosed in International Patent Publication No. WO 2014 / 134165.

[0274] Moreover, in certain embodiments, the cells are engineered to express IL-18. In certain embodiments, the cells further comprise an exogenous IL-18 polypeptide. In certain embodiments, the exogenous IL-18 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 118, provided below. MGYRMQLLSCIALSLALVTNSGYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED [SEQ ID NO: 118]

[0275] In certain embodiments, the cell further comprises a nucleic acid molecule encoding an IL-18 polypeptide. In certain embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO: 119, provided below. [SEQ ID NO: 119]

[0276] Alternatively, in certain embodiments, the cells further comprise a modified promoter / enhancer at the IL-18 locus that can increase IL-18 gene expression, e.g., a constitutive or inducible promoter is placed to drive IL-18 gene expression.

[0277] Cells engineered to express IL-18, including an antigen recognition receptor (e.g., CAR) and including, for example, an exogenous IL-18 polypeptide or a modified promoter / enhancer at the IL-18 locus, are disclosed in International Patent Publication No. WO 2018 / 027155, which is incorporated by reference in its entirety.

[0278] Additionally or alternatively, the cells are engineered to express IL-33. In certain embodiments, the cells further comprise an exogenous IL-33 polypeptide. In certain embodiments, the exogenous IL-33 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 120, provided below. MYRMQLLSCIALSLALVTNSSITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET [SEQ ID NO: 120]

[0279] In certain embodiments, the cell further comprises a nucleic acid molecule encoding an IL-33 polypeptide. In certain embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO: 121, provided below. [SEQ ID NO: 121]

[0280] Alternatively, in certain embodiments, the cells further comprise a modified promoter / enhancer at the IL-33 locus that can increase IL-33 gene expression, e.g., a constitutive or inducible promoter is positioned to drive IL-33 gene expression. Cells engineered to express IL-33, including antigen recognition receptors (e.g., CARs), e.g., including exogenous IL-33 polypeptides or modified promoters / enhancers at the IL-33 locus, are disclosed in International Patent Publication No. WO 2019 / 099479, which is incorporated by reference in its entirety.

[0281] Additionally or alternatively, the cells are engineered to express IL-36. In certain embodiments, the cells further comprise an exogenous IL-36 polypeptide. In certain embodiments, the cells further comprise a modified promoter / enhancer at the IL-36 locus that can increase IL-36 gene expression, e.g., a constitutive or inducible promoter is placed to drive IL-36 gene expression. Cells engineered to express IL-36, including antigen recognition receptors (e.g., CARs), e.g., including exogenous IL-36 polypeptides or modified promoters / enhancers at the IL-36 locus, are disclosed in International Patent Publication No. WO 2019 / 099483, which is incorporated by reference in its entirety.

[0282] 5.5. Nucleic Acid Compositions and Vectors The subject matter disclosed herein provides nucleic acids encoding the CD33-targeting antigen-recognizing receptors disclosed herein (e.g., those disclosed in Section 5.3). Nucleic acid compositions comprising the nucleic acids disclosed herein are further provided. Also provided are cells comprising such nucleic acid compositions.

[0283] In certain embodiments, the nucleic acid composition further comprises a promoter operably linked to the CD33-targeting antigen recognition receptor disclosed herein.

[0284] In certain embodiments, the promoter is endogenous or exogenous. In certain embodiments, the exogenous promoter is selected from the elongation factor (EF)-1 promoter, the cytomegalovirus immediate early promoter (CMV) promoter, the simian virus 40 early promoter (SV40) promoter, the phosphoglycerate kinase (PGK) promoter, and the metallothionein promoter. In certain embodiments, the promoter is an inducible promoter. In certain embodiments, the inducible promoter is selected from the NFAT transcription response element (TRE) promoter, the CD69 promoter, the CD25 promoter, and the IL-2 promoter. The compositions and nucleic acid compositions can be administered to a subject and / or delivered to cells by methods known in the art or as described herein. Genetic modification of cells (e.g., T cells or NK cells) can be achieved by transducing a substantially homogenous cell composition with a recombinant DNA construct. In certain embodiments, a retroviral vector (e.g., a gamma-retroviral vector or a lentiviral vector) is used to introduce the DNA construct into the cells. For example, a polynucleotide encoding an antigen-recognizing receptor can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, the retroviral long terminal repeat, or a promoter specific for the target cell type of interest. Non-viral vectors can be used as well.

[0285] For the initial genetic modification of cells to contain the CD33-targeted antigen-recognizing receptor (e.g., CAR) disclosed herein, retroviral vectors can be used for transduction, but any other suitable viral vector or non-viral delivery system can be used. Antigen-recognizing receptors can be constructed in a single multicistronic expression cassette, in multiple expression cassettes in a single vector, or in multiple vectors. Examples of elements that create polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-κB IRES, RUNX1 IRES, p53 IRES, Hepatitis A IRES, Hepatitis C IRES, Pestivirus IRES, Aphthovirus IRES, Picornavirus IRES, Poliovirus IRES, and Encephalomyocarditis virus IRES), and cleavable linkers (e.g., 2A peptides, e.g., P2A, T2A, E2A, and F2A peptides). Also suitable are combinations of retroviral vectors with appropriate packaging lines, in which the capsid proteins will function to infect human cells. A variety of amphotropic virus-producing cell lines are known, including PA12 (Miller et al., (1985) Mol Cell Biol (1985); 5:431-437), PA317 (Miller., et al., Mol Cell Biol (1986); 6:2895-2902), and CRIP (Danos et al., Proc Natl Acad Sci USA (1988); 85:6460-6464). Non-amphotropic particles, such as particles pseudotyped with VSVG, RD114 or GALV envelopes and any others known in the art, are also suitable.

[0286] Possible methods of transduction also include direct co-culture of the cells with producer cells (Bregni et al., Blood (1992); 80:1418-1422), or culture with viral supernatant alone or concentrated vector stocks with or without appropriate growth factors and polycations (Xu et al., Exp Hemat (1994); 22:223-230, and Hughes et al. J Clin Invest (1992); 89:1817).

[0287] Other transducing viral vectors can be used to modify cells. In certain embodiments, the selected vector exhibits high infection efficiency and stable integration and expression (see, for example, Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. USA 94:10319, 1997). Other viral vectors that can be used include, for example, adenovirus, lentivirus, and adeno-associated viral vectors, vaccinia virus, bovine papilloma virus, or herpes viruses such as Epstein-Barr virus (see, e.g., Miller, Human Gene Thera (1990); 15-14; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques (1988); 6:608-614; Tolstoshev et al., Cur Opin Biotechnol (1990); 1:55-61; Sharp, The Lancet (1991); 337:1277-78; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-22, 1987; Anderson, Science (1984); 226:401-409; Moen, Blood Cells 17:407-16, 1991; Miller et al., Biotechnol (1989); 7:980-90; LeGal La Salle et al., Science (1993); 259:988-90; and Johnson, Chest (1995) 107:77S-83S.Retroviral vectors have been particularly well developed and have been used in clinical settings (Rosenberg et al., N Engl J Med (1990);323:370,1990; Anderson et al., U.S. Patent No. 5,399,346).

[0288] Non-viral approaches can also be used for genetic modification of cells. For example, nucleic acid molecules can be introduced into cells by administering nucleic acid in the presence of lipofection (Feigner et al., Proc Natl Acad Sci USA (1987); 84: 7413; Ono et al., Neurosci Lett (1990); 17: 259; Brigham et al., Am J Med Sci (1989); 298: 278; Staubinger et al., Methods in Enzymol (1983); 101: 512; Wu et al., J Biol Chem (1988); 263: 14621; Wu et al., J Biol Chem (1989); 264: 16985) or by microinjection under surgical conditions (Wolff et al., Science (1990); 247: 1465). Other non-viral means for gene transfer include in vitro transfection using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also potentially be useful for delivery of DNA into cells. Transplantation of normal genes into diseased tissues of subjects can also be achieved by transferring normal nucleic acids into ex vivo cultivable cell types (e.g., autologous or heterologous primary cells or their progeny), and then injecting the cells (or their progeny) into target tissues or systemically. Recombinant receptors can also be induced or obtained using transposases or targeted nucleases (e.g., zinc finger nucleases, meganucleases, or TALE nucleases, CRISPR). Transient expression can be obtained by RNA electroporation.

[0289] Any targeted genome editing method can also be used to deliver the antigen-recognizing receptor disclosed herein to cells or subjects.In certain embodiments, the CRISPR system is used to deliver the antigen-recognizing receptor disclosed herein.In certain embodiments, the zinc finger nuclease is used to deliver the antigen-recognizing receptor disclosed herein.In certain embodiments, the TALEN system is used to deliver the antigen-recognizing receptor disclosed herein.

[0290] The clustered regularly interspaced short palindromic repeats (CRISPR) system is a genome editing tool discovered in prokaryotic cells. When utilized for genome editing, this system includes Cas9 (a protein that can modify DNA using crRNA as its guide), CRISPR RNA (crRNA contains RNA used by Cas9 to guide it to the correct part of the host DNA, along with a region that binds to tracrRNA (generally in the form of a hairpin loop) that forms an active complex with Cas9), transactivating crRNA (tracrRNA binds to crRNA and forms an active complex with Cas9), and an optional part of DNA repair template (DNA that induces a cellular repair process that allows the insertion of a specific DNA sequence). CRISPR / Cas9 often uses a plasmid to transfect the target cell. The crRNA needs to be designed for each application, since it is the sequence that Cas9 uses to identify and directly bind to the target DNA in the cell. The repair template carrying the CAR expression cassette also needs to be designed for each application, since it must overlap with the sequences on either side of the cut and code for the insertion sequence. Multiple crRNAs and tracrRNAs can be packaged together to form a single guide RNA (sgRNA), which can be linked together with a Cas9 gene and made into a plasmid for transfection into cells.

[0291] Zinc finger nucleases (ZFNs) are artificial restriction enzymes generated by combining a zinc finger DNA binding domain with a DNA cleavage domain. The zinc finger domain can be engineered to target specific DNA sequences, allowing the zinc finger nuclease to target desired sequences in the genome. The DNA binding domain of an individual ZFN typically contains multiple individual zinc finger repeats, each capable of recognizing multiple base pairs. The most common method of generating new zinc finger domains is to combine smaller zinc finger "modules" of known specificity. The most common cleavage domain in ZFNs is the non-specific cleavage domain from the type II restriction endonuclease FokI. Using endogenous homologous recombination (HR) machinery and a homologous DNA template carrying the CAR expression cassette, ZFNs can be used to insert a CAR expression cassette into the genome. Once the targeting sequence is cleaved by the ZFN, the HR machinery searches for homology between the damaged chromosome and the homologous DNA template, and then copies the sequence of the template between the two broken ends of the chromosome, thereby integrating the homologous DNA template into the genome.

[0292] Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific sequences of DNA. TALEN systems work on roughly the same principle as ZFNs. They are generated by combining a transcription activator-like effector DNA binding domain with a DNA cleavage domain. Transcription activator-like effectors (TALEs) are composed of a 33-34 amino acid repeat motif with two variable positions that have strong recognition for specific nucleotides. By assembling an array of these TALEs, the TALE DNA binding domain can be engineered to bind to a desired DNA sequence, thereby directing the nuclease to cleave at a specific location in the genome. cDNA expression for use in methods of polynucleotide therapy can be derived from any suitable promoter (e.g., human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters) and regulated by any suitable mammalian regulatory element or intron (e.g., elongation factor 1a enhancer / promoter / intron structure). For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct expression of the nucleic acid. Enhancers used can include, but are not limited to, those characterized as tissue- or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.

[0293] 5.5.1 The method of delivery method for delivering the genome editing agent / system may vary as needed. In certain embodiments, the components of the selected genome editing method are delivered as DNA constructs in one or more plasmids. In certain embodiments, the components are delivered via a viral vector. Common delivery methods include, but are not limited to, electroporation, microinjection, gene guns, imperfection, hydrostatic pressure, continuous injection, sonication, magnetic coupling, adeno-associated viruses, envelope protein pseudotypes of viral vectors, replication-competent vector cis and trans-acting elements, herpes simplex viruses, and chemical vehicles (e.g., oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles, and cell-penetrating peptides).

[0294] In certain embodiments, the delivery method involves the use of a colloid. As used herein, the term "colloid" refers to a system in which there are two or more phases, where one phase (e.g., a dispersed phase) is distributed in the other phase (e.g., a continuous phase). Furthermore, at least one of the phases has a small dimension (approximately 10 -9 ~about 10 -6 Non-limiting examples of colloids encompassed by the presently disclosed subject matter include macromolecule complexes, nanocapsules, microparticles, beads, and lipid-based systems (e.g., micelles, liposomes, and lipid nanoparticles).

[0295] In certain embodiments, the delivery method comprises the use of liposomes. As used herein, the term "liposome" refers to a single or multi-layered spherical lipid bilayer structure produced from lipids dissolved in an organic solvent and then dispersed in an aqueous medium. Used experimentally and therapeutically to deliver active pharmaceutical ingredients (e.g., the nucleic acid compositions disclosed herein) to cells, liposomes fuse with the cell membrane, thus transferring the contents into the cytoplasm.

[0296] In certain embodiments, the delivery method involves the use of lipid nanoparticles. As used herein, the term "lipid nanoparticle" refers to a particle having at least one dimension on the order of a nanometer (e.g., about 1 nm to about 1,000 nm) and comprising at least one lipid. In certain embodiments, the lipid nanoparticle can comprise an active pharmaceutical ingredient (e.g., a nucleic acid composition disclosed herein) for delivery to a cell. The morphology of lipid nanoparticles can differ from liposomes. Liposomes are characterized by a lipid bilayer surrounding a hydrophilic core, whereas lipid nanoparticles have an electron-dense core in which cationic lipids and / or ionizable lipids are organized into inverted micelles around the active pharmaceutical ingredient (e.g., a nucleic acid composition disclosed herein). Further information on the morphology and properties of lipid nanoparticles and liposomes can be found in Wilczewska, et al., Pharmacological reports 64, no. 5 (2012): 1020-1037, Eygeris et al., Accounts of Chemical Research 55, no. 1 (2021): 2-12, Zhang et al., Chemical Reviews 121, no. 20 (2021): 12181-12277, and Fan et al., Journal of pharmaceutical and biomedical analysis 192 (2021): 113642.

[0297] In certain embodiments, the lipid nanoparticles have an average diameter of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm.

[0298] In certain embodiments, lipid nanoparticles can include cationic lipids or ionizable lipids. The term "cationic lipid" refers to lipids that include a head group with a permanent positive charge. Non-limiting examples of cationic lipids encompassed by the subject matter disclosed herein include 1,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), and ethylphosphatidylcholine (ePC).

[0299] As used herein, the term "ionizable lipid" refers to lipids that are protonated at low pH and neutral at physiological pH. The pH sensitivity of ionizable lipids is particularly beneficial for in vivo delivery (e.g., delivery of the nucleic acid composition disclosed herein) because neutral lipids interact less with the anionic membrane of blood cells, thus improving the biocompatibility of lipid nanoparticles. When trapped in endosomes, ionizable lipids are protonated, promoting membrane destabilization and allowing nanoparticles to escape endosomal.Non-limiting examples of ionizable lipids encompassed by the presently disclosed subject matter include tetrakis(8-methylnonyl) 3,3',3'',3'''-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate, decyl(2-(dioctylammonio)ethyl)phosphate, ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), bis(2-( dodecyldisulfanyl)ethyl) 3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosyl)azanediyl) dipropionate, 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl) bis(dodecan-2-ol), cKK-E12, 3,6-bis(4-(bis(2-hydroxydodecyl)amino)ethyl) (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, hexa(octan-3-yl)9,9',9'',9''',9'''',9'''''-(((benzene-1,3,5-tricarbonyl)iris(azanediyl))tris(propane-3,1-diyl))tris(azanetriyl))hexanonanoate , heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, and (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl)bis(azanetriyl))tetrakis(ethane-2,1-diyl)(9Z,9'Z,9''Z,9'''Z,12Z,12'Z,12''Z,12'''Z)-tetrakis(octadeca-9,12-dienoate).

[0300] Additionally, in certain embodiments, lipid nanoparticles can include other lipids. For example, but not limited to, lipid nanoparticles of the subject matter disclosed herein can include phospholipids, cholesterol, polyethylene glycol (PEG)-functionalized lipids (PEG-lipids). These lipids can improve certain properties of lipid nanoparticles (e.g., stability, biodistribution, etc.). For example, cholesterol enhances the stability of lipid nanoparticles by regulating their integrity and rigidity. Non-limiting examples of other lipids present in the lipid nanoparticles include cholesterol, DC-cholesterol, β-sitosterol, BHEM-cholesterol, ALC-0159, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (DPPG), dioleoyl ... and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearioyl-2-oleoyl-phosphatidiethanolamine (SOPE), and 1,2-dielideyl-sn-glycero-3-phosphoethanolamine (transDOPE).

[0301] In certain embodiments, the lipid nanoparticles can include a targeting moiety that binds to a ligand. The use of a targeting moiety allows for selective delivery of an active pharmaceutical ingredient (e.g., a nucleic acid composition disclosed herein) to a target cell (e.g., a T cell) that expresses the ligand. In certain embodiments, the targeting moiety can be an antibody or an antigen-binding fragment thereof that binds to a cell surface receptor. For example, but not limited to, the targeting domain is an antibody or an antigen-binding fragment thereof that binds to a receptor expressed on the surface of a T cell (e.g., CD3, CD4, CD8, CD16, CD40L, CD95, FasL, CTLA-4, OX40, GITR, LAG3, ICOS, and PD-1).

[0302] In certain embodiments, the delivery method is an in vivo delivery method. In certain embodiments, the delivery method is an ex vivo delivery method.

[0303] Polypeptides The subject matter disclosed herein provides methods for optimizing an amino acid or nucleotide sequence by generating changes in the sequence. Such changes may include specific mutations, deletions, insertions, or post-translational modifications. The subject matter disclosed herein further includes analogs of any naturally occurring polypeptide disclosed herein, including but not limited to CD33, CD8, CD28, 4-1BB, and CD3zeta. Analogs may differ from the naturally occurring polypeptides disclosed herein by differences in amino acid sequence, post-translational modifications, or both. Analogs may exhibit at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more homology or identity with all or a portion of the naturally occurring amino acid sequence of the subject matter disclosed herein. The length of sequence comparison is at least 5, 10, 15, or 20 amino acid residues, for example, at least 25, 50, or 75 amino acid residues, or more than 100 amino acid residues. Again, in an exemplary approach to determine the degree of identity, the BLAST program may be used, e.g. -3 ~e -100A probability score of 0.01 indicates closely related sequences. Modifications include in vivo and in vitro chemical derivatization of polypeptides, such as acetylation, carboxylation, phosphorylation, or glycosylation, which may occur during synthesis, processing, or after treatment with isolated modifying enzymes. Analogs may also differ from naturally occurring polypeptides by changes in the primary sequence. These include both natural and induced genetic variants (generated by random mutagenesis by exposure to radiation or ethane methyl sulfate, or site-directed mutagenesis, as described, for example, in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press, 1989, or Ausubel et al., supra). Also included are cyclized peptides, molecules, and analogs that contain residues other than L-amino acids, such as D-amino acids, or non-naturally occurring or synthetic amino acids, such as β or γ amino acids.

[0304] In addition to full-length polypeptides, the subject matter disclosed herein also provides fragments of any of the polypeptides disclosed herein. As used herein, the term "fragment" refers to at least 5, 10, 13, or 15 amino acids. In certain embodiments, a fragment comprises at least 20 contiguous amino acids, at least 30 contiguous amino acids, or at least 50 contiguous amino acids. In certain embodiments, a fragment comprises at least 60-80, 100, 200, 300, or more contiguous amino acids. Fragments may be generated by methods known to those of skill in the art or may result from normal protein processing (e.g., removal of amino acids from a nascent polypeptide that are not required for biological activity, or removal of amino acids by alternative mRNA splicing or alternative protein processing events).

[0305] 5.7. Formulation and Administration The subject matter disclosed herein provides a composition comprising the cells disclosed herein. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier. The composition comprising the cells disclosed herein can be conveniently provided as a sterile liquid preparation, for example, an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which may be buffered to a selected pH. Liquid preparations are usually easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, particularly by injection. Viscous compositions, on the other hand, can be formulated within an appropriate viscosity range to provide a longer contact period with a particular tissue. The liquid or viscous composition can include a carrier, which can be a solvent or dispersion medium, including, for example, water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0306] Sterile injectable solutions can be prepared by incorporating the genetically modified cells in the required amount of an appropriate solvent with various amounts of other ingredients as required. Such compositions may be in admixture with suitable carriers, diluents, or excipients, such as sterile water, saline, glucose, dextrose, and the like. The compositions can also be lyophilized. Depending on the route of administration and the desired preparation, the compositions can contain auxiliary substances, such as wetting agents, dispersing agents, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, dyes, and the like. Standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985 (hereby incorporated by reference), may be consulted to prepare suitable preparations without undue experimentation.

[0307] Various additives can be added to improve the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of microbial activity can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Prolonged absorption of injectable pharmaceutical forms can be brought about by the use of agents that delay absorption, such as aluminum monostearate and gelatin. However, according to the subject matter disclosed herein, any vehicle, diluent, or additive used must be compatible with the genetically modified cells.

[0308] The compositions can be isotonic, i.e., they can have the same osmotic pressure as blood and tears. The desired isotonicity of the composition can be achieved using sodium chloride or other pharma- ceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes. Sodium chloride can be used especially for buffers containing sodium ions.

[0309] The viscosity of the composition can be maintained at a selected level, if desired, by using a pharma- ceutically acceptable thickening agent. For example, methylcellulose is readily and economically available and easy to handle. Other suitable thickening agents include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, etc. The concentration of the thickening agent can depend on the selected drug. The important point is to use an amount that achieves the selected viscosity. Obviously, the selection of suitable carriers and other additives depends on the exact route of administration and the nature of the particular dosage form, for example, the liquid dosage form (for example, whether the composition is formulated into a solution, suspension, gel, or another liquid form such as a time-release form or liquid-filled form).

[0310] The composition comprising the cells disclosed herein can be provided systemically or directly to a subject to treat or ameliorate a disease or disorder. In certain embodiments, the cells disclosed herein or a composition comprising the same are directly injected into an organ of interest (e.g., an organ affected by a tumor). Alternatively, the cells disclosed herein or a composition comprising the same are provided indirectly to an organ of interest, for example, by administration into the circulatory system (e.g., tumor vasculature). Proliferative and differentiation agents can be provided before, during, or after administration of the cells or compositions to increase production of cells (e.g., T cells or NK cells) in vitro or in vivo.

[0311] The cells disclosed herein can be administered in any physiologically acceptable vehicle, usually intravascularly, although they can also be introduced into bone or other convenient sites where the cells can find a suitable site for regeneration and differentiation (e.g., the thymus).

[0312] The amount of cells administered may vary depending on the subject being treated. In certain embodiments, the amount of cells administered is about 10 4 ~about 10 10 , about 10 4 ~about 10 7 , about 10 5 ~about 10 7 , about 10 5 ~about 10 9 , or about 10 6 ~about 10 8 of the cells disclosed herein are administered to the subject. More effective cells may be administered in smaller numbers. Typically, at least about 1×10 5 The final dose is approximately l × l0 10 In certain embodiments, the number of 5 , 5×10 5 , 1×10 6 , about 5×10 6 , about 1×10 7 , about 5×10 7 , about 1×10 8 , or about 5 × 10 8The cells disclosed herein are administered to a subject. In certain embodiments, about 1×10 6 The cells disclosed herein are administered to the subject. The exact determination of what is considered an effective dose may be based on individual factors for each subject, including the size, age, sex, weight, and condition of the particular subject. Dosage amounts can be easily ascertained by those skilled in the art from this disclosure and knowledge in the art.

[0313] The cells disclosed herein may include purified cell populations. One of skill in the art can readily determine the percentage of cells disclosed herein in a population using a variety of well-known methods, such as fluorescence-activated cell sorting (FACS). Suitable ranges of purity in a population containing immunoresponsive cells disclosed herein are about 50% to about 55%, about 5% to about 60%, and about 65% to about 70%. In certain embodiments, the purity is about 70% to about 75%, about 75% to about 80%, or about 80% to about 85%. In certain embodiments, the purity is about 85% to about 90%, about 90% to about 95%, and about 95% to about 100%. The dosage can be readily adjusted by one of skill in the art (e.g., decreasing the purity may require increasing the dosage). The cells can be introduced by injection, catheter, etc.

[0314] One of skill in the art can readily determine the amount of cells and optional additives, vehicles, and / or carriers in the composition and administered in the method. Typically, any additives (in addition to the active cell(s) and / or drug(s)) are present in an amount of 0.001 to 50% (by weight) solution in phosphate buffered saline solution, and the active ingredient is present in the order of micrograms to milligrams, such as about 0.0001 to about 5% by weight, about 0.0001 to about 1% by weight, about 0.0001 to about 0.05% by weight, or about 0.001 to about 20% by weight, about 0.01 to about 10% by weight, or about 0.05 to about 5% by weight. For any composition administered to animals or humans, the following can be determined: toxicity, such as by determining the lethal dose (LD) and LD50 in a suitable animal model, e.g., rodents such as mice, the dosage of the composition(s) that induces a suitable response, the concentration of the components therein, and the timing of administration of the composition(s). Such determinations do not require undue experimentation from the knowledge of one of ordinary skill in the art, this disclosure, and documents cited herein. Also, the time of sequential administration can be ascertained without undue experimentation.

[0315] In certain embodiments, the composition is a pharmaceutical composition comprising the cells disclosed herein and a pharma- ceutically acceptable carrier.

[0316] The administration of the composition can be autologous or xenogeneic. For example, cells can be obtained from one subject and administered to the same subject or a different compatible subject. Peripheral blood-derived cells or their progeny (e.g., in vivo, ex vivo, or in vitro derived) can be administered. When administering the composition disclosed herein (e.g., the pharmaceutical composition comprising the cells disclosed herein), it can be formulated in a unit dosage injectable form (solution, suspension, emulsion).

[0317] The cells and compositions disclosed herein may be administered by any method known in the art, including, but not limited to, oral administration, intravenous administration, subcutaneous administration, intranodal administration, intratumoral administration, intrathecal administration, intravitreal administration, intrapleural administration, intraosseous administration, intraperitoneal administration, pleural administration, and direct administration to a subject.

[0318] Additionally or alternatively, the subject matter disclosed herein also provides compositions comprising lipid nanoparticles (e.g., as described in Section 5.5.1) comprising a nucleic acid or nucleic acid composition disclosed herein. The compositions comprising lipid nanoparticles disclosed herein can be conveniently provided as sterile and / or pyrogen-free. The compositions can be prepared to meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0319] The composition comprising the lipid nanoparticles disclosed herein can comprise pharmaceutically acceptable excipients.Non-limiting examples of pharmaceutically acceptable excipients include inert diluents, dispersants, granulating agents, surface active agents, emulsifying agents, disintegrating agents, binders, preservatives, buffers, lubricants, and / or oils.Furthermore, excipients such as cocoa butter and suppository wax, coloring agents, coating agents, sweeteners, flavoring agents, and / or perfumes can be present in the composition.

[0320] In certain embodiments, compositions comprising lipid nanoparticles disclosed herein can be prepared as injectable preparations. These injectable preparations can include pharma- ceutically acceptable vehicles and solvents, including, but not limited to, water, Ringer's solution, USP, isotonic sodium chloride solution, and / or oils (e.g., oleic acid). In certain embodiments, injectable preparations comprising lipid nanoparticles disclosed herein can include liquid suspensions of crystalline or amorphous materials with low water solubility. The use of these low water solubility materials allows for delayed absorption from subcutaneous or intramuscular injection. Alternatively or additionally, compositions comprising lipid nanoparticles disclosed herein can be prepared for rectal or vaginal, oral, topical and / or transdermal, intradermal, pulmonary, nasal, buccal, or ophthalmic administration. Additional information regarding various methods for formulating and preparing pharmaceutical compositions comprising the lipid nanoparticles disclosed herein can be found in Remington: The Science and Practice of Pharmacy, 22nd Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, Md., 2012.

[0321] In certain embodiments, compositions comprising lipid nanoparticles disclosed herein can be formulated for controlled release or sustained release. As used herein, the term "controlled release" refers to a pharmaceutical composition or compound release profile that conforms to a specific release pattern to produce a therapeutic outcome. As used herein, the term "sustained release" refers to a pharmaceutical composition or compound that conforms to a release rate over a specific period of time. The period of time can include, but is not limited to, hours, days, weeks, months, and years.

[0322] A composition comprising the lipid nanoparticles disclosed herein can be provided to a subject systemically or directly to induce and / or enhance an immune response to an antigen and / or treat and / or prevent a tumor, e.g., a tumor associated with CD33. In certain embodiments, the lipid nanoparticles disclosed herein or a composition comprising the same are provided to an immunoresponsive cell in vivo. In certain embodiments, the lipid nanoparticles disclosed herein or a composition comprising the same are directly injected into an organ of interest (e.g., an organ affected by a tumor). Alternatively, the lipid nanoparticles of the present disclosure or a composition comprising the same are provided indirectly to an organ of interest, e.g., by administration to the circulatory system (e.g., tumor vasculature). In certain embodiments, the lipid nanoparticles disclosed herein or a composition comprising the same are provided to an immunoresponsive cell ex vivo. Proliferative and differentiation agents can be provided before, during, or after administration of the lipid nanoparticles or compositions to increase production of cells (e.g., T cells or NK cells) ex vivo or in vivo.

[0323] The lipid nanoparticles disclosed herein can be administered in any physiologically acceptable vehicle, usually intravascularly, but they can also be introduced into bone or other convenient sites where cells can find a suitable site for regeneration and differentiation (e.g., the thymus).

[0324] The amount of cells administered may vary depending on the subject being treated. In certain embodiments, the amount of cells administered is about 0.001 mg / kg to about 10 mg / kg, about 0.005 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.05 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 10 mg / kg, about 2 mg / kg to about 10 mg / kg, about 5 mg / kg to about 10 mg / kg, about 0.0001 mg / kg to about 5 mg / kg, about 0.001 mg / kg~about 5mg / kg, about 0.005mg / kg~about 5mg / kg, about 0.01mg / kg~about 5mg / kg, about 0.05mg / kg~about 5mg / kg, about 0.1mg / kg~about 5mg / kg, about 1mg / kg~about 5mg / kg, about 2mg / kg~about 5mg / kg, about 0.0001mg / kg~about 2.5mg / kg, about 0.001mg / kg~about 2.5mg / kg, about 0.005mg / kg~about 2.5mg / kg, Approximately 0.01mg / kg to approximately 2.5mg / kg, approximately 0.05mg / kg to approximately 2.5mg / kg, approximately 0.1mg / kg to approximately 2.5mg / kg, approximately 1mg / kg to approximately 2.5mg / kg, approximately 2mg / kg to approximately 2.5mg / kg, about 0.0001mg / kg to about 1mg / kg, about 0.001mg / kg to about 1mg / kg, about 0.005mg / kg to about 1mg / kg, about 0.01mg / kg to about 1mg / kg, about 0.05mg / kg About 1 mg / kg, about 0.1 mg / kg to about 1 mg / kg, about 0.0001 mg / kg to about 0.25 mg / kg, about 0.001 mg / kg to about 0.25 mg / kg, about 0.005 mg / kg to about 0.25 mg / kg, about 0.01 mg / kg to about 0.25 mg / kg, about 0.05 mg / kg to about 0.25 mg / kg, or about 0.1 mg / kg to about 0.25 mg / kg of the lipid nanoparticles disclosed herein are administered to the subject. In certain embodiments, about 0.005 mg / kg to about 2.5 mg / kg, about 0.1 mg / kg to about 1 mg / kg, or about 0.05 mg / kg to about 1 mg / kg of the cells disclosed herein are administered to the subject. The exact determination of what is considered an effective dose may be based on individual factors for each subject, including the size, age, sex, weight, and condition of the particular subject. Dosages can be readily ascertained by one of ordinary skill in the art from this disclosure and knowledge in the art.The dosage can be readily adjusted by one of skill in the art (eg, decreasing purity may require increased dosage).

[0325] 5.8. Treatment method The subject matter disclosed herein provides methods for inducing and / or increasing an immune response in a subject in need thereof. The cells of the present disclosure and compositions comprising the same may be used in therapy or medicine. The subject matter disclosed herein provides various methods of using the cells (e.g., T cells) or compositions comprising the same. For example, the cells disclosed herein and compositions comprising the same can be used to reduce tumor burden in a subject. The cells disclosed herein can reduce the number of tumor cells, reduce tumor size, and / or eradicate tumors in a subject. The cells disclosed herein and compositions comprising the same can be used to treat and / or prevent tumors in a subject. The cells disclosed herein and compositions comprising the same can be used to treat or ameliorate a disease or disorder in a subject. In certain embodiments, the disease or disorder is associated with CD33. In certain embodiments, the disease or disorder is associated with overexpression of CD33. In certain embodiments, the disease or disorder is a tumor. The cells disclosed herein and compositions comprising the same can be used to extend the survival time of a subject suffering from a tumor.

[0326] Such methods include administering the cells disclosed herein or compositions (e.g., pharmaceutical compositions) comprising same to achieve a desired effect, e.g., alleviation of an existing condition or prevention of recurrence. For treatment, the dosage is an amount effective to produce the desired effect. An effective amount can be provided in one administration or a series of administrations. An effective amount can be provided in a bolus or by continuous perfusion.

[0327] The subject matter disclosed herein provides various methods of using the cells (e.g., T cells) or compositions comprising the same. For example, the subject matter disclosed herein provides a method of reducing tumor burden in a subject. In certain embodiments, the method of reducing tumor burden comprises administering to a subject the cells disclosed herein or compositions comprising the same. The cells disclosed herein can reduce the number of tumor cells, reduce tumor size, and / or eradicate tumors in a subject.

[0328] The subject matter disclosed herein also provides a method of increasing or extending the survival time of a subject bearing a tumor. In certain embodiments, the method of increasing or extending the survival time of a subject bearing a tumor or neoplasm comprises administering to the subject an immunoresponsive cell disclosed herein or a composition comprising the same. This method can reduce or eradicate the tumor burden in the subject.

[0329] Additionally, the presently disclosed subject matter provides a method for increasing an immune response in a subject, comprising administering to the subject a cell disclosed herein or a composition comprising same.

[0330] The subject matter disclosed herein further provides a method for treating and / or preventing a tumor in a subject, the method comprising administering to the subject a cell disclosed herein or a composition comprising the same.

[0331] In certain embodiments, the method comprises administering to a subject in need thereof a cell disclosed herein or a composition comprising the same. In certain embodiments, the cell is a T cell. The T cell is a CD4 + T cells or CD8 + In certain embodiments, the T cells are CD4 + T cells.

[0332] In certain embodiments, the tumor is a cancer. In certain embodiments, the cancer is a hematological cancer or a solid tissue cancer. Non-limiting examples of hematological cancers include acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), myeloproliferative neoplasms (MPN), and chronic myeloid neoplasms. In certain embodiments, the hematological cancer is AML.

[0333] The subject may have an advanced form of the disease, in which case the treatment objectives may include slowing or reversing disease progression and / or ameliorating side effects. The subject may have a history of a condition that has already been treated, in which case the treatment objectives typically include reducing or delaying the risk of recurrence.

[0334] As a result of surface expression of the CD33 target antigen-recognition receptor disclosed herein, the adoptively transferred cells (e.g., immunoresponsive cells, e.g., T cells or NK cells) are endowed with enhanced selective cytolytic activity at the tumor site. Furthermore, following localization to the tumor or viral infection and their proliferation, the cells transform the tumor or viral infection site into a highly conductive environment for a wide range of immune cells (tumor-infiltrating lymphocytes, NK cells, NKT cells, dendritic cells, and macrophages) that participate in the physiological anti-tumor or anti-viral response.

[0335] Further modifications can be introduced into engineered immune cells (e.g., T cells) expressing CD33-specific CARs to avoid or minimize the risk of immunological complications (known as "malignant T cell transformation"), such as graft-versus-host disease (GvHD). Modification of engineered immune cells can include engineering a suicide gene into CD33-specific CAR-expressing T cells. Suitable suicide genes include, but are not limited to, herpes simplex virus thymidine kinase (hsv-tk), inducible caspase 9 suicide gene (iCasp-9), and truncated human epidermal growth factor receptor (EGFRt) polypeptide. In certain embodiments, the suicide gene is an EGFRt polypeptide. The EGFRt polypeptide can enable T cell elimination by administering an anti-EGFR monoclonal antibody (e.g., cetuximab). The EGFRt can be covalently linked to the C-terminus of the intracellular domain of the CD33-specific CAR. The suicide gene can be included in a vector that includes a nucleic acid encoding a CD33-specific CAR as disclosed herein. The incorporation of a suicide gene into the CD33-specific CAR disclosed herein provides an additional level of safety with the ability to eliminate most CAR T cells within a very short period of time. Engineered immune cells (e.g., T cells) disclosed herein that have been incorporated with a suicide gene can be preemptively eliminated at a given time point after CAR T cell infusion or eradicated at the earliest sign of toxicity.

[0336] 5.9.Kit The subject matter disclosed herein provides kits for ameliorating a disease or disorder in a subject, inducing and / or enhancing an immune response in a subject, treating and / or preventing a tumor in a subject, reducing tumor burden in a subject, and / or increasing or extending survival time of a subject with a tumor in a subject. In certain embodiments, the kit comprises a cell disclosed herein or a composition comprising the same. In certain embodiments, the kit comprises a sterile container, which may be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container form known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding pharmaceuticals. In certain non-limiting embodiments, the kit comprises a nucleic acid molecule encoding a CD33-targeting antigen recognition receptor (e.g., CAR) disclosed herein.

[0337] Optionally, the cells and / or nucleic acid molecules are provided with instructions for administering the cells or nucleic acid molecules to a subject having or at risk of developing a disease or disorder (e.g., a tumor). The instructions generally include information regarding the use of the composition to treat and / or prevent a tumor. In certain embodiments, the instructions include at least one of the following: description of the therapeutic agent, dosing schedule and administration for the treatment or prevention of a tumor or neoplasm, precautions, warnings, indications, counterindications, overdosage information, adverse reactions, animal pharmacology, clinical studies, and / or references. The instructions may be printed directly on the container (if present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

[0338] 5.10. ILLUSTRATIVE EMBODIMENTS A1. In certain non-limiting embodiments, the subject matter disclosed herein provides an antigen-recognizing receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain specifically binds to CD33, the extracellular antigen-binding domain comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: (a) CDR1 comprising the amino acid sequence set forth in SEQ ID NO:2 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:3 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:4 or a conservative modification thereof; (b) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14 or a conservative modification thereof; (c) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24 or a conservative modification thereof; (d) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 or a conservative modification thereof; (e) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39 or a conservative modification thereof; (f) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 47 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 48 or a conservative modification thereof; (g) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 56 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 57 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 58 or a conservative modification thereof; (h) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 66 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 67 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 68 or a conservative modification thereof; (i) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 76 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 57 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 77 or a conservative modification thereof; or (j) comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 85 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 86 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 87 or a conservative modification thereof.

[0339] A2. An antigen-recognizing receptor according to A1, wherein the extracellular antigen-binding domain is a single-chain variable fragment (scFv).

[0340] A3. An antigen recognition receptor according to A2, wherein the extracellular antigen-binding domain is a human scFv.

[0341] A4. An antigen recognizing receptor according to A1, wherein the extracellular antigen binding domain is an optionally cross-linked Fab.

[0342] A5. An antigen recognition receptor according to A1, wherein the extracellular antigen-binding domain is F(ab)2.

[0343] A6. An antigen-recognizing receptor according to any one of A2 to A5, wherein one or more of scFv, Fab and F(ab)2 are contained in a fusion protein having a heterologous sequence to form an extracellular antigen-binding domain.

[0344] A7. The heavy chain variable region is (a) CDR1 comprising the amino acid sequence set forth in SEQ ID NO:2 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:3 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:4 or a conservative modification thereof; (b) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14 or a conservative modification thereof; or (c) An antigen recognition receptor according to any one of A1 to A6, comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 22 or a conservative modification thereof, CDR2 having the amino acid sequence set forth in SEQ ID NO: 23 or a conservative modification thereof, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 24 or a conservative modification thereof.

[0345] A8. The light chain variable region is (a) CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7 or a conservative modification thereof; (b) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16 or a conservative modification thereof, and CDR3 comprising SEQ ID NO: 17 or a conservative modification thereof; (c) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27 or a conservative modification thereof; (d) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 40 or a conservative modification thereof, CDR2 comprising SEQ ID NO: 41 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42 or a conservative modification thereof; (e) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 49 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 50 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 51 or a conservative modification thereof; (f) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 59 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 60 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 61 or a conservative modification thereof; (g) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 69 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 70 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 71 or a conservative modification thereof; (h) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 78 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 79 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 80 or a conservative modification thereof; or (i) An antigen recognition receptor according to any one of A1 to A7, comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 5 or a conservative modification thereof, CDR2 having the amino acid sequence set forth in SEQ ID NO: 41 or a conservative modification thereof, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 42 or a conservative modification thereof.

[0346] A9. The light chain variable region is (a) CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7 or a conservative modification thereof; (b) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16 or a conservative modification thereof, and a CDR3 comprising SEQ ID NO: 17 or a conservative modification thereof; or (c) An antigen recognition receptor according to any one of A1 to A8, comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 25 or a conservative modification thereof, CDR2 having the amino acid sequence set forth in SEQ ID NO: 26 or a conservative modification thereof, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 27 or a conservative modification thereof.

[0347] A10. (a) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:2, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:3, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:4, and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7; or (b) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17; or (c) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27; or (d) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27; or (e) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 40, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 41, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42; or (f) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 47, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 48, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 49, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 50, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 51; or (g) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 56, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 57, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 58, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 59, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 60, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 61; or (h) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 66, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 67, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 68, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 69, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 70, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 71; or (i) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 76, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 57, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 77, and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 79, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 80; or (j) An antigen recognition receptor according to any one of A1 to A9, wherein the heavy chain variable region comprises CDR1 having the amino acid sequence set forth in SEQ ID NO: 85, CDR2 having the amino acid sequence set forth in SEQ ID NO: 86, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 87, and the light chain variable region comprises CDR1 having the amino acid sequence set forth in SEQ ID NO: 5, CDR2 having the amino acid sequence set forth in SEQ ID NO: 41, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 42.

[0348] A11. (a) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:2, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:3, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:4, and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7; or (b) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14, and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17; or (c) An antigen recognition receptor described in any one of A1 to A10, wherein the heavy chain variable region comprises CDR1 having the amino acid sequence described in SEQ ID NO: 22, CDR2 having the amino acid sequence described in SEQ ID NO: 23, and CDR3 having the amino acid sequence described in SEQ ID NO: 24, and the light chain variable region comprises CDR1 having the amino acid sequence described in SEQ ID NO: 25, CDR2 having the amino acid sequence described in SEQ ID NO: 26, and CDR3 having the amino acid sequence described in SEQ ID NO: 27.

[0349] A12. An antigen recognition receptor according to any one of A1 to A11, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:52, SEQ ID NO:62, SEQ ID NO:72, SEQ ID NO:81, or SEQ ID NO:88.

[0350] A13. An antigen recognition receptor according to any one of A1 to A12, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 35, SEQ ID NO: 43, SEQ ID NO: 52, SEQ ID NO: 62, SEQ ID NO: 72, SEQ ID NO: 81, or SEQ ID NO: 88.

[0351] A14. An antigen recognition receptor according to any one of A1 to A10, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 18, or SEQ ID NO: 28.

[0352] A15. An antigen recognition receptor according to any one of A1 to A14, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:19, SEQ ID NO:29, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:63, SEQ ID NO:73, SEQ ID NO:82, SEQ ID NO:89, or SEQ ID NO:92.

[0353] A16. An antigen recognition receptor described in any one of A1 to A15, wherein the light chain variable region comprises the amino acid sequence described in SEQ ID NO: 9, SEQ ID NO: 19, SEQ ID NO: 29, SEQ ID NO: 44, SEQ ID NO: 53, SEQ ID NO: 63, SEQ ID NO: 73, SEQ ID NO: 82, SEQ ID NO: 89, or SEQ ID NO: 92.

[0354] A17. An antigen recognition receptor according to any one of A1 to A16, wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 9, SEQ ID NO: 19, or SEQ ID NO: 29.

[0355] A18. (a) the heavy chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to a selected amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:52, SEQ ID NO:62, SEQ ID NO:72, SEQ ID NO:81, or SEQ ID NO:88; (b) An antigen recognition receptor according to any one of A1 to A17, wherein the light chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:19, SEQ ID NO:29, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:63, SEQ ID NO:73, SEQ ID NO:82, SEQ ID NO:89, or SEQ ID NO:92.

[0356] A19. (a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:52, SEQ ID NO:62, SEQ ID NO:72, SEQ ID NO:81, or SEQ ID NO:88; (b) An antigen recognition receptor according to any one of A1 to A18, wherein the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 9, SEQ ID NO: 19, SEQ ID NO: 29, SEQ ID NO: 44, SEQ ID NO: 53, SEQ ID NO: 63, SEQ ID NO: 73, SEQ ID NO: 82, SEQ ID NO: 89, or SEQ ID NO: 92.

[0357] A20. (a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:18, or SEQ ID NO:28; (b) An antigen recognition receptor according to any one of A1 to A19, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 19, or SEQ ID NO: 29.

[0358] A21. (a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:9; or (b) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 19; or (c) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:28 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:29; or (d) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 35 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 29; or (e) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 43 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 44; or (f) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:52 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:53; or (g) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:62 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:63; or (h) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 72 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 73; or (i) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 81 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 82; or (j) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 88 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 89; or (k) An antigen recognition receptor according to any one of A1 to A20, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 93.

[0359] A22. (a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:9; or (b) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 19; or (c) An antigen recognition receptor according to any one of A1 to A21, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 28 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 29.

[0360] A23. An antigen recognition receptor according to any one of A1 to A22, wherein the extracellular antigen-binding domain comprises a linker between the heavy chain variable region and the light chain variable region.

[0361] A24. The antigen recognition receptor according to A23, wherein the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, or SEQ ID NO:100.

[0362] A25. An antigen recognition receptor according to any one of A1 to A24, wherein the signal peptide is covalently bound to the 5' end of the extracellular antigen-binding domain.

[0363] A26. An antigen recognition receptor according to any one of A1 to A25, wherein the transmembrane domain comprises a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, or a combination thereof.

[0364] A27. An antigen recognition receptor according to any one of A1 to A26, wherein the intracellular signaling domain comprises a CD3ζ polypeptide.

[0365] A28. An antigen recognition receptor according to any one of A1 to A27, wherein the intracellular signaling domain further comprises at least one costimulatory signaling region.

[0366] A29. The antigen recognition receptor of A28, wherein at least one costimulatory signaling region comprises a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, or a combination thereof.

[0367] A30. The antigen recognition receptor according to any one of A1 to A29, wherein the antigen recognition receptor is a chimeric antigen receptor (CAR) or a T cell-like fusion protein.

[0368] A31. The antigen recognition receptor according to any one of A1 to A30, wherein the antigen recognition receptor is a CAR.

[0369] A32. An antigen recognition receptor according to any one of A1 to A31, wherein the antigen recognition receptor is recombinantly expressed.

[0370] A33. The antigen recognition receptor according to any one of A1 to A32, wherein the antigen recognition receptor is expressed from a vector.

[0371] A34. The antigen recognition receptor according to A33, wherein the vector is a γ-retrovirus vector.

[0372] B1. In certain non-limiting embodiments, the presently disclosed subject matter provides a cell comprising an antigen recognition receptor according to any one of A1-A34.

[0373] B2. The cell of B1, wherein the cell is transduced with an antigen-recognizing receptor.

[0374] B3. The cell of B1 or B2, wherein the antigen-recognizing receptor is constitutively expressed on the surface of the cell.

[0375] B4. The cell according to any one of B1 to B3, wherein th...

Claims

1. An antigen-recognizing receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain specifically binds to CD33, and the extracellular antigen-binding domain comprises a heavy chain variable region and a light chain variable region; (a) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:2, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:3, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:4, and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7; or (b) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17; or (c) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27; or (d) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27; or (e) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 40, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 41, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42; or (f) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO:47, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:38, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:48, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO:49, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:50, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:51; or (g) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO:56, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:57, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:58, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO:59, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:60, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:61; or (h) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 66, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 67, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 68, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 69, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 70, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 71; or (i) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 76, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 57, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 77, and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 79, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 80; or (j) An antigen-recognizing receptor, wherein the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 85, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 86, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 87, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 41, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

42.

2. The antigen-recognizing receptor of claim 1 , wherein the extracellular antigen-binding domain is a single-chain variable fragment (scFv).

3. The antigen-recognizing receptor according to claim 2 , wherein the extracellular antigen-binding domain is a human scFv.

4. The antigen-recognizing receptor of claim 1, wherein the extracellular antigen-binding domain is an optionally cross-linked Fab.

5. The extracellular antigen-binding domain is F(ab) ２ The antigen-recognition receptor according to claim 1,

6. The scFv, Fab and F(ab) ２ The antigen-recognizing receptor according to any one of claims 2 to 5, wherein one or more of the following are included in a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain.

7. (a) the heavy chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the selected amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:52, SEQ ID NO:62, SEQ ID NO:72, SEQ ID NO:81, or SEQ ID NO:88; and / or (b) the light chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:19, SEQ ID NO:29, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:63, SEQ ID NO:73, SEQ ID NO:82, SEQ ID NO:89, or SEQ ID NO:

92.

8. (a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:52, SEQ ID NO:62, SEQ ID NO:72, SEQ ID NO:81, or SEQ ID NO:88; and / or (b) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 9, SEQ ID NO: 19, SEQ ID NO: 29, SEQ ID NO: 44, SEQ ID NO: 53, SEQ ID NO: 63, SEQ ID NO: 73, SEQ ID NO: 82, SEQ ID NO: 89, or SEQ ID NO:

92. The antigen-recognizing receptor of claim 7.

9. (a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 9; or (b) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 19; or (c) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:28 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:29; or (d) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 35 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 29; or (e) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 43 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 44; or (f) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 52 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 53; or (g) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 62 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 63; or (h) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 72 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 73; or (i) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 81 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 82; or (j) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 88 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 89; or (k) The antigen-recognizing receptor of claim 7, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

93. (a) the extracellular antigen-binding domain comprises a linker between the heavy chain variable region and the light chain variable region, optionally the linker comprising or consisting of an amino acid sequence set forth in SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, or SEQ ID NO:100; and / or (b) a signal peptide is covalently linked to the 5' end of the extracellular antigen-binding domain. (a) the transmembrane domain comprises a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, or a combination thereof; (b) the intracellular signaling domain comprises a CD3ζ polypeptide; and / or (c) the intracellular signaling domain further comprises at least one costimulatory signaling region, and optionally, the at least one costimulatory signaling region comprises a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, or a combination thereof.

12. The antigen recognition receptor according to any one of claims 1 to 5 and 7 to 10, wherein the antigen recognition receptor is a chimeric antigen receptor (CAR) or a T cell-like fusion protein.

13. A cell comprising the antigen-recognizing receptor of claim 1.

14. the cells are transduced with the antigen-recognizing receptor, and / or The cell of claim 13 , wherein the antigen-recognizing receptor is constitutively expressed on the surface of the cell.

15. 14. The cell of claim 13, wherein the cell is (a) an immunoresponsive cell, (b) a lymphoid or myeloid cell, (c) a cell selected from the group consisting of a T cell, a natural killer (NK) cell, and a stem cell from which a lymphoid cell can differentiate, (d) a T cell, (e) a cytotoxic T lymphocyte (CTL) or a regulatory T cell, and / or (f) a pluripotent stem cell, optionally wherein the pluripotent stem cell is an embryonic-like stem cell or an induced pluripotent stem cell.

16. A nucleic acid encoding the antigen-recognizing receptor of claim 13.

17. A vector comprising the nucleic acid of claim 16, optionally wherein the vector is a gamma-retroviral vector.

18. A host cell that expresses the nucleic acid of claim 16.

19. A lipid nanoparticle comprising the nucleic acid of claim 16.

20. A pharmaceutical composition comprising the cells described in claim 13 or the lipid nanoparticles described in claim 19.

21. 21. A kit for treating or ameliorating a disease or disorder in a subject, reducing tumor burden in a subject, treating and / or preventing a tumor in a subject, and / or increasing or extending survival time of a subject having a tumor, comprising the composition of claim 20, and optionally the kit further comprising written instructions for using the cells or composition to treat or ameliorate a disease or disorder in a subject, reducing tumor burden in a subject, treating and / or preventing a tumor in a subject, and / or increasing or extending survival time of a subject having a tumor.

22. A method for producing a CD33-targeting antigen-recognizing receptor according to any one of claims 1 to 5 and 7 to 10, comprising introducing into the cell a nucleic acid encoding the antigen-recognizing receptor.

23. 21. The composition of claim 20 for use in treating or ameliorating a disease or disorder in a subject.

24. 24. The composition for use of claim 23, wherein the disease or disorder is selected from the group consisting of neuroendocrine tumors of the lung, extrapulmonary neuroendocrine carcinoma, melanoma, neuroendocrine prostate cancer, breast cancer, neuroendocrine tumors of the gastrointestinal tract, pancreatic cancer, medullary thyroid carcinoma, small cell bladder cancer, small cell ovarian carcinoma, low-grade glioma, glioblastoma, and neuroblastoma.

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