WO2023086983A1 - D-domain containing polypeptides and uses thereof - Google Patents

D-domain containing polypeptides and uses thereof Download PDF

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Publication number
WO2023086983A1
WO2023086983A1 PCT/US2022/079796 US2022079796W WO2023086983A1 WO 2023086983 A1 WO2023086983 A1 WO 2023086983A1 US 2022079796 W US2022079796 W US 2022079796W WO 2023086983 A1 WO2023086983 A1 WO 2023086983A1
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Prior art keywords
domain
cell
adapter
car
ddpp
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PCT/US2022/079796
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English (en)
French (fr)
Inventor
David Lafleur
Jeffrey S. SWERS
Justin EDWARDS
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Arcellx Inc
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Arcellx Inc
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Priority to CN202280088084.1A priority Critical patent/CN118679180A/zh
Priority to CA3237239A priority patent/CA3237239A1/en
Priority to EP22893904.7A priority patent/EP4433497A4/en
Priority to IL312586A priority patent/IL312586A/en
Priority to KR1020247019906A priority patent/KR20240105452A/ko
Priority to MX2024005673A priority patent/MX2024005673A/es
Priority to JP2024528494A priority patent/JP2024544559A/ja
Priority to AU2022388811A priority patent/AU2022388811A1/en
Publication of WO2023086983A1 publication Critical patent/WO2023086983A1/en
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4214—Receptors for cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4224—Molecules with a "CD" designation not provided for elsewhere
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4715—Pregnancy proteins, e.g. placenta proteins, alpha-feto-protein, pregnancy specific beta glycoprotein
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/11—Antigen recognition domain
    • A61K2239/15—Non-antibody based
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/23—On/off switch
    • A61K2239/24—Dimerizable CARs; CARs with adapter
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2318/00—Antibody mimetics or scaffolds
    • C07K2318/20—Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the field of the invention generally relates to D domain containing polypeptides, including multifunctional chimeric antigen receptors and Adapters comprising the D domains, and their use in methods of treatment, for example, by directing immune responses to target cells.
  • proteins comprising a D Domain (DD) target binding domain (DDpp) wherein the DD specifically binds a target of interest.
  • the target of interest is human CD123 (SEQ ID NO: 1), or a fragment thereof.
  • the DDpp are monovalent or multivalent.
  • the DDpp are monospecific or multispecific.
  • the DDpp are monospecific and multivalent.
  • the DDpp are multispecific and multivalent. Fusion proteins comprising one or more DD are also provided, as are methods of making and using the fusion proteins. Nucleic acids encoding the DDpps and vectors and host cells containing the nucleic acids are also provided.
  • the DDpp comprises a CD123-binding DD comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 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 NO: 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.
  • the disclosure provides a chimeric antigen receptor (CAR) which comprises a target binding domain comprising a DD disclosed.
  • the DD binds CD123 and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100.
  • the CD123-specific DD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 13, 14, 31, 32, and 33.
  • the CD123-specific DD comprises the amino acid sequence of SEQ ID NO: 14.
  • the DD binds AFP p26 (SEQ ID NO: 37) and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94.
  • 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 than 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 cell is a T cell (CAR-T cell) or a natural killer (NK) cell (CAR-NK cell). In some embodiments, the cell is an autologous immune cell. In some embodiments the cell is an autologous T cell (CAR-T cell) or an autologous natural killer (NK) cell.
  • the cell is an allogenic immune cell.
  • the cell is an allogenic T cell (CAR-T cell) or an allogenic natural killer (NK) cell.
  • CAR-T cell an allogenic T cell
  • NK an allogenic natural killer
  • the CAR is expressed in an immune cell derived from human embryonic stem cells (CAR- hESCs) or induced pluripotent stem cells (CAR-iPSC cell).
  • Nucleic acids encoding the disclosed DDpp are also provided.
  • vectors e.g., plasmids, viral vectors, and non- viral vectors
  • the vector comprises a nucleotide sequence which regulates the expression of the polypeptide encoded by the nucleic acid molecule.
  • the vector comprises an inducible promoter sequence.
  • the vector includes one or more additional standard components for expression of a protein encoded a nucleic acid (e.g., promoters, packaging components, etc.).
  • the vector is a lentiviral vector.
  • the disclosure also provides host cells that comprise the nucleic acid molecules encoding a target-binding DDpp disclosed herein.
  • the host cells e.g., cells of a cell line
  • the host cells are engineered to express a protein containing a DD disclosed herein (e.g., a DD having the amino acid sequence of SEQ ID NO: 8-33).
  • the expression of the DDpp e.g., DDpp fusion protein, or Adapter
  • the expression results in the DDpp (e.g., CAR) being expressed on the surface and/or integral to the membrane of the host cells.
  • the host cell is a bacterial, yeast, fungal, or plant cell. In other embodiments, the host cell is a mammalian cell. In a further embodiment, 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 the DDpp (e.g., CAR) on its cell surface.
  • DDpp e.g., CAR
  • the disclosure further provides a host cell expressing a protein comprising a DD disclosed herein.
  • the host cell expresses a chimeric antigen receptor (CAR) comprising a DD disclosed herein.
  • CAR comprises a target binding domain that comprises a DD comprising an amino acid sequence selected from SEQ ID NO: 8-33 and a transmembrane domain.
  • the CD123-specific DD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 13, 14, 31, 32, and 33.
  • the CD123-specific DD comprises the amino acid sequence of SEQ ID NO: 14.
  • the CAR further comprise an intracellular domain (comprising a signaling domain).
  • the CAR immune cell is a T cell.
  • the CAR immune effector cell is a NK cell.
  • the CAR immune effector cell is not a T cell or an NK cell.
  • the CAR immune cell is an autologous immune cell. In some embodiments, the CAR immune cell is an allogenic immune cell.
  • the host cell is an immune effector cell that further comprises a second CAR polypeptide having a DD or other binding domain (e.g., scFv) that specifically binds the same or a different target e.g., a different epitope of the same target, or a second target of interest) expressed by the cancer cell) as the first CAR expressed by the host immune cell.
  • a second CAR polypeptide having a DD or other binding domain e.g., scFv
  • compositions containing a protein comprising a DD disclosed herein, nucleic acids disclosed herein encoding the proteins, vectors disclosed herein containing the nucleic acids, viruses encoding the proteins, and host cells disclosed herein containing the nucleic acids and or vectors are also provided.
  • kits containing one or more of the disclosed target-binding DDpps e.g., DDpp fusion proteins such as DD- Fc and DD-CAR, or Adapter
  • nucleic acid molecules, vectors, and host cells e.g., a therapeutic kit, a diagnostic kit, a kit for research use, etc.
  • DDpp provided herein possess activities that include but are not limited to the ability to specifically bind a target of interest (e.g., CD 123) in vitro or in vivo and the ability to serve as a reactive site for linking or associating a protein such as a DDpp fusion protein with one or more additional moieties (e.g., a solid support), and/or other modifications.
  • a target of interest e.g., CD 123
  • additional moieties e.g., a solid support
  • the DDpp provided herein can also possess additional desirable properties and/or functionalities useful in manufacturing, formulation and biological, diagnostic, and therapeutic applications.
  • the 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, CAR and/or Adapter) that specifically binds a therapeutic target of interest (e.g., CD123) to a subject in need thereof.
  • a DDpp e.g., a DDpp fusion protein, CAR and/or Adapter
  • a therapeutic target of interest e.g., CD123
  • the disease or disorder is cancer, a B cell malignancy, a disease or disorder of the immune system, or an infection.
  • 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 neoplasm (BPDCN).
  • AML acute myeloid leukemia
  • myelodysplastic syndrome e.g., high-risk myelodysplastic syndrome
  • B-cell acute lymphoblastic leukemia e.g., hairy cell leukemia
  • Hodgkin's lymphoma or blastic plasmacytoid dendritic neoplasm BPDCN
  • the disease or disorder is acute myeloid leukemia.
  • the disclosure provides:
  • a protein comprising a D Domain target binding domain that specifically binds CD 123 and comprises the amino acid sequence of SEQ ID NO: 8-32 or 33.
  • [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.
  • heterologous polypeptide comprises the extracellular domain, or a fragment of an extracellular domain, of a receptor selected from the group consisting of: BCMA, CD123, CSl, and CD19.
  • a chimeric antigen receptor which comprises a target binding domain comprising the protein according to any one of [1] to [3].
  • the CAR of [14] which comprises, a target binding domain, a transmembrane domain, and an intracellular signaling domain.
  • transmembrane domain comprises a CD8a, 41BB, or CD28 transmembrane domain.
  • the intracellular signaling domain comprises the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, 0X40, CD30, CD40, PD1, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, and any combination thereof.
  • a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, 0X40, CD30, CD40, PD1, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, and any combination thereof.
  • An Adapter comprising (a) a D domain target binding domain that specifically binds CD 123 and comprises the amino acid sequence of SEQ ID NO: 8-32 or 33, and (b) an antigenic determinant (AD).
  • AFP p26 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 37-43 and 44.
  • a vector comprising the polynucleotide of [38].
  • a host cell comprising the polynucleotide of [38] or the vector of [39] or [40].
  • a host cell comprising the polynucleotide of [43] or the vector of any one of [44] to [46].
  • NK natural killer
  • a pharmaceutical composition comprising the protein according to any one of [1] to [13] or [22] and a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprising the vector of [44] and a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprising a cell expressing the CAR of any one of [14] to [22] and a pharmaceutically acceptable excipient.
  • [54.] The pharmaceutical composition of [53], wherein the cell is a T cell or a natural killer (NK) cell.
  • a kit comprising the Adapter of any one of [23] to [37].
  • a kit comprising a cell expressing the CAR of any one of [14] to [22].
  • a method of delivering an immune response to one or more target cells 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.
  • CAR chimeric antigen receptor
  • 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 CD 123, (ii) a transmembrane domain, and (iii) an intracellular domain.
  • CAR chimeric antigen receptor
  • transmembrane domain comprises a CD8a, 41BB or CD28 transmembrane domain.
  • intracellular signaling domain is selected from the group consisting of a domain of a human T cell receptor alpha, beta, or zeta chain; a human 4 IBB domain; a human CD28 domain; and any combination thereof.
  • the intracellular signaling domain comprises the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, 0X40, CD30, CD40, PD1, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, and any combination thereof.
  • the CAR comprising a D domain that binds to CD123 comprises the amino acid sequence of SEQ ID NO: 62-66 or 67.
  • the target cell is an acute myeloid leukemia (AML) cell, myelodysplasia cell, B-cell acute lymphoblastic leukemia cell, hairy cell leukemia cell, Hodgkin's lymphoma cell or blastic plasmacytoid dendritic neoplasm (BPDCN) cell, preferably an acute myeloid leukemia (AML) cell.
  • AML acute myeloid leukemia
  • myelodysplasia cell myelodysplasia cell
  • B-cell acute lymphoblastic leukemia cell hairy cell leukemia cell
  • Hodgkin's lymphoma cell or blastic plasmacytoid dendritic neoplasm (BPDCN) cell preferably an acute myeloid leukemia (AML) cell.
  • AML acute myeloid leukemia
  • a method of delivering an immune response to a target cell comprising: contacting a composition comprising the target cell with an Adapter, wherein (a) the composition comprising the target cell further comprises a cell 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 Adapter comprises (i) a D domain that binds to CD123 and (ii) AFP p26 AD.
  • ADBD antigenic determinant binding domain
  • AD AFP p26 antigenic determinant
  • AD AFP p26 antigenic determinant
  • a method of delivering an immune response to a target cell comprising: contacting a composition comprising the target cell with an Adapter, wherein (a) the composition comprising the target cell further comprises a cell expressing a CAR, wherein the CAR comprises (i) 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 CD 123 and (ii) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
  • AD AFP p26 antigenic determinant
  • ADBD antigenic determinant binding domain
  • a method of delivering an immune response to a target cell comprising: contacting a composition comprising the target cell with an Adapter, wherein (a) the composition comprising the target cell further comprises a cell expressing a CAR, wherein the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CD 123, (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the Adapter comprises (i) a D domain that binds to CD 123 and (ii) an the AD bound by the CAR.
  • ADBD antigenic determinant binding domain
  • AD antigenic determinant
  • a method of delivering an immune response to a target cell comprising: contacting a composition comprising the target cell with an Adapter, wherein (a) the composition comprising the target cell further comprises a cell expressing a CAR, wherein the CAR comprises (i) 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 a target AD and (ii) an second antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
  • ADBD first antigenic determinant binding domain
  • ADBD second antigenic determinant binding domain
  • 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, 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 Adapter comprising (i) a D domain that binds to CD 123 and (ii) an AFP p26 AD.
  • ADBD antigenic determinant binding domain
  • AD AFP p26 antigenic determinant
  • AD AFP p26 antigenic determinant
  • 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, 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 Adapter comprising (i) a D domain that binds to CD 123 and (ii) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
  • AD AFP p26 antigenic determinant
  • ADBD antigenic determinant binding domain
  • 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, wherein (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CD 123, (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the composition comprising the target cell further comprises an Adapter comprising (i) a D domain that binds to CD 123 and (ii) the AD bound by the CAR.
  • ADBD antigenic determinant binding domain
  • AD antigenic determinant
  • 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, 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 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 the AFP p26 antigenic determinant (AD).
  • ADBD first antigenic determinant binding domain
  • ADBD second antigenic determinant binding domain
  • a method of killing a target cell comprising: contacting a composition comprising the target cell with an Adapter, wherein (a) the composition comprising the target cell further comprises a cell 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 Adapter comprises (i) a D domain that binds to CD123 and (ii) an AFP p26 AD.
  • ADBD antigenic determinant binding domain
  • AD AFP p26 antigenic determinant
  • AD AFP p26 antigenic determinant
  • a method of killing a target cell comprising: contacting a composition comprising the target cell with an Adapter, wherein (a) the composition comprising the target cell further comprises a cell 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).
  • AD AFP p26 antigenic determinant
  • ADBD antigenic determinant binding domain
  • a method of killing a target cell comprising: contacting a composition comprising the target cell with an Adapter, wherein (a) the composition comprising the target cell further comprises a cell expressing a CAR, wherein the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CD 123, (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the Adapter comprises (i) a D domain that binds to CD 123 and (ii) an the AD bound by the CAR.
  • ADBD antigenic determinant binding domain
  • AD antigenic determinant
  • a method of killing a target cell comprising: contacting a composition comprising the target cell with an Adapter, wherein (a) the composition comprising the target cell further comprises a cell 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 a target AD and (ii) an second antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
  • ADBD first antigenic determinant binding domain
  • ADBD second antigenic determinant binding domain
  • a method of killing a target cell comprising: contacting a composition comprising the target cell with a cell expressing a CAR, 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 Adapter comprising (i) a D domain that binds to CD123 and (ii) an AFP p26 AD.
  • ADBD antigenic determinant binding domain
  • AD AFP p26 antigenic determinant
  • AD AFP p26 antigenic determinant
  • a method of killing a target cell comprising: contacting a composition comprising the target cell with a cell expressing a CAR, 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 Adapter comprising (i) a D domain that binds to CD 123 and (ii) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
  • AD AFP p26 antigenic determinant
  • ADBD antigenic determinant binding domain
  • a method of killing a target cell comprising: contacting a composition comprising the target cell with a cell 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 (b) the composition comprising the target cell further comprises an Adapter comprising (i) a D domain that binds to CD 123 and (ii) the AD bound by the CAR.
  • ADBD antigenic determinant binding domain
  • AD antigenic determinant
  • the composition comprising the target cell further comprises an Adapter comprising (i) a D domain that binds to CD 123 and (ii) the AD bound by the CAR.
  • a method of killing a target cell comprising: contacting a composition comprising the target cell with a cell expressing a CAR, 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 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 the AFP p26 antigenic determinant (AD).
  • ADBD first antigenic determinant binding domain
  • ADBD second antigenic determinant binding domain
  • transmembrane domain comprises a CD8a, 41BB or CD28 transmembrane domain.
  • intracellular signaling domain is selected from the group consisting of a domain of a human T cell receptor alpha, beta, or zeta chain; a human 4 IBB domain; a human CD28 domain; and any combination thereof.
  • the intracellular signaling domain comprises the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, 0X40, CD30, CD40, PD1, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, and any combination thereof.
  • a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, 0X40, CD30, CD40, PD1, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, and any combination thereof.
  • tumor antigen is selected from the group: BCMA, CD19, CD22, CS1, HER2, TACI, BAFFR, and PDE1.
  • tumor antigen is selected from the group: CD45, CD26, CD30, CD33, and CD38.
  • [123.] The method of any one of [77] to [121], wherein the target AD is selected from: CD19, CD22, CD123, BCMA, CS1, HER2, TACI, BAFFR, and PDL1.
  • AML acute myeloid leukemia
  • BPDCN blastic plasmacytoid dendritic neoplasm
  • a method of delivering an immune response to a target cell in a patient comprising: administering to the patient a cell expressing a chimeric antigen receptor (CAR) comprising (i) a D domain that binds to CD 123, (ii) a transmembrane domain, and (iii) an intracellular domain.
  • a method of killing a target cell in a patient in need thereof comprising: administering to the patient a cell expressing a chimeric antigen receptor (CAR) comprising (i) a D domain that binds to CD 123, (ii) a transmembrane domain, and (iii) an intracellular domain.
  • the target cell is an acute myeloid leukemia (AML) cell, myelodysplasia cell, B-cell acute lymphoblastic leukemia cell, hairy cell leukemia cell, Hodgkin's lymphoma cell or blastic plasmacytoid dendritic neoplasm (BPDCN) cell, preferably an acute myeloid leukemia (AML) cell.
  • AML acute myeloid leukemia
  • myelodysplasia cell myelodysplasia cell
  • B-cell acute lymphoblastic leukemia cell hairy cell leukemia cell
  • Hodgkin's lymphoma cell or blastic plasmacytoid dendritic neoplasm (BPDCN) cell preferably an acute myeloid leukemia (AML) cell.
  • AML acute myeloid leukemia
  • a method of depleting lymphocytes in a patient in need thereof comprising: administering to the patient a cell expressing a chimeric antigen receptor (CAR) comprising (i) a D domain that binds to CD 123, (ii) a transmembrane domain, and (iii) an intracellular domain.
  • CAR chimeric antigen receptor
  • lymphocytes are B lymphocytes.
  • a method of treating cancer comprising: administering to a patient in need thereof a cell expressing a chimeric antigen receptor (CAR) comprising (i) a D domain that binds to CD 123, (ii) a transmembrane domain, and (iii) an intracellular domain.
  • CAR chimeric antigen receptor
  • hematological cancer is acute leukemia, chronic leukemia, polycythemia vera, lymphoma, Hodgkin’s disease, non-Hodgkin’s lymphoma, multiple myeloma, Waldenstrom’s macroglobulinemia, heavy chain disease, myelodysplastic syndrome (e.g., high-risk myelodysplastic syndrome), hairy cell leukemia, or myelodysplasia.
  • myelodysplastic syndrome e.g., high-risk myelodysplastic syndrome
  • hairy cell leukemia or myelodysplasia.
  • hematological 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 neoplasm (BPDCN).
  • AML acute myeloid leukemia
  • myelodysplastic syndrome e.g., high-risk myelodysplastic syndrome
  • B-cell acute lymphoblastic leukemia e.g., hairy cell leukemia
  • Hodgkin’ s lymphoma hedritic neoplasm
  • a method of treating an autoimmune disease or disorder comprising: administering to a patient in need thereof a cell expressing a chimeric antigen receptor (CAR) comprising (i) a D domain that binds to CD 123, (ii) a transmembrane domain, and (iii) an intracellular domain.
  • CAR chimeric antigen receptor
  • transmembrane domain comprises a CD8a, 41BB or CD28 transmembrane domain.
  • intracellular signaling domain is selected from the group consisting of a domain of a human T cell receptor alpha, beta, or zeta chain; a human 4 IBB domain; a human CD28 domain; and any combination thereof.
  • the intracellular signaling domain comprises the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, 0X40, CD30, CD40, PD1, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, and any combination thereof.
  • a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, 0X40, CD30, CD40, PD1, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, and any combination thereof.
  • administering the cell expressing the CAR comprises administering a pharmaceutical composition comprising the cell expressing the CAR.
  • a method of delivering an immune response to a target cell in a patient comprising: administering to the patient an Adapter comprising (i) a D domain that binds to CD123 and (ii) an AFP p26 AD.
  • a method of killing a target cell 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 AFP p26 AD.
  • AML acute myeloid leukemia
  • BPDCN blastic plasmacytoid dendritic neoplasm
  • a method of depleting 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 AFP p26 AD.
  • lymphocytes are B lymphocytes.
  • 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 AFP p26 AD.
  • hematological cancer is acute leukemia, chronic leukemia, polycythemia vera, lymphoma, Hodgkin’s disease, non-Hodgkin’s lymphoma, multiple myeloma, Waldenstrom’s macroglobulinemia, heavy chain disease, myelodysplastic syndrome (e.g., high-risk myelodysplastic syndrome), hairy cell leukemia, or myelodysplasia.
  • myelodysplastic syndrome e.g., high-risk myelodysplastic syndrome
  • hairy cell leukemia or myelodysplasia.
  • hematological 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 neoplasm (BPDCN)
  • AML acute myeloid leukemia
  • myelodysplastic syndrome e.g., high-risk myelodysplastic syndrome
  • B-cell acute lymphoblastic leukemia e.g., hairy cell leukemia
  • Hodgkin’ s lymphoma hedgkin’ s lymphoma or blastic plasmacytoid dendritic neoplasm
  • 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 AFP p26 AD.
  • a method of delivering an immune response to a target cell in a patient comprising: administering to the patient an Adapter comprising (i) a D domain that binds to CD 123 and (ii) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
  • an Adapter comprising (i) a D domain that binds to CD 123 and (ii) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
  • a method of killing a target cell in a patient in need thereof comprising: administering to the patient an Adapter comprising (i) a D domain that binds to CD 123 and (ii) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
  • an Adapter comprising (i) a D domain that binds to CD 123 and (ii) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
  • the target cell is an acute myeloid leukemia (AML) cell, myelodysplasia cell, B-cell acute lymphoblastic leukemia cell, hairy cell leukemia cell, Hodgkin’s lymphoma cell or blastic plasmacytoid dendritic neoplasm (BPDCN) cell, preferably an acute myeloid leukemia (AML) cell.
  • AML acute myeloid leukemia
  • a method of depleting lymphocytes in a patient in need thereof comprising: administering to the patient an Adapter comprising (i) a D domain that binds to CD 123 and (ii) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
  • an Adapter comprising (i) a D domain that binds to CD 123 and (ii) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
  • lymphocytes are B lymphocytes.
  • a method of treating cancer comprising: administering to a patient in need thereof an Adapter comprising (i) a D domain that binds to CD 123 and (ii) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
  • an Adapter comprising (i) a D domain that binds to CD 123 and (ii) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
  • hematological cancer is acute leukemia, chronic leukemia, polycythemia vera, lymphoma, Hodgkin’s disease, non-Hodgkin’s lymphoma, multiple myeloma, Waldenstrom’s macroglobulinemia, heavy chain disease, myelodysplastic syndrome (e.g., high-risk myelodysplastic syndrome), hairy cell leukemia, or myelodysplasia.
  • myelodysplastic syndrome e.g., high-risk myelodysplastic syndrome
  • hairy cell leukemia or myelodysplasia.
  • hematological 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 neoplasm (BPDCN).
  • AML acute myeloid leukemia
  • myelodysplastic syndrome e.g., high-risk myelodysplastic syndrome
  • B-cell acute lymphoblastic leukemia e.g., hairy cell leukemia
  • Hodgkin’ s lymphoma hedritic neoplasm
  • 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 CD 123 and (ii) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
  • an Adapter comprising (i) a D domain that binds to CD 123 and (ii) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
  • a method of delivering an immune response to a target cell in a patient comprising: administering to the patient an Adapter comprising (i) a D domain that binds to CD 123 and (ii) the AD bound by a CAR, wherein the patient has been administered a cell expressing the CAR, wherein (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CD 123, (ii) a transmembrane domain, and (iii) an intracellular domain.
  • ADBD antigenic determinant binding domain
  • a method of killing a target cell in a patient in need thereof comprising: administering to the patient an Adapter comprising (i) a D domain that binds to CD 123 and (ii) the AD bound by a CAR, wherein the patient has been administered a cell expressing the CAR, wherein (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CD 123, (ii) a transmembrane domain, and (iii) an intracellular domain, wherein the patient has been administered a cell 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.
  • ADBD antigenic determinant binding domain
  • ADBD antigenic determinant binding domain
  • ADBD antigenic
  • the target cell is an acute myeloid leukemia (AML) cell, myelodysplasia cell, B-cell acute lymphoblastic leukemia cell, hairy cell leukemia cell, Hodgkin’s lymphoma cell or blastic plasmacytoid dendritic neoplasm (BPDCN) cell, preferably an acute myeloid leukemia (AML) cell.
  • AML acute myeloid leukemia
  • a method of depleting lymphocytes in a patient in need thereof comprising: administering to the patient an Adapter comprising (i) a D domain that binds to CD 123 and (ii) the AD bound by a CAR, wherein the patient has been administered a cell expressing the CAR, wherein (a) the CAR comprises
  • ADBD antigenic determinant binding domain
  • AD antigenic determinant
  • lymphocytes are B lymphocytes.
  • a method of treating cancer comprising: administering to a patient in need thereof an Adapter comprising (i) a D domain that binds to CD 123 and (ii) the AD bound by a CAR, wherein the patient has been administered a cell expressing the CAR, wherein (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CD123,
  • ADBD antigenic determinant binding domain
  • hematological cancer is acute leukemia, chronic leukemia, polycythemia vera, lymphoma, Hodgkin’s disease, non-Hodgkin’s lymphoma, multiple myeloma, Waldenstrom’s macroglobulinemia, heavy chain disease, myelodysplastic syndrome (e.g., high-risk myelodysplastic syndrome), hairy cell leukemia, or myelodysplasia.
  • myelodysplastic syndrome e.g., high-risk myelodysplastic syndrome
  • hairy cell leukemia or myelodysplasia.
  • hematological 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 neoplasm (BPDCN).
  • AML acute myeloid leukemia
  • myelodysplastic syndrome e.g., high-risk myelodysplastic syndrome
  • B-cell acute lymphoblastic leukemia e.g., hairy cell leukemia
  • Hodgkin’ s lymphoma hedritic neoplasm
  • 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 CD 123 and (ii) the AD bound by a CAR, wherein the patient has been administered a cell expressing the CAR, wherein (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CD 123, (ii) a transmembrane domain, and (iii) an intracellular domain.
  • ADBD antigenic determinant binding domain
  • a method of delivering an immune response to a target cell in a patient comprising: administering to the patient an Adapter comprising (i) a D domain that binds to CD 123 and (ii) the AD bound by a CAR, wherein the patient comprise a cell expressing the CAR, wherein (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CD 123, (ii) a transmembrane domain, and (iii) an intracellular domain.
  • ADBD antigenic determinant binding domain
  • a method of killing a target cell in a patient in need thereof comprising: administering to the patient an Adapter comprising (i) a D domain that binds to CD 123 and (ii) the AD bound by a CAR, wherein the patient comprise a cell expressing the 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.
  • ADBD antigenic determinant binding domain
  • the target cell is an acute myeloid leukemia (AML) cell, myelodysplasia cell, B-cell acute lymphoblastic leukemia cell, hairy cell leukemia cell, Hodgkin’s lymphoma cell or blastic plasmacytoid dendritic neoplasm (BPDCN) cell, preferably an acute myeloid leukemia (AML) cell.
  • AML acute myeloid leukemia
  • a method of depleting lymphocytes in a patient in need thereof comprising: administering to the patient an Adapter comprising (i) a D domain that binds to CD 123 and (ii) the AD bound by a CAR, wherein the patient comprise a cell expressing the CAR, wherein (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CD 123, (ii) a transmembrane domain, and (iii) an intracellular domain.
  • ADBD antigenic determinant binding domain
  • lymphocytes are B lymphocytes.
  • a method of treating cancer comprising: administering to a patient in need thereof an Adapter comprising (i) a D domain that binds to CD 123 and (ii) the AD bound by a CAR, wherein the patient comprise a cell expressing the CAR, wherein (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CD 123, (ii) a transmembrane domain, and (iii) an intracellular domain.
  • ADBD antigenic determinant binding domain
  • hematological cancer is acute leukemia, chronic leukemia, polycythemia vera, lymphoma, Hodgkin’s disease, non-Hodgkin’s lymphoma, multiple myeloma, Waldenstrom’s macroglobulinemia, heavy chain disease, myelodysplastic syndrome (e.g., high-risk myelodysplastic syndrome), hairy cell leukemia, or myelodysplasia.
  • myelodysplastic syndrome e.g., high-risk myelodysplastic syndrome
  • hairy cell leukemia or myelodysplasia.
  • hematological 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 neoplasm (BPDCN).
  • AML acute myeloid leukemia
  • myelodysplastic syndrome e.g., high-risk myelodysplastic syndrome
  • B-cell acute lymphoblastic leukemia e.g., hairy cell leukemia
  • Hodgkin's lymphoma Hodgkin's lymphoma or blastic plasmacytoid dendritic neoplasm
  • 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 CD 123 and (ii) the AD bound by a CAR, wherein the patient comprise a cell expressing the CAR, wherein (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CD 123, (ii) a transmembrane domain, and (iii) an intracellular domain.
  • ADBD antigenic determinant binding domain
  • a method of delivering an immune response to a target cell 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 the AFP p26 antigenic determinant (AD).
  • ADBD first antigenic determinant binding domain
  • ADBD second antigenic determinant binding domain
  • a method of killing a target cell 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) an second antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
  • ADBD first antigenic determinant binding domain
  • ADBD second antigenic determinant binding domain
  • a method of depleting 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) an second antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
  • ADBD first antigenic determinant binding domain
  • ADBD second antigenic determinant binding domain
  • lymphocytes are B lymphocytes or T lymphocytes.
  • 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) an second antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
  • ADBD first antigenic determinant binding domain
  • ADBD second antigenic determinant binding domain
  • the method of [243], wherein the cancer is hematological cancer.
  • the hematological cancer is acute leukemia, chronic leukemia, polycythemia vera, lymphoma, Hodgkin’s disease, non-Hodgkin’s lymphoma, multiple myeloma, Waldenstrom’s macroglobulinemia, heavy chain disease, myelodysplastic syndrome (e.g., high-risk myelodysplastic syndrome), hairy cell leukemia, or myelodysplasia.
  • hematological 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 neoplasm (BPDCN).
  • AML acute myeloid leukemia
  • myelodysplastic syndrome e.g., high-risk myelodysplastic syndrome
  • B-cell acute lymphoblastic leukemia e.g., hairy cell leukemia
  • Hodgkin's lymphoma Hodgkin's lymphoma or blastic plasmacytoid dendritic neoplasm
  • 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) an second antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).
  • ADBD first antigenic determinant binding domain
  • ADBD second antigenic determinant binding domain
  • transmembrane domain comprises a CD8a, 41BB or CD28 transmembrane domain.
  • intracellular signaling domain is selected from the group consisting of a domain of a human T cell receptor alpha, beta, or zeta chain; a human 4 IBB domain; a human CD28 domain; and any combination thereof.
  • the intracellular signaling domain comprises the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, 0X40, CD30, CD40, PD1, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, and any combination thereof.
  • a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, 0X40, CD30, CD40, PD1, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, and any combination thereof.
  • tumor antigen is selected from the group: BCMA, CD19, CD22, CS1, HER2, TACI, BAFFR, and PDL1.
  • AML acute myeloid leukemia
  • BPDCN blastic plasmacytoid dendritic neoplasm
  • administering the Adapter comprises administering a pharmaceutical composition comprising the Adapter.
  • administering the Adapter and cell expressing the CAR comprises administering a pharmaceutical composition comprising the Adapter and a pharmaceutical composition comprising the cell expressing the CAR.
  • FIG. 1 Screen of 8W9C mutants as MBP fusions. Assorted 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 (8W9C), high-affinity (5B7L), low-affinity (5S5W) and non-binding (a3D) controls were assayed for comparison.
  • FIG. 1 CD123 binding of 8W9C mutants as MBP fusions. 8W9C mutants (6Z5E & 4H2H) were assayed by ELISA for binding to CD123 (CD123 Binding). Wild-type (8W9C), high-affinity (5B7L) and low- affinity (5S5W) controls were assayed for comparison.
  • FIG. 3 CD123 binding of 8W9C mutants as MBP fusions. 8W9C mutant (1J1K) was assayed by ELISA for binding to CD123 (CD123 Binding). Wild-type (8W9C), high-affinity (5B7L) and low-affinity (5S5W) controls were assayed for comparison.
  • FIG. 4 Cell binding of 6T1D mutants as Adapters. Assorted variant 6T1D mutants were assayed at various concentrations by flow cytometry for binding to CD123-expressing OCI-AML2 cells. Binding was measured by mean fluorescent intensity (MFI).
  • MFI mean fluorescent intensity
  • FIG. 1 NFAT activation of CAR T cells by sparX proteins. JNL1O/8G8V-CAR are assayed for activation in the presence CD123 expressing M0LM13 target cells and various concentrations of CD123 specific Adapter proteins. Samples included affinity modified variants of 6T1D (e.g. 3F4N & 4R8U). The parental D domain (6T1D) and non-binding (a3D) controls are assayed for comparison.
  • 6T1D affinity modified variants of 6T1D
  • the parental D domain (6T1D) and non-binding (a3D) controls are assayed for comparison.
  • FIG. NFAT activation of CAR T cells cultured with CD123-specific Adapter proteins.
  • JNL1O/8G8V-CAR are assayed for activation in the presence M0LM13 cells lacking CD 123 expression (M0LM13 (CD123-/-)) and various concentrations of sparX proteins.
  • Samples included affinity modified variants of 6T1D (e.g. 3F4N & 4R8U).
  • the parental D domain (6T1D) and non-binding (a3D) controls were assayed for comparison.
  • FIG. 7 IL-2 production of CAR T cells cultured with CD123-specific Adapter proteins.
  • Primary T cells expressing 8G8V-CAR were assayed for IL-2 expression in the presence of M0LM13 target cells and various concentrations of Adapter proteins.
  • the parental D domain (6T1D) and non-binding (a3D) controls were assayed for comparison.
  • FIG. 8 IFNy production of CAR T cells cultured with CD123-specific Adapter proteins.
  • Primary T cells expressing 8G8V-CAR were assayed for IFNy expression in the presence of M0LM13 target cells and various concentrations of Adapter proteins.
  • Samples included affinity modified variants of 6T1D (e.g. 3F4N & 4R8U). The parental D domain (6T1D) and non-binding (a3D) controls were assayed for comparison.
  • FIG. 9 NF AT activation of 8G8V-CAR T cells cultured with CD123-specific Adapter proteins and target cells. JNL1O/8G8V-CAR were assayed for activation in the presence of M0LM13 target cells and various concentrations of Adapter proteins. Samples included affinity modified variants of 6T1D (e.g. 3F4N, 4R8U, 0C8S & 5B1Q). The parental D domain (6T1D) and non-binding (a3D) controls were assayed for comparison. [0023] Figure 10. Lysis of target cells by 8G8V-CAR T cells cultured with CD123-specific Adapter proteins.
  • 6T1D affinity modified variants of 6T1D
  • 6T1D The parental D domain
  • a3D non-binding
  • M0LM13 target cell lysis by primary 8G8V-CAR T cells in the presence of various concentrations of Adapter proteins were assayed.
  • Samples included affinity modified variants of 6T1D (e.g. 3F4N, 4R8U, 0C8S & 5B IQ).
  • the parental D domain (6T1D) and non-binding (a3D) controls were assayed for comparison.
  • FIG. 11 IFNy production by 8G8V-CAR T cells.
  • Primary 8G8V-CAR T cells were assayed by ELISA for IFNy production in the presence of M0LM13 target cells and various concentrations of CD123- specific Adapter protein.
  • Samples included affinity modified variants of 6T1D (e.g. 3F4N, 4R8U, 0C8S & 5B1Q).
  • the parental D domain (6T1D) and non-binding (a3D) controls were assayed for comparison.
  • FIG. 12 IL-2 production by 8G8V-CAR T cells.
  • Primary 8G8V-CAR T cells were assayed by ELISA for IL-2 production in the presence of M0LM13 target cells and various concentrations of CD123- specific Adapter protein.
  • Samples included affinity modified variants of 6T1D (e.g. 3F4N, 4R8U, 0C8S & 5B1Q).
  • the parental D domain (6T1D) and non-binding (a3D) controls are assayed for comparison.
  • FIG. 13 Lysis of target cells by 8G8V-CAR T and CD123-specific Adapter proteins.
  • NALM6- CD123+ cells were lysed by primary 8G8V-CAR T cells in the presence of various concentrations of Adapter proteins.
  • Samples included both mono and bivalent formats of Adapter proteins comprised of low affinity (4G0D), high affinity (5B1Q) and parental D domain (6T1D).
  • Non-binding (a3D) controls is assayed for comparison.
  • Figure 14 In vivo efficacy of the Dd-X CD 123 specific D domain were assessed using a M0LM14- GFP/Luciferase tumor model. Shaded area indicates Dd-X Adapter withdrawal, q.o.d. every other day; q.d. every day.
  • Figure 15 In vivo efficacy of the Dd-X CD 123 specific D domain were assessed using a disseminated MV4-11 tumor model. Shaded area indicates Dd-X Adapter withdrawal, q.d. every day.
  • Figure 16 Patient-derived AML xenograft models.
  • AML xenografts The proportion of the live cells within the bone marrow which represented engrafted AML cells (hCD45+CD3-) following treatment is shown. DETAILED DESCRIPTION
  • Chimeric antigen receptor or "CAR” or “CARs” as used herein refers to an engineered receptor, which grafts an antigen or target specificity onto a cell (for example T cells such as naive T cells, central memory T cells, effector memory T cells, NK cells, NKT cells or combination thereof).
  • CARs are also known as artificial T cell receptors, chimeric T cell receptors or chimeric immunoreceptors.
  • Adapter refers to a multi-domain soluble protein that comprises an antigenic determinant (AD) and an antigenic determinant binding domain (ADBD), wherein the ADBD binds to a second AD.
  • AD antigenic determinant
  • ADBD antigenic determinant binding domain
  • an Adapter can comprise additional AD, additional ADBD, and/or other additional domains.
  • ADBD antigenic determinant binding domain
  • a polypeptide e.g., an Adapter or CAR
  • the ADBD is an antigen-binding antibody fragment, a scFv, or an antigen-binding peptide that is not based on an antibody or antibody fragment sequence (e.g., a D domain or an affibody).
  • the ADBD comprises 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).
  • the ADBD is a D domain.
  • the ADBD is an antibody-based binding sequence.
  • the ADBD is a scFv or a domain antibody (dAb).
  • the ADBD has the ability to bind to a target antigen on the surface of a cell.
  • the ADBD has the ability to bind to a target antigen on the surface of an immune effector cell.
  • the ADBD has the ability to bind a growth factor receptor, an immunoregulatory receptor, or a hormone receptor.
  • the ADBD is a non antibody-scaffold based polypeptide sequence that is sufficient to confer recognition and specific binding to a target antigenic determinant.
  • non-antibody based ADBD is a polypeptide that has the ability to bind to target antigen on the surface of a cell.
  • the non-antibody based ADBD has the ability to bind a growth factor receptor, an immunoregulatory receptor, or a hormone receptor.
  • the ADBD is a D domain-based polypeptide.
  • the ADBD is a D domain-based polypeptide that is sufficient to confer recognition and specific binding to a target antigenic determinant.
  • the ADBD is a D domain-based polypeptide that has the ability to bind to 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 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 a target antigen on a serum protein.
  • D domain refers to a target binding polypeptide sharing certain sequence and certain structural features of the reference scaffold sequence: MGSWAEFKQRLAAIK
  • the reference scaffold is a variant of a non-naturally occurring and targetless antiparallel three helical bundle reference polypeptide originally engineered as an exercise in protein folding (see, Walsh et al., PNAS 96: 5486-5491 (1999) incorporated by reference herein in its entirety).
  • a D domain or a molecule comprising a D domain, can specifically (non-randomly) bind to a target molecule. While not wishing to be bound by theory, it is believed that in designing the D domain, the structural constraints of surface-exposed residues (that can be modified) confer the ability of the surface exposed residues to specifically bind a target of interest. In some embodiments, a D domain generally consists of 70-75 amino acid residues.
  • a D domain comprises an amino acid sequence that differs (e.g., due to amino acid modifications) from that of a reference scaffold having the sequence of SEQ ID NO: 2 by up to 20 substitutions.
  • the D domain does not contain the sequence LAAIKTRLQ (SEQ ID NO: 49).
  • protein and “polypeptide” are used interchangeably herein to refer to a biological polymer comprising units derived from amino acids linked via peptide bonds; a protein can be composed of two or more polypeptide chains.
  • antibody or “immunoglobulin,” as used interchangeably herein, include full-length antibodies and antibody fragments including any functional domain of an antibody such as an antigen-binding fragment or single chains thereof, an effector domain, salvage receptor binding epitope, or portion thereof.
  • a typical antibody comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CHI, CH2, and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, Cl.
  • the VH and VL regions can be further subdivided into regions of hypervariablity, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FW).
  • CDRs Complementarity Determining Regions
  • FW framework regions
  • Each VH and VL is composed of three CDRs and four FWs, arranged from amino-terminus to carboxyl-terminus in the following order: FW 1 , CDR1 , FW2, CDR2, FW3, CDR3, FW4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • antibodies of the present disclosure include typical antibodies, scFvs, and combinations thereof where, for example, a DDpp is covalently linked (e.g., via peptide bonds or via a chemical linker) to the N-terminus of either the heavy chain and/or the light chain of a typical whole (full-length) antibody, or intercalated in the H chain and/or the L chain of a full- length antibody.
  • antibody fragment refers to a portion of an intact antibody and refers to any functional domain of an antibody such as an antigen-binding fragment or single chains thereof, an effector domain or a portion thereof, and a salvage receptor binding epitope or a portion thereof.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, single chain antibodies, and multi-specific antibodies formed from antibody fragments.
  • “Antibody fragment” as used herein comprises an antigen-binding site or epitope binding site.
  • the DDpp fusion protein comprises an effector domain or portion thereof.
  • the DDpp fusion protein comprises a salvage receptor binding epitope, or portion thereof.
  • single chain variable fragment(s),” or “scFv” antibodies refer to forms of antibodies (e.g., antibody fragments) comprising the variable regions of only the heavy and light chains, connected by a linker peptide.
  • the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.
  • ScFv antibodies are generally 220-250 amino acids in length and contain linkers 10-25 amino acids in length.
  • a DDpp fusion protein comprises a DDpp and a scFv.
  • an immunoglobulin Fc region or simply “Fc” is understood to mean the carboxyl-terminal portion of an immunoglobulin chain constant region, preferably an immunoglobulin heavy chain constant region, or a portion thereof.
  • an immunoglobulin Fc region may comprise (1) a CHI domain, a CH2 domain, and a CH3 domain, (2) a CHI domain and a CH2 domain, (3) a CHI 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.
  • 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, and the flexible hinge N-terminal to these domains.
  • IgA and IgM Fc may include the J chain.
  • Fc comprises immunoglobulin domains Cy2 and Cy3 and the hinge between Cyl and Cy2.
  • the immunoglobulin Fc region comprises at least an immunoglobulin hinge region a CH2 domain and a CH3 domain, and preferably lacks the CHI domain.
  • the class of immunoglobulin from which the heavy chain constant region is derived is IgG (Igy) (y subclasses 1, 2, 3, or 4).
  • IgG immunoglobulin
  • IgA Igoe
  • IgD Ig5
  • IgE Iga
  • IgM Igp
  • the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to comprise residues C226 or p260 to its carboxyl-terminus, wherein the 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.
  • Fc may refer to this region in isolation, or this region 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, and thus slight differences between the presented sequence and sequences in the prior art may exist. The choice of appropriate immunoglobulin heavy chain constant region is discussed in detail in U.S. Pat. Nos. 5,541,087, and 5,726,044, each of which is herein incorporated by reference in its entirety.
  • the portion of the DNA construct encoding the immunoglobulin Fc region preferably comprises at least a portion of a hinge domain, and preferably at least a portion of a CH3 domain of Fc gamma or the homologous domains in any of IgA, IgD, IgE, or IgM.
  • substitution or deletion of amino acids within the immunoglobulin heavy chain constant regions may be useful in the practice of the methods and compositions disclosed herein.
  • One example would be to introduce amino acid substitutions in the upper CH2 region to create an Fc variant with reduced affinity for Fc receptors (Cole, J. Immunol. 159: 3613 (1997)).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcRs Fc receptors
  • NK Natural Killer
  • macrophages e.g., NK cells, neutrophils, and macrophages
  • lysis or other cytotoxic effects of the target cell.
  • any in vitro ADCC assay known in the art can be used, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. PNAS 95: 652-656 (1998).
  • linker refers to a peptide or other chemical linkage located between two or more otherwise independent functional domains of a DDpp fusion protein, Adapter or CAR.
  • a linker may be located between an antigenic determinant (AD) domain and an antigenic determinant binding domain (ADBD) of an Adapter.
  • a linker may be located between two antigenic determinant binding domains or an antigenic binding domain and a transmembrane domain of a CAR.
  • a linker is a peptide or other chemical linkage located between a DDpp and another polypeptide of a DDpp fusion protein. Suitable linkers for coupling the two or more domains of an Adapter are described herein and/or will otherwise be clear to a person skilled in the art.
  • operably linked indicates that two molecules are attached so as to each retain at least some level of functional activity that each molecule had alone (assuming that each molecule had a function activity). In embodiments, when one molecule was without functional activity, it is operably linked with another molecule if the other molecule retains at least some level of its functional activity. Operably linked can also refer to linkage of two non-functional molecules. Two molecules can be "operably linked” whether they are attached directly or indirectly e.g., via a linker).
  • binding agent such as a DDpp reacts or associates more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above, to the epitope, protein, or target molecule than with alternative substances, including proteins unrelated to the target epitope, protein, or target molecule.
  • a binding agent such as a DDpp reacts or associates more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above, to the epitope, protein, or target molecule than with alternative substances, including proteins unrelated to the target epitope, protein, or target molecule.
  • specific binding can, in some embodiments, include a binding agent that recognizes a protein or target in more than one species.
  • specific binding can include a binding agent that recognizes more than one protein or target.
  • a binding agent that specifically binds a first target may or may not specifically bind a second target.
  • “specific binding” does not necessarily require (although it can include) exclusive binding, e.g., binding to a single target.
  • a binding agent may, in certain embodiments, specifically bind more than one target.
  • multiple targets may be bound by the same antigen-binding site on the binding agent.
  • Target refers to any molecule or combination of molecules that can be bound by a DDpp such as a DDpp fusion protein, by other component of the DDpp fusion protein such as an antibody or antibody variable domain fragment, by an Adapter or CAR, or by a component of the DDpp fusion protein, Adapter or CAR such as antigenic determinant binding domain.
  • epitopes can be formed from contiguous amino acids and noncontiguous amino acids and/or other chemically active surface groups of molecules (such as carbohydrates) juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding are typically lost upon protein denaturing.
  • An epitope typically includes at least 3 amino acids, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.
  • a "peptide tag” as used herein refers to a peptide sequence that is part of or attached (for instance through genetic engineering) to another protein, to provide a function to the resultant fusion.
  • Peptide tags are usually relatively short in comparison to a protein to which they are fused; by way of example, peptide tags are, in some embodiments, four or more amino acids in length, such as, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more amino acids.
  • the DDpp is a fusion protein that contains a peptide tag. In other embodiments, the DDpp specifically binds a peptide tag. Numerous peptide tags that have uses as provided herein are known in the art.
  • peptide tags that may be a component of a DDpp fusion protein or a target bound by a DDpp (e.g., a DDpp fusion protein) include but are not limited to HA (hemagglutinin), c-myc, the Herpes Simplex virus glycoprotein D (gD), T7, GST, GFP, MBP, Strep-tags, His-tags, Myc-tags, TAP-tags and FLAG® tag (Eastman Kodak, Rochester, N.Y.)
  • antibodies to the tag epitope allow detection and localization of the fusion protein using techniques known in the art, such as, Western blots, ELISA assays, and immunostaining of cells.
  • Derived from indicates a relationship between a first and a second molecule. It generally refers to structural similarity between the first molecule and a second molecule and does not connote or include a process or source limitation on a first molecule that is derived from a second molecule. For example, in the case of an intracellular signaling domain that is derived from a CD3zeta molecule, the intracellular signaling domain retains sufficient CD3zeta structure such that is has the required function, namely, the ability to generate a signal under the appropriate conditions.
  • substitution refers to a replacement of a particular amino acid residue with a different amino acid residue at a corresponding amino acid position of the reference sequence.
  • 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, including 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)), nonpolar 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
  • substitution of a phenylalanine for a tyrosine is a conservative substitution.
  • conservative substitutions in the sequences of the DDpp result in the altered or unaltered specific binding of the DDpp containing the substitution to the target of interest (e.g., CD123, or AFP p26) to which it binds.
  • conservative substitutions in the sequences of the DDpp do not abrogate the binding of the DDpp containing the substitution to the target of interest to which it binds.
  • non-conservative amino acid substitution is one in which one amino acid residue is replaced with another amino acid residue having a dissimilar side chain.
  • non-conservative substitutions in the sequences of the DDpp result in the specific binding of the DDpp containing the substitution to the target of interest (e.g., CD123 or AFP p26) to which it binds.
  • non-conservative substitutions in the sequences of the DDpp do not abrogate the binding of the DDpp containing the substitution to the target of interest to which it binds.
  • non-conservative substitutions in the sequences of the DDpp, Adapter or CAR result in a retained specific binding of the DDpp, Adapter or CAR containing the substitution to the target of interest to which it binds.
  • Non-natural amino acids “amino acid analogs” and “non-standard amino acid residues” are used interchangeably herein.
  • Non-natural amino acids that can be substituted in a DDpp as provided herein are known in the art.
  • the non-natural amino acid is 4-hydroxyproline which can be substituted for proline; 5 -hydroxy lysine 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.
  • non-natural amino acids that can be substituted in a DDpp disclosed herein include, but are not limited to molecules such as: D-isomers of the common amino acids, 2,4-diaminobutyric acid, alpha-amino isobutyric acid, A-aminobutyric acid, Abu, 2-amino butyric acid, gamma-Abu, epsilon- Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, beta-alanine, lanthionine, dehydroalanine, y-aminobutyric acid,
  • non-natural amino acids can include for example, 4-amino butyric acid, 4-amino-3-hydroxy-5- phenylpentanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine, and/or D-isomers of amino acids.
  • non-natural amino acids or amino acid analogs can include deletion of one or more amino acids from a sequence.
  • polynucleotide and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. These terms include, but are not limited to, DNA, RNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA), shRNA (small hairpin RNA), snRNA (small nuclear RNA), snoRNA (short nucleolar RNA), miRNA (microRNA), genomic DNA, synthetic DNA, synthetic RNA, and/or tRNA.
  • an isolated polynucleotide is a modified mRNA comprising non-naturally occurring nucleosides or nucleotides.
  • a modified mRNA comprises 2-thiouridine, pseudouridine, or 1 -methylpseudouridine.
  • vector refers to the vehicle by which a nucleic acid sequence (e.g., a disclosed DDpp, Adapter or CAR coding sequence) can be maintained or amplified in a host cell (e.g., cloning vector) or introduced into a host cell, so as to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence.
  • Vectors include plasmids, phages, viruses, etc.
  • a "host cell” includes an individual cell or cell culture which can be or has been a recipient of nucleic acids encoding a disclosed DDpp, Adapter or CAR.
  • Host cells includes but are not limited to bacteria, yeast plant, animal, and mammalian cells.
  • Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change.
  • a host cell includes cells transfected or infected in vivo, in vitro, or ex vivo with nucleic acids encoding a disclosed DDpp, Adapter or CAR.
  • the host cell is capable of expressing and displaying a disclosed DDpp or CAR on its surface, such as for example, in phage display or a CAR T cell.
  • the host cell is capable of expressing an Adapter.
  • the host cell is capable of expressing and secreting an Adapter.
  • the host cell is capable of expressing a CAR.
  • the host cell is capable of expressing and displaying a CAR on its surface. "Expression" includes transcription and/or translation.
  • solid support As used herein, the terms “solid support,” “support,” “matrices,” and “resins” are used interchangeably and refer to, without limitation, any column (or column material), bead, test tube, microtiter dish, solid particle (for example, agarose or sepharose), microchip (for example, silicon, silicon-glass, or gold chip), or membrane (e.g., biologic or filter membrane) to which a DDpp, antibody, or other protein may be attached (e.g., coupled, linked, or adhered), either directly or indirectly (for example, through other binding partner intermediates such as other antibodies or Protein A), or in which a DDpp or antibody may be embedded (for example, through a receptor or channel).
  • any column or column material
  • test tube for example, microtiter dish
  • solid particle for example, agarose or sepharose
  • microchip for example, silicon, silicon-glass, or gold chip
  • membrane e.g., biologic or filter membrane
  • Suitable solid supports include, but are not limited to, a chromatographic resin or matrix (e.g., SEPHAROSE-4 FF agarose beads), the wall or floor of a well in a plastic microtiter dish, a silica based biochip, polyacrylamide, agarose, silica, nitrocellulose, paper, plastic, nylon, metal, and combinations thereof.
  • DDpp and other compositions may be attached on a support material by a non-covalent association or by covalent bonding, using reagents and techniques known in the art.
  • the DDpp is coupled to a chromatography material using a linker.
  • compositions, carriers, diluents and reagents are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of therapeutically prohibitive undesirable physiological effects such as nausea, dizziness, gastric upset and the like.
  • Parenteral administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.
  • Modulate means adjustment or regulation of amplitude, frequency, degree, or activity.
  • modulation may be positively modulated (e.g., an increase in frequency, degree, or activity) or negatively modulated e.g., a decrease in frequency, degree, or activity).
  • modulation in a positive or negative direction is referenced as compared to the cell, tissue, or organ function prior to administration of a therapeutic.
  • modulation in a positive or negative direction is referenced with respect to a normal, healthy cell, tissue or organ.
  • an "effective amount" of a DDpp (such as a DDpp fusion protein), CAR cell, Adapter, and/or CAR cell/ Adapter composition as provided herein, is an amount sufficient to carry out a specifically stated purpose such as to bring about an observable change in the level of one or more biological activities related to the target to which the DDpp (e.g., a DDpp fusion protein), CAR cell and/or Adapter binds.
  • the change increases the level of target activity.
  • the change decreases the level of target activity.
  • An "effective amount" can be determined empirically and in a routine manner, in relation to the stated purpose.
  • terapéuticaally effective amount refers to an amount of a DDpp (such as a DDpp fusion protein), a CAR cell and/or Adapter, or other therapeutic agent effective to "treat” (e.g., reduce symptoms of) a disease or disorder in a subject (mammal).
  • therapeutically effective amount also refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result.
  • "Patient,” “subject,” “animal” and “mammal” are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, and other animals. Animals include all vertebrates, e.g., mammals and non-mammals, such as chickens, amphibians, and reptiles.
  • “Mammal” as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like.
  • the patient is a human.
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as embryos and fetuses, whether male or female, are intended to be included within the scope of this term.
  • treat refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen or delay) the symptoms, complications, or biochemical indicia of a disease, condition, or disorder, alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder.
  • Treatment can be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease, condition, or disorder targeted pathologic condition, prevent the pathologic condition, pursue or obtain beneficial results, or lower the chances of the individual developing the condition even if the treatment is ultimately unsuccessful.
  • Treatment can be with a DDpp fusion protein, CAR cell, Adapter, and/or CAR cell/ Adapter composition, alone or in combination with an additional therapeutic agent.
  • the terms "treat,” “treatment,” and “treating,” are used herein to refer to therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen or delay) the symptoms, complications, or biochemical indicia of a proliferative disorder, or the amelioration of one or more symptoms (preferably, one or more discernible symptoms) of a proliferative disorder.
  • the terms “treat”, “treatment” and “treating” refer to the amelioration of at least one measurable physical parameter of a proliferative disorder, such as growth of a tumor, not necessarily discernible by the patient.
  • the terms “treat”, “treatment” and “treating” refer to the inhibition of the progression of a proliferative disorder, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both.
  • the terms “treat”, “treatment” and “treating” refer to the reduction or stabilization of tumor size, tumor cell proliferation or survival, or cancerous cell count.
  • Cancer refers to any of a number of diseases that are characterized by uncontrolled, abnormal proliferation of cells, the ability of affected cells to spread locally or through the bloodstream and lymphatic system to other parts of the body (metastasize) as well as any of a number of characteristic structural and/or molecular features.
  • Tuor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • a "cancerous tumor,” or “malignant cell” is understood as a cell having specific structural properties, lacking differentiation and being capable of invasion and metastasis.
  • Cancers that can be treated using a DDpp fusion protein, CAR cell, Adapter, and/or CAR cell/ Adapter composition provided herein include without limitation, breast, lung, brain, cervical, skin, bone, liver, pancreatic, colorectal, renal, head and neck, ovarian, hematopoietic e.g., leukemia), and prostate cancer, and lymphoma.
  • Other types of cancer and tumors that may be treated using a DDpp fusion protein, CAR cell, Adapter, and/or CAR cell/ Adapter composition are described herein or otherwise known in the art.
  • a reference to cancers, tumors, or tumor cells of a particular "type" is understood to mean cancer, tumors, or tumor cells characterized by a specific disease.
  • a first and second cancer of the same type is mixed cellularity Hodgkin’s lymphoma and lymphocyte rich Hodgkin’s lymphoma.
  • a first and second cancer of the same type is precursor B cell acute lymphoblastic leukemia (ALL) and mature B cell ALL.
  • ALL B cell acute lymphoblastic leukemia
  • Examples of a first and second cancer of a different type include, for example, Hodgkin’s lymphoma and ALL.
  • tumor antigen refers to an antigen that is common to a specific hyperproliferative disorder such as cancer.
  • tumor antigen or “cancer antigen” are used interchangeably herein.
  • antigens are derived from cancers 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, leukemias, multiple myeloma, glioblastoma, neuroblastoma, uterine cancer, cervical cancer, renal cancer, thyroid cancer, bladder cancer, kidney cancer, mesothelioma, and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colon cancer and other cancers known in the art.
  • the cancer is acute myeloid leukemia (AML), B-cell acute lymphoblastic leukemia, hairy cell leukemia, Hodgkin's lymphoma or blastic plasmacytoid dendritic neoplasm (BPDCN).
  • AML acute myeloid leukemia
  • BPDCN blastic plasmacytoid dendritic neoplasm
  • the cancer is B-cell acute lymphoid leukemia ("BALL”), T cell acute lymphoid leukemia (“TALL”), acute lymphoid leukemia (ALL), acute myeloid leukemia (AML); one or more chronic leukemias including but not limited to chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL); additional hematologic cancers or hematologic conditions including, but not limited to B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelody
  • Tumor and cancer antigens may be further defined as “tumor-specific antigens (TSA)", “cancerspecific antigens (CSA)", “tumor-associated antigens (TAA)”, or “cancer-associated antigens (CAA)”.
  • TSA tumor-specific antigens
  • CSA cancerspecific antigens
  • TAA tumor-associated antigens
  • CAA cancer-associated antigens
  • a TSA is an antigen that is unique to tumor cells and does not occur on other cells in the body.
  • a TAA is an antigen that is found on both tumor and some normal cells.
  • a TAA may be expressed on normal cells under conditions that fail to induce a state of immunologic tolerance to the antigen. The expression of the TAAs on the tumor may occur under conditions that enable the immune system to respond to the antigen.
  • TAAs may be expressed on normal cells during fetal development when the immune system is immature and unable to respond or may be normally present at extremely low levels on normal cells but which are expressed at much higher levels on tumor cells. Because of the dynamic nature of tumors, in some instances, tumor cells may express unique antigens at certain stages, and at others also express antigens that are also expressed on nontumor cells. Thus, inclusion of a certain marker as a TAA does not preclude it being considered a TSA.
  • the TAA and/or TSA that contains an antigenic determinant specifically bound by a CAR cell, Adapter, and/or CAR cell/ Adapter composition provided herein is selected from: BCMA, CD19, CD20, CD22, CD30, CD33/lL3Ra, CD70, CD123, CD171 (Ll-CAM), CS1, EGFRvIII, GD2, Lewis Y , ROR 1, mesothelin, IL13Ra2, cMet, PSMA, folate receptor alpha (FR-alpha), CEA, ErbB2 (HER-2/neu); EGFR (HER), PSCA, PSA, MUC1, MUC16, CD44v6, CD44v6/7, CD44v7/8, CD55, ILl lRa, EphA2, EGP40, TAG72, CAIX, HMW-MAA (CSPG4), MAGEA4, NKG2D ligands, beta-HCG, Glycolipid F77, HLA
  • autoimmune disease is defined as a disorder that results from an autoimmune response.
  • An autoimmune disease is the result of an inappropriate and excessive response to a self-antigen.
  • autoimmune diseases include but are not limited to, Addison’ s disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondylo
  • target cell refers to a cell or cells which are involved in a disease and can be targeted by DDpp containing compositions or by a CAR, an Adapter, and/or CAR/ Adapter composition provided herein.
  • Target cells include any cell in a subject (e.g., a human or animal) that can be targeted by a DDPP, a CAR, an Adapter, and/or CAR/ Adapter composition.
  • the target cell can be a cell expressing or overexpressing a target specifically bound by a CAR, Adapter, and/or CAR/ Adapter composition.
  • the target cell can be a cell expressing or overexpressing a target specifically bound by a DDpp fusion protein, a CAR, Adapter, and/or CAR/ Adapter composition.
  • the cells express at least Fc(RIII and perform ADCC effector function.
  • human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred in certain embodiments.
  • PBMC peripheral blood mononuclear cells
  • NK natural killer cells
  • the effector cells can be isolated from native source thereof, e.g., from blood or PBMCs as described herein or otherwise known in the art. In a specific embodiment, the effector cells are human effector cells.
  • effector function refers to the specialized immune function of a differentiated cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.
  • immune cell refers to the cells of the 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.
  • T cell and "T lymphocyte” are interchangeable and used synonymously herein. Examples include but are not limited to naive T cells, central memory T cells, effector memory T cells or combinations thereof.
  • immune response refers to immunities including but not limited to innate immunity, humoral immunity, cellular immunity, immunity, inflammatory response, acquired (adaptive) immunity, autoimmunity and/or overactive immunity.
  • transduction refers to the introduction of a foreign nucleic acid into a cell using a viral vector.
  • Transfection refers to the introduction of a foreign nucleic acid into a cell using recombinant DNA technology.
  • transformation means the introduction of a "foreign” e.g., extrinsic, extracellular, or otherwise non-endogenous) nucleic acid (DNA or RNA) sequence to a host cell, so that the host cell will express the introduced nucleic acid to produce a desired substance, such as a protein or enzyme coded by the introduced coding sequence.
  • the introduced nucleic acid sequence can also be called a "cloned” or “foreign” gene or sequence, can include regulatory or control sequences, such as start, stop, promoter, signal, secretion, or other sequences used by a cell's genetic machinery.
  • the nucleic acid sequence can include nonfunctional sequences or sequences with no known function.
  • a host cell that receives and expresses introduced nucleic acid e.g., DNA or RNA
  • the DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell, or cells of a different genus or species or may be non-naturally occurring.
  • Co-express refers to expression of two or more protein coding sequences by the same cell or cell population.
  • the coding sequences may be for example, nucleic acids that each encode a single protein or a chimeric protein as a single polypeptide chain.
  • Cell surface receptor refers to molecules and complexes of molecules capable of receiving a signal and the transmission of such a signal 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 av[>3 integrin receptor on a metastatic cell.
  • cell surface receptor also includes a molecule expressed on a cell surface that contains a DDpp capable of binding a target of interest (e.g., CD123, or AFP p26).
  • receptor denotes 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.
  • a molecule e.g., a ligand
  • the molecule that interacts with a receptor is a bioactive molecule.
  • Membrane-bound cell-surface receptors are typically characterized by a multi-domain structure comprising an extracellular ligand-binding domain, a membrane spanning domain, and an intracellular effector domain that is typically involved in signal transduction.
  • Antigen loss escape variants refer to cells which exhibit reduced or loss of expression of the target antigen, which antigens are targeted by a CAR provided herein.
  • ADs Antigenic Determinants
  • ADs Antigenic determinants
  • ADBDs antigenic determinant binding regions
  • ASBDs antigen-binding fragments of an antibody or alternative scaffold binding domains (e.g., D domains)
  • the ADs in the Adapters and on the target cells provided herein can be bound by the CARs discussed below.
  • the AD e.g., in an Adapter and/or on a target cell
  • the AD is an AD that is present in a naturally occurring protein or other molecule.
  • the AD is an AD that is endogenous to humans.
  • the AD in the Adapter is an AD that is present on a target cell.
  • the AD (e.g., in an Adapter and/or on a target cell) is an AD that is present in a transmembrane protein, e.g., an AD that is present in the extracellular portion of a transmembrane protein.
  • the AD is a tumor antigen.
  • the AD is a tumor-associated antigen.
  • the AD is a tumor-specific antigen.
  • the AD (e.g., in an Adapter and/or on a target cell) is a cancer antigen.
  • the AD is a cancer-associated antigen.
  • the AD is a cancer-specific antigen.
  • the AD (e.g., in an Adapter and/or on a target cell) is an epitope of BCMA.
  • the AD is an epitope of CD19.
  • the AD is an epitope of CD20.
  • the AD is an epitope of CD22.
  • the AD is an epitope of CD123.
  • the AD is an epitope of CD37.
  • the AD is an epitope of CS1.
  • the AD is an epitope of CS1 that is bound by elotuzumab.In some embodiments, the AD is an epitope of HER2.
  • 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 that is bound by the UCHE-1, A6, or ODP4 antibody. In some embodiments, the AD is an epitope of human CD45 that is bound by the 4KB5, MB1, KiB3, 2H4, 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.
  • the AD is an epitope of CD123.
  • 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 of the amino acid residues of SEQ ID NO: 1.
  • the AD is an epitope of AFP p26.
  • 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 of the amino acid residues of SEQ ID NO: 37.
  • the AD comprises the amino acid residues of SEQ ID NO: 37.
  • 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 of the amino acid residues of SEQ ID NO: 39.
  • the AD comprises the amino acid residues of SEQ ID NO: 39.
  • the AD comprises the amino acid residues of SEQ ID NO: 37-43 or 44.
  • the AD e.g., in an Adapter and/or on a target cell
  • a chimeric antigen receptor CAR
  • the AD is bound by a cell expressing a chimeric antigen receptor.
  • the AD (e.g., on a target cell) is bound by an Adapter.
  • the AD is bound by a scFv.
  • the AD is bound by an alternative scaffold binding domain (ASBD).
  • ASBD alternative scaffold binding domain
  • the AD is bound by a D domain.
  • the AD is bound by an antibody or an antigen-binding fragment thereof.
  • ADs suitable for use in connection with the DDpp have been disclosed in Int'l. Appl. Pub. Nos. WO 2016164305, WO 2016164308A1, WO 2019099440, and WO 2019099433, US Patent Nos. 10,662,248, and 10,647,775, and US Pat. Appl. Nos. 20200223934, and 20210002381, each of which is incorporated herein by reference for all purposes.
  • ADBDs Antigenic Determinant Binding Domains
  • ADBD antigenic- determinant binding domain
  • ADBD antigenic- determinant binding domain
  • the ADBD described herein can be present in a DDpp fusion protein, Adapter and/or a chimeric antigen receptor (CAR)).
  • the ADBD (e.g., of a DDpp fusion protein, Adapter and/or CAR) is an antibody or an antigen-binding fragment thereof.
  • the ADBD is a scFv.
  • the ADBD is an alternative scaffold binding domain.
  • the ADBD is a D domain.
  • ADBDs suitable for use in connection with the DDpp have been disclosed in Int'l. Appl. Pub. Nos. WO 2016164305, WO 2016164308A1, WO 2019099440, and WO 2019099433, US Patent Nos. 10,662,248, and 10,647,775, and US Pat. Appl. Nos. 20200223934, and 20210002381, each of which is incorporated herein by reference for all purposes.
  • ADBD Antibody-derived Antigenic Determinant Binding Domains
  • one or more ADBDs can be derived from an antibody molecule, e.g., one or more of monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-domain antibodies e.g., a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) from, e.g., human or camelid origin.
  • 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 may be beneficial for Adapter and/or CAR to comprise a human or a humanized ADBD. Compositions and techniques for routinely generating such ADBDs are known in the art.
  • the ADBD e.g., of a DDpp fusion protein, Adapter and/or CAR
  • the ADBD comprises a fragment of an antibody that is sufficient to confer recognition and specific binding to the target antigen.
  • an antibody fragment include, but are not limited to, an Fab, Fab', F(ab')2, or Fv fragment, an scFv antibody fragment, a linear antibody, single domain antibody such as an sdAb (either VU or VH), a camelid VHH domain, and multi-specific antibodies formed from antibody fragments.
  • the ADBD (e.g., of a DDpp fusion protein, Adapter and/or CAR) is a "scFv," which can comprise a fusion protein comprising a VU chain and a VH chain of an antibody, wherein the VH and VL are, e.g., linked via a short flexible polypeptide linker, e.g., a linker described herein.
  • scFvs can routinely be 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)).
  • the ADBD e.g., of a DDpp fusion protein, Adapter and/or CAR
  • SDAB single domain antigen binding
  • a SDAB molecule includes molecules containing complementary determining regions that are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain variable domains, binding molecules naturally devoid of light chains, single domains derived from conventional 4-chain antibodies, engineered domains and single domain scaffolds other than those derived from antibodies. SDAB molecules can be derived from any species including, but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, and bovine. This term also includes naturally occurring single domain antibody molecules from species other than Camelidae and sharks.
  • the ADBD (e.g., of a DDpp fusion protein, Adapter and/or CAR) comprises a human antibody or a fragment thereof. In some embodiments, the ADBD (e.g., of a DDpp fusion protein, Adapter and/or CAR) comprises a humanized antibody or a fragment thereof.
  • the ADBD(s) is an alternative scaffold binding domain (ASBD).
  • ASBD alternative scaffold binding domain
  • An "alternative scaffold binding domain” or “ASBD” as used herein, is an antigenic determinant binding domain that is derived from, or corresponds to, a non-antibody- based binding scaffold.
  • the disclosure provides a CAR comprising an ADBD that is an ASBD.
  • the disclosure provides a cell comprising a CAR that comprises an ADBD that is an ASBD.
  • an immune effector cell that comprises a CAR comprising and ASBD is provided.
  • the disclosure provides an Adapter comprising an ADBD that is an ASBD.
  • the disclosure provides a composition comprising an Adapter and a CAR that each comprise an ASBD.
  • the binding of the ASBD (e.g., of a DDpp fusion protein, Adapter and/or CAR) to the target AD is mediated by secondary structures of the binding scaffold, such as alpha helices or beta sheets.
  • the ASBD is a three-helix bundle-based binding domain.
  • the ASBD is a D domain-based binding domain.
  • the ASBD is a Z-domain (Affibody)-based binding domain.
  • 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.
  • the D domain comprises a sequence selected from the group: SEQ ID NO: 8-33, 99 and 100.
  • the D domain comprises a sequence selected from the group: SEQ ID NO: 8, 13, 14, 31, 32, and 33.
  • the D domain comprises the sequence of SEQ ID NO: 14.
  • the D domain comprises a sequence selected from the group: SEQ ID NO: 74-93 and 94.
  • the D domain comprises a sequence selected from the group: SEQ ID NO: 70-73 and 92-94.
  • the D domain comprises the sequence of SEQ ID NO: 73.
  • the ASBD e.g., of a DDpp fusion protein, Adapter and/or CAR
  • Z-domain scaffold-based binding domains generally consist of 58 amino acid residues in which substitutions of up to 13 positions located in the first and second of three alpha helices, confer binding confer target (AD) recognition and binding specificity for the target (AD) of interest.
  • Z- domain (Affibody) scaffold-based binding domains are further described in U.S. Pat. No. 5,831,012, the entire contents of which are herein incorporated by reference in their entirety.
  • the ASBD e.g., of a DDpp fusion protein, Adapter and/or CAR
  • the ASBD is a DARPin- based AD binding domain.
  • the ASBD is an adnectin-based AD binding domain.
  • the adnectin-based binding domain is derived from the tenth domain of fibronectin type III (10Fn3).
  • This ADBD is generally a 94 amino acid binding domain that adopts a beta sandwich fold containing seven strands that are connected by six loops. Substitutions in three surface-exposed loops on one side of the adnectin domain generate target (AD) specific binding moieties.
  • the ASBD e.g., of a DDpp fusion protein, Adapter and/or CAR
  • the anticalin scaffold displays a conserved P-barrel structure made up of eight anti-parallel [Tstrands and generally consists of 160-180 amino acids.
  • the ligand binding pocket of the anticallin-based binding scaffold is composed of four loops, each containing up to 24 substitutions, that collectively confer target (AD) recognition and binding specificity.
  • 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. Appl. Publ. Nos. 20040175756, 20050053973, 20050048512, and 20060008844, the entire contents of each of which are herein incorporated by reference in their entireties.
  • the ASBD (e.g., of a DDpp fusion protein, Adapter and/or CAR) is a fynomer scaffold-based AD binding domain.
  • the fynomer binding domain is generally 60-75 amino acids in length and is composed of a pair of anti-parallel beta sheets joined by two flexible loops. Substitutions/insertions in the loops confer AD target recognition and binding specificity.
  • 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 anti-parallel [5-strands connected by loops of variable length and multiple disulfide bonds.
  • 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 in length.
  • the hydrophobic core of this ADBD is composed of a twisted two-stranded antiparallel P-sheet and two a-helices stabilized by three pairs of disulfide bonds. Substitutions and insertions in the three loops confer AD target recognition and binding specificity.
  • Kunitz scaffold-based binding domains are further described in Inti. Appl. Publ. No. WO 2004063337, the entire contents of which are herein incorporated by reference in their entirety.
  • the ASBD (e.g., of a DDpp fusion protein, Adapter and/or CAR) is a WW domain-based AD-binding domain.
  • Linkers are peptide or other chemical linkages located between two or more otherwise independent functional domains of the DDpp fusion protein, Adapter or CAR.
  • Suitable linkers for operably linking a DDpp and an additional component of a DDpp fusion protein or two or more functional domains of the Adapter in a single-chain amino acid sequence include but are not limited to, polypeptide linkers such as glycine linkers, serine linkers, mixed glycine/serine linkers, glycine- and serine- rich linkers or linkers composed of largely polar polypeptide fragments.
  • the linker is made up of a majority of amino acids selected from glycine, alanine, proline, asparagine, glutamine, and lysine.
  • one or more linkers in the DDpp fusion protein, Adapter or CAR is made up 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 the DDpp fusion protein, Adapter or CAR is made up 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 the DDpp fusion protein, Adapter or CAR is made up of a majority of amino acids that are sterically unhindered.
  • a linker in which the majority of amino acids are glycine, serine, and/or alanine.
  • the peptide linker is selected from polyglycines (such as (Gly)s (SEQ ID NO: 45), and (Gly)s (SEQ ID NO: 46), poly (Gly- Ala), and polyalanines.
  • the peptide linker contains the sequence of Gly-Gly-Gly-Gly-Thr-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 47).
  • the peptide linker contains the sequence of 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.
  • the DDpp fusion protein, Adapter or CAR comprises an ADBD (e.g., a D domain) directly attached (i.e., without a linker) to another component of the DDpp fusion protein, Adapter or CAR, respectively.
  • the DDpp fusion protein, Adapter or CAR contains at least 2, at least 3, at least 4, or at least 5 ADBDs (e.g., D domains) directly attached to another domain of the DDpp fusion protein, Adapter or CAR, respectively.
  • an ADBD (e.g., D domain) can be operably linked to another component of the DDpp fusion protein, Adapter or CAR through a linker.
  • DDpp fusion proteins, Adapters or CARs can contain a single linker, multiple linkers, or no linkers.
  • the DDpp fusion protein, Adapter or CAR comprises an ADBD (e.g., D domain) operably linked to another component of the DDpp fusion protein, Adapter or CAR, respectively, through a linker peptide.
  • the DDpp fusion protein, Adapter or CAR contains at least 2, at least 3, at least 4, or at least 5 ADBDs (e.g., D domains) operably linked to another domain of the DDpp fusion protein, Adapter or CAR, respectively, through the same or different linkers.
  • ADBDs e.g., D domains
  • Linkers can be of any size or composition so long as they are able to operably link a functional domain of the DDpp fusion protein, Adapter or CAR in a manner that enables the functional domain to function e.g., the ability of an antigenic determinant binding domain to bind a target of interest).
  • linker(s) are 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.
  • linker(s) may have some influence on the properties of the final polypeptide of the invention, including but not limited to the affinity, specificity or avidity for a target of interest, or for one or more other target proteins of interest.
  • these linkers may be the same or different.
  • a person skilled in the art will be able to routinely determine the optimal linker composition and length for the purpose of operably linking the functional domains of a DDpp fusion protein, Adapter or CAR.
  • the linker can also be a non-peptide linker such as an alkyl linker, or a PEG linker.
  • These alkyl linkers may further be substituted by any non-sterically hindering group such as lower alkyl e.g., C1-C6) lower acyl, halogen (e.g., CI, Br), CN, NH2, phenyl, etc.
  • An exemplary non-peptide linker is a PEG linker.
  • the PEG linker has a molecular weight of about 100 to 5000 kDa, or about 100 to 500 kDa.
  • Suitable linkers for coupling DDpp fusion protein, Adapter or CAR functional domains by chemical cross-linking include, but are not limited to, homo-bifunctional chemical cross-linking compounds such as glutaraldehyde, imidoesters such as dimethyl adipimidate (DMA), dimethyl suberimidate (DMS) and dimethyl pimelimidate (DMP) or N-hydroxy succinimide (NHS) esters such as dithiobis(succinimidylpropionate)(DSP) and dithiobis (sulfosuccini- midylpropionate)(DTSSP).
  • homo-bifunctional chemical cross-linking compounds such as glutaraldehyde, imidoesters such as dimethyl adipimidate (DMA), dimethyl suberimidate (DMS) and dimethyl pimelimidate (DMP) or N-hydroxy succinimide (NHS) esters such as dithiobis(succinimidylpropionate)
  • linkers for coupling DDpp fusion protein, Adapter or CAR functional domains include but are not limited to cross-linkers with one amine -reactive end and a sulfhydryl-reactive moiety at the other end, or with a NHS ester at one end and an SH-reactive group (e.g., a maleimide or pyridyl).
  • one or more of the linkers in the DDpp fusion protein, Adapter or CAR is cleavable.
  • cleavable linkers include, include but are not limited to a peptide sequence recognized by proteases (in vitro or in vivo) of varying type, such as Tev, thrombin, factor Xa, plasmin (blood proteases), metalloproteases, cathepsins e.g., GFLG, etc.), and proteases found in other corporeal compartments.
  • the linker is a "cleavable linker" that facilitates the release of a DDpp fusion protein functional domain, Adapter functional domain or cytotoxic agent in a cell or at the cell surface.
  • a DDpp fusion protein functional domain e.g., Adapter functional domain or cytotoxic agent in a cell or at the cell surface.
  • an acid-labile linker e.g., hydrazone
  • protease-sensitive linker e.g., peptidase-sensitive
  • photolabile linker e.g., peptidase-sensitive linker
  • dimethyl linker or disulfide-containing linker see, e.g., Chari, Can. Res. 52: 127-131 (1992); U.S. Pat. No. 5,208,020; and U.S. Appl. Pub. No.
  • one or more of the linkers in the CAR is cleavable.
  • cleavable linkers include, include but are not limited to a peptide sequence recognized by proteases (in vitro or in vivo) of varying type, such as Tev, thrombin, factor Xa, plasmin (blood proteases), metalloproteases, cathepsins (e.g., GFLG, etc.), and proteases found in other corporeal compartments.
  • a short oligo- or polypeptide linker is used to link together any of the domains of a CAR.
  • Linkers can be composed of flexible residues like glycine and serine (or any other amino acid) so that the adjacent protein domains are free to move relative to one another.
  • the amino acids sequence composition of the linker may be selected to minimize potential immunogenicity of the CAR. Longer linkers can be used when it is desirable to ensure that two adjacent domains do not sterically interfere with one another.
  • the linker is between 10 and 15 amino acids in length, or between 15 and 20, or between 20 and 30, or between 30 and 60, or between 60 and 100 amino acids in length (or any range in between those listed).
  • the linker is a glycine-serine doublet sequence.
  • the Extracellular Spacer Domain corresponds to the human T cell surface glycoprotein CD8 alpha-chain ESD region e.g., amino acid residues 138 to 182 CD8 alpha chain; Swiss-Prot Acc. No. P01732).
  • the ESD corresponds to the CD8 ESD region that has been further modified, through amino acid substitution, to improve expression function or immunogenicity.
  • the ESD corresponds to the CD28 ESD or sequences containing modifications of the CD28 ESD that confer improved expression function or immunogenicity.
  • Linker optimization can be evaluated using techniques described herein and/or otherwise known in the art.
  • linkers do not disrupt the ability of a DDpp fusion protein, Adapter or CAR to bind a target antigenic determinant and/or another Adapter or CAR functional domain to function appropriately (e.g., the ability of an effector functional domain in the Adapter to elicit an effector function or the ability of an FcRn binding domain in the Adapter to bind FcRn).
  • the disclosure provides a DDpp that specifically binds to CD123.
  • the DDpp comprises a D Domain (DD) that specifically binds CD123 and comprises the amino acid sequence of SEQ ID NO: 8-32 or 33.
  • 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.
  • the D domain that binds to CD123 comprises the amino acid sequence of SEQ ID NO: 14. Proteins comprising variants of the D domains that retain the ability to specifically bind their respective targets are also provided.
  • the DDpp comprises a D Domain (DD) that specifically binds AFP p26 and comprises the amino acid sequence of SEQ ID NO: 74-93 or 94. Proteins comprising variants of the D domains that retain the ability to specifically bind their respective targets are also provided.
  • DD D Domain
  • the DDpp is fused to a heterologous polypeptide.
  • the heterologous polypeptide comprises a full-length antibody or an antibody fragment.
  • the 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.
  • the DD is fused to an antibody fragment which is an Fc.
  • the heterologous polypeptide comprises a member selected from the group consisting of: (i) a transmembrane domain; (ii) a membrane associating domain; (iii) human serum albumin or a fragment thereof; (iv) AFP or a fragment thereof; (v) AFP p26 or a fragment thereof; (vi) the extracellular domain of a receptor or a fragment thereof; and (vii) the extracellular domain of an intracellular receptor (e.g., a nuclear protein) or a fragment thereof.
  • an intracellular receptor e.g., a nuclear protein
  • the protein contains a heterologous polypeptide that comprises the extracellular domain, or a fragment of an extracellular domain of BCMA (SEQ ID NO: 34) or CD123 (SEQ ID NO: 1) or CD19 (SEQ ID NO: 95) or CS1 (SEQ ID NO: 35).
  • the protein contains a heterologous polypeptide that comprises the extracellular domain, or a fragment of an extracellular domain, of a receptor selected from the group consisting of: CD19, CD20, CD22, HVEM, BTLA, DR3, CD37; TSLPR, IL7R, and gp96.
  • the protein contains a heterologous polypeptide that comprises an antigenic portion of a serum protein (e.g., AFP, and AFP p26). In some embodiments, the protein contains a heterologous polypeptide that comprises 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 with major histocompatibility complex (MHC) class I or class II complexes.
  • MHC major histocompatibility complex
  • a DD of the DDpp is a variant of a CD 123 -binding DD reference sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100, that retains the ability to specifically bind CD123.
  • the sequence of the variant DD comprises the amino acid sequence of a variant that has 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 NO: 8-33, 99 and 100, and the variant DD retains the ability to specifically bind CD 123.
  • a DD of the DDpp is a variant of a AFP p26-binding DD reference sequence selected from the group consisting of SEQ ID NO: 74-93 and 94, that retains the ability to specifically bind AFP p26.
  • the sequence of the variant DD comprises the amino acid sequence of a variant that has 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 NO: 74-93 and 94, and the variant DD retains the ability to specifically bind AFP p26.
  • the identity between a variant DD (query) sequence and a reference DD sequence is determined using the FASTDB computer program based on the algorithm of Brutlag et al. Comp. App. Biosci. 6: 237-245 (1990).
  • the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the reference sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence.
  • a determination of whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of this embodiment.
  • the disclosed DDpp (e.g., a DDpp fusion protein) is labeled.
  • Eabels that can be used to label the DDpp include but are not limited to an enzymatic label, a fluorescent label, a luminescent label, and a bioluminescent label.
  • the label is a biotin moiety.
  • the label is a streptavidin moiety.
  • the label is a His-tag or a FEAG tag.
  • the label is luciferase, green fluorescent protein, red fluorescent protein, or other similar agent.
  • the DDpp comprises a CD123-binding DD comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 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 NO: 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 NO: 74-93 and 94.
  • the DDpp fusion protein is attached to a solid support.
  • the solid support is selected from the group consisting of: a bead, a glass slide, a chip, a gelatin, and an agarose.
  • the DDpp comprises a CD123-binding DD comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100.
  • the DDpp comprises a DD comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 13, 14, 31, 32, and 33.
  • the DDpp comprises a DD comprising the amino acid sequence of SEQ ID NO: 14.
  • the DDpp comprises a p26-binding DD comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94.
  • the DDpp (e.g., a DDpp fusion protein) is associated with a liposome. In some embodiments, the DDpp is associated with the liposome through covalent binding. In some embodiments, DDpp is a fusion protein. In further embodiments, the DDpp is a CAR. In additional embodiments, the DDpp is associated with the liposome through ionic binding but not covalent binding. In some embodiments, the DDpp comprises a CD123-binding DD comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100.
  • the DDpp comprises a DD comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 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 NO: 74-93 and 94.
  • the target-binding DDpp is conjugated to a therapeutic or cytotoxic agent (e.g., a chemotherapeutic agent or a radiotherapeutic agent).
  • the DDpp comprises a CD123- binding DD comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100.
  • the DDpp comprises a DD comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 13, 14, 31, 32, and 33.
  • the DDpp comprises a DD comprising the amino acid sequence of SEQ ID NO: 14.
  • the DDpp comprises a p26- binding DD comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94.
  • the disclosure provides compositions comprising one or more of the DD sequences disclosed on Table 1.
  • the disclosure provides compositions comprising one or more DDs comprising a sequence with 60-70%, 70-75%, 75-80%, 80-85%, 85-90%, 95-99% homology (and overlapping ranges therein) with a sequence disclosed in Table 1.
  • the DD(s) having such homology are functionally similar or identical as compared to the respective reference sequence in Table 1.
  • the disclosure provides a polypeptide that comprises one or more DD that compete with (wholly or partially) one or more of the DD sequences disclosed in Table 1 (reference sequence) for its respective target.
  • polypeptide The ability of one polypeptpide to compete with a reference polypeptide for binding to a respective target can routinely be determined using a standard competition assay known in the art. In some embodiments, competition does not require that the polypeptide competes for the same epitope as a polypeptide (DD) of Table 1, rather the polypeptide can compete by binding a sterically inhibiting epitope, an overlapping epitope, etc.
  • DD polypeptide
  • the disclosure provides a protein comprising a D Domain (DD) target binding domain (DDpp) that specifically binds CD123 (SEQ ID NO: 1) and comprises the amino acid sequence of SEQ ID NO: 8-32 or 33.
  • the DDpp comprises a DD comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 13, 14, 31, 32, and 33.
  • the DDpp comprises a DD comprising the amino acid sequence of SEQ ID NO: 14.
  • a DD of the DDpp specifically binds CD123.
  • the DD specifically binds CD123 having an amino acid sequence consisting of SEQ ID NO: 8.
  • the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NO: SEQ ID NO: 8-33, 99 and 100.
  • the DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NO: SEQ ID NO: 8-33, 99 and 100.
  • the DDpp specifically binds CD123 (SEQ ID NO: 1) and comprises the amino acid sequence of SEQ ID NO: 8, 13, 14, 31, 32, or 33.
  • 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.
  • the CD123-binding DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NO: 8-32, and 33.
  • the CD123-binding DDpp comprises multiple target-binding domains that bind a single target (e.g., dimers, trimers, etc.).
  • the DDpp comprises 2, 3, 4, 5, or more than 5, DD that specifically bind CD123 and that have an amino acid sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100.
  • the DDpp comprises 2, 3, 4, 5, or more than 5, DD that have the same sequence.
  • the DDpp comprises 2, 3, 4, 5 or more than 5, DD that specifically bind to different epitopes of CD123 and that have an amino acid sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100.
  • the DDpp comprises a DD that specifically binds CD123 and further comprises 2, 3, 4, 5 or more than 5, additional different DDs or target-binding binding domains (e.g., scFvs) that specifically bind to CD123 or a different target antigen.
  • the DDpp comprises a DD that specifically binds CD123 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NO: 8- 33, 99 and 100) and further comprises one or more additional DDs or other target-binding binding domains that bind one or more antigens expressed on the surface of a B -lineagecell.
  • the DDpp comprises a DD that specifically binds CD123 e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100) and further comprises one or more additional DDs or other target-binding binding domains that bind one or more cancer antigens.
  • the DDpp comprises a DD that specifically binds CD123 e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100) and specifically binds 2, 3, 4, 5, or more than 5, different targets.
  • the DDpp comprises a DD that specifically binds CD123 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100) and specifically binds 2, 3, 4, 5, or more than 5, different cancer antigens.
  • the DDpp comprises a DD that specifically binds CD123 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100) and specifically binds 2, 3, 4, 5, or more than 5, different cancer antigens expressed on the surface of a cancer cell.
  • the DDpp comprises a DD that specifically binds CD123 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NO: 8- 33, 99 and 100) and specifically binds 2, 3, 4, 5, or more than 5, cancer antigens expressed on the surface of different cancer cells.
  • the CD123-specific DD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 13, 14, 31, 32, and 33.
  • the CD123- specific DD comprises the amino acid sequence of SEQ ID NO: 14.
  • the DDpp comprises a variant of a CD123-binding DD disclosed herein (reference DD) that retains the ability to specifically bind CD123.
  • the sequence of the CD123-binding DD variant contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1-3, 1-5, or 1-10, conservative or nonconservative substitutions compared to a reference CD123-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100.
  • the sequence of the CD123- binding DD variant contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1-3, 1-5, or 1-10, conservative substitutions compared to a reference CD123-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NO: 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, 1-3, 1-5, or 1-10, non-conservative substitutions compared to a reference CD123- binding DD having an amino acid sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100.
  • the reference CD123-binding DD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 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.
  • 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 in 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 NO: 8-33, 99 and 100.
  • 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 in 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 NO: 8-33, 99 and 100.
  • 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 in 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 NO: 8-33, 99 and 100.
  • the reference CD123-binding DD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 13, 14, 31, 32, and 33.
  • the reference CD123-binding DD comprises the amino acid sequence of SEQ ID NO: 14.
  • 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 in 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 CD123-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100.
  • 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 in 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 NO: 8-33, 99 and 100.
  • 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 in 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 NO: 8-33, 99 and 100.
  • the reference CD123-binding DD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 13, 14, 31, 32, and 33.
  • the reference CD123-binding DD comprises the amino acid sequence of SEQ ID NO: 14.
  • the sequence of the CD123-binding DD variant contains a total of 1, 2, 3, 4, 5,
  • the sequence of the CD123-binding DD variant contains a total of 1, 2, 3, 4, 5, 6,
  • 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 in 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 NO: 8-33, 99 and 100.
  • the reference CD123-binding DD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 13, 14, 31, 32, and 33.
  • the reference CD123-binding DD comprises the amino acid sequence of SEQ ID NO: 14.
  • the disclosure provides CD123-binding DDpp that completely or partially (e.g., overlap with an epitope) block binding of a reference DD to CD 123, wherein the reference DD has an amino acid sequence selected from SEQ ID NO: 8-33, 99 and 100.
  • the disclosure provides CD123-binding DDpp that bind to the same epitope of CD123as a a reference DD consisting of an amino acid sequence selected from SEQ ID NO: 8-33, 99 and 100.
  • the reference CD123-binding DD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 13, 14, 31, 32, and 33.
  • the reference CD123-binding DD comprises the amino acid sequence of SEQ ID NO: 14.
  • the DDpp is a fusion protein comprising a DD that specifically binds CD123.
  • a DD of the DDpp fusion protein specifically binds CD123 having an amino acid sequence consisting of of SEQ ID NO: 1.
  • the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100.
  • the DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100.
  • the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 13, 14, 31, 32, and 33.
  • 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 some embodiments, the DDpp fusion protein comprises a full-length antibody or a portion (fragment) of an antibody.
  • the DDpp fusion protein comprises a full length IgG antibody (e.g., IgGl, IgG2, IgG2, or IgG4). In further embodiments, the DDpp fusion protein comprises a full length antibody that specifically binds a cancer antigen. In further embodiments, the DDpp comprises a commercially 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.
  • the CD123- binding DDpp is an Fc fusion protein.
  • the DDpp is a fusion protein comprising a CD123-binding DD operably linked to a serum protein.
  • the CD123-binding DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100.
  • the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 13, 14, 31, 32, and 33.
  • the DDpp comprises the amino acid sequence of SEQ ID NO: 8.
  • the DDpp comprises the amino acid sequence of SEQ ID NO: 13.
  • the DDpp comprises the amino acid sequence of SEQ ID NO: 14.
  • 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 NO: 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.
  • the CD123-binding DDpp fusion protein comprises AFP p26 (SEQ ID NO: 37), or a fragment thereof.
  • the CD123-bindng DDpp fusion protein comprises a polypeptide having the sequence of SEQ ID NO: 37-43 or 44.
  • the DDpp fusion protein contains a fragment of a serum protein or an antigenic fragment of a serum protein (e.g., AFP, and AFP p26).
  • the DDpp fusion protein comprises a fragment consisting 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.
  • the CD123-specific DD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 13, 14, 31, 32, and 33.
  • the CD123-specific DD comprises the amino acid sequence of SEQ ID NO: 14.
  • the CD123-binding DDpp fusion protein comprises the extracellular domain of a receptor or a fragment thereof.
  • the DDpp fusion protein comprises a DD that comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100.
  • the CD123-binding DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 13, 14, 31, 32, and 33.
  • the CD123-binding DDpp comprises the amino acid sequence of SEQ ID NO: 8.
  • the CD123-binding DDpp comprises the amino acid sequence of SEQ ID NO: 13.
  • the CD123-binding DDpp comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the CD123-binding DDpp comprises 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 an amino acid sequence selected from the group consisting of SEQ ID NO: 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.
  • 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.
  • the CD123-binding DDpp fusion protein contains a fragment consisting 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 an extracellular domain, of a cell surface receptor.
  • the DDpp fusion protein contains a fragment of an extracellular domain of BCMA (SEQ ID NO: 34). In some embodiments, the DDpp fusion protein contains a fragment of an extracellular domain of CD123 (SEQ ID NO: 1). In some embodiments, the DDpp fusion protein contains a fragment of an extracellular domain of CS1 (SEQ ID NO: 35). In some embodiments, the DDpp contains a fragment of an extracellular domain, of a receptor selected from the group consisting of: CD19, CD20, CD22, HVEM, BTLA, DR3, CD37; TSLPR, IL7R, and gp96.
  • the CD123-binding DDpp fusion protein comprises an intracellular protein (e.g., a nuclear protein) or a fragment thereof.
  • the DDpp fusion protein comprises a DD that comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100.
  • the CD123-binding DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 13, 14, 31, 32, and 33.
  • the CD123-binding DDpp comprises the amino acid sequence of SEQ ID NO: 8.
  • the CD123-binding DDpp comprises the amino acid sequence of SEQ ID NO: 13.
  • 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 an amino acid sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100.
  • the CD123-binding DDpp fusion protein comprises a fragment consisting 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).
  • an intracellular protein e.g., a nuclear protein
  • the disclosure provides a protein comprising a D Domain (DD) target binding domain (DDpp) that specifically binds AFP p26 (SEQ ID NO: 37) and comprises the amino acid sequence of SEQ ID NO: 74-93 or 94.
  • DD D Domain
  • DDpp target binding domain
  • a DD of the DDpp specifically binds AFP p26. In further embodiments, a DD of the DDpp specifically binds AFP p26 having an amino acid sequence consisting of SEQ ID NO: 37. In some embodiments, the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 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 NO: 74-93 and 94. In some embodiments, the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 92, 93 and 94.
  • the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 70-73, 92, 93 and 94. In some embodiments, the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 73.
  • the DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94.
  • the DD specifically binds AFP p26 having an amino acid sequence consisting of SEQ ID NO: 37, but does not specifically bind AFP having an amino acid sequence consisting of SEQ ID NO: 36.
  • the AFP p26-binding DDpp comprises multiple target-binding domains that bind a single target (e.g., dimers, trimers, etc.).
  • the DDpp comprises 2, 3, 4, 5, or more than 5, DD that specifically bind AFP p26 and that have an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94. In some embodiments, the DDpp comprises 2, 3, 4, 5, or more than 5, DD that have the same sequence. In some embodiments, the DDpp comprises 2, 3, 4, 5 or more than 5, DD that specifically bind to different epitopes of AFP p26 and that have an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94.
  • the DDpp comprises a DD that specifically binds AFP p26 and further comprises 2, 3, 4, 5 or more than 5, additional different DDs or target-binding binding domains (e.g., scFvs) that specifically bind to AFP p26 or a different target antigen.
  • the DDpp comprises a DD that specifically binds AFP p26 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94) and further comprises one or more additional DDs or other target-binding binding domains that bind one or more antigens expressed on the surface of a B cell.
  • the DDpp comprises a DD that specifically binds AFP p26 e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94) and further comprises one or more additional DDs or other target-binding binding domains that bind one or more cancer antigens.
  • the DDpp comprises a DD that specifically binds AFP p26 e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94) and specifically binds 2, 3, 4, 5, or more than 5, different targets.
  • the DDpp comprises a DD that specifically binds AFP p26 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94) and specifically binds 2, 3, 4, 5, or more than 5, different cancer antigens.
  • the DDpp comprises a DD that specifically binds AFP p26 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94) and specifically binds 2, 3, 4, 5, or more than 5, different cancer antigens expressed on the surface of a cancer cell.
  • the DDpp comprises a DD that specifically binds AFP p26 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94) and specifically binds 2, 3, 4, 5, or more than 5, cancer antigens expressed on the surface of different cancer cells.
  • the DDpp comprises a variant of a AFP p26-binding DD disclosed herein (reference DD) that retains the ability to specifically bind AFP p26.
  • the sequence of the AFP p26-binding DD variant contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1-3, 1-5, or 1-10, conservative or nonconservative substitutions compared to a reference AFP p26-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NO: 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, 1-3, 1-5, or 1-10, conservative substitutions compared to a reference AFP p26-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94.
  • the sequence of the AFP p26-binding DD variant contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1-3, 1-5, or 1-10, non-conservative substitutions compared to a reference AFP p26-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94.
  • 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 in 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 NO: 74-93 and 94.
  • 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 in 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 NO: 74-93 and 94.
  • 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 in 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 NO: 74-93 and 94.
  • the reference p26-binding DD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 70-73 and 92-94.
  • the reference p26-binding DD comprises the amino acid sequence of SEQ ID NO: 14.
  • 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 in 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 NO: 74-93 and 94.
  • 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 in 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 NO: 74-93 and 94.
  • 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 in 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 NO: 74-93 and 94.
  • the reference AFP p26-binding DD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 70-73 and 92-94. In some embodiments, the reference AFP p26-binding DD comprises the amino acid sequence of SEQ ID NO: 73.
  • 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 in positions corresponding to amino acid residues: 7, 11, 14, 18, 21, 28, 31, 35, 38 tone 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 NO: 74-93 and 94.
  • 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 in 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 NO: 74-93 and 94.
  • 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 in 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 NO: 74-93 and 94.
  • the reference AFP p26-binding DD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 70-73 and 92-94.
  • the reference AFP p26-binding DD comprises the amino acid sequence of SEQ ID NO: 73.
  • the disclosure provides an AFP p26-binding DDpp that completely or partially (e.g., overlap with an epitope) block binding of a reference DD to AFP p26, wherein the reference DD has an amino acid sequence selected from SEQ ID NO: 74-93, and 94.
  • the disclosure provides AFP p26-binding DDpp that bind to the same epitope of AFP p26 as a a reference DD consisting of an amino acid sequence selected from SEQ ID NO: 74-93, and 94.
  • the reference p26-binding DD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 70-73 and 92-94.
  • the reference p26-binding DD comprises the amino acid sequence of SEQ ID NO: 73.
  • the DDpp is a fusion protein comprising a DD that specifically binds AFP p26.
  • the DD specifically binds AFP p26 having an amino acid sequence consisting of SEQ ID NO: 37.
  • the DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94.
  • a DD of the DDpp fusion protein specifically binds AFP p26 but does not specifically bind AFP having an amino acid sequence consisting of SEQ ID NO: 36.
  • the DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94.
  • the AFP p26-binding DDpp is an Fc fusion protein.
  • the DDpp is a fusion protein comprising a DD that specifically binds AFP p26.
  • the DDpp is a fusion protein comprising a DD that specifically binds AFP p26 having an amino acid sequence consisting of SEQ ID NO: 37.
  • the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94.
  • the DDpp is a fusion protein comprising a AFP p26-binding DD that is a variant of a DD comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94.
  • the DDpp is a fusion protein comprising a DD that specifically binds AFP p26 operably linked to a full-length antibody or a portion (fragment) of an antibody.
  • the DDpp is an Fc fusion protein.
  • the DDpp fusion protein comprises a full length IgG antibody (e.g., IgGl, IgG2, IgG2, or IgG4).
  • the DDpp fusion protein comprises a full length antibody that specifically binds a cancer antigen.
  • the DDpp comprises a commercially approved therapeutic antibody (e.g., rituximab, ofatumumab, ocrelizumab, veltuzumab, MEDI-551, epratuzumab, belimumab, tabalumab, AMG-557, MEDI-570, NN882, elotuzumab, and daratumumab).
  • the AFP p26-binding DDpp is an Fc fusion protein.
  • the Fc fusion protein comprises a variant human Fc domain.
  • the DDpp is a fusion protein comprising an AFP p26-binding DD operably linked to a serum protein.
  • the DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94.
  • the AFP p26-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94.
  • the DDpp fusion protein comprises human serum albumin or a fragment thereof.
  • the DDpp fusion protein contains a fragment of a serum protein or an antigenic fragment of a serum protein.
  • the DDpp fusion protein comprises a fragment consisting 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.
  • the AFP p26-binding DDpp fusion protein comprises the extracellular domain of a receptor or a fragment thereof.
  • the DDpp fusion protein comprises a DD that comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94.
  • the AFP p26-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94.
  • 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.
  • the AFP p26-binding DDpp fusion protein comprises the extracellular domain of BCMA (SEQ ID NO: 34), or CD123 (SEQ ID NO: 1), or CS1 (SEQ ID NO: 35), or a fragment thereof.
  • the AFP p26-binding DDpp fusion protein comprises the extracellular domain of of a receptor selected from the group consisting of: CD19, CD20, CD22, HVEM, BTLA, DR3, CD37; TSLPR, IL7R, and gp96, or a fragment thereof.
  • the AFP p26-binding DDpp fusion protein contains a fragment consisting 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 an extracellular domain, of a cell surface receptor.
  • the DDpp fusion protein contains a fragment of an extracellular domain of BCMA (SEQ ID NO: 34) or CD123 (SEQ ID NO: 1).
  • the DDpp fusion protein contains a fragment of an extracellular domain of BCMA (SEQ ID NO: 34), or CD123 (SEQ ID NO: 1), or CS1 (SEQ ID NO: 35).
  • the DDpp contains a fragment of an extracellular domain, of a receptor selected from the group consisting of: CD19, CD20, CD22, HVEM, BTLA, DR3, CD37; TSLPR, IL7R, and gp96.
  • the AFP p26-binding DDpp fusion protein comprises an intracellular protein (e.g., a nuclear protein) or a fragment thereof.
  • the DDpp fusion protein comprises a DD that comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94.
  • the AFP p26-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94.
  • the AFP p26-binding DDpp fusion protein comprises a fragment consisting 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).
  • an intracellular protein e.g., a nuclear protein
  • the AFP p26-binding DDpp fusion protein comprises a fragment of a serum protein (e.g., HSA), an extracellular domain of a receptor (e.g., BCMA, CS1, CD123, and CD19), or an intracellular protein (e.g., a nuclear protein), consisting 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.
  • a serum protein e.g., HSA
  • an extracellular domain of a receptor e.g., BCMA, CS1, CD123, and CD19
  • an intracellular protein e.g., a nuclear protein
  • Nucleic acids encoding the DDpp and vectors containing the nucleic acids are also provided.
  • Host cells containing the nucleic acids and vectors containing the nucleic acids are also provided.
  • the host cell is a prokaryote or a eukaryote that display the variant DD on its surface.
  • the host cell displays the variant DD on its surface.
  • the host cell is a phage that displays the variant DD on its surface.
  • the host cell is a human immune cell that expresses a variant DD fusion protein on its surface.
  • a DDpp agonist refers to a DDpp that in some way increases or enhances the biological activity of the DDpp target (e.g., CD123) or has biological activity comparable to a known agonist of the DDpp target.
  • the DDpp is an antagonist of the target 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 the DDpp target protein or has biological activity comparable to a known antagonist or inhibitor of the DDpp target protein.
  • DDpp fusion proteins are provided herein.
  • the two polypeptides may be operably attached directly or indirectly.
  • a "DDpp fusion protein” comprises at least one DDpp disclosed herein that specifically binds 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).
  • 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.
  • the DDpp fusion protein contains one DDpp.
  • the DDpp fusion protein is a soluble protein comprising one or more targetbinding DDpp and a p26 protein (e.g., having the sequence of SEQ ID NO: 37-43 or 44).
  • the soluble DDpp fusion protein has a plasma half-life in vivo 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.
  • the soluble 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, or 1-10 hours, 2-10 hours, 4-10 hours, 6-10 hours, or 6-9 hours in a mouse.
  • 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, or 1-10 hours, 2-10 hours, 4-10 hours, 6-10 hours, or 6-9 hours, in a human.
  • the disclosure provides a method for modifying the in vivo half-life (e.g., in a mouse or human) of a soluble fusion protein comprising a p26 protein (e.g., having the sequence of SEQ ID NO: 37-43 or 44).
  • the soluble p26 fusion protein comprises one or more target-binding DDpp.
  • the half-life of the p26 soluble fusion protein is increased or decreased by substituting or deleting one or more amino acid residues normally found in the human p26 protein, or by inserting one or more amino acid residues not normally found in the human p26 protein.
  • 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 nonconservative substitutions), deletions, and/or insertions so as to increase or decrease the in vivo half-life of the soluble fusion protein.
  • amino acid residue corresponding to the glutamine (Gin, Q) at position 217 of SEQ ID NO: 37 of p26 is substituted with another amino acid residues.
  • the substitution is Gln217Pro.
  • the p26 sequence of the soluble fusion protein is modified through deletion of 1-150, 1-100, 1- 50, 1-25 or 1-10 amino acid residues so as to increase or decrease the in vivo half-life of the soluble fusion protein.
  • 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 nonconservative substitutions), deletions, and/or insertions so as to increase or decrease the interaction of the soluble fusion protein with FcRn.
  • the DDpp fusion protein comprises more than one DDpp, wherein two or more DDpp have the same or different specificities.
  • the DDpp fusion protein comprises a tandem repeat of the same or different DD that allow a DDpp fusion protein to bind multiple targets and/or repeating epitopes or different epitopes on the same target.
  • the DDpp fusion protein comprises at least 2, 3, 4, or 5, or more than 5, DDpp.
  • the DDpp fusion protein contains 1-3, 1-4, 1-5, or more than 5, different DDpp.
  • the DDpp fusion protein contains at least 2, 3, 4, or 5, or more than 5, different DDpp.
  • a DDpp fusion protein can be a monomeric DDpp (i.e., containing one DDpp) or multimeric DDpp (i.e., containing more than one DDpp in tandem optionally operably connected by a linker).
  • the use of multimeric DDpp provides enhanced (e.g., synergistic) target binding.
  • multimeric DDpp allows targeting of more than one target using a single DDpp construct (e.g., bi-, tri-specific, etc.).
  • the linkage of two or more identical DDpp results in a multivalent molecule that provides distinct advantages (e.g., increased binding avidity, target clustering and receptor activation) over monovalent compositions.
  • the linkage of two or more different DDpp results in a multivalent and multi-specific molecule that has the potential to bind more than one target antigen, either independently or simultaneously.
  • the multimeric DDpp fusion protein can be a DDpp homo-multimeric (i.e., containing more than one of the same DDpp in tandem optionally connected by linker(s) (e.g., homodimers, homotrimers, homotetramers etc.) or DDpp hetero-multimeric (i.e., containing two or more DDpp in which there are at least two different DDpp proteins.
  • linker(s) e.g., homodimers, homotrimers, homotetramers etc.
  • DDpp hetero-multimeric i.e., containing two or more DDpp in which there are at least two different DDpp proteins.
  • the number of monomeric DDpp included within 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.
  • the fusion proteins may comprises multimers 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 numbers in between those listed as well as endpoints).
  • multiple tandem components of a DDpp fusion protein can contain the same or different DDpp.
  • the DDpp are present as a monomer, or in homomultimers or heteromers such as, homodimers or heterodimers, homotrimers or heterotrimers, homotetramers or heterotetramers.
  • a DDpp fusion protein can be "monospecific” or “multi-specific.”
  • a DDpp fusion protein that is “multispecific” e.g., bispecific, trispecific or of greater multi-specificity
  • recognizes and binds to two or more different epitopes present on one or more different molecules e.g., proteins, solid support structures, etc.
  • two or more DDs are fused together as a multivalent DDpp.
  • the DD of the multivalent DDpp may be the same or different.
  • the disclosure provides a DDpp homo-dimer (i.e., a DDpp comprising two identical DD), a DDpp homo-multimer (i.e., a DDpp comprising three or more identical DD), a DDpp hetero-dimer (i.e., a DDpp comprising two different DD), and DDpp hetero-multimer (i.e., a DDpp comprising three or more DD, wherein at least two of the DD are different) comprising any of the DD described herein, optionally attached by one or more linkers.
  • two or more DDs are linked by a multimerization domain or attached via chemical linkage, to generate a multivalent DD complex.
  • the DD of the multivalent DD complex may be the same or different.
  • the disclosure provides a DD homo-dimer complex (i.e., a DD complex comprising two identical DD), a DD homo-multimer complex (i.e., a DD complex comprising three or more identical DD), a DD hetero-dimer complex (i.e., a DD complex comprising two different DD), and DD hetero-multimer complex (i.e., a DD complex comprising three or more DD, wherein at least two of the DD are different) comprising any of the DD described herein, optionally attached by one or more linkers.
  • a multi-specific DDpp fusion protein contains at least two DDpp that bind to at least two different epitopes on a single target of interest (e.g., CD123, CD33, LeY, CD38, BCMA, or CS1, preferably CD123, CD33, LeY, or CD38).
  • the DDpp fusion is bispecific and specifically binds to two different targets expressed on the surface of two different cell types.
  • the bispecific DDpp fusion protein specifically binds to a target on a cancer cell and a target on an immune effector cell.
  • the bispecific DDpp fusion protein specifically binds 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 CD123 and CD33. In one embodiment the bispecific DDpp fusion protein specifically binds CD 123 and CD38. In one embodiment the bispecific DDpp fusion protein specifically binds CD 123 and LeY.
  • a multi-specific DDpp fusion protein comprises at least one DDpp that specifically binds one epitope on a target of interest and at least one other domain or sequence conferring function (e.g., an antibody fragment or domain such as an scFv) that specifically binds to a different epitope on the same target of interest.
  • a multi-specific DDpp fusion protein comprises at least one DDpp that specifically binds to an epitope on a target of interest and at least one domain or sequence conferring function e.g., an antibody fragment or domain (e.g., scFv), that specifically binds to an epitope on a different target of interest.
  • the multi-specific DDpp fusion protein comprises at least one DDpp that specifically binds to an epitope on a target of interest and at least one domain or sequence that specifically binds to an epitope on a different target on the same cell.
  • a DDpp fusion protein comprises at least one DDpp and at least one other DDpp or domain sequence conferring function, e.g., an antibody fragment or domain that specifically binds to a solid support.
  • the multimeric DDpp fusion comprising 2 or more DDpp are in turn fused with other heterologous proteins (or their subdomains) and in so doing, impart the multivalent and multi-specific properties to the fusion partner.
  • fusion partners of a DDpp include but are not limited to, antibodies, antibody subdomains (e.g., scFv or Fc domains), serum albumin, serum albumin subdomains, cell surface receptors, an alpha chain of a T cell receptor (TCR), a beta chain of a T cell receptor, cell surface receptor subdomains, peptides, peptide tags e.g., FEAG or myc), fibronectin type III repeats, z-domains, elastin-like polypeptides.
  • TCR T cell receptor
  • peptides peptide tags e.g., FEAG or myc
  • fibronectin type III repeats e.g., FEAG or myc
  • the number and location of DDpp and their respective positions within the fusion protein can vary.
  • DDpp(s) can be located at one or all termini of a fusion partner and/or interspersed within heterologous subunits within the DDpp fusion partner.
  • a DDpp fusion protein comprises a DDpp and a polypeptide sequence containing an additional domain.
  • the DDpp fusion protein comprises a DDpp and a member selected from: an antibody, an antibody fragment (e.g., an antigen binding domain or portion thereof (e.g., an scFv), an effector domain or portion thereof, an FcRn binding domain or portion thereof, and an Fc or a portion thereof), a serum protein (e.g., albumin or a portion thereof), a cytokine, a growth factor, a hormone, an imaging agent, a labeling agent, and a peptide tag.
  • the DDpp fusion protein comprises an Fc domain of an immunoglobulin (e.g., a human Fc domain) or a portion thereof.
  • the Fc domain is a variant human Fc domain.
  • the DDpp is fused to a heterologous polypeptide.
  • the heterologous polypeptide comprises a full-length antibody or an antibody fragment.
  • the 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.
  • the DD is fused to an antibody fragment which is an Fc.
  • the heterologous polypeptide comprises a member selected from the group consisting of: (i) a transmembrane domain; (ii) a membrane associating domain; (iii) human serum albumin or a fragment thereof; (iv) AFP or a fragment thereof; (v) AFP p26 or a fragment thereof; (vi) the extracellular domain of a receptor or a fragment thereof; and (vii) the extracellular domain of an intracellular receptor (e.g., a nuclear protein) or a fragment thereof.
  • the DDpp contains a heterologous polypeptide comprising the extracellular domain, or a fragment of an extracellular domain, of a cell surface receptor.
  • the DDpp of a DDpp fusion protein is incorporated into a larger, multi-domain molecular complex (e.g., a monomeric or multimeric DDpp fusion protein) and in so doing, imparts the functional attributes of the incorporated DDpp to the resultant fusion protein.
  • the DDpp fusion protein comprises a DDpp and a polypeptide sequence from an antibody, an antibody fragment, a serum protein (e.g., human serum albumin) or serum protein fragment, or a cell surface receptor, an alpha chain of a T cell receptor (TCR), a beta chain of a T cell receptor, cytokine, growth factor, hormone, or enzyme, or fragment thereof.
  • DDpp fusion proteins can additionally contain other optional components such as linkers and other components described herein.
  • a DDpp fusion protein described herein is an Adapter protein.
  • the Adapter comprises an antigenic determinant (AD) and an antigenic determinant binding domain (ADBD).
  • the Adapter can further comprise additional ADs, additional ADBDs, and/or other additional domains.
  • an Adapter provided herein comprises at least one ADBD comprising a D domain.
  • an Adapter provided herein comprises (a) a D domain (DD) that binds to CD 123 and (b) an antigenic determinant (AD).
  • the D domain that binds to CD123 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100.
  • 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.
  • the D domain that binds to CD123 comprises the amino acid sequence of SEQ ID NO: 14.
  • the Adapter is a monovalent Adapter comprising a single D domain that binds CD123.
  • the Adapter is a bivalent Adapter comprising two D domains that bind CD123. In some embodiments, the two D domains that bind CD123 are the same. In some embodiments, the two D domains that bind CD123 are different. In some embodiments, the Adapter is a bivalent Adapter comprising a first D domain that binds CD123 and a second D domain that binds a second AD. In some embodiments, the second AD is CD33 or LeY. In some embodiments, a monovalent Adapter comprises a D domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 13, 14, 31, 32, and 33.
  • the monovalent Adapter comprises the D domain comprising the amino acid sequence of SEQ ID NO: 14.
  • a bivalent Adapter comprises a DD comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 13, 14, 31, 32, and 33.
  • the bivalent Adapter comprises the D domain comprising the amino acid sequence of SEQ ID NO: 14.
  • a bivalent Adapter comprises two identical D domains comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 13, 14, 31, 32, and 33.
  • the bivalent Adapter comprises two identical D domains comprising the amino acid sequence of SEQ ID NO: 14.
  • the Adapter comprises an AFP p26 antigenic determinant (AD).
  • the AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 37-43 and 44.
  • the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 37.
  • the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 38.
  • the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 39.
  • the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 40.
  • the monovalent Adapter comprises the amino acid sequence of SEQ ID NO: 50-54 or 55.
  • the monovalent Adapter comprises the amino acid sequence of SEQ ID NO: 50.
  • the bivalent Adapter comprises the amino acid sequence of SEQ ID NO: 56-60 or 61.
  • the monovalent Adapter comprises the amino acid sequence of SEQ ID NO: 61.
  • the Adapter comprises one or more linkers.
  • the Adapter is capable of directing an immune response to a cell expressing CD 123 in an in vitro assay comprising the Adapter, a cell expressing CD 123 and an immune effector cell expressing a CAR comprising an ADBD that binds the AD comprised by the Adapter, e.g., a CAR comprising a D domain that binds AFP p26.
  • an Adapter provided herein comprising (a) a D domain that binds to CD123 and (b) an antigenic determinant binding domain (ADBD) that binds an AFP p26 AD.
  • the AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 37-43 and 44.
  • the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 37.
  • the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 39.
  • the D domain that binds to CD 123 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100.
  • 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. 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 an AFP p26 AD comprises a D domain that binds to the AFP p26 AD. In some embodiments, the D domain that binds to an AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 70-73 and 92-94. In some embodiments, the D domain that binds to an AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 73.
  • the Adapter comprises one or more linkers. .
  • the Adapter is capable of directing an immune response to a cell expressing CD 123 in an in vitro assay comprising the Adapter, a cell expressing CD 123 and an immune effector cell expressing a CAR comprising an AFP p26 AD.
  • the AD can be N-terminal to ADBD.
  • the ADBD can be N-terminal to the AD.
  • the AD and ADBD are directly fused.
  • the AD and the ADBD are fused via a linker (a protein linker or chemical linker) or another protein domain (e.g., a functional domain).
  • an Adapter provided herein comprises at least one ADBD comprising a D domain.
  • the Adapter comprises a linker located between an ADBD and another functional domain of the Adapter. In some embodiments, the linker is located between two ADBDs of the Adapter. In some embodiments, the linker is located between the AD and an ADBD of the Adapter.
  • Suitable linkers for coupling the two or more functional domains of the Adapter will be clear to persons 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 particular embodiments, the linker(s) is suitable for constructing proteins or polypeptides that are intended for pharmaceutical use.
  • an Adapter provided herein comprises at least one ADBD comprising a D domain.
  • an Adapter provided herein can further comprise an additional domain or additional domains, e.g., a domain that confers an extended half-life.
  • the Adapter or the ADBD in the Adapter, is deimmunized.
  • the Adapters provided herein have uses that include but are not limited to diagnostic, analytic, and therapeutic applications.
  • the Adapters are used in combination with chimeric antigen receptors (CARs) provided herein expressed on the surface of cells, e.g., to kill a target cell.
  • CARs chimeric antigen receptors
  • Adapters suitable for use in connection with the DDpp have been disclosed in Int'l. Appl. Pub. Nos. WO 2016164305, WO 2016164308A1, WO 2019099440, and WO 2019099433, US Patent Nos. 10,662,248, and 10,647,775, and US Pat. Appl. Nos. 20200223934, and 20210002381, each of which is incorporated herein by reference for all purposes.
  • ADs Antigenic Determinants
  • An Adapter provided herein comprises at least one antigenic determinant (AD).
  • the Adapter comprises a single AD.
  • the Adapter comprises two or more ADs. Where an Adapter comprises two or more ADs, the ADs can be the same or different.
  • the AD is an epitope of AFP p26.
  • 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 of the amino acid residues of SEQ ID NO: 37. .
  • the AD comprises the amino acid sequence of SEQ ID NO: 37.
  • 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 of the amino acid residues of SEQ ID NO: 39.
  • the AD comprises the amino acid sequence of SEQ ID NO: 39.
  • the AD comprises the amino acid residues of SEQ ID NO: 37-43 or 44.
  • the Adapter comprises the extracellular domain of BCM A (e.g., a polypeptide comprising the sequence of SEQ ID NO: 34.
  • 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 of the amino acid residues of SEQ ID NO: 34.
  • Adapter comprises the extracellular domain of CD123 (e.g., a polypeptide comprising the sequence of SEQ ID NO: 1.
  • 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 of the amino acid residues of SEQ ID NO: 1.
  • the Adapter comprises the extracellular domain of CD 19 (e.g., a polypeptide comprising the sequence of SEQ ID NO: 95.
  • 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 of the amino acid residues of SEQ ID NO: 95.
  • 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 of the 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.
  • CS1 e.g., a polypeptide comprising the sequence of SEQ ID NO: 35. In some embodiments, the Adapter comprises 5-25, 5-50
  • the AD is an epitope of AFP.
  • 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 of the amino acid residues of SEQ ID NO: 36.
  • Adapter comprises a p26 protein (e.g., having the sequence of SEQ ID NO: 37- 43 or 44).
  • the Adapter has a plasma half-life in vivo 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.
  • the Adapter 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, or 1-10 hours, 2-10 hours, 4-10 hours, 6-10 hours, or 6-9 hours in a mouse.
  • the Adapter 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, or 1-10 hours, 2-10 hours, 4-10 hours, 6-10 hours, or 6-9 hours, in a human.
  • the disclosure provides a method for modifying the in vivo half-life (e.g., in a mouse or human) of an Adapter comprising a p26 protein (e.g., having the sequence of SEQ ID NO: 37-43 or 44).
  • the Adapter comprises one or more target-binding DDpp.
  • the half-life of the Adapter is increased or decreased by substituting or deleting one or more amino acid residues normally found in the human p26 protein, or by inserting one or more amino acid residues not normally found in the human p26 protein.
  • the p26 sequence of the Adapter 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 nonconservative substitutions), deletions, and/or insertions so as to increase or decrease the in vivo half-life of the Adapter.
  • amino acid residue corresponding to the glutamine (Gin, Q) at position 217 of p26 (SEQ ID NO: 37) is substituted with another amino acid residues.
  • the substitution is Gln217Pro.
  • the p26 sequence of the Adapter is modified through deletion of 1-150, 1- 100, 1-50, 1-25 or 1-10 amino acid residues so as to increase or decrease the in vivo half-life of the Adapter.
  • the p26 sequence of the Adapter 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 nonconservative substitutions), deletions, and/or insertions so as to increase or decrease the interaction of the Adapter with FcRn.
  • the AD (e.g., in an Adapter and/or on a target cell) is an AD that is present in a naturally occurring protein or other molecule. In some embodiments, the AD is an AD that is endogenous to humans.
  • the AD is an epitope of a human intracellular protein.
  • the AD is an epitope of a human intracellular protein selected from: Tyk2, Jakl, Jak2, Jak3, LCK, ZAP-70, and GRB2.
  • 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 of the amino acid residues of the intracellular protein.
  • the target of interest specifically bound by the ADBD of an Adapter is itself an AD of another Adapter, having a different sequence.
  • ADBDs Antigenic Determinant Binding Domains
  • An Adapter provided herein comprises at least one antigenic determinant binding domain (ADBD).
  • the Adapter contains one ADBD.
  • the Adapter contains at least 2, 3, 4, or 5, or more than 5 ADBDs.
  • the Adapter contains 1-3, 1-4, 1-5, or more than 5 different ADBDs.
  • the Adapter contains at least 2, 3, 4, or 5, or more than 5 different ADBDs.
  • an Adapter can comprise a monomeric ADBD (i.e., containing one antigenic determinant binding domain) or multimeric ADBDs i.e., containing more than one antigenic determinant binding domains in tandem optionally operably connected by a linker).
  • the use of a multimeric Adapter provides enhanced (e.g., synergistic) target binding.
  • the use of a multimeric Adapter allows for targeting of more than one target using a single Adapter construct (e.g., bi-, tri-specific, etc.).
  • an Adapter provided herein comprises at least one ADBD comprising a D domain.
  • an Adapter provided herein comprises more than one ADBD comprising a D domain.
  • all ADBDs of an Adapter provided herein comprise a D domain.
  • the multimeric Adapter is homo-multimeric (i.e., containing more than one of the same ADBD optionally connected by linker(s)(e.g., homodimers, homotrimers, homotetramers etc.) or Adapter heteromultimeric (i.e., containing two or more antigenic determinant binding domains in which there are at least two different antigenic determinant binding domains).
  • linker(s) e.g., homodimers, homotrimers, homotetramers etc.
  • Adapter heteromultimeric i.e., containing two or more antigenic determinant binding domains in which there are at least two different antigenic determinant binding domains.
  • the number of ADBDs included in any particular Adapter may vary, depending on the embodiment, and may be defined, at least in part, by the expression system in which the Adapter is produced.
  • the fusion proteins may comprise multimers 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 numbers in between those listed as well as endpoints).
  • multiple domains of an Adapter can contain the same or different ADBD(s). In some embodiments, 2, 3, 4, 5, or more than 5 domains are in tandem.
  • an Adapter provided herein comprises at least one ADBD comprising a D domain. In some embodiments, an Adapter provided herein comprises more than one ADBD comprising a D domain. In some embodiments, all ADBDs of an Adapter provided herein comprise a D domain.
  • the Adapter comprises two or more ADBDs that are operably linked. In one embodiment, the Adapter comprises two ADBDs that bind to the same or different ADs on a target antigen. The linkage of 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 and receptor activation) over compositions that only contain 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.
  • the Adapter comprises two ADBDs that bind to different antigens on different cells.
  • the linkage of two or more ADBDs results in a multivalent and multi-specific Adapter that has the potential to bind more than one target antigen, either independently or simultaneously.
  • the multivalent Adapter is able to bind the same target antigen simultaneously.
  • the multivalent Adapter is able to bind different target antigens simultaneously.
  • the Adapter comprises two or more operably linked ADBDs that are separated by an Antigenic Determinant.
  • the Antigenic Determinant is at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids in length.
  • 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.
  • the Adapter comprises two or more operably linked ADBDs that are separated by a BCMA Antigenic Determinant.
  • the Adapter comprises two or more operably linked ADBDs that are separated by an AFP P26 Antigenic Determinant.
  • an Adapter provided herein comprises at least one ADBD comprising a D domain.
  • an Adapter provided herein comprises more than one ADBD comprising a D domain.
  • all ADBDs of an Adapter provided herein comprise a D domain.
  • an ADBD in the Adapter provided herein can bind to any AD.
  • the ADBD binds to CD123 (e.g., a polypeptide comprising the sequence of SEQ ID NO: 1).
  • 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).
  • the ADBD binds to BCMA (e.g., a polypeptide comprising the sequence of SEQ ID NO: 34).
  • the ADBD binds to CD22.
  • the ADBD binds to CD19.
  • 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 in the Adapter provided herein specifically binds to an AD of human CD26, CD30, CD33, or CD38. An Adapter can be "monospecific" or “multi-specific.” An Adapter that is "multi-specific" (e.g., bispecific, trispecific or of greater multi-specificity) recognizes and binds to two or more different epitopes present on one or more different molecules.
  • multi-specific e.g., bispecific, trispecific or of greater multi-specificity
  • an Adapter provided herein comprises at least one ADBD comprising a D domain. In some embodiments, an Adapter provided herein comprises more than one ADBD comprising a D domain. In some embodiments, all ADBDs of an Adapter provided herein comprise a D domain.
  • Adapter comprises a domain (e.g., the extracellular domain) of CD123 (e.g., a polypeptide comprising the sequence of SEQ ID NO: 1).
  • the Adapter comprises a domain (e.g., the extracellular domain) of BCMA (e.g., a polypeptide comprising the sequence of SEQ ID NO 34).
  • the Adapter comprises a domain (e.g., the extracellular domain) of CD22.
  • the Adapter comprises a domain (e.g., the extracellular domain) of CD19.
  • the Adapter comprises a domain (e.g., the extracellular domain) of CS1.
  • the Adapter comprises a domain (e.g., the extracellular domain) of HER2. In some embodiments, the Adapter comprises a domain (e.g., the extracellular domain) of CD45. In some embodiments, the Adapter comprises a domain (e.g., the extracellular domain) of CD26, CD30, CD33, or CD38. In some embodiments, the Adapter 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.
  • 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.
  • an Adapter provided herein comprises at least one ADBD comprising a D domain.
  • an Adapter provided herein comprises more than one ADBD comprising a D domain.
  • all ADBDs of an Adapter provided herein comprise a D domain.
  • the Adapter contains at least two ADBDs that bind and cross-link one or more target antigens bound by the ADBDs and/or complexes containing the target antigen(s).
  • the cross-linked antigen(s) is on the same cell.
  • the cross-linked antigen(s) is on different cells.
  • the Adapter comprises two or more operably linked ADBDs that are separated by an Antigenic Determinant (e.g., a domain described above).
  • the Antigenic Determinant is at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids in length.
  • 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.
  • the Adapter comprises two or more operably linked ADBDs that are separated by a BCMA Antigenic Determinant.
  • the Adapter comprises two or more operably linked ADBDs that are separated by a CD123 Antigenic Determinant.
  • the Adapter comprises two or more operably linked ADBDs that are separated by a CD26, CD30, CD33, or CD38 Antigenic Determinant. In further embodiments, the Adapter comprises two or more operably linked ADBDs that are separated by an AFP p26 Antigenic Determinant. In some embodiments, an Adapter provided herein comprises at least one ADBD comprising a D domain. In some embodiments, an Adapter provided herein comprises more than one ADBD comprising a D domain. In some embodiments, all ADBDs of an Adapter provided herein comprise a D domain.
  • the Adapter contains at least two of the same ADBDs (i.e., is multivalent). In some embodiments, the multivalent Adapter is able to bind two or more of the same target antigens simultaneously. In some embodiments, the Adapter is multivalent and is able to bind the same target antigen simultaneously. In some embodiments, the multi-multivalent Adapter comprises two or more operably linked ADBDs that are 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.
  • 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.
  • the Adapter comprises two or more operably linked ADBDs that are separated by a CD123 Antigenic Determinant.
  • the multivalent Adapter comprises two or more operably linked ADBDs that are separated by a BCMA Antigenic Determinant.
  • the multivalent Adapter comprises two or more operably linked ADBDs that are separated by an AFP p26 Antigenic Determinant.
  • an Adapter provided herein comprises at least one ADBD comprising a D domain.
  • an Adapter provided herein comprises more than one ADBD comprising a D domain.
  • all ADBDs of an Adapter provided herein comprise a D domain.
  • the Adapter contains at least two ADBDs that bind to different antigens (i.e., is multispecific). In some embodiments, the multi-specific Adapter is able to bind the different target antigens simultaneously. In some embodiments, the Adapter is also multivalent and is able to bind the same target antigen simultaneously. In some embodiments, the multi-specific Adapter comprises two or more operably linked ADBDs that are 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.
  • 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.
  • the Adapter comprises two or more operably linked ADBDs that are separated by a CD123 Antigenic Determinant.
  • the Adapter comprises two or more operably linked ADBDs that are separated by a BCMA Antigenic Determinant.
  • the Adapter comprises two or more operably linked ADBDs that are separated by an AFP p26 Antigenic Determinant.
  • an Adapter provided herein comprises at least one ADBD comprising a D domain.
  • an Adapter provided herein comprises more than one ADBD comprising a D domain.
  • all ADBDs of an Adapter provided herein comprise a D domain.
  • a multi-specific Adapter contains at least two ADBDs that bind to at least two different epitopes on a single target of interest (e.g., CD123).
  • a multi-specific Adapter comprises at least one ADBD that specifically binds one epitope on a target of interest and at least one other ADBD that specifically binds to a different epitope on the same target antigen.
  • a multi-specific Adapter comprises at least one ADBD that specifically binds to an epitope on a first target antigen and at least one ADBD that specifically binds to an epitope on a second antigen.
  • the Adapter comprises at least one ADBD that specifically binds to an epitope on a first target antigen on a cell and at least one ADBD that specifically binds to an epitope on a second antigen on the same cell. In some embodiments, the Adapter comprises at least one ADBD that specifically binds to an epitope on a first target antigen on a cell and at least one ADBD that specifically binds to an epitope on a second antigen on a different cell. In some embodiments, an Adapter provided herein comprises at least one ADBD comprising a D domain. In some embodiments, an Adapter provided herein comprises more than one ADBD comprising a D domain.
  • all ADBDs of an Adapter provided herein comprise a D domain.
  • the Adapter comprises 2 or more ADBDs that are operably linked with other heterologous proteins (or their subdomains) and in so doing, impart the multivalent, multi-specific, and/or functional properties (e.g., pharmacokinetics such as increased half-life or pharmacodynamics such as increased function) of the fusion partner to the Adapter fusion protein.
  • fusion partners of an Adapter include but are not limited to, antibodies, antibody subdomains (e.g., scFv or Fc domains), serum albumin, serum albumin subdomains, cell surface receptors, an alpha chain of a T cell receptor (TCR), a beta chain of a T cell receptor, cell surface receptor subdomains, peptides, peptide tags (e.g., FEAG or myc).
  • TCR T cell receptor
  • TCR alpha chain of a T cell receptor
  • beta chain of a T cell receptor cell surface receptor subdomains
  • peptides e tags
  • FEAG or myc e.g., FEAG or myc.
  • the number and location of ADBDs and their respective positions within the Adapter can vary. For example, ADBDs can be located at one or all termini of a fusion partner and/or interspersed within heterologous subunits within the Adapter fusion partner.
  • the Adapter comprises 2 or more ADBDs that are separated by a heterologous protein (e.g., Antigenic Determinant).
  • the heterologous protein is at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids in length.
  • the heterologous protein 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, 50-10050-75, 100-500, 100-400, 100-300, 100-200, or 100-150 amino acids in length.
  • an Adapter provided herein comprises at least one ADBD comprising a D domain. In some embodiments, an Adapter provided herein comprises more than one ADBD comprising a D domain. In some embodiments, all ADBDs of an Adapter provided herein comprise a D domain.
  • the Adapter is bispecific and contains ADBDs that specifically bind to two different target antigens.
  • the bispecific Adapter specifically binds to two different target antigens expressed on the surface of two different cell types.
  • the bispecific Adapter specifically binds to two different target antigens expressed on the surface of a tumor cell.
  • the bispecific Adapter specifically binds to two different target antigens expressed on the surface of an AME cell (e.g., CD123 and CD33 or LeY).
  • the bispecific Adapter binds to target antigens expressed on different cells.
  • the bispecific Adapter binds to target antigens expressed on different cells of a tumor. In another embodiment, the bispecific Adapter binds to target antigens expressed on different cells within a 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 a target expressed on a cancer cell (e.g. CD 123) and a target expressed on the surface of a T lymphocyte e.g., CD3). In some embodiments, the bispecific Adapter is able to bind the different target antigens simultaneously.
  • a cancer cell e.g. CD 123
  • a target expressed on the surface of a T lymphocyte e.g., CD3
  • the bispecific Adapter comprises two or more operably linked ADBDs that are separated by an Antigenic Determinant.
  • the Antigenic Determinant is at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids in length.
  • 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.
  • the Adapter comprises two or more operably linked ADBDs that are separated by a CD123 Antigenic Determinant. In further embodiments, the Adapter comprises two or more operably linked ADBDs that are separated by a BCMA Antigenic Determinant. In further embodiments, the Adapter comprises two or more operably linked ADBDs that are separated by an AFP P26 Antigenic Determinant. In some embodiments, an Adapter provided herein comprises at least one ADBD comprising a D domain. In some embodiments, an Adapter provided herein comprises more than one ADBD comprising a D domain. In some embodiments, all ADBDs of an Adapter provided herein comprise a D domain.
  • the ADBD can be any of the types of ADBD discussed herein.
  • an ADBD can be an antibody, an antigen-binding fragment thereof, a ScFv, an alternative scaffold binding domain, a D domain, a T cell receptor, or an antigen-binding fragment thereof.
  • an Adapter provided herein comprises at least one ADBD comprising a D domain.
  • an Adapter provided herein comprises more than one ADBD comprising a D domain.
  • all ADBDs of an Adapter provided herein comprise a D domain.
  • an Adapter comprises more than one ADBD
  • those ADBD can be the same types of antigen-binding molecules or can be different.
  • an Adapter can comprise two ADBD that are D domains. The two ADBD that are D domains can be the same or different.
  • An Adapter can also comprise an ADBD that is a D domain and an ADBD that is a scFv.
  • An Adapter can also comprise an ADBD that is a T cell receptor or antigen-binding fragment thereof and an ADBD that is a scFv.
  • the Adapter comprises two or more operably linked ADBDs that are separated by an Antigenic Determinant.
  • 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 that are separated by a CD 123 Antigenic Determinant.
  • the Adapter comprises two or more operably linked ADBDs that are separated by a BCMA Antigenic Determinant. In further embodiments, the Adapter comprises two or more operably linked ADBDs that are separated by an AFP P26 Antigenic Determinant. In some embodiments, an Adapter provided herein comprises at least one ADBD comprising a D domain. In some embodiments, an Adapter provided herein comprises more than one ADBD comprising a D domain. In some embodiments, all ADBDs of an Adapter provided herein comprise a D domain. [0224] In some embodiments, the ADBD of the Adapter is deimmunized. In some embodiments, the ADBD of the Adapter is deimmunized by replacing one or more amino acid residues in predicted T cell epitopes to decrease binding to host MHC molecules.
  • the Adapter comprises an ADBD that binds to an antigen target containing an AD of interest, and has no discernable impact on the function of the target.
  • the Adapter 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 determined using or routinely modifying assays, bioassays, and/or animal models known in the art for evaluating such activity.
  • An Adapter agonist refers to an Adapter that in some way increases or enhances the biological activity of the Adapter target or has biological activity comparable to a known agonist of the Adapter target.
  • the Adapter is an antagonist of the target it binds.
  • An Adapter antagonist refers to an Adapter that completely or partially blocks or in some way interferes with the biological activity of the Adapter target or has biological activity comparable to a known antagonist or inhibitor of the Adapter target.
  • an Adapter specifically binds a target of interest that is a serum protein.
  • an Adapter specifically binds a serum protein selected from: serum albumin (e.g., human serum albumin (HSA)), thyroxin-binding protein, transferrin, fibrinogen, and an immunoglobulin (e.g., IgG, IgE and IgM).
  • serum albumin e.g., human serum albumin (HSA)
  • thyroxin-binding protein thyroxin-binding protein
  • transferrin thyroxin-binding protein
  • fibrinogen e.g., transferrin, fibrinogen
  • an immunoglobulin e.g., IgG, IgE and IgM
  • Adapter functional domain(s) Adapter functional domain(s)
  • the Adapter comprises a first antigenic determinant (an AD), a domain that binds to a second antigenic determinant (an 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 functional domain of the Adapter can be located between the AD and the ADBD.
  • the Adapter can also be located N-terminal to both the AD and ADBD or C- terminal to both the AD and ADBD.
  • the functional domain of the Adapter can be located between two or more ADs, N-terminal to two or more ADs, or C-terminal to two or more ADs. In some embodiments, where the Adapter comprises two or more ADBDs, the functional domain of the Adapter can be located between two or more ADBDs, N-terminal to two or more ADBDs, or C-terminal to two or more ADBDs.
  • the Adapter comprises a functional domain selected from: an Fc or variant Fc (e.g., a human Fc or 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, growth factor, hormone, or enzyme; an imaging agent; a labeling agent; and a peptide tag.
  • Fc or variant Fc e.g., a human Fc or variant Fc domain
  • a serum protein e.g., human serum albumin
  • the functional domain(s) of the Adapter can be naturally derived or the result of recombinant engineering (e.g., phage display, xenomouse, or synthetic).
  • 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.
  • ADCC antibody dependent cellular cytotoxicity
  • CDC complement dependent cytotoxicity
  • the Adapter comprises a functional domain selected from: an Fc or variant Fc (e.g., a human Fc or 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.
  • Fc or variant Fc e.g., a human Fc or variant human Fc domain
  • a serum protein e.g., human serum albumin
  • FcRn binding domain e.g., a fragment or derivative thereof
  • an Adapter comprises a functional domain that comprises an antibody effector domain or derivative of an antibody effector domain that confers one or more effector functions to the Adapter, such as the ability to bind to one or more Fc receptors.
  • the functional domain comprises one or more CH2 and or CH3 domains of an antibody having effector function provided by the CH2 and CH3 domains.
  • the functional domain comprises one or more derivatives of CH2 and/or CH3 domains of an antibody having effector function provided by the CH2 and CH3 domains.
  • the Adapter comprises a functional domain that increases the antibody dependent cellular cytotoxicity (ADCC) conferred by the Adapter (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.
  • ADCC antibody dependent cellular cytotoxicity
  • fragment engineering modifications of effector function conferring portions of an Fc contained in the functional domain of an Adapter that increases ADCC include one or more modifications corresponding to: IgGl- S298A, E333A, K334A; IgGl-S239D, I332E; IgGl-S239D, A330L, I332E; IgGl-P247I, A339D or Q; IgGl-D280H, K290S with or without S298D or V; IgGl-F243L, R292P, Y300L; IgGl-F243L, R292P, Y300L, P396L; and IgGl-F243L, R292P, Y300L, V305I, P396L; wherein the numbering of the residues in the Fc region is that of the EU index of Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest, 1991 Fifth edition).
  • the Adapter comprises a functional domain that comprises an antibody fragment that confers upon the Adapter a biological or biochemical characteristic of an immunoglobulin.
  • the antibody fragment confers a characteristic selected from: the ability to non-covalently dimerize, the ability to localize at the site of a tumor, and an increased serum half-life when compared to an Adapter without the antibody fragment.
  • 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.
  • Adapter protein stability can be measured using established methods, including, for example, ELISA techniques.
  • 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 between those listed).
  • the Adapter contains an immunoglobulin effector domain or half-life influencing domain that corresponds to an immunoglobulin domain or fragment in which at least a fraction of one or more of the constant region domains has been altered so as to provide desired biochemical characteristics such as reduced or increased effector functions, the ability to non-covalently dimerize, increased ability to localize at the site of a tumor, reduced serum half-life, or increased serum half-life when compared with an immunoglobulin fragment having the corresponding unaltered immunoglobulin sequence.
  • These alterations of the constant region domains can be amino acid substitutions, insertions, or deletions.
  • the Adapter comprises a functional domain that comprises an 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 Adapter.
  • ADCC antibody dependent cellular cytotoxicity
  • the Adapter comprises a sequence of an immunoglobulin effector domain that has been modified to increase ADCC see, 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.
  • immunoglobulin fragment engineering modifications contained in an amino acid sequence in the Adapter that increases ADCC include immunoglobulin effector domain sequences having one or more modifications corresponding to: IgGl-S298A, E333A, K334A; IgGl- S239D, I332E; IgGl-S239D, A330L, I332E; IgGl-P247I, A339D or Q; IgGl-D280H, K290S with or without S298D or V; IgGl-F243L, R292P, Y300L; IgGl-F243L, R292P, Y300L, P396L; and IgGl-F243L, R292P, Y300L, V305I, P396L; wherein the numbering of the residues in the Fc region is that of the EU index of Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest,
  • the Adapter comprises a functional domain that comprises the amino acid sequence of an immunoglobulin effector domain, or a derivative of an immunoglobulin effector domain, that confers antibody-dependent cell phagocytosis (ADCP) to the Adapter.
  • the Adapter comprises a sequence of an immunoglobulin effector domain that has been modified to increase antibodydependent cell phagocytosis (ADCP); (see, e.g., Shields et al., J. Biol. Chem. 276: 6591-6604 (2001); Eazar et al., PNAS 103: 4005-4010 (2006); Stavenhagen et al., Cancer Res.
  • immunoglobulin fragment engineering modifications contained in an amino acid sequence in the Adapter that increases ADCP include immunoglobulin effector domain sequences having one or more modifications corresponding to: IgGl-S298A, E333A, K334A; IgGl-S239D, I332E; IgGl- S239D, A330E, I332E; IgGl-P247I, A339D or Q; IgGl-D280H, K290S with or without S298D or V; IgGl- F243L, R292P, Y300E; IgGl-F243E, R292P, Y300E, P396E; IgGl-F243E, R292P, Y300E, V305I, P396E; and IgGl-G236A, S239D, I332E; wherein the numbering of the residues is that of the EU index of Kabat et al. (Kabat e
  • the Adapter comprises a functional domain that comprises an amino acid sequence of an immunoglobulin effector domain, or a derivative of an immunoglobulin effector domain, that confers complement-dependent cytotoxicity (CDC) to the Adapter.
  • the Adapter comprises a sequence of an immunoglobulin effector domain that has been modified to increase complementdependent 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.
  • Adapters can contain an antibody fragment or domain that contains one or more of the following modifications that increase CDC: IgGl- K326A, E333A; IgGl-K326W, E333S, IgG2-E333S; wherein the numbering of the residues is that of the EU index of Kabat et al. (Sequences of proteins of Immunological Interest, 1991 Fifth edition, herein incorporated by reference).
  • the Adapter comprises a functional domain that comprises an amino acid sequence of an immunoglobulin effector domain, or a derivative of an immunoglobulin effector domain, that confers the ability to bind FcgammaRIIb receptor to the Adapter.
  • the Adapter comprises a sequence of an immunoglobulin effector domain that has been modified to increase inhibitory binding to FcgammaRIIb receptor (see, e.g., Chu et al., Mol. Immunol. 45: 3926-3933 (2008)).
  • An example of an immunoglobulin fragment engineering modification contained in an amino acid sequence in the Adapter that increases binding to inhibitory FcgammaRIIb receptor is IgGl- S267E, L328F.
  • the Adapter contains a functional domain that comprises an amino acid sequence of an immunoglobulin effector domain, or a derivative of an immunoglobulin effector domain, that confers the ability to bind neonatal receptor FcRn to the Adapter.
  • the Adapter contains a functional domain that comprises a sequence of an immunoglobulin FcRn binding domain that has been modified to enhance binding to FcRn (see, e.g., Petkova et al., Int. Immunol. 18: 1759-1769 (2006); Dall'Acqua et al., J.
  • the Adapter comprises a functional domain that comprises a sequence of an immunoglobulin effector domain that has been modified to have a selective affinity for FcRn at pH 6.0, but not pH 7.4.
  • the Adapter functional domain can contain an antibody fragment or domain that contains one or more of the following modifications that increase half-life: IgGl-M252Y, S254T, T256E; IgGl-T250Q, M428L; IgGl-H433K, N434Y; IgGl-N434A; and IgGl-T307A, E380A, N434A; wherein 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, herein incorporated by reference).
  • the Adapter comprises a functional domain that comprises an amino acid sequence corresponding to a immunoglobulin effector domain that has been modified to contain at least one substitution in its sequence corresponding to the Fc region (e.g., Fc gamma) position selected from: 238,
  • the Adapter contains a functional domain that comprises a sequence of an immunoglobulin effector domain derivative wherein at least one residue corresponding to position 434 is a residue selected from: A, W, Y, F and H.
  • the Adapter comprises a sequence of an immunoglobulin effector fragment derivative having the following respective substitutions S298A/E333A/K334A.
  • the Adapter comprises an immunoglobulin effector domain derivative having a substitution corresponding to K322A.
  • the Adapter comprises a sequence of an immunoglobulin effector domain derivative having one or any combination of the following substitutions K246H, H268D, E283E, S324G, S239D and I332E.
  • the Adapter comprises a sequence of an immunoglobulin effector domain derivative having substitutions corresponding to D265A/N297A.
  • the Adapter comprises a functional domain that comprises a sequence of an immunoglobulin effector domain that has been glycoengineered or mutated to increase effector function using techniques known in the art.
  • the inactivation (through point mutations or other means) of a constant region domain sequence contained in the Adapter may reduce Fc receptor binding of the circulating Adapter thereby increasing tumor localization.
  • constant region modifications consistent with certain embodiments of the instant invention moderate complement binding and thus reduce the serum half-life and nonspecific association of a conjugated cytotoxin.
  • modifications of the constant region may be used to modify disulfide linkages or oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or antibody flexibility.
  • the resulting physiological profile, bioavailability and other biochemical effects of the modifications, such as tumor localization, biodistribution and serum half-life, can easily be measured and quantified using well know immunological techniques without undue experimentation.
  • the production of the Adapter useful in practicing the provided methods, may be carried out using a variety of standard techniques for chemical synthesis, semi-synthetic methods, and recombinant DNA methodologies known in the art.
  • the overall production scheme for producing the Adapter comprises obtaining a reference protein scaffold and identifying a plurality of residues within the scaffold for modification.
  • the reference scaffold may comprise a protein structure with one or more alpha-helical regions, or other tertiary structure.
  • the plurality of residues can be modified, for example by substitution of an amino acid. In some embodiments substitution is conservative, while in other embodiments non-conservative substitutions are made.
  • 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
  • the modifications do not include substituting in either a cysteine or a proline.
  • the resulting modified polypeptides can be recombinantly expressed, for example in a plasmid, bacteria, phage, or other vector (e.g. to increase the number of each of the modified polypeptides).
  • the modified polypeptides can then be purified and screened to identify those modified polypeptides that have specific binding to a particular target of interest.
  • certain modified polypeptides will show enhanced binding specificity for a target of interest vis-a-vis the reference scaffold, which in some embodiments may exhibit little or no binding to a given target of interest.
  • the reference scaffold may show some interaction (e.g.
  • modified polypeptides will exhibit at least about two fold, at least about five fold, at least about 10 fold, at least about 20 fold, at least about 50 fold, or at least about 100 fold (or more) increased binding specificity for the target of interest.
  • the reference sequence and/or the modified polypeptides can be de-immunized. For example, residues or motifs that are potentially immunogenic can be identified and modified in order to reduce or eliminate potential immune responses to the Adapter. Additional details regarding various embodiments of the production, selection, and isolation of Adapter are provided in more detail below.
  • the disclosure provides compositions comprising one or more of the Adapters disclosed on Table 2.
  • the disclosure provides compositions comprising one or more Adapters comprising a sequence with 60-70%, 70-75%, 75-80%, 80-85%, 85-90%, 95-99% homology (and overlapping ranges therein) with a sequence disclosed in Table 2.
  • the Adapters having such homology are functionally similar or identical as compared to the respective reference sequence in Table 2.
  • the disclosure provides a polypeptide that comprises one or more Adapters that compete with (wholly or partially) one or more of the Adapters disclosed in Table 2 (reference sequence) for CD 123 binding.
  • the ability of one polypeptpide to compete with a reference polypeptide for binding to a respective target can routinely be determined using a standard competition assay known in the art.
  • competition does not require that the Adapter competes for the same epitope as an Adapters of Table 2, rather the polypeptide can compete by binding a sterically inhibiting epitope, an overlapping epitope, etc.
  • chimeric antigen receptors comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain.
  • a CAR disclosed herein comprises an extracellular domain comprising an Antigenic Determinant (AD).
  • a CAR disclosed herein comprises an extracellular domain comprising at least one D domain (DD) disclosed herein to impart binding specificity.
  • DD D domain
  • a CAR disclosed herein comprises an extracellular domain comprising an ADBD.
  • CARs may be expressed by any cell type.
  • a chimeric antigen receptor (CAR) disclosed herein comprises comprises a D domain that binds to CD 123, (ii) a transmembrane domain, and (iii) an intracellular domain.
  • the D domain that binds to CD123 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100.
  • 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.
  • the D domain that binds to CD123 comprises the amino acid sequence of SEQ ID NO: 14.
  • the CAR comprises the amino acid sequence of SEQ ID NO: 62-66 or 67.
  • the CAR comprises the amino acid sequence of SEQ ID NO: 67.
  • a chimeric antigen receptor (CAR) disclosed herein comprises comprises an antigenic determinant binding domain (ADBD) that binds to an AFP p26 AD, (ii) a transmembrane domain, and (iii) an intracellular domain.
  • ADBD antigenic determinant binding domain
  • the ADBD that binds to AFP p26 AD comprises an scFv that binds to AFP p26 AD.
  • the ADBD that binds to AFP p26 AD comprises a D domain that binds to AFP p26 AD.
  • the AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 37-43 and 44.
  • 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 an AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94. In some embodiments, the DD that binds to an AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 70-73 or 92-94. In some embodiments, the DD that binds to an 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.
  • a chimeric antigen receptor (CAR) disclosed herein comprises comprises an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain.
  • the AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 37-43 and 44.
  • the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 37.
  • the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 39.
  • a CAR disclosed herein comprises an extracellular domain comprising an Antigenic Determinant (AD).
  • AD Antigenic Determinant
  • Such CARs can be expressed on the surface of cells (e.g., immune cell or immune effector cell) and used in combination with Adapters comprising a first ADBD (e.g., a D domain) that binds the AD and a second ADBD (e.g., a D domain) that binds a target on a target cell, for example, to kill a target cell.
  • the AD comprises AFP or a fragment thereof.
  • the AD comprises p26 or a fragment thereof.
  • the AD comprises an amino acid sequence of SEQ ID NO: 37-43 or 44.
  • the AD comprises the amino acid sequence of SEQ ID NO: 39.
  • the AD comprises the amino acid sequence of SEQ ID NO: 40.
  • a CAR disclosed herein comprises an extracellular domain comprising at least one D domain (DD).
  • the D domain can recognize an Antigenic Determinant (AD) (e.g., CD123) on a target cell or an AD (e.g., p26) comprised by an Adapter.
  • AD Antigenic Determinant
  • CARs comprising a DD capable of binding an AD on a target cell can be expressed on the surface of cells (e.g., immune cell or immune effector cell) and used, for example, to kill a target cell expressing the AD.
  • 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 NO: 8-33, 99 and 100.
  • the CD123-binding DD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 13, 14, 31, 32, and 33.
  • the CD123-binding DD comprises the amino acid sequence of SEQ ID NO: 14.
  • CARs comprising a DD capable of binding a first AD can be expressed on the surface of cells e.g., immune cell or immune effector cell) and used in combination with an Adapter comprising the first AD and an ADBD (e.g., a D domain) that binds a second AD on a target cell, for example, to kill the target cell.
  • 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 NO: 74-93 and 94.
  • 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 NO: 70-73 and 92-94.
  • the p26-binding DD comprises the amino acid sequence of SEQ ID NO: 73.
  • p26 comprises an amino acid sequence of SEQ ID NO: 37-43 or 44.
  • p26 comprises the amino acid sequence of SEQ ID NO: 39.
  • p26 comprises the amino acid sequence of SEQ ID NO: 40.
  • a CAR disclosed herein comprises an extracellular domain comprising an ADBD.
  • the ADBD is made up of, at least in part, a target-binding polypeptide (e.g., D domain) as disclosed herein.
  • the ADBD can recognize an Antigenic Determinant (AD) (e.g., CD123) on a target cell or an AD (e.g., p26) comprised by an Adapter.
  • AD Antigenic Determinant
  • CARs comprising an ADBD capable of binding an AD on a target cell can be expressed on the surface of cells (e.g., immune cell or immune effector cell) and used, for example, to kill a target cell expressing the AD.
  • CARs comprising a first ADBD capable of binding a first AD can be expressed on the surface of cells (e.g., immune cell or immune effector cell) and used in combination with an Adapter comprising the first AD and a second ADBD (e.g., a D domain) that binds a second AD on a target cell, for example, to kill the target cell.
  • p26 comprises an amino acid sequence of SEQ ID NO: 37-43 or 44.
  • p26 comprises the amino acid sequence of SEQ ID NO: 39.
  • p26 comprises the amino acid sequence of SEQ ID NO: 40.
  • the present invention further provides a means by which to create cell-associated DDpp, comprised of at least one DDpp designed to impart binding specificity a membrane bound fusion protein.
  • DDpp-receptors may be expressed by any cell type.
  • the DDpp-receptor fusion protein comprises a chimeric antigen receptor (CAR), or DDpp-CAR, that comprises: an extracellular targeting domain, a transmembrane domain, and an intracellular signaling domain.
  • 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.
  • the DDpp-CAR extracellular domain comprises one or more DDpp, in which each DDpp constitutes a specific binding domain with the same or different specificities.
  • the target-specific domain is directed to one (or more) of the cancer or tumor antigens disclosed herein, such as BCMA, CD123, and CSl, as non-limiting examples.
  • the target-specific domain is directed to AFP p26.
  • the intracellular signaling domain or fragment thereof is selected from the group: a human CD3 zeta domain, 41BB domain, a CD28 domain and any combination thereof.
  • the intracellular signaling domain contains the sequence of SEQ ID NO: 115 , 116 or combination thereof.
  • the intracellular signaling domain contains the sequence of SEQ ID NO: 117.
  • the costimulatory signaling region comprises the intracellular domain of a costimulatory molecule selected from the group: CD27, CD28, 41BB, 0X40, CD30, CD40, PD1, lymphocyte function-associated antigen-1 (LFA1), CD2, CD7, LIGHT, NKG2C, B7H3, a ligand that specifically binds with CD83, and any combination thereof.
  • a costimulatory molecule selected from the group: CD27, CD28, 41BB, 0X40, CD30, CD40, PD1, lymphocyte function-associated antigen-1 (LFA1), CD2, CD7, LIGHT, NKG2C, B7H3, a ligand that specifically binds with CD83, and any combination thereof.
  • the CAR comprises a fusion protein that includes an additional target-binding polypeptide.
  • the ADBD of a CAR comprises at least one alternative scaffold binding domain (e.g., a D domain or affibody) designed to impart binding specificity to a membrane bound CAR.
  • a receptor comprising an alternative scaffold binding domain may be expressed by any cell type.
  • the 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.
  • the CAR is composed of an extracellular domain and a transmembrane domain.
  • the CAR is comprised of an extracellular domain composed of one or more ADBDs (e.g., D domain) with the same or different specificities.
  • the intracellular domain (e.g., the cytoplasmic domain) of the CAR comprises the intracellular domain of CD3 zeta chain.
  • the intracellular signaling domain of the CAR is comprised of part of the intracellular domain of CD3 zeta chain.
  • the intracellular domain of the CAR comprises the intracellular domain of CD3 zeta chain and a costimulatory signaling region.
  • the costimulatory signaling region refers to a portion of the CAR comprising all or part of the intracellular domain of a costimulatory molecule. Costimulatory molecules and portions of these molecules that are able to confer costimulatory properties to a CAR are known in the art and can routinely be incorporated into the CAR. In addition, truncations or mutation to these intracellular signaling and costimulatory domains may be incorporated to further enhance or reduce receptor signaling.
  • a T cell is genetically modified to stably express a CAR.
  • the cytoplasmic domain of the CAR can be designed to comprise the CD28 and/or 41BB signaling domain by itself or be combined with any other desired cytoplasmic domain(s) useful in the context of the invention.
  • the cytoplasmic domain of the CAR can be designed to further comprise the signaling domain of CD3-zeta.
  • the cytoplasmic domain contains the sequence of SEQ ID NO: 115, 116 or combination thereof.
  • the cytoplasmic domain contains the sequence of SEQ ID NO: 117.
  • the CAR comprises an extracellular domain, an extracellular protein linker with a transmembrane domain that passes through the cellular membrane (such as found in T cells or NK cells), and a cytoplasmic domain, optionally comprising multiple signaling modules.
  • the CAR may also comprise an epitope tag.
  • the cytoplasmic domain of the CAR can include but is not limited to CD3-zeta, 41BB and CD28 signaling modules and combinations thereof.
  • the cytoplasmic domain contains the sequence of SEQ ID NO: 115, 116 or combination thereof.
  • the cytoplasmic domain contains the sequence of SEQ ID NO: 117.
  • Also provided for are isolated nucleic acid sequences encoding CARs that include the target-binding polypeptides as part (or all) of the targeting region.
  • the targeting domain of the CAR comprises a plurality of binding domains (e.g., DDs, or one or more DD and a scFv) that includes an additional target-binding polypeptide
  • the disclosure also provides cells comprising a nucleic acid sequence encoding a CAR, wherein the CAR comprises an antigen binding domain made up of, at least in part, a disclosed DDpp that binds a target of interest (e.g., CD123 and AFP p26), a transmembrane domain, and a signaling domain.
  • the CAR binds specifically to a tumor antigen (and thus functions to deliver the cell expressing the CAR to the tumor.
  • the tumor antigen is associated with a hematologic malignancy.
  • the tumor antigen is CD123.
  • the cell expressing the CAR is a T cell, a natural killer (NK) cell or other immune cell type. In some embodiments, the cell expressing the CAR (whether T cell, NK cell or other cell type) exhibits an anti-tumor immunity when the polypeptide binds to its corresponding tumor antigen. i. Extracellular Domain
  • the CARs provided herein comprise one or more antigenic determinant binding domains (ADBDs) (e.g., D domains).
  • the ADBD of the CAR can be any ADBD (e.g., D domain) described herein.
  • An exemplary ADBD comprises a polypeptide, e.g., an antibody molecule (which includes an antibody, and antigen binding fragments thereof, e.g., an immunoglobulin, single domain antibody (sdAb), and a scFv), or a non-antibody scaffold (e.g., a D domain, or affibody).
  • the extracellular domain of the CAR can be engineered to include one or more antigenic determinant binding domains (ADBDs) that specifically bind the desired antigen target(s).
  • ADBDs antigenic determinant binding domains
  • 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.
  • an extracellular domain of a CAR may include more than one ADBD, thereby imparting multispecificity or multi-valency to the CAR.
  • the choice of ADBDs in the extracellular domain of the CAR depends upon the identity of the cell or cells to be targeted.
  • the extracellular domain of the CAR may be engineered to specifically bind to cell surface proteins, such as a receptor, on the same cell or another cell.
  • the extracellular domain of the CAR is engineered to specifically bind to a soluble molecule, such as an immunoglobulin.
  • the extracellular domain of the CAR contains one or more ADBDs (e.g., D domain) that bind a ligand that acts as a cell surface marker on target cells associated with a cancer.
  • ADBD(s) target and bind a tumor or cancer antigen e.g., a TAA, TSA, CAA, CSA or other tumor antigen described herein or otherwise known in the art). Accordingly, provided herein are methods for creating CAR, their use in creating chimeric cells such as, human T cells and natural killer cells, and the use of these chimeric T and NK cells in adoptive immunotherapy.
  • an ADBD (e.g., D domain) can depend upon the type and number of ligands or receptors that define the surface of a target cell.
  • the ADBD may be chosen to recognize a ligand or receptor that acts as a cell surface marker on target cells associated with a particular disease state.
  • cell surface markers that may act as ligands or receptors include a cell surface marker associated with a particular disease state, e.g., cell surface makers for viral diseases, bacterial diseases parasitic infections, autoimmune diseases and disorders associated with unwanted cell proliferation, e.g., a cancer, such as, a cancer described herein.
  • the ADBD binds to CD123 (e.g., a polypeptide comprising the sequence of SEQ ID NO: 1).
  • the ADBD comprises a DD sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100.
  • the CD123-specific DD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 13, 14, 31, 32, and 33.
  • the CD123-specific DD comprises the amino acid sequence of SEQ ID NO: 14.
  • the ADBD (e.g., 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., 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., 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.
  • the DD binds AFP p26 and comprises the amino acid sequence of SEQ ID NO: 70-73 or 92-94. In some embodiments, the DD binds AFP p26 and comprises the amino acid sequence of SEQ ID NO: 73.
  • 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 a 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.
  • the CAR comprises a plurality of ADBDs. In some embodiments, the CAR comprises a plurality of the same ADBD. In some embodiments, the CAR comprises a plurality of different ADBDs. In some embodiments, the CAR comprises a plurality of ADBDs that bind the same antigenic determinant. In some embodiments, the CAR comprises a plurality of ADBDs, wherein the binding domains bind to different ADs. In some embodiments, the CAR comprises a plurality of ADBDs, wherein the binding domains bind to different ADs on the same cell. In some embodiments, the CAR comprises a plurality of ADBDs, wherein the binding domains bind to different ADs on different cells.
  • a CAR comprises a plurality of, e.g., 2, 3, 4, 5, or more than 5, ADBDs e.g., D domains, affibodies, or scFvs), wherein each ADBD(s) are able to bind to a target antigen.
  • ADBDs e.g., D domains, affibodies, or scFvs
  • two or more of the ADBDs of a CAR can bind to different ADs.
  • two or more of the ADBDs of the CAR can bind to the same antigen, e.g., the same or different epitopes on the same antigen.
  • a plurality of ADBDs of the CAR are linked to each other, e.g., the C-terminus of a first ADBD is linked to the N-terminus of a second ADBD.
  • the C-terminus of a first ADBD is linked to the N-terminus of a second ADBD by a covalent bond, e.g., a peptide bond.
  • a linker or hinge region is contained between one or more of the ADBDs, e.g., a linker or hinge region is located between the C-terminus of a first ADBD and the N-terminus of a second ADBD.
  • an antigen binding member comprising two ADBDs (e.g., ADBDi and ADBD2) can be arranged in the following configuration: [ADBD i]-[linker/hinge]-[ADBD2]. Additional ADBDs can be added in a similar manner, optionally with linker or hinge regions located between the C-terminus of an ADBD and the N-terminus of the next ADBD.
  • Linkers or hinge regions suitable for use in linking a plurality of antigen binding members are flexible, non-cleavable, and allow near-free motion of each ADBD independent from the other ADBDs to encourage binding with multiple target ADs simultaneously.
  • Any flexible linker or hinge region known in the art can be used.
  • the peptide linker contains the sequence of SEQ ID NO: 45- 48, 118 or 119.
  • the peptide linker contains the sequence of SEQ ID NO: 45-48, 118 or 119.
  • the CAR comprises a CD123-binding DD. In some embodiments, the CAR comprises a BCMA-binding DD and a CD123-binding DD.
  • the antigen binding moiety portion of the CAR specifically binds CD123 and CD33. In some embodiments, the antigen binding moiety portion of the CAR specifically binds CD123 and LeY. In additional embodiments, the antigen binding moiety portion of the CAR further binds a target selected from: BCMA, CS1, HVEM, BTLA, DR3, CD19, CD20, and CD22.
  • the antigen binding moiety portion of the CAR further binds a tumor antigen. In additional embodiments, the antigen binding moiety portion of the CAR further binds CD33 or LeY. In additional embodiments, the antigen binding moiety portion of the CAR further binds a target selected from: BCMA, CD123, CS1, HER2, HVEM, BTLA, DR3, CD19, CD20, and CD22.
  • the CAR comprises 2, 3, 4, 5, or more than 5, DD and/or other binding domains (e.g., scFv) that specifically bind a target of interest e.g., CD123) expressed on the surface of the cancer cell.
  • the CAR comprises 2, 3, 4, 5, or more than 5, DD or other binding domains (e.g., scFv) that specifically bind a second, different target of interest, expressed on the surface of the cancer cell.
  • the administered CAR further comprises 2, 3, 4, 5, or more than 5, DD or other binding domains (e.g., scFv) that specifically binds a second, different target of interest, expressed by a second, different cancer cell or a vascular endothelial cell.
  • the CAR comprises 2, 3, 4, 5, or more than 5, DD and/or other binding domains (e.g., scFv) that specifically bind AFP p26.
  • the CAR comprises 2, 3, 4, 5, or more than 5, DD and/or other binding domains (e.g., scFv) that specifically bind CD123.
  • the CD123-binding DD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 13, 14, 31, 32, and 33. In some embodiments, the CD123-binding DD comprises the amino acid sequence of SEQ ID NO: 14. ii. Extracellular Spacer Domain [0275]
  • the CARs comprise an extracellular spacer domain.
  • extracellular spacer domain or “ESD” refers to a polypeptide sequence of a CAR positioned between the ADBD and the transmembrane domain. In an embodiment the extracellular spacer domain allows sufficient distance from the outer surface of the cell and the ADBD as well as flexibility to minimize steric hindrance between the cell and the ADBD.
  • the extracellular spacer domain is sufficiently short or flexible that it does not interfere with engagement of the cell that includes the CAR with a cell bearing an AD, e.g., a target cell.
  • the extracellular spacer domain is from 2 to 20, 5 to 15, 7 to 12, or 8 to 10 amino acids in length.
  • the ESD domain includes at least 50, 20, or 10 residues. In some embodiments the ESD is 10 to 300, 10 to 250, or 10 to 200 residues in length.
  • the distance from which the ESD extends from the cell is sufficiently short that the hinge does not hinder engagement of the CAR ADBD with the surface of a target cell.
  • the ESD extends less than 20, 15, or 10 nanometers from the surface of the cytotoxic cell.
  • suitability for an ESD can be influenced by both linear length, the number of amino acid residues and flexibility of the ESD.
  • an IgG4 ESD can be as long as 200 amino acids in length, but the distance it extends from the surface of the cytotoxic cell is smaller due to Ig-domain folding.
  • a CD8 alpha ESD which is ⁇ 43 amino acids at ⁇ 8 nm in length.
  • the IgG4 C2 & C3 ESD is ⁇ 200 amino acids in length, but has a distance from the cytotoxic cell surface that is comparable to that of the CD8 alpha ESD. While not wishing to be bound by theory, the similarity in extension is influenced by flexibility.
  • the extracellular spacer domains include but are not limited to Fc fragments of antibodies or fragments or derivatives thereof, hinge regions of antibodies or fragments or derivatives thereof, CH2 regions of antibodies, CH3 regions of antibodies, artificial spacer sequences or combinations thereof. Additional examples of extracellular spacer domains include but are not limited to CD8a hinge, and artificial spacers made of polypeptides which may be as small as, for example, Gly3 or CHI and CH3 domains of IgGs (such as human IgG4).
  • the extracellular spacer domain is any one or more of (i) a hinge, CH2 and CH3 regions of IgG4, (ii) a hinge region of IgG4, (iii) a hinge and CH2 of IgG4, (iv) a hinge region of CD8a, (v) a hinge, CH2 and CH3 regions of IgGl, (vi) a hinge region of IgGl or (vi) a hinge and CH2 region of IgGl.
  • Other extracellular spacer domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments provided herein.
  • the ESD is a naturally occurring sequence.
  • the ESD of the CAR corresponds to an ESD from a human protein, a fragment thereof, or a short oligo- or polypeptide linker.
  • the CAR ESD corresponds to a human Ig (immunoglobulin) ESD (hinge), or fragment thereof.
  • the ESD comprises e.g., consists of) the amino acid sequence of the IgG4 ESD.
  • the hinge comprises (e.g., consists of) the amino acid sequence of the IgD hinge.
  • the hinge can be a human CD8 hinge, or fragment thereof.
  • the hinge comprises (e.g., consists of) the amino acid sequence of the CD8 hinge.
  • the ESD is an artificial sequence.
  • the ESD is a short oligopeptide linker comprising a glycine-serine doublet.
  • the CAR comprises the CD8a extracellular spacer domain.
  • the CAR does not contain an extracellular spacer domain. iii. Transmembrane Domain
  • transmembrane domain refers to the region of a cell surface expressed protein, such as a CAR, which spans the plasma membrane.
  • the TMD links an extracellular sequence (e.g., an extracellular ADBD or an extracellular AD), and an intracellular sequence, such as an intracellular signaling domain.
  • the transmembrane domain of the CAR is the transmembrane region of a transmembrane protein (for example Type I transmembrane proteins), an artificial hydrophobic sequence or a combination thereof.
  • Other transmembrane domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention.
  • the extracellular ADBD is a CD123-binding ADBD (e.g., D domain).
  • the extracellular ADBD comprises the amno acid sequence of SEQ ID NO 8, 13, 14, 31, 32, or 33.
  • the extracellular ADBD comprises the amno acid sequence of SEQ ID NO 14.
  • the extracellular ADBD is a p26-binding ADBD (e.g., D domain).
  • the extracellular ADBD comprises the amno acid sequence of SEQ ID NO: 70-73 or 92-94.
  • the extracellular ADBD comprises the amno acid sequence of SEQ ID NO 73.
  • the extracellular AD comprises p26.
  • the extracellular AD comprises the amno acid sequence of SEQ ID NO 37-43 or 44.
  • the CAR can be designed to contain a transmembrane domain that is fused to the extracellular domain of the receptor. As described above, the fusion of the extracellular and transmembrane domains can be accomplished with or without a linker. In one embodiment, the 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 the CD8a transmembrane domain. In some embodiments, the CD8a transmembrane domain comprises the amino acid sequence of SEQ ID NO: 113. In some embodiments, the CD 8 a transmembrane domain comprises the amino acid sequence of SEQ ID NO: 114.
  • the transmembrane domain of the CAR comprises the CD8a hinge domain.
  • the CD8a hinge domain also referred to as extracellular spacer domain (ESD)
  • ESD extracellular spacer domain
  • the transmembrane domain is selected or modified by amino acid substitution to promote or inhibit association with other surface membrane proteins.
  • the transmembrane domain can be derived either from a natural or from a synthetic source. Where the source is natural, the domain can be derived from any membrane-bound or transmembrane protein.
  • Transmembrane regions of particular use for the purposes herein may be derived from (i.e., comprise at least the transmembrane region(s) of) a member selected from the group: the alpha, beta or zeta chain of the T cell receptor; CD28, CD3 epsilon, CD45, CD4, CD5, CD8a, CD8b, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD 137, and CD 154.
  • the transmembrane domain is derived from the transmembrane region(s) of a NKR. In some embodiments, the transmembrane domain is derived from the transmembrane region of CD 8 a. In some embodiments, the CD 8 a 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.
  • the transmembrane domain is derived from the transmembrane region of a molecule selected from the group consisting of, KIRDS2, 0X40, TNFR2, LFA1 (CDl la, CD18), ICOS, 41BB, GITR, LTBR, BAFFR, HVEM, NKp80 (KLRF1), IL2R beta, IL2R gamma, IL7R a, ITGA1, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGB7, VLA1, VLA6, IA4, ITGAX, CDllc, ITGB1 CD27, CD29, ITGB2, CD2, CDl la, CDl lb, CDlld, CD18, CD19, CD40, CD49a, CD49d, CD49f, CD84, CD96, CD100, CD103, CD160, CD162, CD226, CD229, CEACAM1, CRTAM, PSGL1, SLAM (SLAMF1),
  • the transmembrane domain can be synthetic, and preferably predominantly comprises hydrophobic residues such as leucine and valine.
  • the transmembrane domain comprises the triplet of FWV (phenylalanine, tryptophan and valine) at each end of the transmembrane domain.
  • NKR domains e.g., transmembrane, hinge or stem, or intracellular (e.g., cytoplasmic) domains (identified by the NKR from which the domain is derived)
  • Killer immunoglobulin KIR2DL1 receptors include KIR2DL2/L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1/S1, KIR3DL2, KIR3DL3, KIR2DP1, KIR2DP1, NCRs: , NKp30, NKp44, NKp46, SLAM; Receptors SLAM, CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, CD2F-10, SLAMF6, SLAMF7; Fc-binding Receptors CD16, FcgRIII, CD64, Ly49; Re
  • Intracellular signaling domains that can be used in a chimeric antigen receptor (CAR) according to the present invention.
  • ISD Intracellular signaling domain
  • cytoplasmic domain refers to the portion of the CAR which transduces the effector function signal and directs the cell to perform its specialized function (e.g., cytolytic activity and helper activity, including cytokine secretion).
  • the cytoplasmic domain (i.e., intracellular signaling domain) of a CAR is responsible for activation of at least one of the normal effector functions of an immune cell engineered to express a CAR.
  • effector function refers to a specialized function of a cell.
  • the effector function of a T cell for example, includes cytolytic activity and helper activity including the secretion of cytokines.
  • intracellular signaling domain refers to the portion of a CAR protein which transduces the effector function signal and directs the cell to perform a specialized function. While typically the entire intracellular signaling domain corresponding to a naturally occurring receptor can be employed, in many cases it is not necessary to use the entire chain.
  • an intracellular signaling domain in the CAR includes the cytoplasmic sequences of the T cell receptor (TCR) and also the sequence of co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, or any derivative or variant of these sequences that has functional capability.
  • TCR T cell receptor
  • domains that transduce an effector function signal include but are not limited to the chain of the T cell receptor complex or any of its homologues (e.g., r
  • chain, FcsRly and P chains e.g., r
  • human CD3 zeta chain CD3 polypeptides (A, 5 and a)
  • the intracellular signaling domain of the CAR produces an intracellular signal when an extracellular domain (e.g., an ADBD) to which it is fused, binds a cognate AD.
  • the Intracellular signaling domains of the CAR can include primary intracellular signaling domains and costimulatory signaling domains.
  • the CAR is constructed for expression in an immune cell (e.g., a T or NK cell), such that the expressed CAR comprises a domain such as a primary intracellular signaling domain and/or costimulatory signaling domain, that is derived from a polypeptide typically associated with the immune cell.
  • the CAR is for expression in a T cell and comprises a 41BB domain and a CD3 zeta domain.
  • the CAR molecule is constructed for expression in an immune cell such that the expressed CAR comprises a domain that is derived from a polypeptide that is not typically associated with the immune cell.
  • the CAR for expression in a T cell comprises a KIR domain derived from a NK cell.
  • the CAR for expression in an NK cell comprises a 41BB domain and a CD3 zeta domain derived from a T cell (See e.g. WO2013/033626, incorporated herein by reference).
  • the intracellular signaling domain of the CAR comprises sufficient primary stimulatory molecule sequence to produce an intracellular signal, e.g., when an ADBD to which it is fused binds a cognate AD.
  • the intracellular signal of the CAR mediates a T cell response selected from the group: proliferation, cytokine secretion, killing, activation, and differentiation.
  • the intracellular signaling region of the CAR comprises a domain that contains an immunoreceptor tyrosine-based activation motif (ITAM).
  • ITAM immunoreceptor tyrosine-based activation motif
  • the CAR intracellular signaling region comprises one or more ITAM containing domains from a molecule selected from: TCR zeta (CD3 zeta), FcR gamma (FCER1G), Fc gamma Rlla, FcR beta (FCER1B), CD3 gamma, CD3 delta, CD3 epsilon, CD3 gamma, CD5, CD22, CD79a, CD79b, DAP10, DAP12, CD32 (Fc gamma Rlla), CD79a, and CD79b.
  • ITAM immunoreceptor tyrosine-based activation motif
  • the intracellular signaling domain of the CAR comprises a CD3 zeta signaling domain. In another specific embodiment, the intracellular signaling domain of the CAR comprises a DAP12 signaling domain.
  • the ITAM containing signaling domain has at least 70, 75, 80, 85, 90, 95, 98, or 99% sequence identity with, or differs by no more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residues from the corresponding residues of a naturally occurring ITAM containing domain.
  • ITAM containing primary cytoplasmic signaling sequences examples include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD22, CD79a, CD79b, and CD66d. It is particularly preferred that a cytoplasmic signaling molecule in the CAR comprises a cytoplasmic signaling sequence derived from CD3 zeta.
  • the cytoplasmic domain of the CAR comprises the CD3-zeta signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR.
  • the cytoplasmic domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling region.
  • the costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule.
  • a costimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen.
  • Examples of such molecules include CD27, CD28, 41BB (CD 137), 0X40, CD30, CD40, PD1, ICOS, lymphocyte function- associated antigen-1 (LFA1), CD2, CD7, LIGHT, NKG2C, B7H3, TIM1, and LAG3.
  • Co-stimulatory domain refers to the portion of the CAR which enhances the proliferation, survival and/or development of memory cells.
  • the CAR may comprise one or more costimulatory domains.
  • Each co-stimulatory domain comprises the costimulatory domain of any one or more of, for example, a member of the TNFR superfamily, selected from CD28, CD137 (41BB), CD134 (0X40), DaplO, CD27, CD2, CD5, ICAM1, LFA1(CD1 la/CD18), Lek, TNFRI, TNFRII, Fas, CD30, and CD40 or a combination thereof.
  • Other co-stimulatory domains e.g., from other proteins will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention.
  • the intracellular domain of the CAR comprises an IT AM containing domain and a costimulatory signaling domain that comprises a functional fragment or analog of a costimulatory molecule that is sufficient to produce an intracellular signal when an extracellular ADBD to which it is fused, binds cognate ligand.
  • the CAR comprises a costimulatory signaling domain corresponding to that found in a molecule selected from: CD137 (41BB), 0X40, LIGHT, TNFR2, TRANCE/RANKL, GITR, BAFFR, HVEM, B7H3, CDS, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, VLA1, VLA6, C49f, IA4, LFA1, CD2, CD4, CD7, CD8 alpha, CD8 beta, CD11A, CD11B, CD11C, CD11D, CD18, CD19, CD27, CD28, CD29, CD30, CD40, CD49A, CD49D, CD69, CD84, CD96, CD100, CD103, CD150, CD160, CD162, CD226, CD229, CD278, ICAM1, CEACAM1, CRT AM,
  • the CAR comprises a costimulatory domain corresponding to that found in a molecule selected from the group consisting 41BB, CD28, CD27, ICOS, and 0X40.
  • the CAR comprises a plurality of costimulatory domains.
  • the CAR comprises the following costimulatory signaling domains, from the extracellular to intracellular direction: 41BB-CD27, CD27-41BB, 41BB-CD28, CD28-41BB, OX40-CD28, CD28-OX40, CD28-41BB; or 41BB-CD28.
  • the costimulatory signaling domain of the CAR has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity with, or differs by no more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residues from the corresponding residues of a naturally occurring costimulatory domain.
  • the CAR contains an intracellular signaling domain having the sequence of SEQ ID NO: 115, 116 or combination thereof. In some embodiments, the CAR contains an intracellular signaling domain having the sequence of SEQ ID NO: 117. In some embodiments, the CAR contains an intracellular signaling domain having a sequence that has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity with of SEQ ID NO: 115, 116 or 117. In some embodiments, the CAR contains an intracellular signaling domain having the sequence of SEQ ID NO: 115, 116 or 117 comprising 1, 2, 3, 4, 5, 7, 8, 9, 10, 15 or 20 insertions, deletions or substitutions.
  • the CAR contains an intracellular signaling domain having the sequence of SEQ ID NO: 115, 116 or 117 comprising 1, 2, 3, 4, 5, 7, 8, 9, or 10 insertions, deletions or substitutions. In some embodiments, the CAR contains an intracellular signaling domain having the sequence of SEQ ID NO: 115, 116 or 117 comprising 1, 2, 3, 4, 5, 7, 8, 9, or 10 substitutions.
  • Polypeptide linkers may be positioned between adjacent elements of the CAR. For example linkers may be positioned between adjacent ADBDs or between an ADBD and the transmembrane domain or between the transmembrane domain and the cytoplasmic domain or between adjacent cytoplasmic domains.
  • the cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR may be linked to each other in a random or specified order.
  • a short linker preferably between 2 and 10 amino acids in length may form the linkage.
  • a glycine-serine doublet provides a particularly suitable linker.
  • the CAR comprises, a target binding domain, a transmembrane domain, and an intracellular signaling domain.
  • the CAR transmembrane domain comprises a CD8a, 41BB or CD28 transmembrane domain.
  • the CAR transmembrane domain comprises the CD8a transmenrane domain.
  • the CAR comprises the CD8a transmenrane domain and CD8a extracellular spacer domain.
  • the CD8a transmembrane domain comprises the amino acid sequence of SEQ ID NO: 113.
  • the CD8a transmembrane domain comprises the amino acid sequence of SEQ ID NO: 114.
  • the CD8a extracellular spacer domain (ESD), also referred to as hinge domain, comprises the amino acid sequence of SEQ ID NO: 112.
  • the CAR comprises an intracellular signaling domain selected from the group consisting of a domain of a human T cell receptor alpha, beta, or zeta chain; a human 41BB domain; a human CD28 domain; and any combination thereof.
  • the CAR contains an intracellular signaling domain having the sequence of SEQ ID NO: 115, 116 or combination thereof.
  • the CAR contains an intracellular signaling domain having the sequence of SEQ ID NO: 117.
  • the CAR contains an intracellular signaling domain having a sequence that has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity with of SEQ ID NO: 115, 116 or 117. In some embodiments, the CAR contains an intracellular signaling domain having the sequence of SEQ ID NO: 115, 116 or 117 comprising 1, 2, 3, 4, 5, 7, 8, 9, 10, 15 or 20 insertions, deletions or substitutions. In some embodiments, the CAR contains an intracellular signaling domain having the sequence of SEQ ID NO: 115, 116 or 117 comprising 1, 2, 3, 4, 5, 7, 8, 9, or 10 insertions, deletions or substitutions.
  • the CAR contains an intracellular signaling domain having the sequence of SEQ ID NO: 115, 116 or 117 comprising 1, 2, 3, 4, 5, 7, 8, 9, or 10 substitutions.
  • the CAR intracellular signaling domain comprises the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, 0X40, CD30, CD40, PD1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and any combination thereof.
  • the CAR further comprises a second target binding domain having the same or a different target than the DD target binding domain.
  • the CAR comprises a first target binding domain that binds CD 123 and a second target binding domain that binds a different target. In some embodiments, the CAR comprises first and second target binding domains that bind CD123. In some embodiments, the CAR is expressed in an immune effector cell. In some embodiments, the immune effector cell is a T cell (CAR-T cell) or a natural killer (NK) cell (CAR-NK cell). In some embodiments, the immune effector cell is an autologous cell. In some embodiments, the immune effector cell is an allogenic cell. In some embodiments, the CAR is associated with a liposome.
  • the CAR comprises a target binding domain comprising a DD disclosed herein (e.g., a DD comprising the amino acid sequence of SEQ ID NO: 8-33 or 74-94).
  • the DD binds CD123 and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100.
  • the CD123-specific DD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 13, 14, 31, 32, and 33.
  • the CD123-specific DD comprises the amino acid sequence of SEQ ID NO: 14.
  • the DD binds AFP p26 and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94. In some embodiments, the DD binds AFP p26 and comprises the amino acid sequence of SEQ ID NO: 70-73 or 92-94. In some embodiments, the DD binds AFP p26 and comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, the CAR comprises an extracellular AD comprising p26. In some embodiments, the extracellular AD comprises the amno acid sequence of SEQ ID NO 37-43 or 44.
  • CARs suitable for use in connection with the DDpp have been disclosed in Int'l. Appl. Pub. Nos. WO 2016164305, WO 2016164308A1, WO 2019099440, and WO 2019099433, US Patent Nos. 10,662,248, and 10,647,775, and US Pat. Appl. Nos. 20200223934, and 20210002381, each of which is incorporated herein by reference for all purposes.
  • the DDpp contains a heterologous polypeptide comprising a fragment consisting 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 an extracellular domain, of a cell surface receptor.
  • the DDpp contains a heterologous polypeptide that comprises the extracellular domain, or a fragment of an extracellular domain of BCMA (SEQ ID NO: 34) or CD123 (SEQ ID NO: 1).
  • the DDpp contains a heterologous polypeptide that comprises the extracellular domain, or a fragment of an extracellular domain of BCMA (SEQ ID NO: 34), or CD123 (SEQ ID NO: 1), or CS1 (SEQ ID NO: 35).
  • the DDpp contains a heterologous polypeptide that comprises the extracellular domain, or a fragment of an extracellular domain, of a receptor selected from the group consisting of: CD19, CD20, CD22, HVEM, BTLA, DR3, CD37; TSLPR, IL7R, and gp96.
  • the protein contains a heterologous polypeptide that comprises a serum protein or an antigenic fragment of a serum protein (e.g., AFP, and AFP p26).
  • the DDpp contains a heterologous polypeptide comprising a fragment consisting 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.
  • the protein contains a heterologous polypeptide that comprises an intracellular protein or an antigenic portion of an intracellular protein (e.g., a nuclear protein).
  • the DDpp contains a heterologous polypeptide comprising a fragment consisting 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 an intracellular protein.
  • the DDpp contains a heterologous polypeptide having the sequence of SEQ ID NO: 37-43 or 44.
  • the DDpp fusion protein specifically binds CD123 and/or AFP p26, and further binds one or more additional targets of interest.
  • the targets of interest specifically bound by a DDpp fusion protein can be any molecule for which it is desirable for a DDpp to bind.
  • the targets specifically bound by the DDpp fusion protein can be CD123 and/or AFP p26, and additionally any additional target of manufacturing, formulation, therapeutic, diagnostic, or prognostic relevance or value.
  • a number of exemplary additional targets are provided herein, by way of example, and are intended to be illustrative and not limiting.
  • the additional target of interest bound by the DDpp fusion protein can be naturally occurring or synthetic.
  • the additional target of interest can be an extracellular component or an intracellular component, a soluble factor (e.g., an enzyme, hormone, cytokine, and growth factor, toxin, venom, pollutant, etc.), or a transmembrane protein (e.g., a cell surface receptor).
  • the target of interest bound by the DDpp fusion protein is a human protein.
  • a DDpp e.g., a DDpp fusion protein
  • the additional target of interest is BCMA, CS1, or HER2
  • a DDpp fusion protein specifically binds CD123 and/or AFP p26, and a serum protein.
  • the DDpp fusion protein specifically binds a serum protein selected from: serum albumin (e.g., human serum albumin (HSA)), thyroxin-binding protein, transferrin, fibrinogen, and an immunoglobulin (e.g., IgG, IgE and IgM).
  • serum albumin e.g., human serum albumin (HSA)
  • thyroxin-binding protein thyroxin-binding protein
  • transferrin transferrin
  • fibrinogen e.g., fibrinogen
  • an immunoglobulin e.g., IgG, IgE and IgM
  • the binding of a DDpp to a carrier protein is believed to confer upon the DDpp (or a fusion thereof) an improved pharmacodynamic profile that includes, but is not limited to, improved tumor targeting, tumor penetration, diffusion within the tumor, and enhanced therapeutic activity compared to the DDpp fusion protein in which the carrier protein binding sequence is missing (see, e.g., WOOl/45746, the contents of which is herein incorporated by reference in its entirety).
  • the DDpp fusion protein comprises a full-length antibody or a fragment or subdomain of an antibody.
  • the DDpp fusion protein comprises a full length IgG antibody (e.g., IgGl, IgG2, IgG2, or IgG4).
  • the DDpp fusion protein comprises a full length antibody that specifically binds a cancer antigen.
  • the DDpp comprises a commercially approved therapeutic antibody (e.g., rituximab, ofatumumab, ocrelizumab, veltuzumab, MEDI-551, epratuzumab, belimumab, tabalumab, AMG-557, MEDI-570, and NN882).
  • the DDpp comprises an antibody or antigen-binding fragment thereof that binds to the T-cell receptor (TCR) complex on T cells (e.g., binds to the CD3 epsilon chain).
  • the DDpp is an Fc fusion protein.
  • the Fc protein comprises a variant human Fc domain.
  • the DDpp fusion protein comprises a full-length antibody or an antibody fragment or subdomain (e.g., an IgGl antibody, IgG3 antibody, antibody variable region, CDR3, scFv, Fc, FcRn binding subdomain, and other antibody subdomains).
  • DDpp proteins can be operably linked to one another and/or to one or more termini of an antibody, antibody chain, antibody fragment, or antibody subdomain to form a DDpp fusion protein.
  • the antibody component of a DDpp fusion protein can be any suitable full-length immunoglobulin or antibody fragment (e.g., an antigen binding domain and/or effector domain) or a fragment thereof.
  • the DDpp-antibody fusion protein retains the structural and functional properties of a traditional monoclonal antibody.
  • the DDpp-antibody fusion protein retains the epitope binding properties, but advantageously also incorporate, via the DDpp fusion, one or more additional targetbinding specificities.
  • Antibodies that can be used in the DDpp fusions include, but are not limited to, monoclonal, multi-specific, human, humanized, primatized, and chimeric antibodies.
  • Immunoglobulin or antibody molecules provided herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
  • the antibodies are Fc optimized antibodies.
  • Antibodies can be from or derived from any animal origin including birds and mammals or generated synthetically.
  • the antibody component of the DDpp-antibody fusion protein can be naturally derived or the result of recombinant engineering (e.g., phage display, xenomouse, and synthetic).
  • the antibody component of the antibody-DDpp fusion enhances half-life, and increase or decrease antibody dependent cellular cytotoxicity (ADCC), and/or complement dependent cytotoxicity (CDC) activity.
  • ADCC antibody dependent cellular cytotoxicity
  • CDC complement dependent cytotoxicity
  • the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In specific embodiments, the antibodies are human.
  • the constant region mediates several effector functions.
  • binding of the Cl component of complement to antibodies activates the complement system.
  • Activation of complement is important in the opsonization and lysis of cell pathogens.
  • the activation of complement also stimulates the inflammatory response and can also be involved in autoimmune hypersensitivity.
  • antibodies bind to cells via the Fc region, with an Fc receptor site on the antibody Fc region binding to an Fc receptor (FcR) on a cell.
  • Fc receptor Fc receptor
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the DDpp-Fc fusion protein has an altered effector function that, in turn, affects the biological profile of the administered DDpp-Fc fusion protein.
  • the deletion or inactivation (through point mutations or other means) of a constant region subdomain can reduce Fc receptor binding of the circulating modified antibody.
  • the constant region modifications can moderate complement binding and thus reduce the serum half-life and nonspecific association of a conjugated cytotoxin.
  • modifications of the constant region can be used to eliminate disulfide linkages or oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or antibody flexibility.
  • modifications to the constant region in accordance with this disclosure can easily be made using biochemical or molecular engineering techniques known to those of ordinary skill in the art.
  • the DDpp-Fc fusion protein does not have one or more effector functions.
  • the DDpp-Fc fusion protein has no antibody-dependent cellular cytoxicity (ADCC) activity and/or no complement-dependent cytoxicity (CDC) activity.
  • the DDpp-Fc fusion protein does not bind to an Fc receptor and/or complement factors.
  • the DDpp-Fc fusion protein has no effector function. Examples of Fc sequence engineering modifications that reduce or eliminate ADCC and/or CDC activity and Fc receptor and/or complement factor binding are described herein or otherwise know in the art, as are assays and procedures for testing the same.
  • DDpp-Fc fusion protein is engineered to fuse the CH3 domain directly to the hinge region of the respective modified antibody.
  • a peptide spacer is inserted between the hinge region and the modified CH2 and/or CH3 domains.
  • compatible constructs can be expressed in which the CH2 domain has been deleted and the remaining CH3 domain (modified or unmodified) is joined to the hinge region with a 5-20 amino acid spacer.
  • Such a spacer can be added, for instance, to ensure that the regulatory elements of the constant domain remain free and accessible or that the hinge region remains flexible.
  • Amino acid spacers can, in some cases, prove to be immunogenic and elicit an unwanted immune response against the construct. Accordingly, in certain embodiments, any spacer added to the construct can be relatively non-immunogenic, or even omitted altogether, so as to maintain the desired biochemical qualities of the modified DDpp-Fc fusion protein.
  • the DDpp-Fc fusion protein is modified by the partial deletion or substitution of a few or even a single amino acid in a constant region.
  • the mutation of a single amino acid in selected areas of the CH2 domain can be enough to substantially reduce Fc binding and thereby.
  • one or more constant region domains that control the effector function e.g., complement C1Q binding
  • Such partial deletions of the constant regions can improve selected characteristics of the DDpp-Fc fusion protein (e.g., serum half-life) while leaving other desirable functions associated with the corresponding constant region domain intact.
  • the constant region of the DDpp-Fc fusion protein is modified through the mutation or substitution of one or more amino acids that enhances the profile of the resulting construct.
  • a conserved binding site e.g., Fc binding
  • the disclosure also provides an DDpp-Fc fusion protein that contains the addition of one or more amino acids to the constant region to enhance desirable characteristics such, as decreasing or increasing effector function or providing attachments sites for one or more cytotoxin, labeling or carbohydrate moieties.
  • the DDpp is operably linked to an antibody fragment or subdomain (e.g., scFv, diabody, EP 404,097; WO93/111161; WO14/028776; and Holliger et al., PNAS 90: 6444-6448 (1993), the contents of each of which is herein incorporated by reference in its entirety).
  • the antibody fragment or subdomain can be any fragment or domain of an antibody. See for example, W004/058820, WO99/42077 and W005/017148, the contents of each of which is herein incorporated by reference in its entirety.
  • a DDpp fusion protein can contain an antibody effector domain or derivative of an antibody effector domain that confers one or more effector functions to the DDpp and/or confers upon the DDpp fusion protein the ability to bind to one or more Fc receptors.
  • a DDpp-antibody fusion protein contains an antigenbinding fragment of an antibody or a fragment thereof.
  • a DDpp-antibody fusion protein contains an immunoglobulin effector domain that comprises one or more CH2 and or CH3 domains of an antibody having effector function provided by the CH2 and CH3 domains.
  • the DDpp fusion contains a full-length antibody or an antibody fragment that is an antigen-binding fragment.
  • the antibody or antibody fragment binds a disease-related antigen.
  • the DDpp fusion protein comprises an antibody or an antibody fragment that specifically binds a cancer antigen.
  • the DDpp fusion protein comprises an antibody or an antibody fragment that specifically binds a particular pathogen (e.g., a bacterial cell e.g., tuberculosis, smallpox, anthrax)), a virus (e.g., HIV), a parasite (e.g., malaria, leishmaniosis), a fungal infection, a mold, a mycoplasm, a prion antigen
  • the DDpp fusion protein comprises an antibody or an antibody fragment that specifically binds a particular pathogen (e.g., a bacterial cell (e.g., tuberculosis, smallpox, anthrax)), a virus (e.g., HIV), a parasite (e.g., malaria, leishmaniosis), a fungal infection, a mold, a mycoplasm, or a prion antigen.
  • a particular pathogen e.g., a bacterial cell e.g., tuber
  • the DDpp fusion protein comprises an antibody or an antibody fragment that specifically binds an antigen associated with a disease or disorder of the immune system.
  • the DDpp fusion protein containing an antibody fragment or domain retains activities of the parent antibody.
  • the DDpp fusion protein containing an antibody fragment or domain is capable of inducing complement dependent cytotoxicity.
  • the DDpp fusion protein containing an antibody fragment or domain is capable of inducing antibody dependent cell mediated cytotoxicity (ADCC).
  • ADCC antibody dependent cell mediated cytotoxicity
  • the DDpp fusion protein comprises an antibody fragment that confers upon the DDpp fusion protein a biological or biochemical characteristic of an immunoglobulin.
  • the antibody fragment confers a characteristic selected from: the ability to non-covalently dimerize, the ability to localize at the site of a tumor, and an increased serum half-life when compared to the DDpp fusion protein in which said one or more DDpp have been deleted.
  • the DDpp fusion protein is at least as stable as the corresponding antibody without the attached DDpp. In certain embodiments, the DDpp fusion protein is more stable than the corresponding antibody without the attached DDpp.
  • the DDpp fusion protein stability can be measured using established methods, including, for example, ELISA techniques.
  • the DDpp fusion protein 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 between those listed).
  • a DDpp fusion contains an immunoglobulin effector domain or half-life influencing domain that corresponds to an immunoglobulin domain or fragment in which at least a fraction of one or more of the constant region domains has been altered so as to provide desired biochemical characteristics such as reduced or increased effector functions, the ability to non-covalently dimerize, increased ability to localize at the site of a tumor, reduced serum half-life, or increased serum halflife when compared with an immunoglobulin fragment having the corresponding unaltered immunoglobulin sequence.
  • These alterations of the constant region domains can be amino acid substitutions, insertions, or deletions.
  • a DDpp fusion protein comprises an 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 DDpp fusion protein.
  • a DDpp fusion protein comprises a sequence of an immunoglobulin effector domain that has been modified to increase ADCC (see, 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.
  • immunoglobulin fragment engineering modifications contained in an amino acid sequence in a DDpp fusion protein that increases ADCC include immunoglobulin effector domain sequences having one or more modifications corresponding to: IgGl-S298A, E333A, K334A; IgGl-S239D, I332E; IgGl-S239D, A330L, I332E; IgGl-P247I, A339D or Q; IgGl-D280H, K290S with or without S298D or V; IgGl-F243L, R292P, Y300L; IgGl-F243L, R292P, Y300L, P396L; and IgGl-F243L, R292P, Y300L, V305I, P396L; wherein the numbering of the residues in the Fc region is that of the EU index of Kabat et al. (Kabat et al., Sequences of proteins of
  • a DDpp fusion protein comprises a sequence of an immunoglobulin effector domain that has been modified to decrease ADCC (see, e.g., Idusogie et al.. ⁇ . Immunol. 166: 2571-2575 (2001); Sazinsky et al., PNAS 105: 20167-20172 (2008); Davis et al., J. Rheumatol. 34: 2204-2210 (2007); Bolt et al., Eur. J. Immunol. 23: 403-411 (1993); Alegre et al., Transplantation 57: 1537-1543 (1994); Xu et al., Cell Immunol.
  • immunoglobulin fragment sequence engineering modifications contained in an amino acid sequence in a DDpp fusion protein that decreases ADCC include immunoglobulin effector domain sequences having one or more modifications corresponding to: IgGl-K326W, E333S; IgG2-E333S; IgGl-N297A; IgGl-L234A, L235A; IgG2-V234A, G237A; IgG4-L235A, G237A, E318A; IgG4-S228P, L236E; IgG2- 118-260; IgG4- 261-447; IgG2-H268Q, V309L, A330S, A331S; IgGl- C220S, C226S, C229S, p268S; IgGl-C226S, C229S, E233P, L234V, L235A; or IgGl-L234F, L235E, P3
  • a DDpp fusion protein comprises an amino acid sequence of an immunoglobulin effector domain, or a derivative of an immunoglobulin effector domain, that confers antibodydependent cell phagocytosis (ADCP) to the DDpp fusion protein.
  • a DDpp fusion protein comprises a sequence of an immunoglobulin effector domain that has been modified to increase antibody-dependent cell phagocytosis (ADCP); (see, 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.
  • immunoglobulin fragment engineering modifications contained in an amino acid sequence in a DDpp fusion protein that increases ADCP include immunoglobulin effector domain sequences having one or more modifications corresponding to: IgGl-S298A, E333A, K334A; IgGl-S239D, I332E; IgGl-S239D, A330L, I332E; IgGl-P247I, A339D or Q; IgGl-D280H, K290S with or without S298D or V; IgGl-F243L, R292P, Y300L; IgGl-F243L, R292P, Y300L, P396L; IgGl-F243L, R292P, Y300L, V305I, P396L; and IgGl-G236A, S239D, I332E; wherein the numbering of the residues is that of the EU index of Kabat et al. (
  • a DDpp fusion protein comprises a sequence of an immunoglobulin effector domain that has been modified to decrease ADCP (see, e.g., Sazinsky et al., PNAS 105: 20167-20172 (2008); Davis et al., J. Rheumatol. 34: 2204-2210 (2007); Bolt et al., Eur. J. Immunol. 23: 403-411 (1993); Alegre et al., Transplantation 57: 1537-1543 (1994); Xu et al., Cell Immunol.
  • DDpp fusion proteins can contain an antibody fragment or domain that contains one or more of the following modifications that decrease ADCC: IgGl-N297A; IgGl-L234A, L235A; IgG2-V234A, G237A; IgG4-L235A, G237A, E318A; IgG4-S228P, L236E; IgG2 EU sequence 118-260; IgG4-EU sequence 261-447; IgG2-H268Q, V309L, A330S, A331S; IgGl-C220S, C226S, C229S, p268S; IgGl-C226S, C229S, E233P, L234V, L235A; and IgGl-L234F, L235E, P331S; wherein the numbering of the residues is that of the EU index of Kabat et al. (Kabat et al.
  • a DDpp fusion protein comprises an amino acid sequence of an immunoglobulin effector domain, or a derivative of an immunoglobulin effector domain, that confers complement-dependent cytotoxicity (CDC) to the DDpp fusion protein.
  • a DDpp fusion protein 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.
  • DDpp fusion proteins can contain an antibody fragment or domain that contains one or more of the following modifications that increase CDC: IgGl-K326A, E333A; IgGl-K326W, E333S, IgG2-E333S; wherein 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, the contents of which is herein incorporated by reference in its entirety).
  • a DDpp fusion protein comprises an amino acid sequence of an immunoglobulin effector domain, or a derivative of an immunoglobulin effector domain, that confers the ability to bind FcgammaRIIb receptor to the DDpp fusion.
  • a DDpp fusion protein comprises a sequence of an immunoglobulin effector domain that has been modified to increase inhibitory binding to FcgammaRIIb receptor (see, e.g., Chu et al., Mol. Immunol. 45: 3926-3933 (2008)).
  • An example of an immunoglobulin fragment engineering modification contained in an amino acid sequence in a DDpp fusion protein that increases binding to inhibitory FcgammaRIIb receptor is IgGl- S267E, L328F.
  • a DDpp fusion protein comprises a sequence of an immunoglobulin effector domain that has been modified to decrease CDC (see, e.g., WO97/11971; W007/106585; US 2007/0148167A1; McEarchern etal., Blood 109: 1185-1192 (2007); Hayden-Ledbetter et al., Clin. Cancer 15: 2739-2746 (2009); Lazar et al., PNAS 103: 4005-4010 (2006); Bruckheimer et al., Neoplasia 11: 509-517 (2009); Strohl, Curr. Op. Biotechnol.
  • DDpp fusion proteins can contain an antibody fragment or domain that contains one or more of the following modifications that decrease CDC: IgGl-S239D, A330L, I332E; IgG2- 118-260; IgG4- 261-447; IgG2-H268Q, V309L, A330S, A331S; IgGl-C226S, C229S, E233P, L234V, L235A; IgGl-L234F, L235E, P331S; and IgGl-C226S, p260S; wherein 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, the contents of which is here
  • a DDpp fusion protein comprises an amino acid sequence of an immunoglobulin effector domain, or a derivative of an immunoglobulin effector domain, that confers the ability to bind neonatal receptor FcRn to the to the DDpp fusion.
  • a DDpp fusion protein comprises a sequence of an immunoglobulin FcRn binding domain that has been modified to enhance binding to FcRn (see, e.g., Petkova et al., Int. Immunol. 18: 1759-1769 (2006); Dall'Acqua et al., J.
  • a DDpp fusion protein comprises a sequence of an immunoglobulin effector domain that has been modified to have a selective affinity for FcRn at pH 6.0, but not pH 7.4.
  • DDpp fusion proteins can contain an antibody fragment or domain that contains one or more of the following modifications that increase half-life: IgGl-M252Y, S254T, T256E; IgGl-T250Q, M428E; IgGl- H433K, N434Y; IgGl-N434A; and IgGl-T307A, E380A, N434A; wherein 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, the contents of which is herein incorporated by reference in its entirety).
  • a DDpp fusion protein comprises a sequence of an immunoglobulin effector domain that has been modified to decrease binding to FcRn (see, e.g., Petkova et al., Int. Immunol. 18: 1759- 1769 (2006); Datta-Mannan et al., Drug Metab. Dispos. 35: 86-94 (2007); Datta-Mannan et al., J. Biol. Chem. 282: 1709-1717 (2007); Strohl, Curr. Op. Biotechnol. 20: 685-691 (2009); and Vaccaro et al., Nat. Biotechnol.
  • DDpp fusion proteins can contain an antibody fragment or domain that contains one or more of the following modifications that decrease half-life: IgGl-M252Y, S254T, T256E; H433K, N434F, 436H; IgGl- 1253 A; and IgGl-P257I, N434H and D376V, N434H; wherein 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, the contents of which is herein incorporated by reference in its entirety).
  • DDpp fusion protein comprises an amino acid sequence corresponding to a immunoglobulin effector domain that has been modified to contain at least one substitution in its sequence corresponding to the Fc region (e.g., FC gamma) position selected from the group consisting of: 238, 239, 246, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286,
  • Fc region e.g., FC gamma
  • the DDpp fusion protein comprises a sequence of an immunoglobulin effector domain derivative wherein at least one residue corresponding to position 434 is a residue selected from the group consisting of: A, W, Y, F and H.
  • the DDpp fusion protein comprises a sequence of an immunoglobulin effector fragment derivative having the following respective substitutions S298A/E333A/K334A.
  • the DDpp fusion protein comprises an immunoglobulin effector domain derivative having a substitution corresponding to K322A.
  • the DDpp fusion protein comprises a sequence of an immunoglobulin effector domain derivative having one or any combination of the following substitutions K246H, H268D, E283L, S324G, S239D and I332E.
  • a DDpp fusion protein comprises a sequence of an immunoglobulin effector domain derivative having substitutions corresponding to D265A/N297A.
  • a DDpp fusion protein comprises a sequence of an immunoglobulin effector domain that has been glycoengineered or mutated to increase effector function using techniques known in the art.
  • the inactivation (through point mutations or other means) of a constant region domain sequence contained in a DDpp may reduce Fc receptor binding of the circulating DDpp fusion protein thereby increasing tumor localization.
  • constant region modifications consistent with certain provided embodiments, moderate complement binding and thus reduce the serum half-life and nonspecific association of a conjugated cytotoxin.
  • modifications of the constant region may be used to modify disulfide linkages or oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or antibody flexibility.
  • the resulting physiological profile, bioavailability and other biochemical effects of the modifications, such as tumor localization, biodistribution and serum half-life, can easily be measured and quantified using well know immunological techniques without undue experimentation.
  • an immune effector cell comprises a cell surface receptor for an immunoglobulin or other peptide binding molecule, such as a receptor for an immunoglobulin constant region and including the class of receptors commonly referred to as "Fc receptors" (“FcR"s).
  • Fc receptors Fc receptors
  • a number of FcRs have been structurally and/or functionally characterized and are known in the art, including FcR having specific abilities to interact with a restricted subset of immunoglobulin heavy chain isotypes, or that interact with Fc domains with varying affinities, and/or which may be expressed on restricted subsets of immune effector cells under certain conditions (e.g., Kijimoto-Ochichai etal., Cell Mol. Life. Sci.
  • Immunoeffector cells that are capable of mediating ADCC are examples of immune effector cells.
  • Other immune effector cells include Natural Killer cells, tumor-infiltrating T lymphocytes (TILs), cytotoxic T lymphocytes, and granulocytic cells such as cells that comprise allergic response mechanisms.
  • TILs tumor-infiltrating T lymphocytes
  • cytotoxic T lymphocytes cytotoxic T lymphocytes
  • granulocytic cells such as cells that comprise allergic response mechanisms.
  • Immune effector cells thus include, but are not limited to, cells of hematopoietic origin including cells at various stages of differentiation within myeloid and lymphoid lineages and which may (but need not) express one or more types of functional cell surface FcR, such as T lymphocytes, B lymphocytes, NK cells, monocytes, macrophages, dendritic cells, neutrophils, basophils, eosinophils, mast cells, platelets, erythrocytes, and precursors, progenitors (e.g., hematopoietic stem cells), as well as quiescent, activated, and mature forms of such cells.
  • FcR functional cell surface FcR
  • immune effector cells may include cells of non-hematopoietic origin that are capable of mediating immune functions, for example, endothelial cells, keratinocytes, fibroblasts, osteoclasts, epithelial cells, and other cells.
  • Immune effector cells can also include cells that mediate cytotoxic or cytostatic events, or endocytic, phagocytic, or pinocytotic events, or that effect induction of apoptosis, or that effect microbial immunity or neutralization of microbial infection, or cells that mediate allergic, inflammatory, hypersensitivity and/or autoimmune reactions.
  • the disclosed DDpp can be fused or complexed to a second peptide domain increases the half-life or stability of the DDpp.
  • the DDpp further comprises one or more amino acids that facilitate synthesis, handling, or use of the peptide, including, but not limited to, one or two lysines at the N-terminus and/or C-terminus to increase solubility of the polypeptide.
  • Suitable fusion proteins include, but are not limited to, proteins comprising a DDpp linked to one or more polypeptides, polypeptide fragments, or amino acids not generally recognized to be part of the protein sequence.
  • a fusion peptide comprises the entire amino acid sequences of two or more peptides or, alternatively, comprises portions (fragments) of two or more peptides.
  • a peptide e.g., Protein S-binding peptide
  • a marker protein e.g., a peptide that facilitates purification
  • a peptide sequence that promotes formation of multimeric proteins e.g., a fragment of any of the foregoing.
  • Suitable fusion partners include, but are not limited to, a His tag, a FLAG tag, a strep tag, and a myc tag.
  • the DDpp is fused to one or more moieties that enhance the half-life of the polypeptide.
  • Half-life can be increased by for example, increasing the molecular weight of the DDpp to avoid renal clearance and/or incorporating a binding domain for FcRn-mediated recycling pathway.
  • the DDpp is fused to, or chemically conjugated to, an albumin polypeptide or a fragment thereof (e.g., human serum albumin (HSA)).
  • HSA human serum albumin
  • the fused or chemically conjugated albumin fragment comprises 10%, 25%, 50%, or 75% of the full length albumin protein.
  • the DDpp is fused to or complexed with an albumin binding domain or fatty acid that binds albumin when administered in vivo.
  • an albumin binding domain is "albu-tag," a moiety derived from on 4-(p-iodophenyl)-butanoic acid (Dumelin et al., Angew Chem. Int. Ed Engl. 47: 3196-3201 (2008)).
  • the DDpp is fused to, or chemically conjugated to, a transferrin polypeptide or a fragment thereof (e.g., human transferrin).
  • the fused or chemically conjugated transferrin fragment comprises 10%, 25%, 50%, or 75% of the full length transferrin protein.
  • the DDpp is fused to or complexed with a transferrin binding domain that binds transferrin when administered in vivo.
  • the DDpp is fused to, or chemically conjugated to a proline-alanine-serine multimer (PASylation; XL-Protein GmbH), a non-exact repeat peptide sequence (XTENylation, rPEG), a homopolymer of glycine residues (HAPylation), elastin-like repeat(s) sequences (ELPylation; see for example, U.S. Pat. Appl. No. 61/442,106, the contents of which is herein incorporated by reference in its entirety), an artificial GLK (GLK fusion; Huang et al., Eur. J. Pharm. Biopharm. 72: 435-41 (2010)), or a CTP peptide from human CG beta-subunit (CTP fusion).
  • PASylation proline-alanine-serine multimer
  • XTENylation XTENylation, rPEG
  • HAPylation homopolymer of glycine residues
  • the DDpp fusion protein specifically binds CD123 and/or AFP p26, and further binds a disease-related antigen.
  • the disease-related antigen can be an antigen characteristic of a cancer, and/or of a particular cell type (e.g., a hyperproliferative cell), and/or of a pathogen e.g., a bacterial cell (e.g., tuberculosis, smallpox, and anthrax), a virus (e.g., HIV), a parasite (e.g., malaria and leishmaniosis), a fungal infection, a mold, a mycoplasm, a prion antigen, or an antigen associated with a disorder of the immune system.
  • the DDpp fusion protein is conjugated to a therapeutic or cytotoxic agent.
  • a DDpp fusion protein is linked to one or more chemical moieties (e.g., labels) that facilitate detection, multimerization, binding with an interaction partner, or characterization of DDpp activity.
  • An exemplary chemical moiety is biotin.
  • Other moieties suitable for conjugation to the DDpp include, but are not limited to, a photosensitizer, a dye, a fluorescence dye, a radionuclide, a radionuclide- containing complex, an enzyme, a toxin, and a cytotoxic agent.
  • Photosensitizers include, e.g., Photofrin, Visudyne, Levulan, Foscan, Metvix, Hexvix.RTM., Cysview.TM., Laserphyrin, Antrin, Photochlor, Photosens, Photrex, Lumacan, Cevira, Visonac, BF-200 ALA, and Amphinex.
  • a His tag, a FLAG tag, a strep tag, or a myc tag is conjugated to the DDpp.
  • the DDpp fusion protein comprises a DD that binds CD123, AFP p26, or a fragment thereof, and further binds a peptide tag present on a target of interest.
  • peptide tags provide a useful means by which to detect and/or attach targets of interest containing the peptide tags.
  • the DDpp fusion protein specifically binds a peptide tag selected from the group: a hexahistidyl (His6) tag, a myc tag or a FLAG tag.
  • His6 hexahistidyl
  • myc tag a myc tag
  • FLAG tag FLAG tag
  • the DDpp fusion protein comprises a peptide epitope tag.
  • the peptide tag is selected from the group consisting of a hexahistidyl (His6) tag, a myc tag and a FLAG tag.
  • peptide tags include, but are not limited to, avitag (allows biotinylation of the tag and isolation with streptavidin), calmodulin, E-tag, hemagglutinin (HA), S-tag, SBP-tag, softag 1, streptavidin, tetra or poly-cysteine, V5, VSV, and Xpress tag.
  • covalent peptide tags include, but are not limited to, isopeptag (covalently binds pilinC protein), Spytag (covalently binds to the SpyCatcher protein), and Snooptag (covalently binds to the SnoopCatcher protein).
  • protein tags including but not limited to biotin carboxyl carrier protein (BCCP), glutathione-s- transferase, green fluorescent protein (or other fluorophore), Halo tag, Nus tag, thioredoxin, and Fc tags may optionally be used.
  • the DDpp fusion protein comprises a removable tag. Any combination of extracellular, transmembrane and intracellular domains disclosed herein may be used, depending on the embodiment.
  • DDpp fusion proteins e.g., an Adapter
  • DDpp fusion protein conjugates have uses that include but are not limited to diagnostic, analytic, manufacturing and therapeutic applications.
  • the DDpp fusion protein (e.g., an Adapter) contains at least one reactive residue.
  • Reactive residues are useful, for example, as sites for the attachment of conjugates such as chemotherapeutic drugs.
  • the reactive residue can be, for example, a cysteine, a lysine, or another reactive residue.
  • a cysteine can be added to a DDpp at either the N- or C- terminus, or within the DDpp sequence.
  • a cysteine can be substituted for another amino acid in the sequence of a DDpp.
  • a lysine can be added to a DDpp at either end or within the DDpp sequence and/or a lysine can be substituted for another amino acid in the sequence of a DDpp.
  • a reactive residue e.g., cysteine, lysine, etc.
  • a reactive residue is located in a loop sequence of a DDpp e.g., amino acid residues corresponding to residues 22-24 and 46-49 of SEQ ID NO: 2).
  • a reactive residue is located between components of a DDpp fusion, e.g., in a linker located between a DDpp and other component of a DDpp fusion protein.
  • the reactive residue can also be located within the sequence of a DDpp, or other component of the DDpp fusion protein.
  • a DDpp or a DDpp fusion protein comprises at least one, at least two, at least three reactive residues.
  • a DDpp such as a DDpp fusion protein comprises at least one, at least two, or at least three, cysteine residues.
  • binding affinity for a target refers to a property of a polypeptide which may be directly measured through the determination of the affinity constants, e.g., the amount of DDpp that associates and dissociates at a given antigen concentration.
  • affinity constants e.g., the amount of DDpp that associates and dissociates at a given antigen concentration.
  • Different methods can be used to characterize the molecular interaction, including but not limited to, competition analysis, equilibrium analysis and microcalorimetric analysis, and real-time interaction analysis based on surface plasmon resonance interaction (for example using a Biacore® instrument). These methods are known to the skilled person and are described, for example, in Neri et al., Tibtech 14: 465-470 (1996) and Jansson et al., J Biol Chem 272: 8189- 8197 (1997).
  • DDpp bind a target of interest (e.g., CD123 or AFP p26) with a dissociation constant (KD) of less than or equal to 5xl0“ 3 M, 10“ 3 M, 5xl0“ 4 M, 10" 4 M, 5xl0“ 5 M, or 10“ 5 M.
  • KD dissociation constant
  • a DDpp binds a target of interest with a KD of less than or equal to 5xl0“ 6 M, 10“ 6 M, 5xl0“ 7 M, 10“ 7 M, 5xl0“ 8 M, or 10“ 8 M.
  • a DDpp binds a target of interest with a KD of less than or equal to 5xl0“ 9 M, 10“ 9 M, 5xl0“ 10 M, IO’ 10 M, 5xl0 -11 M, 10’ 11 M, 5xl0’ 12 M, IO’ 12 M, 5xl0’ 13 M, 10’ 13 M, 5xl0’ 14 M, 10’ 14 M, 5x10-15 M, or 10“ 15 M.
  • the provided DDpp has a dissociation constant selected from the group consisting of between 10 4 M and 10 5 M, between 10 5 M and 10 6 M, between 10 6 M and 10 7 M, between 10 7 M and 10 8 M, between 10 8 M and 10 9 M, between 10 9 M and 10 10 M, between 10 10 M and 10 11 M and between 10 11 M and 10 12 M.
  • the DDpp binds a target of interest (e.g., CD123 or AFP p26) in active form. In one embodiment the DDpp reversibly binds the target of interest in active form and also releases the bound target in active form. In some embodiments, the DDpp binds a target of interest in the native form.
  • a target of interest e.g., CD123 or AFP p26
  • DDpp binds a target of interest with an off-rates or K o ff of greater than or equal to 10 -10 sec -1 , 5xl0 -9 sec -1 , 10 -9 sec -1 , 5xl0 -8 sec -1 , 10 -8 sec -1 , 5xl0 -7 sec -1 , 10 -7 sec , 5xl0 -6 sec -1 , 10 -6 sec -1 , 5xl0 -5 sec -1 , 10 -5 sec -1 , 5xl0 -4 sec -1 , 10 -4 sec -1 , 5xl0 -3 sec -1 , 10 -3 sec -1 , 5xl0 -2 sec -1 , 10 -2 sec -1 , 5xl0 -1 sec -1 , or 10 -1 sec -1 .
  • Binding experiments to determine KD and off-rates can routinely be performed in a number of conditions including, but not limited to, [pH 6.0, 0.01% Tween 2, [pH 6.0, 0.1% gelatin], [p H5.0, 0.01% Tween 2, [pH 9.0, 0.1% Tween 2, [pH 6.0, 15% ethylene glycol, 0.01% Tween 2, [pH 5.0, 15% ethylene glycol, 0.01% Tween 2, and [pH 9.0, 15% ethylene glycol, 0.01% Tween 2.
  • the buffers in which to make these solutions can routinely be determined by one skilled in the art, and depend largely on the desired pH of the final solution.
  • Low pH solutions can be made, for example, in citrate buffer, glycine-HCl buffer, or in succinic acid buffer.
  • High pH solutions can be made, for example, in Tris-HCl, phosphate buffers, or sodium bicarbonate buffers.
  • a number of conditions may routinely be used by those skilled in the art to determine KD and off-rates for the purpose of determining, for example, optimal pH and/or salt concentrations.
  • the DDpp specifically binds a target of interest (e.g., CD 123 or AFP p26) with a Koff ranging from 0.1 to 10 7 sec 1 , 10 2 to 10 7 sec or 0.5 X 10 2 to 10 7 sec
  • a target of interest e.g., CD 123 or AFP p26
  • the DDpp e.g., a DDpp fusion protein
  • a DDpp binds a target of interest with an off rate (Koff) of less than 5 X 10 4 sec 1 , 10 4 sec 1 , 5 X 10 5 sec 4 , or 10 5 sec 4 , 5 X10 6 sec 4 , 10 6 sec 4 , 5 X 10 7 sec 1 , or 10 7 sec 1 .
  • Koff off rate
  • the DDpp specifically binds a target of interest (e.g., CD123 or AFP p26) with a Kon ranging from 10 3 to 10 7 M 'sec , 10 3 to 10 6 M 'sec , or 10 3 to 10 5 M 'sec .
  • the DDpp e.g., a DDpp fusion protein
  • the target of interest with an on rate (Ko n ) of greater than 10 3 M 'sec ', 5 X 10 3 M 'sec , 10 4 M 'sec 4 , or 5 X 10 4 M 'sec 4 .
  • the DDpp binds the target of interest with a Kon of greater than 10 5 M 'sec 4 , 5 X 10 5 M 'sec 4 , 10 6 M 1 sec 1 , or 5 X 10 6 M 'sec 4 , or 10 7 M 1 sec
  • Nucleic acid molecules encoding the disclosed DDpp are encompassed herein, as are vectors containing these nucleic acids, host cells containing these nucleic acids vectors, and methods of making the DDpp-albumin fusion proteins and using these nucleic acids, vectors, and/or host cells.
  • the invention also encompasses pharmaceutical formulations comprising a DDpp-albumin fusion protein and a pharmaceutically acceptable diluent or carrier. Such formulations can be used in methods of treating, preventing, ameliorating or diagnosing a disease or disease symptom in a patient, preferably a mammal, most preferably a human, comprising the step of administering the pharmaceutical formulation to the patient.
  • a DDpp fusion protein may be linked to other organic or inorganic molecules or substrates through the use of chemically conjugation.
  • DDpp-drug conjugates are intended to facilitate the local delivery of cytotoxic agents through the targeting specificity of the DDpp.
  • This combination of targeting specificity and cytotoxic agent allows targeted delivery of the drug to tumors, and intracellular accumulation therein, where systemic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells as well as the tumor cells sought to be eliminated (Baldwin et al., Lancet pages 603-605 (1986); Thorpe, "Antibody Carriers Of Cytotoxic agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al., (ed.s), pp. 475-506 (1985)).
  • Cytotoxic agents include chemotherapeutic agents, growth inhibitory agents, toxins (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), radioactive isotopes (i.e., a radioconjugate), etc.
  • Chemotherapeutic agents useful in the generation of such immunoconjugates include, for example, methotrexate, adriamicin, doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents.
  • Chemotherapeutic agents useful in the generation of such immunoconjugates also include antitubulin drugs, such as auristatins, including monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).
  • auristatins including monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).
  • Enzymatically active toxins and fragments thereof that can be used according to the disclosed methods include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phen
  • a DDpp e.g., a DDpp fusion protein
  • a radioisotope e.g., a DDpp fusion protein
  • a DDpp is conjugated to an isotope selected from 90 Y, 125 1, 131 1, 123 I, ni In, 105 Rh, 153 Sm, 67 Cu, 67 Ga, 166 HO, 177 LU, 186 Re and 188 Re using anyone of a number of known chelators or direct labeling.
  • the DDpp is coupled to drugs, prodrugs or lymphokines such as interferon.
  • Conjugates of the DDpp and cytotoxin can routinely be made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyidithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazo-niumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6- diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene).
  • SPDP N-s
  • the toxin is conjugated to a DDpp fusion protein through an enzyme-cleavable linker system (e.g., such as that present in SGN-35).
  • an enzyme-cleavable linker system e.g., such as that present in SGN-35.
  • Conjugates of a DDpp and one or more small molecule toxins, such as a calicheamicin, maytansinoids, a trichothene, and CC1065 can also be used.
  • the cytotoxic agent is covalently attached to a DDpp by a linker.
  • the linker attaching the DDpp and the cytotoxic agent is cleavable by a protease.
  • Affinity maturation strategies can be used to generate high affinity DD and DDpp that can be used in the DDpp fusion proteins described herein.
  • An improved DD and DDpp that specifically binds a desired target e.g., BCAM, CD123, CS1, HER2, AFP, and AFP p26
  • a desired target e.g., BCAM, CD123, CS1, HER2, AFP, and AFP p26
  • At least one, two, three, four, five, or more amino acid mutations e.g., conservative or non-conservative substitutions
  • deletions or insertions can be introduced into a DD sequence disclosed in Table 1 (i.e., a reference sequence) and the resulting DDpp can be screened for binding to the respective target and biological activity, such as the ability to antagonize the biological activity of the respective target or agonize the biological activity of the respective target.
  • the discosed DDpp are preferably not antigenic with respect to the subject (e.g., human).
  • the sequence of the DDpp does not contain a human HFA-DR binding motif or cleavage sites for proteasomes and immune -proteasomes.
  • the DDpp sequence does not contain an antigenic sequence as determined by a computer prediction model version existent on the filing date of this specification.
  • the DDpp sequence does not contain an MHC (class I or class II) binding site sequence as predicted by an algorithm selected from ProPred (see, e.g., Singh, Bioinformatics 17(12): 1236-1237 (2001)), ProPredl (Singh, Bioinformatics 19(8): 1009-14 (2003)), SYFPEITHI (see, e.g., Schuler, Immunoinf. Meth, in Mol. Biol. 409(1): 75-93 (2007)), SMM-align (see, e.g., Nielsen, BMC Bioinformatics 8: 238 (2007)), RANKPEP (see, e.g., Reche, Hum Immunol 63: 701-709.
  • ProPred see, e.g., Singh, Bioinformatics 17(12): 1236-1237 (2001)
  • ProPredl Ster, Bioinformatics 19(8): 1009-14 (2003)
  • SYFPEITHI see, e.g., Schul
  • the DDpp does not contain a sequence that shares characteristics with a high affinity (binding threshold less than 6%) T cell epitope. (Singh, Bioinformatics 17: 1236-1237 (2001)). In some embodiments, the DDpp does not contain a sequence that shares characteristics with a promiscuous (present in greater than 50% of relevant alleles) T cell epitope (Singh, Bioinformatics 17: 1236-1237 (2001)).
  • the DDpp does not contain a sequence that shares characteristics with a high affinity or a promiscuous T cell epitope.
  • the DDpp does not contain the sequence LAAIKTRLQ (SEQ ID NO: 49).
  • Polynucleotides comprising a nucleotide sequence encoding a DDpp (e.g., an Adapter or CAR) are also provided. Such polynucleotides optionally further comprise, one or more expression control elements.
  • the polynucleotide can comprise one or more promoters or transcriptional enhancers, ribosomal binding sites, transcription termination signals, and polyadenylation signals, as expression control elements.
  • the polynucleotide can be inserted within any suitable vector, which can be contained within any suitable host cell for expression.
  • a polynucleotide disclosed herein encodes a polypeptide comprising a D domain that binds to CD123.
  • the D domain that binds to CD123 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100.
  • the D domain that binds to CD 123 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 13, 14, 31, 32, and 33.
  • the D domain that binds to CD123 comprises the amino acid sequence of SEQ ID NO: 14.
  • a polynucleotide disclosed herein encodes a chimeric antigen receptor (CAR), wherein the CAR comprises a D domain that binds to CD 123, (ii) a transmembrane domain, and (iii) an intracellular domain.
  • the D domain that binds to CD123 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100.
  • the D domain that binds to CD 123 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 13, 14, 31, 32, and 33.
  • the D domain that binds to CD123 comprises the amino acid sequence of SEQ ID NO: 14.
  • the CAR comprises the amino acid sequence of SEQ ID NO: 62-66 or 67.
  • the CAR comprises the amino acid sequence of SEQ ID NO: 67.
  • a polynucleotide disclosed herein encodes a chimeric antigen receptor (CAR), wherein the CAR comprises (i) a D domain that binds to AFP p26 AD, (ii) a transmembrane domain, and (iii) an intracellular domain.
  • the DD that binds to an AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94.
  • the DD that binds to an AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 70-73 or 92-94.
  • the DD that binds to an AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 73.
  • the CAR comprises the amino acid sequence of SEQ ID NO: 68.
  • the CAR comprises the amino acid sequence of SEQ ID NO: 69.
  • a polynucleotide disclosed herein encodes a chimeric antigen receptor (CAR), wherein the CAR comprises (i) an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain.
  • the AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 37-43 and 44.
  • the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 37.
  • the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 39.
  • a polynucleotide disclosed herein encodes an Adapter comprising (a) a D domain (DD) that binds to CD123 and (b) an antigenic determinant (AD).
  • the D domain that binds to CD123 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8- 33, 99 and 100.
  • 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.
  • the D domain that binds to CD123 comprises the amino acid sequence of SEQ ID NO: 14.
  • the Adapter is a monovalent Adapter comprising a single D domain that binds CD123. In some embodiments, the Adapter is a bivalent Adapter comprising two D domains that bind CD123. In some embodiments, the two D domains that bind CD123 are the same. In some embodiments, the two D domains that bind CD123 are different. In some embodiments, the Adapter is a bivalent Adapter comprising a first D domain that binds CD123 and a second D domain that binds a second AD. In some embodiments, the second AD is CD33 or LeY.
  • a monovalent Adapter comprises a D domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 13, 14, 31, 32, and 33. In some embodiments, the monovalent Adapter comprises the D domain comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, a bivalent Adapter comprises a DD comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 13, 14, 31, 32, and 33. In some embodiments, the bivalent Adapter comprises the D domain comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, a bivalent Adapter comprises two identical D domains comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 13, 14, 31, 32, and 33.
  • the bivalent Adapter comprises two identical D domains comprising the amino acid sequence of SEQ ID NO: 14.
  • the Adapter comprises an AFP p26 antigenic determinant (AD).
  • the AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 37-43 and 44.
  • the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 37.
  • the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 39.
  • the monovalent Adapter comprises the amino acid sequence of SEQ ID NO: 50-54 or 55.
  • the monovalent Adapter comprises the amino acid sequence of SEQ ID NO: 50.
  • the bivalent Adapter comprises the amino acid sequence of SEQ ID NO: 56-60 or 61.
  • the monovalent Adapter comprises the amino acid sequence of SEQ ID NO: 61.
  • the Adapter comprises one or more linkers.
  • a polynucleotide disclosed herein encodes an Adapter comprising (a) a D domain that binds to CD 123 and (b) an antigenic determinant binding domain (ADBD) that binds an AFP p26 AD.
  • the AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 37-43 and 44.
  • the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 37.
  • the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 39.
  • the D domain that binds to CD123 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 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 NO: 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 an AFP p26 AD comprises a D domain that binds to the AFP p26 AD. In some embodiments, the D domain that binds to an AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 70-73 and 92-94. In some embodiments, the D domain that binds to an AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, the Adapter comprises one or more linkers.
  • a polynucleotide disclosed herein encodes an Adapter comprising (a) a first antigenic determinant binding domain (ADBD) that binds to a target antigenic determinant (AD) on a target cell and (b) an antigenic determinant binding domain (ADBD) that binds to an AFP p26 AD.
  • the AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 37-43 and 44.
  • the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 37.
  • the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 39.
  • the ADBD that binds to an AFP p26 AD comprises a D domain that binds to AFP p26.
  • the D domain that binds to an AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 70-73 and 92-94.
  • the D domain that binds to an AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 73.
  • the Adapter comprises one or more linkers.
  • the polynucleotide is DNA.
  • the polynucleotide is RNA. In some embodiments, the polynucleotide is mRNA. In some embodiment the RNA, e.g., mRNA comprises modified ribonucleotides. [0369] In some embodiments, an mRNA disclosed herein comprises a coding region encoding a polypeptide disclosed herein, and additionally comprises one or more of a 5' untranslated region, 3' untranslated region, 5' cap, and polyadenylation signal.
  • an mRNA disclosed herein comprises a coding region encoding a polypeptide disclosed herein, a 5' untranslated region, a 3' untranslated region, a 5' cap, and a polyadenylation signal.
  • an mRNA disclosed herein comprises modified ribonucleotides.
  • the mRNA comprises N1 -methylpseudouridine or N1 -ethylpseudouridine.
  • the 5' terminal cap is 7mG(5')ppp(5')NlmpNp. See, e.g., US20200261572, US20190351040, and US20190211065, each of which is incorporated herein by reference in its entirety.
  • the polynucleotide is a vector comprising a polynucleotide described herein.
  • the vector is a transfer vector suitable for use in the production of a recombinant lenti virus.
  • the vector is a lentiviral vector encoding a polypeptide disclosed herein (e.g., a CAR).
  • the lentiviral vector is suitable for transducing an immune cell (e.g., a T cell or NK cell) to produce a cell expressing the polypeptide (e.g., CAR).
  • the polynucleotide is a recombinant virus comprising a polynucleotide described herein.
  • the polynucleotide is a recombinant lentivirus comprising a polynucleotide encoding a polypeptide (e.g., a CAR) described herein.
  • the polynucleotide is a recombinant adenovirus vectors or adeno-associated virus (AAV).
  • the disclosed DDpp can routinely be made using commercially available reagents and techniques known in the art.
  • the DDpp are synthesized by solid phase synthesis techniques known in the art, such as, Merrifield, J. Am. Chem. Soc. 85: 2149 (1963); Davis et al., Biochem. Inti. 10: 394-414 (1985); Larsen et al., J. Am. Chem. Soc. 115: 6247 (1993); Smith et al., J. Peptide Protein Res. 44: 183 (1994); O'Donnell et al., J. Am. Chem. Soc.
  • the disclosure contemplates synthetic peptides.
  • the peptide is expressed recombinantly by introducing a nucleic acid encoding the disclosed DDpp into host cells, which are cultured to express the peptide. Such peptides are purified from the culture media or cell pellets.
  • the production of the DDpp useful in practicing the provided methods, may be carried out using a variety of standard techniques for chemical synthesis, semi-synthetic methods, and recombinant DNA methodologies known in the art. Also provided is a method for producing a DDpp, individually or as part of multi-domain fusion protein, as soluble agents and cell associated proteins.
  • the reference sequence and/or the modified polypeptides e.g., DDpp can be deimmunized. For example, residues or motifs that are potentially immunogenic can be identified and modified in order to reduce or eliminate potential immune responses to the DDpp. Additional details regarding various embodiments, of the production, selection, and isolation of DDpp are provided in more detail below.
  • a DDpp such as a DDpp fusion protein (e.g., an Adapter) is "recombinantly produced,” (i.e., produced using recombinant DNA technology).
  • exemplary recombinant methods available for synthesizing DDpp fusion proteins include, but are not limited to polymerase chain reaction (PCR) based synthesis, concatemerization, seamless cloning, and recursive directional ligation (RDL) (see, e.g., Meyer et al., Biomacromolecules 3: 357-367 (2002); Kurihara et al., Biotechnol. Lett.
  • PCR polymerase chain reaction
  • RDL recursive directional ligation
  • Nucleic acids comprising a polynucleotide sequence encoding a DDpp (e.g., an Adapter or CAR) are also provided.
  • Such polynucleotides optionally further comprise, one or more expression control elements.
  • the polynucleotide can comprise one or more promoters or transcriptional enhancers, ribosomal binding sites, transcription termination signals, and polyadenylation signals, as expression control elements.
  • the polynucleotide can be inserted within any suitable vector, which can be contained within any suitable host cell for expression.
  • nucleic acids encoding DDpp is typically achieved by operably linking a nucleic acid encoding the DDpp to a promoter in an expression vector.
  • Typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.
  • Methods known in the art can be used to routinely construct expression vectors containing the nucleic acid sequence encoding a DDpp along with appropriate transcriptional/ translational control signals. These methods include, but are not limited to in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination.
  • a nucleic acid sequence encoding a DDpp is operably linked to a suitable promoter sequence such that the nucleic acid sequence is transcribed and/or translated into DDpp in a host.
  • a vector comprising a DDpp e.g., an Adapter
  • the vector can remain episomal or become chromosomally integrated, as long as the insert encoding therapeutic agent can be transcribed.
  • Vectors can be constructed by standard recombinant DNA technology. Vectors can be plasmids, phages, cosmids, phagemids, viruses, or any other types known in the art, which are used for replication and expression in prokaryotic or eukaryotic cells.
  • a wide variety of components known in the art can be included in such vectors, including a wide variety of transcription signals, such as promoters and other sequences that regulate the binding of RNA polymerase onto the promoter.
  • Any promoter known or demonstrated to be effective in the cells in which the vector will be expressed can be used to initiate expression of the DDpp.
  • Suitable promoters can be inducible e.g., regulated) or constitutive.
  • Nonlimiting examples of suitable promoters include the SV40 early promoter region, the promoter contained in the 3' long terminal repeat of Rous sarcoma virus, the HSV-1 (herpes simplex virus-1) thymidine kinase promoter, the regulatory sequences of the metallothionein gene, etc., as well as the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells; insulin gene control region which is active in pancreatic beta cells, mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells, albumin gene control region which is active in liver, alpha-fetoprotein gene control region which is active in liver, alpha 1 -antitrypsin gene control region which is active in the liver, beta-globin gene control region which is active in erythroid cells, myelin basic protein gene control region which is active in oligodendrocyte
  • one or several nucleic acids encoding a DDpp is expressed under the control of a constitutive promoter or, alternately, a regulated expression system.
  • Suitable regulated expression systems include, but are not limited to, a tetracycline-regulated expression system, an ecdysone inducible expression system, a lac-switch expression system, a glucocorticoid-inducible expression system, a temperature-inducible promoter system, and a metallothionein metal-inducible expression system.
  • nucleic acids encoding a DDpp may be expressed under the control of a constitutive promoter, while others may be expressed under the control of a regulated promoter. Expression levels may be determined by methods known in the art, including Western blot analysis and Northern blot analysis.
  • a variety of host-expression vector systems can be utilized to express a nucleic acid encoding a DDpp (e.g., an Adapter).
  • Vectors containing the nucleic acids encoding the DDpp include plasmid vectors, a single and double-stranded phage vectors, as well as single and double-stranded RNA or DNA viral vectors.
  • Phage and viral vectors may also be introduced into host cells in the form of packaged or encapsulated virus using known techniques for infection and transduction.
  • viral vectors may be replication competent or alternatively, replication defective.
  • cell-free translation systems may also be used to produce the protein using RNAs derived from the DNA expression constructs (see, e.g., W086/05807 and W089/01036; and U.S. Pat. No. 5,122,464; the contents of each of which is herein incorporated by reference in its entirety).
  • any type of cells or cultured cell line can be used to express a DDpp (e.g., an Adapter) provided herein.
  • the background cell line used to generate an engineered host cells is a phage, a bacterial cell, a yeast cell or a mammalian cell.
  • a variety of host-expression vector systems may be used to express the coding sequence a DDpp fusion protein.
  • Mammalian cells can be used as host cell systems transfected with recombinant plasmid DNA or cosmid DNA expression vectors containing the coding sequence of the target of interest and the coding sequence of the fusion polypeptide.
  • the cells can be primary isolates from organisms (including human), cultures, or cell lines of transformed or transgenic nature.
  • the host cell is a human cell.
  • the host cell is human T cell.
  • the host cell is derived from a human patient.
  • Useful host cells include but are not limited to microorganisms such as, bacteria (e.g., E. coli, B. subtilis, P. fluorescens) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing DDpp (e.g., an Adapter) coding sequences; yeast (e.g., Saccharomyces, Pichid) transformed with recombinant yeast expression vectors containing DDpp coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculo virus) containing DDpp coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing DDpp coding sequences.
  • bacteria e.g., E. coli, B
  • the mammalian cell systems are used to produce the DDpp.
  • Mammalian cell systems typically utilize recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
  • promoters derived from the genome of mammalian cells
  • mammalian viruses e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter.
  • Prokaryotes useful as host cells in producing a DDpp such as DDpp fusion protein (e.g., an Adapter), include gram negative or gram positive organisms such as, E. coli and B. subtilis.
  • Expression vectors for use in prokaryotic host cells generally contain one or more phenotypic selectable marker genes (e.g., genes encoding proteins that confer antibiotic resistance or that supply an autotrophic requirement).
  • prokaryotic host expression vectors examples include the pKK223-3 (Pharmacia, Uppsala, Sweden), pGEMl (Promega, Wis., USA), pET (Novagen, Wis., USA) and pRSET (Invitrogen, Calif., USA) series of vectors (see, e.g., Studier, J. Mol. Biol. 219: 37 (1991) and Schoepfer, Gene 124: 83 (1993)).
  • Exemplary promoter sequences frequently used in prokaryotic host cell expression vectors include T7, (Rosenberg et al., Gene 56: 125-135 (1987)), beta-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275: 615 (1978)); and Goeddel et al., Nature 281: 544 (1979)), tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8: 4057 (1980)), and tac promoter (Sambrook et al. , 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
  • a eukaryotic host cell systems including yeast cells transformed with recombinant yeast expression vectors containing the coding sequence of a DDpp (e.g., an Adapter), such as, the expression systems taught in U.S. Appl. No. 60/344,169 and W003/056914 (methods for producing humanlike glycoprotein in a non-human eukaryotic host cell) (the contents of each of which is herein incorporated by reference in its entirety).
  • Exemplary yeast that can be used to produce the provided compositions, such as, DD include yeast from the genus Saccharomyces, Pichia, Actinomycetes and Kluyveromyces.
  • Yeast vectors typically contain an origin of replication sequence from a 2mu yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene.
  • ARS autonomously replicating sequence
  • promoter sequences in yeast expression constructs include, promoters from metallothionein, 3 -phosphoglycerate kinase (Hitzeman, J. Biol. Chem.
  • glycolytic enzymes such as, enolase, glyceraldehyde-3 -phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phospho glycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • Additional suitable vectors and promoters for use in yeast expression as well as yeast transformation protocols are known in the art. See, e.g., Fleer, Gene 107: 285-195 (1991) and Hinnen, PNAS 75: 1929 (1978).
  • Insect and plant host cell culture systems are also useful for producing the compositions encompassed by the disclosure.
  • host cell systems include for example, insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the coding sequence of a DD; plant cell systems infected with recombinant virus expression vectors e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the coding sequence of a DD, including, but not limited to, the expression systems taught in U.S. Pat. No. 6,815,184; U.S. Publ. Nos. 60/365,769, and 60/368,047; and W004/057002, WO04/024927, and W003/078614; the contents of each of which is herein incorporated by reference in its entirety.
  • the host cell systems may be used, including animal cell systems infected with recombinant virus expression vectors (e.g., adenoviruses, retroviruses, adeno-associated viruses, herpes viruses, lentiviruses) including cell lines engineered to contain multiple copies of the DNA encoding a DDpp either stably amplified (CHO/dhfr) or unstably amplified in double-minute chromosomes (e.g., murine cell lines).
  • the vector comprising the polynucleotide(s) encoding the DDpp is polycistronic.
  • Exemplary mammalian cells useful for producing these compositions include 293 cells (e.g., 293T and 293F), CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 (Crucell, Netherlands) cells VERY, Hela cells, COS cells, MDCK cells, 3T3 cells, W138 cells, BT483 cells, Hs578T cells, HTB2 cells, BT20 cells, T47D cells, CRL7O30 cells, HsS78Bst cells, hybridoma cells, and other mammalian cells.
  • 293 cells e.g., 293T and 293F
  • CHO cells e.g., 293T and 293F
  • BHK cells e.g., NS0 cells, SP2/0 cells
  • YO myeloma cells e.g., P3X63 mouse myel
  • Additional exemplary mammalian host cells that are useful in practicing the the provided embodiments include but are not limited, to T cells.
  • Some examples of expression systems and selection methods are described in the following references and references cited therein: Borth et al., Biotechnol. Bioen. 71(4): 266-73 (2000), in Werner et al., Arzneistoff-aba/Drug Res. 48(8): 870-80 (1998), Andersen etal., Curr. Op. Biotechnol. 13: 117-123 (2002), Chadd et al., Curr. Op. Biotechnol. 12: 188- 194 (2001), and Giddings, Curr. Op. Biotechnol. 12: 450-454 (2001).
  • Transcriptional and translational control sequences for mammalian host cell expression vectors are frequently derived from viral genomes.
  • Commonly used promoter sequences and enhancer sequences in mammalian expression vectors include, sequences derived from Polyoma virus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus (CMV).
  • Exemplary commercially available expression vectors for use in mammalian host cells include pCEP4 (Invitrogen) and pcDNA3 (Invitrogen).
  • a nucleic acid into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like.
  • Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).
  • Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
  • Viral vectors, and especially retroviral vectors have become the most widely used method for inserting genes into mammalian (e.g., human) cells.
  • Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat, Nos. 5,350,674 and 5,585,362, the contents of each of which is herein incorporated by reference in its entirety.
  • Methods for introducing a DNA and RNA polynucleotides of interest into a host cell include electroporation of cells, in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing chemicals, drugs, or polynucleotides to be introduced into the cell.
  • DDpp containing DNA or RNA constructs may be introduced into mammalian or prokaryotic cells using electroporation.
  • Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oILin-water emulsions, micelles, mixed micelles, and liposomes.
  • An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).
  • an exemplary delivery vehicle is a liposome.
  • the use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo).
  • the nucleic acid can be associated with a lipid.
  • the nucleic acid associated with a lipid can be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they can be present in a bilayer structure, as micelles, or with a "collapsed" structure. They can also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape.
  • Lipids are fatty substances which can be naturally occurring or synthetic lipids.
  • lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • Lipids suitable for use can be obtained from commercial sources.
  • DMPC dimyristoyl phosphatidylcholine
  • DCP dicetyl phosphate
  • Choi cholesterol
  • DMPG dimyristoyl phosphatidylglycerol
  • Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20°C.
  • Liposome is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al.
  • compositions that have different structures in solution than the normal vesicular structure are also encompassed.
  • the lipids can assume a micellar structure or merely exist as non-uniform aggregates of lipid molecules.
  • lipofectamine-nucleic acid complexes are also contemplated.
  • assays include, for example, "molecular biological” assays known in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the provided embodiments.
  • molecular biological assays known in the art, such as Southern and Northern blotting, RT-PCR and PCR
  • biochemical assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the provided embodiments.
  • Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient organism, tissue, or cell and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
  • reporter genes can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., FEBS Lett. 479: 79-82 (2000)).
  • Suitable expression systems are known in the art and can be prepared using known techniques or obtained commercially.
  • the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter.
  • Such promoter regions can routinely be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.
  • a number of selection systems can be used in mammalian host-vector expression systems, including, but not limited to, the herpes simplex virus thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase (Lowy et al., Cell 22: 817 (1980)) genes, which can be employed in tk , hgprt or aprt cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for e.g., dhfr, gpt, neo, hygro, trpB, hisD, ODC (ornithine decarboxylase), and the glutamine synthase system.
  • a DDpp e.g., an Adapter
  • it can be purified by any method known in the art for purification of a recombinant protein, for example, by chromatography e.g., ion exchange, hydrophobic interaction, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • the DDpp is optionally fused to a heterologous polypeptide sequences (e.g., FLAg tag or HIS tag) described herein or otherwise known in the art to facilitate purification. More particularly, it is envisioned that ligands (e.g., antibodies and other affinity matrices) for for affinity purification are used and that optionally, the component of the DDpp fusion composition (e.g., an Adapter) that are bound by these ligands (e.g., FLAg tag or HIS tag) are removed from the composition prior to final preparation of the DDpp using techniques known in the art.
  • ligands e.g., antibodies and other affinity matrices
  • the component of the DDpp fusion composition e.g., an Adapter
  • these ligands e.g., FLAg tag or HIS tag
  • CARs e.g., CARs comprising an extracellular D domain disclosed herein or an extracellular p26
  • CARs are intentionally cell associated and used in the context of the cell in which they are expressed.
  • One particular embodiment relates to a strategy of adoptive cell transfer of T cells which have been transduced to express a CAR.
  • the cell can be genetically modified to stably express a CAR on its surface, conferring novel target specificity that is MHC independent.
  • viruses can be used in applications in which viruses are used for transfection and integration into a mammalian cell genome.
  • Viruses which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • Lentiviral vectors are particularly suitable to achieving long-term gene transfer (e.g., adoptive T cell immune therapy) since they allow long-term, stable integration of a transgene and its propagation in daughter cells.
  • Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., Inti. Appl. Publ. Nos. WO 01/96584 and WO 01/29058; and U.S. Pat. No. 6,326,193).
  • Several vector promoter sequences are available for expression of the transgenes.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • CMV immediate early cytomegalovirus
  • This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
  • Another example of a suitable promoter is EF- la.
  • constitutive promoter sequences can also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter.
  • Inducible promoters include, but are not limited to a metallothionein promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
  • the expression vector introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
  • the selectable marker may be carried on a separate piece of DNA and used in a co- transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells.
  • Useful selectable markers include, for example, antibiotic -resistance genes, such as neo and are otherwise known in the art.
  • T cells Prior to expansion and genetic modification of the T cells of the invention, a source of T cells is obtained from a subject.
  • T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • any number of T cell lines available in the art may be used.
  • a host cell comprising nucleic acids encoding a CAR described herein.
  • a composition comprising a nucleic acid sequence encoding the CAR.
  • electroporation of cells results in the expression of a DDpp-CAR on the surface of T cells, NK cells, and/or NKT cells. Such expression may be transient or stable over the life of the cell. Electroporation may be accomplished with methods known in the art including MaxCyte GT® and STX® Transfection Systems (MaxCyte, Gaithersburg, MD, USA).
  • the expression of a CAR disclosed herein results in cell associated compositions.
  • the CAR comprises a target binding domain comprising a DD disclosed herein (e.g., a DD comprising the amino acid sequence of SEQ ID NO: 8-33 or 74-94).
  • the DD binds CD123 and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100.
  • the CD123-specific DD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 13, 14, 31, 32, and 33.
  • the CD123-specific DD comprises the amino acid sequence of SEQ ID NO: 14.
  • the DD binds AFP p26 and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94. In some embodiments, the DD binds AFP p26 and comprises the amino acid sequence of SEQ ID NO: 70-73 or 92-94. In some embodiments, the DD binds AFP p26 and comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, the CAR comprises an extracellular AD comprising p26. In some embodiments, the extracellular AD comprises the amno acid sequence of SEQ ID NO 37-43 or 44.
  • DDpp e.g., an Adapter
  • production may also be carried out using organic chemical synthesis of the desired polypeptide using a variety of liquid and solid phase chemical processes known in the art.
  • Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Tam et al., J. Am. Chem. Soc. 105: 6442 (1983); Merrifield, Science 232: 341-347 (1986); Barany and Merrifield, The Peptides, Gross and Meienhofer, eds, Academic Press, New York, 1- 284; Barany et al., Int. J. Pep. Protein Res.
  • the DDpp (e.g., an Adapter) that are used in the methods ecompassed herein may be modified during or after synthesis or translation, e.g., by glycosylation, acetylation, benzylation, phosphorylation, amidation, pegylation, formylation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, ubiquitination, etc.
  • the peptides are acetylated at the N-terminus and/or amidated at the C-terminus.
  • the disclosure also provides DDpp (e.g., an Adapter) derivatives and include polypeptides that have been chemically modified in some manner distinct from addition, deletion, or substitution of amino acids.
  • a DDpp is chemically bonded with polymers, lipids, other organic moieties, and/or inorganic moieties. Examplary polypeptide modifications are provided in Hermanson, Bioconjugate Techniques, Academic Press, (1996).
  • the DDpp optionally comprise a functional group that facilitates conjugation to another moiety e.g., a peptide moiety).
  • Exemplary functional groups include, but are not limited to, isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride, aldehyde, epoxide, oxirane, carbonate, arylating agent, imidoester, carbodiimide, anhydride, alkyl halide derivatives (e.g., haloacetyl derivatives), maleimide, aziridine, acryloyl derivatives, arylating agents, thiol-disulfide exchange reagents (e.g., pyridyl disulfides or TNB thiol), diazoalkane, carboyldiimadazole, N,N'-Disuccinyl carbonate, N- Hydroxysuccinimidyl chloroformate, and hydrazine derivatives.
  • Maleimide is useful, for example, for generating a DDpp that binds albumin in viv
  • the DDpp e.g., an Adapter
  • the DDpp is covalently modified to include one or more water soluble polymer attachments.
  • the water soluble polymer (or other chemical moiety) is attached to any amino acid residue, although attachment to the N- or C-terminus is preferred in some embodiments.
  • Useful polymers include, but are not limited to, PEG (e.g., PEG approximately 40 kD, 30 kD, 20 kD, 10 kD, 5 kD, or 1 kD in size), polyoxyethylene glycol, polypropylene glycol, monomethoxy-polyethylene glycol, dextran, hydroxyethyl starch, cellulose, poly-(N-vinyl pyrrolidone) -polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polysialic acid (PSA), polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures of any of the foregoing.
  • PEG e.g., PEG approximately 40 kD, 30 kD, 20 kD, 10 kD, 5 kD, or 1 kD in size
  • polyoxyethylene glycol polypropylene
  • the DDpp is PEGylated.
  • PEG moieties are available in different shapes, e.g., linear or branched.
  • water soluble polymer attachments see U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; and 4,179,337.
  • moieties useful for improving peptide half-life or stability include, for instance, albumin (optionally modified to allow conjugation to the DDpp), fatty acid chains (e.g., C12-C18 fatty acid, such as a C14 fatty acid, or dicarboxylic acids, such as octadecane dicarboxylic acid (oddc)), an antibody or fragment thereof e.g., an Fc portion of an antibody), and proline- alanine-serine multimers.
  • albumin optionally modified to allow conjugation to the DDpp
  • fatty acid chains e.g., C12-C18 fatty acid, such as a C14 fatty acid, or dicarboxylic acids, such as octadecane dicarboxylic acid (oddc)
  • oddc octadecane dicarboxylic acid
  • an antibody or fragment thereof e.g., an Fc portion of an antibody
  • the DDpp (e.g., an Adapter) is conjugated to a polyethylene glycol (PEG) moiety, human serum albumin (HSA), an antibody or antibody fragment, hydroxy ethyl starch, a proline- alanine-serine multimer (PASylation), a C12-C18 fatty acid, or polysialic acid.
  • PEG polyethylene glycol
  • HSA human serum albumin
  • PASylation proline- alanine-serine multimer
  • C12-C18 fatty acid or polysialic acid
  • the DDpp (e.g., an Adapter) are acylated at the N-terminal amino acid of the peptide.
  • the DDpp are amidated at the C-terminal amino acid of the polypeptide.
  • the peptides are acylated at the N-terminal amino acid of the peptide and are amidated at the C- terminal amino acid of the peptide.
  • the DDpp (e.g., an Adapter) comprises one or more modified or non- proteinogenic amino acids or a modified linker group (see, e.g., Grant, Synthetic Peptides: A User's Guide, Oxford University Press (1992)).
  • Modified amino acids include, for example, amino acids wherein the amino and/or carboxyl group is replaced by another group.
  • Non-limiting examples include modified amino acids incorporating thioamides, ureas, thioureas, acylhydrazides, esters, olefines, sulfonamides, phosphoric acid amides, ketones, alcohols, boronic acid amides, benzodiazepines and other aromatic or non-aromatic heterocycles (see, Estiarte et al., Burgers Medicinal Chemistry, 6.sup.th edition, Volume 1, Part 4, John Wiley & Sons, New York (2002)).
  • Non-proteinogenic amino acids include, but are not limited, to beta-alanine (Bal), norvaline (Nva), norleucine (Nle), 4-aminobutyric acid (gamma-Abu), 2-aminoisobutyric acid (Aib), 6- aminohexanoic acid (epsilon- Ahx), ornithine (Orn), hydroxyproline (Hyp), taurine, sarcosine, citrulline (Cit), cysteic acid (Coh), cyclohexylalanine (Cha), methioninesulfoxide (Meo), methioninesulfone (Moo), homoserine-methylester (Hsm), propargylglycine (Eag), 5-fluorotryptophan (5Fw), 6-fluorotryptophan (6Fw), 3',4'-dimethoxyphenyl-alanine (Ear), 3',4'-difluorophenylalanine (Dff
  • modified linkers include but are not limited to the flexible linker 4,7,10-trioxa-l,13-tridecanediamine (Ttds), glycine, 6-aminohexanoic acid, beta-alanine (Bal), pentynoic acid (Pyn), and combinations of Ttds, glycine, 6-aminohexanoic acid and Bal.
  • any of numerous chemical modifications may be carried out by known techniques, including, but not limited to acetylation, formylation, etc. Additionally, the derivative may contain one or more non-classical amino acids.
  • cells are engineered to express one or more of the CARs described herein.
  • CAR-containing cells referred to as "CAR cells” have uses in monotherapy and in combination therapies that include other therapeutic agents, such as the Adapters described herein, for example, to kill a target cell.
  • a cell expressing a chimeric antigen receptor comprising a D domain that binds to CD 123, (ii) a transmembrane domain, and (iii) an intracellular domain.
  • the D domain that binds to CD123 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8-33, 99 and 100.
  • 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.
  • the D domain that binds to CD123 comprises the amino acid sequence of SEQ ID NO: 14.
  • the CAR comprises the amino acid sequence of SEQ ID NO: 62-66 or 67. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 67.
  • the cell is an immune cell. In some embodiments, the cell is an immune effector cell. In some embodiments, the cell is a T cell (CAR-T cell) or a natural killer (NK) cell (CAR-NK cell). In some embodiments, the cell is an autologous immune effector cell. In some embodiments the cell is an autologous T cell (CAR-T cell) or an autologous natural killer (NK) cell. In some embodiments, the cell is an allogenic immune effector cell.
  • the cell is an allogenic T cell (CAR-T cell) or an allogenic natural killer (NK) cell.
  • an immune effector cell expressing the CAR is capable of directing an immune response to a cell expressing CD 123 in an in vitro assay.
  • a cell expressing a chimeric antigen receptor comprising an antigenic determinant binding domain (ADBD) that binds to an AFP p26 AD, (ii) a transmembrane domain, and (iii) an intracellular domain.
  • ADBD antigenic determinant binding domain
  • the ADBD that binds to AFP p26 AD comprises an scFv that binds to AFP p26 AD.
  • the ADBD that binds to AFP p26 AD comprises a D domain that binds to AFP p26 AD.
  • the AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 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 an AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 74-93 and 94. In some embodiments, the DD that binds to an AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 70-73 or 92-94.
  • the DD that binds to an AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 73.
  • the CAR comprises the amino acid sequence of SEQ ID NO: 68.
  • the CAR comprises the amino acid sequence of SEQ ID NO: 69.
  • the cell is an immune cell.
  • the cell is an immune effector cell.
  • the cell is a T cell (CAR-T cell) or a natural killer (NK) cell (CAR-NK cell).
  • the cell is an autologous immune effector cell.
  • the cell is an autologous T cell (CAR-T cell) or an autologous natural killer (NK) cell.
  • the cell is an allogenic immune effector cell. In some embodiments the cell is an allogenic T cell (CAR-T cell) or an allogenic natural killer (NK) cell. In some embodiments, the cell is capable of directing an immune response to a cell expressing CD 123 in an in vitro assay comprising the cell, a cell expressing CD 123 and an Adapter comprising (1) a D domain that binds to CD123 and (b) an AFP p26 AD, e.g., an Adapter comprising the amino acid sequence of SEQ ID NO: 50.
  • a cell expressing a chimeric antigen receptor comprising an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain.
  • the AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 37-43 and 44.
  • the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 37.
  • the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 39.
  • the cell is an immune cell. In some embodiments, the cell is an immune effector cell.
  • the cell is a T cell (CAR-T cell) or a natural killer (NK) cell (CAR-NK cell).
  • the cell is an autologous immune effector cell.
  • the cell is an autologous T cell (CAR-T cell) or an autologous natural killer (NK) cell.
  • the cell is an allogenic immune effector cell.
  • the cell is an allogenic T cell (CAR-T cell) or an allogenic natural killer (NK) cell.

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PCT/US2022/079796 2021-11-15 2022-11-14 D-domain containing polypeptides and uses thereof Ceased WO2023086983A1 (en)

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CN202280088084.1A CN118679180A (zh) 2021-11-15 2022-11-14 包含d结构域的多肽及其用途
CA3237239A CA3237239A1 (en) 2021-11-15 2022-11-14 D-domain containing polypeptides and uses thereof
EP22893904.7A EP4433497A4 (en) 2021-11-15 2022-11-14 D-DOMAIN POLYPEPTIDES AND ASSOCIATED USES
IL312586A IL312586A (en) 2021-11-15 2022-11-14 D-domain containing polypeptides and uses thereof
KR1020247019906A KR20240105452A (ko) 2021-11-15 2022-11-14 D-도메인 함유 폴리펩티드 및 그의 용도
MX2024005673A MX2024005673A (es) 2021-11-15 2022-11-14 Polipeptidos que contienen el dominio d y usos de los mismos.
JP2024528494A JP2024544559A (ja) 2021-11-15 2022-11-14 Ｄドメイン含有ポリペプチドおよびその使用法
AU2022388811A AU2022388811A1 (en) 2021-11-15 2022-11-14 D-domain containing polypeptides and uses thereof

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EP4545092A1 (en) * 2023-10-27 2025-04-30 Technische Universität München Computationally designed binders for immune cell therapies
WO2025111382A3 (en) * 2023-11-21 2025-08-07 Board Of Regents, The University Of Texas System Anti-lamp5 antibodies, engineered immune cells expressing anti-lamp5 antibodies, and uses thereof

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RS61907B1 (sr) 2015-04-06 2021-06-30 Subdomain Llc Polipeptidi koji sadrže de novo vezujući domen i njihova primena
WO2025174812A1 (en) * 2024-02-12 2025-08-21 University Of Pittsburgh-Of The Commonwealth System Of Higher Education Chimeric antigen receptor and/or synnotch receptor systems and cells and methods of their use

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IL274677B2 (en) 2017-11-14 2025-01-01 Arcellx Inc Therapies with multifunctional immune system cells
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MX2024005673A (es) 2021-11-15 2024-07-09 Arcellx Inc Polipeptidos que contienen el dominio d y usos de los mismos.
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Cited By (5)

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US11827716B2 (en) 2021-11-15 2023-11-28 Arcellx, Inc. D-domain containing polypeptides and uses thereof
US12338294B2 (en) 2021-11-15 2025-06-24 Arcellx, Inc. D-domain containing polypeptides and uses thereof
EP4545092A1 (en) * 2023-10-27 2025-04-30 Technische Universität München Computationally designed binders for immune cell therapies
WO2025088214A1 (en) * 2023-10-27 2025-05-01 Technische Universität München, in Vertretung des Freistaates Bayern Computationally designed binders for immune cell therapies
WO2025111382A3 (en) * 2023-11-21 2025-08-07 Board Of Regents, The University Of Texas System Anti-lamp5 antibodies, engineered immune cells expressing anti-lamp5 antibodies, and uses thereof

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EP4433497A1 (en) 2024-09-25
CA3237239A1 (en) 2023-05-19
EP4433497A4 (en) 2026-02-25
US20250382380A1 (en) 2025-12-18
AU2022388811A1 (en) 2024-05-16
IL312586A (en) 2024-07-01
US20240124605A1 (en) 2024-04-18
US20230250185A1 (en) 2023-08-10
JP2024544559A (ja) 2024-12-03
CN118679180A (zh) 2024-09-20
MX2024005673A (es) 2024-07-09
KR20240105452A (ko) 2024-07-05
US11827716B2 (en) 2023-11-28

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