D domain-containing polypeptides and methods of use thereof
Patent Information
- Application Number
- JP2024528494
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2021-11-15
- Filing Date
- 2022-11-14
- Publication Date
- 2025-10-30
Smart Images
Figure 00000179_0000 
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Figure 00000180_0000
Abstract
Description
[Technical Field]
[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 63 / 279,489, filed November 15, 2021, which is incorporated by reference in its entirety.
[0002] Reference to an electronically submitted sequence listing The contents of the electronically submitted Sequence Listing (Name: 6666_0301_Sequence_Listing.xml; Size: 163,868 bytes; Creation Date: November 6, 2022) submitted with this application are incorporated herein by reference in their entirety.
[0003] FIELD OF THE INVENTION The field of the invention relates generally to D domain-containing polypeptides, such as multifunctional chimeric antigen receptors and adaptors comprising D domains, and their use in methods of treatment, such as by directing an immune response to target cells. [Background technology]
[0004] background Adoptive transfer of genetically modified T cells is a rapidly evolving, innovative treatment for cancer. Chimeric antigen receptor (CAR)-engineered T cells are renewable agents capable of providing sustained functional immunity. Clinical efficacy with CD19 CAR T has been demonstrated in a variety of hematologic cancers, and promising early clinical data have been reported for other genetically modified CAR T in solid tumors. However, considerable challenges must be overcome before the substantial potential of CAR technology can be more fully realized. Summary of the Invention
[0005] overview In one aspect, provided herein is a protein (DDpp) comprising a D domain (DD) target-binding domain, wherein the DD specifically binds to a target of interest. In some embodiments, the target of interest is human CD123 (SEQ ID NO: 1) or a fragment thereof. In some embodiments, the DDpp is monovalent or multivalent. In some embodiments, the DDpp is monospecific or multispecific. In further embodiments, the DDpp is monospecific and multivalent. In other embodiments, the DDpp is multispecific and multivalent. Fusion proteins comprising one or more DDpps are also provided, as are methods of making and using the fusion proteins. Nucleic acids encoding the DDpps, as well as vectors and host cells containing the nucleic acids, are also provided. Non-limiting examples of such uses include, but are not limited to, target analysis and diagnostic and therapeutic applications. In some embodiments, the DDpp comprises a CD123-binding DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-33, 99, and 100. In some embodiments, the DDpp comprises a DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 13, 14, 31, 32, and 33. In some embodiments, the DDpp comprises a DD comprising the amino acid sequence of SEQ ID NO: 14.
[0006] In one aspect, the present disclosure provides a chimeric antigen receptor (CAR) comprising a target binding domain comprising the disclosed DD. In some embodiments, the DD binds to CD123 and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-33, 99, and 100. In some embodiments, the CD123-specific DD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 13, 14, 31, 32, and 33. In some embodiments, the CD123-specific DD comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the DD binds to AFP p26 (SEQ ID NO: 37) and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-93 and 94. In some embodiments, the CAR comprises a target binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the CAR further comprises a second target binding domain having the same or a different target as the DD target binding domain. In some embodiments, the CAR is expressed in an immune cell. In some embodiments, the CAR is expressed in an immune effector cell. In some embodiments, the immune cells are T cells (CAR-T cells) or natural killer (NK) cells (CAR-NK cells). In some embodiments, the cells are autologous immune cells. In some embodiments, the cells are autologous T cells (CAR-T cells) or autologous natural killer (NK) cells. In some embodiments, the cells are allogeneic immune cells. In some embodiments, the cells are allogeneic T cells (CAR-T cells) or allogeneic natural killer (NK) cells. In some embodiments, the CAR is expressed in immune cells derived from human embryonic stem cells (CAR-hESC) or induced pluripotent stem cells (CAR-iPSC cells).
[0007] Nucleic acids encoding the disclosed DDpp (e.g., DDpp fusion proteins, CARs, or adapters) are also provided. Additionally, vectors (e.g., plasmids, viral vectors, and non-viral vectors) containing nucleic acids encoding DDpp (e.g., DDpp fusion proteins, CARs, or adapters), as well as host cells containing the nucleic acids and vectors, are provided. In some embodiments, the vector comprises a nucleotide sequence that controls expression of a polypeptide encoded by the nucleic acid molecule. In further embodiments, the vector comprises an inducible promoter sequence. In additional embodiments, the vector comprises one or more additional standard components (e.g., promoters, packaging components, etc.) for expression of a protein encoded by the nucleic acid. In some embodiments, the vector is a lentiviral vector.
[0008] In one aspect, the present disclosure also provides a host cell comprising a nucleic acid molecule encoding a target-binding DDpp disclosed herein. In some embodiments, the host cell (e.g., a cell of a cell line) has been engineered to express a protein containing a DD disclosed herein (e.g., a DD having the amino acid sequence of SEQ ID NOs:8-33). In some embodiments, expression of a DDpp (e.g., a DDpp fusion protein or adapter) by the host cell allows for the production and isolation of the DDpp. In some embodiments, expression results in expression of the DDpp (e.g., a CAR) on the surface and / or integrally in the membrane of the host cell. In some embodiments, the host cell is a bacterial, yeast, fungal, or plant cell. In other embodiments, the host cell is a mammalian cell. In further embodiments, the mammalian cell is an immune cell. In one embodiment, the host cell is a human immune cell. In some embodiments, the human immune cell is a T cell. In other embodiments, the human immune cell is a natural killer (NK) cell. In some embodiments, the human immune cell displays a DDpp (e.g., a CAR) on its cell surface.
[0009] In one aspect, the present disclosure further provides a host cell expressing a protein comprising a DD disclosed herein. In some embodiments, the host cell expresses a chimeric antigen receptor (CAR) comprising a DD disclosed herein. In some embodiments, the CAR comprises a target binding domain comprising a DD comprising an amino acid sequence selected from SEQ ID NOs:8-33 and a transmembrane domain. In some embodiments, the CD123-specific DD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:8, 13, 14, 31, 32, and 33. In some embodiments, the CD123-specific DD comprises the amino acid sequence of SEQ ID NO:14. In some embodiments, the CAR further comprises an intracellular domain (including a signaling domain). In some embodiments, the CAR immune cell is a T cell. In some embodiments, the CAR immune effector cell is an NK cell. In some embodiments, the CAR immune effector cell is not a T cell or an NK cell. In some embodiments, the CAR immune cell is an autologous immune cell. In some embodiments, the CAR immune cell is an allogeneic immune cell. In some embodiments, the host cell is an immune effector cell that further comprises a second CAR polypeptide having a DD or other binding domain (e.g., an scFv) that specifically binds to a target expressed by the cancer cell that is the same or different from the first CAR expressed by the host immune cell (e.g., a different epitope of the same target or a second target of interest).
[0010] Pharmaceutical compositions containing the proteins comprising the DDs disclosed herein, the nucleic acids encoding the proteins disclosed herein, the vectors disclosed herein containing the nucleic acids, the viruses encoding the proteins, and the host cells containing the nucleic acids and / or vectors disclosed herein are also provided. Kits (e.g., therapeutic kits, diagnostic kits, research kits, etc.) containing one or more of the disclosed target-binding DDpps (e.g., DDpp fusion proteins, e.g., DD-Fc and DD-CAR, or adapters), nucleic acid molecules, vectors, and host cells are also provided.
[0011] The DDpp provided herein possess activities including, but not limited to, the ability to specifically bind to a target of interest (e.g., CD123) in vitro or in vivo, and the ability to serve as a reactive site for linking or association with one or more additional moieties (e.g., solid supports) and / or other modifications of proteins such as DDpp fusion proteins. The DDpp provided herein may also possess additional desirable properties and / or functionalities useful in manufacture, formulation, and biological, diagnostic, and therapeutic applications.
[0012] Methods of using DDpp in diagnostic and therapeutic applications are also provided. In one embodiment, the present disclosure provides a method of treating a disease or disorder, comprising administering a therapeutically effective amount of a DDpp (e.g., a DDpp fusion protein, a CAR, and / or an adapter) that specifically binds to a therapeutic target of interest (e.g., CD123) to a subject in need thereof. In some embodiments, the disease or disorder is cancer, a B-cell malignancy, a disease or disorder of the immune system, or an infection. Also provided is a method of treating a disease or disorder, comprising co-administering an additional therapeutic agent with the disclosed DDpp. In some embodiments, the disease or disorder is acute myeloid leukemia (AML), myelodysplastic syndrome (e.g., high-risk myelodysplastic syndrome), B-cell acute lymphoblastic leukemia, hairy cell leukemia, Hodgkin's lymphoma, or blastic plasmacytoid dendritic cell neoplasm (BPDCN). In some embodiments, the disease or disorder is acute myeloid leukemia.
[0013] In some aspects, the present disclosure provides: [1] A protein that specifically binds to CD123 and comprises a D domain target binding domain comprising the amino acid sequence of SEQ ID NO: 8-32 or 33. [2] The protein of [1], wherein the D domain comprises the amino acid sequence of SEQ ID NO: 8, 13, 14, or 31 to 33. [3] The protein of [1], wherein the D domain comprises the amino acid sequence of SEQ ID NO:14. [4] Any of the proteins [1] to [3], in which the D domain is fused to a heterologous polypeptide. [5] The protein of [4], wherein the heterologous polypeptide comprises a full-length antibody or an antibody fragment. [6] The protein of [4], wherein the D domain is fused to the amino terminus of a full-length antibody heavy chain; the amino terminus of a full-length antibody light chain; the carboxyl terminus of a full-length antibody heavy chain; or the carboxyl terminus of a full-length antibody light chain. [7] The protein of [4], wherein the heterologous polypeptide is an Fc domain. [8] The heterologous polypeptide is (a) transmembrane domain; (b) membrane-associated domain; (c) human serum albumin or a fragment thereof; (d) AFP or a fragment thereof; (e) AFP p26 or a fragment thereof; and (f) the extracellular domain of a receptor or a fragment thereof The protein of [4], comprising a member selected from the group consisting of: [9] The protein of [4], wherein the heterologous polypeptide comprises an extracellular domain, or a fragment of an extracellular domain, of a receptor selected from the group consisting of BCMA, CD123, CS1, and CD19.
[10] A protein according to any one of [2] to [9], further comprising a peptide linker.
[11] A protein labeled with any of [1] to
[10] .
[12] The protein of
[11] , wherein the label is selected from the group consisting of an enzyme label, a fluorescent label, a luminescent label, a bioluminescent label, and a biotin moiety.
[13] Any of the proteins described in [1] to
[12] conjugated to a therapeutic or cytotoxic substance.
[14] [1]-[3] Chimeric antigen receptors (CARs) containing target-binding domains containing proteins.
[15] CAR,
[14] which contains a target binding domain, a transmembrane domain, and an intracellular signaling domain.
[16] CARs whose transmembrane domains include those of CD8a, 41BB, or CD28,
[14] or
[15] .
[17] A CAR according to any one of
[14] to
[16] , wherein the intracellular signaling domain is selected from the group consisting of a domain of the human T cell receptor α chain, β chain, or ζ chain; a human 41BB domain; a human CD28 domain; and any combination thereof.
[18] A CAR according to any one of
[14] to
[17] , wherein the intracellular signaling domain comprises an intracellular domain of a costimulatory molecule selected from the group consisting of a ligand that specifically binds to CD27, CD28, 41BB, OX40, CD30, CD40, PD1, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, CD83, and any combination thereof.
[19] A CAR according to any one of
[14] to
[18] , further comprising a peptide linker.
[20] The CAR of
[14] , comprising the amino acid sequence of SEQ ID NO: 62-66 or 67.
[21] The CAR of
[14] , comprising the amino acid sequence of SEQ ID NO:67.
[22] Any of the proteins [1]-
[13] or any of the CARs
[14] -
[21] further comprising a second target binding domain having the same or a different target as the D domain target binding domain.
[23] An adapter comprising: (a) a D domain target binding domain that specifically binds to CD123 and comprises the amino acid sequence of SEQ ID NO: 8 to 32 or 33; and (b) an antigenic determinant (AD).
[24] The adapter of
[23] , wherein the D domain comprises the amino acid sequence of SEQ ID NO: 8, 13, 14, or 31-33.
[25] The adapter of
[23] , wherein the D domain comprises the amino acid sequence of SEQ ID NO:14.
[26] Any of the adaptors
[23] -
[25] , in which the AD contains AFP p26 or a fragment thereof.
[27] The adapter of
[26] , wherein AFP p26 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 37 to 43 and 44.
[28] The adapter of
[26] , wherein AFP p26 comprises the amino acid sequence of SEQ ID NO:37.
[29] The adaptor of
[26] , wherein AFP p26 comprises the amino acid sequence of SEQ ID NO:39.
[30] Any of the adapters
[23] to
[25] , wherein the AD comprises BCMA (SEQ ID NO: 34) or a fragment thereof.
[31] An adapter according to any one of
[23] to
[30] , further comprising a peptide linker.
[32] Any of the adaptors
[23] –
[31] containing a single D domain that specifically binds to CD123.
[33] The adapter of
[32] , comprising the amino acid sequence of SEQ ID NO: 50-54 or 55.
[34] The adapter of
[32] , comprising the amino acid sequence of SEQ ID NO:50.
[35] Any of the adaptors
[23] –
[31] that contain two D domains that specifically bind to CD123.
[36] The adapter of
[35] , comprising the amino acid sequence of SEQ ID NO: 56-60 or 61.
[37] The adapter of
[35] , comprising the amino acid sequence of SEQ ID NO:61.
[38] An isolated polynucleotide encoding any one of the proteins [1] to
[10] or
[22] or any one of the adaptors
[23] to
[37] .
[39]
[38] A vector containing the polynucleotide.
[40] The vector of
[39] , wherein the polynucleotide is operably linked to a nucleotide sequence that controls expression of the protein encoded by the polynucleotide.
[41] A host cell containing the polynucleotide of
[38] or the vector of
[39] or
[40] .
[42] A method for producing any of the proteins [1] to
[10] or
[22] or any of the adaptors
[23] to
[37] , comprising the step of culturing the host cell of
[41] under appropriate conditions so as to produce the protein or adaptor. An isolated polynucleotide encoding any of the CARs
[43]
[14] to
[22] .
[44]
[43] A vector containing the polynucleotide.
[45] The vector of
[44] , wherein the polynucleotide is operably linked to a nucleotide sequence that controls expression of the protein encoded by the polynucleotide.
[46] The vector of
[45] , which is a lentiviral vector. A host cell containing the polynucleotide of
[47]
[43] or any of the vectors of
[44] -
[46] .
[48]
[14] -
[22] Cells engineered to express any of the CARs.
[49] T cells or natural killer (NK) cells,
[47] or
[48] cells.
[50] A pharmaceutical composition comprising a protein according to any one of [1] to
[13] or
[22] and a pharmaceutically acceptable excipient.
[51]
[44] A pharmaceutical composition comprising the vector and a pharmaceutically acceptable excipient.
[52] The pharmaceutical composition of
[51] , wherein the vector is a lentiviral vector. A pharmaceutical composition comprising cells expressing any of the CARs of
[53]
[14] to
[22] and a pharmaceutically acceptable excipient.
[54] The pharmaceutical composition of
[53] , wherein the cells are T cells or natural killer (NK) cells. A kit containing one of the adapters
[55]
[23] to
[37] .
[56]
[44] kit containing the vector. A kit containing cells expressing any of the CARs
[57]
[14] –
[22] .
[58] The kit of
[57] , wherein the cells are T cells or natural killer (NK) cells.
[59] A method of delivering an immune response to one or more target cells, comprising contacting a composition comprising the target cells with a cell expressing a chimeric antigen receptor (CAR) comprising (i) a D domain that binds to CD123, (ii) a transmembrane domain, and (iii) an intracellular domain.
[60] A method of killing a target cell, comprising contacting a composition comprising the target cell with a cell expressing a chimeric antigen receptor (CAR) comprising (i) a D domain that binds to CD123, (ii) a transmembrane domain, and (iii) an intracellular domain.
[61] Methods such as
[59] or
[60] in which target cells express CD123.
[62] Any of the methods of
[59] to
[61] , wherein the D domain that binds to CD123 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 32 and 338 to 33, 99, and 100.
[63] Any of the methods of
[59] to
[61] , wherein the D domain that binds to CD123 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8, 13, 14, 31, 32, and 33.
[64] Any of the methods
[59] to
[61] , wherein the D domain that binds to CD123 comprises the amino acid sequence of SEQ ID NO:14.
[65] Any of the methods
[59] -
[64] , wherein the transmembrane domain comprises the transmembrane domain of CD8a, 41BB, or CD28.
[66] Any of the methods
[59] to
[65] , wherein the intracellular signaling domain is selected from the group consisting of a domain of the human T cell receptor α chain, β chain, or ζ chain; a human 41BB domain; a human CD28 domain; and any combination thereof.
[67] Any of the methods
[59] to
[66] , wherein the intracellular signaling domain comprises an intracellular domain of a costimulatory molecule selected from the group consisting of a ligand that specifically binds to CD27, CD28, 41BB, OX40, CD30, CD40, PD1, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, CD83, and any combination thereof.
[68] Any of the methods
[59] to
[61] , wherein the CAR containing a D domain that binds to CD123 comprises the amino acid sequence of SEQ ID NO: 62 to 66 or 67.
[69] Any of the methods
[59] to
[61] , wherein the CAR containing a D domain that binds to CD123 comprises the amino acid sequence of SEQ ID NO:67.
[70] Any of the methods
[59] -
[61] , in which the cells expressing CAR are immune cells.
[71] The method of
[70] , wherein the cells expressing the CAR are immune effector cells.
[72] The method of
[70] , wherein the CAR-expressing cells are T cells.
[73] The method of
[70] , wherein the CAR-expressing cells are natural killer (NK) cells.
[74] Any of the methods
[58] to
[73] , wherein the target cells are cancer cells.
[75] The method of
[74] , wherein the target cells are acute myeloid leukemia (AML) cells, myelodysplastic cells, B-cell acute lymphoblastic leukemia cells, hairy cell leukemia cells, Hodgkin's lymphoma cells, or blastic plasmacytoid dendritic cell neoplasm (BPDCN) cells, preferably acute myeloid leukemia (AML) cells.
[76] Any of the methods
[58] to
[73] , wherein the contacting step is carried out in a human patient.
[77] A method of delivering an immune response to a target cell, comprising contacting a composition comprising the target cell with an adaptor, A method wherein (a) the composition comprising the target cells further comprises cells expressing a CAR, wherein the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the adaptor comprises (i) a D domain that binds to CD123 and (ii) an AFP p26 AD.
[78] A method of delivering an immune response to a target cell, comprising contacting a composition comprising the target cell with an adaptor, A method wherein (a) the composition comprising the target cells further comprises cells expressing a CAR, wherein the CAR comprises (i) an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the adapter comprises (i) a D domain that binds to CD123 and (ii) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
[79] A method of delivering an immune response to a target cell, comprising contacting a composition comprising the target cell with an adaptor, A method wherein (a) the composition comprising the target cells further comprises cells expressing a CAR, wherein the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CD123, (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the adapter comprises (i) a D domain that binds to CD123 and (ii) an AD that is bound by the CAR.
[80] A method of delivering an immune response to a target cell, comprising contacting a composition comprising the target cell with an adaptor, A method, wherein (a) the composition comprising the target cells further comprises cells expressing a CAR, wherein the CAR comprises (i) an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the adapter comprises (i) a first antigenic determinant binding domain (ADBD) that binds to the target AD and (ii) a second antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
[81] A method of delivering an immune response to a target cell, comprising contacting a composition comprising the target cell with a cell expressing a CAR, A method, wherein (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the composition comprising the target cell further comprises an adaptor comprising (i) a D domain that binds to CD123 and (ii) an AFP p26 AD.
[82] A method of delivering an immune response to a target cell, comprising contacting a composition comprising the target cell with a cell expressing a CAR, A method, wherein: (a) the CAR comprises (i) an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the composition comprising the target cell further comprises an adaptor comprising (i) a D domain that binds to CD123 and (ii) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
[83] A method of delivering an immune response to a target cell, comprising contacting a composition comprising the target cell with a cell expressing a CAR, A method wherein (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CD123, (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the composition comprising the target cell further comprises an adaptor comprising (i) a D domain that binds to CD123 and (ii) an AD bound by the CAR.
[84] A method of delivering an immune response to a target cell, comprising contacting a composition comprising the target cell with a cell expressing a CAR, A method, wherein: (a) the CAR comprises (i) an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the composition comprising the target cell further comprises an adaptor comprising (i) a first antigenic determinant binding domain (ADBD) that binds to the target AD and (ii) a second antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
[85] A method of killing a target cell, comprising contacting a composition comprising the target cell with an adaptor, A method wherein (a) the composition comprising the target cells further comprises cells expressing a CAR, wherein the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the adaptor comprises (i) a D domain that binds to CD123 and (ii) an AFP p26 AD.
[86] A method of killing a target cell, comprising contacting a composition comprising the target cell with an adaptor, A method wherein (a) the composition comprising the target cells further comprises cells expressing a CAR, wherein the CAR comprises (i) an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the adapter comprises (i) a D domain that binds to CD123 and (ii) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
[87] A method of killing a target cell, comprising contacting a composition comprising the target cell with an adaptor, A method wherein (a) the composition comprising the target cells further comprises cells expressing a CAR, wherein the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CD123, (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the adapter comprises (i) a D domain that binds to CD123 and (ii) an AD that is bound by the CAR.
[88] A method of killing a target cell, comprising contacting a composition comprising the target cell with an adaptor, A method, wherein (a) the composition comprising the target cells further comprises cells expressing a CAR, wherein the CAR comprises (i) an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the adapter comprises (i) a first antigenic determinant binding domain (ADBD) that binds to the target AD and (ii) a second antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
[89] A method of killing a target cell, comprising contacting a composition comprising the target cell with a cell expressing a CAR, A method, wherein (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the composition comprising the target cell further comprises an adaptor comprising (i) a D domain that binds to CD123 and (ii) an AFP p26 AD.
[90] A method of killing a target cell, comprising contacting a composition comprising the target cell with a cell expressing a CAR, A method, wherein: (a) the CAR comprises (i) an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the composition comprising the target cell further comprises an adaptor comprising (i) a D domain that binds to CD123 and (ii) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
[91] A method of killing a target cell, comprising contacting a composition comprising the target cell with a cell expressing a CAR, A method wherein (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CD123, (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the composition comprising the target cell further comprises an adaptor comprising (i) a D domain that binds to CD123 and (ii) an AD bound by the CAR.
[92] A method of killing a target cell, comprising contacting a composition comprising the target cell with a cell expressing a CAR, A method, wherein: (a) the CAR comprises (i) an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the composition comprising the target cell further comprises an adaptor comprising (i) a first antigenic determinant binding domain (ADBD) that binds to the target AD and (ii) a second antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
[93] Target cells express CD123, either
[77] –
[79] ,
[81] –
[83] ,
[85] –
[87] , or
[89] –
[91] .
[94] Either
[80] ,
[84] ,
[88] , or
[92] methods in which the target cells express the target AD.
[95] Any of the methods of
[77] to
[94] , wherein the D domain that binds to CD123 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 32 and 338 to 33, 99, and 100.
[96] Any of the methods of
[77] to
[94] , wherein the D domain that binds to CD123 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 13, 14, 31, 32, and 33.
[97] Any of the methods
[77] to
[94] , wherein the D domain that binds to CD123 comprises the amino acid sequence of SEQ ID NO:14.
[98] Any of the methods
[77] -
[97] , wherein the transmembrane domain comprises the transmembrane domain of CD8a, 41BB, or CD28.
[99] Any of the methods
[77] to
[98] , wherein the intracellular signaling domain is selected from the group consisting of a domain of the human T cell receptor α chain, β chain, or ζ chain; a human 41BB domain; a human CD28 domain; and any combination thereof.
[0100] Any of the methods described in
[77] to
[99] , wherein the intracellular signaling domain comprises an intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, OX40, CD30, CD40, PD1, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds to CD83, and any combination thereof.
[0101] Any of the methods
[77] to
[94] , wherein the CAR containing a D domain that binds to CD123 comprises the amino acid sequence of SEQ ID NO: 62 to 66 or 67.
[0102] Any of the methods
[77] to
[94] , wherein the CAR containing a D domain that binds to CD123 comprises the amino acid sequence of SEQ ID NO:67.
[0103] Any of the methods
[77] to
[0102] , wherein the AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 37 to 43 and 44.
[0104] Any of the methods
[77] to
[0103] , wherein the AFP p26 AD comprises the amino acid sequence of SEQ ID NO:37.
[0105] Any of the methods
[77] to
[0103] , wherein the AFP p26 AD comprises the amino acid sequence of SEQ ID NO:39.
[0106] The method according to any one of
[77] to
[0105] , wherein the ADBD that binds to AFP p26 AD comprises an scFv that binds to AFP p26 AD.
[0107] Any of the methods described in
[77] to
[0105] , wherein the ADBD that binds to AFP p26 AD includes a D domain that binds to AFP p26 AD.
[0108] The method of
[0107] , wherein the D domain that binds to AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NO:74-93 and 94.
[0109] A method according to
[0107] , wherein the D domain that binds to AFP p26 AD comprises the amino acid sequence of SEQ ID NO:70-73 or 92-94.
[0110] A method according to
[0107] , wherein the D domain that binds to AFP p26 AD comprises the amino acid sequence of SEQ ID NO:73.
[0111] A method according to
[0107] , in which a CAR containing a D domain that binds to AFP p26 AD comprises the amino acid sequence of SEQ ID NO:68.
[0112] A method according to
[0107] , in which a CAR containing a D domain that binds to AFP p26 AD comprises the amino acid sequence of SEQ ID NO:69.
[0113] Any of the methods
[77] to
[0112] , wherein the CAR comprises an ADBD that binds to an AD other than CD123, and the ADBD binds to a tumor antigen.
[0114] The method of
[0113] , wherein the tumor antigen is selected from the group consisting of BCMA, CD19, CD22, CS1, HER2, TACI, BAFFR, and PDL1.
[0115] The method of
[0113] , wherein the tumor antigen is BCMA.
[0116] The method of
[0113] , wherein the tumor antigen is CD19.
[0117] The method of
[0113] , wherein the tumor antigen is selected from the group consisting of CD45, CD26, CD30, CD33, and CD38.
[0118] Any of the methods
[77] to
[0117] , wherein the adapter comprising (i) a D domain that binds to CD123 and (ii) an AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 50 to 54 or 55.
[0119] Any of the methods
[77] to
[0117] , wherein the adapter comprising (i) a D domain that binds to CD123 and (ii) an AFP p26 AD comprises the amino acid sequence of SEQ ID NO:50.
[0120] Any of the methods
[77] to
[0117] , wherein the adapter comprising (i) a D domain that binds to CD123 and (ii) an AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 56 to 60 or 61.
[0121] Any of the methods
[77] to
[0117] , wherein the adapter comprising (i) a D domain that binds to CD123 and (ii) an AFP p26 AD comprises the amino acid sequence of SEQ ID NO:61.
[0122] Any of the methods
[77] to
[0121] , wherein the target AD is selected from CD45, CD26, CD30, CD33, and CD38.
[0123] Any of the methods
[77] to
[0121] , wherein the target AD is selected from CD19, CD22, CD123, BCMA, CS1, HER2, TACI, BAFFR, and PDL1.
[0124] Any of the methods
[77] to
[0121] , wherein the target AD is BCMA.
[0125] Any of the methods
[77] to
[0121] , wherein the target AD is CD19.
[0126] Any of the methods
[77] to
[0125] , wherein the CAR-expressing cells are immune cells.
[0127] The method of
[0126] , wherein the cells expressing CAR are immune effector cells.
[0128] The method of
[0126] , wherein the CAR-expressing cells are T cells.
[0129] The method of
[0126] , wherein the CAR-expressing cells are natural killer (NK) cells.
[0130] Any of the methods
[77] to
[0129] , wherein the target cells are cancer cells.
[0131] The method of
[0130] , wherein the target cells are acute myeloid leukemia (AML) cells, myelodysplastic cells, B-cell acute lymphoblastic leukemia cells, hairy cell leukemia cells, Hodgkin's lymphoma cells, or blastic plasmacytoid dendritic cell neoplasm (BPDCN) cells.
[0132] The method of
[0130] , wherein the target cells are acute myeloid leukemia (AML) cells.
[0133] Any of the methods
[77] to
[0132] , wherein the contacting step is carried out in a human patient.
[0134] A method according to
[0133] , comprising administering CAR-expressing cells and an adapter to a human patient in any order.
[0135] A method according to
[0133] , comprising administering the adapter to a human patient, wherein the human patient has previously been administered cells expressing a CAR.
[0136] A method according to
[0133] , comprising administering the adapter to a human patient, wherein the human patient comprises cells expressing the CAR.
[0137] 1. A method of delivering an immune response to target cells in a patient, comprising administering to the patient cells expressing a chimeric antigen receptor (CAR) comprising: (i) a D domain that binds to CD123; (ii) a transmembrane domain; and (iii) an intracellular domain.
[0138] 1. A method of killing target cells in a patient in need thereof, comprising administering to the patient cells expressing a chimeric antigen receptor (CAR) comprising: (i) a D domain that binds to CD123, (ii) a transmembrane domain, and (iii) an intracellular domain.
[0139] A method according to
[0137] or
[0138] , wherein the target cells express CD123.
[0140] Any of the methods
[0137] to
[0139] , wherein the target cells are cancer cells.
[0141] The method of
[0140] , wherein the target cells are acute myeloid leukemia (AML) cells, myelodysplastic cells, B-cell acute lymphoblastic leukemia cells, hairy cell leukemia cells, Hodgkin's lymphoma cells, or blastic plasmacytoid dendritic cell neoplasm (BPDCN) cells, preferably acute myeloid leukemia (AML) cells.
[0142] 1. A method of lymphodepleting lymphocytes in a patient in need thereof, comprising administering to the patient cells expressing a chimeric antigen receptor (CAR) comprising: (i) a D domain that binds to CD123; (ii) a transmembrane domain; and (iii) an intracellular domain.
[0143] A method according to
[0142] , in which lymphocytes express CD123.
[0144] A method according to
[0142] or
[0143] , wherein the lymphocytes are B lymphocytes.
[0145] 1. A method of treating cancer, comprising administering to a patient in need thereof cells expressing a chimeric antigen receptor (CAR) comprising (i) a D domain that binds to CD123, (ii) a transmembrane domain, and (iii) an intracellular domain.
[0146] The method of
[0145] , wherein the cancer is a blood cancer.
[0147] The method of
[0146] , wherein the blood cancer is acute leukemia, chronic leukemia, polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's hypergammaglobulinemia, heavy chain disease, myelodysplastic syndrome (e.g., high-risk myelodysplastic syndrome), hairy cell leukemia, or myelodysplasia.
[0148] The method of
[0146] , wherein the blood cancer is acute myeloid leukemia (AML), myelodysplastic syndrome (e.g., high-risk myelodysplastic syndrome), B-cell acute lymphoblastic leukemia, hairy cell leukemia, Hodgkin's lymphoma, or blastic plasmacytoid dendritic cell neoplasm (BPDCN).
[0149] The method of
[0146] , wherein the blood cancer is AML.
[0150] The method of
[0146] , wherein the blood cancer is BPDCN.
[0151] 1. A method of treating an autoimmune disease or disorder, comprising administering to a patient in need thereof cells expressing a chimeric antigen receptor (CAR) comprising: (i) a D domain that binds to CD123, (ii) a transmembrane domain, and (iii) an intracellular domain.
[0152] The method of
[0146] , wherein the autoimmune disease or disorder is lupus erythematosus.
[0153] Any of the methods of
[0137] to
[0152] , wherein the D domain that binds to CD123 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:8 to 32 and 338 to 33, 99, and 100.
[0154] Any of the methods of
[0137] to
[0152] , wherein the D domain that binds to CD123 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8, 13, 14, 31, 32, and 33.
[0155] Any of the methods of
[0137] to
[0152] , wherein the D domain that binds to CD123 comprises the amino acid sequence of SEQ ID NO:14.
[0156] Any of the methods of
[0137] to
[0155] , wherein the transmembrane domain comprises the transmembrane domain of CD8a, 41BB, or CD28.
[0157] Any of the methods of
[0137] to
[0156] , wherein the intracellular signaling domain is selected from the group consisting of a domain of the human T cell receptor alpha chain, beta chain, or zeta chain; a human 41BB domain; a human CD28 domain; and any combination thereof.
[0158] Any of the methods of
[0137] to
[0157] , wherein the intracellular signaling domain comprises an intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, OX40, CD30, CD40, PD1, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds to CD83, and any combination thereof.
[0159] Any of the methods of
[0137] to
[0152] , wherein the CAR containing a D domain that binds to CD123 comprises the amino acid sequence of SEQ ID NO: 62 to 66 or 67.
[0160] Any of the methods of
[0137] to
[0152] , wherein the CAR containing a D domain that binds to CD123 comprises the amino acid sequence of SEQ ID NO:67.
[0161] Any of the methods of
[0137] to
[0160] , wherein the CAR-expressing cells are immune cells.
[0162] The method of
[0161] , wherein the cells expressing CAR are immune effector cells.
[0163] The method of
[0161] , wherein the CAR-expressing cells are T cells.
[0164] The method of
[0161] , wherein the CAR-expressing cells are natural killer (NK) cells.
[0165] Any of the methods of
[0137] to
[0164] , wherein the step of administering cells expressing CAR comprises administering a pharmaceutical composition comprising cells expressing CAR.
[0166] A method of delivering an immune response to target cells in a patient, comprising administering to the patient an adapter comprising (i) a D domain that binds to CD123 and (ii) AFP p26 AD.
[0167] A method of killing target cells in a patient in need thereof, comprising administering to the patient an adapter comprising (i) a D domain that binds to CD123 and (ii) AFP p26 AD.
[0168] A method according to
[0166] or
[0167] , wherein the target cells express CD123.
[0169] Any of the methods
[0166] to
[0168] , wherein the target cells are cancer cells.
[0170] The method of
[0168] , wherein the target cells are acute myeloid leukemia (AML) cells, myelodysplastic cells, B-cell acute lymphoblastic leukemia cells, hairy cell leukemia cells, Hodgkin's lymphoma cells, or blastic plasmacytoid dendritic cell neoplasm (BPDCN) cells, preferably acute myeloid leukemia (AML) cells.
[0171] A method of lymphodepleting lymphocytes in a patient in need thereof, comprising administering to the patient an adapter comprising (i) a D domain that binds to CD123 and (ii) AFP p26 AD.
[0172] A method according to
[0171] , in which lymphocytes express CD123.
[0173] A method according to
[0171] or
[0172] , wherein the lymphocytes are B lymphocytes.
[0174] A method of treating cancer, comprising administering to a patient in need thereof an adapter comprising (i) a D domain that binds to CD123 and (ii) AFP p26 AD.
[0175] The method of
[0174] , wherein the cancer is a blood cancer.
[0176] The method of
[0175] , wherein the blood cancer is acute leukemia, chronic leukemia, polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's hypergammaglobulinemia, heavy chain disease, myelodysplastic syndrome (e.g., high-risk myelodysplastic syndrome), hairy cell leukemia, or myelodysplasia.
[0177] The method of
[0175] , wherein the blood cancer is acute myeloid leukemia (AML), myelodysplastic syndrome (e.g., high-risk myelodysplastic syndrome), B-cell acute lymphoblastic leukemia, hairy cell leukemia, Hodgkin's lymphoma, or blastic plasmacytoid dendritic cell neoplasm (BPDCN).
[0178] The method of
[0175] , wherein the blood cancer is AML.
[0179] The method of
[0175] , wherein the blood cancer is BPDCN.
[0180] A method of treating an autoimmune disease or disorder, comprising administering to a patient in need thereof an adapter comprising (i) a D domain that binds to CD123 and (ii) AFP p26 AD.
[0181] The method of
[0180] , wherein the autoimmune disease or disorder is lupus erythematosus.
[0182] Any of the methods of
[0166] to
[0181] , wherein the patient has been administered cells expressing a CAR, and the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain.
[0183] Methods
[0166] to
[0181] , wherein the patient comprises cells expressing a CAR, and the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain.
[0184] Any of the methods of
[0166] to
[0181] further comprising the step of administering cells expressing a CAR, wherein the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain.
[0185] A method for delivering an immune response to target cells in a patient, comprising administering to the patient an adapter comprising (i) a D domain that binds to CD123 and (ii) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
[0186] 1. A method of killing target cells in a patient in need thereof, comprising administering to the patient an adapter comprising (i) a D domain that binds to CD123 and (ii) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
[0187] A method according to
[0185] or
[0186] , wherein the target cells express CD123.
[0188] Any of the methods
[0185] to
[0187] , wherein the target cells are cancer cells.
[0189] The method of
[0187] , wherein the target cells are acute myeloid leukemia (AML) cells, myelodysplastic cells, B-cell acute lymphoblastic leukemia cells, hairy cell leukemia cells, Hodgkin's lymphoma cells, or blastic plasmacytoid dendritic cell neoplasm (BPDCN) cells, preferably acute myeloid leukemia (AML) cells.
[0190] 1. A method for lymphodepleting lymphocytes in a patient in need thereof, comprising administering to the patient an adapter comprising (i) a D domain that binds to CD123 and (ii) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
[0191] A method according to
[0190] , in which lymphocytes express CD123.
[0192] A method according to
[0190] or
[0191] , wherein the lymphocytes are B lymphocytes.
[0193] 1. A method of treating cancer, comprising administering to a patient in need thereof an adapter comprising (i) a D domain that binds to CD123 and (ii) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
[0194] The method of
[0193] , in which the cancer is a blood cancer.
[0195] The method of
[0194] , wherein the blood cancer is acute leukemia, chronic leukemia, polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's hypergammaglobulinemia, heavy chain disease, myelodysplastic syndrome (e.g., high-risk myelodysplastic syndrome), hairy cell leukemia, or myelodysplasia.
[0196] The method of
[0194] , wherein the blood cancer is acute myeloid leukemia (AML), myelodysplastic syndrome (e.g., high-risk myelodysplastic syndrome), B-cell acute lymphoblastic leukemia, hairy cell leukemia, Hodgkin's lymphoma, or blastic plasmacytoid dendritic cell neoplasm (BPDCN).
[0197] The method of
[0194] , wherein the blood cancer is AML.
[0198] The method of
[0194] , wherein the blood cancer is BPDCN.
[0199] 1. A method of treating an autoimmune disease or disorder, comprising administering to a patient in need thereof an adapter comprising (i) a D domain that binds to CD123 and (ii) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
[0200] The method of
[0199] , wherein the autoimmune disease or disorder is lupus erythematosus.
[0201] Any of the methods of
[0185] to
[0200] , wherein the patient has been administered cells expressing a CAR, and (a) the CAR comprises (i) an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain.
[0202] Any of the methods of
[0185] to
[0200] , wherein the patient comprises cells expressing a CAR, and (a) the CAR comprises (i) an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain.
[0203] Any of the methods of
[0185] to
[0200] further comprising a step of administering cells expressing a CAR, wherein (a) the CAR comprises (i) an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain.
[0204] 1. A method of delivering an immune response to target cells in a patient, comprising administering to the patient an adapter comprising (i) a D domain that binds to CD123 and (ii) an AD that is bound by a CAR, A method in which the patient has been administered cells expressing a CAR, and (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CD123, (ii) a transmembrane domain, and (iii) an intracellular domain.
[0205] 1. A method of killing target cells in a patient in need thereof, comprising administering to the patient an adapter comprising (i) a D domain that binds to CD123 and (ii) an AD that is bound by a CAR, A method in which the patient has been administered cells expressing a CAR, wherein (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CD123, (ii) a transmembrane domain, and (iii) an intracellular domain; and the patient has been administered cells expressing a CAR, wherein (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CD123, (ii) a transmembrane domain, and (iii) an intracellular domain.
[0206] A method according to
[0204] or
[0205] , wherein the target cells express CD123.
[0207] Any of the methods
[0204] to
[0206] , wherein the target cells are cancer cells.
[0208] The method of
[0206] , wherein the target cells are acute myeloid leukemia (AML) cells, myelodysplastic cells, B-cell acute lymphoblastic leukemia cells, hairy cell leukemia cells, Hodgkin's lymphoma cells, or blastic plasmacytoid dendritic cell neoplasm (BPDCN) cells, preferably acute myeloid leukemia (AML) cells.
[0209] 1. A method of lymphodepleting lymphocytes in a patient in need thereof, comprising administering to the patient an adaptor comprising (i) a D domain that binds to CD123 and (ii) an AD that is bound by a CAR, A method in which the patient has been administered cells expressing a CAR, and (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CD123, (ii) a transmembrane domain, and (iii) an intracellular domain.
[0210] A method according to
[0209] , in which lymphocytes express CD123.
[0211] A method according to
[0209] or
[0210] , wherein the lymphocytes are B lymphocytes.
[0212] 1. A method of treating cancer comprising administering to a patient in need thereof an adapter comprising (i) a D domain that binds to CD123 and (ii) an AD that is bound by a CAR, A method in which the patient has been administered cells expressing a CAR, and (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CD123, (ii) a transmembrane domain, and (iii) an intracellular domain.
[0213] Method
[0212] , where the cancer is blood cancer.
[0214] The method of
[0213] , wherein the blood cancer is acute leukemia, chronic leukemia, polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's hypergammaglobulinemia, heavy chain disease, myelodysplastic syndrome (e.g., high-risk myelodysplastic syndrome), hairy cell leukemia, or myelodysplasia.
[0215] The method of
[0213] , wherein the blood cancer is acute myeloid leukemia (AML), myelodysplastic syndrome (e.g., high-risk myelodysplastic syndrome), B-cell acute lymphoblastic leukemia, hairy cell leukemia, Hodgkin's lymphoma, or blastic plasmacytoid dendritic cell neoplasm (BPDCN).
[0216] The method of
[0213] , wherein the blood cancer is AML.
[0217] The method of
[0213] , wherein the blood cancer is BPDCN.
[0218] 1. A method of treating an autoimmune disease or disorder, comprising administering to a patient in need thereof an adapter comprising (i) a D domain that binds to CD123 and (ii) an AD that is bound by a CAR, A method in which the patient has been administered cells expressing a CAR, and (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CD123, (ii) a transmembrane domain, and (iii) an intracellular domain.
[0219] The method of
[0218] , wherein the autoimmune disease or disorder is lupus erythematosus.
[0220] 1. A method of delivering an immune response to target cells in a patient, comprising administering to the patient an adapter comprising (i) a D domain that binds to CD123 and (ii) an AD that is bound by a CAR, A method wherein the patient comprises cells expressing a CAR, and (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CD123, (ii) a transmembrane domain, and (iii) an intracellular domain.
[0221] 1. A method of killing target cells in a patient in need thereof, comprising administering to the patient an adapter comprising (i) a D domain that binds to CD123 and (ii) an AD that is bound by a CAR, A method wherein the patient comprises cells expressing a CAR, and (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CD123, (ii) a transmembrane domain, and (iii) an intracellular domain.
[0222] Methods
[0220] or
[0221] in which the target cells express CD123. Can any of methods
[0220] to
[0222] in which the target cells are endothelial cells be included?
[0223] Any of the methods of
[0220] to
[0222] , wherein the target cells are cancer cells or endothelial cells.
[0224] The method of
[0222] , wherein the target cells are acute myeloid leukemia (AML) cells, myelodysplastic cells, B-cell acute lymphoblastic leukemia cells, hairy cell leukemia cells, Hodgkin's lymphoma cells, or blastic plasmacytoid dendritic cell neoplasm (BPDCN) cells, preferably acute myeloid leukemia (AML) cells.
[0225] 1. A method of lymphodepleting lymphocytes in a patient in need thereof, comprising administering to the patient an adaptor comprising (i) a D domain that binds to CD123 and (ii) an AD that is bound by a CAR, A method wherein the patient comprises cells expressing a CAR, and (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CD123, (ii) a transmembrane domain, and (iii) an intracellular domain.
[0226] A method according to
[0225] , in which lymphocytes express CD123.
[0227] A method according to
[0225] or
[0226] , wherein the lymphocytes are B lymphocytes.
[0228] 1. A method of treating cancer comprising administering to a patient in need thereof an adapter comprising (i) a D domain that binds to CD123 and (ii) an AD that is bound by a CAR, A method wherein the patient comprises cells expressing a CAR, and (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CD123, (ii) a transmembrane domain, and (iii) an intracellular domain.
[0229] Method
[0228] , in which the cancer is blood cancer.
[0230] The method of
[0229] , wherein the blood cancer is acute leukemia, chronic leukemia, polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's hypergammaglobulinemia, heavy chain disease, myelodysplastic syndrome (e.g., high-risk myelodysplastic syndrome), hairy cell leukemia, or myelodysplasia.
[0231] The method of
[0229] , wherein the blood cancer is acute myeloid leukemia (AML), myelodysplastic syndrome (e.g., high-risk myelodysplastic syndrome), B-cell acute lymphoblastic leukemia, hairy cell leukemia, Hodgkin's lymphoma, or blastic plasmacytoid dendritic cell neoplasm (BPDCN).
[0232] The method of
[0229] , wherein the blood cancer is AML.
[0233] The method of
[0229] , wherein the blood cancer is BPDCN.
[0234] 1. A method of treating an autoimmune disease or disorder, comprising administering to a patient in need thereof an adapter comprising (i) a D domain that binds to CD123 and (ii) an AD that is bound by a CAR, A method wherein the patient comprises cells expressing a CAR, and (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CD123, (ii) a transmembrane domain, and (iii) an intracellular domain.
[0235] The method of
[0234] , wherein the autoimmune disease or disorder is lupus erythematosus.
[0236] 1. A method of delivering an immune response to target cells in a patient, comprising administering to the patient an adapter comprising: (i) a first antigenic determinant binding domain (ADBD) that binds to a target AD; and (ii) a second antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD).
[0237] 1. A method of killing target cells in a patient in need thereof, comprising administering to the patient an adapter comprising: (i) a first antigenic determinant binding domain (ADBD) that binds to a target AD; and (ii) a second antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD).
[0238] A method according to
[0236] or
[0237] , wherein the target cells express the target AD.
[0239] Any of the methods
[0236] to
[0238] , wherein the target cells are cancer cells.
[0240] 1. A method of lymphodepleting lymphocytes in a patient in need thereof, comprising administering to the patient an adapter comprising: (i) a first antigenic determinant binding domain (ADBD) that binds to a target AD; and (ii) a second antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD).
[0241] A method in which lymphocytes express a target AD.
[0242] A method according to
[0240] or
[0241] , wherein the lymphocytes are B lymphocytes or T lymphocytes.
[0243] 1. A method of treating cancer, comprising administering to a patient in need thereof an adapter comprising (i) a first antigenic determinant binding domain (ADBD) that binds to a target AD and (ii) a second antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD).
[0244] Method
[0243] , in which the cancer is a blood cancer.
[0245] The method of
[0244] , wherein the blood cancer is acute leukemia, chronic leukemia, polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's hypergammaglobulinemia, heavy chain disease, myelodysplastic syndrome (e.g., high-risk myelodysplastic syndrome), hairy cell leukemia, or myelodysplasia.
[0246] The method of
[0244] , wherein the blood cancer is acute myeloid leukemia (AML), myelodysplastic syndrome (e.g., high-risk myelodysplastic syndrome), B-cell acute lymphoblastic leukemia, hairy cell leukemia, Hodgkin's lymphoma, or blastic plasmacytoid dendritic cell neoplasm (BPDCN).
[0247] The method of
[0244] , wherein the blood cancer is AML.
[0248] The method of
[0244] , wherein the blood cancer is BPDCN.
[0249] 1. A method of treating an autoimmune disease or disorder, comprising administering to a patient in need thereof an adapter comprising: (i) a first antigenic determinant binding domain (ADBD) that binds to a target AD; and (ii) a second antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD).
[0250] The method of
[0249] , wherein the autoimmune disease or disorder is rheumatoid arthritis.
[0251] Any of the methods of
[0236] to
[0250] , wherein the patient has been administered cells expressing a CAR, and the CAR comprises (i) an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain.
[0252] Any of the methods of
[0236] to
[0250] , wherein the patient comprises cells expressing a CAR, and the CAR comprises (i) an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain.
[0253] A method according to
[0236] to
[0250] , further comprising the step of administering cells expressing a CAR, wherein the CAR comprises (i) an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain.
[0254] Any of the methods of
[0166] to
[0253] , wherein the D domain that binds to CD123 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:8-32 and 338-33, 99, and 100.
[0255] Any of the methods of
[0166] to
[0253] , wherein the D domain that binds to CD123 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8, 13, 14, 31, 32, and 33.
[0256] Any of the methods of
[0166] to
[0253] , wherein the D domain that binds to CD123 comprises the amino acid sequence of SEQ ID NO:14.
[0257] Any of the methods of
[0166] to
[0256] , wherein the transmembrane domain comprises the transmembrane domain of CD8a, 41BB, or CD28.
[0258] Any of the methods of
[0166] to
[0257] , wherein the intracellular signaling domain is selected from the group consisting of a domain of the human T cell receptor alpha chain, beta chain, or zeta chain; a human 41BB domain; a human CD28 domain; and any combination thereof.
[0259] Any of the methods of
[0166] to
[0258] , wherein the intracellular signaling domain comprises an intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, OX40, CD30, CD40, PD1, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds to CD83, and any combination thereof.
[0260] Any of the methods of
[0166] to
[0259] , wherein the AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NO:37 to 43 and 44.
[0261] Any of the methods of
[0166] to
[0260] , wherein the AFP p26 AD comprises the amino acid sequence of SEQ ID NO:37.
[0262] Any of the methods of
[0166] to
[0260] , wherein the AFP p26 AD comprises the amino acid sequence of SEQ ID NO:39.
[0263] Any of the methods of
[0166] to
[0262] , wherein the ADBD that binds to AFP p26 AD comprises an scFv that binds to AFP p26 AD.
[0264] Any of the methods of
[0166] to
[0262] , wherein the ADBD that binds to AFP p26 AD includes a D domain that binds to AFP p26 AD.
[0265] The method of
[0264] , wherein the D domain that binds to AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NO:74 to 93 and 94.
[0266] A method according to
[0264] , wherein the D domain that binds to AFP p26 AD comprises the amino acid sequence of SEQ ID NO:70-73 or 92-94.
[0267] A method according to
[0264] , wherein the D domain that binds to AFP p26 AD comprises the amino acid sequence of SEQ ID NO:73.
[0268] A method according to
[0264] , in which a CAR containing a D domain that binds to AFP p26 AD comprises the amino acid sequence of SEQ ID NO:68.
[0269] A method according to
[0264] , in which a CAR containing a D domain that binds to AFP p26 AD comprises the amino acid sequence of SEQ ID NO:69.
[0270] Any of the methods of
[0166] to
[0269] , wherein the CAR comprises an ADBD that binds to an AD other than CD123, and the ADBD binds to a tumor antigen.
[0271] The method of
[0270] , wherein the tumor antigen is selected from the group consisting of BCMA, CD19, CD22, CS1, HER2, TACI, BAFFR, and PDL1.
[0272] The method of
[0270] , wherein the tumor antigen is BCMA.
[0273] The method of
[0270] , wherein the tumor antigen is CD19.
[0274] The method of
[0270] , wherein the tumor antigen is selected from the group consisting of CD45, CD26, CD30, CD33, and CD38.
[0275] Any of the methods of
[0166] to
[0274] , wherein the adapter comprising (i) a D domain that binds to CD123 and (ii) AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 50 to 54 or 55.
[0276] Any of the methods of
[0166] to
[0274] , wherein the adapter comprising (i) a D domain that binds to CD123 and (ii) an AFP p26 AD comprises the amino acid sequence of SEQ ID NO:50.
[0277] Any of the methods of
[0166] to
[0274] , wherein the adapter comprising (i) a D domain that binds to CD123 and (ii) an AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 56 to 60 or 61.
[0278] Any of the methods of
[0166] to
[0274] , wherein the adapter comprising (i) a D domain that binds to CD123 and (ii) an AFP p26 AD comprises the amino acid sequence of SEQ ID NO:61.
[0279] Any of the methods of
[0166] to
[0278] , wherein the target AD is selected from CD45, CD26, CD30, CD33, and CD38.
[0280] Any of the methods of
[0166] to
[0278] , wherein the target AD is selected from CD19, CD22, CD123, BCMA, CS1, HER2, TACI, BAFFR, and PDL1.
[0281] Any of the methods
[0166] to
[0278] , wherein the target AD is BCMA.
[0282] Any of the methods
[0166] to
[0278] , wherein the target AD is CD19.
[0283] Any of the methods
[0166] to
[0278] , wherein the target AD is CD45.
[0284] Any of the methods
[0166] to
[0278] , wherein the CAR-expressing cells are immune cells.
[0285] The method of
[0284] , wherein the cells expressing CAR are immune effector cells.
[0286] The method of
[0284] , wherein the CAR-expressing cells are T cells.
[0287] The method of
[0284] , wherein the CAR-expressing cells are natural killer (NK) cells.
[0288] Any of the methods
[0166] to
[0287] , wherein the target cells are cancer cells.
[0289] The method of
[0288] , wherein the target cells are acute myeloid leukemia (AML) cells, myelodysplastic cells, B-cell acute lymphoblastic leukemia cells, hairy cell leukemia cells, Hodgkin's lymphoma cells, or blastic plasmacytoid dendritic cell neoplasm (BPDCN) cells.
[0290] The method of
[0288] , wherein the target cells are acute myeloid leukemia (AML) cells.
[0291] Any of the methods of
[0166] to
[0290] , wherein the cells expressing the CAR and the adapter are administered separately in any order.
[0292] Any of the methods of
[0166] to
[0291] , wherein the step of administering the adaptor comprises administering a pharmaceutical composition containing the adaptor.
[0293] Any of the methods of
[0166] to
[0291] , wherein administering the adaptor and cells expressing the CAR comprises administering a pharmaceutical composition comprising the adaptor, and a pharmaceutical composition comprising cells expressing the CAR. [Brief explanation of the drawings]
[0014] [Figure 1] Screening of 8W9C mutants as MBP fusions. Various clones of 8W9C mutants (mutant 8W9C) were screened by ELISA for binding to CD123 (CD123 binding) and expression (MBP binding). One or more replicates of wild-type control (8W9C), high-affinity control (5B7L), low-affinity control (5S5W), and non-binding control (a3D) were assayed for comparison. [Figure 2]CD123 binding of 8W9C mutants as MBP fusions. 8W9C mutants (6Z5E and 4H2H) were assayed for binding to CD123 (CD123 binding) by ELISA. Wild-type control (8W9C), high-affinity control (5B7L), and low-affinity control (5S5W) were assayed for comparison. [Figure 3] CD123 binding of 8W9C mutants as MBP fusions. The 8W9C mutant (1J1K) was assayed for binding to CD123 (CD123 binding) by ELISA. Wild-type control (8W9C), high-affinity control (5B7L), and low-affinity control (5S5W) were assayed for comparison. [Figure 4] Cellular binding of 6T1D mutants as adaptors. Various variant 6T1D mutants were assayed at various concentrations for binding to CD123-expressing OCI-AML2 cells by flow cytometry. Binding was measured by mean fluorescence intensity (MFI). [Figure 5] NFAT activation of CAR T cells by sparX protein. JNL10 / 8G8V-CAR is assayed for activation in the presence of CD123-expressing MOLM13 target cells and various concentrations of CD123-specific adaptor proteins. Samples include affinity-modified variants of 6T1D (e.g., 3F4N and 4R8U). Parent D domain control (6T1D) and non-binding control (a3D) are assayed for comparison. [Figure 6] NFAT activation of CAR T cells cultured with CD123-specific adaptor proteins. JNL10 / 8G8V-CAR is assayed for activation in the presence of MOLM13 cells lacking CD123 expression (MOLM13 (CD123- / -)) and various concentrations of sparX protein. Samples included affinity-modified variants of 6T1D (e.g., 3F4N and 4R8U). A parent D domain control (6T1D) and a non-binding control (a3D) were assayed for comparison. [Figure 7]IL-2 production of CAR T cells cultured with CD123-specific adaptor proteins. Primary T cells expressing 8G8V-CAR were assayed for IL-2 expression in the presence of MOLM13 target cells and various concentrations of adaptor proteins. Samples included affinity-engineered variants of 6T1D (e.g., 3F4N and 4R8U). A parental D domain control (6T1D) and a non-binding control (a3D) were assayed for comparison. [Figure 8] IFNγ production of CAR T cells cultured with CD123-specific adaptor proteins. Primary T cells expressing 8G8V-CAR were assayed for IFNγ expression in the presence of MOLM13 target cells and various concentrations of adaptor proteins. Samples included affinity-engineered variants of 6T1D (e.g., 3F4N and 4R8U). A parental D domain control (6T1D) and a non-binding control (a3D) were assayed for comparison. [Figure 9] NFAT activation of 8G8V-CAR T cells cultured with CD123-specific adaptor proteins and target cells. JNL10 / 8G8V-CAR was assayed for activation in the presence of MOLM13 target cells and various concentrations of adaptor proteins. Samples included affinity-modified variants of 6T1D (e.g., 3F4N, 4R8U, 0C8S, and 5B1Q). A parent D domain control (6T1D) and a non-binding control (a3D) were assayed for comparison. [Figure 10] Lysis of target cells by 8G8V-CAR T cells cultured with CD123-specific adaptor proteins. MOLM13 target cell lysis by primary 8G8V-CAR T cells was assayed in the presence of various concentrations of adaptor proteins. Samples included affinity-engineered variants of 6T1D (e.g., 3F4N, 4R8U, 0C8S, and 5B1Q). A parental D-domain control (6T1D) and a non-binding control (a3D) were assayed for comparison. [Figure 11]IFNγ production by 8G8V-CAR T cells. Primary 8G8V-CAR T cells were assayed for IFNγ production by ELISA in the presence of MOLM13 target cells and various concentrations of CD123-specific adaptor proteins. Samples included affinity-engineered variants of 6T1D (e.g., 3F4N, 4R8U, 0C8S, and 5B1Q). A parental D domain control (6T1D) and a non-binding control (a3D) were assayed for comparison. [Figure 12] IL-2 production by 8G8V-CAR T cells. Primary 8G8V-CAR T cells were assayed for IL-2 production by ELISA in the presence of MOLM13 target cells and various concentrations of CD123-specific adaptor proteins. Samples included affinity-engineered variants of 6T1D (e.g., 3F4N, 4R8U, 0C8S, and 5B1Q). A parent D domain control (6T1D) and a non-binding control (a3D) were assayed for comparison. [Figure 13] Lysis of target cells by 8G8V-CAR T and CD123-specific adaptor proteins. NALM6-CD123+ cells were lysed by primary 8G8V-CAR T cells in the presence of various concentrations of adaptor proteins. Samples included both monovalent and bivalent formats of adaptor proteins composed of low-affinity D domains (4G0D), high-affinity D domains (5B1Q), and parental D domains (6T1D). A non-binding control (a3D) was assayed for comparison. [Figure 14] The in vivo efficacy of the Dd-X CD123-specific D domain was assessed using a MOLM14-GFP / luciferase tumor model. The shaded area indicates Dd-X adapter withdrawal. qod every other day; qd every day. [Figure 15] The in vivo efficacy of the Dd-X CD123-specific D domain was assessed using a disseminated MV4-11 tumor model. The shaded area indicates Dd-X adapter withdrawal. qd daily. [Figure 16] Patient-derived AML xenograft model. [Figure 17]Dd-X CD123-specific D domain eliminates or controls multiple patient-derived AML xenografts. The percentage of viable cells in the bone marrow representing engrafted AML cells (hCD45+CD3-) after treatment is shown. DETAILED DESCRIPTION OF THE INVENTION
[0015] Detailed Description The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described in any way.
[0016] Definition of Terms Whenever an embodiment is described herein by the word "comprising," it is understood that otherwise similar embodiments described by the terms "consisting of" and / or "consisting essentially of" are also provided. However, when used in the claims as transitional phrases, each should be interpreted separately and in the appropriate legal and factual context (e.g., "comprising" is considered the more open-ended phrase, while "consisting of" is more exclusive, and "consisting essentially of" is the middle ground).
[0017] As used herein, the singular forms "a," "an," and "the" include plural referents unless otherwise indicated.
[0018] As used herein, the term "and / or" as used in phrases such as "A and / or B" is intended to include both A and B; A or B; A alone; and B alone. Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.
[0019] The term "about," as used herein, when referring to measurable values, such as amounts, durations, and other measurable values known in the art, is intended to encompass variations of ±20%, or in some embodiments, ±10%, or in some embodiments, ±5%, or in some embodiments, ±1%, or in some embodiments, ±0.1% of the stated value, as such variations are appropriate for practicing the disclosed methods.
[0020] "Chimeric antigen receptor" or "CAR," as used herein, refers to an engineered receptor that transfers specificity for an antigen or target into a cell (e.g., a T cell, e.g., a naive T cell, a central memory T cell, an effector memory T cell, an NK cell, an NKT cell, or a combination thereof). CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors.
[0021] The term "adapter," as used herein, refers to a multidomain soluble protein comprising an antigenic determinant (AD) and an antigenic determinant binding domain (ADBD), where the ADBD binds to a second AD. In addition to that AD and its ADBD, an adapter may contain additional ADs, additional ADBDs, and / or other additional domains.
[0022] The term "antigenic determinant binding domain" or "ADBD," as used herein, refers to a sequence of a polypeptide (e.g., an adapter or CAR) sufficient to confer recognition and specific binding to a target antigenic determinant (AD). In some embodiments, the ADBD is an antigen-binding antibody fragment, scFv, or an antigen-binding peptide (e.g., a D domain or affibody) that is not based on the sequence of an antibody or antibody fragment. In some embodiments, the ADBD includes a non-antibody-based binding scaffold (e.g., a D domain, affibody, fibronectin domain, nanobody, lipocalin domain, ankyrin domain, maxybody, protein A domain, or affilin domain). In some embodiments, the ADBD is a D domain. In some embodiments, the ADBD is an antibody-based binding sequence. In some embodiments, the ADBD is an scFv or domain antibody (dAb). In some embodiments, the ADBD is capable of binding to a target antigen on the surface of a cell. In some embodiments, the ADBD is capable of binding to a target antigen on the surface of an immune effector cell. In some embodiments, the ADBD has the ability to bind to a growth factor receptor, an immunoregulatory receptor, or a hormone receptor.
[0023] In specific embodiments, the ADBD is a non-antibody scaffold-based polypeptide sequence that is sufficient to confer recognition and specific binding to a target antigen determinant. In some embodiments, the non-antibody-based ADBD is a polypeptide that has the ability to bind to a target antigen on the surface of a cell. In some embodiments, the non-antibody-based ADBD has the ability to bind to a growth factor receptor, an immunoregulatory receptor, or a hormone receptor. In some embodiments, the ADBD is a D domain-based polypeptide. In specific embodiments, the ADBD is a D domain-based polypeptide that is sufficient to confer recognition and specific binding to a target antigen on the surface of a cell. In some embodiments, the ADBD is a D domain-based polypeptide that has the ability to bind to a growth factor receptor, an immunoregulatory receptor, or a hormone receptor. In some embodiments, the ADBD is a D domain-based polypeptide that has the ability to bind to a target antigen on a serum protein.
[0024] The term "D domain" refers to the reference scaffold sequence: TIFF2024544559000001.tif17170 refers to target-binding polypeptides that share certain sequence and structural features (see WO 2016 / 164305 and WO 2016 / 164308, each of which is incorporated herein by reference in its entirety). This reference scaffold is a variant of a non-naturally occurring, targetless antiparallel three-helix bundle reference polypeptide that was originally engineered as an exercise in protein folding (see Walsh et al., PNAS 96:5486-5491 (1999), which is incorporated herein by reference in its entirety). This reference scaffold does not have known target-binding activity, but it has been discovered that polypeptides containing modifications of the reference scaffold having the amino acid sequence of SEQ ID NO: 1 can specifically bind to targets of interest. Thus, D domains, or molecules containing D domains, can specifically (non-randomly) bind to target molecules. Without wishing to be bound by theory, it is believed that in designing a D domain, structural constraints on surface-exposed residues (which may be modified) confer the ability of the surface-exposed residues to specifically bind to a target of interest. In some embodiments, a D domain generally consists of 70-75 amino acid residues. In some embodiments, a D domain comprises an amino acid sequence that differs (e.g., due to amino acid modifications) from the amino acid sequence of a reference scaffold having the sequence of SEQ ID NO:2 by up to 20 substitutions. In a specific embodiment, the D domain does not contain the sequence LAAIKTRLQ (SEQ ID NO:49).
[0025] The terms "protein" and "polypeptide" are used interchangeably herein to refer to biological polymers comprising units derived from amino acids linked through peptide bonds; a protein may be composed of two or more polypeptide chains.
[0026] The terms "antibody" or "immunoglobulin," as used interchangeably herein, include full-length antibodies and antibody fragments, e.g., functional domains of antibodies, e.g., antigen-binding fragments or single chains thereof, effector domains, salvage receptor-binding epitopes, or portions thereof. A typical antibody comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, Cl. The VH and VL regions are further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FWs). Each VH and VL is composed of three CDRs and four FWs, arranged from the amino terminus to the carboxyl terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, such as various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Examples of antibodies of the present disclosure include typical antibodies, scFvs, and combinations thereof, in which, for example, DDpp is covalently linked (e.g., via a peptide bond or a chemical linker) to the N-terminus of either the heavy chain and / or light chain of a typical complete (full-length) antibody, or intercalated into the H chain and / or L chain of a full-length antibody.
[0027] The term "antibody fragment" refers to a portion of an intact antibody, including any functional domain of an antibody, such as an antigen-binding fragment or single chain thereof, an effector domain or portion thereof, and a salvage receptor-binding epitope or portion thereof. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments, linear antibodies, single-chain antibodies, and multispecific antibodies formed from antibody fragments. As used herein, "antibody fragment" includes an antigen-binding site or an epitope-binding site. In one embodiment, the DDpp fusion protein includes an effector domain or portion thereof. In one embodiment, the DDpp fusion protein includes a salvage receptor-binding epitope or portion thereof.
[0028] The term "single-chain variable fragment" or "scFv" antibody, as used herein, refers to a form of antibody (e.g., an antibody fragment) that comprises only the variable regions of the heavy and light chains connected by a linker peptide. An scFv may comprise VL-linker-VH or VH-linker-VL. An scFv antibody is generally 220-250 amino acids in length and contains a linker of 10-25 amino acids in length. In one embodiment, a DDpp fusion protein comprises DDpp and an scFv.
[0029] As used herein, the term "Fc region" or simply "Fc" is understood to refer to the carboxyl-terminal portion of an immunoglobulin chain constant region, preferably an immunoglobulin heavy chain constant region or a portion thereof. For example, an immunoglobulin Fc region can include (1) a CH1 domain, a CH2 domain, and a CH3 domain; (2) a CH1 domain and a CH2 domain; (3) a CH1 domain and a CH3 domain; (4) a CH2 domain and a CH3 domain; or (5) a combination of two or more domains and an immunoglobulin hinge region. Thus, in various embodiments, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, along with the flexible hinge N-terminal to these domains. In the case of IgA and IgM, Fc can include the J chain. In the case of IgG, Fc includes immunoglobulin domains Cγ2 and Cγ3, and the hinge between Cγ1 and Cγ2. In preferred embodiments, the immunoglobulin Fc region comprises at least the hinge region, CH2 domain, and CH3 domain of an immunoglobulin, and preferably lacks the CH1 domain. In one embodiment, the class of immunoglobulin from which the heavy chain constant region is derived is IgG (Igγ) (γ subclasses 1, 2, 3, or 4). Other classes of immunoglobulins, such as IgA (Igα), IgD (Igδ), IgE (Igε), and IgM (Igμ), may also be used. Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is generally defined to include residues C226 or p260 through the carboxyl terminus, where numbering is according to the EU index as set forth in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NIH, Bethesda, Md. (1991)). Fc can refer to this region in isolation or in the context of a full-length antibody, antibody fragment, or Fc fusion protein.Polymorphisms have been observed at a number of different Fc positions, including, but not limited to, positions 270, 272, 312, 315, 356, and 358, as numbered by the EU index; therefore, slight differences may exist between the presented sequence and prior art sequences. The selection of appropriate immunoglobulin heavy chain constant regions is described in detail in U.S. Patent Nos. 5,541,087 and 5,726,044, each of which is incorporated herein by reference in its entirety. Selection of a particular immunoglobulin heavy chain constant region sequence from a particular immunoglobulin class and subclass to achieve a particular result is considered to be within the level of ordinary skill in the art. The portion of the DNA construct encoding the immunoglobulin Fc region preferably includes at least a portion of the hinge domain and, preferably, at least a portion of the CH3 domain of Fcγ or a homologous domain in either IgA, IgD, IgE, or IgM. Furthermore, it is contemplated that substitutions or deletions of amino acids within the immunoglobulin heavy chain constant region may be useful in practicing the methods and compositions disclosed herein. One example would be introducing amino acid substitutions into the upper CH2 region to create Fc variants with reduced affinity for Fc receptors (Cole, J. Immunol. 159:3613 (1997)).
[0030] "Antibody-dependent cellular cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize antibodies bound to target cells and subsequently cause lysis (or other cytotoxic effects) of the target cells. To assess the ADCC activity of a molecule of interest, any in vitro ADCC assay known in the art can be used, for example, those described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337. Useful effector cells for such assays include, but are not limited to, peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, the ADCC activity of a molecule of interest can be assessed in vivo, for example, in an animal model, such as that disclosed in Clynes et al. PNAS 95:652-656 (1998).
[0031] The terms "linker," "spacer," and "hinge" are used interchangeably herein to refer to a peptide or other chemical linkage positioned between two or more otherwise independent functional domains of a DDpp fusion protein, an adapter, or a CAR. For example, a linker may be positioned between the antigenic determinant (AD) domain and the antigenic determinant binding domain (ADBD) of an adapter. Similarly, a linker may be positioned between two antigenic determinant binding domains of a CAR, or between the antigen binding domain and the transmembrane domain. In some embodiments, the linker is a peptide or other chemical linkage positioned between the DDpp of a DDpp fusion protein and another polypeptide. Linkers suitable for coupling two or more domains of an adapter are described herein and / or will otherwise be apparent to one of skill in the art.
[0032] The term "operably linked," as used herein, refers to the attachment of two molecules such that each molecule retains at least some level of the functional activity that each molecule has alone (assuming each molecule has functional activity). In some embodiments, a molecule is operably linked to another molecule if the other molecule retains at least some level of its functional activity when the other molecule does not have the functional activity. Operatively linked can also refer to the attachment of two non-functional molecules. Two molecules can be "operably linked" whether they are attached directly or indirectly (e.g., via a linker).
[0033] The terms "specifically bind," "having selective affinity for," "bind," or "binding" are used interchangeably to mean that a binding agent, e.g., DDpp, reacts with or associates with an epitope, protein, or target molecule more frequently, rapidly, for a longer duration, with greater affinity, or some combination of the above, than another substance, e.g., a protein unrelated to the target epitope, protein, or target molecule. Due to sequence identity between homologous proteins of different species, specific binding may, in some embodiments, include a binding agent that recognizes proteins or targets from multiple species. Similarly, due to homology in certain regions of the polypeptide sequences of different proteins, specific binding may include a binding agent that recognizes multiple proteins or targets. It is understood that, in certain embodiments, a binding agent that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific binding" does not necessarily require exclusive binding, e.g., binding to a single target (although it can include such binding). Thus, a binding agent may, in certain embodiments, specifically bind to multiple targets. In certain embodiments, multiple targets may be bound by the same antigen binding site on the binding agent.
[0034] "Target" refers to any molecule or combination of molecules that can be bound by a DDpp, e.g., a DDpp fusion protein, by another component of a DDpp fusion protein, e.g., an antibody or antibody variable domain fragment, by an adaptor or CAR, or by a component of a DDpp fusion protein, adaptor, or CAR, e.g., an antigenic determinant binding domain.
[0035] The terms "epitope" and "antigenic determinant" are used interchangeably herein to refer to a portion of any molecule (e.g., a target of interest, e.g., CD123 or AFP p26) that can be recognized and specifically bound by a specific binding agent (e.g., DDpp or an antibody). When the molecule being recognized is a polypeptide, the epitope may be formed from contiguous amino acids or from non-contiguous amino acids juxtaposed by tertiary folding of the protein and / or other chemically active surface groups of the molecule (e.g., carbohydrates). Epitopes formed from contiguous amino acids are typically retained upon protein denaturation, whereas epitopes formed by tertiary folding are typically lost upon protein denaturation. An epitope typically comprises at least three amino acids, more commonly at least five or eight to ten amino acids, in a unique spatial conformation.
[0036] As used herein, "peptide tag" refers to a peptide sequence that is part of or attached to another protein (e.g., through genetic engineering) to provide a function to the resulting fusion. Peptide tags are generally relatively short compared to the protein to which they are fused; for example, peptide tags in some embodiments are 4 amino acids or more in length, e.g., 5, 6, 7, 8, 9, 10, 15, 20, or 25 amino acids or more in length. In some embodiments, DDpp is a fusion protein containing a peptide tag. In other embodiments, DDpp specifically binds to the peptide tag. Numerous peptide tags with the uses provided herein are known in the art. Examples of peptide tags that can be components of a DDpp fusion protein, or a target to which a DDpp (e.g., a DDpp fusion protein) binds, include, but are not limited to, HA (hemagglutinin), c-myc, herpes simplex virus glycoprotein D (gD), T7, GST, GFP, MBP, Strep tag, His tag, Myc tag, TAP tag, and FLAG® tag (Eastman Kodak, Rochester, NY). Similarly, antibodies against the tag epitope allow for detection and localization of the fusion protein using techniques known in the art, such as Western blots, ELISA assays, and cell immunostaining.
[0037] The term "derived from," as used herein, indicates the relationship between a first molecule and a second molecule. It generally refers to the structural similarity between the first and second molecules and does not imply or include a limitation on the process or origin of the first molecule derived from the second molecule. For example, in the case of an intracellular signaling domain derived from the CD3ζ molecule, the intracellular signaling domain retains sufficient CD3ζ structure to have the required function, i.e., the ability to generate a signal under appropriate conditions. It does not imply or include a limitation to a particular process for generating the intracellular signaling domain, e.g., it does not mean that one must start from the CD3ζ sequence and delete or mutate unnecessary sequences to arrive at the intracellular signaling domain to provide the intracellular signaling domain.
[0038] The term "naturally occurring," when used in reference to biological materials, e.g., nucleic acid molecules, polypeptides, antigenic determinants, and host cells, refers to those found in nature and that have not been modified by humans. Conversely, "non-natural" or "synthetic," when used in reference to biological materials, refers to those that are not found in nature and that have been modified by humans.
[0039] As used herein, "modifications" to the sequence of a reference sequence include substitutions, deletions, insertions, and / or additions to the sequence of corresponding amino acid positions in the reference sequence (e.g., a DD disclosed herein).
[0040] A "substitution" with respect to a sequence of a reference sequence refers to the replacement of a particular amino acid residue with a different amino acid residue at a corresponding amino acid position in the reference sequence.
[0041] A "conservative" amino acid substitution is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, and include basic side chains (e.g., lysine (K), arginine (R), histidine (H)), acidic side chains (e.g., aspartic acid (D), glutamic acid (E)), uncharged polar side chains (e.g., glycine (G), asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), cysteine (C)). Conservative substitutions include non-polar side chains (e.g., alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W)), beta-branched side chains (e.g., threonine (T), valine (V), isoleucine (I)), and aromatic side chains (e.g., tyrosine (Y), phenylalanine (F), tryptophan (W), histidine (H)). For example, a phenylalanine-tyrosine substitution is considered a conservative substitution. In a specific embodiment, a conservative substitution in the sequence of a DDpp results in altered or unchanged specific binding of the DDpp containing the substitution to the target of interest to which it binds (e.g., CD123 or AFP p26). In one embodiment, a conservative substitution in the sequence of a DDpp does not eliminate the binding of the DDpp containing the substitution to the target of interest to which it binds. Methods for identifying conservative and non-conservative nucleotide and amino acid substitutions that confer, alter, or maintain selective binding affinity are known in the art (see, e.g., Brummell, Biochem. 32:1180-1187 (1993); Kobayashi, Protein Eng. 12(10):879 884 (1999); and Burks, PNAS 94:412-417 (1997)).
[0042] " Non-conservative " amino acid substitution refers to the replacement of an amino acid residue with another amino acid residue that has a dissimilar side chain.In one embodiment, non-conservative substitution in the sequence of DDpp allows the DDpp containing substitution to bind with the target of interest (for example, CD123 or AFP p26) specifically.In one embodiment, non-conservative substitution in the sequence of DDpp does not eliminate the binding of the DDpp containing substitution with the target of interest that it binds.In one embodiment, non-conservative substitution in the sequence of DDpp, adaptor or CAR allows the DDpp containing substitution, adaptor or CAR to retain the specific binding of the DDpp containing substitution with the target of interest that it binds.
[0043] The terms "unnatural amino acid," "amino acid analog," and "non-standard amino acid residue" are used interchangeably herein. Unnatural amino acids that can be substituted in the DDpp provided herein are known in the art. In one embodiment, the unnatural amino acid is 4-hydroxyproline, which can be substituted for proline; 5-hydroxylysine, which can be substituted for lysine; 3-methylhistidine, which can be substituted for histidine; homoserine, which can be substituted for serine; and ornithine, which can be substituted for lysine. Additional examples of unnatural amino acids that can be substituted in the DDpps disclosed herein include, but are not limited to, molecules such as D-isomers of the common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, A-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, lanthionine, dehydroalanine, γ-aminobutyric acid, selenocysteine, and pyrrolidine fluoroamino acids, designer amino acids such as β-methyl amino acids, Cα-methyl amino acids, and Nα-methyl amino acids, or combinations of unnatural amino acids. Additional unnatural amino acids can include, for example, 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine, and / or D-isomers of amino acids. As described herein, in some embodiments, unnatural amino acids or amino acid analogs can include deletions of one or more amino acids from the sequence.
[0044] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to any polymeric form of nucleotides, either ribonucleotides or deoxyribonucleotides, of any length. These terms include, but are not limited to, DNA, RNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA), shRNA (short hairpin RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), miRNA (microRNA), genomic DNA, synthetic DNA, synthetic RNA, and / or tRNA. In some embodiments, the isolated polynucleotide is a modified mRNA containing non-naturally occurring nucleosides or nucleotides. In some embodiments, the modified mRNA contains 2-thiouridine, pseudouridine, or 1-methylpseudouridine.
[0045] The terms "vector," "cloning vector," and "expression vector," as used herein, refer to a vehicle (e.g., a cloning vector) that can maintain or amplify a nucleic acid sequence (e.g., a sequence encoding a disclosed DDpp, adapter, or CAR) in a host cell, or a vehicle that can be introduced into a host cell to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, phages, viruses, and the like.
[0046] A "host cell" includes an individual cell or cell culture that can be or has been a recipient of a nucleic acid encoding the disclosed DDpp, adapter, or CAR. Host cells include, but are not limited to, bacterial, yeast, plant, animal, and mammalian cells. A host cell includes the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or DNA totality) to the original parent cell due to natural, accidental, or deliberate mutation and / or variation. A host cell includes cells transfected or infected in vivo, in vitro, or ex vivo with a nucleic acid encoding the disclosed DDpp, adapter, or CAR. In some examples, for example, in phage display or CAR T cells, the host cell can express and display the disclosed DDpp or CAR on its surface. In some embodiments, the host cell can express the adapter. In some embodiments, the host cell can express and secrete the adapter. In some embodiments, the host cell can express the CAR. In some embodiments, the host cell can express and display the CAR on its surface. "Expression" includes transcription and / or translation.
[0047] As used herein, the terms "solid support," "support," "matrix," and "resin" are used interchangeably and refer, without limitation, to any column (or column material), bead, test tube, microtiter dish, solid particle (e.g., agarose or sepharose), microchip (e.g., silicon, silicon-glass, or gold chip), or membrane (biological membrane or filter membrane) to which DDpp, antibodies, or other proteins can be attached (e.g., coupled, linked, or adhered) directly or indirectly (e.g., through other binding partner intermediates, such as other antibodies or protein A), or to which DDpp or antibodies can be embedded (e.g., through a receptor or channel). Reagents and techniques for attaching polypeptides to solid supports (e.g., matrices, resins, plastics, etc.) are well known in the art. Suitable solid supports include, but are not limited to, chromatographic resins or matrices (e.g., SEPHAROSE-4 FF agarose beads), the walls or bottoms of wells in plastic microtiter dishes, silica-based biochips, polyacrylamide, agarose, silica, nitrocellulose, paper, plastic, nylon, metal, and combinations thereof. DDpp and other compositions can be attached to the support material by non-covalent association or covalent bonding using reagents and techniques known in the art. In one embodiment, DDpp is coupled to the chromatographic material using a linker.
[0048] As used herein, the terms "pharmaceutically acceptable" or "physiologically tolerable" and grammatical variations thereof are used interchangeably when referring to compositions, carriers, diluents, and reagents and indicate that the material may be administered to a human without producing therapeutically prohibited undesirable physiological effects, such as nausea, dizziness, stomach upset, etc.
[0049] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, or infusion techniques.
[0050] "Modulate" means adjusting or controlling the magnitude, frequency, extent, or activity. In another related aspect, such modulation can be a positive modulation (e.g., an increase in frequency, extent, or activity) or a negative modulation (e.g., a decrease in frequency, extent, or activity). In some embodiments, positive or negative modulation is referred to as compared to the function of a cell, tissue, or organ prior to administration of the therapeutic agent. In additional embodiments, positive or negative modulation is referred to as compared to a normal, healthy cell, tissue, or organ.
[0051] An "effective amount" of a DDpp (e.g., a DDpp fusion protein), CAR cell, adaptor, and / or CAR cell / adaptor composition provided herein is an amount sufficient to achieve a specifically stated purpose, e.g., to cause an observable change in the level of one or more biological activities associated with the target to which the DDpp (e.g., a DDpp fusion protein), CAR cell, and / or adaptor binds. In certain embodiments, the change increases the level of the target activity. In other embodiments, the change decreases the level of the target activity. An "effective amount" can be determined empirically and routinely with respect to the stated purpose. The term "therapeutically effective amount" refers to an amount of a DDpp (e.g., a DDpp fusion protein), CAR cell, and / or adaptor, or other therapeutic agent, effective to "treat" (e.g., alleviate symptoms of) a disease or disorder in a subject (mammal). The term "therapeutically effective amount" also refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result.
[0052] The terms "patient," "subject," "animal," and "mammal" are used interchangeably and refer to mammals, e.g., human patients and non-human primates, as well as laboratory animals, e.g., rabbits, rats, and mice, and other animals. Animals include all vertebrates, e.g., mammals and non-mammals, e.g., chickens, amphibians, and reptiles. "Mammal," as used herein, refers to any member of the class Mammalia, including, but not limited to, humans and non-human primates, e.g., chimpanzees and other ape and monkey species; livestock, e.g., cows, sheep, pigs, goats, and horses; domestic animals, e.g., dogs and cats; laboratory animals, e.g., rodents, e.g., mice, rats, and guinea pigs. In a specific embodiment, the patient is a human. The term does not denote a particular age or sex. Thus, adult and neonatal subjects, as well as embryos and fetuses, whether male or female, are intended to be within the scope of this term.
[0053] The terms "treat," "treatment," and "treating," as used herein, refer to both therapeutic treatment and prophylactic or preventative measures, where the objective is to prevent or slow (alleviate or delay) symptoms, complications, or biochemical manifestations of a disease, condition, or disorder, to alleviate symptoms of a disease, condition, or disorder, or to arrest or inhibit further development. Treatment may be prophylactic (to prevent or delay the onset of a disease or to prevent the manifestation of its clinical or subclinical symptoms) or therapeutic (to suppress or alleviate symptoms after the manifestation of a disease, condition, or disorder, to target a pathological condition, to prevent a pathological condition, to pursue or obtain a beneficial result, or to reduce the likelihood that an individual will develop a condition, even if treatment is ultimately unsuccessful). Those in need of treatment include those already with the condition, as well as those prone to have the condition, or those in whom the condition is to be prevented. Treatment can be with a DDpp fusion protein, CAR cell, adaptor, and / or CAR cell / adaptor composition, alone or in combination with an additional therapeutic agent. In some embodiments, the terms "treat," "treatment," and "treating" are used herein to refer to therapeutic treatment and prophylactic or preventative measures, where the objective is to prevent or slow (alleviate or delay) a symptom, complication, or biochemical manifestation of a proliferative disorder, or to ameliorate one or more symptoms (preferably one or more discernible symptoms) of a proliferative disorder. In specific embodiments, the terms "treat," "treatment," and "treating" refer to the amelioration of at least one measurable physical parameter of a proliferative disorder, e.g., tumor growth, which is not necessarily discernible by the patient.In other embodiments, the terms "treat," "treatment," and "treating" refer to inhibiting the progression of a proliferative disorder physically, e.g., by stabilization of a discernible symptom, physiologically, e.g., by stabilization of a physical parameter, or both. In other embodiments, the terms "treat," "treatment," and "treating" refer to a reduction or stabilization of tumor size, tumor cell growth or survival, or cancer cell number.
[0054] "Cancer," "tumor," or "malignant tumor" are used synonymously and refer to any of a number of diseases characterized by unregulated, abnormal growth of cells, the ability of affected cells to spread (metastasize) locally or through the bloodstream and lymphatic system to other parts of the body, and any of a number of characteristic structural and / or molecular features. "Tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. A "cancerous tumor" or "malignant cell" is understood to be a cell that has specific structural characteristics, lacks differentiation, and is capable of invasion and metastasis. Cancers that can be treated using the DDpp fusion proteins, CAR cells, adapters, and / or CAR cell / adapter compositions provided herein include, but are not limited to, breast cancer, lung cancer, brain cancer, cervical cancer, skin cancer, bone cancer, liver cancer, pancreatic cancer, colorectal cancer, kidney cancer, head and neck cancer, ovarian cancer, hematopoietic cancer (e.g., leukemia), and prostate cancer, as well as lymphoma. Other types of cancers and tumors that can be treated using the DDpp fusion proteins, CAR cells, adapters, and / or CAR cell / adapter compositions are described herein or otherwise known in the art. Reference to a particular "type" of cancer, tumor, or tumor cell is understood to mean a cancer, tumor, or tumor cell characterized by a particular disease. For example, in some embodiments, the first and second cancers of the same type are mixed-cell type Hodgkin's lymphoma and lymphocyte-rich type Hodgkin's lymphoma. In other embodiments, the first and second cancers of the same type are precursor B-cell acute lymphoblastic leukemia (ALL) and mature B-cell ALL. Examples of first and second cancers of different types include, for example, Hodgkin's lymphoma and ALL.
[0055] The term "tumor antigen" refers to an antigen common to certain hyperproliferative disorders, such as cancer. The terms "tumor antigen" and "cancer antigen" are used interchangeably herein. In certain aspects, the antigen is derived from cancer, including but not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer (e.g., NSCLC or SCLC), liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, multiple myeloma, glioblastoma, neuroblastoma, uterine cancer, cervical cancer, renal cancer, thyroid cancer, bladder cancer, kidney cancer, mesothelioma, and adenocarcinoma, such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colon cancer, and other cancers known in the art. In some embodiments, the cancer is acute myeloid leukemia (AML), B-cell acute lymphoblastic leukemia, hairy cell leukemia, Hodgkin's lymphoma, or blastic plasmacytoid dendritic cell neoplasm (BPDCN). In some embodiments, the cancer is selected from the group consisting of B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML); one or more chronic leukemias, such as, but not limited to, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL); additional hematological cancers or conditions, such as, but not limited to, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic leukemia, and leukemia. cell tumors, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndromes (e.g., high-risk myelodysplastic syndrome), non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's hypergammaglobulinemia.
[0056] Tumor antigens and cancer antigens can be further defined as "tumor-specific antigens (TSAs)," "cancer-specific antigens (CSAs)," "tumor-associated antigens (TAAs)," or "cancer-associated antigens (CAAs)." TSAs are antigens unique to tumor cells and not present in other cells in the body. TAAs are antigens found on both tumor cells and some normal cells. TAAs can be expressed in normal cells under conditions that do not induce a state of immune tolerance to the antigen. Expression of TAAs in tumors can occur under conditions that allow the immune system to respond to the antigen. TAAs can be expressed in normal cells during fetal development, when the immune system is immature and unable to respond, or they can be normally present at very low levels in normal cells but are expressed at much higher levels in tumor cells. Due to the dynamic nature of tumors, in some cases, tumor cells express unique antigens at certain stages and antigens that are also expressed in non-tumor cells at other stages. Therefore, the inclusion of a particular marker as a TAA does not exclude it from being considered a TSA. In some embodiments, the TAAs and / or TSAs containing antigenic determinants specifically bound by the CAR cells, adaptors, and / or CAR cell / adaptor compositions provided herein include, but are not limited to, BCMA, CD19, CD20, CD22, CD30, CD33 / IL3Ra, CD70, CD123, CD171 (L1-CAM), CS1, EGFRvIII, GD2, Lewis YROR1, ringworm, IL13Ra2, cMet, PSMA, free radical α(FR-α), CEA, ErbB2(HER-2 / neu); EGFR(HER), PSCA, PSA, MUC1, MUC16, CD44v6, CD44v6 / 7, CD44v7 / 8 CD55, IL11Ra, EphA2, EGP40, TAG72, CAIX, HMW-MAA(CSPG4), MAGEA4, N KG2D fragment, β-HCG, activator F77, HLA-A2(NY-ESO-1), HMW-MAA, GD3, TCR, MAGE A3, TUESDAY, WT1, MARCH, gp100(Pmel 17) TRP1, TRP2, HLA-A1, MAGE1, MAGE3, BAGE, GAGE1 GAGE2, pi5, p53, Ras, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MY L-RAR;VEGFR2, FAP, FAR, EBVA, HPV vaccine E6, HPV vaccine E7, TSP-180, M AGE4, MAGE5, MAGE6, RAGE, pl85erbB2, pl80erbB3, nm-23Hl, CA 19-9, CA72-4, CAM 17.1 NuMa, K-ras, β-linkage, CDK4, Mum-1, p15, p16, 43-9F, α-glycoprotein, BCA225, BTAA, CA125, CA 15-3, CA 27.29(BCAA), CA195, CA242, CA50, CAM43, CD68, CO-029, FGF5, G250, HTgp-175, M344, MA50, MG7-Ag, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, M2BP, TAAL6, TLP, TPS, FcRH5, GPCR5d, LILBR4, CLL1, and FLT3.
[0057] The term "autoimmune disease" as used herein is defined as a disorder caused by an autoimmune response. Autoimmune diseases are the result of an inappropriate and excessive response to self-antigens. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes mellitus (type 1), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis.
[0058] The term "target cell," as used herein, refers to a cell that is involved in a disease and can be targeted by a DDpp-containing composition provided herein or by a CAR, adapter, and / or CAR / adapter composition. Target cells include any cell of a subject (e.g., a human or animal) that can be targeted by a DDPP, CAR, adapter, and / or CAR / adapter composition. A target cell can be a cell that expresses or overexpresses a target that is specifically bound by a CAR, adapter, and / or CAR / adapter composition. A target cell can be a cell that expresses or overexpresses a target that is specifically bound by a DDpp fusion protein, a CAR, adapter, and / or CAR / adapter composition.
[0059] The term "autologous," as used herein, refers to any material derived from the same individual into which it is subsequently reintroduced.
[0060] The term "allogeneic," as used herein, refers to any material derived from a different animal of the same species as the individual into which it is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material derived from individuals of the same species may be sufficiently genetically distinct to interact antigenically.
[0061] The term "effector cell," as used herein, refers to a leukocyte that expresses one or more FcRs and performs effector function. Preferably, the cell expresses at least Fc(RIII) and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; in certain embodiments, PBMCs and NK cells are preferred. Effector cells can be isolated from their native source, e.g., blood or PBMCs, as described herein or otherwise known in the art. In a specific embodiment, the effector cells are human effector cells.
[0062] The term "effector function" refers to the specialized immune function of a differentiated cell. The effector function of a T cell can be, for example, cytolytic activity, or helper activity, such as the secretion of cytokines.
[0063] The term "immune cell," as used herein, refers to cells of a mammalian immune system, including, but not limited to, antigen-presenting cells, B cells, basophils, cytotoxic T cells, dendritic cells, eosinophils, granulocytes, helper T cells, leukocytes, lymphocytes, macrophages, mast cells, memory cells, monocytes, natural killer cells, neutrophils, phagocytes, plasma cells, and T cells.
[0064] The terms "T cells" and "T lymphocytes" are interchangeable and are used synonymously herein. Examples include, but are not limited to, naive T cells, central memory T cells, effector memory T cells, or combinations thereof.
[0065] The term "immune response," as used herein, refers to immunity including, but not limited to, innate immunity, humoral immunity, cellular immunity, immunity, inflammatory response, acquired (adaptive) immunity, autoimmunity, and / or hyperactive immunity.
[0066] The term "transduction," as used herein, refers to the introduction of foreign nucleic acid into a cell using a viral vector. "Transfection," as used herein, refers to the introduction of foreign nucleic acid into a cell using recombinant DNA technology. The term "transformation" means the introduction of a "foreign" (e.g., foreign, extracellular, or otherwise non-endogenous) nucleic acid (DNA or RNA) sequence into a host cell, such that the host cell expresses the introduced nucleic acid to produce a desired substance, e.g., a protein or enzyme, encoded by the introduced coding sequence. The introduced nucleic acid sequence may also be referred to as a "cloned" or "foreign" gene or sequence and may include control or regulatory sequences, such as initiation sequences, termination sequences, promoter sequences, signal sequences, secretion sequences, or other sequences used by the cell's genetic machinery. The nucleic acid sequence may include nonfunctional sequences or sequences of unknown function. A host cell that receives and expresses introduced nucleic acid (e.g., DNA or RNA) has been "transformed" and is a "transformant" or "clone." The DNA or RNA introduced to a host cell can come from any source, for example, cells of the same genus or species as the host cell, or cells of a different genus or species, or it can be non-naturally occurring.
[0067] "Co-express," as used herein, refers to the expression of two or more protein-coding sequences by the same cell or cell population. The coding sequences can be, for example, nucleic acids that each encode a single protein or encode a chimeric protein as a single polypeptide chain.
[0068] "Cell surface receptor" refers to molecules and molecular complexes that can receive signals and transmit such signals across the plasma membrane of a cell. An example of a cell surface receptor provided herein is an activated integrin receptor, for example, an activated αvβ3 integrin receptor on a metastatic cell. As used herein, "cell surface receptor" also includes molecules expressed on the cell surface that contain DDpp that can bind to a target of interest (e.g., CD123 or AFP p26). The term "receptor" refers to a cell-associated protein that binds to or otherwise interacts with a molecule (e.g., a ligand) and mediates the effect of the ligand on the cell. In some embodiments, the molecule that interacts with the receptor is a bioactive molecule. Membrane-bound cell surface receptors are typically characterized by a multidomain structure that includes an extracellular ligand-binding domain, a transmembrane domain, and an intracellular effector domain that typically participates in signal transduction.
[0069] "Antigen loss escape variant," as used herein, refers to a cell that exhibits reduced or lost expression of the target antigen targeted by a CAR provided herein.
[0070] A. Antigenic determinant (AD) An antigenic determinant (AD) is an epitope that can be recognized and specifically bound by an antigenic determinant binding domain (ADBD) (e.g., an antigen-binding fragment of an antibody or an alternative scaffold binding domain (ASBD) (e.g., a D domain)). The ADs in the adapters and on target cells provided herein can be bound by the CARs described below.
[0071] In some embodiments, the AD (e.g., in the adapter and / or on the target cell) is an AD present in a naturally occurring protein or other molecule. In some embodiments, the AD is an endogenous AD in humans.
[0072] In some embodiments, the AD in the adaptor is an AD present on a target cell.
[0073] In some embodiments, the AD (e.g., in the adapter and / or on the target cell) is an AD present in a transmembrane protein, e.g., an AD present in the extracellular portion of a transmembrane protein. In some embodiments, the AD is a tumor antigen. In some embodiments, the AD is a tumor-associated antigen. In some embodiments, the AD is a tumor-specific antigen.
[0074] In some embodiments, the AD (e.g., in the adapter and / or on the target cell) is a cancer antigen. In some embodiments, the AD is a cancer-associated antigen. In some embodiments, the AD is a cancer-specific antigen.
[0075] In some embodiments, the AD (e.g., in the adapter and / or on the target cell) is an epitope of BCMA. In some embodiments, the AD is an epitope of CD19. In some embodiments, the AD is an epitope of CD20. In some embodiments, the AD is an epitope of CD22. In some embodiments, the AD is an epitope of CD123. In some embodiments, the AD is an epitope of CD37. In some embodiments, the AD is an epitope of CS1. In further embodiments, the AD is an epitope of CS1 bound by elotuzumab. In some embodiments, the AD is an epitope of HER2. In some embodiments, the AD is an epitope of AFP. In some embodiments, the AD is an epitope of AFP p26. In some embodiments, the AD is an epitope of CD45. In some embodiments, the AD is an epitope of human CD45 bound by the UCHL-1 antibody, the A6 antibody, or the ODP4 antibody. In some embodiments, the AD is an epitope of human CD45 bound by the 4KB5 antibody, MB1 antibody, KiB3 antibody, 2H4 antibody, or MT2 antibody. In some embodiments, the AD is an epitope of CD26. In some embodiments, the AD is an epitope of CD30. In some embodiments, the AD is an epitope of CD33. In some embodiments, the AD is an epitope of CD38.
[0076] In some embodiments, the AD is an epitope of CD 123. In further embodiments, the AD comprises 5-25, 5-50, 5-75, 5-100, 5-125, or 5-150 amino acid residues, more than 150 amino acid residues, or all amino acid residues of SEQ ID NO:1.
[0077] In some embodiments, the AD is an epitope of AFP p26. In further embodiments, the AD comprises 5-25, 5-50, 5-75, 5-100, 5-125, or 5-150 amino acid residues, more than 150 amino acid residues, or all amino acid residues of SEQ ID NO:37. In further embodiments, the AD comprises amino acid residues of SEQ ID NO:37. In further embodiments, the AD comprises 5-25, 5-50, 5-75, 5-100, 5-125, or 5-150 amino acid residues, more than 150 amino acid residues, or all amino acid residues of SEQ ID NO:39. In further embodiments, the AD comprises amino acid residues of SEQ ID NO:39. In further embodiments, the AD comprises amino acid residues of SEQ ID NO:37-43 or 44.
[0078] In some embodiments, the AD (e.g., in the adapter and / or on the target cell) is bound by a chimeric antigen receptor (CAR). In some embodiments, the AD is bound by a cell expressing a chimeric antigen receptor. In some embodiments, the AD (e.g., on the target cell) is bound by an adapter. In some embodiments, the AD is bound by an scFv. In some embodiments, the AD is bound by an alternative scaffold binding domain (ASBD). In some embodiments, the AD is bound by a D domain. In some embodiments, the AD is bound by an antibody or antigen-binding fragment thereof.
[0079] Suitable ADs for use in connection with the DDpps (e.g., adapters and CARs) disclosed herein are essentially those disclosed in International Application Publications WO 2016164305, WO 2016164308A1, WO 2019099440, and WO 2019099433, U.S. Patent Nos. 10,662,248 and 10,647,775, and U.S. Patent Application Nos. 20200223934 and 20210002381, each of which is incorporated herein by reference.
[0080] B. Antigenic Determinant Binding Domain (ADBD) A protein domain that binds to an antigenic determinant (AD) is referred to herein as an "antigenic determinant binding domain" or "ADBD." In some embodiments, the ADBD is sufficient to confer recognition and specific binding to a target of interest. The ADBDs described herein may be present in a DDpp fusion protein, an adapter, and / or a chimeric antigen receptor (CAR).
[0081] In some embodiments, the ADBD (e.g., of a DDpp fusion protein, adapter, and / or CAR) is an antibody or antigen-binding fragment thereof. In some embodiments, the ADBD is an scFv. In some embodiments, the ADBD is an alternative scaffold-binding domain. In some embodiments, the ADBD is a D domain.
[0082] ADBDs suitable for use in connection with the DDpps (e.g., adaptors and CARs) disclosed herein are substantially as disclosed in International Application Publication Nos. WO 2016164305, WO 2016164308A1, WO 2019099440, and WO 2019099433, U.S. Patent Nos. 10,662,248 and 10,647,775, and U.S. Patent Application Nos. 20200223934 and 20210002381, each of which is incorporated herein by reference.
[0083] i. Antibody-derived antigenic determinant binding domain (ADBD) In some embodiments, one or more ADBDs (e.g., of a DDpp fusion protein, an adapter, and / or a CAR) can be derived from an antibody molecule, e.g., a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, e.g., a single-domain antibody, e.g., a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain (VHH), derived from a human or camelid origin. In some embodiments, the ADBD is derived from the same species in which the adapter or CAR will ultimately be used, e.g., for use in humans. It is beneficial for the adapter and / or CAR to comprise a human or humanized ADBD. Compositions and techniques for routinely producing such ADBDs are known in the art.
[0084] In some embodiments, the ADBD (e.g., of a DDpp fusion protein, adapter, and / or CAR) comprises a fragment of an antibody that is sufficient to confer recognition and specific binding to a target antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, or Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies such as sdAbs (either VL or VH), camelid VHH domains, and multispecific antibodies formed from antibody fragments.
[0085] In some embodiments, the ADBD (e.g., of a DDpp fusion protein, adapter, and / or CAR) is an "scFv," which may comprise, for example, a fusion protein comprising the VL and VH chains of an antibody, with the VH and VL linked via a short, flexible polypeptide linker, e.g., a linker described herein. scFvs can be routinely prepared according to methods known in the art (see, e.g., Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)).
[0086] In some embodiments, the ADBD (e.g., of a DDpp fusion protein, adapter, and / or CAR) is a single-domain antigen-binding (SDAB) molecule. SDAB molecules include molecules containing a complementarity-determining region that is part of a single-domain polypeptide. Examples include, but are not limited to, binding molecules that naturally lack a heavy chain variable domain, a light chain, a single domain derived from a traditional four-chain antibody, an engineered domain, and a single-domain scaffold other than that derived from an antibody. SDAB molecules can be derived from any species, including, but not limited to, mouse, human, camel, llama, fish, shark, goat, rabbit, and cow. The term also includes naturally occurring single-domain antibody molecules derived from species other than camelids and sharks.
[0087] In some embodiments, the ADBD (e.g., of the DDpp fusion protein, adapter, and / or CAR) comprises a human antibody or fragment thereof. In some embodiments, the ADBD (e.g., of the DDpp fusion protein, adapter, and / or CAR) comprises a humanized antibody or fragment thereof.
[0088] Antibody humanization is well known in the art and essentially involves substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody, i.e., CDR grafting (EP 239,400; International Patent Publication WO 91 / 09967; and U.S. Pat. Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; and 6,548,640, the contents of which are incorporated herein by reference in their entireties), as described by Winter et al. (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)). Antibody humanization can also be achieved by veneering or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., Protein Engineering 7(6):805-814 (1994); and Roguska et al., PNAS 91:969-973 (1994)) or chain shuffling (U.S. Pat. No. 5,565,332), the contents of which are incorporated herein by reference in their entireties.
[0089] ii. Alternative scaffold-binding domains In some embodiments, the ADBD (e.g., of a DDpp fusion protein, adapter, and / or CAR) is an alternative scaffold-binding domain (ASBD). An "alternative scaffold-binding domain" or "ASBD," as used herein, is an antigenic determinant-binding domain derived from or corresponding to a non-antibody-based binding scaffold.
[0090] In some embodiments, the present disclosure provides a CAR comprising an ADBD that is an ASBD. In some embodiments, the present disclosure provides a cell comprising a CAR comprising an ADBD that is an ASBD. In further embodiments, an immune effector cell is provided comprising a CAR comprising an ASBD. In some embodiments, the present disclosure provides an adaptor comprising an ADBD that is an ASBD.
[0091] In a further embodiment, the present disclosure provides a composition comprising an adaptor and a CAR, each comprising an ASBD.
[0092] In some embodiments, the binding of the ASBD (e.g., of a DDpp fusion protein, an adapter, and / or a CAR) to the target AD is mediated by the secondary structure of the binding scaffold, for example, an α-helix or a β-sheet. In some embodiments, the ASBD is a three-helix bundle-based binding domain. In some embodiments, the ASBD is a D domain-based binding domain. In other embodiments, the ASBD is a Z domain (affibody)-based binding domain.
[0093] In some embodiments, the ASBD (e.g., of a DDpp fusion protein, adapter, and / or CAR) is a D domain (de novo binding domain)-based AD binding domain. In some embodiments, the D domain comprises a sequence selected from the group of SEQ ID NOs: 8-33, 99, and 100. In some embodiments, the D domain comprises a sequence selected from the group of SEQ ID NOs: 8, 13, 14, 31, 32, and 33. In some embodiments, the D domain comprises the sequence of SEQ ID NO: 14. In some embodiments, the D domain comprises a sequence selected from the group of SEQ ID NOs: 74-93 and 94. In some embodiments, the D domain comprises a sequence selected from the group of SEQ ID NOs: 70-73 and 92-94. In some embodiments, the D domain comprises the sequence of SEQ ID NO: 73.
[0094] In some embodiments, the ASBD (e.g., of a DDpp fusion protein, adapter, and / or CAR) is a Z-domain scaffold (affibody)-based AD binding domain. Z-domain scaffold-based binding domains generally consist of 58 amino acid residues, with substitutions at up to 13 positions located in the first and second of three α-helices conferring recognition and binding specificity for the target of interest (AD). Z-domain (affibody) scaffold-based binding domains are further described in U.S. Patent No. 5,831,012, the entire contents of which are incorporated herein by reference.
[0095] Additional examples of ASBDs that exhibit secondary structure-mediated target binding include DARPins, affilins, and armadillo repeat-based binding scaffolds.
[0096] In some embodiments, the ASBD (eg, of a DDpp fusion protein, an adaptor, and / or a CAR) is a DARPin-based AD-binding domain.
[0097] In some embodiments, the ASBD is an adnectin-based AD binding domain. The adnectin-based binding domain is derived from the 10th domain of fibronectin type III (10Fn3). This ADBD is a 94-amino acid binding domain that generally adopts a beta-sandwich fold containing seven chains connected by six loops. Substitutions in three surface-exposed loops on one side of the adnectin domain generate target (AD)-specific binding moieties.
[0098] In some embodiments, the ASBD (e.g., of a DDpp fusion protein, adaptor, and / or CAR) is a lipocalin-, affilin-, or anticalin-based AD-binding domain. The anticalin scaffold exhibits a conserved β-barrel structure composed of eight antiparallel β-strands and generally consists of 160-180 amino acids. The ligand-binding pocket of anticalin-based binding scaffolds is composed of four loops, each containing up to 24 substitutions, that collectively confer target (AD) recognition and binding specificity.
[0099] In some embodiments, the ASBD (e.g., of a DDpp fusion protein, adapter, and / or CAR) is an avimer scaffold-based AD binding domain. Avimer scaffold-based binding domains are derived from the A domain of cell surface receptors and are generally 35 amino acids in length. Avimer scaffold-based binding domains are further described in U.S. Patent Application Publication Nos. 20040175756, 20050053973, 20050048512, and 20060008844 (the entire contents of each of which are incorporated herein by reference).
[0100] In some embodiments, the ASBD (e.g., of a DDpp fusion protein, adapter, and / or CAR) is a fynomer-scaffold-based AD-binding domain. The fynomer-binding region is generally 60-75 amino acids long and consists of a pair of antiparallel β-sheets joined by two flexible loops. Substitutions / insertions in the loops confer AD target recognition and binding specificity.
[0101] In some embodiments, the ASBD (e.g., of a DDpp fusion protein, adapter, and / or CAR) is a knottin scaffold-based AD-binding domain. Knottin scaffold-based binding domains correspond to a 30 amino acid protein fold composed of three antiparallel β-strands connected by loops of variable length and multiple disulfide bonds.
[0102] In some embodiments, the ASBD (e.g., of a DDpp fusion protein, adapter, and / or CAR) is a Kunitz domain-based AD binding domain. Kunitz domain-based binding domains are derived from the active motif of Kunitz-type protease inhibitors and are generally about 60 amino acids long. The hydrophobic core of this ADBD is composed of a twisted two-stranded antiparallel β-sheet and two α-helices stabilized by three pairs of disulfide bonds. Substitutions and insertions in three loops confer AD target recognition and binding specificity. Kunitz scaffold-based binding domains are further described in International Application Publication No. WO 2004063337, the entire contents of which are incorporated herein by reference.
[0103] In some embodiments, the ASBD (eg, of a DDpp fusion protein, an adaptor, and / or a CAR) is a WW domain-based AD-binding domain.
[0104] C. Linker A linker is a peptide or other chemical linkage positioned between otherwise independent functional domains of a DDpp fusion protein, an adapter, or a CAR.
[0105] Linkers suitable for operably linking two or more functional domains of a DDpp and additional component of a DDpp fusion protein, or an adaptor, into a single amino acid sequence include, but are not limited to, polypeptide linkers, such as glycine linkers, serine linkers, mixed glycine / serine linkers, glycine-rich linkers, serine-rich linkers, or linkers composed of predominantly polar polypeptide fragments.
[0106] In one embodiment, the linker is composed of a majority of amino acids selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In one embodiment, one or more linkers in a DDpp fusion protein, adapter, or CAR are composed of a majority of amino acids selected from glycine, alanine, proline, asparagine, aspartic acid, threonine, glutamine, and lysine. In one embodiment, one or more linkers in a DDpp fusion protein, adapter, or CAR are composed of one or more amino acids selected from glycine, alanine, proline, asparagine, aspartic acid, threonine, glutamine, and lysine. In another embodiment, one or more linkers in a DDpp fusion protein, adapter, or CAR are composed of a majority of amino acids that are sterically unhindered. In another embodiment, a linker in which the majority of amino acids are glycine, serine, and / or alanine. In some embodiments, the peptide linker is selected from polyglycines, such as (Gly)5 (SEQ ID NO:45) and (Gly)8 (SEQ ID NO:46), poly(Gly-Ala), and polyalanines. In some embodiments, the peptide linker contains the sequence Gly-Gly-Gly-Gly-Thr-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:47). In some embodiments, the peptide linker contains the sequence Gly-Gly-Gly-Gly-Asp-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:48). In some embodiments, the peptide linker contains the sequence of SEQ ID NO:118 or 119.
[0107] In one embodiment, the DDpp fusion protein, adapter, or CAR comprises an ADBD (e.g., D domain) attached directly (i.e., without a linker) to another component of the DDpp fusion protein, adapter, or CAR, respectively. In one embodiment, the DDpp fusion protein, adapter, or CAR contains at least two, at least three, at least four, or at least five ADBDs (e.g., D domains) attached directly to another domain of the DDpp fusion protein, adapter, or CAR, respectively.
[0108] In another embodiment, an ADBD (e.g., a D domain) may be operably linked to another component of a DDpp fusion protein, adapter, or CAR through a linker. A DDpp fusion protein, adapter, or CAR may contain a single linker, multiple linkers, or no linker. In one embodiment, a DDpp fusion protein, adapter, or CAR comprises an ADBD (e.g., a D domain) operably linked to another component of a DDpp fusion protein, adapter, or CAR, respectively, through a linker peptide. In one embodiment, a DDpp fusion protein, adapter, or CAR contains at least two, at least three, at least four, or at least five ADBDs (e.g., D domains) operably linked to another domain of a DDpp fusion protein, adapter, or CAR, respectively, through the same or different linkers.
[0109] The linker can be of any size or composition, so long as it is capable of operably linking the functional domains of the DDpp fusion protein, adapter, or CAR in a manner that allows the functional domains to function (e.g., the ability of an antigenic determinant binding domain to bind to a target of interest). In some embodiments, the linker is about 1 to about 100 amino acids, about 1 to 50 amino acids, about 1 to 20 amino acids, about 1 to 15 amino acids, about 1 to 10 amino acids, about 1 to 5 amino acids, about 2 to 20 amino acids, about 2 to 15 amino acids, about 2 to 10 amino acids, or about 2 to 5 amino acids. It should be apparent that the length, degree of flexibility, and / or other characteristics of the linker can have some effect on the properties of the final polypeptide of the invention, such as, but not limited to, affinity, specificity, or avidity for a target of interest or one or more other target proteins of interest. When more than one linker is used in a DDpp fusion protein, adapter, or CAR, these linkers can be the same or different. In the context and disclosure provided herein, one of skill in the art will be able to routinely determine the optimal linker composition and length for operably linking the functional domains of a DDpp fusion protein, an adapter, or a CAR.
[0110] The linker may be a non-peptide linker, such as an alkyl linker or a PEG linker. For example, an alkyl linker, such as -NH-(CH2)sC(0)-, where s=2 to 20, may be used. These alkyl linkers may be further substituted with a non-sterically hindering group, such as lower alkyl, e.g., C1 to C6 lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc. An exemplary non-peptide linker is a PEG linker. In certain embodiments, the PEG linker has a molecular weight of about 100 to 5,000 kDa or about 100 to 500 kDa.
[0111] Linkers suitable for coupling functional domains of DDpp fusion proteins, adapters, or CARs by chemical cross-linking include, but are not limited to, homobifunctional chemical cross-linking compounds such as glutaraldehyde, imidoesters such as dimethyl adipimidate (DMA), dimethyl suberimidate (DMS), and dimethyl pimelimidate (DMP), or N-hydroxysuccinimide (NHS) esters such as dithiobis(succinimidyl propionate) (DSP) and dithiobis(sulfosuccinimidyl propionate) (DTSSP). Examples of linkers suitable for coupling functional domains of DDpp fusion proteins, adapters, or CARs include, but are not limited to, cross-linkers with one amine-reactive end and a sulfhydryl-reactive moiety at the other end, or cross-linkers with an NHS ester at one end and an SH-reactive group (e.g., maleimide or pyridyl).
[0112] In additional embodiments, one or more of the linkers in the DDpp fusion protein, adapter, or CAR are cleavable. Examples of cleavable linkers include, but are not limited to, peptide sequences recognized by various types of proteases (in vitro or in vivo), such as Tev, thrombin, Factor Xa, plasmin (a blood protease), metalloproteases, cathepsins (e.g., GFLG, etc.), and proteases found in other body compartments.
[0113] In some embodiments, the linker is a "cleavable linker" that facilitates release of the DDpp fusion protein functional domain, adapter functional domain, or cytotoxic substance within or on the cell surface. For example, an acid-labile linker (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (see, e.g., Chari, Can. Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020; and U.S. Patent Application Publication No. 20090110753, the contents of each of which are incorporated herein by reference in their entirety) can be used, where the covalent bond between the DDpp or cytotoxic substance and the fusion partner is desirably cleaved intracellularly when the composition is transferred into the cell. The terms "cleaved intracellularly" and "intracellular cleavage" refer to an intracellular metabolic process or reaction to a DDpp drug conjugate in which the covalent attachment, i.e., the linkage via a linker, between the DDpp and the cytotoxic agent, between the DDpp and the fusion partner, or between two DDpps is broken down intracellularly, resulting in a dissociated free DDpp and / or cytotoxic agent.
[0114] In additional embodiments, one or more of the linkers in the CAR are cleavable. Examples of cleavable linkers include, but are not limited to, peptide sequences recognized by various types of proteases (in vitro or in vivo), such as Tev, thrombin, Factor Xa, plasmin (a blood protease), metalloproteases, cathepsins (e.g., GFLG, etc.), and proteases found in other body compartments.
[0115] In some embodiments, short oligopeptide or polypeptide linkers, approximately 1 to 100 amino acids in length, are used to link any of the domains of the CAR. The linker may be composed of flexible residues, such as glycine and serine (or other amino acids), so that adjacent protein domains can move freely relative to one another. The amino acid sequence composition of the linker may be selected to minimize the potential immunogenicity of the CAR. Longer linkers may be used when it is desirable to ensure that two adjacent domains do not sterically interfere with one another.
[0116] In some embodiments, preferably, 2 to 10 amino acids in length form the link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. In further embodiments, the linker is 10 to 15 amino acids in length, or 15 to 20, or 20 to 30, or 30 to 60, or 60 to 100 amino acids in length (or any range intermediate between the recited ranges). In further embodiments, the linker is a glycine-serine doublet sequence. In some embodiments, the extracellular spacer domain (ESD) corresponds to the human T-cell surface glycoprotein CD8 α-chain ESD region (e.g., amino acid residues 138-182 of the CD8 α-chain (Swiss-Prot Acc. No. P01732)). In some embodiments, the ESD corresponds to a CD8 ESD region that has been further modified through amino acid substitutions to improve expression function or immunogenicity. In further embodiments, the ESD corresponds to the CD28 ESD or a sequence containing modifications of the CD28 ESD that confer improved expression function or immunogenicity.
[0117] Linker optimization can be evaluated using techniques described herein and / or otherwise known in the art. In some embodiments, the linker does not interfere with the ability of the DDpp fusion protein, adapter, or CAR to bind to a target antigenic determinant and / or a functional domain of another adapter or CAR to function properly (e.g., the ability of an effector function domain in the adapter to elicit effector function, or the ability of an FcRn-binding domain in the adapter to bind to FcRn).
[0118] D domain polypeptide (DDpp) In various embodiments, the present disclosure provides a DDpp that specifically binds to CD123. In some embodiments, the DDpp comprises a D domain (DD) that specifically binds to CD123 and comprises the amino acid sequence of SEQ ID NOs: 8-32 or 33. In some embodiments, the D domain that binds to CD123 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 13, 14, 31, 32, and 33. In some embodiments, the D domain that binds to CD123 comprises the amino acid sequence of SEQ ID NO: 14. Proteins comprising variants of the D domain that retain the ability to specifically bind to their respective targets are also provided.
[0119] In some embodiments, DDpp comprises a D domain (DD) that specifically binds to AFP p26 and comprises the amino acid sequence of SEQ ID NOs: 74-93 or 94. Proteins comprising variants of the D domain that retain the ability to specifically bind to their respective targets are also provided.
[0120] In some embodiments, DDpp is fused to a heterologous polypeptide. In some embodiments, the heterologous polypeptide comprises a full-length antibody or an antibody fragment. In some embodiments, DD is fused to the amino terminus of a full-length antibody heavy chain; the amino terminus of a full-length antibody light chain; the carboxyl terminus of a full-length antibody heavy chain; or the carboxyl terminus of a full-length antibody light chain. In other embodiments, DD is fused to an antibody fragment that is Fc. In additional embodiments, the heterologous polypeptide comprises a member selected from the group consisting of: (i) a transmembrane domain; (ii) a membrane-associated domain; (iii) human serum albumin or a fragment thereof; (iv) an AFP or a fragment thereof; (v) an AFP p26 or a fragment thereof; (vi) an extracellular domain of a receptor or a fragment thereof; and (vii) an extracellular domain of an intracellular receptor (e.g., a nuclear protein) or a fragment thereof. In some embodiments, the protein contains a heterologous polypeptide comprising the extracellular domain, or a fragment of the extracellular domain, of BCMA (SEQ ID NO:34), CD123 (SEQ ID NO:1), CD19 (SEQ ID NO:95), or CS1 (SEQ ID NO:35). In some embodiments, the protein contains a heterologous polypeptide comprising the extracellular domain, or a fragment of the extracellular domain, of a receptor selected from the group consisting of CD19, CD20, CD22, HVEM, BTLA, DR3, CD37; TSLPR, IL7R, and gp96. In some embodiments, the protein contains a heterologous polypeptide comprising an antigenic portion of a serum protein (e.g., AFP and AFP p26). In some embodiments, the protein contains a heterologous polypeptide comprising an antigenic portion of an intracellular protein (e.g., a nuclear protein). In some embodiments, the protein is labeled. In further embodiments, the label is selected from the group consisting of an enzymatic label, a fluorescent label, a luminescent label, a bioluminescent label, and a biotin moiety. In additional embodiments, the protein is conjugated to a therapeutic or cytotoxic agent. In some embodiments, the protein contains a heterologous polypeptide that binds to one or more major histocompatibility complexes (MHC) class I or class II complexes.
[0121] In some embodiments, the DD of the DDpp is a variant of a CD123-binding DD reference sequence selected from the group consisting of SEQ ID NOs: 8-33, 99, and 100, which retains the ability to specifically bind to CD123. In some embodiments, the sequence of the variant DD comprises a variant amino acid sequence having at least 75%, 80%, 85%, 87%, 89%, 90%, 92%, 94%, 96%, or 98% sequence identity to a reference DD sequence selected from the group consisting of SEQ ID NOs: 8-33, 99, and 100, and wherein the variant DD retains the ability to specifically bind to CD123.
[0122] In some embodiments, the DD of the DDpp is a variant of an AFP p26-binding DD reference sequence selected from the group consisting of SEQ ID NOs:74-93 and 94, which retains the ability to specifically bind to AFP p26. In some embodiments, the sequence of the variant DD comprises a variant amino acid sequence having at least 75%, 80%, 85%, 87%, 89%, 90%, 92%, 94%, 96%, or 98% sequence identity to a reference DD sequence selected from the group consisting of SEQ ID NOs:74-93 and 94, and wherein the variant DD retains the ability to specifically bind to AFP p26.
[0123] In a specific embodiment, the identity between a variant DD (query) sequence and a reference DD sequence, also referred to as a global sequence alignment, is determined using the FASTDB computer program, which is based on the algorithm of Brutlag et al. Comp. App. Biosci. 6:237-245 (1990). Preferred parameters used in FASTDB amino acid alignment are: matrix=PAM 0, k-tuple=2, mismatch penalty=1, joining penalty=20, randomization group length=0, cutoff score=1, window size=sequence length, gap penalty=5, gap size penalty=0.05, window size=500 or the length of the subject amino acid sequence, whichever is shorter. According to this embodiment, if the reference DD sequence is shorter than the variant DD query sequence due to N- or C-terminal deletions rather than internal deletions, a manual correction is made to the results to take into account the fact that the FASTDB program does not account for the N- and C-terminal truncations of the reference DD sequence when calculating the overall percent identity. In the case of a reference sequence that is N- and C-terminally truncated compared to the query sequence, the percent identity is corrected by calculating the number of query sequence residues at the N- and C-termini of the reference sequence that are not matched / aligned with the corresponding target residues as a percentage of the total bases in the query sequence. Whether a residue is matched / aligned is determined by the results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity calculated by the FASTDB program using the specified parameters to arrive at a final percent identity score. This final percent identity score is the one used for purposes of this embodiment.
[0124] In some embodiments, the disclosed DDpp (e.g., DDpp fusion protein) is labeled. Labels that can be used to label a DDpp include, but are not limited to, enzymatic labels, fluorescent labels, luminescent labels, and bioluminescent labels. In some embodiments, the label is a biotin moiety. In some embodiments, the label is a streptavidin moiety. In some embodiments, the label is a His tag or a FLAG tag. In some embodiments, the label is luciferase, green fluorescent protein, red fluorescent protein, or other similar agent. In some embodiments, the DDpp comprises a CD123-binding DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-33, 99, and 100. In some embodiments, the DDpp comprises a DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 13, 14, 31, 32, and 33. In some embodiments, the DDpp comprises a DD comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the DDpp comprises a p26-binding DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-93 and 94.
[0125] In other embodiments, the DDpp fusion protein is attached to a solid support. In some embodiments, the solid support is selected from the group consisting of beads, glass slides, chips, gelatin, and agarose. In some embodiments, the DDpp comprises a CD123-binding DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-33, 99, and 100. In some embodiments, the DDpp comprises a DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 13, 14, 31, 32, and 33. In some embodiments, the DDpp comprises a DD comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the DDpp comprises a p26-binding DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-93 and 94.
[0126] In some embodiments, the DDpp (e.g., a DDpp fusion protein) is associated with a liposome. In some embodiments, the DDpp is associated with a liposome through a covalent bond. In some embodiments, the DDpp is a fusion protein. In further embodiments, the DDpp is a CAR. In additional embodiments, the DDpp is associated with a liposome through an ionic bond rather than a covalent bond. In some embodiments, the DDpp comprises a CD123-binding DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-33, 99, and 100. In some embodiments, the DDpp comprises a DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 13, 14, 31, 32, and 33. In some embodiments, the DDpp comprises a DD comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the DDpp comprises a p26-binding DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-93 and 94.
[0127] In some embodiments, the target-binding DDpp is conjugated to a therapeutic or cytotoxic agent (e.g., a chemotherapeutic or radiotherapeutic agent). In some embodiments, the DDpp comprises a CD123-binding DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-33, 99, and 100. In some embodiments, the DDpp comprises a DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 13, 14, 31, 32, and 33. In some embodiments, the DDpp comprises a DD comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the DDpp comprises a p26-binding DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-93 and 94.
[0128] Table 1: Exemplary target-specific binding DDs TIFF2024544559000002.tif15160TIFF2024544559000003.tif221160TIFF2024544559000004.tif221160TIFF2024544559000005.tif105160
[0129] In some embodiments, the present disclosure provides compositions comprising one or more of the DD sequences disclosed in Table 1. In other embodiments, the present disclosure provides compositions comprising one or more DDs comprising sequences having 60-70%, 70-75%, 75-80%, 80-85%, 85-90%, 95-99% (and overlapping ranges therein) homology to a sequence disclosed in Table 1. In some embodiments, DDs with such homology are functionally similar or identical compared to their respective reference sequences in Table 1. In some embodiments, the present disclosure provides polypeptides comprising one or more DDs that compete (fully or partially) with one or more of the DD sequences (reference sequences) disclosed in Table 1 for their respective targets. The ability of a polypeptide to compete with a reference polypeptide for binding to its respective target can be routinely determined using standard competition assays known in the art. In some embodiments, competition does not require that the polypeptide compete for the same epitope as polypeptide (DD) of Table 1; rather, the polypeptide may compete by binding to a sterically inhibitory epitope, an overlapping epitope, etc.
[0130] A. CD123-bound DDpp In some embodiments, the present disclosure provides a protein (DDpp) that specifically binds to CD123 (SEQ ID NO:1) and comprises a D domain (DD) target binding domain comprising the amino acid sequence of SEQ ID NOs:8-32 or 33. In some embodiments, the DDpp comprises a DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:8, 13, 14, 31, 32, and 33. In some embodiments, the DDpp comprises a DD comprising the amino acid sequence of SEQ ID NO:14.
[0131] In some embodiments, DD of DDpp specifically binds to CD123. In further embodiments, DD has an amino acid sequence consisting of SEQ ID NO:8 and specifically binds to CD123. In some embodiments, DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:8-33, 99, and 100. In other embodiments, DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs:8-33, 99, and 100. In some embodiments, DDpp specifically binds to CD123 (SEQ ID NO:1) and comprises the amino acid sequence of SEQ ID NO:8, 13, 14, 31, 32, or 33. In some embodiments, DDpp comprises the amino acid sequence of SEQ ID NO:8. In some embodiments, DDpp comprises the amino acid sequence of SEQ ID NO:13. In some embodiments, DDpp comprises the amino acid sequence of SEQ ID NO:14. In some embodiments, DDpp comprises the amino acid sequence of SEQ ID NO:31. In some embodiments, the DDpp comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the DDpp comprises the amino acid sequence of SEQ ID NO: 33.
[0132] In other embodiments, the CD123-binding DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-32 and 33. In some embodiments, the CD123-binding DDpp comprises multiple target-binding domains (e.g., dimers, trimers, etc.) that bind to a single target. In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs that specifically bind to CD123 and have amino acid sequences selected from the group consisting of SEQ ID NOs: 8-33, 99, and 100. In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs that have the same sequence. In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs that specifically bind to different epitopes of CD123 and have amino acid sequences selected from the group consisting of SEQ ID NOs: 8-33, 99, and 100. In some embodiments, the DDpp comprises a DD that specifically binds to CD123 and further comprises two, three, four, five, or more than five additional different DDs or target binding domains (e.g., scFvs) that specifically bind to CD123 or different target antigens. In some embodiments, the DDpp comprises a DD that specifically binds to CD123 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs:8-33, 99, and 100) and further comprises one or more additional DDs or other target binding domains that bind to one or more antigens expressed on the surface of B-cell lineage cells. In some embodiments, the DDpp comprises a DD that specifically binds to CD123, e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs:8-33, 99, and 100, and further comprises one or more additional DDs or other target binding domains that bind to one or more cancer antigens. In some embodiments, the DDpp comprises a DD that specifically binds to CD123 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs:8-33, 99, and 100) and specifically binds to two, three, four, five, or more than five different targets.In further embodiments, the DDpp comprises a DD that specifically binds to CD123 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-33, 99, and 100) and specifically binds to two, three, four, five, or more than five different cancer antigens. In some embodiments, the DDpp comprises a DD that specifically binds to CD123 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-33, 99, and 100) and specifically binds to two, three, four, five, or more than five different cancer antigens expressed on the surface of cancer cells. In some embodiments, the DDpp comprises a DD that specifically binds to CD123 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-33, 99, and 100) and specifically binds to two, three, four, five, or more than five different cancer antigens expressed on the surface of different cancer cells. In some embodiments, the CD123-specific DD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 13, 14, 31, 32, and 33. In some embodiments, the CD123-specific DD comprises the amino acid sequence of SEQ ID NO: 14.
[0133] In some embodiments, the DDpp comprises a variant of a CD123-binding DD (reference DD) disclosed herein that retains the ability to specifically bind to CD123. In some embodiments, the sequence of the CD123-binding DD variant contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 conservative or non-conservative substitutions compared to a reference CD123-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-33, 99, and 100. In some embodiments, the sequence of the CD123-binding DD variant contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 conservative substitutions compared to a reference CD123-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-33, 99, and 100. In some embodiments, the sequence of the CD123-binding DD variant contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 non-conservative substitutions compared to a reference CD123-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-33, 99, and 100. In some embodiments, the reference CD123-binding DD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 13, 14, 31, 32, and 33. In some embodiments, the reference CD123-binding DD comprises the amino acid sequence of SEQ ID NO: 14.
[0134] In some embodiments, the sequence of the CD123-binding DD variant contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1-3, 1-5, or 1-10 conservative or non-conservative substitutions at positions corresponding to amino acid residues 1-22, 29-46, and 52-72 of a reference CD123-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-33, 99, and 100. In some embodiments, the sequence of the CD123-binding DD variant contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1-3, 1-5, or 1-10 conservative substitutions at positions corresponding to amino acid residues 1-22, 29-46, and 52-72 of a reference CD123-DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-33, 99, and 100. In some embodiments, the sequence of the CD123-binding DD variant contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 non-conservative substitutions at positions corresponding to amino acid residues 1 to 22, 29 to 46, and 52 to 72 of a reference CD123-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-33, 99, and 100. In some embodiments, the reference CD123-binding DD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 13, 14, 31, 32, and 33. In some embodiments, the reference CD123-binding DD comprises the amino acid sequence of SEQ ID NO: 14.
[0135] In some embodiments, the sequence of the CD123-binding DD variant contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 conservative or non-conservative substitutions at positions corresponding to amino acid residues 2 to 6, 8 to 10, 12, 13, 15 to 17, 19, 20, 29, 30, 32 to 34, 36, 37, 39 to 41, 43, 44, 52 to 55, 57 to 59, 61, 62, 64 to 66, and 68 to 70 of a reference CD123-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 33, 99, and 100. In some embodiments, the sequence of the CD123-binding DD variant contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 conservative substitutions at positions corresponding to amino acid residues 2 to 6, 8 to 10, 12, 13, 15 to 17, 19, 20, 29, 30, 32 to 34, 36, 37, 39 to 41, 43, 44, 52 to 55, 57 to 59, 61, 62, 64 to 66, and 68 to 70 of a reference DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-33, 99, and 100. In some embodiments, the sequence of the CD123-binding DD variant contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1-3, 1-5, or 1-10 non-conservative substitutions at positions corresponding to amino acid residues 2-6, 8-10, 12, 13, 15-17, 19, 20, 29, 30, 32-34, 36, 37, 39-41, 43, 44, 52-55, 57-59, 61, 62, 64-66, and 68-70 of a reference DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-33, 99, and 100. In some embodiments, the reference CD123-binding DD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 13, 14, 31, 32, and 33. In some embodiments, the reference CD123-binding DD comprises the amino acid sequence of SEQ ID NO: 14.
[0136] In some embodiments, the sequence of the CD123-binding DD variant contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 conservative or non-conservative substitutions at positions corresponding to amino acid residues 7, 11, 14, 18, 21, 28, 31, 35, 38, 42, 45, 53, 56, 60, 63, and 67 of a reference CD123-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs:8-33, 99, and 100. In some embodiments, the sequence of the CD123-binding DD variant contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 conservative substitutions at positions corresponding to amino acid residues 7, 11, 14, 18, 21, 28, 31, 35, 38, 42, 45, 53, 56, 60, 63, and 67 of a reference DD having an amino acid sequence selected from the group consisting of SEQ ID NOs:8-33, 99, and 100. In some embodiments, the sequence of the CD123-binding DD variant contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 non-conservative substitutions at positions corresponding to amino acid residues 7, 11, 14, 18, 21, 28, 31, 35, 38, 42, 45, 53, 56, 60, 63, and 67 of a reference DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-33, 99, and 100. In some embodiments, the reference CD123-binding DD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 13, 14, 31, 32, and 33. In some embodiments, the reference CD123-binding DD comprises the amino acid sequence of SEQ ID NO: 14.
[0137] In some embodiments, the present disclosure provides a CD123-binding DDpp that completely or partially blocks binding of (e.g., overlaps with) an epitope of) a reference DD to CD123, wherein the reference DD has an amino acid sequence selected from SEQ ID NOs: 8-33, 99, and 100. In other embodiments, the present disclosure provides a CD123-binding DDpp that binds to the same epitope of CD123 as a reference DD consisting of an amino acid sequence selected from SEQ ID NOs: 8-33, 99, and 100. In some embodiments, the reference CD123-binding DD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 13, 14, 31, 32, and 33. In some embodiments, the reference CD123-binding DD comprises the amino acid sequence of SEQ ID NO: 14.
[0138] In some embodiments, DDpp is a fusion protein comprising a DD that specifically binds to CD123. In some embodiments, the DD of the DDpp fusion protein specifically binds to CD123 having the amino acid sequence of SEQ ID NO:1. In some embodiments, DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:8-33, 99, and 100. In other embodiments, DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs:8-33, 99, and 100. In some embodiments, DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:8, 13, 14, 31, 32, and 33. In some embodiments, DDpp comprises the amino acid sequence of SEQ ID NO:8. In some embodiments, DDpp comprises the amino acid sequence of SEQ ID NO:13. In some embodiments, DDpp comprises the amino acid sequence of SEQ ID NO:14. In some embodiments, DDpp comprises the amino acid sequence of SEQ ID NO:31. In some embodiments, the DDpp comprises the amino acid sequence of SEQ ID NO:32. In some embodiments, the DDpp comprises the amino acid sequence of SEQ ID NO:33. In some embodiments, the DDpp fusion protein comprises a full-length antibody or a portion (fragment) of an antibody. In some embodiments, the DDpp fusion protein comprises a full-length IgG antibody (e.g., IgG1, IgG2, IgG2, or IgG4). In further embodiments, the DDpp fusion protein comprises a full-length antibody that specifically binds to a cancer antigen. In further embodiments, the DDpp comprises a commercially available, approved therapeutic antibody (e.g., rituximab, ofatumumab, ocrelizumab, veltuzumab, MEDI-551, epratuzumab, belimumab, tabalumab, AMG-557, MEDI-570, and NN882). In other embodiments, the CD123-binding DDpp is an Fc-fusion protein.
[0139] In some embodiments, the DDpp is a fusion protein comprising a CD123-binding DD operably linked to a serum protein. In some embodiments, the CD123-binding DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-33, 99, and 100. In some embodiments, the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 13, 14, 31, 32, and 33. In some embodiments, the DDpp comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the DDpp comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the DDpp comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the DDpp comprises the amino acid sequence of SEQ ID NO: 31. In some embodiments, the DDpp comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the DDpp comprises the amino acid sequence of SEQ ID NO: 33. In other embodiments, the CD123-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs:8-33, 99, and 100. In some embodiments, the CD123-binding DDpp fusion protein comprises all or a portion of human serum albumin. In some embodiments, the DDpp fusion protein comprises AFP (SEQ ID NO:36) or a fragment thereof. In some embodiments, the CD123-binding DDpp fusion protein comprises AFP p26 (SEQ ID NO:37) or a fragment thereof. In some embodiments, the CD123-binding DDpp fusion protein comprises a polypeptide having the sequence of SEQ ID NOs:37-43 or 44. In some embodiments, the DDpp fusion protein contains a fragment of a serum protein (e.g., AFP and AFP p26), or an antigenic fragment of a serum protein. In some embodiments, the DDpp fusion protein comprises a fragment of a serum protein consisting of between 5 and 500, 5 and 400, 5 and 300, 5 and 200, 5 and 100, 5 and 50, 10 and 500, 10 and 400, 10 and 300, 10 and 200, 10 and 100, or 10 and 50 amino acids.In some embodiments, the CD123-specific DD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 13, 14, 31, 32, and 33. In some embodiments, the CD123-specific DD comprises the amino acid sequence of SEQ ID NO: 14.
[0140] In some embodiments, the CD123-binding DDpp fusion protein comprises the extracellular domain of a receptor or a fragment thereof. In some embodiments, the DDpp fusion protein comprises a DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:8-33, 99, and 100. In some embodiments, the CD123-binding DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:8, 13, 14, 31, 32, and 33. In some embodiments, the CD123-binding DDpp comprises the amino acid sequence of SEQ ID NO:8. In some embodiments, the CD123-binding DDpp comprises the amino acid sequence of SEQ ID NO:13. In some embodiments, the CD123-binding DDpp comprises the amino acid sequence of SEQ ID NO:14. In some embodiments, the CD123-binding DDpp comprises the amino acid sequence of SEQ ID NO:31. In some embodiments, the CD123-binding DDpp comprises the amino acid sequence of SEQ ID NO:32. In some embodiments, the CD123-binding DDpp comprises the amino acid sequence of SEQ ID NO:33. In other embodiments, the CD123-binding DDpp fusion protein comprises a variant of the amino acid sequence selected from the group consisting of SEQ ID NOs:8-33, 99, and 100. In further embodiments, the CD123-binding DDpp fusion protein comprises the extracellular domain of CD123 (SEQ ID NO:1) or a fragment thereof. In some embodiments, the CD123-binding DDpp fusion protein comprises the extracellular domain of a receptor selected from the group consisting of CD19, CD20, CD22, HVEM, BTLA, DR3, CD37; TSLPR, IL7R, NKG2D, and gp96, or a fragment thereof.
[0141] In some embodiments, the CD123-binding DDpp fusion protein contains a fragment of 5-500, 5-400, 5-300, 5-200, 5-100, 5-50, 10-500, 10-400, 10-300, 10-200, 10-100, or 10-50 amino acids of the extracellular domain of a cell surface receptor. In some embodiments, the DDpp fusion protein contains a fragment of the extracellular domain of BCMA (SEQ ID NO:34). In some embodiments, the DDpp fusion protein contains a fragment of the extracellular domain of CD123 (SEQ ID NO:1). In some embodiments, the DDpp fusion protein contains a fragment of the extracellular domain of CS1 (SEQ ID NO:35). In some embodiments, the DDpp contains a fragment of the extracellular domain of a receptor selected from the group consisting of CD19, CD20, CD22, HVEM, BTLA, DR3, CD37; TSLPR, IL7R, and gp96.
[0142] In additional embodiments, the CD123-binding DDpp fusion protein comprises an intracellular protein (e.g., a nuclear protein) or a fragment thereof. In some embodiments, the DDpp fusion protein comprises a DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:8-33, 99, and 100. In some embodiments, the CD123-binding DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:8, 13, 14, 31, 32, and 33. In some embodiments, the CD123-binding DDpp comprises the amino acid sequence of SEQ ID NO:8. In some embodiments, the CD123-binding DDpp comprises the amino acid sequence of SEQ ID NO:13. In some embodiments, the CD123-binding DDpp comprises the amino acid sequence of SEQ ID NO:14. In some embodiments, the CD123-binding DDpp comprises the amino acid sequence of SEQ ID NO:31. In some embodiments, the CD123-binding DDpp comprises the amino acid sequence of SEQ ID NO:32. In some embodiments, the CD123-binding DDpp comprises the amino acid sequence of SEQ ID NO: 33. In other embodiments, the CD123-binding DDpp fusion protein comprises a variant of the amino acid sequence selected from the group consisting of SEQ ID NOs: 8-33, 99, and 100. In some embodiments, the CD123-binding DDpp fusion protein comprises a fragment of an intracellular protein (e.g., a nuclear protein) consisting of 5 to 500, 5 to 400, 5 to 300, 5 to 200, 5 to 100, 5 to 50, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, or 10 to 50 amino acid residues.
[0143] B. AFP p26 binding DDpp In some embodiments, the present disclosure provides a protein (DDpp) that specifically binds to AFP p26 (SEQ ID NO:37) and comprises a D domain (DD) target binding domain comprising the amino acid sequence of SEQ ID NOs:74-93 or 94.
[0144] In some embodiments, DD of the DDpp specifically binds to AFP p26. In further embodiments, DD of the DDpp specifically binds to AFP p26 having the amino acid sequence of SEQ ID NO:37. In some embodiments, the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:74-93 and 94. In additional embodiments, the AFP p26-binding DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs:74-93 and 94. In some embodiments, the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:92, 93, and 94. In some embodiments, the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:70-73, 92, 93, and 94. In some embodiments, the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:73.
[0145] In further embodiments, the DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs:74-93 and 94. In further embodiments, the DD specifically binds to AFP p26 having an amino acid sequence consisting of SEQ ID NO:37, but does not specifically bind to AFP having an amino acid sequence consisting of SEQ ID NO:36. In some embodiments, the AFP p26-binding DDpp comprises multiple target-binding domains (e.g., dimers, trimers, etc.) that bind to a single target. In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs that specifically bind to AFP p26 and have amino acid sequences selected from the group consisting of SEQ ID NOs:74-93 and 94. In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs with the same sequence. In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs that specifically bind to different epitopes of AFP p26 and have amino acid sequences selected from the group consisting of SEQ ID NOs:74-93 and 94. In some embodiments, the DDpp comprises a DD that specifically binds to AFP p26 and further comprises two, three, four, five, or more than five additional different DDs or target binding domains (e.g., scFvs) that specifically bind to AFP p26 or different target antigens. In some embodiments, the DDpp comprises a DD that specifically binds to AFP p26 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs:74-93 and 94) and further comprises one or more additional DDs or other target binding domains that bind to one or more antigens expressed on the surface of B cells. In some embodiments, the DDpp comprises a DD that specifically binds to AFP p26, e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs:74-93 and 94, and further comprises one or more additional DDs or other target binding domains that bind to one or more cancer antigens.In some embodiments, the DDpp comprises a DD that specifically binds to AFP p26 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-93 and 94) and specifically binds to two, three, four, five, or more than five different targets. In further embodiments, the DDpp comprises a DD that specifically binds to AFP p26 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-93 and 94) and specifically binds to two, three, four, five, or more than five different cancer antigens. In some embodiments, the DDpp comprises a DD that specifically binds to AFP p26 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-93 and 94) and specifically binds to two, three, four, five, or more than five different cancer antigens expressed on the surface of cancer cells. In some embodiments, the DDpp comprises a DD that specifically binds to AFP p26 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-93 and 94) and specifically binds to two, three, four, five, or more than five cancer antigens expressed on the surface of different cancer cells.
[0146] In some embodiments, the DDpp comprises a variant of an AFP p26-binding DD (reference DD) disclosed herein that retains the ability to specifically bind to AFP p26. In some embodiments, the sequence of the AFP p26-binding DD variant contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 conservative or non-conservative substitutions compared to a reference AFP p26-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-93 and 94. In some embodiments, the sequence of the AFP p26-binding DD variant contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 conservative substitutions compared to a reference AFP p26-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-93 and 94. In some embodiments, the sequence of the AFP p26-binding DD variant contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 non-conservative substitutions compared to a reference AFP p26-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-93 and 94.
[0147] In some embodiments, the sequence of the AFP p26-binding DD variant contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1-3, 1-5, or 1-10 conservative or non-conservative substitutions at positions corresponding to amino acid residues 1-22, 29-46, and 52-72 of a reference AFP p26-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-93 and 94. In some embodiments, the sequence of the AFP p26-binding DD variant contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1-3, 1-5, or 1-10 conservative substitutions at positions corresponding to amino acid residues 1-22, 29-46, and 52-72 of a reference AFP p26-DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-93 and 94. In some embodiments, the sequence of the AFP p26-binding DD variant contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 non-conservative substitutions at positions corresponding to amino acid residues 1 to 22, 29 to 46, and 52 to 72 of a reference AFP p26-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-93 and 94. In some embodiments, the reference p26-binding DD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 70-73 and 92-94. In some embodiments, the reference p26-binding DD comprises the amino acid sequence of SEQ ID NO: 14.
[0148] In some embodiments, the sequence of the AFP p26-binding DD variant contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 conservative or non-conservative substitutions at positions corresponding to amino acid residues 2-6, 8-10, 12, 13, 15-17, 19, 20, 29, 30, 32-34, 36, 37, 39-41, 43, 44, 52-55, 57-59, 61, 62, 64-66, and 68-70 of a reference AFP p26-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-93 and 94. In some embodiments, the sequence of the AFP p26-binding DD variant contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1-3, 1-5, or 1-10 conservative substitutions at positions corresponding to amino acid residues 2-6, 8-10, 12, 13, 15-17, 19, 20, 29, 30, 32-34, 36, 37, 39-41, 43, 44, 52-55, 57-59, 61, 62, 64-66, and 68-70 of a reference DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-93 and 94. In some embodiments, the sequence of the AFP p26-binding DD variant contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1-3, 1-5, or 1-10 non-conservative substitutions at positions corresponding to amino acid residues 2-6, 8-10, 12, 13, 15-17, 19, 20, 29, 30, 32-34, 36, 37, 39-41, 43, 44, 52-55, 57-59, 61, 62, 64-66, and 68-70 of a reference DD having an amino acid sequence selected from the group consisting of SEQ ID NOs:74-93 and 94. In some embodiments, the reference AFP p26-binding DD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:70-73 and 92-94. In some embodiments, the reference AFP p26-binding DD comprises the amino acid sequence of SEQ ID NO:73.
[0149] In some embodiments, the sequence of the AFP p26-binding DD variant contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 conservative or non-conservative substitutions at positions corresponding to amino acid residues 7, 11, 14, 18, 21, 28, 31, 35, 38, 42, 45, 53, 56, 60, 63, and 67 of a reference AFP p26-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-93 and 94. In some embodiments, the sequence of the AFP p26-binding DD variant contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 conservative substitutions at positions corresponding to amino acid residues 7, 11, 14, 18, 21, 28, 31, 35, 38, 42, 45, 53, 56, 60, 63, and 67 of a reference DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-93 and 94. In some embodiments, the sequence of the AFP p26-binding DD variant contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 non-conservative substitutions at positions corresponding to amino acid residues 7, 11, 14, 18, 21, 28, 31, 35, 38, 42, 45, 53, 56, 60, 63, and 67 of a reference DD having an amino acid sequence selected from the group consisting of SEQ ID NOs:74-93 and 94. In some embodiments, the reference AFP p26-binding DD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:70-73 and 92-94. In some embodiments, the reference AFP p26-binding DD comprises the amino acid sequence of SEQ ID NO:73.
[0150] In some embodiments, the present disclosure provides an AFP p26-binding DDpp that completely or partially blocks (e.g., overlaps with) the binding of a reference DD to AFP p26, wherein the reference DD has an amino acid sequence selected from SEQ ID NOs:74-93 and 94. In other embodiments, the present disclosure provides an AFP p26-binding DDpp that binds to the same epitope of AFP p26 as a reference DD consisting of an amino acid sequence selected from SEQ ID NOs:74-93 and 94. In some embodiments, the reference p26-binding DD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:70-73 and 92-94. In some embodiments, the reference p26-binding DD comprises the amino acid sequence of SEQ ID NO:73.
[0151] In some embodiments, the DDpp is a fusion protein comprising a DD that specifically binds to AFP p26. In some embodiments, the DD specifically binds to AFP p26 having the amino acid sequence of SEQ ID NO:37. In some embodiments, the DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:74-93 and 94. In some embodiments, the DD of the DDpp fusion protein specifically binds to AFP p26 but does not specifically bind to an AFP having the amino acid sequence of SEQ ID NO:36. In other embodiments, the DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs:74-93 and 94. In other embodiments, the AFP p26-binding DDpp is an Fc fusion protein.
[0152] In some embodiments, DDpp is a fusion protein comprising a DD that specifically binds to AFP p26. In some embodiments, DDpp is a fusion protein comprising a DD that specifically binds to AFP p26 having the amino acid sequence of SEQ ID NO:37. In further embodiments, DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:74-93 and 94. In other embodiments, DDpp is a fusion protein comprising an AFP p26-binding DD that is a variant of a DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:74-93 and 94. In some embodiments, DDpp is a fusion protein comprising a DD that specifically binds to AFP p26 operably linked to a full-length antibody or a portion (fragment) of an antibody. In some embodiments, DDpp is an Fc-fusion protein. In some embodiments, the DDpp fusion protein comprises a full-length IgG antibody (e.g., IgG1, IgG2, IgG2, or IgG4). In further embodiments, the DDpp fusion protein comprises a full-length antibody that specifically binds to a cancer antigen. In further embodiments, the DDpp comprises a commercially available approved therapeutic antibody (e.g., rituximab, ofatumumab, ocrelizumab, veltuzumab, MEDI-551, epratuzumab, belimumab, tabalumab, AMG-557, MEDI-570, NN882, elotuzumab, and daratumumab). In other embodiments, the AFP p26-binding DDpp is an Fc-fusion protein. In further embodiments, the Fc-fusion protein comprises a variant human Fc domain.
[0153] In some embodiments, DDpp is a fusion protein comprising an AFP p26-binding DD operably linked to a serum protein. In some embodiments, the DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-93 and 94. In other embodiments, the AFP p26-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-93 and 94. In further embodiments, the DDpp fusion protein comprises human serum albumin or a fragment thereof. In some embodiments, the DDpp fusion protein contains a fragment of a serum protein or an antigenic fragment of a serum protein. In some embodiments, the DDpp fusion protein comprises a fragment of 5-500, 5-400, 5-300, 5-200, 5-100, 5-50, 10-500, 10-400, 10-300, 10-200, 10-100, or 10-50 amino acids of a serum protein.
[0154] In some embodiments, the AFP p26-binding DDpp fusion protein comprises the extracellular domain of a receptor or a fragment thereof. In some embodiments, the DDpp fusion protein comprises a DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:74-93 and 94. In other embodiments, the AFP p26-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs:74-93 and 94. In further embodiments, the AFP p26-binding DDpp fusion protein comprises the extracellular domain of BCMA (SEQ ID NO:34) or CD123 (SEQ ID NO:1), or a fragment thereof. In still further embodiments, the AFP p26-binding DDpp fusion protein comprises the extracellular domain of BCMA (SEQ ID NO:34), CD123 (SEQ ID NO:1), or CS1 (SEQ ID NO:35), or a fragment thereof. In some embodiments, the AFP p26-binding DDpp fusion protein comprises the extracellular domain of a receptor selected from the group consisting of CD19, CD20, CD22, HVEM, BTLA, DR3, CD37; TSLPR, IL7R, and gp96, or a fragment thereof.
[0155] In some embodiments, the AFP p26-binding DDpp fusion protein contains a fragment of 5-500, 5-400, 5-300, 5-200, 5-100, 5-50, 10-500, 10-400, 10-300, 10-200, 10-100, or 10-50 amino acids of the extracellular domain of a cell surface receptor. In some embodiments, the DDpp fusion protein contains a fragment of the extracellular domain of BCMA (SEQ ID NO:34) or CD123 (SEQ ID NO:1). In some embodiments, the DDpp fusion protein contains a fragment of the extracellular domain of BCMA (SEQ ID NO:34) or CD123 (SEQ ID NO:1) or CS1 (SEQ ID NO:35). In some embodiments, the DDpp contains a fragment of the extracellular domain of a receptor selected from the group consisting of CD19, CD20, CD22, HVEM, BTLA, DR3, CD37; TSLPR, IL7R, and gp96.
[0156] In additional embodiments, the AFP p26-binding DDpp fusion protein comprises an intracellular protein (e.g., a nuclear protein) or a fragment thereof. In some embodiments, the DDpp fusion protein comprises a DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-93 and 94. In other embodiments, the AFP p26-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-93 and 94. In some embodiments, the AFP p26-binding DDpp fusion protein comprises a fragment of 5-500, 5-400, 5-300, 5-200, 5-100, 5-50, 10-500, 10-400, 10-300, 10-200, 10-100, or 10-50 amino acid residues of an intracellular protein (e.g., a nuclear protein). In some embodiments, the AFP p26-binding DDpp fusion protein comprises a fragment of the extracellular domain of a serum protein (e.g., HSA), a receptor (e.g., BCMA, CS1, CD123, and CD19), or an intracellular protein (e.g., a nuclear protein) consisting of 5 to 500, 5 to 400, 5 to 300, 5 to 200, 5 to 100, 5 to 50, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, or 10 to 50 amino acid residues.
[0157] Nucleic acids encoding DDpp and vectors containing the nucleic acids are also provided. Host cells containing the nucleic acids and vectors containing the nucleic acids (including viral particles) are also provided. In some embodiments, the host cell is a prokaryotic or eukaryotic cell that displays a variant DD on its surface. In some embodiments, the host cell displays a variant DD on its surface. In further embodiments, the host cell is a phage that displays a variant DD on its surface. In further embodiments, the host cell is a human immune cell that expresses a variant DD fusion protein on its surface.
[0158] A DDpp agonist refers to a DDpp that in some way increases or enhances the biological activity of a DDpp target (e.g., CD123), or has biological activity comparable to that of a known agonist of the DDpp target. In another embodiment, the DDpp is an antagonist of the target to which it binds (e.g., CD123). A DDpp antagonist refers to a DDpp that completely or partially blocks or in some way interferes with the biological activity of a DDpp target protein, or has biological activity comparable to that of a known antagonist or inhibitor of the DDpp target protein.
[0159] DDpp fusion protein DDpp fusion proteins are provided herein. "Fusion proteins," "chimeric polypeptides," "chimeric proteins," "chimeric antigens," and DDpps containing / containing heterologous polypeptides are polypeptides that contain at least two polypeptides, optionally including a linker to operably link the two polypeptides into a single continuous polypeptide, for example, produced by recombinant processes. The two polypeptides may be operably attached directly or indirectly.
[0160] The "DDpp fusion proteins" provided herein comprise at least one DDpp disclosed herein that specifically binds to a target of interest (e.g., BCMA (SEQ ID NO:34), CD123 (SEQ ID NO:1), CS1 (SEQ ID NO:35), HER2, AFP (SEQ ID NO:36), AFP p26 (SEQ ID NO:37), or a fragment thereof). In one embodiment, the DDpp fusion protein contains one DDpp.
[0161] In some embodiments, the DDpp fusion protein is a soluble protein comprising one or more target-binding DDpp and a p26 protein (e.g., having the sequence of SEQ ID NOs: 37-43 or 44). In some embodiments, the soluble DDpp fusion protein has an in vivo plasma half-life of at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 16 hours, at least 32 hours, at least 64 hours, or more than 65 hours. In some embodiments, the soluble fusion protein has an in vivo plasma half-life in mice of at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 16 hours, at least 32 hours, at least 64 hours, or greater than 65 hours, or 1-10 hours, 2-10 hours, 4-10 hours, 6-10 hours, or 6-9 hours. In some embodiments, the soluble DDpp fusion protein has an in vivo plasma half-life in humans of at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 16 hours, at least 32 hours, at least 64 hours, or greater than 65 hours, or 1-10 hours, 2-10 hours, 4-10 hours, 6-10 hours, or 6-9 hours.
[0162] In some embodiments, the present disclosure provides methods for modifying the in vivo half-life (e.g., in mice or humans) of a soluble fusion protein comprising a p26 protein (e.g., having a sequence of SEQ ID NO:37-43 or 44). In some embodiments, the soluble p26 fusion protein comprises one or more target-binding DDpp. In some embodiments, the half-life of the p26 soluble fusion protein is increased or decreased by substitution or deletion of one or more amino acid residues normally found in human p26 protein, or by insertion of one or more amino acid residues not normally found in human p26 protein. In another embodiment, the p26 sequence of the soluble fusion protein is modified through 1, 2, 3, 4, 5, 10, or 1-20, 1-10, 3-10, or 3-5 amino acid substitutions (conservative and / or non-conservative substitutions), deletions, and / or insertions to increase or decrease the in vivo half-life of the soluble fusion protein. In a specific embodiment, the amino acid residue corresponding to glutamine (Gln, Q) at position 217 of SEQ ID NO:37 of p26 is substituted with another amino acid residue. In a further embodiment, the substitution is Gln217Pro. In another embodiment, the p26 sequence of the soluble fusion protein is modified through the deletion of 1 to 150, 1 to 100, 1 to 50, 1 to 25, or 1 to 10 amino acid residues to increase or decrease the in vivo half-life of the soluble fusion protein. In additional embodiments, the p26 sequence of the soluble fusion protein is modified through 1, 2, 3, 4, 5, 10, or 1 to 20, 1 to 10, 3 to 10, or 3 to 5 amino acid substitutions (conservative and / or non-conservative substitutions), deletions, and / or insertions to increase or decrease the interaction of the soluble fusion protein with FcRn.
[0163] A. Multimeric DDpp fusion proteins In one embodiment, the DDpp fusion protein comprises multiple DDpps, where the two or more DDpps have the same or different specificities. In additional embodiments, the DDpp fusion protein comprises tandem repeats of the same or different DDpps, allowing the DDpp fusion protein to bind to multiple targets and / or repeated or different epitopes on the same target. In some embodiments, the DDpp fusion protein comprises at least two, three, four, or five, or more than five DDpps. In some embodiments, the DDpp fusion protein contains one to three, one to four, one to five, or more than five different DDpps. In some embodiments, the DDpp fusion protein contains at least two, three, four, or five, or more than five different DDpps. Thus, the DDpp fusion protein may be a monomeric DDpp (i.e., it may contain one DDpp) or a multimeric DDpp (i.e., it may contain multiple DDpps in tandem, optionally operably connected by a linker). In some embodiments, the use of multimeric DDpp provides enhanced (e.g., synergistic) target binding. In additional embodiments, multimeric DDpp allow a single DDpp construct to be used to target multiple targets (e.g., bispecific, trispecific, etc.). Linking two or more identical DDpp results in multivalent molecules that offer distinct advantages over monovalent compositions (e.g., increased binding avidity, target clustering, and receptor activation). Linking two or more different DDpp results in multivalent and multispecific molecules capable of binding to multiple target antigens independently or simultaneously.
[0164] A multimeric DDpp fusion protein may be a DDpp homomultimer (e.g., a homodimer, homotrimer, homotetramer, etc.) (i.e., containing multiple identical DDpps in tandem, optionally connected by a linker) or a DDpp heteromultimer (i.e., containing two or more DDpps, at least two of which may be different DDpp proteins). The number of monomeric DDpps included in a multimeric composition may vary depending on the embodiment and may be defined, at least in part, by the expression system in which the DDpp is produced. However, in some embodiments, the fusion protein may contain a multimer of about 5 to about 10 DDpp subunits, about 10 to about 15 subunits, about 15 to about 20 subunits, about 20 to about 25 subunits, or about 25 to about 30 subunits (including intermediate numbers and endpoints within the ranges listed). Furthermore, the multiple tandem components of a DDpp fusion protein may contain the same or different DDpps. In some DDpp fusions, DDpp exists as a monomer or in homo- or hetero-multimers, eg, homo- or hetero-dimers, homo- or hetero-trimers, homo- or hetero-tetramers.
[0165] DDpp fusion proteins can be "monospecific" or "multispecific." A DDpp fusion protein that is "multispecific" (e.g., bispecific, trispecific, or of greater multispecificity) recognizes and binds to two or more different epitopes present on one or more different molecules (e.g., proteins, solid support structures, etc.).
[0166] In some embodiments, two or more DDs are fused together as a multivalent DDpp. The DDs of a multivalent DDpp may be the same or different. Thus, the present disclosure provides DDpp homodimers (i.e., DDpps containing two identical DDs), DDpp homomultimers (i.e., DDpps containing three or more identical DDs), DDpp heterodimers (i.e., DDpps containing two different DDs), and DDpp heteromultimers (i.e., DDpps containing three or more DDs, where at least two of the DDs are different), optionally comprising any of the DDs described herein attached by one or more linkers.
[0167] In some embodiments, two or more DDs are linked by a multimerization domain or attached via chemical linkage to generate a multivalent DD complex. The DDs of a multivalent DD complex may be the same or different. Thus, the present disclosure provides DD homodimer complexes (i.e., DD complexes containing two identical DDs), DD homomultimer complexes (i.e., DD complexes containing three or more identical DDs), DD heterodimer complexes (i.e., DD complexes containing two different DDs), and DD heteromultimer complexes (i.e., DD complexes containing three or more DDs, where at least two of the DDs are different), optionally including any of the DDs described herein attached by one or more linkers.
[0168] In one embodiment, the multispecific DDpp fusion protein contains at least two DDpps that bind to at least two different epitopes of a single target of interest (e.g., CD123, CD33, LeY, CD38, BCMA, or CS1, preferably CD123, CD33, LeY, or CD38). In a further embodiment, the DDpp fusion is bispecific and specifically binds to two different targets expressed on the surface of two different cell types. In one embodiment, the bispecific DDpp fusion protein specifically binds to a target on a cancer cell and a target on an immune effector cell. In one embodiment, the bispecific DDpp fusion protein specifically binds to a target expressed on a cancer cell (e.g., CD123) and a target expressed on the surface of a T lymphocyte (e.g., CD3). In one embodiment, the bispecific DDpp fusion protein specifically binds to CD123 and CD33. In one embodiment, the bispecific DDpp fusion protein specifically binds to CD123 and CD38. In one embodiment, the bispecific DDpp fusion protein specifically binds to CD123 and LeY.
[0169] In additional embodiments, the multispecific DDpp fusion protein comprises at least one DDpp that specifically binds to an epitope of a target of interest and at least one other domain or sequence (e.g., an antibody fragment or domain, e.g., an scFv) that confers the function of specifically binding to a different epitope of the same target of interest. In one embodiment, the multispecific DDpp fusion protein comprises at least one DDpp that specifically binds to an epitope of a target of interest and at least one domain or sequence, e.g., an antibody fragment or domain (e.g., an scFv), that confers the function of specifically binding to an epitope of a different target of interest. In one embodiment, the multispecific DDpp fusion protein comprises at least one DDpp that specifically binds to an epitope of a target of interest and at least one domain or sequence that specifically binds to an epitope of a different target of the same cell. In another embodiment, the DDpp fusion protein comprises at least one DDpp and at least one other DDpp or domain sequence, e.g., an antibody fragment or domain, that confers the function of specifically binding to a solid support.
[0170] In further embodiments, multimeric DDpp fusions containing two or more DDpps are then fused to other heterologous proteins (or their subdomains), thereby conferring multivalent and multispecific properties to the fusion partner. Examples of DDpp fusion partners include, but are not limited to, antibodies, antibody subdomains (e.g., scFv or Fc domains), serum albumin, serum albumin subdomains, cell surface receptors, T cell receptor (TCR) α chains, T cell receptor β chains, cell surface receptor subdomains, peptides, peptide tags (e.g., FLAG or myc), fibronectin type III repeats, z domains, and elastin-like polypeptides. The number and location of DDpps and their respective locations within the fusion protein can vary. For example, DDpps can be located at one or all ends of the fusion partner and / or can be interspersed within heterologous subunits within the DDpp fusion partner.
[0171] In additional embodiments, the DDpp fusion protein comprises DDpp and a polypeptide sequence containing an additional domain. In some embodiments, the DDpp fusion protein comprises DDpp and a member selected from an antibody, an antibody fragment (e.g., an antigen-binding domain or portion thereof (e.g., scFv), an effector domain or portion thereof, an FcRn-binding domain or portion thereof, and an Fc or portion thereof), a serum protein (e.g., albumin or portion thereof), a cytokine, a growth factor, a hormone, an imaging agent, a labeling agent, and a peptide tag. In some embodiments, the DDpp fusion protein comprises an Fc domain of an immunoglobulin (e.g., a human Fc domain) or a portion thereof. In further embodiments, the Fc domain is a variant human Fc domain.
[0172] In some embodiments, DDpp is fused to a heterologous polypeptide. In some embodiments, the heterologous polypeptide comprises a full-length antibody or an antibody fragment. In some embodiments, DD is fused to the amino terminus of a full-length antibody heavy chain; the amino terminus of a full-length antibody light chain; the carboxyl terminus of a full-length antibody heavy chain; or the carboxyl terminus of a full-length antibody light chain. In other embodiments, DD is fused to an antibody fragment that is Fc. In additional embodiments, the heterologous polypeptide comprises a member selected from the group consisting of: (i) a transmembrane domain; (ii) a membrane-associated domain; (iii) human serum albumin or a fragment thereof; (iv) an AFP or a fragment thereof; (v) an AFP p26 or a fragment thereof; (vi) an extracellular domain of a receptor or a fragment thereof; and (vii) an extracellular domain of an intracellular receptor (e.g., a nuclear protein) or a fragment thereof. In some embodiments, DDpp contains a heterologous polypeptide comprising the extracellular domain of a cell surface receptor or a fragment of the extracellular domain.
[0173] In some embodiments, the DDpp of the DDpp fusion protein is incorporated into a larger multidomain molecular complex (e.g., a monomeric or multimeric DDpp fusion protein), thereby conferring the functional attributes of the incorporated DDpp to the resulting fusion protein. In some embodiments, the DDpp fusion protein comprises a DDpp and a polypeptide sequence derived from an antibody, antibody fragment, serum protein (e.g., human serum albumin) or serum protein fragment, or cell surface receptor, T cell receptor (TCR) alpha chain, T cell receptor beta chain, cytokine, growth factor, hormone, or enzyme, or fragment thereof. Incorporation of a DD into a multidomain and / or multifunctional complex can be routinely achieved by recombinant fusion with another polypeptide, conjugation to another chemical moiety, and covalent chemical linkage to another polypeptide (or other desired compound) using techniques known in the art. The DDpp fusion protein may further contain other optional components, such as linkers and other components described herein.
[0174] B. Adapter In some embodiments, the DDpp fusion proteins described herein are adapter proteins. The adapters comprise an antigenic determinant (AD) and an antigenic determinant binding domain (ADBD). The adapters may further comprise additional ADs, additional ADBDs, and / or other additional domains. In some embodiments, the adapters provided herein comprise at least one ADBD comprising a D domain.
[0175] In some embodiments, the adaptor provided herein comprises (a) a D domain (DD) that binds to CD123 and (b) an antigenic determinant (AD). In some embodiments, the D domain that binds to CD123 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:8-33, 99, and 100. In some embodiments, the D domain that binds to CD123 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:8, 13, 14, 31, 32, and 33. In some embodiments, the D domain that binds to CD123 comprises the amino acid sequence of SEQ ID NO:14. In some embodiments, the adaptor is a monovalent adaptor comprising a single D domain that binds to CD123. In some embodiments, the adaptor is a bivalent adaptor comprising two D domains that bind to CD123. In some embodiments, the two D domains that bind to CD123 are the same. In some embodiments, the two D domains that bind to CD123 are different. In some embodiments, the adaptor is a bivalent adaptor comprising a first D domain that binds to CD123 and a second D domain that binds to a second AD. In some embodiments, the second AD is CD33 or LeY. In some embodiments, the monovalent adaptor comprises a D domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 13, 14, 31, 32, and 33. In some embodiments, the monovalent adaptor comprises a D domain comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the bivalent adaptor comprises a DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 13, 14, 31, 32, and 33. In some embodiments, the bivalent adaptor comprises a D domain comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the bivalent adaptor comprises two identical D domains comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 8, 13, 14, 31, 32, and 33. In some embodiments, the bivalent adaptor comprises two identical D domains comprising the amino acid sequence of SEQ ID NO:14.In some embodiments, the adapter comprises an AFP p26 antigenic determinant (AD). In some embodiments, the AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-43 and 44. In some embodiments, the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, the monovalent adapter comprises the amino acid sequence of SEQ ID NOs: 50-54 or 55. In some embodiments, the monovalent adapter comprises the amino acid sequence of SEQ ID NO: 50. In some embodiments, the bivalent adapter comprises the amino acid sequence of SEQ ID NOs: 56-60 or 61. In some embodiments, the monovalent adapter comprises the amino acid sequence of SEQ ID NO: 61. In some embodiments, the adapter comprises one or more linkers. In some embodiments, the adapter can direct an immune response to a CD123-expressing cell in an in vitro assay comprising the adapter, a CD123-expressing cell, and an immune effector cell expressing a CAR comprising an ADBD that binds to the AD included in the adapter, for example, a CAR comprising a D domain that binds to AFP p26.
[0176] In some embodiments, provided herein is an adapter comprising (a) a D domain that binds to CD123 and (b) an antigenic determinant binding domain (ADBD) that binds to AFP p26 AD. In some embodiments, the AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-43 and 44. In some embodiments, the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the D domain that binds to CD123 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-33, 99, and 100. In some embodiments, the D domain that binds to CD123 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 13, 14, 31, 32, and 33. In some embodiments, the D domain that binds to CD123 comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the ADBD that binds to AFP p26 AD comprises a D domain that binds to AFP p26 AD. In some embodiments, the D domain that binds to AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 70-73 and 92-94. In some embodiments, the D domain that binds to AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, the adaptor comprises one or more linkers. In some embodiments, the adaptor is capable of directing an immune response to CD123-expressing cells in an in vitro assay comprising the adaptor, CD123-expressing cells, and immune effector cells expressing a CAR comprising AFP p26 AD.
[0177] In the adaptor provided herein, the AD may be located at the N-terminal side of the ADBD. Alternatively, the ADBD may be located at the N-terminal side of the AD. In some embodiments, the AD and the ADBD are directly fused. In some embodiments, the AD and the ADBD are fused via a linker (a protein linker or a chemical linker) or another protein domain (e.g., a functional domain). In some embodiments, the adaptor provided herein comprises at least one ADBD comprising a D domain.
[0178] In some embodiments, the adapter comprises a linker located between the ADBD of the adapter and another functional domain. In some embodiments, the linker is located between the two ADBDs of the adapter. In some embodiments, the linker is located between the AD and ADBD of the adapter. Linkers suitable for coupling two or more functional domains of the adapter will be apparent to those skilled in the art and may generally be any linker used in the art to link peptides, proteins, or other organic molecules. Exemplary linkers are provided herein. In specific embodiments, the linker is suitable for constructing proteins or polypeptides for pharmaceutical use. In some embodiments, the adapters provided herein comprise at least one ADBD comprising a D domain.
[0179] In addition to an AD (or multiple ADs) and an ADBD (or multiple ADBDs), the adapters provided herein can further comprise additional domains, for example, domains that confer extended half-life.
[0180] In some embodiments, the adaptor, or the ADBD within the adaptor, is deimmunized.
[0181] The adapters provided herein have uses including, but not limited to, diagnostic, analytical, and therapeutic applications. In a specific embodiment, the adapters are used in combination with the chimeric antigen receptors (CARs) provided herein expressed on the surface of cells, for example, to kill target cells.
[0182] Suitable adapters for use in connection with the DDpps (e.g., adapters and CARs) disclosed herein are substantially as disclosed in International Application Publication Nos. WO 2016164305, WO 2016164308A1, WO 2019099440, and WO 2019099433, U.S. Patent Nos. 10,662,248 and 10,647,775, and U.S. Patent Application Nos. 20200223934 and 20210002381, each of which is incorporated herein by reference.
[0183] i. Antigenic determinant (AD) The adapters provided herein comprise at least one antigenic determinant (AD). In some embodiments, the adapter comprises a single AD. In some embodiments, the adapter comprises two or more ADs. When the adapter comprises two or more ADs, the ADs may be the same or different.
[0184] In some embodiments, the AD is an epitope of AFP p26. In further embodiments, the AD comprises 5-25, 5-50, 5-75, 5-100, 5-125, or 5-150 amino acid residues, more than 150 amino acid residues, or all amino acid residues of SEQ ID NO:37. In further embodiments, the AD comprises the amino acid sequence of SEQ ID NO:37. In further embodiments, the AD comprises 5-25, 5-50, 5-75, 5-100, 5-125, or 5-150 amino acid residues, more than 150 amino acid residues, or all amino acid residues of SEQ ID NO:39. In further embodiments, the AD comprises the amino acid sequence of SEQ ID NO:39. In further embodiments, the AD comprises amino acid residues of SEQ ID NO:37-43 or 44.
[0185] In some embodiments, the adaptor comprises the extracellular domain of BCMA (e.g., a polypeptide comprising the sequence of SEQ ID NO: 34). In some embodiments, the adaptor comprises 5-25, 5-50, 5-75, 5-100, 5-125, or 5-150 amino acid residues, more than 150 amino acid residues, or all amino acid residues of SEQ ID NO: 34.
[0186] In some embodiments, the adaptor comprises the extracellular domain of CD123 (e.g., a polypeptide comprising the sequence of SEQ ID NO:1). In some embodiments, the adaptor comprises between 5 and 25, between 5 and 50, between 75, between 5 and 100, between 5 and 125, or between 5 and 150 amino acid residues, more than 150 amino acid residues, or all amino acid residues of SEQ ID NO:1.
[0187] In some embodiments, the adaptor comprises the extracellular domain of CD19 (e.g., a polypeptide comprising the sequence of SEQ ID NO:95). In some embodiments, the adaptor comprises 5-25, 5-50, 5-75, 5-100, 5-125, or 5-150 amino acid residues, more than 150 amino acid residues, or all amino acid residues of SEQ ID NO:95.
[0188] In some embodiments, the adapter comprises the extracellular domain of CS1 (e.g., a polypeptide comprising the sequence of SEQ ID NO:35). In some embodiments, the adapter comprises 5-25, 5-50, 5-75, 5-100, 5-125, or 5-150 amino acid residues, more than 150 amino acid residues, or all amino acid residues of SEQ ID NO:35. In some embodiments, the adapter comprises the extracellular domain of CD20. In some embodiments, the adapter comprises the extracellular domain of CD22. In some embodiments, the adapter comprises the extracellular domain of CD37. In some embodiments, the adapter comprises the extracellular domain of HER2. In some embodiments, the adapter comprises the extracellular domain of CD45. In some embodiments, the adapter comprises the extracellular domain of CD26, CD30, CD33, or CD38.
[0189] In some embodiments, the AD is an epitope of AFP. In further embodiments, the AD comprises 5-25, 5-50, 5-75, 5-100, 5-125, or 5-150 amino acid residues, more than 150 amino acid residues, or all amino acid residues of SEQ ID NO:36.
[0190] In some embodiments, the adaptor comprises a p26 protein (e.g., having a sequence of SEQ ID NO:37-43 or 44). In some embodiments, the adaptor has an in vivo plasma half-life of at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 16 hours, at least 32 hours, at least 64 hours, or more. In some embodiments, the adaptor has an in vivo plasma half-life in mice of at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 16 hours, at least 32 hours, at least 64 hours, or greater than 65 hours, or 1-10 hours, 2-10 hours, 4-10 hours, 6-10 hours, or 6-9 hours. In some embodiments, the adapter has an in vivo plasma half-life in humans of at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 16 hours, at least 32 hours, at least 64 hours, or greater than 65 hours, or between 1 and 10 hours, between 2 and 10 hours, between 4 and 10 hours, between 6 and 10 hours, or between 6 and 9 hours.
[0191] In some embodiments, the present disclosure provides methods for modifying the in vivo half-life (e.g., in a mouse or a human) of an adaptor comprising a p26 protein (e.g., having a sequence of SEQ ID NO:37-43 or 44). In some embodiments, the adaptor comprises one or more target-binding DDpp. In some embodiments, the half-life of the adaptor is increased or decreased by substitution or deletion of one or more amino acid residues normally found in the human p26 protein, or by insertion of one or more amino acid residues not normally found in the human p26 protein. In another embodiment, the p26 sequence of the adaptor is modified through substitution (conservative and / or non-conservative), deletion, and / or insertion of 1, 2, 3, 4, 5, 10, or 1-20, 1-10, 3-10, or 3-5 amino acids to increase or decrease the in vivo half-life of the adaptor. In a specific embodiment, the amino acid residue corresponding to glutamine (Gln, Q) at position 217 of p26 (SEQ ID NO:37) is substituted with another amino acid residue. In a further embodiment, the substitution is Gln217Pro. In another embodiment, the p26 sequence of the adapter is modified through the deletion of 1 to 150, 1 to 100, 1 to 50, 1 to 25, or 1 to 10 amino acid residues to increase or decrease the in vivo half-life of the adapter. In additional embodiments, the p26 sequence of the adapter is modified through the substitution (conservative and / or non-conservative substitution), deletion, and / or insertion of 1, 2, 3, 4, 5, 10, or 1 to 20, 1 to 10, 3 to 10, or 3 to 5 amino acids to increase or decrease the interaction of the adapter with FcRn.
[0192] In some embodiments, the AD (e.g., in the adapter and / or on the target cell) is an AD present in a naturally occurring protein or other molecule. In some embodiments, the AD is an endogenous AD in humans.
[0193] In some embodiments, the AD is an epitope of a human intracellular protein. In further embodiments, the AD is an epitope of a human intracellular protein selected from Tyk2, Jak1, Jak2, Jak3, LCK, ZAP-70, and GRB2. In further embodiments, the AD includes 5 to 25, 5 to 50, 5 to 75, 5 to 100, 5 to 125, or 5 to 150 amino acid residues, more than 150 amino acid residues, or all amino acid residues of the intracellular protein.
[0194] In some embodiments, the target of interest specifically bound by the ADBD of an adaptor is itself the AD of another adaptor with a different sequence.
[0195] ii. Antigenic determinant binding domain (ADBD) The adapters provided herein comprise at least one antigenic determinant binding domain (ADBD). In some embodiments, the adapter contains one ADBD. In some embodiments, the adapter contains at least two, three, four, or five, or more than five ADBDs. In some embodiments, the adapter contains one to three, one to four, one to five, or more than five different ADBDs. In some embodiments, the adapter contains at least two, three, four, or five, or more than five different ADBDs. Thus, the adapter may comprise a monomeric ADBD (i.e., it may contain one antigenic determinant binding domain) or a multimeric ADBD (i.e., it may contain multiple antigenic determinant binding domains in tandem, optionally operably connected by a linker). In some embodiments, the use of a multimeric adapter provides enhanced (e.g., synergistic) target binding. In additional embodiments, the use of a multimeric adapter allows for targeting of multiple targets using a single adapter construct (e.g., bispecific, trispecific, etc.). In some embodiments, the adapters provided herein comprise at least one ADBD comprising a D domain. In some embodiments, the adapters provided herein comprise multiple ADBDs comprising a D domain. In some embodiments, all of the ADBDs of the adapters provided herein comprise a D domain.
[0196] Multimeric adapters can be homomultimers (e.g., homodimers, homotrimers, homotetramers, etc.) (i.e., containing multiple identical ADBDs, optionally connected by a linker) or adapter heteromultimers (i.e., containing two or more antigenic determinant binding domains, at least two of which are different). The number of ADBDs contained in a particular adapter can vary depending on the embodiment and can be defined, at least in part, by the expression system in which the adapter is produced. However, in some embodiments, the fusion protein can contain a multimer of about 5 to about 10 ADBDs, about 10 to about 15 ADBDs, about 15 to about 20 ADBDs, about 20 to about 25 ADBDs, or about 25 to about 30 ADBDs (including intermediate numbers and endpoints within the above range). Furthermore, the multiple domains of an adapter can contain the same ADBD or different ADBDs. In some embodiments, two, three, four, five, or more than five domains are present in tandem. In some embodiments, the adapters provided herein comprise at least one ADBD comprising a D domain. In some embodiments, the adapters provided herein comprise multiple ADBDs comprising a D domain. In some embodiments, all of the ADBDs of the adapters provided herein comprise a D domain.
[0197] In one embodiment, the adapter comprises two or more operably linked ADBDs. In one embodiment, the adapter comprises two ADBDs that bind to the same or different ADs of a target antigen. Linking two or more identical ADBDs that bind to the same target antigen results in a multivalent molecule that provides distinct advantages (e.g., increased binding avidity, target clustering, receptor activation) over compositions containing only one ADBD for a target antigen. In another embodiment, the adapter comprises two ADBDs that bind to different antigens. In some embodiments, the adapter comprises two ADBDs that bind to different antigens on the same cell. In some embodiments, the adapter comprises two ADBDs that bind to different antigens on different cells. Linking two or more ADBDs results in a multivalent and multispecific adapter capable of binding to multiple target antigens independently or simultaneously. In some embodiments, the multivalent adapter can simultaneously bind to the same target antigen. In some embodiments, the multivalent adapter can simultaneously bind to different target antigens. In some embodiments, the adapter comprises two or more operably linked ADBDs separated by antigenic determinants. In some embodiments, the antigenic determinant is at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids in length. In some embodiments, the antigenic determinant is 5 to 500, 5 to 400, 10 to 300, 5 to 200, 50 to 100, 5 to 50, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 50, 50 to 500, 50 to 400, 50 to 300, 50 to 200, 50 to 100, 50 to 75, 100 to 500, 100 to 400, 100 to 300, 100 to 200, or 100 to 150 amino acids in length. In further embodiments, the adapter comprises two or more operably linked ADBDs separated by a BCMA antigenic determinant. In further embodiments, the adapter comprises two or more operably linked ADBDs separated by an AFP P26 antigenic determinant. In some embodiments, the adapter provided herein comprises at least one ADBD comprising a D domain.In some embodiments, the adapters provided herein comprise multiple ADBDs that comprise a D domain. In some embodiments, all of the ADBDs of the adapters provided herein comprise a D domain.
[0198] The ADBD of the adapter provided herein can bind to any AD. In some embodiments, the ADBD binds to CD123 (e.g., a polypeptide comprising the sequence of SEQ ID NO:1). In some embodiments, the ADBD binds to AFP p26 (e.g., a polypeptide comprising the sequence of SEQ ID NO:37-43 or 44, preferably SEQ ID NO:37 or 39). In some embodiments, the ADBD binds to BCMA (e.g., a polypeptide comprising the sequence of SEQ ID NO:34). In some embodiments, the ADBD binds to CD22. In some embodiments, the ADBD binds to CD19. In some embodiments, the ADBD binds to CD20. In some embodiments, the ADBD binds to CD37. In some embodiments, the ADBD binds to CS1. In some embodiments, the ADBD binds to HER2. In some embodiments, the ADBD binds to CD45. In some embodiments, the ADBD of the adapter provided herein specifically binds to an AD of human CD26, CD30, CD33, or CD38. Adapters may be "monospecific" or "multispecific." Adapters that are "multispecific" (e.g., bispecific, trispecific, or more multispecific) recognize and bind to two or more different epitopes present on one or more different molecules. In some embodiments, the adapters provided herein comprise at least one ADBD that comprises a D domain. In some embodiments, the adapters provided herein comprise multiple ADBDs that comprise D domains. In some embodiments, all of the ADBDs of the adapters provided herein comprise a D domain.
[0199] In some embodiments, the adaptor comprises a domain (e.g., extracellular domain) of CD123 (e.g., a polypeptide comprising the sequence of SEQ ID NO:1). In some embodiments, the adaptor comprises a domain (e.g., extracellular domain) of BCMA (e.g., a polypeptide comprising the sequence of SEQ ID NO: 34). In some embodiments, the adaptor comprises a domain (e.g., extracellular domain) of CD22. In some embodiments, the adaptor comprises a domain (e.g., extracellular domain) of CD19. In some embodiments, the adaptor comprises a domain (e.g., extracellular domain) of CS1. In some embodiments, the adaptor comprises a domain (e.g., extracellular domain) of HER2. In some embodiments, the adaptor comprises a domain (e.g., extracellular domain) of CD45. In some embodiments, the adaptor comprises a domain (e.g., extracellular domain) of CD26, CD30, CD33, or CD38. In some embodiments, the adaptor comprises a fragment of a domain. In some embodiments, the adapter comprises a fragment of a domain that is at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids in length. In some embodiments, the antigenic determinant is 5 to 500, 5 to 400, 10 to 300, 5 to 200, 50 to 100, 5 to 50, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 50, 50 to 500, 50 to 400, 50 to 300, 50 to 200, 50 to 100, 50 to 75, 100 to 500, 100 to 400, 100 to 300, 100 to 200, or 100 to 150 amino acids in length. In some embodiments, the adapters provided herein comprise at least one ADBD comprising a D domain. In some embodiments, the adapters provided herein comprise multiple ADBDs comprising a D domain. In some embodiments, all of the ADBDs of the adapters provided herein comprise a D domain.
[0200] In some embodiments, the adapter contains at least two ADBDs that bind to and bridge one or more target antigens to which the ADBDs bind, and / or complexes containing the target antigens. In some embodiments, the antigens to be bridged are present on the same cell. In some embodiments, the antigens to be bridged are present on different cells. In some embodiments, the adapter comprises two or more operably linked ADBDs (e.g., the domains described above) separated by an antigenic determinant. In some embodiments, the antigenic determinant is at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids in length. In some embodiments, the antigenic determinant is 5-500, 5-400, 10-300, 5-200, 50-100, 5-50, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 50-500, 50-400, 50-300, 50-200, 50-100, 50-75, 100-500, 100-400, 100-300, 100-200, or 100-150 amino acids in length. In further embodiments, the adapter comprises two or more operably linked ADBDs separated by a BCMA antigenic determinant. In further embodiments, the adapter comprises two or more operably linked ADBDs separated by a CD123 antigenic determinant. In further embodiments, the adapter comprises two or more operably linked ADBDs separated by an antigenic determinant of CD26, CD30, CD33, or CD38. In further embodiments, the adapter comprises two or more operably linked ADBDs separated by an AFP p26 antigenic determinant. In some embodiments, the adapter provided herein comprises at least one ADBD comprising a D domain. In some embodiments, the adapter provided herein comprises multiple ADBDs comprising a D domain. In some embodiments, all of the ADBDs of the adapter provided herein comprise a D domain.
[0201] In some embodiments, the adapter contains at least two of the same ADBDs (i.e., is multivalent). In some embodiments, the multivalent adapter can simultaneously bind to two or more of the same target antigens. In some embodiments, the adapter is multivalent and can simultaneously bind to the same target antigen. In some embodiments, the multivalent adapter comprises two or more operably linked ADBDs separated by an antigenic determinant. In some embodiments, the antigenic determinant is at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids in length. In some embodiments, the antigenic determinant is 5-500, 5-400, 10-300, 5-200, 50-100, 5-50, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 50-500, 50-400, 50-300, 50-200, 50-100, 50-75, 100-500, 100-400, 100-300, 100-200, or 100-150 amino acids in length. In further embodiments, the adapter comprises two or more operably linked ADBDs separated by a CD123 antigenic determinant. In further embodiments, the multivalent adapter comprises two or more operably linked ADBDs separated by a BCMA antigenic determinant. In further embodiments, the multivalent adapter comprises two or more operably linked ADBDs separated by an AFP p26 antigenic determinant. In some embodiments, the adapter provided herein comprises at least one ADBD comprising a D domain. In some embodiments, the adapter provided herein comprises multiple ADBDs comprising a D domain. In some embodiments, all of the ADBDs of the adapter provided herein comprise a D domain.
[0202] In some embodiments, the adapter contains at least two ADBDs that bind to different antigens (i.e., is multispecific). In some embodiments, the multispecific adapter can simultaneously bind to different target antigens. In some embodiments, the adapter is also multivalent and can simultaneously bind to the same target antigen. In some embodiments, the multispecific adapter comprises two or more operably linked ADBDs separated by an antigenic determinant. In some embodiments, the antigenic determinant is at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids in length. In some embodiments, the antigenic determinant is 5-500, 5-400, 10-300, 5-200, 50-100, 5-50, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 50-500, 50-400, 50-300, 50-200, 50-100, 50-75, 100-500, 100-400, 100-300, 100-200, or 100-150 amino acids in length. In further embodiments, the adapter comprises two or more operably linked ADBDs separated by a CD123 antigenic determinant. In further embodiments, the adapter comprises two or more operably linked ADBDs separated by a BCMA antigenic determinant. In further embodiments, the adapter comprises two or more operably linked ADBDs separated by an AFP p26 antigenic determinant. In some embodiments, the adapter provided herein comprises at least one ADBD comprising a D domain. In some embodiments, the adapter provided herein comprises multiple ADBDs comprising a D domain. In some embodiments, all of the ADBDs of the adapter provided herein comprise a D domain.
[0203] In one embodiment, the multispecific adapter contains at least two ADBDs that bind to at least two different epitopes of a single target of interest (e.g., CD123). In additional embodiments, the multispecific adapter comprises at least one ADBD that specifically binds to one epitope of the target of interest and at least one other ADBD that specifically binds to a different epitope of the same target antigen. In one embodiment, the multispecific adapter comprises at least one ADBD that specifically binds to an epitope of a first target antigen and at least one ADBD that specifically binds to an epitope of a second antigen. In some embodiments, the adapter comprises at least one ADBD that specifically binds to an epitope of a first target antigen of a cell and at least one ADBD that specifically binds to an epitope of a second antigen of the same cell. In some embodiments, the adapter comprises at least one ADBD that specifically binds to an epitope of a first target antigen of a cell and at least one ADBD that specifically binds to an epitope of a second antigen of a different cell. In some embodiments, the adapters provided herein comprise at least one ADBD comprising a D domain. In some embodiments, the adapters provided herein comprise multiple ADBDs comprising a D domain. In some embodiments, all of the ADBDs of the adapters provided herein comprise a D domain.
[0204] In further embodiments, the adapter comprises two or more ADBDs operably linked to other heterologous proteins (or subdomains thereof), thereby conferring the multivalent, multispecific, and / or functional properties of the fusion partner (e.g., pharmacokinetics, e.g., increased half-life, or pharmacodynamics, e.g., increased function) to the adapter fusion protein. Examples of adapter fusion partners include, but are not limited to, antibodies, antibody subdomains (e.g., scFv or Fc domains), serum albumin, serum albumin subdomains, cell surface receptors, T cell receptor (TCR) α chains, T cell receptor β chains, cell surface receptor subdomains, peptides, peptide tags (e.g., FLAG or myc). The number and location of ADBDs, as well as their respective locations within the adapter, can vary. For example, ADBDs can be located at one or all termini of the fusion partner and / or can be interspersed within heterologous subunits within the adapter fusion partner. In some embodiments, the adapter comprises two or more ADBDs separated by a heterologous protein (e.g., an antigenic determinant). In some embodiments, the heterologous protein is at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids in length. In some embodiments, the heterologous protein is 5 to 500, 5 to 400, 10 to 300, 5 to 200, 50 to 100, 5 to 50, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 50, 50 to 500, 50 to 400, 50 to 300, 50 to 200, 50 to 100, 50 to 75, 100 to 500, 100 to 400, 100 to 300, 100 to 200, or 100 to 150 amino acids in length. In some embodiments, the adapters provided herein comprise at least one ADBD comprising a D domain. In some embodiments, the adapters provided herein comprise multiple ADBDs that comprise a D domain. In some embodiments, all of the ADBDs of the adapters provided herein comprise a D domain.
[0205] In one embodiment, the adapter is bispecific and contains an ADBD that specifically binds to two different target antigens. In a further embodiment, the bispecific adapter specifically binds to two different target antigens expressed on the surface of two different cell types. In a further embodiment, the bispecific adapter specifically binds to two different target antigens expressed on the surface of tumor cells. In a further embodiment, the bispecific adapter specifically binds to two different target antigens expressed on the surface of AML cells (e.g., CD123 and CD33 or LeY). In one embodiment, the bispecific adapter binds to target antigens expressed on different cells. In a further embodiment, the bispecific adapter binds to target antigens expressed on different cells in a tumor. In another embodiment, the bispecific adapter binds to target antigens expressed on different cells within the tumor vasculature or tumor microenvironment. In one embodiment, the bispecific adapter specifically binds to a cancer cell target and an immune effector cell target. In one embodiment, the bispecific adapter specifically binds to a target expressed on a cancer cell (e.g., CD123) and a target expressed on the surface of a T lymphocyte (e.g., CD3). In some embodiments, the bispecific adapter can simultaneously bind to different target antigens. In some embodiments, the bispecific adapter comprises two or more operably linked ADBDs separated by an antigenic determinant. In some embodiments, the antigenic determinant is at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids in length. In some embodiments, the antigenic determinant is 5 to 500, 5 to 400, 10 to 300, 5 to 200, 50 to 100, 5 to 50, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 50, 50 to 500, 50 to 400, 50 to 300, 50 to 200, 50 to 100, 50 to 75, 100 to 500, 100 to 400, 100 to 300, 100 to 200, or 100 to 150 amino acids in length. In further embodiments, the adapter comprises two or more operably linked ADBDs separated by a CD123 antigenic determinant.In further embodiments, the adapter comprises two or more operably linked ADBDs separated by a BCMA antigenic determinant. In further embodiments, the adapter comprises two or more operably linked ADBDs separated by an AFP P26 antigenic determinant. In some embodiments, the adapter provided herein comprises at least one ADBD comprising a D domain. In some embodiments, the adapter provided herein comprises multiple ADBDs comprising a D domain. In some embodiments, all of the ADBDs of the adapter provided herein comprise a D domain.
[0206] In some embodiments where an adapter comprises multiple ADBDs, the ADBDs can be any of the types of ADBDs described herein.For example, the ADBDs can be an antibody, an antigen-binding fragment thereof, an ScFv, an alternative scaffold-binding domain, a D domain, a T cell receptor, or an antigen-binding fragment thereof.In some embodiments, the adapters provided herein comprise at least one ADBD comprising a D domain.In some embodiments, the adapters provided herein comprise multiple ADBDs comprising a D domain.In some embodiments, all of the ADBDs of the adapters provided herein comprise a D domain.
[0207] In some embodiments, where an adapter comprises multiple ADBDs, the ADBDs may be the same type of antigen-binding molecule or may be different. For example, an adapter may comprise two ADBDs that are D domains. The two D domain ADBDs may be the same or different. An adapter may comprise an ADBD that is a D domain and an ADBD that is an scFv. An adapter may comprise an ADBD that is a T cell receptor or an antigen-binding fragment thereof and an ADBD that is an scFv. In some embodiments, an adapter comprises two or more operably linked ADBDs separated by an antigenic determinant. In some embodiments, the antigenic determinant is at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids in length. In some embodiments, the antigenic determinant is 5-500, 5-400, 10-300, 5-200, 50-100, 5-50, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 50-500, 50-400, 50-300, 50-200, 50-100, 50-75, 100-500, 100-400, 100-300, 100-200, or 100-150 amino acids in length. In further embodiments, the adapter comprises two or more operably linked ADBDs separated by a CD123 antigenic determinant. In further embodiments, the adapter comprises two or more operably linked ADBDs separated by a BCMA antigenic determinant. In further embodiments, the adapter comprises two or more operably linked ADBDs separated by an AFP P26 antigenic determinant. In some embodiments, the adapter provided herein comprises at least one ADBD comprising a D domain. In some embodiments, the adapter provided herein comprises multiple ADBDs comprising a D domain. In some embodiments, all of the ADBDs of the adapter provided herein comprise a D domain.
[0208] In some embodiments, the ADBD of the adaptor is deimmunized by replacing one or more amino acid residues in a predicted T cell epitope to reduce binding to a host MHC molecule.
[0209] In some embodiments, the adaptor comprises an ADBD that binds to an antigen target containing an AD of interest, and does not have a discernible effect on the function of the target. Alternatively, in some embodiments, the adaptor comprises an ADBD that binds to an antigen target containing an AD of interest, and completely or partially inhibits, antagonizes, agonizes, blocks, increases, stimulates, or interferes with the biological activity of the target.Binding can be identified as agonistic or antagonistic, and can be determined using or routinely modified assays, bioassays, and / or animal models known in the art for evaluating such activity.
[0210] An adapter agonist refers to an adapter that in some way increases or enhances the biological activity of an adapter target, or an adapter that has biological activity comparable to that of a known agonist of the adapter target. In another embodiment, an adapter is an antagonist of the target to which it binds. An adapter antagonist refers to an adapter that completely or partially blocks or in some way interferes with the biological activity of an adapter target, or an adapter that has biological activity comparable to that of a known antagonist or inhibitor of the adapter target.
[0211] In one embodiment, the adaptor specifically binds to the target of interest, which is a serum protein. In one embodiment, the adaptor specifically binds to a serum protein selected from serum albumin (e.g., human serum albumin (HSA)), thyroxine-binding protein, transferrin, fibrinogen, and immunoglobulins (e.g., IgG, IgE, and IgM). Without being bound by theory, it is believed that the binding of the adaptor to a carrier protein provides the adaptor with an improved pharmacodynamic profile, including but not limited to, improved tumor targeting, tumor penetration, intratumoral diffusion, and enhanced therapeutic activity, compared to an adaptor that lacks a carrier protein binding sequence (see, for example, WO 01 / 45746, the contents of which are incorporated herein by reference in their entirety).
[0212] iii. Adaptor functional domain In some embodiments, the adaptor comprises a first antigenic determinant (AD) and a second antigenic determinant-binding domain (ADBD), and further comprises a functional domain that confers one or more additional desirable properties (e.g., improved manufacturing) and / or pharmacokinetic or pharmacodynamic properties (e.g., improved half-life). The adaptor's functional domain may be located between the AD and the ADBD. The adaptor may be located N-terminally to both the AD and the ADBD, or C-terminally to both the AD and the ADBD. In some embodiments where the adaptor comprises two or more ADs, the adaptor's functional domain may be located between the two or more ADs, N-terminally to the two or more ADs, or C-terminally to the two or more ADs. In some embodiments where the adaptor comprises two or more ADBDs, the adaptor's functional domain may be located between the two or more ADBDs, N-terminally to the two or more ADBDs, or C-terminally to the two or more ADBDs.
[0213] In some embodiments, the adapter comprises a functional domain selected from Fc or a variant Fc (e.g., a human Fc or a variant Fc domain) or a fragment thereof, a serum protein (e.g., human serum albumin) or a fragment thereof; an FcRn-binding domain; a serum protein-binding domain; a cytokine, a growth factor, a hormone, or an enzyme; an imaging agent; a labeling agent; and a peptide tag.
[0214] The functional domain of the adapter may be naturally derived or may be the result of recombinant engineering (phage display, xenomouse, or synthetic). In certain embodiments, the functional domain of the adapter enhances half-life, increases or decreases antibody-dependent cellular cytotoxicity (ADCC), and / or increases or decreases complement-dependent cytotoxicity (CDC) activity.
[0215] In some embodiments, the adapter comprises a functional domain selected from Fc or a variant Fc (e.g., a human Fc or a variant human Fc domain) or a fragment or derivative thereof, a serum protein (e.g., human serum albumin) or a fragment or derivative thereof (e.g., a serum protein binding domain); an FcRn binding domain; and a serum protein binding domain.
[0216] In one embodiment, the adaptor comprises a functional domain comprising an antibody effector domain, or a derivative of an antibody effector domain, that confers one or more effector functions to the adaptor, e.g., the ability to bind to one or more Fc receptors. In some embodiments, the functional domain comprises one or more CH2 domains and / or CH3 domains of an antibody having an effector function provided by the CH2 domain and CH3 domain. In some embodiments, the functional domain comprises one or more derivatives of the CH2 domain and / or CH3 domain of an antibody having an effector function provided by the CH2 domain and CH3 domain. Other sequences that can be included in an adaptor to provide effector function and are encompassed by the present invention will be apparent to those of skill in the art and can be routinely selected and engineered into the adaptors encompassed herein based on the desired effector function.
[0217] In one embodiment, the adaptor comprises a functional domain that increases antibody-dependent cellular cytotoxicity (ADCC) conferred by the adaptor (see, e.g., Bruhns et al., Blood 113:3716-3725 (2009); Shields et al., J. Biol. Chem. 276:6591-6604 (2001); Lazar et al., PNAS 103:4005-4010 (2006); Stavenhagen et al., Cancer Res. 67:8882-8890 (2007); Horton et al., Cancer Res. 68:8049-8057 (2008); Zalevsky et al., Blood 113:3735-3743 (2009); Bruckheimer, Neoplasia 11:509-517 (2009); WO2006 / 020114; Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and WO2004 / 074455). Examples of fragment-engineered modifications of the effector function-conferring portion of Fc contained in the adaptor functional domain that increase ADCC include one or more modifications corresponding to IgG1-S298A, E333A, K334A; IgG1-S239D, I332E; IgG1-S239D, A330L, I332E; IgG1-P247I, A339D or Q; IgG1-D280H, K290S with or without S298D or V; IgG1-F243L, R292P, Y300L; IgG1-F243L, R292P, Y300L, P396L; and IgG1-F243L, R292P, Y300L, V305I, P396L, where the numbering of residues in the Fc region is according to the EU index of Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 1991 Fifth edition).
[0218] Thus, in some embodiments, the adapter comprises a functional domain comprising an antibody fragment that confers a biological or biochemical characteristic of an immunoglobulin to the adapter. In some embodiments, the antibody fragment confers a characteristic selected from the ability to non-covalently dimerize, the ability to localize to tumor sites, and increased serum half-life when compared to an adapter without the antibody fragment. In certain embodiments, the adapter is at least as stable as the corresponding antibody fragment without the adapter. In certain embodiments, the adapter is more stable than the corresponding antibody fragment without the adapter. The stability of the adapter protein can be measured using established methods, including, for example, ELISA techniques. In some embodiments, the adapter is stable in whole blood (in vivo or ex vivo) at 37°C for at least about 10 hours, at least about 15 hours, at least about 20 hours, at least about 24 hours, at least about 25 hours, at least about 30 hours, at least about 35 hours, at least about 40 hours, at least about 45 hours, at least about 48 hours, at least about 50 hours, at least about 55 hours, at least about 60 hours, at least about 65 hours, at least about 70 hours, at least about 72 hours, at least about 75 hours, at least about 80 hours, at least about 85 hours, at least about 90 hours, at least about 95 hours, or at least about 100 hours (including any time periods in between those recited). In one embodiment, the adapter contains an immunoglobulin effector domain or half-life-affecting domain corresponding to a domain or fragment of an immunoglobulin in which at least a fraction of one or more of the constant region domains has been altered to provide a desired biochemical characteristic, such as decreased or increased effector function, ability to non-covalently dimerize, increased ability to localize to tumor sites, shortened serum half-life, or increased serum half-life, when compared to an immunoglobulin fragment having a corresponding unaltered immunoglobulin sequence. These alterations in the constant region domains can be amino acid substitutions, insertions, or deletions.
[0219] In one embodiment, the adaptor comprises a functional domain comprising the amino acid sequence of an immunoglobulin effector domain, or a derivative of an immunoglobulin effector domain, that confers antibody-dependent cellular cytotoxicity (ADCC) to the adaptor. In additional embodiments, the adapter comprises a sequence of an immunoglobulin effector domain that has been modified to increase ADCC (e.g., Bruhns, Blood 113:3716-3725 (2009); Shields, J. Biol. Chem. 276:6591-6604 (2001); Lazar, PNAS 103:4005-4010 (2006); Stavenhagen, Cancer Res. 67:8882-8890 (2007); Horton, Cancer Res. 68:8049-8057 (2008); Zalevsky, Blood 113:3735-3743 (2009); Bruckheimer, Neoplasia 11:509-517 (2009); WO 06 / 020114; Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and WO 04 / 074455, the contents of each of which are incorporated herein by reference in their entirety. Examples of immunoglobulin fragment engineering modifications contained in the amino acid sequence within the adapter that increase ADCC include immunoglobulin effector domain sequences having one or more modifications corresponding to IgG1-S298A, E333A, K334A; IgG1-S239D, I332E; IgG1-S239D, A330L, I332E; IgG1-P247I, A339D or Q; IgG1-D280H, K290S with or without S298D or V; IgG1-F243L, R292P, Y300L; IgG1-F243L, R292P, Y300L, P396L; and IgG1-F243L, R292P, Y300L, V305I, P396L; wherein the numbering of residues in the Fc region is according to the EU index of Kabat et al. (Kabat et al., incorporated herein by reference). et al., Sequences of proteins of Immunological Interest, 1991 Fifth edition).
[0220] In additional embodiments, the adaptor comprises a functional domain comprising the amino acid sequence of an immunoglobulin effector domain, or a derivative of an immunoglobulin effector domain, that confers antibody-dependent cellular phagocytosis (ADCP) to the adaptor. In additional embodiments, the adapter comprises a sequence of an immunoglobulin effector domain that has been modified to increase antibody-dependent cellular phagocytosis (ADCP) (e.g., Shields et al., J. Biol. Chem. 276:6591-6604 (2001); Lazar et al., PNAS 103:4005-4010 (2006); Stavenhagen et al., Cancer Res. 67:8882-8890 (2007); Richards et al., Mol. Cancer Ther. 7:2517-2527 (2008); Horton et al., Cancer Res. 68:8049-8057 (2008), Zalevsky et al., Blood 113:3735-3743 (2009); Bruckheimer et al., Neoplasia 11:509-517 (2009); WO 06 / 020114; Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and WO 04 / 074455 (the contents of each of which are incorporated herein by reference in their entirety).Examples of immunoglobulin fragment engineering modifications contained in the amino acid sequence within the adapter that increase ADCP include IgG1-S298A, E333A, K334A; IgG1-S239D, I332E; IgG1-S239D, A330L, I332E; IgG1-P247I, A339D or Q; IgG1-D280H, K290S with or without S298D or V; IgG1-F243L, R292P, Y3 00L; IgG1-F243L, R292P, Y300L, P396L; IgG1-F243L, R292P, Y300L, V305I, P396L; and IgG1-G236A, S239D, I332E; where the numbering of the residues is that of the EU index of Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest, 1991 Fifth edition, incorporated herein by reference).
[0221] In additional embodiments, the adaptor comprises a functional domain comprising the amino acid sequence of an immunoglobulin effector domain, or a derivative of an immunoglobulin effector domain, that confers complement-dependent cytotoxicity (CDC) to the adaptor. In additional embodiments, the adaptor comprises a sequence of an immunoglobulin effector domain that has been modified to increase complement-dependent cytotoxicity (CDC) (see, e.g., Idusogie et al., J. Immunol. 166:2571-2575 (2001); Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and Natsume et al., Cancer Res. 68:3863-3872 (2008) (the contents of each of which are incorporated herein by reference in their entirety)). For example, the adapter may contain an antibody fragment or domain containing one or more of the following modifications that increase CDC: IgG1-K326A, E333A; IgG1-K326W, E333S, IgG2-E333S; where the numbering of residues is that of the EU index of Kabat et al. (Sequences of proteins of Immunological Interest, 1991 Fifth edition, incorporated herein by reference).
[0222] In additional embodiments, the adapter comprises a functional domain comprising the amino acid sequence of an immunoglobulin effector domain, or a derivative of an immunoglobulin effector domain, that confers to the adapter the ability to bind to the FcγRIIb receptor. In additional embodiments, the adapter comprises a sequence of an immunoglobulin effector domain that has been modified to increase inhibitory binding to the FcγRIIb receptor (see, e.g., Chu et al., Mol. Immunol. 45:3926-3933 (2008)). An example of an immunoglobulin fragment-engineered modification contained in the amino acid sequence of the adapter that increases binding to the inhibitory FcγRIIb receptor is IgG1-S267E, L328F.
[0223] The half-life of IgG is mediated by pH-dependent binding to the fetal receptor FcRn. In certain embodiments, the adapter contains a functional domain comprising the amino acid sequence of an immunoglobulin effector domain, or a derivative of an immunoglobulin effector domain, that confers on the adapter the ability to bind to the fetal receptor FcRn. In certain embodiments, the adapter contains a functional domain comprising the sequence of an immunoglobulin FcRn-binding domain that has been modified to enhance binding to FcRn (e.g., Petkova et al., Int. Immunol. 18:1759-1769 (2006); Dall'Acqua et al., J. Immunol. 169:5171-5180 (2002); Oganesyan et al., Mol. Immunol. 46:1750-1755 (2009); Dall'Acqua et al., J. Biol. Chem. 281:23514-23524 (2006); Hinton et al., J. Immunol. 176:346-356 (2006); Datta-Mannan et al., Drug Metab. Dispos. 35:86-94 (2007); Datta-Mannan et al., J. Biol. Chem. 282:1709-1717 (2007); WO 06 / 130834; Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and Yeung et al., J. Immunol. 182:7663-7671 (2009) (the contents of each of which are incorporated herein by reference in their entirety).
[0224] In additional embodiments, the adapter comprises a functional domain comprising the sequence of an immunoglobulin effector domain that has been modified to have selective affinity for FcRn at pH 6.0 but not at pH 7.4. For example, the adapter functional domain may comprise an antibody fragment or domain containing one or more of the following modifications that increase half-life: IgG1-M252Y, S254T, T256E; IgG1-T250Q, M428L; IgG1-H433K, N434Y; IgG1-N434A; and IgG1-T307A, E380A, N434A, where the numbering of residues is that of the EU index of Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest, 1991 Fifth edition, incorporated herein by reference).
[0225] According to another aspect, the adapters are 238, 239, 246, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 4 and a functional domain comprising an amino acid sequence corresponding to an immunoglobulin effector domain that has been modified to contain at least one substitution in a sequence corresponding to an Fc region (e.g., Fcγ) position selected from: 73, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, and 439, where the numbering of residues in the Fc region is according to the EU numbering system of Kabat et al. (Sequences of proteins of Immunological Interest, 1991 Fifth edition, incorporated herein by reference). In a specific embodiment, the adapter contains a functional domain comprising the sequence of an immunoglobulin effector domain derivative, in which at least one residue corresponding to position 434 is selected from A, W, Y, F, and H. According to another embodiment, the adapter comprises the sequence of an immunoglobulin effector fragment derivative having the following respective substitutions: S298A / E333A / K334A. In additional embodiments, the adapter comprises an immunoglobulin effector domain derivative having a substitution equivalent to K322A. In another embodiment, the adapter comprises the sequence of an immunoglobulin effector domain derivative having one or any combination of the following substitutions: K246H, H268D, E283L, S324G, S239D, and I332E. According to yet another embodiment, the adapter comprises the sequence of an immunoglobulin effector domain derivative having substitutions equivalent to D265A / N297A.
[0226] In certain embodiments, the adapter comprises a functional domain comprising the sequence of an immunoglobulin effector domain that has been glycosylated or mutated to increase effector function using techniques known in the art. For example, inactivation (through point mutation or other means) of the constant region domain sequence contained in the adapter can reduce Fc receptor binding of the circulating adapter, thereby increasing tumor localization. In other cases, constant region modifications consistent with certain embodiments of the present invention can moderate complement binding and therefore reduce the serum half-life and nonspecific association of the conjugated cytotoxin. Still other modifications of the constant region can be used to modify disulfide bonds or oligosaccharide moieties, which allow for enhanced localization through increased antigen specificity or antibody flexibility. The resulting physiological profile, bioavailability, and other biochemical effects, such as tumor localization, biodistribution, and serum half-life, can be easily measured and quantified using well-known immunological techniques without undue experimentation.
[0227] The production of adaptors useful in carrying out the provided methods can be carried out using a variety of standard techniques for chemical synthesis, semi-synthetic methods, and recombinant DNA methodologies known in the art. In some embodiments, the overall production scheme for producing adaptors involves obtaining a reference protein scaffold and identifying multiple residues within the scaffold for modification. Depending on the embodiment, the reference scaffold may include a protein structure containing one or more alpha-helical regions or other tertiary structures. After identification, multiple residues can be modified, for example, by amino acid substitution. In some embodiments, the substitutions are conservative, while in other embodiments, non-conservative substitutions are made. In some embodiments, a natural amino acid (e.g., one of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine) is substituted at the targeted position for modification of the reference scaffold. In certain embodiments, the modification does not involve substitution of either cysteine or proline. In specific embodiments, after modifications are made at all of the desired identified positions, the resulting modified polypeptides (e.g., candidate adapters) can be recombinantly expressed, for example, in a plasmid, bacteria, phage, or other vector (e.g., to increase the number of each modified polypeptide). The modified polypeptides can then be purified and screened to identify modified polypeptides that have specific binding to a particular target of interest. In some embodiments, compared to a reference scaffold that may show little or no binding to a given target of interest, in some embodiments, certain modified polypeptides show enhanced binding specificity for the target of interest.In additional embodiments, depending on the target of interest, the reference scaffold may exhibit some interaction (e.g., non-specific interaction) with the target of interest, while certain modified polypeptides exhibit increased binding specificity for the target of interest by at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, or at least about 100-fold (or more). Optionally, the reference sequence and / or modified polypeptide (e.g., adapter) may be deimmunized. For example, potentially immunogenic residues or motifs can be identified and modified to reduce or eliminate potential immune responses against the adapter. Additional details regarding various aspects of adapter generation, selection, and isolation are provided in more detail below.
[0228] Table 2. Exemplary AFP p26-containing adaptors TIFF2024544559000006.tif86160TIFF2024544559000007.tif218160TIFF2024544559000008.tif218160TIFF2024544559000009.tif90160
[0229] In some embodiments, the present disclosure provides compositions comprising one or more of the adaptors disclosed in Table 2. In other embodiments, the present disclosure provides compositions comprising one or more adaptors comprising sequences having 60-70%, 70-75%, 75-80%, 80-85%, 85-90%, 95-99% (and overlapping ranges therein) homology to a sequence disclosed in Table 2. In some embodiments, adaptors having such homology are functionally similar or identical compared to their respective reference sequences in Table 2. In some embodiments, the present disclosure provides polypeptides comprising one or more adaptors that compete (fully or partially) for CD123 binding with one or more of the adaptors (reference sequences) disclosed in Table 2. The ability of a polypeptide to compete with a reference polypeptide for binding to its respective target can be routinely determined using standard competition assays known in the art. In some embodiments, competition does not require that the adapters compete for the same epitope as the adapters in Table 2; rather, the polypeptides may compete by binding to sterically hindering epitopes, overlapping epitopes, etc.
[0230] C. Chimeric Antigen Receptor Also provided herein is a chimeric antigen receptor (CAR) comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the CAR disclosed herein comprises an extracellular domain comprising an antigenic determinant (AD). In some embodiments, the CAR disclosed herein comprises an extracellular domain comprising at least one D domain (DD) disclosed herein to confer binding specificity. In some embodiments, the CAR disclosed herein comprises an extracellular domain comprising an ADBD. CARs can be expressed by any cell type.
[0231] In some embodiments, a chimeric antigen receptor (CAR) disclosed herein comprises a D domain that binds to CD123, (ii) a transmembrane domain, and (iii) an intracellular domain. In some embodiments, the D domain that binds to CD123 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:8-33, 99, and 100. In some embodiments, the D domain that binds to CD123 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:8, 13, 14, 31, 32, and 33. In some embodiments, the D domain that binds to CD123 comprises the amino acid sequence of SEQ ID NO:14. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NOs:62-66 or 67. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO:67.
[0232] In some embodiments, the chimeric antigen receptor (CAR) disclosed herein comprises an antigenic determinant binding domain (ADBD) that binds to AFP p26 AD, (ii) a transmembrane domain, and (iii) an intracellular domain. In some embodiments, the ADBD that binds to AFP p26 AD comprises an scFv that binds to AFP p26 AD. In some embodiments, the ADBD that binds to AFP p26 AD comprises a D domain that binds to AFP p26 AD. In some embodiments, the AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-43 and 44. In some embodiments, the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the DD that binds to AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-93 and 94. In some embodiments, the DD that binds to AFP p26 AD comprises the amino acid sequence of SEQ ID NOs: 70-73 or 92-94. In some embodiments, the DD that binds to the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 68. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 69.
[0233] In some embodiments, a chimeric antigen receptor (CAR) disclosed herein comprises an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain. In some embodiments, the AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-43 and 44. In some embodiments, the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 39.
[0234] In some embodiments, the CARs disclosed herein comprise an extracellular domain comprising an antigenic determinant (AD). Such CARs are expressed on the surface of cells (e.g., immune cells or immune effector cells) and can be used in combination with an adaptor comprising a first ADBD (e.g., a D domain) that binds to the AD and a second ADBD (e.g., a D domain) that binds to a target on the target cell, e.g., to kill the target cell. In some embodiments, the AD comprises AFP or a fragment thereof. In some embodiments, the AD comprises p26 or a fragment thereof. In some embodiments, the AD comprises the amino acid sequence of SEQ ID NO: 37-43 or 44. In some embodiments, the AD comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the AD comprises the amino acid sequence of SEQ ID NO: 40.
[0235] In some embodiments, a CAR disclosed herein comprises an extracellular domain comprising at least one D domain (DD). In some embodiments, the D domain can recognize an antigenic determinant (AD) on a target cell (e.g., CD123) or an AD contained in an adaptor (e.g., p26). In some embodiments, a CAR comprising a DD capable of binding to an AD on a target cell can be expressed on the surface of a cell (e.g., an immune cell or immune effector cell) and used, for example, to kill target cells expressing an AD. In some embodiments, a CAR disclosed herein comprises an extracellular domain comprising at least one CD123-binding D domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-33, 99, and 100. In some embodiments, the CD123-binding DD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 13, 14, 31, 32, and 33. In some embodiments, the CD123-binding DD comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, a CAR comprising a DD capable of binding to a first AD (e.g., p26) can be expressed on the surface of a cell (e.g., an immune cell or immune effector cell) and used in combination with an adaptor comprising the first AD and an ADBD (e.g., a D domain) that binds to a second AD on a target cell, e.g., to kill the target cell. In some embodiments, a CAR disclosed herein comprises an extracellular domain comprising at least one p26-binding D domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-93 and 94. In some embodiments, a CAR disclosed herein comprises an extracellular domain comprising at least one p26-binding D domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 70-73 and 92-94. In some embodiments, the p26-binding DD comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, p26 comprises the amino acid sequence of SEQ ID NOs: 37-43 or 44. In some embodiments, p26 comprises the amino acid sequence of SEQ ID NO: 39.In some embodiments, p26 comprises the amino acid sequence of SEQ ID NO:40.
[0236] In some embodiments, a CAR disclosed herein comprises an extracellular domain comprising an ADBD. In some embodiments, the ADBD is at least partially composed of a target-binding polypeptide (e.g., a D domain) disclosed herein. In some embodiments, the ADBD can recognize an antigenic determinant (AD) on a target cell (e.g., CD123) or an AD contained in an adaptor (e.g., p26). In some embodiments, a CAR comprising an ADBD capable of binding to an AD on a target cell can be expressed on the surface of a cell (e.g., an immune cell or immune effector cell) and used, for example, to kill target cells expressing an AD. In some embodiments, a CAR comprising a first ADBD capable of binding to a first AD (e.g., p26) can be expressed on the surface of a cell (e.g., an immune cell or immune effector cell) and used, for example, to kill target cells, in combination with an adaptor comprising the first AD and a second ADBD (e.g., a D domain) that binds to a second AD on a target cell. In some embodiments, p26 comprises the amino acid sequence of SEQ ID NOs: 37-43 or 44. In some embodiments, p26 comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, p26 comprises the amino acid sequence of SEQ ID NO: 40.
[0237] The present invention further provides a means for producing cell-associated DDpps composed of at least one DDpp designed to confer binding specificity to a membrane-bound fusion protein. The DDpp-receptor can be expressed by any cell type. In one embodiment, the DDpp-receptor fusion protein comprises a chimeric antigen receptor (CAR) or DDpp-CAR comprising an extracellular targeting domain, a transmembrane domain, and an intracellular signaling domain. In another embodiment, the DDpp-CAR is composed of an extracellular targeting domain, a transmembrane domain, and a cytoplasmic domain, wherein the cytoplasmic domain comprises the signaling domain. In further embodiments, the DDpp-CAR extracellular domain comprises one or more DDpps, wherein each DDpp constitutes a specific binding domain with the same or different specificity. In some embodiments, the target-specific domain is directed against one (or more) of the cancer antigens or tumor antigens disclosed herein, such as, but not limited to, BCMA, CD123, and CS1. In some embodiments, the target-specific domain is directed against AFP p26.
[0238] In some embodiments, the intracellular signaling domain or fragment thereof is selected from the group consisting of a human CD3 zeta domain, a 41BB domain, a CD28 domain, and any combination thereof. In some embodiments, the intracellular signaling domain contains the sequence of SEQ ID NO: 115, 116, or a combination thereof. In some embodiments, the intracellular signaling domain contains the sequence of SEQ ID NO: 117. Depending on the embodiment, the costimulatory signaling region comprises the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, OX40, CD30, CD40, PD1, lymphocyte function-associated antigen 1 (LF A1), CD2, CD7, LIGHT, NKG2C, B7H3, a ligand that specifically binds to CD83, and any combination thereof.
[0239] In some embodiments, the CAR comprises a fusion protein comprising an additional target-binding polypeptide.
[0240] In some embodiments, the ADBD of the CAR comprises at least one alternative scaffold-binding domain (e.g., a D domain or an affibody) designed to confer binding specificity to the membrane-bound CAR. Receptors comprising alternative scaffold-binding domains can be expressed by any cell type.
[0241] In one embodiment, a CAR is composed of the following elements: an extracellular domain, a transmembrane domain, and a cytoplasmic domain, wherein the cytoplasmic domain comprises a signaling domain. In another embodiment, a CAR is composed of an extracellular domain and a transmembrane domain. In a further embodiment, a CAR is composed of an extracellular domain composed of one or more ADBDs (e.g., D domains) with the same or different specificities. In one embodiment, the intracellular domain (e.g., cytoplasmic domain) of a CAR comprises the intracellular domain of the CD3 zeta chain. In another embodiment, the intracellular signaling domain of a CAR is composed of a portion of the intracellular domain of the CD3 zeta chain. In a further embodiment, the intracellular domain of a CAR comprises the intracellular domain of the CD3 zeta chain and a costimulatory signaling region. A costimulatory signaling region refers to a portion of a CAR that comprises all or part of the intracellular domain of a costimulatory molecule. Costimulatory molecules and portions of these molecules that can confer costimulatory properties to a CAR are known in the art and can be routinely incorporated into CARs. Furthermore, truncations or mutations can be incorporated into these intracellular signaling domains and costimulatory domains to further enhance or reduce receptor signaling. In a preferred embodiment, T cells are genetically modified to stably express a CAR. In such embodiments, the cytoplasmic domain of the CAR may be designed to contain the signaling domain of CD28 and / or 41BB alone, or may be combined with any other desired cytoplasmic domain useful in the present invention. In one embodiment, the cytoplasmic domain of the CAR may be designed to further contain the signaling domain of CD3ζ. In some embodiments, the cytoplasmic domain contains the sequence of SEQ ID NO: 115, 116, or a combination thereof. In some embodiments, the cytoplasmic domain contains the sequence of SEQ ID NO: 117. In one embodiment, the CAR comprises an extracellular domain, an extracellular protein linker comprising a transmembrane domain that crosses the cell membrane (e.g., found in T cells or NK cells), and optionally, a cytoplasmic domain comprising multiple signaling modules. In some embodiments, the CAR may also comprise an epitope tag.In some embodiments, the cytoplasmic domain of the CAR can include, but is not limited to, the signaling modules of CD3ζ, 41BB, and CD28, and combinations thereof. In some embodiments, the cytoplasmic domain contains the sequence of SEQ ID NO: 115, 116, or a combination thereof. In some embodiments, the cytoplasmic domain contains the sequence of SEQ ID NO: 117.
[0242] Also provided is an isolated nucleic acid sequence encoding a CAR that includes a target-binding polypeptide as part (or all) of its targeting region.
[0243] In some embodiments, the targeting domain of the CAR comprises multiple binding domains (e.g., a DD, or one or more DDs and scFvs) that comprise additional target-binding polypeptides.
[0244] The present disclosure also provides cells comprising a nucleic acid sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain composed at least in part of the disclosed DDpp that binds to a target of interest (e.g., CD123 and AFP p26), a transmembrane domain, and a signaling domain. In some embodiments, the CAR specifically binds to a tumor antigen (and thus functions to deliver CAR-expressing cells to tumors). In some embodiments, the tumor antigen is associated with a hematological malignancy. In some embodiments, the tumor antigen is CD123. In some embodiments, the CAR-expressing cells are T cells, natural killer (NK) cells, or other immune cell types. In some embodiments, the CAR-expressing cells (which may be T cells, NK cells, or other cell types) exhibit anti-tumor immunity when the polypeptide binds to the corresponding tumor antigen.
[0245] i. Extracellular domain The CARs provided herein comprise one or more antigenic determinant binding domains (ADBDs) (e.g., D domains). The ADBDs of the CARs can be any ADBDs (e.g., D domains) described herein. Exemplary ADBDs include polypeptides, such as antibody molecules (including antibodies and antigen-binding fragments thereof, e.g., immunoglobulins, single-domain antibodies (sdAbs), and scFvs), or non-antibody scaffolds (e.g., D domains or affibodies).
[0246] Depending on the desired antigen to be targeted, the extracellular domain of the CAR can be engineered to contain one or more antigenic determinant binding domains (ADBDs) that specifically bind to the desired antigen target. For example, in one embodiment, the CAR is engineered to target CD123, and a CD123-binding ADBD (e.g., D domain) is incorporated into the extracellular domain of the CAR. Alternatively, the extracellular domain of the CAR may contain multiple ADBDs, thereby conferring multispecificity or multivalency to the CAR.
[0247] The choice of ADBD in the extracellular domain of CAR depends on the identity of the cell to be targeted.For example, the extracellular domain of CAR can be engineered to specifically bind to the cell surface protein, such as receptor, of the same cell or another cell.In other embodiments, the extracellular domain of CAR is engineered to specifically bind to soluble molecules, such as immunoglobulins.
[0248] In other embodiments, the extracellular domain of the CAR contains one or more ADBDs (e.g., D domains) that bind to ligands that act as cell surface markers on target cells associated with cancer. In some embodiments, the ADBDs target and bind to tumor antigens or cancer antigens (e.g., TAA, TSA, CAA, CSA, or other tumor antigens described herein or otherwise known in the art). Thus, provided herein are methods for producing CARs, their use in producing chimeric cells, such as human T cells and natural killer cells, and the use of these chimeric T cells and NK cells in adoptive immunotherapy.
[0249] The selection of an ADBD (e.g., a D domain) can depend on the type and number of ligands or receptors that define the surface of target cells. For example, an ADBD can be selected to recognize a ligand or receptor that acts as a cell surface marker on target cells associated with a particular disease state. Examples of cell surface markers that can act as ligands or receptors include cell surface markers associated with particular disease states, such as viral diseases, bacterial diseases, parasitic infections, autoimmune diseases, and disorders associated with unwanted cell proliferation, such as cancer, for example, the cancers described herein.
[0250] In some embodiments, the ADBD binds to CD123 (e.g., a polypeptide comprising the sequence of SEQ ID NO:1). In some embodiments, the ADBD comprises a DD sequence selected from the group consisting of SEQ ID NOs:8-33, 99, and 100. In some embodiments, the CD123-specific DD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:8, 13, 14, 31, 32, and 33. In some embodiments, the CD123-specific DD comprises the amino acid sequence of SEQ ID NO:14.
[0251] In some embodiments, the ADBD (e.g., the D domain) binds to AFP (e.g., a polypeptide comprising the sequence of SEQ ID NO:36) or a fragment thereof. In some embodiments, the ADBD (e.g., the D domain) binds to AFP p26 (e.g., a polypeptide comprising the sequence of SEQ ID NO:37-43 or 44). In some embodiments, the ADBD (e.g., the D domain) binds to a polypeptide comprising the sequence of SEQ ID NO:37. In some embodiments, the ADBD comprises a DD sequence selected from the group consisting of SEQ ID NO:74-93 and 94. In some embodiments, the DD binds to AFP p26 and comprises the amino acid sequence of SEQ ID NO:70-73 or 92-94. In some embodiments, the DD binds to AFP p26 and comprises the amino acid sequence of SEQ ID NO:73.
[0252] In some embodiments, the CAR comprises an ADBD that is an antibody or an antigen-binding fragment thereof. In some embodiments, the CAR comprises an ADBD that is an scFv. In some embodiments, the CAR comprises an ADBD that is an alternative scaffold-binding domain. In some embodiments, the CAR comprises an ADBD that is a D domain. In some embodiments, the CAR comprises a T cell receptor or an antigen-binding fragment thereof.
[0253] Also provided herein is a CAR comprising multiple ADBDs. In some embodiments, the CAR comprises multiple identical ADBDs. In some embodiments, the CAR comprises multiple different ADBDs. In some embodiments, the CAR comprises multiple ADBDs that bind to the same antigenic determinant. In some embodiments, the CAR comprises multiple ADBDs, wherein the binding domains bind to different ADs. In some embodiments, the CAR comprises multiple ADBDs, wherein the binding domains bind to different ADs on the same cell. In some embodiments, the CAR comprises multiple ADBDs, wherein the binding domains bind to different ADs on different cells.
[0254] In some embodiments, the CAR comprises multiple ADBDs (e.g., D domains, affibodies, or scFvs), for example, two, three, four, five, or more than five ADBDs, wherein each ADBD can bind to a target antigen. In one embodiment, two or more of the ADBDs of the CAR can bind to different ADs. In additional embodiments, two or more of the ADBDs of the CAR can bind to the same antigen, for example, the same or different epitopes on the same antigen. In one embodiment, the multiple ADBDs of the CAR are linked to each other, for example, the C-terminus of the first ADBD is linked to the N-terminus of the second ADBD. In one embodiment, the C-terminus of the first ADBD is linked to the N-terminus of the second ADBD by a covalent bond, for example, a peptide bond.
[0255] In some embodiments, a linker or hinge region is contained between one or more of the ADBDs, for example, a linker or hinge region is located between the C-terminus of the first ADBD and the N-terminus of the second ADBD. For example, an antigen binding member comprising two ADBDs (e.g., ADBD1 and ADBD2) can be arranged in the following configuration: [ADBD1]-[linker / hinge]-[ADBD2]. Optionally, additional ADBDs can be added similarly by a linker or hinge region located between the C-terminus of one ADBD and the N-terminus of the next ADBD. Linkers or hinge regions suitable for use in linking multiple antigen binding members are flexible and non-cleavable, allowing each ADBD to move largely freely independent of other ADBDs, promoting simultaneous binding to multiple target ADs. Any flexible linker or hinge region known in the art can be used. Exemplary linkers include peptide linkers containing glycine and serine residues, e.g., (GGGGS)n, where n is a positive integer greater than or equal to 1, e.g., n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO:96). In some embodiments, the peptide linker contains the sequence of SEQ ID NO:45-48, 118, or 119. In some embodiments, the peptide linker contains the sequence of SEQ ID NO:45-48, 118, or 119.
[0256] In some embodiments, the CAR comprises a CD123-binding DD. In some embodiments, the CAR comprises a BCMA-binding DD and a CD123-binding DD.
[0257] In some embodiments, the antigen-binding portion of the CAR specifically binds to CD123 and CD33. In some embodiments, the antigen-binding portion of the CAR specifically binds to CD123 and LeY. In additional embodiments, the antigen-binding portion of the CAR further binds to a target selected from BCMA, CS1, HVEM, BTLA, DR3, CD19, CD20, and CD22.
[0258] In some embodiments, the antigen-binding portion of the CAR further binds to a tumor antigen. In additional embodiments, the antigen-binding portion of the CAR further binds to CD33 or LeY. In additional embodiments, the antigen-binding portion of the CAR further binds to a target selected from BCMA, CD123, CS1, HER2, HVEM, BTLA, DR3, CD19, CD20, and CD22.
[0259] In some embodiments, the CAR comprises two, three, four, five, or more than five DD and / or other binding domains (e.g., scFv) that specifically bind to a target of interest (e.g., CD123) expressed on the surface of a cancer cell. In additional embodiments, the CAR comprises two, three, four, five, or more than five DD or other binding domains (e.g., scFv) that specifically bind to a second, different target of interest expressed on the surface of a cancer cell. In additional embodiments, the administered CAR further comprises two, three, four, five, or more than five DD or other binding domains (e.g., scFv) that specifically bind to a second, different target of interest expressed by a second, different cancer cell or vascular endothelial cell. In some embodiments, the CAR comprises two, three, four, five, or more than five DD and / or other binding domains (e.g., scFv) that specifically bind to AFP p26. In some embodiments, the CAR comprises two, three, four, five, or more than five DDs and / or other binding domains (e.g., scFvs) that specifically bind to CD123. In some embodiments, the CD123-binding DDs comprise an amino acid sequence selected from the group consisting of SEQ ID NOs:8, 13, 14, 31, 32, and 33. In some embodiments, the CD123-binding DDs comprise the amino acid sequence of SEQ ID NO:14.
[0260] ii. Extracellular spacer domain In some embodiments, CAR comprises an extracellular spacer domain. As used herein, the term "extracellular spacer domain" or "ESD" refers to the polypeptide sequence of CAR located between the ADBD and the transmembrane domain. In one embodiment, the extracellular spacer domain allows sufficient distance from the outer surface of the cell and the ADBD, and flexibility to minimize steric hindrance between the cell and the ADBD.
[0261] In specific embodiments, the extracellular spacer domain is sufficiently short or flexible so as not to interfere with engagement of cells containing the CAR with cells bearing AD, e.g., target cells. In one embodiment, the extracellular spacer domain is 2-20, 5-15, 7-12, or 8-10 amino acids in length. In some embodiments, the ESD domain comprises at least 50, 20, or 10 residues. In some embodiments, the ESD is 10-300, 10-250, or 10-200 residues in length.
[0262] In some embodiments, the distance the ESD extends from the cell is short enough so that the hinge does not interfere with the engagement of the CAR ADBD with the surface of the target cell. In some embodiments, the ESD extends less than 20, 15, or 10 nanometers from the surface of the cytotoxic cell. Therefore, the suitability of the ESD can be affected by both the linear length, number of amino acid residues, and flexibility of the ESD. For example, an IgG4 ESD may be about 200 amino acids long, but due to the folding of the Ig domain, it extends a shorter distance from the surface of the cytotoxic cell. The CD8α ESD is about 43 amino acids long, or about 8 nm. In contrast, the IgG4 C2 & C3 ESDs are about 200 amino acids long, but have a distance from the surface of the cytotoxic cell comparable to that of the CD8α ESD. Without wishing to be bound by theory, the similarity of extension is affected by flexibility.
[0263] In some embodiments, the extracellular spacer domain includes, but is not limited to, an Fc fragment or a fragment or derivative thereof of an antibody, a hinge region or a fragment or derivative thereof, an antibody CH2 region, an antibody CH3 region, an artificial spacer sequence, or a combination thereof. Additional examples of extracellular spacer domains include, but are not limited to, a CD8a hinge and an artificial spacer consisting of a polypeptide that may be as small as Gly3, or the CH1 and CH3 domains of an IgG (e.g., human IgG4). In some embodiments, the extracellular spacer domain is one or more of: (i) the hinge, CH2, and CH3 regions of IgG4, (ii) the hinge region of IgG4, (iii) the hinge and CH2 of IgG4, (iv) the hinge region of CD8a, (v) the hinge, CH2, and CH3 regions of IgG1, (vi) the hinge region of IgG1, or (vi) the hinge and CH2 regions of IgG1. Other extracellular spacer domains will be apparent to those of skill in the art and can be used in conjunction with the alternative embodiments provided herein.
[0264] In some embodiments, the ESD is a naturally occurring sequence. In some embodiments, the CAR ESD corresponds to an ESD derived from a human protein, a fragment thereof, or a short oligopeptide or polypeptide linker. In some embodiments, the CAR ESD corresponds to a human Ig (immunoglobulin) ESD (hinge) or a fragment thereof. In one embodiment, the ESD comprises (e.g., consists of) the amino acid sequence of an IgG4 ESD. In one embodiment, for example, the hinge comprises (e.g., consists of) the amino acid sequence of an IgD hinge. In some embodiments, the hinge can be a human CD8 hinge or a fragment thereof. In one embodiment, for example, the hinge comprises (e.g., consists of) the amino acid sequence of a CD8 hinge.
[0265] In some embodiments, the ESD is an artificial sequence. In one embodiment, the ESD is a short oligopeptide linker containing a glycine-serine doublet.
[0266] In some embodiments, the CAR comprises a CD8a extracellular spacer domain.
[0267] In some embodiments, the CAR does not contain an extracellular spacer domain.
[0268] iii. Transmembrane domain The term "transmembrane domain" (TMD), as used herein, refers to a region of a protein expressed on the cell surface, e.g., a CAR, that spans the cell membrane. In some embodiments, a TMD connects an extracellular sequence (e.g., an extracellular ADBD or an extracellular AD) to an intracellular sequence, e.g., an intracellular signaling domain. In some embodiments, the transmembrane domain of a CAR is a transmembrane region of a transmembrane protein (e.g., a type I transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. Other transmembrane domains will be apparent to those skilled in the art and may be used in connection with alternative embodiments of the present invention. In some embodiments, the extracellular ADBD is a CD123-binding ADBD (e.g., a D domain). In some embodiments, the extracellular ADBD comprises the amino acid sequence of SEQ ID NO: 8, 13, 14, 31, 32, or 33. In some embodiments, the extracellular ADBD comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the extracellular ADBD is a p26-binding ADBD (e.g., a D domain). In some embodiments, the extracellular ADBD comprises the amino acid sequence of SEQ ID NOs: 70-73 or 92-94. In some embodiments, the extracellular ADBD comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, the extracellular AD comprises p26. In some embodiments, the extracellular AD comprises the amino acid sequence of SEQ ID NOs: 37-43 or 44.
[0269] CARs can be designed to contain a transmembrane domain fused to the extracellular domain of a receptor. As described above, fusion of the extracellular domain and the transmembrane domain may be achieved with or without a linker. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. In a specific embodiment, the transmembrane domain in the CAR is a CD8a transmembrane domain. In some embodiments, the CD8a transmembrane domain comprises the amino acid sequence of SEQ ID NO: 113. In some embodiments, the CD8a transmembrane domain comprises the amino acid sequence of SEQ ID NO: 114. In some cases, the transmembrane domain of a CAR comprises a CD8a hinge domain. In some embodiments, the CD8a hinge domain, also referred to as the extracellular spacer domain (ESD), comprises the amino acid sequence of SEQ ID NO: 112. In some embodiments, the transmembrane domain is selected or modified by amino acid substitution to promote or inhibit association with other surface membrane proteins.
[0270] The transmembrane domain may be derived from either natural or synthetic sources. If the origin is natural, the domain may be derived from any membrane-bound or transmembrane protein. Particularly useful transmembrane regions for the purposes herein may be derived from (i.e., comprise at least the transmembrane region of) a member selected from the group consisting of the α, β, or ζ chain of the T cell receptor; CD28, CD3ε, CD45, CD4, CD5, CD8a, CD8b, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In some embodiments, the transmembrane domain is derived from the transmembrane region of NKR. In some embodiments, the transmembrane domain is derived from the transmembrane region of CD8a. In some embodiments, the CD8a transmembrane domain comprises the amino acid sequence of SEQ ID NO:113. In some embodiments, the CD8a transmembrane domain comprises the amino acid sequence of SEQ ID NO:114. In further embodiments, the transmembrane domain is selected from the group consisting of KIRDS2, OX40, TNFR2, LFA1 (CD11a, CD18), ICOS, 41BB, GITR, LTBR, BAFFR, HVEM, NKp80 (KLRF1), IL2Rβ, IL2Rγ, IL7Rα, ITGA1, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGB7, VLA1, VLA6, IA4, ITGAX, CD11c, ITGB1, CD27, CD29, ITGB2, CD The transmembrane domain is derived from the transmembrane region of a molecule selected from the group consisting of CD11a, CD11b, CD11d, CD18, CD19, CD40, CD49a, CD49d, CD49f, CD84, CD96, CD100, CD103, CD160, CD162, CD226, CD229, CEACAM1, CRTAM, PSGL1, SLAM (SLAMF1), SLAMF4, SLAMF6 (NTB-A, Ly108), SLAMF7, SLAMF8, SELPLG, and PAG / Cbp. Alternatively, the transmembrane domain may be synthetic and preferably contains primarily hydrophobic residues, such as leucine and valine. In a further embodiment, the transmembrane domain contains a triplet of FWV (phenylalanine, tryptophan, and valine) at each end of the transmembrane domain.
[0271] Exemplary NKR domains (identified by the NKR from which the domain is derived), such as a transmembrane domain, a hinge domain or stem domain, or an intracellular (eg, cytoplasmic) domain. Killer immunoglobulin KIR2DL1 receptors (KIR) include KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1 / S1, KIR3DL2, KIR3DL3, KIR2DP1, KIR2DP1, NCR, NKp30, NKp44, NKp46, and SLAM receptors; SLAM receptors, CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, CD2F-10, SLAMF6, and SLAMF7; Fc-binding receptors CD16, FcgRIII, CD64, and Ly49; and lectin-associated NK receptors Ly49 and Ly49. Ly49A cell receptor, Ly49C; other NK receptors include NKG2D, CD160 (TM-containing splice variant), DNAM1, CRTAM, CD27, PSGL1, CD96, CD100, NKp80, CEACAM1, and CD244.
[0272] iv. Intracellular signaling domain Intracellular signaling domains that can be used in chimeric antigen receptors (CARs) according to the present invention are described herein.
[0273] "Intracellular signaling domain" (ISD) or "cytoplasmic domain," as used herein, refers to the portion of a CAR that transmits effector function signals and directs the cell to perform specialized functions (e.g., cytolytic activity and helper activity, e.g., cytokine secretion).
[0274] The cytoplasmic domain of a CAR (i.e., the intracellular signaling domain) is responsible for activating at least one of the normal effector functions of immune cells engineered to express the CAR. The term "effector function" refers to a specialized function of a cell. Effector functions of T cells include, for example, cytolytic activity and helper activity, such as cytokine secretion. Thus, the term "intracellular signaling domain" refers to a portion of a CAR protein that transmits an effector function signal and directs the cell to perform a specialized function. Typically, the entire intracellular signaling domain corresponding to a naturally occurring receptor can be used, although in many cases it is not necessary to use the entire chain. When a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chai...
Claims
1. A D domain target binding domain polypeptide that specifically binds to CD123 and comprises the amino acid sequence of SEQ ID NO: 14, 8-13, 15-32, or 33.
2. comprising the D domain of claim 1 fused to a heterologous polypeptide, Optionally, the heterologous polypeptide is (a) a full-length antibody or antibody fragment; (b) Fc domain; (c) transmembrane domain; (d) membrane-associated domain; (e) human serum albumin or a fragment thereof; (f) AFP or a fragment thereof; (g) AFP p26 or a fragment thereof; or (h) the extracellular domain of a receptor or a fragment thereof Including, Polypeptide.
3. 3. The polypeptide of claim 1 or claim 2, which is labeled or conjugated to a therapeutic or cytotoxic substance.
4. a target-binding domain comprising the D domain of claim 1 that specifically binds to CD123, a transmembrane domain, and an intracellular signaling domain; Optionally, (a) the transmembrane domain comprises the transmembrane domain of CD8a, 41BB, or CD28; (b) the intracellular signaling domain is selected from the group consisting of a domain of the human T cell receptor alpha chain, beta chain, or zeta chain; a human 41BB domain; a human CD28 domain; and any combination thereof; and / or (c) the intracellular signaling domain comprises an intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, OX40, CD30, CD40, PD1, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds to CD83, and any combination thereof; Chimeric antigen receptor (CAR).
5. The CAR of claim 4, comprising the amino acid sequence of SEQ ID NO: 62-66 or 67.
6. 10. An adaptor comprising: (a) the D domain target binding domain of claim 1 that specifically binds to CD123; and (b) an antigenic determinant (AD), optionally wherein the AD comprises an AFP p26 polypeptide, and optionally wherein the AFP p26 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-43 and 44.
7. An adapter as described in claim 6, comprising two D domains that specifically bind to CD123.
8. 8. An isolated polynucleotide encoding the polypeptide of claim 1 or 2 or the adapter of claim 6 or 7.
9. An isolated polynucleotide encoding a CAR of claim 4 or claim 5.
10. 6. A cell engineered to express the CAR of claim 4 or claim 5, and optionally, the cell being a T cell or a natural killer (NK) cell.
11. A pharmaceutical composition comprising the polypeptide of claim 1 or 2 or the adapter of claim 6 or 7, and a pharmaceutically acceptable excipient.
12. 10. A pharmaceutical composition comprising a cell expressing the CAR of claim 4 or claim 5 and a pharmaceutically acceptable excipient, optionally wherein the cell is a T cell or a natural killer (NK) cell.
13. 1. A composition for use in a method of delivering an immune response to or killing one or more target cells, comprising: The composition comprises the target cell, and the method comprises contacting the composition with a cell expressing the chimeric antigen receptor (CAR) of claim 4, comprising (i) a D domain that binds to CD123, (ii) a transmembrane domain, and (iii) an intracellular domain.
14. A composition for use in a method of delivering an immune response to or killing a target cell, comprising the target cell, the method comprising contacting the composition with an adaptor; (a) the composition comprising the target cells further comprises cells expressing a CAR, the CAR comprising (i) an antigenic determinant binding domain (ADBD) that binds to an AFPp26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the adapter comprising (i) the D domain of claim 1 that binds to CD123 and (ii) an AFPp26 AD.
15. A composition for use in a method of killing a target cell, comprising the target cell, the method comprising contacting the composition with a cell expressing a CAR; (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the composition comprising the target cell further comprises an adaptor comprising (i) the D domain of claim 1 that binds to CD123 and (ii) an AFP p26 AD.
16. A composition for use in a method of killing a target cell, comprising the target cell, the method comprising contacting the composition with a cell expressing a CAR and an adaptor; The composition, wherein (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the adapter comprises (i) the D domain of claim 1 that binds to CD123 and (ii) an AFP p26 AD.
17. 1. A composition for delivering an immune response to, killing target cells, or treating cancer in a patient, comprising:
5. A composition comprising a cell expressing the chimeric antigen receptor (CAR) of claim 4, wherein the CAR comprises (i) a D domain that binds to CD123, (ii) a transmembrane domain, and (iii) an intracellular domain.
18. 1. A composition for delivering an immune response to, killing target cells, or treating cancer in a patient, comprising: The composition comprising: (i) an adaptor comprising the D domain of claim 1 that binds to CD123; and (ii) AFP p26 AD.
19. (a) the patient has been administered a cell expressing a CAR, wherein the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an AFPp26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain; (b) the patient comprises cells expressing a CAR, wherein the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain; or (c) the composition is administered in combination with a cell expressing a CAR, the CAR comprising (i) an antigenic determinant binding domain (ADBD) that binds to an AFPp26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain; 19. The composition of claim 18.
20. (a) the D domain that binds to CD123 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 13, 14, 31, 32, and 33; (b) the D domain that binds to CD123 comprises the amino acid sequence of SEQ ID NO:14; (c) the transmembrane domain comprises the transmembrane domain of CD8a, 41BB, or CD28; (d) the intracellular signaling domain is selected from the group consisting of a human T cell receptor alpha chain, beta chain, or zeta chain domain; a human 41BB domain; a human CD28 domain; and any combination thereof; (e) the intracellular signaling domain comprises an intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, OX40, CD30, CD40, PD1, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds to CD83, and any combination thereof; (f) the AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-43 and 44; (g) the ADBD that binds to AFP p26 AD comprises a D domain that binds to AFP p26 AD; (h) the ADBD that binds to AFP p26 AD comprises a D domain comprising the amino acid sequence of SEQ ID NO: 73, 70-72, or 92-94; (i) the CAR comprising a D domain that binds to AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 68 or 69; (j) the cell expressing the CAR is an immune cell, optionally a T cell or a natural killer (NK) cell; (k) the contacting step is performed in a human subject; (l) the cell expressing the CAR is an autologous immune cell; (m) the target cell is a cancer cell; and / or (n) the cancer is acute myeloid leukemia (AML) or high-risk myelodysplastic syndrome; 20. The composition of any one of claims 13 to 19.