WO2019173837A1 - Dimeric antigen receptors (dar) - Google Patents

Dimeric antigen receptors (dar) Download PDF

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Publication number
WO2019173837A1
WO2019173837A1 PCT/US2019/021681 US2019021681W WO2019173837A1 WO 2019173837 A1 WO2019173837 A1 WO 2019173837A1 US 2019021681 W US2019021681 W US 2019021681W WO 2019173837 A1 WO2019173837 A1 WO 2019173837A1
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Prior art keywords
region
seq
sequence
heavy chain
amino acid
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Ceased
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PCT/US2019/021681
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English (en)
French (fr)
Inventor
Henry Hongjun Ji
Wenzhong Guo
Yanliang Zhang
Gunnar F. Kaufmann
Bei Bei DING
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Sorrento Therapeutics Inc
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Sorrento Therapeutics Inc
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Priority to CN201980031146.3A priority Critical patent/CN112105649B/zh
Priority to AU2019231315A priority patent/AU2019231315A1/en
Priority to EP19764893.4A priority patent/EP3762430A4/en
Priority to KR1020207028896A priority patent/KR20200130395A/ko
Priority to JP2020546854A priority patent/JP7542439B2/ja
Application filed by Sorrento Therapeutics Inc filed Critical Sorrento Therapeutics Inc
Priority to SG11202008568WA priority patent/SG11202008568WA/en
Priority to CA3092993A priority patent/CA3092993A1/en
Publication of WO2019173837A1 publication Critical patent/WO2019173837A1/en
Priority to US17/013,359 priority patent/US12180294B2/en
Priority to IL277181A priority patent/IL277181A/en
Anticipated expiration legal-status Critical
Priority to JP2023172758A priority patent/JP2023168545A/ja
Ceased legal-status Critical Current

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Definitions

  • DAR Dimeric Antigen Receptors
  • the present disclosure provides dimeric antigen receptors (DAR) protein constructs that bind specifically to a target antigen, nucleic acids that encode the dimeric antigen receptors, vectors comprising the nucleic acids, and host cells harboring the vectors.
  • DAR dimeric antigen receptors
  • Chimeric antigen receptors have been developed to target antigens associated, in particular, with cancer.
  • the first-generation CAR was engineered to contain a signaling domain (TCRz) that delivers an activation stimulus (signal 1) only (Geiger et al., ./. Immunol. 162(10): 5931-5939, 1999; Haynes et al., J. Immunol. 166(1): 182-187, 2001) (Hombach et al. Cancer Res. 61(5): 1976-1982, 2001; Hombach et al., J. Immunol. 167(11): 6123-6131, 2001; Maher et al., Nat. Biotechnol.
  • TCRz signaling domain
  • T cells grafted with the first-generation CARs alone exhibited limited anti -turn or efficacy due to suboptimal activation (Beecham et al., J. Immunother. 23(6): 631-642, 2000).
  • CAR-T cells targeting CD 19, a molecule expressed on B cells, have shown success in treatment of B cell malignancies and have received FDA approval, with some trials showing up to 70% of response, including sustained complete response.
  • the present disclosure provides dimeric antigen receptors (DAR) precursor polypeptide comprising ten regions ordered from the amino terminus to the carboxyl terminus: (1) a heavy chain leader sequence (2) an antibody heavy chain variable region, (3) an antibody heavy chain constant region, (4) an optional hinge region, (5) a transmembrane region, (6) an intracellular signaling region, (7) a T2A cleavage sequence, (8) a light chain leader sequence, (9) an antibody light chain variable region, and (10) an antibody light chain constant region.
  • DAR dimeric antigen receptors
  • the present disclosure provides dimeric antigen receptors (DAR) precursor polypeptide comprising ten regions ordered from the amino terminus to the carboxyl terminus: (1) a light chain leader sequence (2) an antibody light chain variable region, (3) an antibody light chain constant region, (4) an optional hinge region, (5) a transmembrane region, (6) an intracellular signaling region, (7) a T2A cleavage sequence, (8) a heavy chain leader sequence, (9) an antibody heavy chain variable region, and (10) an antibody heavy chain constant region.
  • DAR dimeric antigen receptors
  • the precursor polypeptide optionally comprises a hinge sequence from an antibody selected from a group consisting of IgG, IgA, IgM, IgE and IgD.
  • the precursor polypeptide comprises a hinge sequence from a CD8a or CD28 hinge region.
  • the precursor polypeptide comprises a hinge region comprises a CPPC or SPPC amino acid sequence.
  • the precursor polypeptide comprises a transmembrane region from CD 8 a, CD8p, 4-1BB/CD137, CD28, CD34, CD4, FceRIy, CD16, OX40/CD134, O ⁇ 3z, CD3e, CD3y, CD35, TCRa, TCRp, TCRC, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD33,
  • the precursor polypeptide comprises an intracellular signaling region comprises an intracellular signaling sequence in any order and of any combination of two to five signaling sequences from 4-1BB, CD3zeta, CD28, CD27, 0X40, CD30, CD40, PD-l, ICOS, lymphocyte function-associated antigen-l (LFA-l), CD2, CD7, LIGHT, NKG2C, B7-H3, GITR (TNFRSF18), DR3 (TNFRSF25), TNFR2 and/or CD226.
  • the precursor polypeptide comprises the amino acid sequence of SEQ ID NO: 15 or 18.
  • the present disclosure provides a dimeric antigen receptors (DAR) comprising: (a) a first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region, (ii) an antibody heavy chain constant region, (iii) an optional hinge region, (iv) a transmembrane region, and (v) an intracellular signaling region comprising two or three intracellular signaling sequences; and (b) a second polypeptide chain comprising two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region, and (ii) an antibody light chain constant region, wherein the antibody heavy chain constant region and the antibody light chain constant region form a dimerization domain, and wherein the antibody heavy chain variable region and the antibody light chain variable region form an antigen binding domain.
  • DAR dimeric antigen receptors
  • the present disclosure provides a dimeric antigen receptors (DAR) comprising: (a) a first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region, (ii) an antibody light chain constant region, (iii) an optional hinge region, (iv) a transmembrane region, and (v) an intracellular signaling region comprising two or three intracellular signaling sequences; and (b) a second polypeptide chain comprising two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region, and (ii) an antibody heavy chain constant region, wherein the antibody heavy chain constant region and the antibody light chain constant region form a dimerization domain, and wherein the antibody heavy chain variable region and the antibody light chain variable region form an antigen binding domain.
  • DAR dimeric antigen receptors
  • the dimeric antigen receptor comprises an antibody heavy chain constant region and the antibody light chain constant region that dimerize via one or two disulfide bonds.
  • the dimeric antigen receptor optionally comprises a hinge region which can be derived from an antibody selected from a group consisting of IgG, IgA, IgM, IgE and IgD.
  • the hinge comprises a CD8a or CD28 hinge region.
  • the hinge region comprises a CPPC or SPPC amino acid sequence.
  • the dimeric antigen receptor comprises a transmembrane region from CD 8 a, CD8p, 4-1BB/CD137, CD28, CD34, CD4, FceRIy, CD16, OX40/CD134, O ⁇ 3z, CD3e, CD3y, CD35, TCRa, TCRp, TCRC, CD32, CD64, CD64, CD45, CD5, CD9,
  • the dimeric antigen receptor comprises an intracellular signaling region comprising an intracellular signaling sequence in any order and of any combination of two to five signaling sequences from 4-1BB, CD3zeta, CD28, CD27, 0X40, CD30, CD40, PD-l, ICOS, lymphocyte function-associated antigen-l (LFA-l), CD2, CD7, LIGHT, NKG2C, B7-H3, GITR (TNFRSF 18), DR3 (TNFRSF25), TNFR2 and/or CD226.
  • the present disclosure provides a dimeric antigen receptors (DAR) comprising: (a) a first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) a CD38 antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CD38 antibody heavy chain constant region comprising the amino acid sequence of SEQ ID NO:2, (iii) a CD28 hinge region comprising the amino acid sequence of SEQ ID NO:5 and optionally a second hinge region comprising the amino acid sequence of SEQ ID NO:2l, (iv) a CD28 transmembrane region comprising the amino acid sequence of SEQ ID NO:6, and (v) an intracellular signaling region comprising a 4-1BB intracellular signaling sequence comprising the amino acid sequence of SEQ ID NO:7 and a CD3zeta intracellular signaling sequence comprising the amino acid sequence of SEQ ID NO:9 or 20; and (b) a second polypeptide chain comprising two regions
  • the dimeric antigen receptors comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 13 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 14.
  • the dimeric antigen receptors comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 16 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 17.
  • the present disclosure provides a nucleic acid encoding a precursor polypeptide comprising (1) an antibody heavy chain leader sequence comprising the amino acid sequence of SEQ ID NO: 10; (2) an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO:l; (3) an antibody heavy chain constant region comprising the amino acid sequence of SEQ ID NO:2; (4) an optional hinger region comprising the amino acid sequence of SEQ ID NO:5; (5) a transmembrane region comprising the amino acid sequence of SEQ ID NO:6; (6) an intracellular signaling region comprising any one or any combination of two or more signaling sequences selected from a group consisting of 4-1BB signaling sequence comprising the amino acid sequence of SEQ ID NO:7, CD28 signaling sequence comprising the amino acid sequence of SEQ ID NO:8, CD3zeta (long) signaling sequence comprising the amino acid sequence of SEQ ID NO:9 and/or CD3zeta (short) signaling sequence having an ITAM 3 motif and comprising the amino acid sequence of
  • the present disclosure provides a nucleic acid encoding a precursor polypeptide comprising the amino acid sequence of SEQ ID NO:l5 or 18.
  • the present disclosure provides a nucleic acid encoding (a) a first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) a CD38 antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CD38 antibody heavy chain constant region comprising the amino acid sequence of SEQ ID NO:2, (iii) a CD28 hinge region comprising the amino acid sequence of SEQ ID NO:5 and optionally a second hinge region comprising the amino acid sequence of SEQ ID NO:2l, (iv) a CD28 transmembrane region comprising the amino acid sequence of SEQ ID NO:6, and (v) an intracellular signaling region comprising a 4-1BB intracellular signaling sequence comprising the amino acid sequence of SEQ ID NO:7 and a CD3zeta intracellular signaling sequence comprising the amino acid sequence of SEQ ID NO:9 or 20; and the same nucleic acid or a second nucleic acid encoding (b)
  • the nucleic acid encodes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 13 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 14.
  • the nucleic acid encodes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 16 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 17.
  • the present disclosure provides a vector operably linked to a nucleic acid encoding a precursor polypeptide comprising (1) an antibody heavy chain leader sequence comprising the amino acid sequence of SEQ ID NO: 10; (2) an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1; (3) an antibody heavy chain constant region comprising the amino acid sequence of SEQ ID NO:2; (4) an optional hinger region comprising the amino acid sequence of SEQ ID NO:5; (5) a transmembrane region comprising the amino acid sequence of SEQ ID NO:6; (6) an intracellular signaling region comprising any one or any combination of two or more signaling sequences selected from a group consisting of 4-1BB signaling sequence comprising the amino acid sequence of SEQ ID NO:7, CD28 signaling sequence comprising the amino acid sequence of SEQ ID NO: 8, CD3zeta (long) signaling sequence comprising the amino acid sequence of SEQ ID NO:9 and/or CD3zeta (short) signaling sequence having an IT AM 3 motif and
  • the present disclosure provides a vector operably linked to a nucleic acid encoding a precursor polypeptide having the amino acid sequence of SEQ ID NO: 15 or 18.
  • the vector comprises an expression vector that is operably linked to a nucleic acid that encodes precursor polypeptide comprising the amino acid sequence of SEQ ID NO: 15 or 18.
  • the expression vector in the host cell or the population of host cells directs transient introduction of the nucleic acid encoding the precursor polypeptide into the host cell or the population of host cells.
  • the expression vector in the host cell or the population of host cells directs stable insertion into the host cell’s genome the nucleic acid encoding the precursor polypeptide into the host cells’ genome.
  • the expression vector in the host cell or the population of host cells directs transcription and/or translation of the nucleic acid encoding the precursor polypeptide in the host cell or the population of host cells.
  • the expression vector can include one or more regulatory sequences, such as inducible and/or constitutive promoters and enhancers, which direct transcription and/or translation (e.g., expression) of the nucleic acid encoding the precursor polypeptide in the host cell or population of host cells.
  • the present disclosure provides a vector operably linked to a nucleic acid encoding (a) a first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) a CD38 antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CD38 antibody heavy chain constant region comprising the amino acid sequence of SEQ ID NO:2, (iii) a CD28 hinge region comprising the amino acid sequence of SEQ ID NO:5 and optionally a second hinge region comprising the amino acid sequence of SEQ ID NO:2l, (iv) a CD28 transmembrane region comprising the amino acid sequence of SEQ ID NO:6, and (v) an intracellular signaling region comprising a 4-1BB intracellular signaling sequence comprising the amino acid sequence of SEQ ID NO:7 and a CD3zeta intracellular signaling sequence comprising the amino acid sequence of SEQ ID NO:9 or 20; and the same vector or a second vector operably linked to a nucle
  • the expression vector is operably linked to a nucleic acid that encodes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 13 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 14.
  • a first expression vector is operably linked to a nucleic acid that encodes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 13 and a second vector operably linked to a nucleic acid that encodes a second polypeptide comprising the amino acid sequence of SEQ ID NO: 14.
  • the expression vector is operably linked to a nucleic acid that encodes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 16 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 17.
  • a first expression vector is operably linked to a nucleic acid that encodes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 16 and a second vector operably linked to a nucleic acid that encodes a second polypeptide comprising the amino acid sequence of SEQ ID NO: 17.
  • the expression vector in the host cell or the population of host cells directs transient introduction of the nucleic acid encoding the first and/or second polypeptide into the host cell or the population of host cells.
  • the expression vector in the host cell or the population of host cells directs stable insertion into the host cell’s genome the nucleic acid encoding the first and/or second polypeptide into the host cells’ genome.
  • the expression vector in the host cell or the population of host cells directs transcription and/or translation of the nucleic acid encoding the first and/or second polypeptide in the host cell or the population of host cells.
  • the expression vector can include one or more regulatory sequences, such as inducible and/or constitutive promoters and enhancers, which direct transcription and/or translation (e.g., expression) of the nucleic acid encoding the first and/or second polypeptide in the host cell or population of host cells.
  • the present disclosure provides a host cell or a population of host cells harboring a vector operably linked to a nucleic acid encoding a precursor polypeptide comprising (1) an antibody heavy chain leader sequence comprising the amino acid sequence of SEQ ID NO: 10;
  • an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: l; (3) an antibody heavy chain constant region comprising the amino acid sequence of SEQ ID NO:2; (4) an optional hinger region comprising the amino acid sequence of SEQ ID NO:5;
  • a transmembrane region comprising the amino acid sequence of SEQ ID NO:6; (6) an intracellular signaling region comprising any one or any combination of two or more signaling sequences selected from a group consisting of 4-1BB signaling sequence comprising the amino acid sequence of SEQ ID NO: 7, CD28 signaling sequence comprising the amino acid sequence of SEQ ID NO:8, CD3zeta (long) signaling sequence comprising the amino acid sequence of SEQ ID NO:9 and/or CD3zeta (short) signaling sequence having an IT AM 3 motif and comprising the amino acid sequence of SEQ ID NO:20; (7) a T2A cleavage sequence comprising the amino acid sequence of SEQ ID NO: 12; (8) a light chain leader sequence comprising the amino acid sequence of SEQ ID NO: 11; (9) an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO:3; and (10) an antibody light chain constant region comprising the amino acid sequence of SEQ ID NO:4.
  • the vector harbored by the host cell or the population of host cells comprises an expression vector that directs transient introduction of the nucleic acid encoding the precursor polypeptide into the host cell or the population of host cells.
  • the expression vector in the host cell or the population of host cells directs stable insertion into the host cell’s genome the nucleic acid encoding the precursor polypeptide into the host cells’ genome.
  • the expression vector in the host cell or the population of host cells directs transcription and/or translation of the nucleic acid encoding the precursor polypeptide in the host cell or the population of host cells.
  • the expression vector can include one or more regulatory sequences, such as inducible and/or constitutive promoters and enhancers, which direct transcription and/or translation (e.g., expression) of the nucleic acid encoding the precursor polypeptide in the host cell or population of host cells.
  • the present disclosure provides a host cell or a population of host cells harboring a vector operably linked to a nucleic acid encoding (a) a first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) a CD38 antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CD38 antibody heavy chain constant region comprising the amino acid sequence of SEQ ID NO:2, (iii) a CD28 hinge region comprising the amino acid sequence of SEQ ID NO: 5 and optionally a second hinge region comprising the amino acid sequence of SEQ ID NO:2l, (iv) a CD28 transmembrane region comprising the amino acid sequence of SEQ ID NO:6, and (v) an intracellular signaling region comprising a 4-1BB intracellular signaling sequence comprising the amino acid sequence of SEQ ID NO:7 and a CD3zeta intracellular signaling sequence comprising the amino acid sequence of SEQ ID NO:9 or 20; and
  • the hinge region is optional.
  • the host cell or the population of host cells harbor an expression vector that directs transcription and/or translation (e.g., expression) of the first and/or second polypeptide chain.
  • the host cells expresses the first and/or second polypeptide chain.
  • the host cell or population of host cells harbor a vector operably linked to a nucleic acid that encodes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 13 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 14.
  • the host cell or population of host cells harbor a first vector operably linked to a nucleic acid that encodes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 13 and a second vector operably linked to a nucleic acid that encodes a second polypeptide comprising the amino acid sequence of SEQ ID NO: 14.
  • the host cell or population of host cells harbor the vector operably linked to a nucleic acid that encodes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 16 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: l7.
  • the host cell or population of host cells harbor a first vector operably linked to a nucleic acid that encodes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 16 and a second vector operably linked to a nucleic acid that encodes a second polypeptide comprising the amino acid sequence of SEQ ID NO: 17.
  • the present disclosure provides a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen in the subject, comprising: administering to the subject a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a precursor polypeptide comprising: ten regions ordered from the amino terminus to the carboxyl terminus: (1) a heavy chain leader region, (2) an antibody heavy chain variable region, (3) an antibody heavy chain constant region, (4) an optional hinge region, (5) a transmembrane region, (6) an intracellular signaling region having two to five intracellular signaling sequences, (7) T2A cleavable sequence region, (8) a light chain leader region, (9) an antibody light chain variable region, and (10) an antibody light chain constant region.
  • the host cell harbors an expression vector operably linked to a nucleic acid that encodes a precursor polypeptide which comprises: (1) an antibody heavy chain leader sequence comprising the amino acid sequence of SEQ ID NO: 10; (2) an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: l; (3) an antibody heavy chain constant region comprising the amino acid sequence of SEQ ID NO:2; (4) an optional hinger region comprising the amino acid sequence of SEQ ID NO:5; (5) a transmembrane region comprising the amino acid sequence of SEQ ID NO:6; (6) an intracellular signaling region comprising any one or any combination of two or more signaling sequences selected from a group consisting of 4-1BB signaling sequence comprising the amino acid sequence of SEQ ID NO: 7, CD28 signaling sequence comprising the amino acid sequence of SEQ ID NO:8, CD3zeta (long) signaling sequence comprising the amino acid sequence of SEQ ID NO:9 and/or CD3zet
  • the host cell or the population of host cells, which harbors the expression vector operably linked to a nucleic acid that encodes a precursor polypeptide comprising the amino acid sequence of SEQ ID NO:l5 or 18.
  • the present disclosure provides a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen in the subject, comprising: administering to the subject a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes (a) a first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) a CD38 antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CD38 antibody heavy chain constant region comprising the amino acid sequence of SEQ ID NO:2, (iii) a CD28 hinge region comprising the amino acid sequence of SEQ ID NO:5 and optionally a second hinge region comprising the amino acid sequence of SEQ ID NO:2l, (iv) a CD28 transmembrane region comprising the amino acid sequence of SEQ ID NO:6, and (v) an intracellular signaling region comprising a 4-1BB intracellular signaling sequence comprising
  • the antibody heavy chain constant region and the antibody light chain constant region form a dimerization domain, and wherein the antibody heavy chain variable region and the antibody light chain variable region form an antigen binding domain that binds a CD38 protein.
  • the hinge region is optional.
  • the host cell or the population of host cells are selected from a group consisting of T lymphocytes (e.g., T cells, regulatory T cells, gamma-delta T cells, and cytotoxic T cells), NK (natural killer) cells, macrophages, dendritic cells, mast cells, eosinophils, B lymphocytes and monocytes.
  • T lymphocytes e.g., T cells, regulatory T cells, gamma-delta T cells, and cytotoxic T cells
  • NK natural killer cells
  • macrophages e.g., dendritic cells, mast cells, eosinophils, B lymphocytes and monocytes.
  • the host cells comprise NK cells that comprise cord blood-derived NK cells, or placental derived NK cells.
  • the expression vector in the host cell or the population of host cells directs transient introduction of the nucleic acid encoding the first and/or second polypeptide into the host cell or the population of host cells.
  • the expression vector in the host cell or the population of host cells directs stable insertion into the host cell’s genome the nucleic acid encoding the first and/or second polypeptide into the host cells’ genome.
  • the expression vector in the host cell or the population of host cells directs transcription and/or translation of the nucleic acid encoding the first and/or second polypeptide in the host cell or the population of host cells.
  • the expression vector can include one or more regulatory sequences, such as inducible and/or constitutive promoters and enhancers, which direct transcription and/or translation (e.g., expression) of the nucleic acid encoding the first and/or second polypeptide in the host cell or population of host cells.
  • the disease, disorder or condition associated with detrimental expression of a tumor antigen is a cancer, including, but not limited to hematologic breast cancer, ovarian cancer, prostate cancer, head and neck cancer, lung cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer, liver cancer, renal cancer, esophageal cancer, leiomyoma, leiomyosarcoma, glioma, and glioblastoma.
  • the disease is a hematologic cancer selected from the group consisting of non-Hodgkin's lymphoma (NHL), Burkitf s lymphoma (BL), B chronic
  • B-CLL B and T acute lymphocytic leukemia
  • ALL T cell lymphoma
  • AML acute myeloid leukemia
  • HCL hairy cell leukemia
  • HCL Hodgkin's Lymphoma
  • HL chronic myeloid leukemia
  • CML chronic myeloid leukemia
  • MM multiple myeloma
  • Figure l is a schematic showing an exemplary dimeric antigen receptor comprising two intracellular signaling sequences.
  • Figure 2 is a schematic showing an exemplary dimeric antigen receptor comprising three intracellular signaling sequences.
  • Figure 3 is a schematic showing an exemplary dimeric antigen receptor comprising two intracellular signaling sequences.
  • Figure 4 is a schematic showing an exemplary dimeric antigen receptor comprising three intracellular signaling sequences.
  • Figure 5 is a schematic showing an exemplary precursor polypeptide molecule comprising a T2A cleavage sequence and three intracellular signaling sequences.
  • Figure 6 is a schematic showing an exemplary precursor polypeptide molecule comprising a T2A cleavage sequence and two intracellular signaling sequences.
  • Figure 7 is a schematic showing an exemplary precursor polypeptide molecule comprising a T2A cleavage sequence and three intracellular signaling sequences.
  • Figure 8 is a schematic showing an exemplary precursor polypeptide molecule comprising a T2A cleavage sequence and two intracellular signaling sequences.
  • Figure 9 shows the results of a flow cytometry study comparing T cells expressing a CD38 chimeric antigen receptor (CAR) construct compared to T cells expressing a CD38 dimeric antigen receptor (DAR) construct.
  • the negative control is a cell line carrying a knocked-out TRAC (T-cell receptor alpha constant) gene.
  • the DAR (VI) construct includes a long hinge sequence having CD8 and CD28 hinge sequences, and signaling regions include CD28 and long CD3zeta intracellular signaling sequences.
  • Figure 10 is a graph showing the percent cytotoxicity of T cells expressing CD38 CAR, or CD38 DAR, on RPMI 8226 target cells.
  • the negative control (line A) is a cell line carrying a knocked-out TRAC gene.
  • the CD38 DAR (VI) construct (B dotted line) includes a long hinge sequence having CD8 and CD28 hinge sequences, and signaling regions include CD28 and long CD3zeta intracellular signaling sequences.
  • the CD38 CAR construct is designated (line C).
  • Figure 11 shows the results of a flow cytometry study comparing T cells expressing CD38 CAR VI or V2 constructs.
  • the negative control is a cell line (TRAC KO) is the same that is described in Figure 1.
  • the CD38 DAR (VI) construct includes a long hinge sequence having CD8 and CD28 hinge sequences, and signaling regions include CD28 and long CD3zeta intracellular signaling sequences.
  • the CD38 DAR (V2b) construct includes a short hinge sequence, and the signaling regions include CD28 and long CD3zeta intracellular signaling sequences.
  • Figure 12 is a graph showing the percent cytotoxicity of T cells expressing CD38 CAR, CD38 DAR (VI) or CD38 DAR (V2b) constructs, on RPMI 8226 target cells.
  • the negative control (line A) is a cell line carrying a knocked-out TRAC gene.
  • the CD38 DAR (VI) construct (line B) includes a long hinge sequence having CD8 and CD28 hinge sequences, and signaling regions include CD28 and long CD3zeta intracellular signaling sequences.
  • the CD38 DAR (V2b) construct (C dotted line) includes a short hinge sequence, and the signaling regions include CD28 and long CD3zeta intracellular signaling sequences.
  • the CD38 CAR is designated (line D).
  • Figure 13 is a bar graph showing the level of IFN-gamma release from T cells expressing CD38 CAR, CD38 DAR (VI) or CD38 DAR (V2b) constructs. Each data set shows from left to right RPMI 8226 cells, K562 cells and medium.
  • the negative control is a cell line carrying a knocked-out TRAC (T-cell receptor alpha constant) gene.
  • the CD38 DAR (VI) construct includes a long hinge sequence having CD8 and CD28 hinge sequences, and signaling regions include CD28 and long CD3zeta intracellular signaling sequences.
  • the CD38 DAR (V2b) construct includes a short hinge sequence, and the signaling regions include CD28 and long CD3zeta intracellular signaling sequences.
  • Figure 14 is a bar graph showing the level of TNF-alpha release from T cells expressing CD38 CAR, CD38 DAR (VI) or CD38 DAR (V2b) constructs. Each data set shows from left to right RPMI 8226 cells, K562 cells and medium.
  • the negative control is a cell line carrying a knocked-out TRAC (T-cell receptor alpha constant) gene.
  • the CD38 DAR (VI) construct includes a long hinge sequence having CD8 and CD28 hinge sequences, and signaling regions include CD28 and long CD3zeta intracellular signaling sequences.
  • the CD38 DAR. (V2b) construct includes a short hinge sequence, and the signaling regions include CD28 and long CD3zeta intracellular signaling sequences.
  • Figure 15 is a bar graph showing the level of IL-2 release from T cells expressing CD38 CAR, CD38 DAR (VI) or CD38 DAR (V2b) constructs. Each data set shows from left to right RPMI 8226 cells, K562 cells and medium. The negative control is a cell line carrying a knocked-out TRAC gene.
  • the CD38 DAR (VI) construct includes a long hinge sequence having CD8 and CD28 hinge sequences, and signaling regions include CD28 and long CD3zeta intracellular signaling sequences.
  • the CD38 DAR (V2b) construct includes a short hinge sequence, and the signaling regions include CD28 and long CD3zeta intracellular signaling sequences.
  • Figure 16 shows the results of a flow cytometry study comparing expansion capability of T cells expressing a CAR, or a DAR (VI) or (V2b) construct, when co-cultured with K562 or RPMI 8226 cells.
  • the negative control is a cell line carrying a knocked-out TRAC (T-cell receptor alpha constant) gene.
  • the CD38 DAR (VI) construct includes a long hinge sequence having CD8 and CD28 hinge sequences, and signaling regions include CD28 and long CD3zeta intracellular signaling sequences.
  • the CD38 DAR (V2b) construct includes a short hinge sequence, and the signaling regions include CD28 and long CD3zeta intracellular signaling sequences.
  • Figure 17 is a bar graph showing the fold-change in expansion of T cells expressing a CAR, or a DAR (VI) or (V2b) construct, when co-cultured with K562 or RPMI 8226 cells.
  • the negative control is a cell line carrying a knocked-out TRAC (T-cell receptor alpha constant) gene.
  • the CD38 DAR (VI) construct includes a long hinge sequence having CD8 and CD28 hinge sequences, and signaling regions include CD28 and long CD3zeta intracellular signaling sequences.
  • the CD38 DAR (V2b) construct includes a short hinge sequence, and the signaling regions include CD28 and long CD3zeta intracellular signaling sequences.
  • Figure 18 shows tumoricidal activity of the CD38 DAR T cells assessed in a xenograft animal model.
  • the negative control mice were administered phosphate-buffered saline or a cell line carrying a knocked-out TRAC gene.
  • the test mice were administered T cells expressing CD38 DAR (V2b) construct which include a short hinge sequence, and the signaling regions include CD28 and long CD3zeta intracellular signaling sequences.
  • Figure 19 shows tumoricidal activity of the CD38 DAR (V2a) T cells assessed in a xenograft animal model.
  • the negative control mice were administered phosphate-buffered saline or a cell line carrying a knocked-out TRAC gene.
  • the test mice were administered T cells expressing CD38 DAR (V2a) construct which include a short hinge sequence, and the signaling regions include 4-1BB and long CD3zeta intracellular signaling sequences.
  • Figure 20 shows the results of a flow cytometry study comparing T cells expressing a CD38 DAR (V2a) or (V3) construct.
  • the negative controls include activated T cells, and a cell line carrying a knocked-out TRAC gene.
  • the CD38 DAR (V2a) construct includes a short hinge sequence, and the signaling regions include 4-1BB and long CD3zeta intracellular signaling sequences.
  • the CD38 DAR (V3) construct includes a short hinge sequence, and the signaling region includes 4-1BB and CD3zeta having a shortened IT AM region.
  • Figure 21 is a graph showing the percent cytotoxicity of T cells expressing CD38 DAR (V2a) compared to (V3), on RPMI 8226 target cells.
  • the negative control is a cell line (line A) carrying a knocked-out TRAC gene.
  • the CD38 DAR (V2a) construct (line B) includes a short hinge sequence, and the signaling regions include 4-1BB and long CD3zeta intracellular signaling sequences.
  • the CD38 DAR (V3) construct (line C) includes a short hinge sequence, and the signaling region includes 4-1BB and CD3zeta having a shortened IT AM region.
  • Figure 22 is a bar graph showing the level of IFN-gamma release from T cells expressing CD38 DAR (V2a) compared to DAR (V3) constructs. Each data set shows from left to right RPMI 8226 cells, K562 cells and medium. The negative control is a cell line carrying a knocked-out TRAC gene.
  • the CD38 DAR (V2a) construct includes a short hinge sequence, and the signaling regions include 4-1BB and long CD3zeta intracellular signaling sequences.
  • the CD38 DAR (V3) construct includes a short hinge sequence, and the signaling region includes 4- 1BB and CD3zeta having a shortened IT AM region.
  • Figure 23 is a bar graph showing the level of TNF-alpha release from T cells expressing CD38 DAR (V2a) compared to DAR (V3) constructs. Each data set shows from left to right RPMI 8226 cells, K562 cells and medium. The negative control is a cell line carrying a knocked-out TRAC gene.
  • the CD38 DAR (V2a) construct includes a short hinge sequence, and the signaling regions include 4-1BB and long CD3zeta intracellular signaling sequences.
  • the CD38 DAR (V3) construct includes a short hinge sequence, and the signaling region includes 4- 1BB and CD3zeta having a shortened IT AM region.
  • Figure 24 shows the results of a flow cytometry study to measure the fraction of memory T cells in CD38 DAR (V2a) and DAR (V3) T cells.
  • the CD38 DAR (V2a) construct includes a short hinge sequence, and the signaling regions include 4-1BB and long CD3zeta intracellular signaling sequences.
  • the CD38 DAR (V3) construct includes a short hinge sequence, and the signaling region includes 4-1BB and CD3zeta having a shortened ITAM region.
  • Figure 25 shows the results of a flow cytometry study comparing T cells expressing a CD38 CAR, CD38 DAR second generation and CD38 DAR third generation constructs.
  • the negative control is a cell line carrying a knocked-out TRAC gene.
  • the 28Z second generation construct includes a short hinge sequence, and the signaling region includes CD28 and CD3zeta signaling sequences (e.g., a DAR (V2b) construct).
  • the BBZ second generation construct includes a short hinge sequence, and the signaling region includes 4-1BB and CD3zeta signaling sequences (e.g., a DAR (V2a) construct).
  • the 28BBZ third generation construct includes a short hinge, and the signaling region includes CD28, 4-1BB and CD3zeta signaling sequences (e.g., a DAR (V2c) construct).
  • Figure 26 is a graph showing the percent cytotoxicity of T cells expressing CD38 CAR, one of two different CD38 second generation DAR, or a CD38 third generation DAR construct, on RPMI 8226 target cells.
  • the negative control (line A) is a cell line carrying a knocked-out TRAC gene.
  • the 28Z second generation construct (line E) includes a short hinge sequence, and the signaling region includes CD28 and CD3zeta signaling sequences (e.g., a DAR (V2b construct).
  • the BBZ second generation construct (line C) includes a short hinge sequence, and the signaling region includes 4-1BB and CD3zeta signaling sequences (e.g., a DAR (V2a) construct).
  • the 28BBZ third generation construct (line D dotted line) includes a short hinge, and the signaling region includes CD28, 4-1BB and CD3zeta signaling sequences (e.g., a DAR (V2c) construct).
  • the cells expressing CD38 CAR are designated (line B).
  • Figure 27 is a bar graph showing the level of IL-2 secretion from T cells expressing CD38 CAR, one of two different CD38 second generation DAR, or a CD38 third generation DAR. Each data set shows from left to right K562 cells, RPMI 8226 cells and T cells only.
  • the negative control is a cell line carrying a knocked-out TRAC gene.
  • the 28Z second generation construct includes a short hinge sequence, and the signaling region includes CD28 and CD3zeta signaling sequences (e.g., a DAR (V2b) construct).
  • the BBZ second generation construct includes a short hinge sequence, and the signaling region includes 4-1BB and CD3zeta signaling sequences (e.g., a DAR (V2a) construct).
  • the 28BBZ third generation construct includes a short hinge, and the signaling region includes CD28, 4-1BB and CD3zeta signaling sequences (e.g., a DAR (V2c) construct).
  • Figure 28 is a bar graph showing the level of TNF-alpha secretion from T cells expressing CD38 CAR, one of two different CD38 second generation DAR, or a CD38 third generation DAR. Each data set shows from left to right K562 cells, RPMI 8226 cells and T cells only.
  • the negative control is a cell line carrying a knocked-out TRAC gene.
  • the 28Z second generation construct includes a short hinge sequence, and the signaling region includes CD28 and CD3zeta signaling sequences (e.g., a DAR (V2b) construct).
  • the BBZ second generation construct includes a short hinge sequence, and the signaling region includes 4-1BB and CD3zeta signaling sequences (e.g., a DAR (V2a) construct).
  • the 28BBZ third generation construct includes a short hinge, and the signaling region includes CD28, 4-1BB and CD3zeta signaling sequences (e.g., a DAR (V2c) construct).
  • Figure 29 is a bar graph showing the level of IFN-gamma secretion from T cells expressing CD38 CAR, one of two different CD38 second generation DAR, or a CD38 third generation DAR.
  • Each data set shows from left to right K562 cells, RPMI 8226 cells and T cells only.
  • the negative control is a cell line carrying a knocked-out TRAC gene.
  • the 28Z second generation construct includes a short hinge sequence, and the signaling region includes CD28 and CD3zeta signaling sequences (e.g., a DAR (V2b) construct).
  • the BBZ second generation construct includes a short hinge sequence, and the signaling region includes 4-1BB and CD3zeta signaling sequences (e.g., a DAR (V2a) construct).
  • the 28BBZ third generation construct includes a short hinge, and the signaling region includes CD28, 4-1BB and CD3zeta signaling sequences (e.g., a DAR (V2c) construct).
  • Figure 30 shows tumoricidal activity of the CD38 CAR T cells assessed in a xenograft animal model.
  • the negative control mice were administered phosphate-buffered saline, activated T-cells or a cell line carrying a knocked-out TRAC gene.
  • Test mice were administered T cells expressing either 28Z or BBZ second generation construct, or third generation 28BBZ DAR construct.
  • the 28Z DAR second generation construct includes a short hinge sequence, and the signaling region includes CD28 and CD3zeta signaling sequences (e.g., a DAR (V2b) construct).
  • the BBZ second generation construct includes a short hinge sequence, and the signaling region includes 4-1BB and CD3zeta signaling sequences (e.g., a DAR (V2a) construct).
  • the 28BBZ third generation construct includes a short hinge, and the signaling region includes CD28, 4-1BB and CD3zeta signaling sequences (e.g., a DAR (V2c) construct).
  • Figure 31 shows the results of a flow cytometry study comparing T cells expressing a CD38 DAR V2, V3 or V4 constructs.
  • the negative controls include activated T cells, and a cell line carrying a knocked-out TRAC gene.
  • the CD38 V2 DAR construct includes a short hinge, and the signaling region includes 4-1BB and CD3zeta signaling sequences (e.g., a DAR (V2b) construct).
  • the CD38 V3 DAR construct includes a short hinge sequence, and the signaling region includes 4-1BB and CD3-zeta (with only IT AM 3 motif) signaling sequences (e.g, a DAR (V3) construct).
  • the CD38 DAR V4 construct lacks a hinge sequence, and the signaling region includes 4-1BB and CD3-zeta (with only IT AM 3 motif) signaling sequences (e.g., a DAR (V4) construct).
  • Figure 32 is a graph showing the percent cytotoxicity of T cells expressing CD38 DAR V2, V3 or V4 construct, on RPMI 8226 target cells.
  • the negative control (line A) is a cell line carrying a knocked-out TRAC gene.
  • the CD38 DAR V4 construct (line B) lacks a hinge sequence, and the signaling region includes 4-1BB and CD3-zeta (with only ITAM 3 motif) signaling sequences (e.g., DAR (V4) construct).
  • the CD38 V2 DAR construct (C dotted line) includes a short hinge, and the signaling region includes 4-1BB and CD3zeta signaling sequences (e.g., DAR (V2a) construct).
  • the CD38 V3 DAR construct (line D) includes a short hinge sequence, and the signaling region includes 4-1BB and CD3-zeta (with only ITAM 3 motif) signaling sequences (e.g., DAR (V3) construct).
  • Figure 33 is a bar graph showing the level of IFN-gamma release from T cells expressing CD38 DAR V2, V3 or V4 constructs. Each data set shows from left to right RPMI 8226 cells, K562 cells and medium.
  • the negative control is a cell line carrying a knocked-out TRAC gene.
  • the CD38 V2 DAR construct includes a short hinge, and the signaling region includes 4-1BB and CD3zeta signaling sequences (e.g., DAR (V2b) construct).
  • the CD38 V3 DAR construct includes a short hinge sequence, and the signaling region includes 4-1BB and CD3-zeta (with only ITAM 3 motif) signaling sequences (e.g., DAR (V3) construct).
  • the CD38 DAR V4 construct lacks a hinge sequence, and the signaling region includes 4-1BB and CD3- zeta (with only IT AM 3 motif) signaling sequences (e.g., DAR (V4) construct).
  • Figure 34 is a bar graph showing the level of TNF-alpha release from T cells expressing CD38 DAR V2, V3 or V4 constructs. Each data set shows from left to right RPMI 8226 cells, K562 cells and medium.
  • the negative control is a cell line carrying a knocked-out TRAC gene.
  • the CD38 V2 DAR construct includes a short hinge, and the signaling region includes 4-1BB and CD3zeta signaling sequences (e.g., DAR (V2b) construct).
  • the CD38 V3 DAR construct includes a short hinge sequence, and the signaling region includes 4-1BB and CD3-zeta (with only ITAM 3 motif) signaling sequences (e.g., DAR (V3) construct).
  • the CD38 DAR V4 construct lacks a hinge sequence, and the signaling region includes 4-1BB and CD3- zeta (with only ITAM 3 motif) signaling sequences (e.g., DAR (V4) construct).
  • Figure 35A shows amino acid sequences of different regions of CD38 dimeric antigen receptor (DAR) construct.
  • Figure 35B shows amino acid sequences of a first polypeptide, a second polypeptide and precursor polypeptide of a CD38 dimeric antigen receptor (DAR) construct V2b.
  • DAR dimeric antigen receptor
  • Figure 35C shows amino acid sequences of a first polypeptide, a second polypeptide and precursor polypeptide of a CD38 dimeric antigen receptor (DAR) construct V3.
  • DAR dimeric antigen receptor
  • Figure 35D shows amino acid sequences of a long hinge sequence comprising CD8 and CD28 hinge sequences, and a CD3zeta signaling region having only ITAM 3 motif, a CD8 hinge sequence, and an anti-CD38 CAR construct.
  • Enzymatic reactions and enrichment/purification techniques are also well known and are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein.
  • the terminology used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
  • the term“and/or” used herein is to be taken mean specific disclosure of each of the specified features or components with or without the other.
  • the term“and/or” as used in a phrase such as“A and/or B” herein is intended to include“A and B,”“A or B,”“A” (alone), and“B” (alone).
  • the term“and/or” as used in a phrase such as“A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • the term“about” refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example,“about” or
  • “approximately” can mean within one or more than one standard deviation per the practice in the art.
  • “about” or“approximately” can mean a range of up to 10% (i.e., ⁇ 10%) or more depending on the limitations of the measurement system.
  • about 5 mg can include any number between 4.5 mg and 5.5 mg.
  • the terms can mean up to an order of magnitude or up to 5-fold of a value.
  • polypeptide and “protein” and other related terms used herein are used interchangeably and refer to a polymer of amino acids and are not limited to any particular length. Polypeptides may comprise natural and non-natural amino acids. Polypeptides include recombinant or chemically-synthesized forms. Polypeptides also include precursor molecules that have not yet been subjected to post-translation modification such as proteolytic cleavage, cleavage due dot T2A ribosomal skipping, hydroxylation, methylation, lipidation, acetylation, SUMOylation, ubiquitination, glycosylation, phosphorylation and/or disulfide bond formation.
  • post-translation modification such as proteolytic cleavage, cleavage due dot T2A ribosomal skipping, hydroxylation, methylation, lipidation, acetylation, SUMOylation, ubiquitination, glycosylation, phosphorylation and/or disulfide bond formation
  • nucleic acid refers to polymers of nucleotides and are not limited to any particular length.
  • Nucleic acids include recombinant and chemically-synthesized forms. Nucleic acids include DNA molecules (cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof. Nucleic acid molecule can be single-stranded or double-stranded.
  • nucleic acid molecules of the disclosure comprise a contiguous open reading frame encoding an antibody, or a fragment or scFv, derivative, mutein, or variant thereof.
  • nucleic acids comprise a one type of polynucleotides or a mixture of two or more different types of
  • nucleic acids encoding the dimeric antigen receptors are described herein.
  • An antigen binding protein can have, for example, the structure of an
  • an "immunoglobulin” refers to a tetrameric molecule composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy” chain (about 50-70 kDa).
  • the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa or lambda light chains.
  • Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes).
  • the variable regions of each light/heavy chain pair form the antibody binding site such that an intact immunoglobulin has two antigen binding sites.
  • an antigen binding protein can be a synthetic molecule having a structure that differs from a tetrameric immunoglobulin molecule but still binds a target antigen or binds two or more target antigens.
  • a synthetic antigen binding protein can comprise antibody fragments, 1-6 or more polypeptide chains, asymmetrical assemblies of polypeptides, or other synthetic molecules. Dimeric antigen receptors having immunoglobulin-like properties that bind specifically to target antigens are described herein.
  • an "antibody” and“antibodies” and related terms used herein refers to an intact immunoglobulin or to an antigen binding portion thereof that binds specifically to an antigen.
  • Antigen binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen binding portions include, inter alia , Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide.
  • Antibodies include recombinantly produced antibodies and antigen binding portions.
  • Antibodies include non-human, chimeric, humanized and fully human antibodies.
  • Antibodies include monospecific, multispecific (e.g., bispecific, trispecific and higher order specificities).
  • Antibodies include tetrameric antibodies, light chain monomers, heavy chain monomers, light chain dimers, heavy chain dimers.
  • Antibodies include F(ab’)2 fragments, Fab’ fragments and Fab fragments.
  • Antibodies include single domain antibodies, monovalent antibodies, single chain antibodies, single chain variable fragment (scFv), camelized antibodies, affibodies, disulfide- linked Fvs (sdFv), anti -idiotypic antibodies (anti-id), minibodies.
  • Antibodies include monoclonal and polyclonal populations. Dimeric antigen receptors that behave like antibodies are described herein.
  • An“antigen binding domain,”“antigen binding region,” or“antigen binding site” and other related terms used herein refer to a portion of an antigen binding protein that contains amino acid residues (or other moieties) that interact with an antigen and contribute to the antigen binding protein's specificity and affinity for the antigen. For an antibody that specifically binds to its antigen, this will include at least part of at least one of its CDR domains. Dimeric antigen receptors having antibody heavy chain variable region and the antibody light chain variable region that form an antigen binding domain are described herein.
  • telomere binding refers to non-covalent or covalent preferential binding to an antigen relative to other molecules or moieties (e.g., an antibody specifically binds to a particular antigen relative to other available antigens).
  • an antibody specifically binds to a target antigen if it binds to the antigen with a dissociation constant KD of 10 5 M or less, or 10 6 M or less, or 10 7 M or less, or 10 8 M or less, or 10 9 M or less, or 10 10 M or less.
  • Dimeric antigen receptors that specifically bind target antigens are described herein.
  • a dissociation constant can be measured using a BIACORE surface plasmon resonance (SPR) assay.
  • SPR surface plasmon resonance refers to an optical phenomenon that allows for the analysis of real-time interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIACORE system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ).
  • An "epitope" and related terms as used herein refers to a portion of an antigen that is bound by an antigen binding protein (e.g., by an antibody or an antigen binding portion thereof).
  • An epitope can comprise portions of two or more antigens that are bound by an antigen binding protein.
  • An epitope can comprise non-contiguous portions of an antigen or of two or more antigens (e.g., amino acid residues that are not contiguous in an antigen’s primary sequence but that, in the context of the antigen’s tertiary and quaternary structure, are near enough to each other to be bound by an antigen binding protein).
  • the variable regions, particularly the CDRs, of an antibody interact with the epitope. Dimeric antigen receptors that bind an epitope of a target antigen (e.g., CD38) are described herein.
  • an "antibody fragment”, “antibody portion”, “antigen-binding fragment of an antibody”, or “antigen-binding portion of an antibody” and other related terms used herein refer to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; Fd; and Fv fragments, as well as dAb; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding to the polypeptide.
  • Antigen binding portions of an antibody may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen binding portions include, inter alia, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), and complementarity determining region (CDR) fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer antigen binding properties to the antibody fragment.
  • Dimeric antigen receptors comprising a Fab fragment joined to a hinge, transmembrane and intracellular signaling regions are described herein.
  • the terms“Fab”,“Fab fragment” and other related terms refers to a monovalent fragment comprising a variable light chain region (VL), constant light chain region (CL), variable heavy chain region (VH), and first constant region (CHI).
  • a Fab is capable of binding an antigen.
  • An F(ab')2 fragment is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.
  • a F(Ab’)2 has antigen binding capability.
  • An Fd fragment comprises VH and CHI regions.
  • An Fv fragment comprises VL and VH regions.
  • An Fv can bind an antigen.
  • a dAb fragment has a VH domain, a VL domain, or an antigen-binding fragment of a VH or VL domain
  • U.S. Patents 6,846,634 and 6,696,245 U.S. published Application Nos. 2002/02512, 2004/0202995, 2004/0038291, 2004/0009507, 2003/0039958; and Ward et al., Nature 341 :544- 546, 1989.
  • Dimeric antigen receptors comprising a Fab fragment joined to a hinge
  • transmembrane and intracellular signaling regions are described herein.
  • variable and constant domains are derived from human immunoglobulin sequences (e.g., a fully human antibody).
  • human immunoglobulin sequences e.g., a fully human antibody.
  • These antibodies may be prepared in a variety of ways, examples of which are described below, including through recombinant methodologies or through immunization with an antigen of interest of a mouse that is genetically modified to express antibodies derived from human heavy and/or light chain-encoding genes. Dimeric antigen receptors comprising fully human antibody heavy chain variable region and the antibody light chain variable region are described herein.
  • a hinge region refers to an amino acid segment that is generally found between two domains of a protein and may allow for flexibility of the overall construct and movement of one or both of the domains relative to one another.
  • a hinge region comprises from about 10 to about 100 amino acids, e.g., from about 15 to about 75 amino acids, from about 20 to about 50 amino acids, or from about 30 to about 60 amino acids.
  • the hinge region is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length.
  • the hinge region can be derived from is a hinge region of a naturally-occurring protein, such as a CD8 hinge region or a fragment thereof, a CD8a hinge region, or a fragment thereof, a hinge region of an antibody (e.g., IgG, IgA, IgM, IgE, or IgD antibodies), or a hinge region that joins the constant domains CH1 and CH2 of an antibody.
  • a hinge region of a naturally-occurring protein such as a CD8 hinge region or a fragment thereof, a CD8a hinge region, or a fragment thereof, a hinge region of an antibody (e.g., IgG, IgA, IgM, IgE, or IgD antibodies), or a hinge region that joins the constant domains CH1 and CH2 of an antibody.
  • the hinge region can be derived from an antibody and may or may not comprise one or more constant regions of the antibody, or the hinge region comprises the hinge region of an antibody and the CH3 constant region of the antibody, or the hinge region comprises the hinge region of an antibody and the CH2 and CH3 constant regions of the antibody, or the hinge region is a non-naturally occurring peptide, or the hinge region is disposed between the C-terminus of the scFv and the N-terminus of the transmembrane domain.
  • leader sequence or“leader peptide” or“peptide signal sequence” or “signal peptide” refers to a peptide sequence that is located at the N-terminus of a polypeptide.
  • a leader sequence directs a polypeptide chain to a cellular secretory pathway and can direct integration and anchoring of the polypeptide into the lipid bilayer of the cellular membrane. Typically, a leader sequence is about 10-50 amino acids in length.
  • a leader sequence can direct transport of a precursor polypeptide from the cytosol to the endoplasmic reticulum.
  • a leader sequence include signal sequences comprising CD8a, CD28 or CD 16 leader sequences.
  • CAR Chimeric Antigen Receptor
  • CAR Chimeric Antigen Receptor
  • the CAR extracellular binding domain is a single chain variable fragment (scFv or sFv) derived from fusing the variable heavy and light regions of a monoclonal antibody, such as a human monoclonal antibody.
  • the disclosed constructs are DARs which are distinct from CARs in that DARs do not use a single chain antibody for targeting but instead use separate heavy and light chain variable domain regions.
  • a "vector" and related terms used herein refers to a nucleic acid molecule (e.g., DNA or RNA) which can be operably linked to foreign genetic material (e.g., nucleic acid transgene).
  • Vectors can be used as a vehicle to introduce foreign genetic material into a cell (e.g., host cell).
  • Vectors can include at least one restriction endonuclease recognition sequence for insertion of the transgene into the vector.
  • Vectors can include at least one gene sequence that confers antibiotic resistance or a selectable characteristic to aid in selection of host cells that harbor a vector-transgene construct.
  • Vectors can be single-stranded or double-stranded nucleic acid molecules.
  • Vectors can be linear or circular nucleic acid molecules.
  • a donor nucleic acid used for gene editing methods employing zinc finger nuclease, TALEN or CRISPR/Cas can be a type of a vector.
  • One type of vector is a "plasmid," which refers to a linear or circular double stranded extrachromosomal DNA molecule which can be linked to a transgene, and is capable of replicating in a host cell, and transcribing and/or translating the transgene.
  • a viral vector typically contains viral RNA or DNA backbone sequences which can be linked to the transgene. The viral backbone sequences can be modified to disable infection but retain insertion of the viral backbone and the co-linked transgene into a host cell genome.
  • viral vectors examples include retroviral, lentiviral, adenoviral, adeno-associated, baculoviral, papovaviral, vaccinia viral, herpes simplex viral and Epstein Barr viral vectors.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors comprising a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • An "expression vector” is a type of vector that can contain one or more regulatory sequences, such as inducible and/or constitutive promoters and enhancers.
  • Expression vectors can include ribosomal binding sites and/or polyadenylation sites.
  • Regulatory sequences direct transcription, or transcription and translation, of a transgene linked to the expression vector which is transduced into a host cell.
  • the regulatory sequence(s) can control the level, timing and/or location of expression of the transgene.
  • the regulatory sequence can, for example, exert its effects directly on the transgene, or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequence and/or the nucleic acid).
  • Regulatory sequences can be part of a vector.
  • regulatory sequences are described in, for example, Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif and Baron et ah, 1995, Nucleic Acids Res. 23:3605-3606.
  • Expression vectors comprising nucleic acids that encode dimeric antigen receptors are described herein.
  • a transgene is“operably linked” to a vector when there is linkage between the transgene and the vector to permit functioning or expression of the transgene sequences contained in the vector.
  • a transgene is "operably linked” to a regulatory sequence when the regulatory sequence affects the expression (e.g., the level, timing, or location of expression) of the transgene.
  • transfected or “transformed” or “transduced” or other related terms used herein refer to a process by which exogenous nucleic acid (e.g., transgene) is transferred or introduced into a host cell.
  • a "transfected” or “transformed” or “transduced” host cell is one which has been transfected, transformed or transduced with exogenous nucleic acid (transgene).
  • the host cell includes the primary subject cell and its progeny.
  • Exogenous nucleic acids encoding at least a portion of any of the dimeric antigen receptors are described herein and can be introduced into a host cell.
  • Expression vectors comprising at least a portion of any of the dimeric antigen receptors described herein can be introduced into a host cell, and the host cell can express the first and second polypeptides that dimerize to form the dimeric antigen receptors described herein.
  • the terms "host cell” or“or a population of host cells” or related terms as used herein refer to a cell (or a population thereof) into which foreign (exogenous or transgene) nucleic acids have been introduced.
  • the foreign nucleic acids can include an expression vector operably linked to a transgene, and the host cell can be used to express the nucleic acid and/or polypeptide encoded by the foreign nucleic acid (transgene).
  • a host cell (or a population thereof) can be a cultured cell or can be extracted from a subject.
  • the host cell (or a population thereof) includes the primary subject cell and its progeny without any regard for the number of passages.
  • the host cell (or a population thereof) includes immortalized cell lines.
  • Progeny cells may or may not harbor identical genetic material compared to the parent cell.
  • Host cells encompass progeny cells.
  • a host cell describes any cell (including its progeny) that has been modified, transfected, transduced, transformed, and/or manipulated in any way to express an antibody, as disclosed herein.
  • the host cell (or population thereof) can be introduced with an expression vector operably linked to a nucleic acid encoding the desired antibody, or an antigen binding portion thereof, described herein.
  • Host cells and populations thereof can harbor an expression vector that is stably integrated into the host’s genome or can harbor an extrachromosomal expression vector.
  • host cells and populations thereof can harbor an extrachromosomal vector that is present after several cell divisions or is present transiently and is lost after several cell divisions.
  • a host cell can be a prokaryote, for example, E. coli , or it can be a eukaryote, for example, a single-celled eukaryote (e.g., a yeast or other fungus), a plant cell (e.g., a tobacco or tomato plant cell), an mammalian cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell) or a hybridoma.
  • a prokaryote for example, E. coli
  • a eukaryote for example, a single-celled eukaryote (e.g., a yeast or other fungus)
  • a plant cell e.g., a tobacco or tomato plant cell
  • an mammalian cell e.g., a human cell, a monkey cell, a hamster cell, a rat cell,
  • a host cell can be introduced with an expression vector operably linked to a nucleic acid encoding a desired antibody thereby generating a transfected/transformed host cell which is cultured under conditions suitable for expression of the antibody by the transfected/transformed host cell, and optionally recovering the antibody from the transfected/transformed host cells (e.g., recovery from host cell lysate) or recovery from the culture medium.
  • host cells comprise non-human cells including CHO, BHK, NSO, SP2/0, and YB2/0.
  • host cells comprise human cells including HEK293, HT-1080, Huh-7 and PER.C6.
  • host cells examples include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (see Gluzman et ah, 1981, Cell 23: 175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells or their derivatives such as Veggie CHO and related cell lines which grow in serum- free media (see Rasmussen et ah, 1998, Cytotechnology 28:31) or CHO strain DX-B 11, which is deficient in DHFR (see ETrlaub et ah, 1980, Proc. Natl. Acad. Sci.
  • COS-7 line of monkey kidney cells ATCC CRL 1651
  • L cells C127 cells
  • 3T3 cells ATCC CCL 163
  • CHO Chinese hamster ovary
  • HeLa cells include lymphoid cells such as Y0, NSO or Sp20.
  • a host cell is a mammalian host cell, but is not a human host cell.
  • a host cell is a cultured cell that can be transformed or transfected with a polypeptide-encoding nucleic acid, which can then be expressed in the host cell.
  • the phrase“transgenic host cell” or "recombinant host cell” can be used to denote a host cell that has been introduced (e.g., transformed or transfected) with a nucleic acid to be expressed.
  • a host cell also can be a cell that comprises the nucleic acid but does not express it at a desired level unless a regulatory sequence is introduced into the host cell such that it becomes operably linked with the nucleic acid.
  • host cell refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to, e.g., mutation or environmental influence, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • the host cell or the population of host cells comprise T lymphocytes (e.g., T cells, regulatory T cells, gamma-delta T cells, and cytotoxic T cells), NK (natural killer) cells, macrophages, dendritic cells, mast cells, eosinophils, B lymphocytes, monocytes.
  • T lymphocytes e.g., T cells, regulatory T cells, gamma-delta T cells, and cytotoxic T cells
  • NK natural killer cells
  • macrophages e.g., dendritic cells, mast cells, eosinophils, B lymphocytes, monocytes.
  • the NK cells comprise cord blood-derived NK cells, or placental derived NK cells.
  • Transgenic host cells can be prepared using non-viral methods, including well-known designer nucleases including zinc finger nucleases, TALENS or CRISPR/Cas.
  • a transgene can be introduced into a host cell’s genome using genome editing technologies such as zinc finger nuclease.
  • a zinc finger nuclease includes a pair of chimeric proteins each containing a non specific endonuclease domain of a restriction endonuclease (e.g., Fokl ) fused to a DNA-binding domain from an engineered zinc finger motif.
  • the DNA-binding domain can be engineered to bind a specific sequence in the host’s genome and the endonuclease domain makes a double- stranded cut.
  • the donor DNA carries the transgene, for example any of the nucleic acids encoding a CAR or DAR construct described herein, and flanking sequences that are
  • Transgenic mammalian host cells have been prepared using zinc finger nucleases (U.S. patent Nos. 9,597,357, 9,616,090, 9,816,074 and 8,945,868).
  • a transgenic host cell can be prepared using TALEN (Transcription Activator-Like Effector Nucleases) which are similar to zinc finger nucleases in that they include a non-specific endonuclease domain fused to a DNA- binding domain which can deliver precise transgene insertion.
  • TALEN Transcription Activator-Like Effector Nucleases
  • TALEN Like zinc finger nucleases, TALEN also introduce a double-strand cut into the host’s DNA.
  • Transgenic host cells can be prepared using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats).
  • CRISPR employs a Cas endonuclease coupled to a guide RNA for target specific donor DNA integration.
  • the guide RNA includes a conserved multi -nucleotide containing protospacer adjacent motif (PAM) sequence upstream of the gRNA-binding region in the target DNA and hybridizes to the host cell target site where the Cas endonuclease cleaves the double-stranded target DNA.
  • the guide RNA can be designed to hybridize to a specific target site. Similar to zinc finger nuclease and TALEN, the CRISPR/Cas system can be used to introduce site specific insertion of donor DNA having flanking sequences that have homology to the insertion site. Examples of
  • transgenic host cells can be prepared using zinc finger nuclease, TALEN or CRISPR/Cas system, and the host target site can be a TRAC gene (T Cell Receptor Alpha Constant).
  • the donor DNA can include for example any of the nucleic acids encoding a CAR or DAR construct described herein. Electroporation, nucleofection or lipofection can be used to co-deliver into the host cell the donor DNA with the zinc finger nuclease, TALEN or CRISPR/Cas system.
  • Transgenic host cells can be prepared by transducing T cells with a retroviral vector carrying the CAR or DAR construct.
  • the transduction can be performed essentially as described in Ma et al., 2004 The Prostate 61 : 12-25; and Ma et al., The Prostate 74(3):286-296, 2014 (the disclosures of which are incorporated by reference herein in their entireties).
  • the anti-CD38 CAR or DAR MFG retroviral vector plasmid DNA can be transfected into a Phoenix-Eco cell line (ATCC) using FuGene reagent (Promega, Madison, WI) to produce Ecotropic retrovirus, then harvest transient viral supernatant (Ecotropic virus) can be used to transduce PG13 packaging cells with Gal-V envelope to produce retrovirus to infect human cells. Viral supernatant from the PG13 cells can be used to transduce activated T cells (or PBMCs) two to three days after CD3 or CD3/CD28 activation.
  • ATCC Phoenix-Eco cell line
  • FuGene reagent Promega, Madison, WI
  • Ecotropic virus transient viral supernatant
  • Viral supernatant from the PG13 cells can be used to transduce activated T cells (or PBMCs) two to three days after CD3 or CD3/CD28 activation.
  • Activated human T cells can be prepared by activating normal healthy donor peripheral blood mononuclear cells (PBMC) with 100 ng/ml mouse anti-human CD3 antibody OKT3 (Orth Biotech, Rartian, NJ) or anti-CD3,anti-CD28 TransAct (Miltenyi Biotech, German) as manufacturer’s manual and 300-1000 U/ml IL2 in AIM-V growth medium (GIBCO-Thermo Fisher scientific, Waltham, MA) supplemented with 5% FBS for two days.
  • PBMC peripheral blood mononuclear cells
  • OKT3 Orth Biotech, Rartian, NJ
  • anti-CD3,anti-CD28 TransAct Miltenyi Biotech, German
  • Approximately 5x 10 6 activated human T cells can be transduced in a 10 pg/ml retronectin (Takara Bio ETSA) pre-coated 6-well plate with 3 ml viral supernatant and centrifuged at 1000 g for about 1 hour at approximately 32 °C. After transduction, the transduced T cells can be expanded in AIM-V growth medium supplemented with 5% FBS and 300-1000 U/ml IL2.
  • Polypeptides of the present disclosure can be produced using any methods known in the art.
  • the polypeptides are produced by recombinant nucleic acid methods by inserting a nucleic acid sequence (e.g., DNA) encoding the polypeptide into a recombinant expression vector which is introduced into a host cell and expressed by the host cell under conditions promoting expression.
  • a nucleic acid sequence e.g., DNA
  • nucleic acid e.g., DNA
  • the nucleic acid encoding the polypeptide is operably linked to an expression vector carrying one or more suitable transcriptional or translational regulatory elements derived from mammalian, viral, or insect genes.
  • Such regulatory elements include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation.
  • the expression vector can include an origin or replication that confers replication capabilities in the host cell.
  • the expression vector can include a gene that confers selection to facilitate recognition of transgenic host cells (e.g., transformants).
  • the recombinant DNA can also encode any type of protein tag sequence that may be useful for purifying the protein.
  • protein tags include but are not limited to a histidine tag, a FLAG tag, a myc tag, an HA tag, or a GST tag.
  • Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts can be found in Cloning Vectors: A Laboratory Manual, (Elsevier, N.Y., 1985).
  • the expression vector construct can be introduced into the host cell using a method appropriate for the host cell.
  • a variety of methods for introducing nucleic acids into host cells are known in the art, including, but not limited to, electroporation; transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; viral transfection; non-viral transfection; microprojectile bombardment; lipofection; and infection (e.g., where the vector is an infectious agent).
  • Suitable host cells include prokaryotes, yeast, mammalian cells, or bacterial cells.
  • Suitable bacteria include gram negative or gram positive organisms, for example, E. coli or Bacillus spp. Yeast, such as from the Saccharomyces species, such as S. cerevisiae, may also be used for production of polypeptides.
  • Yeast such as from the Saccharomyces species, such as S. cerevisiae
  • Saccharomyces species such as S. cerevisiae
  • Various mammalian or insect cell culture systems can also be employed to express recombinant proteins. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers,
  • suitable mammalian host cell lines include endothelial cells, COS-7 monkey kidney cells, CV-l, L cells, C127, 3T3, Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293, 293T, and BHK cell lines.
  • Purified polypeptides are prepared by culturing suitable host/vector systems to express the recombinant proteins. The protein is then purified from culture media or cell extracts. Any of the first and second polypeptides that form a dimeric antigen receptor can be expressed by transgenic host cells.
  • Antibodies and antigen binding proteins disclosed herein can also be produced using cell-translation systems.
  • the nucleic acids encoding the polypeptide must be modified to allow in vitro transcription to produce mRNA and to allow cell-free translation of the mRNA in the particular cell-free system being utilized (eukaryotic such as a mammalian or yeast cell-free translation system or prokaryotic such as a bacterial cell-free translation system.
  • Nucleic acids encoding any of the various polypeptides disclosed herein may be synthesized chemically. Codon usage may be selected so as to improve expression in a cell. Such codon usage will depend on the cell type selected. Specialized codon usage patterns have been developed for A. coli and other bacteria, as well as mammalian cells, plant cells, yeast cells and insect cells. See for example: Mayfield et ah, Proc. Natl. Acad. Sci. USA. 2003 l00(2):438-42; Sinclair et al. Protein Expr. Purif. 2002 (l):96-l05; Connell N D. Curr. Opin. Biotechnol. 2001 l2(5):446-9; Makrides et al. Microbiol. Rev. 1996 60(3):512-38; and Sharp et al. Yeast. 1991 7(7):657-78.
  • Antibodies and antigen binding proteins described herein can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, Ill.). Modifications to the protein can also be produced by chemical synthesis.
  • Antibodies and antigen binding proteins described herein can be purified by isolation/purification methods for proteins generally known in the field of protein chemistry.
  • Non-limiting examples include extraction, recrystallization, salting out (e.g., with ammonium sulfate or sodium sulfate), centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal phase chromatography, reversed-phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution or any combinations of these.
  • polypeptides may be exchanged into different buffers and/or concentrated by any of a variety of methods known to the art, including, but not limited to, filtration and dialysis.
  • the purified antibodies and antigen binding proteins described herein are at least 65% pure, at least 75% pure, at least 85% pure, at least 95% pure, or at least 98% pure. Regardless of the exact numerical value of the purity, the polypeptide is sufficiently pure for use as a pharmaceutical product. Any of the dimeric antigen receptors described herein can be expressed by transgenic host cells and then purified to about 65-98% purity or high level of purity using any art-known method.
  • the antibodies and antigen binding proteins herein can further comprise post-translational modifications.
  • post-translational protein modifications include phosphorylation, acetylation, methylation, ADP-ribosylation,
  • glycosylation can be sialylation, which conjugates one or more sialic acid moieties to the polypeptide.
  • Sialic acid moieties improve solubility and serum half-life while also reducing the possible immunogenicity of the protein. See Raju et al. Biochemistry. 2001 31; 40(30):8868-76.
  • the antibodies and antigen binding proteins described herein can be modified to become soluble polypeptides which comprises linking the Antibodies and antigen binding proteins to non-proteinaceous polymers.
  • the non-proteinaceous polymer comprises polyethylene glycol (“PEG”), polypropylene glycol, or polyoxyalkylenes, in the manner as set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
  • compositions comprising any of the dimeric antigen receptors described herein in an admixture with a pharmaceutically-acceptable excipient.
  • An excipient encompasses carriers, stabilizers and excipients.
  • Excipients of pharmaceutically acceptable excipients includes for example inert diluents or fillers (e.g., sucrose and sorbitol), lubricating agents, glidants, and anti-adhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Additional examples include buffering agents, stabilizing agents, preservatives, non-ionic detergents, anti-oxidants and isotonifiers.
  • Therapeutic compositions and methods for preparing them are well known in the art and are found, for example, in“Remington: The Science and Practice of Pharmacy” (20th ed., ed. A. R. Gennaro A R., 2000, Lippincott Williams & Wilkins, Philadelphia, Pa.).
  • Therapeutic compositions can be formulated for parenteral administration may, and can for example, contain excipients, sterile water, saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
  • Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the antibody (or antigen binding protein thereof) described herein.
  • Nanoparticulate formulations may be used to control the biodistribution of the antibody (or antigen binding protein thereof).
  • Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • concentration of the antibody (or antigen binding protein thereof) in the formulation varies depending upon a number of factors, including the dosage of the drug to be administered, and the route of administration.
  • any of the dimeric antigen receptors describe herein may be optionally administered as a pharmaceutically acceptable salt, such as non-toxic acid addition salts or metal complexes that are commonly used in the pharmaceutical industry.
  • acid addition salts include organic acids such as acetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic acids or the like; polymeric acids such as tannic acid, carboxymethyl cellulose, or the like; and inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid, or the like.
  • Metal complexes include zinc, iron, and the like.
  • the antibody or antigen binding portions thereof
  • the antibody is formulated in the presence of sodium acetate to increase thermal stability.
  • any of the dimeric antigen receptors described herein may be formulated for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients.
  • Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium.
  • the present disclosure provides dimeric antigen receptors (DARs) comprising a Fab fragment joined to a transmembrane region and intracellular signaling regions.
  • the DAR construct includes an optional hinge region between the Fab fragment and the transmembrane region.
  • the present disclosure of DAR structures provides unexpected and surprising results that compare a DAR structure having a Fab format antibody to a CAR structure having an scFv format of the same antibody.
  • the DAR and CAR formats directly compare because the hinge regions, transmembrane regions and two intracellular signaling regions are the same. Yet the activity of the DAR format was superior to its corresponding CAR format.
  • the present disclosure provides dimeric antigen receptors (DAR) constructs comprising a heavy chain binding region on one polypeptide chain and a light chain binding region on a separate polypeptide chain.
  • DAR dimeric antigen receptors
  • the two polypeptide chains that make up the dimeric antigen receptors can dimerize to form a protein complex.
  • the dimeric antigen receptors have antibody-like properties as they bind specifically to a target antigen.
  • the dimeric antigen receptors can be used for directed cell therapy.
  • the present disclosure provides a structure for a DAR (dimeric antigen receptor) construct having a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a heavy chain variable region of an antibody and the second polypeptide chain comprises a light chain variable region of an antibody, wherein the first polypeptide chain is linked to the second polypeptide chain by one or a plurality of disulfide bonds at regions outside of a transduced cell when both the first polypeptide chain and the second polypeptide chain are expressed by a same cell.
  • DAR dimeric antigen receptor
  • a DAR construct comprises a first polypeptide chain comprising, in sequence, an antibody heavy chain (or light chain) with a variable domain region and a CH1 region (kappa (K) or lambda (L)) with a corresponding CLICK region, a hinge region, a transmembrane region, and one or two signaling domains and a second polypeptide chain comprising, and an antibody light chain (or heavy chain) variable domain region (kappa (K) or lambda (L)) with a corresponding CLICK region, wherein the CH regions in each first and second polypeptide chains are linked with one or two disulfide bonds at a hinge sequence.
  • the present disclosure provides a structure for a DAR (dimeric antigen receptor) construct having a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a heavy chain variable region of an antibody and the second polypeptide chain comprises a light chain variable region of an antibody, wherein the first polypeptide chain is linked to the second polypeptide chain by one or a plurality of disulfide bonds at regions outside of a transduced cell when both the first polypeptide chain and the second polypeptide chain are expressed by a same cell.
  • DAR dimeric antigen receptor
  • a DAR construct comprises a first polypeptide chain comprising, in sequence, an antibody heavy chain with a variable domain region and a CH1 region (kappa (K) or lambda (L)) with a corresponding CL/CK region, a hinge region, a transmembrane region, and one or two signaling domains and a second polypeptide chain comprising, an antibody light chain variable domain region (kappa or lambda) with a corresponding CL/CK region, wherein the CH regions in each first and second polypeptide chains are linked with one or two disulfide bonds at the hinge sequence.
  • the DAR construct comprises an antibody heavy chain variable region and an antibody light chain variable region on separate polypeptide chains, wherein the heavy chain variable region and the light chain variable region form an antigen binding domain.
  • the hinge region is about 10 to about 100 amino acids in length.
  • the hinge region is independently selected from the group consisting of a CD8 hinge region or a fragment thereof, a CD8a hinge region or a fragment thereof, a hinge region of an antibody (IgG, IgA, IgM, IgE, or IgD) joining the constant domains CH1 and CH2 of an antibody.
  • the hinge region can be derived from an antibody and may or may not comprise one or more constant regions of the antibody.
  • the transmembrane domain can be derived from a membrane protein sequence region selected from the group consisting of CD8a, CD8P, 4-1BB/CD137, CD28, CD34, CD4, FceRIy, CD16, OX40/CD134, O ⁇ 3z, CD3e, CD3y, CD35, TCRa, TCRp, TCRC, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD33, CD37, CD64, CD80, CD86, CD137, CD154, LFA-l T cell co-receptor, CD2 T cell co-receptor/adhesion molecule, CD40, CD40L/CD154, VEGFR2, FAS, and FGFR2B.
  • a membrane protein sequence region selected from the group consisting of CD8a, CD8P, 4-1BB/CD137, CD28, CD34, CD4, FceRIy, CD16, OX40/CD134, O ⁇ 3z, CD3e, CD3y, CD35,
  • the signaling region is selected from the group consisting of signaling regions from CD3-zeta chain, 4-1BB, CD28, CD27, 0X40, CD30, CD40, PD-l, ICOS, lymph oocyte function-associated antigen-l (LFA-l), CD2, CD7, LIGHT, NKG2C, B7-H3, GITR (TNFRSF18), DR3 (TNFRSF25), TNFR2, CD226, and combinations thereof.
  • LFA-l lymph oocyte function-associated antigen-l
  • a general design of a dimeric antigen receptor includes a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an antigen binding region connected to a dimerization region, connected to a hinge region, connected to a transmembrane region, and connected to one or a plurality of intracellular signaling sequence region(s), and wherein the second polypeptide chain comprises an antigen binding domain and a dimerization domain.
  • the antigen binding domain on one or both of the first and the second polypeptide chains is selected from the group consisting of a heavy chain variable region, a light chain variable region, an extracellular region of a cytokine receptor, a single domain antibody, and combinations thereof.
  • the dimerization domain on one or both of the first and second polypeptide chains is selected from the group consisting of a kappa light chain constant region, a lambda light chain constant region, a leucine zipper, myc-max components, and combinations thereof.
  • the“S-S” represents any chemical bond or association that results in dimerization of the first and second polypeptide chains, including disulfide bond, leucine zipper or myc-max components.
  • the present disclosure provides dimeric antigen receptors (DAR) constructs where the first polypeptide chain carries the heavy chain variable (VH) and heavy chain constant regions (CH), and the second polypeptide chain carries the light chain variable (VL) and light chain constant regions (CL) (e.g., Figures 1 and 2).
  • the dimeric antigen receptors (DAR) construct comprises: (a) a first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) an optional hinge region,
  • TM transmembrane region
  • VL antibody light chain variable region
  • CL antibody light chain constant region
  • the present disclosure provides dimeric antigen receptors (DAR) constructs where the first polypeptide chain carries the light chain variable (VL) and light chain constant regions (CL), and the second polypeptide chain carries the heavy chain variable (VH) and heavy chain constant regions (CH) (e.g., Figures 3 and 4).
  • DAR dimeric antigen receptors
  • the dimeric antigen receptors (DAR) constructs comprises (a) a first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region (VL), (ii) an antibody light chain constant region (CL), (iii) an optional hinge region, (iv) a transmembrane region (TM), and (v) an intracellular signaling region; (b) a second polypeptide chain comprising two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region (VH), and (ii) an antibody heavy chain constant region (CH).
  • the antibody heavy chain constant region (CH) and the antibody light chain constant region (CL) can dimerize to form a dimerization domain.
  • the antibody heavy chain constant region and the antibody light chain constant region dimerize via one or two disulfide bonds.
  • the antibody heavy chain variable region (VH) and the antibody light chain variable region (VL) associate with each other to form an antigen binding domain.
  • the antibody heavy chain variable region and the antibody light chain variable region associate with each other when the antibody heavy chain constant region and the antibody light chain constant region dimerize.
  • the antigen binding domain which is formed from the antibody heavy chain variable region and the antibody light chain variable region, binds a target antigen.
  • the antibody heavy chain variable region and the antibody light chain variable region are fully human antibody regions.
  • the hinge region is about 10 to about 100 amino acids in length.
  • the hinge region comprises a hinge region or a fragment thereof from an antibody (e.g., IgG, IgA, IgM, IgE, or IgD).
  • the hinge region comprises a CD8 (e.g., CD8a) or CD28 hinge region or a fragment thereof.
  • the hinge region comprises a CPPC or SPPC amino acid sequence.
  • the hinge region comprises both CD8 and CD28 hinge sequences (e.g., long hinge region), only CD8 sequence (short hinge) or only CD28 hinge sequence (e.g., short hinge region).
  • the transmembrane regions of the first and second polypeptide chains can be independently derived from CD 8 a, CD8p, 4-1BB/CD137, CD28, CD34, CD4, FceRIy, CD16, OX40/CD134, CD3C, CD3e, CD3y, CD35, TCRa, TCRp, TCRC, CD32, CD64, CD64, CD45, CD5, CD9, CD22,
  • the intracellular signaling region of the first polypeptide comprises intracellular signaling sequences in any order and of any combination of two to five signaling sequences from 4-1BB, CD3zeta, CD28, CD27, 0X40, CD30, CD40, PD-l, ICOS, lymphocyte function-associated antigen-l (LFA-l), CD2, CD7, LIGHT, NKG2C, B7-H3, GITR (TNFRSF18), DR3 (TNFRSF25), TNFR2, CD226, and combinations thereof.
  • the intracellular signaling region comprises intracellular signaling sequences from any one or any combination of two or more of CD28, 4-1BB and/or CD3-zeta. In one embodiment, the intracellular signaling region comprises CD28 and CD3-zeta intracellular signaling sequences, or 4-1BB and CD3-zeta intracellular signaling sequences. In one embodiment, the CD3-zeta portion of the intracellular signaling region comprises IT AM (immunoreceptor tyrosine-based activation motif) motifs 1, 2 and 3 (e.g., long CD3-zeta). In one embodiment, the CD3-zeta portion of the intracellular signaling region comprises only one of the ITAM motifs such as only IT AM 1, 2 or 3 (e.g., short CD3- zeta).
  • IT AM immunomunoreceptor tyrosine-based activation motif
  • the present disclosure provides dimeric antigen receptors (DAR) constructs having first and second polypeptide chains that associate with each other to form an antigen binding domain that binds a CD38 protein antigen.
  • the CD38 protein is from human, cynomolgus and/or mouse.
  • the CD38 protein comprises wild type or mutant CD38 protein.
  • the first polypeptide chain of the dimeric antigen receptor comprises an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1.
  • the antibody heavy chain constant region comprises the amino acid sequence of SEQ ID NO:2.
  • the hinge region comprises a CD28 hinge comprising the amino acid sequence of SEQ ID NO:5, or a CD8 hinge comprising the amino acid sequence of SEQ ID NO:2l, or a hinge region comprising a CD28 and CD8 hinge sequences of SEQ ID NO: 19 (e.g., long hinge).
  • the transmembrane region is a CD28 transmembrane region comprising the amino acid sequence of SEQ ID NO:6.
  • the intracellular signaling region comprises any one or any combination of two or more signaling sequences selected from a group consisting of 4-1BB signaling sequence comprising the amino acid sequence of SEQ ID NO:7, CD28 signaling sequence comprising the amino acid sequence of SEQ ID NO:8, CD3zeta (long) signaling sequence comprising the amino acid sequence of SEQ ID NO:9 and/or CD3zeta (short) signaling sequence having an ITAM 3 motif and comprising the amino acid sequence of SEQ ID NO:20.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 13 (e.g., with or without the leader sequence underlined in Figure 35B).
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 13 and a CD28 signaling sequence (SEQ ID NO:8) between the 4-1BB and CD3zeta signaling sequences.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16 (e.g., with or without the leader sequence underlined in Figure 35C).
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16 and a CD28 signaling sequence (SEQ ID NO:8) between the 4-1BB and CD3zeta signaling sequences.
  • the second polypeptide chain of the dimeric antigen receptor comprises an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO:3.
  • the antibody light chain constant region comprises the amino acid sequence of SEQ ID NO:4.
  • the full length of the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 14 (e.g., with or without the leader sequence underlined in Figure 35B).
  • the full length of the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 17 (e.g., with or without the leader sequence underlined in Figure 35C).
  • the present disclosure provides a dimeric antigen receptor (DAR) construct comprising: (a) a first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) a CD38 antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: l, (ii) a CD38 antibody heavy chain constant region comprising the amino acid sequence of SEQ ID NO:2, (iii) a CD28 hinge region comprising the amino acid sequence of SEQ ID NO:5, (iv) a CD28 transmembrane region comprising the amino acid sequence of SEQ ID NO:6, and (v) an intracellular signaling region comprising a 4-1BB intracellular signaling sequence comprising the amino acid sequence of SEQ ID NO:7 and a CD3zeta intracellular signaling sequence comprising the amino acid sequence of SEQ ID NO:9; and (b) a second polypeptide chain comprising two regions ordered from the amino terminus to the carboxyl terminus: (i) a CD38 antibody light
  • the present disclosure provides a dimeric antigen receptor (DAR) construct comprising: (a) a first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) a CD38 antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: l, (ii) a CD38 antibody heavy chain constant region comprising the amino acid sequence of SEQ ID NO:2, (iii) a CD28 hinge region comprising the amino acid sequence of SEQ ID NO:5, (iv) a CD28 transmembrane region comprising the amino acid sequence of SEQ ID NO:6, and (v) an intracellular signaling region comprising a 4-1BB intracellular signaling sequence comprising the amino acid sequence of SEQ ID NO:7 and a CD3zeta intracellular signaling sequence having IT AM 3 motif and comprising the amino acid sequence of SEQ ID NO:20; and (b) a second polypeptide chain comprising two regions ordered from the amino terminus to the carboxyl terminus: (i)
  • the present disclosure provides a Version 1 (e.g., VI) dimeric antigen receptors (DAR) construct comprising a first polypeptide chain carrying heavy chain variable (VH) and heavy chain constant regions (CH), and a second polypeptide chain carrying light chain variable (VL) and light chain constant regions (CL) (e.g., Figure 1), wherein (a) the first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) a long hinge region comprising CD8 and CD28 hinge sequences (e.g., SEQ ID NO: 19), (iv) a transmembrane region (TM) comprising CD28 transmembrane sequence (e.g., SEQ ID NO:6), and (v) an intracellular signaling region comprising CD28 signaling sequence (e.g., SEQ ID NO:8) and CD3-zeta signaling sequence having
  • the antibody heavy chain variable region comprises an anti-CD38 heavy chain variable region sequence (e.g., SEQ ID NO: 1) and the antibody heavy chain constant region (CH) comprises an anti-CD38 heavy chain constant region sequence (e.g., SEQ ID NO:2).
  • the present disclosure provides a Version 2 (e.g., V2) dimeric antigen receptors (DAR) construct comprising a first polypeptide chain carrying heavy chain variable (VH) and heavy chain constant regions (CH), and a second polypeptide chain carrying light chain variable (VL) and light chain constant regions (CL) (e.g., Figures 1 and 2), wherein (a) the first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) a short hinge region comprising a CD28 hinge sequence (e.g., SEQ ID NO:5), (iv) a transmembrane region (TM) comprising CD28 transmembrane sequence (e.g., SEQ ID NO:6), and (v) an intracellular signaling region comprising either (1) a 4-1BB signaling sequence (e.g., SEQ ID NO:7) and CD3-ze
  • VL antibody light chain variable region
  • CL antibody light chain constant region
  • the Version 2a (V2a) DAR construct comprises the intracellular signaling region having the 4-1BB signaling sequence (e.g., SEQ ID NO:7) and CD3-zeta having ITAM motifs 1, 2 and 3 (e.g., SEQ ID NO:9).
  • the Version 2b (V2b) DAR construct comprises the intracellular signaling region having the CD28 (e.g., SEQ ID NO:8) signaling sequence and CD3-zeta having ITAM motifs 1, 2 and 3 (e.g., SEQ ID NO:9).
  • CD28 e.g., SEQ ID NO:8
  • CD3-zeta having ITAM motifs 1, 2 and 3 (e.g., SEQ ID NO:9).
  • the Version 2c (V2c) DAR construct comprises the intracellular signaling region having the 4-1BB (e.g., SEQ ID NO:7) signaling sequence and CD28 (e.g., SEQ ID NO:8) signaling sequence and CD3-zeta having ITAM motifs 1, 2 and 3 (e.g., SEQ ID NO:9).
  • the DAR V2a and V2b are second generation DAR constructs, while the DAR V2c is a third generation DAR construct.
  • the antibody heavy chain variable region comprises an anti-CD38 heavy chain variable region sequence (e.g., SEQ ID NO: l) and the antibody heavy chain constant region (CH) comprises an anti-CD38 heavy chain constant region sequence (e.g., SEQ ID NO:2).
  • the present disclosure provides a Version 3 (e.g., V3) dimeric antigen receptors (DAR) construct comprising a first polypeptide chain carrying heavy chain variable (VH) and heavy chain constant regions (CH), and a second polypeptide chain carrying light chain variable (VL) and light chain constant regions (CL) (e.g., Figure 1), wherein (a) the first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) a short hinge region comprising CD28 hinge sequences (e.g., SEQ ID NO:5), (iv) a transmembrane region (TM) comprising CD28 transmembrane sequence (e.g., SEQ ID NO:6), and (v) an intracellular signaling region comprising 4-1BB signaling sequence (e.g., SEQ ID NO:7) and CD3-zeta signaling sequence having only IT
  • the antibody heavy chain variable region comprises an anti-CD38 heavy chain variable region sequence (e.g., SEQ ID NO: 1) and the antibody heavy chain constant region (CH) comprises an anti-CD38 heavy chain constant region sequence (e.g., SEQ ID NO:2).
  • the present disclosure provides a Version 4 (e.g., V4) dimeric antigen receptors (DAR) construct comprising a first polypeptide chain carrying heavy chain variable (VH) and heavy chain constant regions (CH), and a second polypeptide chain carrying light chain variable (VL) and light chain constant regions (CL), wherein (a) the first polypeptide chain comprising four regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) a
  • transmembrane region comprising CD28 transmembrane sequence (e.g., SEQ ID NO:6), and (iv) an intracellular signaling region comprising 4-1BB signaling sequence (e.g., SEQ ID NO:7) and CD3-zeta signaling sequence having only IT AM motif 3 (e.g., SEQ ID NO:20);
  • a second polypeptide chain comprising two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region (VL) (e.g., kappa or lambda), and (ii) an antibody light chain constant region (CL).
  • VL antibody light chain variable region
  • CL antibody light chain constant region
  • the antibody heavy chain variable region (VH) comprises an anti-CD38 heavy chain variable region sequence (e.g., SEQ ID NO:l) and the antibody heavy chain constant region (CH) comprises an anti-CD38 heavy chain constant region sequence (e.g., SEQ ID NO:2).
  • the present disclosure provides precursor polypeptides.
  • the precursor polypeptide can be processed to become first and second polypeptide chains that associate/assemble to form dimeric antigen receptors (DAR) constructs.
  • DAR dimeric antigen receptors
  • the present disclosure provides precursor polypeptides comprising ten regions ordered from the amino terminus to the carboxyl terminus: (1) a heavy chain leader sequence (2) an antibody heavy chain variable region, (3) an antibody heavy chain constant region, (4) an optional hinge region, (5) a transmembrane region, (6) an intracellular signaling region, (7) a T2A cleavage sequence, (8) a light chain leader sequence, (9) an antibody light chain variable region, and (10) an antibody light chain constant region ( Figures 5 and 6).
  • the intracellular signaling region comprises intracellular signaling sequences of any combination of at least two of 4-1BB, CD3zeta and/or CD28 ( Figures 5 and 6).
  • the T2A cleavage sequence is an amino acid sequence that promotes ribosomal skipping and recommencement of protein translation which generates two separate polypeptides.
  • a population of precursor polypeptides includes a mixture of polypeptides that have been cleaved at the T2A cleavage sequence or not, and/or a mixture of polypeptides that have been cleaved at the heavy chain and/or light chain leader sequences or not.
  • the present disclosure provides precursor polypeptides comprising ten regions ordered from the amino terminus to the carboxyl terminus: (1) a light chain leader sequence (2) an antibody light chain variable region, (3) an antibody light chain constant region, (4) an optional hinge region, (5) a transmembrane region, (6) an intracellular signaling region, (7) a T2A cleavage sequence, (8) a heavy chain leader sequence, (9) an antibody heavy chain variable region, and (10) an antibody heavy chain constant region ( Figures 7 and 8).
  • the intracellular signaling region comprises intracellular signaling sequences of any combination of at least two of 4-1BB, CD3zeta and/or CD28 ( Figures 7 and 8).
  • the T2A cleavage sequence is an amino acid sequence that promotes ribosomal skipping and recommencement of protein translation which generates two separate polypeptides.
  • a population of precursor polypeptides includes a mixture of polypeptides that have been cleaved at the T2A cleavage sequence or not, and/or a mixture of polypeptides that have been cleaved at the heavy chain and/or light chain leader sequences or not.
  • the heavy chain and light chain leader sequences comprise peptide signal sequences that target a polypeptide chain (e.g., first and second polypeptide chains) to the secretory pathway of a cell and will allow for integration and anchoring of the polypeptide into the lipid bilayer of the cellular membrane.
  • the heavy and light chain leader sequence can direct transport of the precursor polypeptide from the cytosol to the endoplasmic reticulum of a host cell.
  • the heavy chain and light chain leader sequences include signal sequences comprising CD8a, CD28 or CD 16 leader sequences.
  • the N- terminal end of a precursor polypeptide includes a first peptide signal sequence (e.g., heavy chain or light chain leader sequence).
  • a first peptide signal sequence e.g., heavy chain or light chain leader sequence
  • the precursor polypeptide can include a second peptide signal sequence (e.g., heavy chain or light chain leader sequence) located after a cleavage sequence.
  • a second peptide signal sequence e.g., heavy chain or light chain leader sequence located after a cleavage sequence.
  • the precursor polypeptide can be cleaved at the cleavage sequence thereby generating first and second polypeptide chains each having a peptide signal sequence at their N-terminal ends.
  • the processing of the precursor polypeptide includes cleaving the precursor into first and second polypeptide chains, secreting the precursor, and/or anchoring the precursor in a cellular membrane.
  • the antibody heavy chain constant region (CH) (of one of the polypeptide chains) and the antibody light chain constant region (CL) (of the other polypeptide chain) can dimerize to form a dimerization domain.
  • the antibody heavy chain constant region and the antibody light chain constant region dimerize via one or two disulfide bonds.
  • the antibody heavy chain variable region (VH) (of one of the polypeptide chains) and the antibody light chain variable region (VL) (of the other polypeptide chain) associate with each other to form an antigen binding domain.
  • VH antibody heavy chain variable region
  • VL antibody light chain variable region
  • the antibody heavy chain variable region and the antibody light chain variable region associate with each other when the antibody heavy chain constant region and the antibody light chain constant region dimerize.
  • the antigen binding domain which is formed from the antibody heavy chain variable region and the antibody light chain variable region, binds a target antigen.
  • the antibody heavy chain variable region and the antibody light chain variable region are fully human antibody regions.
  • the hinge region is about 10 to about 100 amino acids in length.
  • the hinge region comprises a hinge region or a fragment thereof from an antibody (e.g., IgG, IgA, IgM, IgE, or IgD).
  • the hinge region comprises a CD8 (e.g., CD8a) or CD28 hinge region or a fragment thereof.
  • the hinge region comprises a CPPC or SPPC amino acid sequence.
  • the hinge region comprises both CD8 and CD28 hinge sequences (e.g., long hinge region), only CD8 sequence (short hinge) or only CD28 hinge sequence (e.g., short hinge region).
  • the transmembrane regions of the precursor polypeptide chain can be derived from CD8a, CD8P, 4- 1BB/CD137, CD28, CD34, CD4, FceRIy, CD16, OX40/CD134, CD3C, CD3e, CD3y, CD35, TCRa, TCRp, TCRC, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD33, CD37, CD64,
  • the intracellular signaling region of the first polypeptide comprises intracellular signaling sequences in any order and of any combination of two to five signaling sequences from 4-1BB, CD3zeta, CD28, CD27, 0X40, CD30, CD40, PD-l, ICOS, lymphocyte function-associated antigen-l (LFA-l), CD2, CD7, LIGHT, NKG2C, B7-H3, GITR (TNFRSF18), DR3 (TNFRSF25), TNFR2 and/or CD226.
  • the intracellular signaling region comprises intracellular signaling sequences from any one or any combination of two or more of CD28, 4-1BB and/or CD3-zeta. In one embodiment, the intracellular signaling region comprises CD28 and CD3-zeta intracellular signaling sequences, or 4-1BB and CD3-zeta intracellular signaling sequences. In one embodiment, the CD3-zeta portion of the intracellular signaling region comprises IT AM (immunoreceptor tyrosine-based activation motif) motifs 1, 2 and 3 (e.g., long CD3-zeta). In one embodiment, the CD3-zeta portion of the intracellular signaling region comprises only one of the ITAM motifs such as only IT AM 1, 2 or 3 (e.g., short CD3-zeta).
  • IT AM immunomunoreceptor tyrosine-based activation motif
  • the present disclosure provides a precursor polypeptide, comprising ten regions: (1) a heavy chain leader sequence comprising the amino acid sequence of SEQ ID NO: 10; (2) a CD38 antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1; (3) a CD38 antibody heavy chain constant region comprising the amino acid sequence of SEQ ID NO:2; (4) a CD28 hinge region comprising the amino acid sequence of SEQ ID NO:5 and optionally a CD8 hinge comprising the amino acid sequence of SEQ ID NO:2l; (5) a CD28 transmembrane region comprising the amino acid sequence of SEQ ID NO:6; (6) an intracellular signaling region comprising any one or any combination of three or more signaling sequences selected from a group consisting of 4-1BB signaling sequence comprising the amino acid sequence of SEQ ID NO:7, CD28 signaling sequence comprising the amino acid sequence of SEQ ID NO:8, CD3zeta (long) signaling sequence comprising the amino acid sequence of SEQ ID NO:9 and/
  • the full length precursor polypeptide comprises the amino acid sequence of SEQ ID NO: 15 or 18. In one embodiment, the full length precursor polypeptide comprises the amino acid sequence of SEQ ID NO: 15 and a CD28 signaling sequence (SEQ ID NO:8) between the 4-1BB and CD3zeta signaling sequences. In one embodiment, the precursor polypeptide can be processed by cleaving at the T2A cleavable sequence to release the first and second polypeptide chains and secreting the precursor, and/or anchoring the precursor in a cellular membrane.
  • the first and second polypeptide chains can dimerize via at least one disulfide bond between the antibody heavy chain constant region and the antibody light chain constant region, and the antibody heavy chain variable region and the antibody light chain variable region can form an antigen binding domain that binds a CD38 antigen.
  • the hinge region is optional.
  • the present disclosure provides a precursor polypeptide, comprising ten regions: (1) a heavy chain leader sequence comprising the amino acid sequence of SEQ ID NO: 10; (2) a CD38 antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1; (3) a CD38 antibody heavy chain constant region comprising the amino acid sequence of SEQ ID NO:2; (4) a CD28 hinge region comprising the amino acid sequence of SEQ ID NO:5; (5) a CD28 transmembrane region comprising the amino acid sequence of SEQ ID NO:6; (6) an intracellular signaling region comprising a 4-1BB signaling sequence comprising the amino acid sequence of SEQ ID NO:7 and CD3zeta (short) signaling sequence having an IT AM 3 motif and comprising the amino acid sequence of SEQ ID NO:20, and optionally a CD28 signaling sequence comprising the amino acid sequence of SEQ ID NO:8; (7) a T2A cleavage sequence comprising the amino acid sequence of SEQ ID NO: 12; (8)
  • the full length precursor polypeptide comprises the amino acid sequence of SEQ ID NO: 18.
  • the precursor polypeptide can be processed by cleaving at the T2A cleavable sequence to release the first and second polypeptide chains and secreting the precursor, and/or anchoring the precursor in a cellular membrane.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 17.
  • the first and second polypeptide chains can dimerize via at least one disulfide bond between the antibody heavy chain constant region and the antibody light chain constant region, and the antibody heavy chain variable region and the antibody light chain variable region can form an antigen binding domain that binds a CD38 antigen.
  • the present disclosure provides nucleic acids that encode any of the first polypeptide chains, second polypeptide chains, first and second polypeptide chains, dimeric antigen receptors or precursor polypeptides described herein.
  • the present disclosure provides a nucleic acid that encodes a first polypeptide chain comprising: five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region, (ii) an antibody heavy chain constant region, (iii) an optional hinge region, (iv) a transmembrane region, and (v) an intracellular signaling region.
  • the present disclosure provides a nucleic acid that encodes a second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region, and (ii) an antibody light chain constant region.
  • the nucleic acid encodes the first and second polypeptide chains, comprising: (a) a first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region, (ii) an antibody heavy chain constant region, (iii) an optional hinge region, (iv) a transmembrane region, and (v) an intracellular signaling region having two to five intracellular signaling sequences; and (b) a second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region, and (ii) an antibody light chain constant region (e.g., Figures 1 and 2).
  • a first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region, (ii) an antibody heavy chain constant region, (iii) an optional hinge region, (iv) a transmembrane region, and (v) an intracellular signal
  • the present disclosure provides a nucleic acid that encodes a first polypeptide chain comprising: five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region, (ii) an antibody light chain constant region, (iii) an optional hinge region, (iv) a transmembrane region, and (v) an intracellular signaling region.
  • the present disclosure provides a nucleic acid that encodes a second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region, and (ii) an antibody heavy chain constant region.
  • the nucleic acid encodes the first and second polypeptide chains, comprising: (a) a first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region, (ii) an antibody light chain constant region, (iii) an optional hinge region, (iv) a transmembrane region, and (v) an intracellular signaling region having two to five intracellular signaling sequences; and (b) a second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region, and (ii) an antibody heavy chain constant region (e.g., Figures 3 and 4).
  • a first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region, (ii) an antibody light chain constant region, (iii) an optional hinge region, (iv) a transmembrane region, and (v) an intracellular signal
  • the present disclosure provides a nucleic acid that encodes a first polypeptide chain comprising: six regions ordered from the amino terminus to the carboxyl terminus: (i) a heavy chain leader region, (ii) an antibody heavy chain variable region, (iii) an antibody heavy chain constant region, (iv) an optional hinge region, (v) a transmembrane region, and (vi) an intracellular signaling region.
  • the present disclosure provides a nucleic acid that encodes a second polypeptide chain comprising: three regions ordered from the amino terminus to the carboxyl terminus: (i) a light chain leader region, (ii) an antibody light chain variable region, and (iii) an antibody light chain constant region.
  • the nucleic acid encodes the first and second polypeptide chains, comprising: (a) a first polypeptide chain comprising six regions ordered from the amino terminus to the carboxyl terminus: (i) a heavy chain leader sequence, (ii) an antibody heavy chain variable region, (iii) an antibody heavy chain constant region, (iv) an optional hinge region, (v) a transmembrane region, and (vi) an intracellular signaling region having two to five intracellular signaling sequences; and (b) a second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region, and (ii) an antibody light chain constant region.
  • the present disclosure provides a nucleic acid that encodes a first polypeptide chain comprising: six regions ordered from the amino terminus to the carboxyl terminus: (i) a light chain leader region, (ii) an antibody light chain variable region, (iii) an antibody light chain constant region, (iv) an optional hinge region, (v) a transmembrane region, and (vi) an intracellular signaling region.
  • the present disclosure provides a nucleic acid that encodes a second polypeptide chain comprising: three regions ordered from the amino terminus to the carboxyl terminus: (i) a heavy chain leader region, (ii) an antibody heavy chain variable region, and (iii) an antibody light chain constant region.
  • the nucleic acid encodes the first and second polypeptide chains, comprising: (a) a first polypeptide chain comprising six regions ordered from the amino terminus to the carboxyl terminus: (i) a light chain leader sequence, (ii) an antibody light chain variable region, (iii) an antibody light chain constant region, (iv) an optional hinge region, (v) a transmembrane region, and (vi) an intracellular signaling region having two to five intracellular signaling sequences; and (b) a second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region, and (ii) an antibody heavy chain constant region.
  • the present disclosure provides a nucleic acid that encodes a precursor polypeptide comprising: ten regions ordered from the amino terminus to the carboxyl terminus: (1) a heavy chain leader region, (2) an antibody heavy chain variable region, (3) an antibody heavy chain constant region, (4) an optional hinge region, (5) a transmembrane region, (6) an intracellular signaling region having two to five intracellular signaling sequences, (7) T2A cleavable sequence region, (8) a light chain leader region, (9) an antibody light chain variable region, and (10) an antibody light chain constant region (e.g., Figures 5 and 6).
  • the present disclosure provides a nucleic acid that encodes a precursor polypeptide comprising: ten regions ordered from the amino terminus to the carboxyl terminus: (1) a light chain leader region, (2) an antibody light chain variable region, (3) an antibody light chain constant region, (4) an optional hinge region, (5) a transmembrane region, (6) an intracellular signaling region having two to five intracellular signaling sequences, (7) T2A cleavable sequence region, (8) a heavy chain leader region, (9) an antibody heavy chain variable region, and (10) an antibody heavy chain constant region (e.g., Figures 7 and 8).
  • the present disclosure provides nucleic acids that encode a first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) a CD38 antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CD38 antibody heavy chain constant region comprising the amino acid sequence of SEQ ID NO:2, (iii) a CD28 hinge region comprising the amino acid sequence of SEQ ID NO:5 and optionally a CD8 hinge comprising the amino acid sequence of SEQ ID NO:2l, (iv) a CD28 transmembrane region comprising the amino acid sequence of SEQ ID NO:6, and (v) an intracellular signaling region comprising any one or any combination of two or more signaling sequences selected from a group consisting of 4-1BB signaling sequence comprising the amino acid sequence of SEQ ID NO: 7, CD28 signaling sequence comprising the amino acid sequence of SEQ ID NO:8, CD3zeta (long) signaling sequence comprising the amino acid sequence of SEQ ID
  • the nucleic acid encodes a first polypeptide chain which comprises the amino acid sequence of SEQ ID NO: 13 or 16 (e.g., with or without the leader sequence underlined in Figure 35B or 35C). In one embodiment, the nucleic acid encodes the first polypeptide chain which comprises the amino acid sequence of SEQ ID NO: 13 and a CD28 signaling sequence (SEQ ID NO: 8) between the 4- 1BB and CD3zeta signaling sequences. In one embodiment, the nucleic acid encodes the first polypeptide chain which comprises the amino acid sequence of SEQ ID NO: 16 (e.g., with or without the leader sequence underlined in Figure 35C).
  • the nucleic acid encodes the first polypeptide chain which comprises the amino acid sequence of SEQ ID NO: 16 and a CD28 signaling sequence (SEQ ID NO:8) between the 4-1BB and CD3zeta signaling sequences.
  • the hinge region is optional.
  • the nucleic acid encodes the second polypeptide chain of the dimeric antigen receptor which comprises an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO:3. In one embodiment, the nucleic acid encodes the second polypeptide chain of the dimeric antigen receptor which comprises the antibody light chain constant region comprises the amino acid sequence of SEQ ID NO:4. In one embodiment, the nucleic acid encodes the full length of the second polypeptide chain which comprises the amino acid sequence of SEQ ID NO: 14 or 17 (e.g., with or without the leader sequence underlined in Figure 35B or 35C).
  • nucleic acids that encode a second polypeptide chain comprising two regions ordered from the amino terminus to the carboxyl terminus: (i) a CD38 antibody light chain variable region comprising the amino acid sequence of SEQ ID NO:3, and (ii) a CD38 antibody light chain constant region comprising the amino acid sequence of SEQ ID NO:4.
  • the nucleic acid encodes the first polypeptide chain (SEQ ID NO: 13 or 16), a T2A cleavage sequence (SEQ ID NO: 12), and the second polypeptide chain (SEQ ID NO: 14 or 17).
  • the first polypeptide chain (SEQ ID NO: 13 or 16) includes or lacks the leader sequence underlined in Figures 35B and C, respectively.
  • the second polypeptide chain (SEQ ID NO: 14 or 17) includes or lacks the leader sequence underlined in Figures 35B or C, respectively.
  • the present disclosure provides nucleic acids that encode a precursor polypeptide, comprising the amino acid sequence of SEQ ID NO:l5 or 18.
  • the present disclosure provides nucleic acids that encode a Version 1 (e.g., VI) dimeric antigen receptors (DAR) construct comprising a first polypeptide chain carrying heavy chain variable (VH) and heavy chain constant regions (CH), and a second polypeptide chain carrying light chain variable (VL) and light chain constant regions (CL) (e.g., Figure 1), wherein (a) a first nucleic acid encodes the first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) a long hinge region comprising CD8 and CD28 hinge sequences (e.g., SEQ ID NO: 19), (iv) a transmembrane region (TM) comprising CD28 transmembrane sequence (e.g., SEQ ID NO:6), and (v) an intracellular signaling region comprising CD28 signaling sequence (e.g., SEQ ID NO:
  • the antibody heavy chain variable region comprises an anti-CD38 heavy chain variable region sequence (e.g., SEQ ID NO: 1) and the antibody heavy chain constant region (CH) comprises an anti-CD38 heavy chain constant region sequence (e.g., SEQ ID NO:2).
  • the present disclosure provides nucleic acids that encode a Version 2 (e.g., V2) dimeric antigen receptors (DAR) construct comprising a first polypeptide chain carrying heavy chain variable (VH) and heavy chain constant regions (CH), and a second polypeptide chain carrying light chain variable (VL) and light chain constant regions (CL) (e.g., Figures 1 and 2), wherein (a) a first nucleic acid encodes the first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) a short hinge region comprising a CD28 hinge sequence (e.g., SEQ ID NO:5), (iv) a transmembrane region (TM) comprising CD28 transmembrane sequence (e.g., SEQ ID NO:6), and (v) an intracellular signaling region comprising either (1) a 4-1BB signaling sequence (e
  • the nucleic acids encode the Version 2a (V2a) DAR construct comprising the intracellular signaling region having the 4-1BB signaling sequence (e.g., SEQ ID NO:7) and CD3-zeta having IT AM motifs 1, 2 and 3 (e.g., SEQ ID NO:9).
  • the nucleic acids encode the Version 2b (V2b) DAR construct comprising the intracellular signaling region having the CD28 (e.g., SEQ ID NO:8) signaling sequence and CD3-zeta having IT AM motifs 1, 2 and 3 (e.g., SEQ ID NO:9).
  • V2b Version 2b
  • the nucleic acids encode the Version 2c (V2c) DAR construct comprising the intracellular signaling region having the 4-1BB (e.g., SEQ ID NO:7) signaling sequence and CD28 (e.g., SEQ ID NO:8) signaling sequence and CD3-zeta having ITAM motifs 1, 2 and 3 (e.g., SEQ ID NO:9).
  • the DAR V2a and V2b are second generation DAR constructs, while the DAR V2c is a third generation DAR construct.
  • the antibody heavy chain variable region comprises an anti-CD38 heavy chain variable region sequence (e.g., SEQ ID NO: l) and the antibody heavy chain constant region (CH) comprises an anti-CD38 heavy chain constant region sequence (e.g., SEQ ID NO:2).
  • the present disclosure provides nucleic acids that encodes a Version 3 (e.g., V3) dimeric antigen receptors (DAR) construct comprising a first polypeptide chain carrying heavy chain variable (VH) and heavy chain constant regions (CH), and a second polypeptide chain carrying light chain variable (VL) and light chain constant regions (CL) (e.g., Figure 1), wherein (a) a first nucleic acid encodes the first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) a short hinge region comprising CD28 hinge sequences (e.g., SEQ ID NO:5), (iv) a transmembrane region (TM) comprising CD28 transmembrane sequence (e.g., SEQ ID NO:6), and (v) an intracellular signaling region comprising 4-1BB signaling sequence (e.g., SEQ
  • the antibody heavy chain variable region comprises an anti-CD38 heavy chain variable region sequence (e.g., SEQ ID NO: 1) and the antibody heavy chain constant region (CH) comprises an anti-CD38 heavy chain constant region sequence (e.g., SEQ ID NO:2).
  • the present disclosure provides nucleic acids that encode a Version 4 (e.g., V4) dimeric antigen receptors (DAR) construct comprising a first polypeptide chain carrying heavy chain variable (VH) and heavy chain constant regions (CH), and a second polypeptide chain carrying light chain variable (VL) and light chain constant regions (CL), wherein (a) a first nucleic acid encodes the first polypeptide chain comprising four regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) a transmembrane region (TM) comprising CD28 transmembrane sequence (e.g., SEQ ID NO:6), and (iv) an intracellular signaling region comprising 4-1BB signaling sequence (e.g., SEQ ID NO:7) and CD3-zeta signaling sequence having only ITAM motif 3 (e.g., SEQ ID NO:20); (b) a Version 4 (e
  • the DAR V4 construct lacks a hinge sequence.
  • the antibody heavy chain variable region (VH) comprises an anti-CD38 heavy chain variable region sequence (e.g., SEQ ID NO: l) and the antibody heavy chain constant region (CH) comprises an anti-CD38 heavy chain constant region sequence (e.g., SEQ ID NO:2).
  • the present disclosure provides vectors operably linked to nucleic acids that encode any of the first polypeptide chains, second polypeptide chains, first and second polypeptide chains, dimeric antigen receptors or precursor polypeptide described herein.
  • the present disclosure provides a vector operably linked to a nucleic acid that encodes a first polypeptide chain comprising: five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region, (ii) an antibody heavy chain constant region, (iii) an optional hinge region, (iv) a transmembrane region, and (v) an intracellular signaling region.
  • the present disclosure provides a vector operably linked to a nucleic acid that encodes a second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region, and (ii) an antibody light chain constant region.
  • the vector that is operably linked to a nucleic acid encoding the first and second polypeptide chains comprises: (a) a first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region, (ii) an antibody heavy chain constant region, (iii) an optional hinge region, (iv) a transmembrane region, and (v) an intracellular signaling region having two to five intracellular signaling sequences; and (b) a second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region, and (ii) an antibody light chain constant region.
  • the present disclosure provides a vector operably linked to a nucleic acid that encodes a first polypeptide chain comprising: five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region, (ii) an antibody light chain constant region, (iii) an optional hinge region, (iv) a transmembrane region, and (v) an intracellular signaling region.
  • the present disclosure provides a vector operably linked to a nucleic acid that encodes a second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region, and (ii) an antibody heavy chain constant region.
  • the vector that is operably linked to a nucleic acid encoding the first and second polypeptide chains comprises: (a) a first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region, (ii) an antibody light chain constant region, (iii) an optional hinge region, (iv) a transmembrane region, and (v) an intracellular signaling region having two to five intracellular signaling sequences; and (b) a second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region, and (ii) an antibody heavy chain constant region.
  • the present disclosure provides a vector operably linked to a nucleic acid that encodes a first polypeptide chain comprising: six regions ordered from the amino terminus to the carboxyl terminus: (i) a heavy chain leader region, (ii) an antibody heavy chain variable region, (iii) an antibody heavy chain constant region, (iv) an optional hinge region, (v) a transmembrane region, and (vi) an intracellular signaling region.
  • the present disclosure provides a vector operably linked to a nucleic acid that encodes a second polypeptide chain comprising: three regions ordered from the amino terminus to the carboxyl terminus: (i) a light chain leader region, (ii) an antibody light chain variable region, and (iii) an antibody light chain constant region.
  • the vector that is operably linked to a nucleic acid encoding the first and second polypeptide chains comprises: (a) a first polypeptide chain comprising six regions ordered from the amino terminus to the carboxyl terminus: (i) a heavy chain leader sequence, (ii) an antibody heavy chain variable region, (iii) an antibody heavy chain constant region, (iv) an optional hinge region, (v) a transmembrane region, and (vi) an intracellular signaling region having two to five intracellular signaling sequences; and (b) a second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region, and (ii) an antibody light chain constant region.
  • the present disclosure provides a vector operably linked to a nucleic acid that encodes a first polypeptide chain comprising: six regions ordered from the amino terminus to the carboxyl terminus: (i) a light chain leader region, (ii) an antibody light chain variable region, (iii) an antibody light chain constant region, (iv) an optional hinge region, (v) a transmembrane region, and (vi) an intracellular signaling region.
  • the present disclosure provides a vector operably linked to a nucleic acid that encodes a second polypeptide chain comprising: three regions ordered from the amino terminus to the carboxyl terminus: (i) a heavy chain leader region, (ii) an antibody heavy chain variable region, and (iii) an antibody light chain constant region.
  • the vector that is operably linked to a nucleic acid encoding the first and second polypeptide chains comprises: (a) a first polypeptide chain comprising six regions ordered from the amino terminus to the carboxyl terminus: (i) a light chain leader sequence, (ii) an antibody light chain variable region, (iii) an antibody light chain constant region, (iv) an optional hinge region, (v) a transmembrane region, and (vi) an intracellular signaling region having two to five intracellular signaling sequences; and (b) a second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region, and (ii) an antibody heavy chain constant region.
  • the present disclosure provides a vector operably linked to a nucleic acid that encodes a precursor polypeptide comprising: ten regions ordered from the amino terminus to the carboxyl terminus: (1) a heavy chain leader region, (2) an antibody heavy chain variable region, (3) an antibody heavy chain constant region, (4) an optional hinge region, (5) a transmembrane region, (6) an intracellular signaling region having two to five intracellular signaling sequences, (7) T2A cleavable sequence region, (8) a light chain leader region, (9) an antibody light chain variable region, and (10) an antibody light chain constant region.
  • the present disclosure provides a vector operably linked to a nucleic acid that encodes a precursor polypeptide comprising: ten regions ordered from the amino terminus to the carboxyl terminus: (1) a light chain leader region, (2) an antibody light chain variable region, (3) an antibody light chain constant region, (4) an optional hinge region, (5) a transmembrane region, (6) an intracellular signaling region having two to five intracellular signaling sequences, (7) T2A cleavable sequence region, (8) a heavy chain leader region, (9) an antibody heavy chain variable region, and (10) an antibody heavy chain constant region.
  • the present disclosure provides a vector operably linked to a nucleic acid that encodes a first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) a CD38 antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CD38 antibody heavy chain constant region comprising the amino acid sequence of SEQ ID NO:2, (iii) a CD28 hinge region comprising the amino acid sequence of SEQ ID NO:5, (iv) a CD28 transmembrane region comprising the amino acid sequence of SEQ ID NO:6, and (v) an intracellular signaling region comprising a 4-1BB intracellular signaling sequence comprising the amino acid sequence of SEQ ID NO:7 and a CD3zeta intracellular signaling sequence comprising the amino acid sequence of SEQ ID NO:9.
  • the present disclosure provides a vector operably linked to a nucleic acid that encodes a second polypeptide chain comprising two regions ordered from the amino terminus to the carboxyl terminus: (i) a CD38 antibody light chain variable region comprising the amino acid sequence of SEQ ID NO:3, and (ii) a CD38 antibody light chain constant region comprising the amino acid sequence of SEQ ID NO:4.
  • the vector is operably linked to a nucleic acid that encodes the first polypeptide chain (SEQ ID NO: 13 or 16) and the second polypeptide chain (SEQ ID NO: 14 or 17).
  • the vector is operably linked to a nucleic acid that encodes the first polypeptide chain (SEQ ID NO: 13 or 16), a T2A cleavage sequence (SEQ ID NO: 12), and the second polypeptide chain (SEQ ID NO: 14 or 17).
  • the first polypeptide chain (SEQ ID NO: 13 or 16) includes or lacks the leader sequence underlined in Figure 35B and C.
  • the second polypeptide chain (SEQ ID NO: 14 or 17) includes or lacks the leader sequence underlined in Figure 35B and C.
  • the present disclosure provides a vector operably linked to a nucleic acid that encodes a precursor polypeptide, comprising the amino acid sequence of SEQ ID NO: 15 or 18.
  • the present disclosure provides vectors operably linked to nucleic acids that encode a Version 1 (e.g., VI) dimeric antigen receptors (DAR) construct comprising a first polypeptide chain carrying heavy chain variable (VH) and heavy chain constant regions (CH), and a second polypeptide chain carrying light chain variable (VL) and light chain constant regions (CL) (e.g., Figure 1), wherein (a) a first vector is operably linked to a first nucleic acid that encodes the first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) a long hinge region comprising CD8 and CD28 hinge sequences (e.g., SEQ ID NO: 19), (iv) a transmembrane region (TM) comprising CD28 transmembrane sequence (e.g., SEQ ID NO:6), and (v) an intracellular signal
  • the first vector is operably linked to the second nucleic acid that encodes a second polypeptide chain comprising two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region (VL) (e.g., kappa or lambda), and (ii) an antibody light chain constant region (CL).
  • VL antibody light chain variable region
  • CL antibody light chain constant region
  • the antibody heavy chain variable region (VH) comprises an anti-CD38 heavy chain variable region sequence (e.g., SEQ ID NO: 1)
  • the antibody heavy chain constant region (CH) comprises an anti-CD38 heavy chain constant region sequence (e.g., SEQ ID NO:2).
  • the present disclosure provides vectors operably linked to nucleic acids that encode a Version 2 (e.g., V2) dimeric antigen receptors (DAR) construct comprising a first polypeptide chain carrying heavy chain variable (VH) and heavy chain constant regions (CH), and a second polypeptide chain carrying light chain variable (VL) and light chain constant regions (CL) (e.g., Figures 1 and 2), wherein (a) a first vector operably linked to a first nucleic acid that encodes the first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) a short hinge region comprising a CD28 hinge sequence (e.g., SEQ ID NO:5), (iv) a transmembrane region (TM) comprising CD28 transmembrane sequence (e.g., SEQ ID NO:6), and (v) an intracellular signal
  • VH
  • the first vector is operably linked to a second nucleic acid that encodes the second polypeptide chain comprising two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region (VL) (e.g., kappa or lambda), and (ii) an antibody light chain constant region (CL).
  • VL antibody light chain variable region
  • CL antibody light chain constant region
  • the vector is operably linked to a nucleic acid encoding the Version 2a (V2a) DAR construct comprising the intracellular signaling region having the 4-1BB signaling sequence (e.g., SEQ ID NO:7) and CD3-zeta having ITAM motifs 1, 2 and 3 (e.g.,
  • the vector is operably linked to a nucleic acid encoding the Version 2b (V2b) DAR construct comprising the intracellular signaling region having the CD28 (e.g., SEQ ID NO:8) signaling sequence and CD3-zeta having ITAM motifs 1, 2 and 3 (e.g.,
  • the vector is operably linked to a nucleic acid encoding the Version 2c (V2c) DAR construct comprising the intracellular signaling region having the 4-1BB (e.g., SEQ ID NO:7) signaling sequence and CD28 (e.g., SEQ ID NO:8) signaling sequence and CD3-zeta having ITAM motifs 1, 2 and 3 (e.g., SEQ ID NO:9).
  • V2c Version 2c
  • the DAR V2a and V2b are second generation DAR constructs
  • the DAR V2c is a third generation DAR construct.
  • the antibody heavy chain variable region comprises an anti-CD38 heavy chain variable region sequence (e.g., SEQ ID NO: l) and the antibody heavy chain constant region (CH) comprises an anti-CD38 heavy chain constant region sequence (e.g., SEQ ID NO:2).
  • the present disclosure provides vectors operably linked to nucleic acids that encode a Version 3 (e.g., V3) dimeric antigen receptors (DAR) construct comprising a first polypeptide chain carrying heavy chain variable (VH) and heavy chain constant regions (CH), and a second polypeptide chain carrying light chain variable (VL) and light chain constant regions (CL) (e.g., Figure 1), wherein (a) a first vector operably linked to a first nucleic acid that encodes the first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) a short hinge region comprising CD28 hinge sequences (e.g., SEQ ID NO:5), (iv) a transmembrane region (TM) comprising CD28 transmembrane sequence (e.g., SEQ ID NO:6), and (v) an intracellular signaling region comprising
  • VH
  • the first vector operably is linked to a second nucleic acid that encodes the second
  • the polypeptide chain comprising two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region (VL) (e.g., kappa or lambda), and (ii) an antibody light chain constant region (CL).
  • VL antibody light chain variable region
  • CL antibody light chain constant region
  • the antibody heavy chain variable region (VH) comprises an anti-CD38 heavy chain variable region sequence (e.g., SEQ ID NO: 1)
  • the antibody heavy chain constant region (CH) comprises an anti-CD38 heavy chain constant region sequence (e.g., SEQ ID NO:2).
  • the present disclosure provides vectors operably linked to nucleic acids that encode a Version 4 (e.g., V4) dimeric antigen receptors (DAR) construct comprising a first polypeptide chain carrying heavy chain variable (VH) and heavy chain constant regions (CH), and a second polypeptide chain carrying light chain variable (VL) and light chain constant regions (CL), wherein (a) a first vector operably linked to a first nucleic acid that encodes the first polypeptide chain comprising four regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) a transmembrane region (TM) comprising CD28 transmembrane sequence (e.g., SEQ ID NO:6), and (iv) an intracellular signaling region comprising 4-1BB signaling sequence (e.g., V4) dimeric antigen receptors (DAR) construct comprising a first polypeptide chain carrying heavy chain variable (VH
  • the first vector is operably linked to a second nucleic acid that encodes the second polypeptide chain comprising two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region (VL) (e.g., kappa or lambda), and (ii) an antibody light chain constant region (CL).
  • VL antibody light chain variable region
  • CL antibody light chain constant region
  • the antibody heavy chain variable region comprises an anti-CD38 heavy chain variable region sequence (e.g., SEQ ID NO: l) and the antibody heavy chain constant region (CH) comprises an anti-CD38 heavy chain constant region sequence (e.g., SEQ ID NO:2).
  • the present disclosure provides a host cell, or a population of host cells, which harbors one or more expression vectors operably linked to a nucleic acid transgene that encodes any of the first polypeptide chains, second polypeptide chains, first and second polypeptide chains, dimeric antigen receptors or precursor polypeptides described herein.
  • the host cell or population of host cells are introduced with one or more expression vectors, where the vectors are operably linked to a nucleic acid transgene encoding any of the dimeric antigen receptor (DAR) constructs described herein.
  • the host cell or the population of host cells comprise T lymphocytes (e.g., T cells, regulatory T cells, gamma- delta T cells, and cytotoxic T cells), NK (natural killer) cells, macrophages, dendritic cells, mast cells, eosinophils, B lymphocytes, monocytes.
  • the NK cells comprise cord blood-derived NK cells, or placental derived NK cells.
  • the host cell or population of host cells harbor one or more expression vectors that can direct transient introduction of the transgene into the host cells or stable insertion of the transgene into the host cells’ genome.
  • the expression vector(s) can direct transcription and/or translation of the transgene in the host cell.
  • the expression vector can include nucleic acid backbone sequences derived from a retrovirus, lentivirus or adenovirus.
  • the expression vectors can include one or more regulatory sequences, such as inducible and/or constitutive promoters and enhancers.
  • the expression vectors can include ribosomal binding sites and/or polyadenylation sites.
  • the expression vector which is operably linked to the nucleic acid encoding the dimeric antigen receptor (DAR) construct, can direct production of the dimeric antigen receptor (DAR) construct which can be displayed on the surface of the transgenic host cell or the dimeric antigen receptor can be secreted into the cell culture medium.
  • host cells can harbor one or more expression vectors operably linked to the nucleic acid transgene that encodes any of the dimeric antigen receptors, and the host cells can be cultured in an appropriate culture medium to transiently or stably express a dimeric antigen receptor construct.
  • the present disclosure provides a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a first polypeptide chain comprising: five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region, (ii) an antibody heavy chain constant region, (iii) an optional hinge region, (iv) a transmembrane region, and (v) an intracellular signaling region.
  • the present disclosure provides host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region, and (ii) an antibody light chain constant region.
  • the host cell harbors a first expression vector operably linked to a nucleic acid that encodes the first polypeptide chain and harbors a second expression vector operably linked to a nucleic acid that encodes the second polypeptide chain, wherein (a) the first polypeptide chain comprises five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region, (ii) an antibody heavy chain constant region, (iii) an optional hinge region, (iv) a transmembrane region, and (v) an intracellular signaling region having two to five intracellular signaling sequences; and wherein (b) the second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region, and (ii) an antibody light chain constant region.
  • the host cell, or the population of host cells harbors an expression vector operably linked to a nucleic acid that encodes the first and second polypeptide chains, comprising: (a) a first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region, (ii) an antibody heavy chain constant region, (iii) an optional hinge region, (iv) a transmembrane region, and (v) an intracellular signaling region having two to five intracellular signaling sequences; and (b) a second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region, and (ii) an antibody light chain constant region. [00257] In one embodiment, the host cell, or population of host cells, expresses the first and second polypeptide chains.
  • the present disclosure provides host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a first polypeptide chain comprising: five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region, (ii) an antibody light chain constant region, (iii) an optional hinge region, (iv) a transmembrane region, and (v) an intracellular signaling region.
  • the present disclosure provides host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region, and (ii) an antibody heavy chain constant region.
  • the host cell harbors a first expression vector operably linked to a nucleic acid that encodes the first polypeptide chain and harbors a second expression vector operably linked to a nucleic acid that encodes the second polypeptide chain, wherein (a) the first polypeptide chain comprises five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region, (ii) an antibody light chain constant region, (iii) an optional hinge region, (iv) a transmembrane region, and (v) an intracellular signaling region having two to five intracellular signaling sequences; and wherein (b) the second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region, and (ii) an antibody heavy chain constant region.
  • the host cell harbors an expression vector operably linked to a nucleic acid that encodes the first and second polypeptide chains, comprising: (a) a first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region, (ii) an antibody light chain constant region, (iii) an optional hinge region, (iv) a transmembrane region, and (v) an intracellular signaling region having two to five intracellular signaling sequences; and (b) a second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region, and (ii) an antibody heavy chain constant region.
  • the host cell, or population of host cells expresses the first and second polypeptide chains.
  • the present disclosure provides a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a first polypeptide chain comprising: six regions ordered from the amino terminus to the carboxyl terminus: (i) a heavy chain leader region, (ii) an antibody heavy chain variable region, (iii) an antibody heavy chain constant region, (iv) an optional hinge region, (v) a transmembrane region, and (vi) an intracellular signaling region.
  • the present disclosure provides a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a second polypeptide chain comprising: three regions ordered from the amino terminus to the carboxyl terminus: (i) a light chain leader region, (ii) an antibody light chain variable region, and (iii) an antibody light chain constant region.
  • the host cell harbors a first expression vector operably linked to a nucleic acid that encodes the first polypeptide chain and harbors a second expression vector operably linked to a nucleic acid that encodes the second polypeptide chain, wherein (a) the first polypeptide chain comprising six regions ordered from the amino terminus to the carboxyl terminus: (i) a heavy chain leader sequence, (ii) an antibody heavy chain variable region, (iii) an antibody heavy chain constant region, (iv) an optional hinge region, (v) a transmembrane region, and (vi) an intracellular signaling region having two to five intracellular signaling sequences; and wherein (b) the second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region, and (ii) an antibody light chain constant region.
  • the host cell harbors an expression vector operably linked to a nucleic acid that encodes the first and second polypeptide chains, comprising: (a) a first polypeptide chain comprising six regions ordered from the amino terminus to the carboxyl terminus: (i) a heavy chain leader sequence, (ii) an antibody heavy chain variable region, (iii) an antibody heavy chain constant region, (iv) an optional hinge region, (v) a transmembrane region, and (vi) an intracellular signaling region having two to five intracellular signaling sequences; and (b) a second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region, and (ii) an antibody light chain constant region.
  • the host cell expresses the first and second polypeptide chains.
  • the present disclosure provides a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a first polypeptide chain comprising: six regions ordered from the amino terminus to the carboxyl terminus: (i) a light chain leader region, (ii) an antibody light chain variable region, (iii) an antibody light chain constant region, (iv) an optional hinge region, (v) a transmembrane region, and (vi) an intracellular signaling region.
  • the present disclosure provides a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a second polypeptide chain comprising: three regions ordered from the amino terminus to the carboxyl terminus: (i) a heavy chain leader region, (ii) an antibody heavy chain variable region, and (iii) an antibody light chain constant region.
  • the host cell harbors a first expression vector operably linked to a nucleic acid that encodes the first polypeptide chain and harbors a second expression vector operably linked to a nucleic acid that encodes the second polypeptide chain, wherein (a) the first polypeptide chain comprising six regions ordered from the amino terminus to the carboxyl terminus: (i) a light chain leader sequence, (ii) an antibody light chain variable region, (iii) an antibody light chain constant region, (iv) an optional hinge region, (v) a transmembrane region, and (vi) an intracellular signaling region having two to five intracellular signaling sequences; and wherein (b) the second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region, and (ii) an antibody heavy chain constant region.
  • the host cell harbors an expression vector operably linked to a nucleic acid that encodes the first and second polypeptide chains, comprising: (a) a first polypeptide chain comprising six regions ordered from the amino terminus to the carboxyl terminus: (i) a light chain leader sequence, (ii) an antibody light chain variable region, (iii) an antibody light chain constant region, (iv) an optional hinge region, (v) a transmembrane region, and (vi) an intracellular signaling region having two to five intracellular signaling sequences; and (b) a second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region, and (ii) an antibody heavy chain constant region.
  • the host cell expresses the first and second polypeptide chains.
  • the present disclosure provides a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a precursor polypeptide comprising: ten regions ordered from the amino terminus to the carboxyl terminus: (1) a heavy chain leader region, (2) an antibody heavy chain variable region, (3) an antibody heavy chain constant region, (4) an optional hinge region, (5) a transmembrane region, (6) an intracellular signaling region having two to five intracellular signaling sequences, (7) T2A cleavable sequence region, (8) a light chain leader region, (9) an antibody light chain variable region, and (10) an antibody light chain constant region.
  • the host cell expresses the precursor polypeptide.
  • the present disclosure provides a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a precursor polypeptide comprising: ten regions ordered from the amino terminus to the carboxyl terminus: (1) a light chain leader region, (2) an antibody light chain variable region, (3) an antibody light chain constant region, (4) an optional hinge region, (5) a transmembrane region, (6) an intracellular signaling region having two to five intracellular signaling sequences, (7) T2A cleavable sequence region, (8) a heavy chain leader region, (9) an antibody heavy chain variable region, and (10) an antibody heavy chain constant region.
  • the host cell expresses the precursor polypeptide.
  • the present disclosure provides a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) a CD38 antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: l, (ii) a CD38 antibody heavy chain constant region comprising the amino acid sequence of SEQ ID NO:2, (iii) a CD28 hinge region comprising the amino acid sequence of SEQ ID NO:5, (iv) a CD28 transmembrane region comprising the amino acid sequence of SEQ ID NO:6, and
  • an intracellular signaling region comprising a 4-1BB intracellular signaling sequence comprising the amino acid sequence of SEQ ID NO:7 and a CD3zeta intracellular signaling sequence comprising the amino acid sequence of SEQ ID NO:9.
  • the present disclosure provides a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a second polypeptide chain comprising two regions ordered from the amino terminus to the carboxyl terminus: (i) a CD38 antibody light chain variable region comprising the amino acid sequence of SEQ ID NO:3, and (ii) a CD38 antibody light chain constant region comprising the amino acid sequence of SEQ ID NO:4.
  • the vector is operably linked to a nucleic acid that encodes the first and second polypeptide chains (SEQ ID NOS:6 and 7).
  • the vector is operably linked to a nucleic acid that encodes the first polypeptide chain (SEQ ID NO:6), a T2A cleavage sequence (SEQ ID NO: 12), and the second polypeptide chain (SEQ ID NO: 7).
  • the present disclosure provides a vector operably linked to a nucleic acid that encodes a precursor polypeptide, comprising the amino acid sequence of SEQ ID NO: 15.
  • the host cell expresses the first polypeptide chain (SEQ ID NO: 13 or 16) and the second polypeptide chain (SEQ ID NOS:). In one embodiment, the host cell expresses the precursor polypeptide of SEQ ID NO: 14 or 17).
  • the first polypeptide chain (SEQ ID NO: 13 or 16) includes or lacks the leader sequence underlined in Figure 35B or C.
  • the second polypeptide chain (SEQ ID NO: 14 or 17) includes or lacks the leader sequence underlined in Figure 35B or C.
  • the present disclosure provides a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a Version 1 (e.g., VI) dimeric antigen receptors (DAR) construct comprising a first polypeptide chain carrying heavy chain variable (VH) and heavy chain constant regions (CH), and a second polypeptide chain carrying light chain variable (VL) and light chain constant regions (CL) (e.g., Figure 1), wherein the host cell, or a population of host cells harbor (a) a first vector is operably linked to a first nucleic acid that encodes the first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) a long hinge region comprising CD8 and CD28 hinge sequences (e.g., SEQ ID NO: 19), (iv) a transmembrane region (TM
  • the antibody heavy chain variable region comprises an anti-CD38 heavy chain variable region sequence (e.g., SEQ ID NO: l) and the antibody heavy chain constant region (CH) comprises an anti-CD38 heavy chain constant region sequence (e.g., SEQ ID NO:2).
  • the present disclosure provides a the vector is operably linked to a nucleic acid encoding the a Version 2 (e.g., V2) dimeric antigen receptors (DAR) construct comprising a first polypeptide chain carrying heavy chain variable (VH) and heavy chain constant regions (CH), and a second polypeptide chain carrying light chain variable (VL) and light chain constant regions (CL) (e.g., Figures 1 and 2), wherein the host cell, or a population of host cells harbor (a) a first vector operably linked to a first nucleic acid that encodes the first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) a short hinge region comprising a CD28 hinge sequence (e.g., SEQ ID NO:5), (iv) a transmembrane region (TM) comprising CD28 transmembrane sequence (e
  • VH
  • SEQ ID NO:9 or (3) 4-1BB (e.g., SEQ ID NO:7) signaling sequence and CD28 (e.g., SEQ ID NO:8) signaling sequence and CD3-zeta having ITAM motifs 1, 2 and 3 (e.g., SEQ ID NO:9);
  • 4-1BB e.g., SEQ ID NO:7
  • CD28 e.g., SEQ ID NO:8
  • CD3-zeta having ITAM motifs 1, 2 and 3 e.g., SEQ ID NO:9;
  • the first vector is operably linked to a second nucleic acid that encodes the second
  • the vector is operably linked to a nucleic acid encoding the Version 2a (V2a) DAR construct comprising the intracellular signaling region having the 4-1BB signaling sequence (e.g., SEQ ID NO:7) and CD3-zeta having IT AM motifs 1, 2 and 3 (e.g.,
  • the vector is operably linked to a nucleic acid encoding the Version 2b (V2b) DAR construct comprising the intracellular signaling region having the CD28 (e.g., SEQ ID NO:8) signaling sequence and CD3-zeta having ITAM motifs 1, 2 and 3 (e.g.,
  • the vector is operably linked to a nucleic acid encoding the Version 2c (V2c) DAR construct comprising the intracellular signaling region having the 4-1BB (e.g., SEQ ID NO:7) signaling sequence and CD28 (e.g., SEQ ID NO:8) signaling sequence and CD3-zeta having ITAM motifs 1, 2 and 3 (e.g., SEQ ID NO:9).
  • V2c Version 2c
  • the DAR V2a and V2b are second generation DAR constructs
  • the DAR V2c is a third generation DAR construct.
  • the antibody heavy chain variable region comprises an anti-CD38 heavy chain variable region sequence (e.g., SEQ ID NO: l) and the antibody heavy chain constant region (CH) comprises an anti-CD38 heavy chain constant region sequence (e.g., SEQ ID NO:2).
  • the present disclosure provides a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a Version 3 (e.g., V3) dimeric antigen receptors (DAR) construct comprising a first polypeptide chain carrying heavy chain variable (VH) and heavy chain constant regions (CH), and a second polypeptide chain carrying light chain variable (VL) and light chain constant regions (CL) (e.g., Figure 1), wherein the host cell, or a population of host cells harbor (a) a first vector operably linked to a first nucleic acid that encodes the first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) a short hinge region comprising CD28 hinge sequences (e.g., SEQ ID NO:5), (iv) a transmembrane region (TM) comprising CD
  • transmembrane sequence e.g., SEQ ID NO:6
  • an intracellular signaling region comprising 4-1BB signaling sequence (e.g., SEQ ID NO:7) and CD3-zeta signaling sequence having only ITAM motif 3 (e.g., SEQ ID NO:20)
  • the first vector operably is linked to a second nucleic acid that encodes the second polypeptide chain comprising two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region (VL) (e.g., kappa or lambda), and (ii) an antibody light chain constant region (CL).
  • VL antibody light chain variable region
  • CL antibody light chain constant region
  • the antibody heavy chain variable region comprises an anti-CD38 heavy chain variable region sequence (e.g., SEQ ID NO: l) and the antibody heavy chain constant region (CH) comprises an anti-CD38 heavy chain constant region sequence (e.g., SEQ ID NO:2).
  • the present disclosure provides a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a Version 4 (e.g., V4) dimeric antigen receptors (DAR) construct comprising a first polypeptide chain carrying heavy chain variable (VH) and heavy chain constant regions (CH), and a second polypeptide chain carrying light chain variable (VL) and light chain constant regions (CL), wherein the host cell, or a population of host cells harbor (a) a first vector operably linked to a first nucleic acid that encodes the first polypeptide chain comprising four regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) a transmembrane region (TM) comprising CD28 transmembrane sequence (e.g., SEQ ID NO:6), and (iv) an intracellular signaling region comprising 4-1BB signaling sequence
  • the DAR V4 construct lacks a hinge sequence.
  • the antibody heavy chain variable region (VH) comprises an anti- CD38 heavy chain variable region sequence (e.g., SEQ ID NO: l) and the antibody heavy chain constant region (CH) comprises an anti-CD38 heavy chain constant region sequence (e.g., SEQ ID NO:2).
  • the present disclosure further provides methods for conducting adoptive cell therapy by administering to a subject transgenic host cells that have been engineered to express the dimeric antigen receptor constructs.
  • the present disclosure further provides a method of treating a subject having a disease, disorder or condition associated with detrimental expression (e.g., elevated expression) of a tumor antigen.
  • a method includes, for example, administering to a subject a host cell harboring an expression vector operably linked to a nucleic acid that encodes any of the first polypeptide chains or second polypeptide chains, or any of the first and second polypeptide chains, or any of the precursor polypeptide chains described herein.
  • the host cell or the population of host cells express any of the first and second polypeptide chains, or any of the precursor polypeptide chains described herein.
  • the host cell or population of host cells used to treat a subject are introduced with one or more expression vectors, where the vectors are operably linked to a nucleic acid transgene encoding any of the dimeric antigen receptor (DAR) constructs described herein.
  • the host cell or the population of host cells comprise T lymphocytes (e.g., T cells, regulatory T cells, gamma-delta T cells, and cytotoxic T cells), NK (natural killer) cells, macrophages, dendritic cells, mast cells, eosinophils, B lymphocytes, monocytes.
  • the NK cells comprise cord blood-derived NK cells, or placental derived NK cells.
  • the host cell or population of host cells that are used to treat a subject harbor one or more expression vectors that can direct transient introduction of the transgene into the host cells or stable insertion of the transgene into the host cells’ genome.
  • the expression vector(s) can direct transcription and/or translation of the transgene in the host cell.
  • the transgene comprises a nucleic acid that encodes any of the first polypeptide chains or second polypeptide chains, or any of the first and second polypeptide chains, or any of the precursor polypeptide chains described herein.
  • the expression vector can include nucleic acid backbone sequences derived from a retrovirus, lentivirus or adenovirus.
  • the expression vectors can include one or more regulatory sequences, such as inducible and/or constitutive promoters and enhancers.
  • the expression vectors can include ribosomal binding sites and/or polyadenylation sites.
  • the expression vector which is operably linked to the nucleic acid encoding the dimeric antigen receptor (DAR) construct, can direct production of the dimeric antigen receptor (DAR) construct which can be displayed on the surface of the transgenic host cell or the dimeric antigen receptor can be secreted into the cell culture medium.
  • host cells can harbor one or more expression vectors operably linked to the nucleic acid transgene that encodes any of the dimeric antigen receptors, and the host cell can be cultured in an appropriate culture medium to transiently or stably express a dimeric antigen receptor construct.
  • the host cell or population of host cells used to treat the subject are autologous and are derived from the subject receiving the treatment.
  • whole blood can be obtained from the subject and the desired cells (e.g., T lymphocytes, NK cells or macrophages) can be recovered from the whole blood.
  • the host cell or population of host cells used to treat the subject are allogenic and are derived from a different subject.
  • Allogenic cells can be obtained from whole blood from a different subject in the same manner employed for the autologous cells.
  • the allogenic cells are derived from placenta or chord tissue after pregnancy.
  • the desired cells are obtained from the subject to receive treatment, or from a different subject, and are engineered to harbor one or more expression vectors that direct expression of any of the first or second polypeptides, or the precursor polypeptides, thereby generating transgenic host cells.
  • the transgenic host cells can express the first or second polypeptides, or the precursor polypeptide.
  • the host cells can express the first and second polypeptide chains which dimerize to form a dimeric antigen receptor that binds specifically to the tumor antigen in the subject.
  • the host cells can express the precursor polypeptide chain which can be cleaved to form first and second polypeptide chains that dimerize to form a dimeric antigen receptor that binds specifically to the tumor antigen in the subject.
  • the transgenic host cells (e.g., harboring the expression vector(s) or expressing the polypeptide chains) can be administered to the subject to treat the disease, disorder or condition associated with detrimental expression of a tumor antigen.
  • the present disclosure provides a method of treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen, wherein the disorder is cancer, including, but not limited to hematologic breast cancer, ovarian cancer, prostate cancer, head and neck cancer, lung cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer, liver cancer, renal cancer, esophageal cancer, leiomyoma, leiomyosarcoma, glioma, and glioblastoma.
  • cancer including, but not limited to hematologic breast cancer, ovarian cancer, prostate cancer, head and neck cancer, lung cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer, liver cancer, renal cancer, esophageal cancer, leiomyoma, leiomyosarcoma, glioma, and glioblastoma.
  • the cancer is a hematologic cancer selected from the group consisting of non-Hodgkin's lymphoma (NHL), Burkitf s lymphoma (BL), B chronic
  • B-CLL B and T acute lymphocytic leukemia
  • ALL T cell lymphoma
  • AML acute myeloid leukemia
  • HCL hairy cell leukemia
  • HCL Hodgkin's Lymphoma
  • HL chronic myeloid leukemia
  • CML chronic myeloid leukemia
  • MM multiple myeloma
  • the present disclosure further provides a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen, comprises: administering to the subject a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a first polypeptide chain comprising: five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region, (ii) an antibody heavy chain constant region, (iii) an optional hinge region, (iv) a transmembrane region, and (v) an intracellular signaling region (e.g., Figures 1 and 2).
  • the present disclosure further provides a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen, comprises: administering to the subject a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region, and (ii) an antibody light chain constant region (e.g.,
  • the present disclosure further provides a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen, comprises: administering to the subject a host cell, or a population of host cells, which harbors a first and second expression vector, wherein the first expression vector is operably linked to a nucleic acid that encodes the first polypeptide chain and the second expression vector is operably linked to a nucleic acid that encodes the second polypeptide chain, wherein (a) the first polypeptide chain comprises five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region, (ii) an antibody heavy chain constant region, (iii) an optional hinge region, (iv) a transmembrane region, and (v) an intracellular signaling region having two to five intracellular signaling sequences; and wherein (b) the second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region, and (
  • the present disclosure further provides a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen, comprises: administering to the subject a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes the first and second polypeptide chains, comprising: (a) a first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region, (ii) an antibody heavy chain constant region, (iii) an optional hinge region, (iv) a transmembrane region, and (v) an intracellular signaling region having two to five intracellular signaling sequences; and (b) a second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region, and (ii) an antibody light chain constant region (e.g., Figures 1 and 2).
  • a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen comprises: administering to the subject a host cell, or a population of host cells, which expresses the first polypeptide chains, the second polypeptide chains, or the first and second polypeptide chains (e.g., Figures 1 and 2).
  • the first and second polypeptide chains dimerize to form a dimeric antigen receptor that binds specifically to the tumor antigen in the subject.
  • the present disclosure further provides a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen, comprises: administering to the subject a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a first polypeptide chain comprising: five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region, (ii) an antibody light chain constant region, (iii) an optional hinge region, (iv) a transmembrane region, and (v) an intracellular signaling region (e.g., Figures 3 and 4).
  • the present disclosure further provides a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen, comprises: administering to the subject a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region, and (ii) an antibody heavy chain constant region (e.g., Figures 3 and 4).
  • the present disclosure further provides a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen, comprises: administering to the subject a host cell, or a population of host cells, which harbors a first and second expression vector, wherein the first expression vector is operably linked to a nucleic acid that encodes the first polypeptide chain and the second expression vector is operably linked to a nucleic acid that encodes the second polypeptide chain, wherein (a) the first polypeptide chain comprises five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region, (ii) an antibody light chain constant region, (iii) an optional hinge region, (iv) a transmembrane region, and (v) an intracellular signaling region having two to five intracellular signaling sequences; and wherein (b) the second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region, and
  • the present disclosure further provides a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen, comprises: administering to the subject a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes the first and second polypeptide chains, comprising: (a) a first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region, (ii) an antibody light chain constant region, (iii) an optional hinge region, (iv) a transmembrane region, and (v) an intracellular signaling region having two to five intracellular signaling sequences; and (b) a second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region, and (ii) an antibody heavy chain constant region (e.g., Figures 3 and 4).
  • a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen comprises: administering to the subject a host cell, or a population of host cells, which expresses the first polypeptide chains, the second polypeptide chains, or the first and second polypeptide chains (e.g., Figures 3 and 4).
  • the first and second polypeptide chains dimerize to form a dimeric antigen receptor that binds specifically to the tumor antigen in the subject.
  • the present disclosure further provides a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen, comprises: administering to the subject a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a first polypeptide chain comprising: six regions ordered from the amino terminus to the carboxyl terminus: (i) a heavy chain leader region, (ii) an antibody heavy chain variable region, (iii) an antibody heavy chain constant region, (iv) an optional hinge region, (v) a transmembrane region, and (vi) an intracellular signaling region (e.g., Figures 1 and 2).
  • the present disclosure further provides a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen, comprises: administering to the subject a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a second polypeptide chain comprising: three regions ordered from the amino terminus to the carboxyl terminus: (i) a light chain leader region, (ii) an antibody light chain variable region, and (iii) an antibody light chain constant region (e.g., Figures 1 and 2).
  • the present disclosure further provides a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen, comprises: administering to the subject a host cell, or a population of host cells, which harbors a first and second expression vector, wherein the first expression vector is operably linked to a nucleic acid that encodes the first polypeptide chain and the second expression vector is operably linked to a nucleic acid that encodes the second polypeptide chain, wherein (a) the first polypeptide chain comprising six regions ordered from the amino terminus to the carboxyl terminus: (i) a heavy chain leader sequence, (ii) an antibody heavy chain variable region, (iii) an antibody heavy chain constant region, (iv) an optional hinge region, (v) a transmembrane region, and (vi) an intracellular signaling region having two to five intracellular signaling sequences; and wherein (b) the second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus:
  • the present disclosure further provides a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen, comprises: administering to the subject a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes the first and second polypeptide chains, comprising: (a) a first polypeptide chain comprising six regions ordered from the amino terminus to the carboxyl terminus: (i) a heavy chain leader sequence, (ii) an antibody heavy chain variable region, (iii) an antibody heavy chain constant region, (iv) an optional hinge region, (v) a transmembrane region, and (vi) an intracellular signaling region having two to five intracellular signaling sequences; and (b) a second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody light chain variable region, and (ii) an antibody light chain constant region (e.g.,
  • a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen comprises: administering to the subject a host cell, or a population of host cells, which expresses the first polypeptide chains, the second polypeptide chains, or the first and second polypeptide chains (e.g., Figures 1 and 2).
  • the first and second polypeptide chains dimerize to form a dimeric antigen receptor that binds specifically to the tumor antigen in the subject.
  • the present disclosure further provides a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen, comprises: administering to the subject a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a first polypeptide chain comprising: six regions ordered from the amino terminus to the carboxyl terminus: (i) a light chain leader region, (ii) an antibody light chain variable region, (iii) an antibody light chain constant region, (iv) an optional hinge region, (v) a transmembrane region, and (vi) an intracellular signaling region ( Figures 3 and 4).
  • the present disclosure further provides a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen, comprises: administering to the subject a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a second polypeptide chain comprising: three regions ordered from the amino terminus to the carboxyl terminus: (i) a heavy chain leader region, (ii) an antibody heavy chain variable region, and (iii) an antibody light chain constant region ( Figures 3 and 4).
  • the present disclosure further provides a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen, comprises: administering to the subject a host cell, or a population of host cells, which harbors a first and second expression vector, wherein the first expression vector is operably linked to a nucleic acid that encodes the first polypeptide chain and the second expression vector is operably linked to a nucleic acid that encodes the second polypeptide chain, wherein (a) the first polypeptide chain comprising six regions ordered from the amino terminus to the carboxyl terminus: (i) a light chain leader sequence, (ii) an antibody light chain variable region, (iii) an antibody light chain constant region, (iv) an optional hinge region, (v) a transmembrane region, and (vi) an intracellular signaling region having two to five intracellular signaling sequences; and wherein (b) the second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus:
  • the present disclosure further provides a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen, comprises: administering to the subject a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes the first and second polypeptide chains, comprising: (a) a first polypeptide chain comprising six regions ordered from the amino terminus to the carboxyl terminus: (i) a light chain leader sequence, (ii) an antibody light chain variable region, (iii) an antibody light chain constant region, (iv) an optional hinge region, (v) a transmembrane region, and (vi) an intracellular signaling region having two to five intracellular signaling sequences; and (b) a second polypeptide chain comprising: two regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region, and (ii) an antibody heavy chain constant region ( Figures 3 and 4).
  • a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen comprises: administering to the subject a host cell, or a population of host cells, which expresses the first polypeptide chains, the second polypeptide chains, or the first and second polypeptide chains ( Figures 3 and 4).
  • the first and second polypeptide chains dimerize to form a dimeric antigen receptor that binds specifically to the tumor antigen in the subject.
  • the present disclosure further provides a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen, comprises: administering to the subject a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a precursor polypeptide comprising: ten regions ordered from the amino terminus to the carboxyl terminus: (1) a heavy chain leader region, (2) an antibody heavy chain variable region, (3) an antibody heavy chain constant region, (4) an optional hinge region, (5) a transmembrane region, (6) an intracellular signaling region having two to five intracellular signaling sequences, (7) T2A cleavable sequence region, (8) a light chain leader region, (9) an antibody light chain variable region, and (10) an antibody light chain constant region (e.g., Figures 5 and 6).
  • the precursor polypeptide is cleaved to form first and second polypeptide chains that dimerize to form a dimeric antigen receptor that bind
  • the present disclosure further provides a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen, comprises: administering to the subject a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a precursor polypeptide comprising: ten regions ordered from the amino terminus to the carboxyl terminus: (1) a light chain leader region, (2) an antibody light chain variable region, (3) an antibody light chain constant region, (4) an optional hinge region, (5) a transmembrane region, (6) an intracellular signaling region having two to five intracellular signaling sequences, (7) T2A cleavable sequence region, (8) a heavy chain leader region, (9) an antibody heavy chain variable region, and (10) an antibody heavy chain constant region ( Figures 7 and 8).
  • the precursor polypeptide is cleaved to form first and second polypeptide chains that dimerize to form a dimeric antigen receptor that binds specifically to the tumor
  • a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen comprises: administering to the subject a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) a CD38 antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: l, (ii) a CD38 antibody heavy chain constant region comprising the amino acid sequence of SEQ ID NO:2, (iii) a CD28 hinge region comprising the amino acid sequence of SEQ ID NO:5, (iv) a CD28 transmembrane region comprising the amino acid sequence of SEQ ID NO:6, and (v) an intracellular signaling region comprising a 4-1BB intracellular signaling sequence comprising the amino acid sequence of SEQ ID NO:7 and a CD3zeta intracellular signaling sequence comprising the
  • a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen comprises: administering to the subject a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a second polypeptide chain comprising two regions ordered from the amino terminus to the carboxyl terminus: (i) a CD38 antibody light chain variable region comprising the amino acid sequence of SEQ ID NO:3, and (ii) a CD38 antibody light chain constant region comprising the amino acid sequence of SEQ ID NO:4.
  • a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen comprises: administering to the subject a host cell, or a population of host cells, which harbors a first and second expression vector, wherein the first expression vector is operably linked to a nucleic acid that encodes the amino acid sequence of SEQ ID NO: 13 or 16 and the second expression vector is operably linked to a nucleic acid that encode the amino acid sequence of SEQ ID NO: 14 or 17.
  • a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen comprises: administering to the subject a host cell, or a population of host cells, which harbors a vector that is operably linked to a nucleic acid that encode the first and second polypeptide chains wherein the vector comprises the amino acid sequences of SEQ ID NOS: 13 and 14, or SEQ ID NOS: 16 and 17.
  • a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen comprises: administering to the subject a host cell, or a population of host cells, which harbors an expression vector encoding a precursor polypeptide comprising the amino acid sequence of SEQ ID NO: 15 or 18 (e.g., CD38 precursor, 2 nd generation).
  • a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen comprises: administering to the subject a host cell, or a population of host cells, which expresses the first polypeptide chains (SEQ ID NO: 13 or 16), the second polypeptide chains (SEQ ID NO: 14 or 17), the first and second polypeptide chains (SEQ ID NOS: 13 and 14, or SEQ ID NO: 16 and 17) or the precursor polypeptide (SEQ ID NO: 15 or 18).
  • the first polypeptide chain (SEQ ID NO: 13 or 16) includes or lacks the leader sequence underlined in Figure 35B or C.
  • the second polypeptide chain (SEQ ID NO: 14 or 17) includes or lacks the leader sequence underlined in Figure 35B or C.
  • the first and second polypeptide chains dimerize to form a dimeric antigen receptor that binds specifically to the tumor antigen in the subject.
  • the precursor polypeptide is cleaved to form first and second polypeptide chains that dimerize to form a dimeric antigen receptor that binds specifically to the tumor antigen (e.g., CD38) in the subject.
  • the present disclosure provides a method of treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen, wherein the disorder is cancer, including, but not limited to hematologic breast cancer, ovarian cancer, prostate cancer, head and neck cancer, lung cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer, liver cancer, renal cancer, esophageal cancer, leiomyoma, leiomyosarcoma, glioma, and glioblastoma.
  • cancer including, but not limited to hematologic breast cancer, ovarian cancer, prostate cancer, head and neck cancer, lung cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer, liver cancer, renal cancer, esophageal cancer, leiomyoma, leiomyosarcoma, glioma, and glioblastoma.
  • the cancer is a hematologic cancer selected from the group consisting of non-Hodgkin's lymphoma (NHL), Burkitf s lymphoma (BL), B chronic
  • B-CLL B and T acute lymphocytic leukemia
  • ALL T cell lymphoma
  • AML acute myeloid leukemia
  • HCL hairy cell leukemia
  • HCL Hodgkin's Lymphoma
  • HL chronic myeloid leukemia
  • CML chronic myeloid leukemia
  • MM multiple myeloma
  • the present disclosure provides a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen, the method comprising: administering to the subject a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a Version 1 (e.g., VI) dimeric antigen receptors (DAR) construct comprising a first polypeptide chain carrying heavy chain variable (VH) and heavy chain constant regions (CH), and a second polypeptide chain carrying light chain variable (VL) and light chain constant regions (CL) (e.g., Figure 1), wherein the host cell, or a population of host cells harbor (a) a first vector is operably linked to a first nucleic acid that encodes the first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) a long hinge region
  • VH
  • the antibody heavy chain variable region comprises an anti-CD38 heavy chain variable region sequence (e.g., SEQ ID NO: l) and the antibody heavy chain constant region (CH) comprises an anti-CD38 heavy chain constant region sequence (e.g., SEQ ID NO:2).
  • the present disclosure provides a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen, the method comprising: administering to the subject a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a Version 2 (e.g., V2) dimeric antigen receptors (DAR) construct comprising a first polypeptide chain carrying heavy chain variable (VH) and heavy chain constant regions (CH), and a second polypeptide chain carrying light chain variable (VL) and light chain constant regions (CL) (e.g., Figures 1 and 2), wherein the host cell, or a population of host cells harbor (a) a first vector operably linked to a first nucleic acid that encodes the first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) a short hinge
  • the Version 2a (V2a) DAR construct comprises the intracellular signaling region having the 4-1BB signaling sequence (e.g., SEQ ID NO:7) and CD3-zeta having ITAM motifs 1, 2 and 3 (e.g., SEQ ID NO:9).
  • the Version 2b (V2b) DAR construct comprises the intracellular signaling region having the CD28 (e.g., SEQ ID NO:8) signaling sequence and CD3-zeta having ITAM motifs 1, 2 and 3 (e.g., SEQ ID NO:9).
  • the Version 2c (V2c) DAR construct comprises the intracellular signaling region having the 4-1BB (e.g., SEQ ID NO:7) signaling sequence and CD28 (e.g., SEQ ID NO:8) signaling sequence and CD3-zeta having ITAM motifs 1, 2 and 3 (e.g., SEQ ID NO:9).
  • the DAR V2a and V2b are second generation DAR constructs, while the DAR V2c is a third generation DAR construct.
  • the antibody heavy chain variable region comprises an anti-CD38 heavy chain variable region sequence (e.g., SEQ ID NO: l) and the antibody heavy chain constant region (CH) comprises an anti-CD38 heavy chain constant region sequence (e.g., SEQ ID NO:2).
  • the present disclosure provides a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen, the method comprising: administering to the subject a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a Version 3 (e.g., V3) dimeric antigen receptors (DAR) construct comprising a first polypeptide chain carrying heavy chain variable (VH) and heavy chain constant regions (CH), and a second polypeptide chain carrying light chain variable (VL) and light chain constant regions (CL) (e.g., Figure 1), wherein the host cell, or a population of host cells harbor (a) a first vector operably linked to a first nucleic acid that encodes the first polypeptide chain comprising five regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) a short hinge region comprising a first
  • the antibody heavy chain variable region comprises an anti-CD38 heavy chain variable region sequence (e.g., SEQ ID NO: 1) and the antibody heavy chain constant region (CH) comprises an anti-CD38 heavy chain constant region sequence (e.g., SEQ ID NO:2).
  • the present disclosure provides a method for treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen, the method comprising: administering to the subject a host cell, or a population of host cells, which harbors an expression vector operably linked to a nucleic acid that encodes a Version 4 (e.g., V4) dimeric antigen receptors (DAR) construct comprising a first polypeptide chain carrying heavy chain variable (VH) and heavy chain constant regions (CH), and a second polypeptide chain carrying light chain variable (VL) and light chain constant regions (CL), wherein the host cell, or a population of host cells harbor (a) a first vector operably linked to a first nucleic acid that encodes the first polypeptide chain comprising four regions ordered from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) a transmembrane region (TM) comprising CD28
  • VH
  • the DAR V4 construct lacks a hinge sequence.
  • the antibody heavy chain variable region (VH) comprises an anti- CD38 heavy chain variable region sequence (e.g., SEQ ID NO: l) and the antibody heavy chain constant region (CH) comprises an anti-CD38 heavy chain constant region sequence (e.g., SEQ ID NO:2).
  • the present disclosure provides a method of treating a subject having a disease, disorder or condition associated with detrimental expression of a tumor antigen, wherein the disorder is cancer, including, but not limited to hematologic breast cancer, ovarian cancer, prostate cancer, head and neck cancer, lung cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer, liver cancer, renal cancer, esophageal cancer, leiomyoma, leiomyosarcoma, glioma, and glioblastoma.
  • cancer including, but not limited to hematologic breast cancer, ovarian cancer, prostate cancer, head and neck cancer, lung cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer, liver cancer, renal cancer, esophageal cancer, leiomyoma, leiomyosarcoma, glioma, and glioblastoma.
  • the cancer is a hematologic cancer selected from the group consisting of non-Hodgkin's lymphoma (NHL), Burkitf s lymphoma (BL), B chronic
  • B-CLL B and T acute lymphocytic leukemia
  • ALL T cell lymphoma
  • AML acute myeloid leukemia
  • HCL hairy cell leukemia
  • HCL Hodgkin's Lymphoma
  • HL chronic myeloid leukemia
  • CML chronic myeloid leukemia
  • MM multiple myeloma
  • Example 1 Isolation of human PBMC Cells and primary T cells
  • T cells Primary human T cells were isolated from healthy human donors either from huffy coats (San Diego blood bank), fresh blood or leukapheresis products (StemCell). Peripheral blood mononuclear cells were isolated by density gradient centrifugation. T cells were isolated from PBMCs by magnetic negative selection using EasySepTM Human T Cell Isolation Kit (STEMCELL) according to manufacturer’s instructions.
  • Example 2 Primary T cell culture
  • T cells were cultured in CTSTM OpTmizerTM T Cell Expansion SFM supplemented with 5% CTSTM Immune Cell SR (Thermo Fisher Scientific) with 300U/mL IL-2 (Proleukin) at a density of 10 6 cells per mL. Isolated T cells were stimulated freshly or from the frozen tank. Cells were activated with T Cell TransAct (Miltenyi) 3uL/ 10 6 cells per mL for two to three days. Following transfection, T cells were cultured in media with IL-2 at 300U/mL.
  • CTSTM OpTmizerTM T Cell Expansion SFM supplemented with 5% CTSTM Immune Cell SR (Thermo Fisher Scientific) with 300U/mL IL-2 (Proleukin) at a density of 10 6 cells per mL. Isolated T cells were stimulated freshly or from the frozen tank. Cells were activated with T Cell TransAct (Miltenyi) 3uL/ 10 6 cells per mL for two
  • RPMI 8226 Multiple myeloma cell line RPMI 8226 was obtained from ATCC and were transduced using a lentivirus carrying luciferase and GFP genes. A single cell clone with luciferase and GFP expression was selected (RPMI8226-FLuc). K562/RPE cells were made similarly by transducing the K562 cells with lentivirus carrying RPE genes. Both cell lines were cultured in RPMI1640 medium (ATCC) supplemented with 10% fetal bovine serum (Sigma).
  • Activated T cells were introduced with nucleic acids encoding either a CAR or DAR construct.
  • the nucleic acid encoding the anti-CD38 CAR polypeptide included a heavy chain signal peptide (SEQ ID NO: 10) followed by 2 additional amino acid residues Asp and Ile, a myc tag EQKLISEEDL (SEQ ID NO: 22), the anti-CD38 heavy chain variable region (SEQ ID NO: l), 15 amino acid linker GGGGS GGGGS GGGGS (SEQ ID NO:23), the anti-CD38 light chain variable region (SEQ ID NO:3), CD8 hinge region (SEQ ID NO:2l), CD28 hinge region (SEQ ID NO:5), CD28 transmembrane region (SEQ ID NO:6), CD28 intracellular signaling region (SEQ ID NO: 8), and CD3-zeta intracellular signaling region (SEQ ID NO: 9).
  • the full- length anti-CD38 CAR construct has the amino acid sequence of SEQ ID NO:22.
  • nucleic acids encoding the anti-CD38 DAR precursor polypeptides comprise the amino acid sequence of SEQ ID NO: 15 or 18.
  • the CAR, DAR and control T cells were subjected to nutrient starvation overnight with IL-2.
  • the cells were co-cultured with the target cell mixture of CD38 positive RPMI-8226/GFP cells or CD38 negative K562/RPE cells.
  • the ratio of effector to target cell ranged from 5 : 1 to 0.08 : 1.
  • the cells were subjected to flow cytometry to measure the GFP cell population to determine the specific target cell killing by anti-CD38A2 CAR and DAR T cells.
  • Example 7 Detecting effector memory T cells
  • the DAR T cells were washed with DPBS 5% human serum albumin, then stained with anti-CD3-BV42l antibody (SK7, BioLegend) and PE or APC conjugated CD38-Fc protein (Chimerigen Laboratories) for 30-60 minutes at 4 °C.
  • the CD3 and CD38 were detected using iQue Screener Plus (Intellicyte Co). Markers for identifying effector memory T cells and central memory fraction of T cells were CD45RO (BioLegend) and CCR7 (BioLegend).
  • Example 8 In vivo tumor killing
  • RPMI8226 xenograft mouse model Multiple myeloma cell line RPMI8226 obtained from ATCC were transfected by a lentiviral vector with luciferase and GFP genes. A single clone with luciferase and GFP expression was selected (RPMI8226-FLuc). A total 7 x 10 6 cells of RPMI8226-Fluc were suspended in 200 pL PBS, and then injected intravenously into the tail vein of each mouse. The animals with very small or very large tumor burden were excluded based on the bioluminescence from IVIS imaging. The animals selected in study were randomized in different groups.
  • a single treatment of about 10 million engineered CAR or DAR T cells was administered via the tail vein in 200 pL of PBS at three weeks after tumor inoculation.
  • the same amount of ATC or TRAC KO T cells, or 200 pL of PBS, were administered intravenously as the treatment control groups.
  • Tumor growth was monitored by measuring total photon flux with an IVIS Lumina III In Vivo Imaging System (Perkin Elmer Health Sciences, Inc) on the dorsal side of each mouse weekly after tumor cell inoculation until 4 or 5 weeks post treatment. The images were taken about 10 to 20 minutes after 150 mg/kg luciferin intraperitoneal administration.
  • Example 9 Results of T cells expressing DAR VI constructs
  • transgenic T cells expressing either anti-CD38 chimeric antigen receptor (CAR) or anti-CD38 dimeric antigen receptor (DAR) Version 1 were compared using flow cytometry.
  • Transgenic T cells express a higher level of the Version 1 DAR construct compared to transgenic T cells expressing the CAR construct ( Figure 9).
  • Cytokine secretion capability of T cells expressing either anti-CD38 chimeric antigen receptor (CAR) or anti-CD38 dimeric antigen receptor (DAR) Version 1 were compared in an in vitro cytokine secretion assay.
  • Transgenic T cells expressing the Version 1 DAR construct exhibited a higher level of tumor necrosis factor alpha (TNFa) secretion ( Figure 14) and IL-2 secretion (Figure 15), but lower levels of interferon-gamma (IFNy) ( Figure 13), compared to transgenic T cells expressing the CAR construct.
  • TNFa tumor necrosis factor alpha
  • IFNy interferon-gamma
  • Example 10 Results of T cells expressing DAR V2b constructs
  • Transgenic T cells express a higher level of the DAR V2b construct compared to transgenic T cells expressing the DAR VI or CAR construct ( Figure 11).
  • T cells expressing either anti-CD38 chimeric antigen receptor (CAR) or anti-CD38 dimeric antigen receptor (DAR) VI or V2b were compared in an in vitro cytotoxicity assay.
  • Transgenic T cells expressing the DAR V2b construct (C dotted line) exhibited better in vitro cell killing compared to transgenic T cells expressing the DAR VI construct (line B), and the DAR V2b expressing T cells were similar at in vitro cell killing compared to transgenic T cells expressing the CAR construct (line D) ( Figure 12).
  • T cells expressing either anti-CD38 chimeric antigen receptor (CAR) or anti-CD38 dimeric antigen receptor (DAR) V2a, V2b or V2c construct were compared in an in vitro cytotoxicity assay.
  • Transgenic T cells expressing the DAR V2b construct (“28Z” line E) exhibited markedly better in vitro cell killing compared to transgenic T cells expressing the CAR construct (line B) and T cells expressing DAR V2a (“BBZ” line C), and a similar killing capability compared to T cells expressing the DAR V2c (“28BBZ” dotted line D) ( Figure 26).
  • Cytokine secretion capability of T cells expressing either anti-CD38 chimeric antigen receptor (CAR) or anti-CD38 dimeric antigen receptor (DAR) VI or V2b were compared in an in vitro cytokine secretion assay.
  • Transgenic T cells expressing the DAR V2b construct exhibited a higher level of IL-2 secretion compared to transgenic T cells expressing CAR or DAR VI constructs ( Figure 15).
  • Transgenic T cells expressing the DAR V2b construct exhibited a slightly higher level of interferon-gamma (IFNy) secretion compared to transgenic T cells expressing the CAR construct ( Figure 13) and higher levels compared to transgenic T cells expressing the DAR VI construct.
  • IFNy interferon-gamma
  • Transgenic T cells expressing the DAR V2b construct exhibited a lower level of tumor necrosis factor alpha (TNFa) secretion compared to transgenic T cells expressing the CAR or DAR VI construct ( Figure 14).
  • TNFa tumor necrosis factor alpha
  • Flow cytometry was used to measure the capability for in vitro clonal expansion of T cells expressing either anti-CD38 chimeric antigen receptor (CAR) or anti-CD38 dimeric antigen receptor (DAR) VI or V2b, which were co-cultured with either RPMI 8226 or K562 cells.
  • Transgenic T cells expressing the DAR V2b construct exhibited a higher level of fold-change expansion compared to T cells expressing the CAR or DAR VI construct, when co-cultured with RPMI 8226 cells ( Figures 16 and 17).
  • Example 11 Results of T cells expressing DAR V2c constructs
  • transgenic T cells express either anti-CD38 chimeric antigen receptor (CAR) or anti-CD38 dimeric antigen receptor (DAR) V2a, V2b or V2c construct were compared using flow cytometry.
  • Transgenic T cells express a similar level of the DAR V2c (e.g.,“28BBZ”) construct compared to transgenic T cells expressing the DAR V2b (e.g.,
  • T cells expressing either anti-CD38 chimeric antigen receptor (CAR) or anti-CD38 dimeric antigen receptor (DAR) V2a, V2b or V2c construct were compared in an in vitro cytotoxicity assay.
  • Transgenic T cells expressing the DAR V2c construct (“28BBZ” dotted line D) exhibited a markedly higher level of in vitro cell killing compared to transgenic T cells expressing the CAR construct (line B) and DAR V2a construct (line C), but a similar level of cell killing compared to T cells expressing the DAR V2b (“28Z” line E) ( Figure 26).
  • Cytokine secretion capability of T cells expressing either anti-CD38 chimeric antigen receptor (CAR) or anti-CD38 dimeric antigen receptor (DAR) V2a or V2b construct were compared in an in vitro cytokine secretion assay.
  • Transgenic T cells expressing the DAR V2a (“BBZ”) exhibited a slightly higher level of IL-2 secretion compared to T cells expressing the DAR V2b (“28Z”) construct, and a higher level compared to transgenic T cells expressing CAR construct (Figure 27).
  • Transgenic T cells expressing the DAR V2b (“BBZ”) construct exhibited a slightly higher level of tumor necrosis factor alpha (TNFa) secretion compared to transgenic T cells expressing the CAR or DAR V2b (“28Z”) construct ( Figure 28).
  • Transgenic T cells expressing the DAR V2b (“BBZ”) construct exhibited a slightly higher level of interferon- gamma (IFNy) secretion compared to transgenic T cells expressing the CAR construct, but the cell expressing the DAR V2b (“BBZ”) exhibited a slightly lower level of IFNy secretion compared to transgenic T cells expressing the DAR V2b (“28Z”) construct ( Figure 29).
  • IFNy interferon- gamma
  • mice were administered phosphate-buffered saline (“PBS”) or a cell line carrying a knocked-out TRAC gene (“TRAC KO”).
  • PBS phosphate-buffered saline
  • TRAC KO knocked-out TRAC gene
  • the test mice were administered transgenic T cells expressing either CD38 CAR or CD38 DAR (V2a) construct.
  • V2a the mice that were treated with T cells expressing the CD38 DAR (V2a) constructs exhibited significantly less tumor burden compared to mice treated with T cells expressing a CD38 CAR construct (Figure 19).
  • the tumoricidal activity of the T cells expressing CD38 DAR V2a construct were compared directly against T cells expressing either DAR V2b or DAR V2c in another in vivo xenograft animal model.
  • the negative control mice were administered phosphate-buffered saline (“PBS”) or activated T-cells (“ATC”) or a cell line carrying a knocked-out TRAC gene (“TRAC KO”).
  • the test mice were administered transgenic T cells expressing either CD38 DAR V2a, V2b or V2c construct.
  • mice that were treated with T cells expressing the CD38 DAR V2a constructs (“BBZ”) exhibited measurably less tumor burden compared to mice treated with T cells expressing either CD38 CAR V2b (“28Z”) or V2c (“28BBZ”) construct ( Figure 30).
  • Example 12 Results of T cells expressing DAR V2a constructs
  • Transgenic T cells express a higher level of the DAR V2a (e.g., “BBZ”) construct compared to transgenic T cells expressing the DAR V2b (e.g.,“28Z”) or CAR construct (Figure 25).
  • T cells expressing either anti-CD38 chimeric antigen receptor (CAR) or anti-CD38 dimeric antigen receptor (DAR) V2a, V2b or V2c construct were compared in an in vitro cytotoxicity assay.
  • Transgenic T cells expressing the DAR V2a construct (“BBZ” line C) exhibited better in vitro cell killing compared to transgenic T cells expressing the CAR construct (line B), but a lower cell killing level compared to T cells expressing DAR V2b (“28Z” line E) or T cells expressing the DAR V2c (“28BBZ” dotted line D) ( Figure 26).
  • Cytokine secretion capability of T cells expressing either anti-CD38 chimeric antigen receptor (CAR) or anti-CD38 dimeric antigen receptor (DAR) V2a or V2b construct were compared in an in vitro cytokine secretion assay.
  • Transgenic T cells expressing the DAR V2a (“BBZ”) exhibited a slightly higher level of IL-2 secretion compared to T cells expressing the DAR V2b (“28Z”) construct, and a higher level compared to transgenic T cells expressing CAR construct (Figure 27).
  • Transgenic T cells expressing the DAR V2b (“BBZ”) construct exhibited a slightly higher level of tumor necrosis factor alpha (TNFa) secretion compared to transgenic T cells expressing the CAR or DAR V2b (“28Z”) construct ( Figure 28).
  • Transgenic T cells expressing the DAR V2b (“BBZ”) construct exhibited a slightly higher level of interferon- gamma (IFNy) secretion compared to transgenic T cells expressing the CAR construct, but the cell expressing the DAR V2b (“BBZ”) exhibited a slightly lower level of IFNy secretion compared to transgenic T cells expressing the DAR V2b (“28Z”) construct ( Figure 29).
  • IFNy interferon- gamma
  • mice An in vivo xenograft animal model was used to measure the tumoricidal activity of the CD38 DAR T cells in a xenograft animal model.
  • the negative control mice were administered phosphate-buffered saline (“PBS”) or a cell line carrying a knocked-out TRAC gene (“TRAC KO”).
  • the test mice were administered transgenic T cells expressing either CD38 CAR or CD38 DAR (V2a) construct.
  • the mice that were treated with T cells expressing the CD38 DAR (V2a) constructs exhibited significantly less tumor burden compared to mice treated with T cells expressing a CD38 CAR construct (Figure 19).
  • the tumoricidal activity of the T cells expressing CD38 DAR V2a construct were compared directly against T cells expressing either DAR V2b or DAR V2c in another in vivo xenograft animal model.
  • the negative control mice were administered phosphate-buffered saline (“PBS”) or activated T-cells (“ATC”) or a cell line carrying a knocked-out TRAC gene (“TRAC KO”).
  • the test mice were administered transgenic T cells expressing either CD38 DAR V2a, V2B or V2c construct.
  • mice that were treated with T cells expressing the CD38 DAR V2a constructs (“BBZ”) exhibited measurably less tumor burden compared to mice treated with T cells expressing either CD38 CAR V2b (“28Z”) or V2c (“28BBZ”) construct ( Figure 30).
  • Example 13 Results of T cells expressing DAR V3 construct
  • Transgenic T cells expressing anti-CD38 dimeric antigen receptor (DAR) V2a or V3 construct were compared using flow cytometry.
  • Transgenic T cells express a higher level of the DAR V3 construct compared to transgenic T cells expressing the DAR V2a ( Figures 20 and 31).
  • Cytokine secretion capability of T cells expressing anti-CD38 dimeric antigen receptor (DAR) V2a or V3 construct were compared in an in vitro cytokine secretion assay.
  • Transgenic T cells expressing the DAR V3 exhibited a markedly higher level of interferon- gamma (IFNy) secretion compared to T cells expressing the DAR V2a ( Figure 22).
  • Transgenic T cells expressing the DAR V3 exhibited a markedly higher level of tumor necrosis factor alpha (TNFa) secretion compared to transgenic T cells expressing the DAR V2a construct ( Figure 23).
  • IFNy interferon- gamma
  • TNFa tumor necrosis factor alpha
  • Cytokine secretion capability of T cells expressing anti-CD38 dimeric antigen receptor (DAR) V2a or V3 were compared in an in vitro cytokine secretion assay.
  • Transgenic T cells expressing the DAR V3 construct exhibited a markedly higher level of interferon-gamma (IFNY) secretion compared to transgenic T cells expressing the V2a construct ( Figure 33).
  • IFNY interferon-gamma
  • Transgenic T cells expressing the DAR V3 construct exhibited a lower level of tumor necrosis factor alpha (TNFa) secretion compared to transgenic T cells expressing the DAR V2a construct ( Figure 34).
  • TNFa tumor necrosis factor alpha
  • TCM central memory T cells
  • Example 14 Results of T cells expressing DAR V4 constructs
  • Transgenic T cells expressing anti-CD38 dimeric antigen receptor (DAR) V2a, V3 or V4 construct were compared using flow cytometry.
  • Transgenic T cells express a higher level of the DAR V3 construct compared to transgenic T cells expressing the DAR V2a or V4 construct ( Figure 31).
  • the DAR V4 construct lacks a hinge region, and the cell killing results indicate that a DAR construct devoid of a hinge region does not improve the cell killing capability of T cells expressing the DAR V4 construct compared to T cells expressing the V2a or V3 constructs, both of which include a short hinge region (e.g., only CD28 hinge sequence).
  • Cytokine secretion capability of T cells expressing anti-CD38 dimeric antigen receptor (DAR) V2a, V3 or V4 construct were compared in an in vitro cytokine secretion assay.
  • Transgenic T cells expressing the DAR V4 construct exhibited a markedly reduced level of interferon-gamma (IFNy) secretion compared to T cells expressing DAR V3 construct ( Figure 33).
  • Transgenic T cells expressing the DAR V4 construct exhibited a lower level of tumor necrosis factor alpha (TNFa) secretion compared to transgenic T cells expressing the DAR V3 construct ( Figure 34).
  • IFNy interferon-gamma
  • TNFa tumor necrosis factor alpha

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WO2021046445A1 (en) * 2019-09-05 2021-03-11 Sorrento Therapeutics, Inc. Dimeric antigen receptors (dar) that bind bcma
WO2021174124A1 (en) * 2020-02-27 2021-09-02 Sorrento Therapeutics, Inc. Dimeric antigen receptors (dar) that bind cd20
JP2023516595A (ja) * 2020-02-27 2023-04-20 ソレント・セラピューティクス・インコーポレイテッド Ｃｄ２０を結合する二量体抗原受容体（ｄａｒ）
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